why are hla alleles associated with drug hypersensitivity? · pdf file1. introduction: hla...
TRANSCRIPT
Why are HLA alleles associated with drug hypersensitivity
Disclosure
In relation to this presentation I declare that there are no conflicts of interest
A conflict of interest is any situation in which a speaker or immediate family members have interests and those may cause a conflict with the current presentation Conflicts of interest do not preclude the delivery of the talk but should be explicitly declared These may include financial interests (eg owning stocks of a related company having received honoraria consultancy fees) research interests (research support by grants or otherwise) organisational interests and gifts
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Drug HLA
Allele
HLA Carriage Rate Disease
Prevalence
OR NPV PPV NNT to
prevent
ldquo1rdquo
Abacavir
Hypersensitivity
Syndrome
B5701 5-8 Caucasian
lt1 AfricanAsia
25 African
American
8 (3 true
HSR and 2-7
false positive
diagnosis
960 100 for patch
test confirmed
55 13
Allopurinol
SJSTEN and
DRESSDIHS
B5801 9-11 Han Chinese
1-6 Caucasian
1250-11000 gt80
0
100 in Han
Chinese
3 250
Carbamazepine
SJSTEN
B1502 10-15 Han
Chinese
lt01 Caucasian
1-4 Han
Chinese
20 100 in Han
Chinese (with
other B75
serotype)
3 1000
Dapsone
DRESSDHIS
B1301 2-20 Chinese
28
PapuansAustralian
Aboriginals
0
EuropeanAfrican
15 Japanese
1-4 Han
Chinese
20 998 78 84
Flucloxacillin
DILI
B5701 As above 85100 000 81 9999 012
13819
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Identification of peptides recognized by T cells from abacavir
hypersensitivity patients
We identified endogenous peptides that bind HLA-B5701 better in the presence of abacavir
IC50 (nM) for HLA-B5701 bindingPeptide no abacavir + abacavir Source
KVAKVEPAV 402856 02 ribosome binding protein 1 homolog (RRBP1)
KSNGTIIHV 109856 03 CD28
KIYEGQVEV 94033 02 60S ribosomal protein L5
TVAPFNPTV 89929 01 S100P-binding protein isoform A
VTTDIQVKV 73088 02 transcription elongation factor SPT5 isoform A
RVAGIHKKV 500322 1717 60S ribosomal protein L13 isoform 1
KAAKIRVSV 46711 01 GHF-IIH subunit 2-like protein(GTF2H2C)
RTFHHGVRV 3247 01 nucleoporin 37kDa isoform CRA_a
ATIKLQSTV 3194 01 abnormal spindle protein ASP
Donor 1 Donor 2
Abacavir expanded
No drug control
T cells from patients
recognize this peptide
better in the presence
of the drug
How does the self-peptide recognized by the TCR
VTTDIQVKV corresponding to human transcription
elongation factor SPT5 isoform A 976-984 stimulated
CD8+ T cells from abacavir hypersensitivity patients
Self-peptide binds HLA-B5701 gt30000 fold better in
the presence of abacavir
Crystals of HLA-B5701 bound to abacavir and
VTTDIQVKV formed by hanging drop
X-ray data of HLA-B5701 complexed to abacavir and self
peptide
REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) 1942
REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) 28323
REMARK 3 MIN(FOBSSIGMA_FOBS) 134
REMARK 3 COMPLETENESS FOR RANGE () 9455
REMARK 3 NUMBER OF REFLECTIONS 69458
REMARK 3 NUMBER OF REFLECTIONS (NON-ANOMALOUS) 69458
REMARK 3
REMARK 3 FIT TO DATA USED IN REFINEMENT
REMARK 3 R VALUE (WORKING + TEST SET) 01874
REMARK 3 R VALUE (WORKING SET) 01852
REMARK 3 FREE R VALUE 02295
REMARK 3 FREE R VALUE TEST SET SIZE () 500
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
Does abacavir bind HLA-B5701 with the same orientation
with different peptides
Hydroxyl group of abacavir shows alternate conformations
Hydroxyl group of abacavir shows alternate conformations
4 crystal structures show H bonds between abacavir hydroxyl
group and ordered water molecules
HLA-B5701 Ser70 peptide carbonyl and ordered water
molecules form a network H bonded to abacavir hydroxyl
group
Ser70Ordered water
molecule
carbonyl
Abacavir hydroxyl
Do different peptides adopt alternate conformations when
bound to abacavir and HLA-B5701
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Disclosure
In relation to this presentation I declare that there are no conflicts of interest
A conflict of interest is any situation in which a speaker or immediate family members have interests and those may cause a conflict with the current presentation Conflicts of interest do not preclude the delivery of the talk but should be explicitly declared These may include financial interests (eg owning stocks of a related company having received honoraria consultancy fees) research interests (research support by grants or otherwise) organisational interests and gifts
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Drug HLA
Allele
HLA Carriage Rate Disease
Prevalence
OR NPV PPV NNT to
prevent
ldquo1rdquo
Abacavir
Hypersensitivity
Syndrome
B5701 5-8 Caucasian
lt1 AfricanAsia
25 African
American
8 (3 true
HSR and 2-7
false positive
diagnosis
960 100 for patch
test confirmed
55 13
Allopurinol
SJSTEN and
DRESSDIHS
B5801 9-11 Han Chinese
1-6 Caucasian
1250-11000 gt80
0
100 in Han
Chinese
3 250
Carbamazepine
SJSTEN
B1502 10-15 Han
Chinese
lt01 Caucasian
1-4 Han
Chinese
20 100 in Han
Chinese (with
other B75
serotype)
3 1000
Dapsone
DRESSDHIS
B1301 2-20 Chinese
28
PapuansAustralian
Aboriginals
0
EuropeanAfrican
15 Japanese
1-4 Han
Chinese
20 998 78 84
Flucloxacillin
DILI
B5701 As above 85100 000 81 9999 012
13819
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Identification of peptides recognized by T cells from abacavir
hypersensitivity patients
We identified endogenous peptides that bind HLA-B5701 better in the presence of abacavir
IC50 (nM) for HLA-B5701 bindingPeptide no abacavir + abacavir Source
KVAKVEPAV 402856 02 ribosome binding protein 1 homolog (RRBP1)
KSNGTIIHV 109856 03 CD28
KIYEGQVEV 94033 02 60S ribosomal protein L5
TVAPFNPTV 89929 01 S100P-binding protein isoform A
VTTDIQVKV 73088 02 transcription elongation factor SPT5 isoform A
RVAGIHKKV 500322 1717 60S ribosomal protein L13 isoform 1
KAAKIRVSV 46711 01 GHF-IIH subunit 2-like protein(GTF2H2C)
RTFHHGVRV 3247 01 nucleoporin 37kDa isoform CRA_a
ATIKLQSTV 3194 01 abnormal spindle protein ASP
Donor 1 Donor 2
Abacavir expanded
No drug control
T cells from patients
recognize this peptide
better in the presence
of the drug
How does the self-peptide recognized by the TCR
VTTDIQVKV corresponding to human transcription
elongation factor SPT5 isoform A 976-984 stimulated
CD8+ T cells from abacavir hypersensitivity patients
Self-peptide binds HLA-B5701 gt30000 fold better in
the presence of abacavir
Crystals of HLA-B5701 bound to abacavir and
VTTDIQVKV formed by hanging drop
X-ray data of HLA-B5701 complexed to abacavir and self
peptide
REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) 1942
REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) 28323
REMARK 3 MIN(FOBSSIGMA_FOBS) 134
REMARK 3 COMPLETENESS FOR RANGE () 9455
REMARK 3 NUMBER OF REFLECTIONS 69458
REMARK 3 NUMBER OF REFLECTIONS (NON-ANOMALOUS) 69458
REMARK 3
REMARK 3 FIT TO DATA USED IN REFINEMENT
REMARK 3 R VALUE (WORKING + TEST SET) 01874
REMARK 3 R VALUE (WORKING SET) 01852
REMARK 3 FREE R VALUE 02295
REMARK 3 FREE R VALUE TEST SET SIZE () 500
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
Does abacavir bind HLA-B5701 with the same orientation
with different peptides
Hydroxyl group of abacavir shows alternate conformations
Hydroxyl group of abacavir shows alternate conformations
4 crystal structures show H bonds between abacavir hydroxyl
group and ordered water molecules
HLA-B5701 Ser70 peptide carbonyl and ordered water
molecules form a network H bonded to abacavir hydroxyl
group
Ser70Ordered water
molecule
carbonyl
Abacavir hydroxyl
Do different peptides adopt alternate conformations when
bound to abacavir and HLA-B5701
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Drug HLA
Allele
HLA Carriage Rate Disease
Prevalence
OR NPV PPV NNT to
prevent
ldquo1rdquo
Abacavir
Hypersensitivity
Syndrome
B5701 5-8 Caucasian
lt1 AfricanAsia
25 African
American
8 (3 true
HSR and 2-7
false positive
diagnosis
960 100 for patch
test confirmed
55 13
Allopurinol
SJSTEN and
DRESSDIHS
B5801 9-11 Han Chinese
1-6 Caucasian
1250-11000 gt80
0
100 in Han
Chinese
3 250
Carbamazepine
SJSTEN
B1502 10-15 Han
Chinese
lt01 Caucasian
1-4 Han
Chinese
20 100 in Han
Chinese (with
other B75
serotype)
3 1000
Dapsone
DRESSDHIS
B1301 2-20 Chinese
28
PapuansAustralian
Aboriginals
0
EuropeanAfrican
15 Japanese
1-4 Han
Chinese
20 998 78 84
Flucloxacillin
DILI
B5701 As above 85100 000 81 9999 012
13819
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Identification of peptides recognized by T cells from abacavir
hypersensitivity patients
We identified endogenous peptides that bind HLA-B5701 better in the presence of abacavir
IC50 (nM) for HLA-B5701 bindingPeptide no abacavir + abacavir Source
KVAKVEPAV 402856 02 ribosome binding protein 1 homolog (RRBP1)
KSNGTIIHV 109856 03 CD28
KIYEGQVEV 94033 02 60S ribosomal protein L5
TVAPFNPTV 89929 01 S100P-binding protein isoform A
VTTDIQVKV 73088 02 transcription elongation factor SPT5 isoform A
RVAGIHKKV 500322 1717 60S ribosomal protein L13 isoform 1
KAAKIRVSV 46711 01 GHF-IIH subunit 2-like protein(GTF2H2C)
RTFHHGVRV 3247 01 nucleoporin 37kDa isoform CRA_a
ATIKLQSTV 3194 01 abnormal spindle protein ASP
Donor 1 Donor 2
Abacavir expanded
No drug control
T cells from patients
recognize this peptide
better in the presence
of the drug
How does the self-peptide recognized by the TCR
VTTDIQVKV corresponding to human transcription
elongation factor SPT5 isoform A 976-984 stimulated
CD8+ T cells from abacavir hypersensitivity patients
Self-peptide binds HLA-B5701 gt30000 fold better in
the presence of abacavir
Crystals of HLA-B5701 bound to abacavir and
VTTDIQVKV formed by hanging drop
X-ray data of HLA-B5701 complexed to abacavir and self
peptide
REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) 1942
REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) 28323
REMARK 3 MIN(FOBSSIGMA_FOBS) 134
REMARK 3 COMPLETENESS FOR RANGE () 9455
REMARK 3 NUMBER OF REFLECTIONS 69458
REMARK 3 NUMBER OF REFLECTIONS (NON-ANOMALOUS) 69458
REMARK 3
REMARK 3 FIT TO DATA USED IN REFINEMENT
REMARK 3 R VALUE (WORKING + TEST SET) 01874
REMARK 3 R VALUE (WORKING SET) 01852
REMARK 3 FREE R VALUE 02295
REMARK 3 FREE R VALUE TEST SET SIZE () 500
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
Does abacavir bind HLA-B5701 with the same orientation
with different peptides
Hydroxyl group of abacavir shows alternate conformations
Hydroxyl group of abacavir shows alternate conformations
4 crystal structures show H bonds between abacavir hydroxyl
group and ordered water molecules
HLA-B5701 Ser70 peptide carbonyl and ordered water
molecules form a network H bonded to abacavir hydroxyl
group
Ser70Ordered water
molecule
carbonyl
Abacavir hydroxyl
Do different peptides adopt alternate conformations when
bound to abacavir and HLA-B5701
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Drug HLA
Allele
HLA Carriage Rate Disease
Prevalence
OR NPV PPV NNT to
prevent
ldquo1rdquo
Abacavir
Hypersensitivity
Syndrome
B5701 5-8 Caucasian
lt1 AfricanAsia
25 African
American
8 (3 true
HSR and 2-7
false positive
diagnosis
960 100 for patch
test confirmed
55 13
Allopurinol
SJSTEN and
DRESSDIHS
B5801 9-11 Han Chinese
1-6 Caucasian
1250-11000 gt80
0
100 in Han
Chinese
3 250
Carbamazepine
SJSTEN
B1502 10-15 Han
Chinese
lt01 Caucasian
1-4 Han
Chinese
20 100 in Han
Chinese (with
other B75
serotype)
3 1000
Dapsone
DRESSDHIS
B1301 2-20 Chinese
28
PapuansAustralian
Aboriginals
0
EuropeanAfrican
15 Japanese
1-4 Han
Chinese
20 998 78 84
Flucloxacillin
DILI
B5701 As above 85100 000 81 9999 012
13819
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Identification of peptides recognized by T cells from abacavir
hypersensitivity patients
We identified endogenous peptides that bind HLA-B5701 better in the presence of abacavir
IC50 (nM) for HLA-B5701 bindingPeptide no abacavir + abacavir Source
KVAKVEPAV 402856 02 ribosome binding protein 1 homolog (RRBP1)
KSNGTIIHV 109856 03 CD28
KIYEGQVEV 94033 02 60S ribosomal protein L5
TVAPFNPTV 89929 01 S100P-binding protein isoform A
VTTDIQVKV 73088 02 transcription elongation factor SPT5 isoform A
RVAGIHKKV 500322 1717 60S ribosomal protein L13 isoform 1
KAAKIRVSV 46711 01 GHF-IIH subunit 2-like protein(GTF2H2C)
RTFHHGVRV 3247 01 nucleoporin 37kDa isoform CRA_a
ATIKLQSTV 3194 01 abnormal spindle protein ASP
Donor 1 Donor 2
Abacavir expanded
No drug control
T cells from patients
recognize this peptide
better in the presence
of the drug
How does the self-peptide recognized by the TCR
VTTDIQVKV corresponding to human transcription
elongation factor SPT5 isoform A 976-984 stimulated
CD8+ T cells from abacavir hypersensitivity patients
Self-peptide binds HLA-B5701 gt30000 fold better in
the presence of abacavir
Crystals of HLA-B5701 bound to abacavir and
VTTDIQVKV formed by hanging drop
X-ray data of HLA-B5701 complexed to abacavir and self
peptide
REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) 1942
REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) 28323
REMARK 3 MIN(FOBSSIGMA_FOBS) 134
REMARK 3 COMPLETENESS FOR RANGE () 9455
REMARK 3 NUMBER OF REFLECTIONS 69458
REMARK 3 NUMBER OF REFLECTIONS (NON-ANOMALOUS) 69458
REMARK 3
REMARK 3 FIT TO DATA USED IN REFINEMENT
REMARK 3 R VALUE (WORKING + TEST SET) 01874
REMARK 3 R VALUE (WORKING SET) 01852
REMARK 3 FREE R VALUE 02295
REMARK 3 FREE R VALUE TEST SET SIZE () 500
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
Does abacavir bind HLA-B5701 with the same orientation
with different peptides
Hydroxyl group of abacavir shows alternate conformations
Hydroxyl group of abacavir shows alternate conformations
4 crystal structures show H bonds between abacavir hydroxyl
group and ordered water molecules
HLA-B5701 Ser70 peptide carbonyl and ordered water
molecules form a network H bonded to abacavir hydroxyl
group
Ser70Ordered water
molecule
carbonyl
Abacavir hydroxyl
Do different peptides adopt alternate conformations when
bound to abacavir and HLA-B5701
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Identification of peptides recognized by T cells from abacavir
hypersensitivity patients
We identified endogenous peptides that bind HLA-B5701 better in the presence of abacavir
IC50 (nM) for HLA-B5701 bindingPeptide no abacavir + abacavir Source
KVAKVEPAV 402856 02 ribosome binding protein 1 homolog (RRBP1)
KSNGTIIHV 109856 03 CD28
KIYEGQVEV 94033 02 60S ribosomal protein L5
TVAPFNPTV 89929 01 S100P-binding protein isoform A
VTTDIQVKV 73088 02 transcription elongation factor SPT5 isoform A
RVAGIHKKV 500322 1717 60S ribosomal protein L13 isoform 1
KAAKIRVSV 46711 01 GHF-IIH subunit 2-like protein(GTF2H2C)
RTFHHGVRV 3247 01 nucleoporin 37kDa isoform CRA_a
ATIKLQSTV 3194 01 abnormal spindle protein ASP
Donor 1 Donor 2
Abacavir expanded
No drug control
T cells from patients
recognize this peptide
better in the presence
of the drug
How does the self-peptide recognized by the TCR
VTTDIQVKV corresponding to human transcription
elongation factor SPT5 isoform A 976-984 stimulated
CD8+ T cells from abacavir hypersensitivity patients
Self-peptide binds HLA-B5701 gt30000 fold better in
the presence of abacavir
Crystals of HLA-B5701 bound to abacavir and
VTTDIQVKV formed by hanging drop
X-ray data of HLA-B5701 complexed to abacavir and self
peptide
REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) 1942
REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) 28323
REMARK 3 MIN(FOBSSIGMA_FOBS) 134
REMARK 3 COMPLETENESS FOR RANGE () 9455
REMARK 3 NUMBER OF REFLECTIONS 69458
REMARK 3 NUMBER OF REFLECTIONS (NON-ANOMALOUS) 69458
REMARK 3
REMARK 3 FIT TO DATA USED IN REFINEMENT
REMARK 3 R VALUE (WORKING + TEST SET) 01874
REMARK 3 R VALUE (WORKING SET) 01852
REMARK 3 FREE R VALUE 02295
REMARK 3 FREE R VALUE TEST SET SIZE () 500
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
Does abacavir bind HLA-B5701 with the same orientation
with different peptides
Hydroxyl group of abacavir shows alternate conformations
Hydroxyl group of abacavir shows alternate conformations
4 crystal structures show H bonds between abacavir hydroxyl
group and ordered water molecules
HLA-B5701 Ser70 peptide carbonyl and ordered water
molecules form a network H bonded to abacavir hydroxyl
group
Ser70Ordered water
molecule
carbonyl
Abacavir hydroxyl
Do different peptides adopt alternate conformations when
bound to abacavir and HLA-B5701
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Identification of peptides recognized by T cells from abacavir
hypersensitivity patients
We identified endogenous peptides that bind HLA-B5701 better in the presence of abacavir
IC50 (nM) for HLA-B5701 bindingPeptide no abacavir + abacavir Source
KVAKVEPAV 402856 02 ribosome binding protein 1 homolog (RRBP1)
KSNGTIIHV 109856 03 CD28
KIYEGQVEV 94033 02 60S ribosomal protein L5
TVAPFNPTV 89929 01 S100P-binding protein isoform A
VTTDIQVKV 73088 02 transcription elongation factor SPT5 isoform A
RVAGIHKKV 500322 1717 60S ribosomal protein L13 isoform 1
KAAKIRVSV 46711 01 GHF-IIH subunit 2-like protein(GTF2H2C)
RTFHHGVRV 3247 01 nucleoporin 37kDa isoform CRA_a
ATIKLQSTV 3194 01 abnormal spindle protein ASP
Donor 1 Donor 2
Abacavir expanded
No drug control
T cells from patients
recognize this peptide
better in the presence
of the drug
How does the self-peptide recognized by the TCR
VTTDIQVKV corresponding to human transcription
elongation factor SPT5 isoform A 976-984 stimulated
CD8+ T cells from abacavir hypersensitivity patients
Self-peptide binds HLA-B5701 gt30000 fold better in
the presence of abacavir
Crystals of HLA-B5701 bound to abacavir and
VTTDIQVKV formed by hanging drop
X-ray data of HLA-B5701 complexed to abacavir and self
peptide
REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) 1942
REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) 28323
REMARK 3 MIN(FOBSSIGMA_FOBS) 134
REMARK 3 COMPLETENESS FOR RANGE () 9455
REMARK 3 NUMBER OF REFLECTIONS 69458
REMARK 3 NUMBER OF REFLECTIONS (NON-ANOMALOUS) 69458
REMARK 3
REMARK 3 FIT TO DATA USED IN REFINEMENT
REMARK 3 R VALUE (WORKING + TEST SET) 01874
REMARK 3 R VALUE (WORKING SET) 01852
REMARK 3 FREE R VALUE 02295
REMARK 3 FREE R VALUE TEST SET SIZE () 500
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
Does abacavir bind HLA-B5701 with the same orientation
with different peptides
Hydroxyl group of abacavir shows alternate conformations
Hydroxyl group of abacavir shows alternate conformations
4 crystal structures show H bonds between abacavir hydroxyl
group and ordered water molecules
HLA-B5701 Ser70 peptide carbonyl and ordered water
molecules form a network H bonded to abacavir hydroxyl
group
Ser70Ordered water
molecule
carbonyl
Abacavir hydroxyl
Do different peptides adopt alternate conformations when
bound to abacavir and HLA-B5701
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
How does the self-peptide recognized by the TCR
VTTDIQVKV corresponding to human transcription
elongation factor SPT5 isoform A 976-984 stimulated
CD8+ T cells from abacavir hypersensitivity patients
Self-peptide binds HLA-B5701 gt30000 fold better in
the presence of abacavir
Crystals of HLA-B5701 bound to abacavir and
VTTDIQVKV formed by hanging drop
X-ray data of HLA-B5701 complexed to abacavir and self
peptide
REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) 1942
REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) 28323
REMARK 3 MIN(FOBSSIGMA_FOBS) 134
REMARK 3 COMPLETENESS FOR RANGE () 9455
REMARK 3 NUMBER OF REFLECTIONS 69458
REMARK 3 NUMBER OF REFLECTIONS (NON-ANOMALOUS) 69458
REMARK 3
REMARK 3 FIT TO DATA USED IN REFINEMENT
REMARK 3 R VALUE (WORKING + TEST SET) 01874
REMARK 3 R VALUE (WORKING SET) 01852
REMARK 3 FREE R VALUE 02295
REMARK 3 FREE R VALUE TEST SET SIZE () 500
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
Does abacavir bind HLA-B5701 with the same orientation
with different peptides
Hydroxyl group of abacavir shows alternate conformations
Hydroxyl group of abacavir shows alternate conformations
4 crystal structures show H bonds between abacavir hydroxyl
group and ordered water molecules
HLA-B5701 Ser70 peptide carbonyl and ordered water
molecules form a network H bonded to abacavir hydroxyl
group
Ser70Ordered water
molecule
carbonyl
Abacavir hydroxyl
Do different peptides adopt alternate conformations when
bound to abacavir and HLA-B5701
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
X-ray data of HLA-B5701 complexed to abacavir and self
peptide
REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) 1942
REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) 28323
REMARK 3 MIN(FOBSSIGMA_FOBS) 134
REMARK 3 COMPLETENESS FOR RANGE () 9455
REMARK 3 NUMBER OF REFLECTIONS 69458
REMARK 3 NUMBER OF REFLECTIONS (NON-ANOMALOUS) 69458
REMARK 3
REMARK 3 FIT TO DATA USED IN REFINEMENT
REMARK 3 R VALUE (WORKING + TEST SET) 01874
REMARK 3 R VALUE (WORKING SET) 01852
REMARK 3 FREE R VALUE 02295
REMARK 3 FREE R VALUE TEST SET SIZE () 500
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
Does abacavir bind HLA-B5701 with the same orientation
with different peptides
Hydroxyl group of abacavir shows alternate conformations
Hydroxyl group of abacavir shows alternate conformations
4 crystal structures show H bonds between abacavir hydroxyl
group and ordered water molecules
HLA-B5701 Ser70 peptide carbonyl and ordered water
molecules form a network H bonded to abacavir hydroxyl
group
Ser70Ordered water
molecule
carbonyl
Abacavir hydroxyl
Do different peptides adopt alternate conformations when
bound to abacavir and HLA-B5701
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) 1942
REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) 28323
REMARK 3 MIN(FOBSSIGMA_FOBS) 134
REMARK 3 COMPLETENESS FOR RANGE () 9455
REMARK 3 NUMBER OF REFLECTIONS 69458
REMARK 3 NUMBER OF REFLECTIONS (NON-ANOMALOUS) 69458
REMARK 3
REMARK 3 FIT TO DATA USED IN REFINEMENT
REMARK 3 R VALUE (WORKING + TEST SET) 01874
REMARK 3 R VALUE (WORKING SET) 01852
REMARK 3 FREE R VALUE 02295
REMARK 3 FREE R VALUE TEST SET SIZE () 500
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
Does abacavir bind HLA-B5701 with the same orientation
with different peptides
Hydroxyl group of abacavir shows alternate conformations
Hydroxyl group of abacavir shows alternate conformations
4 crystal structures show H bonds between abacavir hydroxyl
group and ordered water molecules
HLA-B5701 Ser70 peptide carbonyl and ordered water
molecules form a network H bonded to abacavir hydroxyl
group
Ser70Ordered water
molecule
carbonyl
Abacavir hydroxyl
Do different peptides adopt alternate conformations when
bound to abacavir and HLA-B5701
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Does abacavir bind HLA-B5701 with the same orientation
with different peptides
Hydroxyl group of abacavir shows alternate conformations
Hydroxyl group of abacavir shows alternate conformations
4 crystal structures show H bonds between abacavir hydroxyl
group and ordered water molecules
HLA-B5701 Ser70 peptide carbonyl and ordered water
molecules form a network H bonded to abacavir hydroxyl
group
Ser70Ordered water
molecule
carbonyl
Abacavir hydroxyl
Do different peptides adopt alternate conformations when
bound to abacavir and HLA-B5701
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Hydroxyl group of abacavir shows alternate conformations
Hydroxyl group of abacavir shows alternate conformations
4 crystal structures show H bonds between abacavir hydroxyl
group and ordered water molecules
HLA-B5701 Ser70 peptide carbonyl and ordered water
molecules form a network H bonded to abacavir hydroxyl
group
Ser70Ordered water
molecule
carbonyl
Abacavir hydroxyl
Do different peptides adopt alternate conformations when
bound to abacavir and HLA-B5701
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Hydroxyl group of abacavir shows alternate conformations
4 crystal structures show H bonds between abacavir hydroxyl
group and ordered water molecules
HLA-B5701 Ser70 peptide carbonyl and ordered water
molecules form a network H bonded to abacavir hydroxyl
group
Ser70Ordered water
molecule
carbonyl
Abacavir hydroxyl
Do different peptides adopt alternate conformations when
bound to abacavir and HLA-B5701
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
4 crystal structures show H bonds between abacavir hydroxyl
group and ordered water molecules
HLA-B5701 Ser70 peptide carbonyl and ordered water
molecules form a network H bonded to abacavir hydroxyl
group
Ser70Ordered water
molecule
carbonyl
Abacavir hydroxyl
Do different peptides adopt alternate conformations when
bound to abacavir and HLA-B5701
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
HLA-B5701 Ser70 peptide carbonyl and ordered water
molecules form a network H bonded to abacavir hydroxyl
group
Ser70Ordered water
molecule
carbonyl
Abacavir hydroxyl
Do different peptides adopt alternate conformations when
bound to abacavir and HLA-B5701
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Do different peptides adopt alternate conformations when
bound to abacavir and HLA-B5701
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Crystal structure of VTTDIQVKV SPT5a 976-984 bound to
abacavir and HLA-B5701
What are the TCR contact residues
VTTDIQVKV SPT5a 976-984
P4 (Asp) and P8 (Lys)
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Are there viral sequences similar to human VTTDIQVKV
SPT5a 976-984
Sequence
Identity
VTTDIQVKV SPT5a 976-984
VTTNIQTKV HIV-1 153-161 778
VTQQAQVRL HSV12 230-238 444
VTTDSVRAL HSV1 12-20 444
Percent Identity Matrix - created by Clustal21
self
virus
This HIV-1 sequence was from NCBI not from an abacavir hypersensitivity patient
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Do viral peptides bound to abacavir and HLA-B5701
resemble VTTDIQVKV SPT5a 976-984
Colored by B factor
Colored sequence
similarity between self
and viral peptides
Blosum62 similarity values are blue 40ndash50 cyan 50ndash60 green 60ndash70 yellow 70ndash90 orange and 90ndash100 red
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
What are most the solvent exposed peptide side chains
available for TCR recognition
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
P4D P8KPotential
TCR
contact
sites
P4 (Asp) and P8 (Lys) in VTTDIQVKV SPT5a 976-984
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Self P4D
Virus P4D
Self P8K
Virus P8KPotential TCR
contact sites
In summary
1) Viruses are capable of generating sequences with the potential to form stable complexes with
drug and HLA
2) Similarity in selfvirus peptide TCR contact residues may influence crossreactive mechanisms
driving T cell responsiveness
Sharing of TCR contact residues between self and viral
epitopes may influence hypersensitivity
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Why does carbamazepine induce severe cutaneous adverse
reactions with different HLA associations
HLA-B1502 Asian
HLA-B3101 Caucasian
Peptide elution studies
show no strong
alteration of peptide
repertoire
Canonical anchors
retained at P2 and P9
in the presence of
drug
Drug does not interact
structural pockets that
accommodate anchor
residues
Different from
abacavir
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
1 Generate HLA complexes in the presence and absence of drugs
2 Characterize HLA complexes in solution (gel filtration SDS-PAGE
mass spectrometry peptide sequencing)
3 Characterize drug binding sites with drug affinity responsive target
stability (DARTS) assay
4 Structure determination by X-ray crystallography
β2-microglobulin
HLA-A3101
HLA-B1502
Codon optimized pET plasmids
Transform BL21(DE3)pLysS
Grow 6-12 liters E coli
Isolate inclusion bodies
SDS-PAGE
Refold + peptides
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Peptides for refolding with HLA-B1502 HLA-A3101 HLA-
B5801
Since the peptide sequence does appear to influence drugHLA interactions we selected
peptides likely to bind each HLA molecule
HLA-B1502
QLAGAGHSY Illing motif not natural sequence
HLASSGHSY irf7 like 195-203 natural sequence
ELRAREESY HSV T cell epitope
ELRAREEAY VZV T cell epitope
HLA-A3101
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
HLA-B5801
KTINALVYF camelpox 11-19 T cell epitope
KTAAAAAAF poly-alanine with anchors for binding
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
HLA-A3101 can be refolded with peptides +-
carbamazepine
0 00 10 00 20 00 30 00M i n T ent h
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U
-0 025
-0 000
0 025
0 050
0 075
0 100
0 125
0 150
0 175
0 200
A U670158 Kd
44 Kd
17 Kd
13 Kd
HLA-A3101
12 kD + 33 kD = 45kD
RLRDLLLIAAR Env769-779 T cell epitope
RLAAAAAAAAR poly-alanine with anchors for binding
Superdex 75
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
HLA-B1502 forms multimeric complexes when refolded with
peptides +- carbamazepine by non-denaturing PAGE
50 Kd
25 Kd
15 Kd
10 Kd
HLA-B1502
ESY peptide
ELRAREESY HSV T cell epitope
75 Kd
HLA-B1502
ESY peptide
+ carbamazepine
Higher molecular
weight complexes
with no drug
consistent with
FPLC
Fractions
from gel
filtration of
HLA
complexes
run on non-
denaturing
gel
Next mass
spec and
peptide
sequence of
multimers
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Can structural interactions between drugs and the associated
HLA molecules be defined with purified components
Takes advantage of a reduction in the protease susceptibility
of the target protein upon drug binding
Universally applicable because it requires no modification of
the drug and is independent of the mechanism of drug action
Target identification using drug affinity responsive target stability (DARTS)
Lomenick B Hao R Jonai N Chin RM Aghajan M Warburton S Wang J Wu
RP Gomez F Loo JA Wohlschlegel JA Vondriska TM Pelletier J Herschman
HR Clardy J Clarke CF Huang J
Proc Natl Acad Sci U S A 2009 Dec 22106(51)21984-9
We are attempting the DARTS method to define the carbamazepine binding site
on HLA-A3101
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Carbamazepine protects fragments of HLA-A3101
complexes from proteolysis
β2-microglobulin
HLA-A3101
+ carbamazepine - carbamazepine
1 mgml complex
Add drug or
vehicle control
Pronase at
different
concentrations
15 minutes RT
Stop reaction
SDS-PAGE
1100
1300
1300011000
110000
1100
1300
1300011000
110000
At 1300 pronase with no drug
complete destruction of HLA
At 1300 pronase with carbamazepine
fragments are protected
Ongoing experiment
sequence fragments
protected by drug
Next DART with
HLA-B5801 and
oxypurinol
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA associations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Why does sulfamethoxazole have multiple HLA
associations
SJSTEN HLA-A29 -B12 -DR7 Roujeau 1986
HLA-B38 Lonjou 2008
Fixed drug eruption HLA-A30 -B13 -Cw6 Ozkaya-Bayazip 2001
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Drug Hypersensitivity How Drugs Stimulate T Cells via Pharmacological Interaction with Immune
Receptors
Pichler WJ Adam J Watkins S Wuillemin N Yun J Yerly D
Int Arch Allergy Immunol 2015168(1)13-24
Does sulfamethoxazole have multiple HLA associations
because different mechanisms are involved
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Can effects of sulfamethoxazole on peptideHLA binding
affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
Mechanistic insights in T cell
recognition of sulfamethoxazole
may be learned from structures
of drug-specific TCRs complexed
to drugpeptideHLA complexes
Few drug specific TCR
transfectants have been
characterized but there are
several sulfamethoxazole
specific TCRs restricted to HLA-
DR1
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Can effects of sulfamethoxazole on peptideHLA-DR1
binding affinity be measured
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
ldquoThe B-LCL used as APC could
be either autologous or MHC-
matched concerning the TCR
transfectants analyzed in this
study HLA-DR1-matched B-LCL
were sufficient to activate the
transfectant cells and induce IL-2
production (data not shown)
However if B-LCL with
unmatched HLA-DR (eg HLA-
DR713) were used no IL-2
production was seen rdquo
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked
to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed
to HLA-DR1
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Is sulfamethoxazole buried in the antigen binding cleft or
bound to the solvent accessible surface
Model of sulfamethoxazole docked to NGTLNGLDYDDYVYP and
NGTLNGLDADDYVYP complexed to HLA-DR1
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Can crystallizable complexes of sulfamethoxazole bound to
peptideHLA-DR1 be generated
Mol Pharmacol 2006 Jul70(1)356-65 Transfection of drug-specific T-
cell receptors into hybridoma cells tools to monitor drug interaction
with T-cell receptors and evaluate cross-reactivity to related
compounds
Schmid DA1 Depta JP Luumlthi M Pichler WJ
TCR αβ from transfectants
+
HLA-DR1 complexed to FVNPVEAFQFKFELL
and sulfamethoxazole
(Kd=79 nM)
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
1 Introduction HLA associations reveal alternative mechanisms for
different pathologies and different drugs
2 Why is abacavir hypersensitivity strongly associated with HLA-
B57011 What is the structural composition of complexes recognized by T cells in patients
2 X-ray crystallography and peptide binding assays
3 Why does carbamazepine induce severe cutaneous adverse reactions
with different HLA assocations in different ethnic groups1 HLA-B1502 generate complexes +- drug
2 HLA-A3101 generate complexes +- drug
3 Gel filtration chromatography
4 Drug affinity responsive target stability (DARTS)
4 Why does sulfamethoxazole have multiple HLA associations 1 Different mechanisms class I class II hapten pi
2 Sulfamethoxazole enhances HLA binding of specific peptides
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Summary
1 Abacavir binds in the cleft of HLA-B5701 in 4 crystal structures
Peptides can be identified from self and viral peptides that bind
HLA with higher affinity in the presence of the drug
2 HLA-A3101 and HLA-B5801 form standard HLA monomers +-
carbamazepine or oxypurinol
3 Carbamazepine appears to alter HLA-B1502 complex formation
4 Sulfamethoxazole enhances the affinity of specific peptides for
HLA-DR1
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Thank you organizers of this meeting
Site common to B44
supertype alleles
Sites different between
B supertypes
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II
Many thanks to
NIHNIAIDThe Structural and Functional basis of HLA-
Associated Drug Hypersensitivity
R01 AI103348 6302014-6302017
Bjoern Peters
Jean Jakoncic
Elizabeth Phillips
Simon Mallal
Andrew Lucas
Don Hunt
Soren Buus
Mikkel Nors Harndahl
Alessandro Sette
Howard Grey
David M Koelle
Wen-Hung Chung Shuen-Iu Hung
Ostrov LabSusana RestrepoAustin BraceyAmzy ZhangRalph CobbMolly Kennedy
Yuri Pompeu
NSLS
NSLS-II