structure-guided design in the discovery of antimicrobials
TRANSCRIPT
Structure-Guided Design in the Discovery of Antimicrobials
Colin FishwickColin Fishwick
Professor of Medicinal ChemistryProfessor of Medicinal ChemistryUniversity of Leeds, School of ChemistryUniversity of Leeds, School of Chemistry
The threat of antibiotic resistance
“A post-antibiotic era – in which common infections and minor injuries can kill – far from being an apocalyptic fantasy, is instead a very real possibility for the 21st century” – WHO 2015
Urgent need for new antibacterial agents Urgent need for new antibacterial agents effective against bacteria resistant to multiple effective against bacteria resistant to multiple antibioticsantibiotics DUE TODUE TO::Overuse of antibioticsOveruse of antibioticsLack of new antibacterial agentsLack of new antibacterial agents SOLUTION:SOLUTION:Find new molecular targets and/or novel Find new molecular targets and/or novel inhibitors in order to combat resistanceinhibitors in order to combat resistance
KJ Simmons, I Chopra, CWG Fishwick. Structure-based discovery of antibacterial drugs Nature Rev Microbiol, 2010, 8, 501-510
In silico assisted inhibitor discovery
docking /virtual high-throughput
screening (vHTS)
X-ray structure ormodel of enzyme
SPROUT de novomolecular design
inhibitorstructures
actual compounds
enzyme assays
compoundlibraries
laboratorysynthesis
structural refinement
Ligand-basedvHTS
Example 1: Topoisomerases
• DNA is compacted by supercoiling.
• Must be relaxed to allow replication and transcription.
• Topoisomerases control supercoiling.
• Gyrase and Topo IV are Type II (ATP dependant) topoisomerases and share 40% sequence identity, though their functions are distinct and independently essential to bacterial cell survival.
• They are (i) Highly conserved; (ii) Essential; (iii) lacking a close mammalian homologue.
• Inhibiting both at same time- ‘dual targeting’ potentially slows onset of resistance
DNA Gyrase and Topoisomerase IVInhibitor binding sites
Known inhibitors, but none in clinical use
Novobiocin
ATP binding site
DNA binding siteFluoroquinolone antibiotics widely used
Moxifloxacin
OO
OH
O
OO
OHO NH2
ONH
O
HO
N
OO
HO
N
F
H
H
OMeNH
• In-house crystallographic data showing a triazolopyridine inhibitor bound to the E. faecalis GyrB ATP binding site was used as a platform in the design of novel inhibitors
SPROUT was used in a bid to mimic these interactions via a novel scaffold
N
N
N
N N
O
ONH
N
H HO
OH
OH
HN
HO
NH2H2N
HN
N55
D82
R85
Bioorg. Med. Chem. Lett., 2009, 19, 894-899
Confidential
Confidential
Primary Scaffold Screening
Compound GyrB IC50 (µM)a MIC (µg / mL)
SA EF SP HI
Triazolopyridine 0.041 8 4 8 -
2 158.3 >64 >64 >64 >64
3 6.0 >64 >64 32 >64
4 >754 >64 >64 >64 >64
5 >716 >64 >64 >64 >64
6 >530 >64 >64 >64 >64
7 >513 >64 >64 >64 >64
8 74 >64 >64 >64 >64
9 1.8 >64 >64 >64 >64
10 1.4 >64 >64 >64 >64
a E. coli GyrB ATPase assay; SA, S. aureus ATCC 29213; EF, E. faecalis ATCC 29212; SP, S. pyogenes ATCC 51339; HI, H. influenzae ATCC 51907
• ca. 5-10 compounds for each scaffold, most active shown.
Ian Yule, Sarah NarramoreIan Yule, Sarah Narramore
Primary Scaffold Screening
Compound GyrB IC50 (µM)a MIC (µg / mL)
SA EF SP HI
Triazolopyridine 0.041 8 4 8 -
2 158.3 >64 >64 >64 >64
3 6.0 >64 >64 32 >64
4 >754 >64 >64 >64 >64
5 >716 >64 >64 >64 >64
6 >530 >64 >64 >64 >64
7 >513 >64 >64 >64 >64
8 74 >64 >64 >64 >64
9 1.8 >64 >64 >64 >64
10 1.4 >64 >64 >64 >64
a E. coli GyrB ATPase assay; SA, S. aureus ATCC 29213; EF, E. faecalis ATCC 29212; SP, S. pyogenes ATCC 51339; HI, H. influenzae ATCC 51907
• ca. 5-10 compounds for each scaffold, most active shown.
Ian Yule, Sarah NarramoreIan Yule, Sarah Narramore
Primary Scaffold Screening
Compound GyrB IC50 (µM)a MIC (µg / mL)
SA EF SP HI
Triazolopyridine 0.041 8 4 8 -
2 158.3 >64 >64 >64 >64
3 6.0 >64 >64 32 >64
4 >754 >64 >64 >64 >64
5 >716 >64 >64 >64 >64
6 >530 >64 >64 >64 >64
7 >513 >64 >64 >64 >64
8 74 >64 >64 >64 >64
9 1.8 >64 >64 >64 >64
10 1.4 >64 >64 >64 >64
a E. coli GyrB ATPase assay; SA, S. aureus ATCC 29213; EF, E. faecalis ATCC 29212; SP, S. pyogenes ATCC 51339; HI, H. influenzae ATCC 51907
• ca. 5-10 compounds for each scaffold, most active shown.
Ian Yule, Sarah NarramoreIan Yule, Sarah Narramore
SAR
Compound 3
N
NH
O
NH
NH
ON
N
N
NN
NH
NH
O
NHO
Exploring ‘Compound 3’ de novo library
• Further examination of the model identified a key location into which compounds could be extended• A substantial route optimization effort was required to access structural variants of 3 which could further probe the pocket. A 35 compound library was prepared.
Optimal region for structural branching; Phosphate channel
C-4 structural Optimisation
R1 R2 GyrB IC50 (µM)* MIC (µg / mL)
SA EF SP
2-thiazolyl NMe-pip 33.2 >64 >64 >64
3-tolyl NMe-pip 18.0 >64 >64 >64
2-thiazolyl 3-pyridyl 0.56 >64 64 64
3-chlorophenyl 1-imidazolyl 0.55 >64 32 64
3-chlorophenyl 1-pyrazolyl 2.96 8 1 4
3-pyridyl NHCH2(3-py) 0.66 >64 8 8
3-tolyl NHC6H11 2.12 >64 >64 >64
2-thiazolyl 3-toluidyl 0.098 >64 >64 >64
3-tolyl 3-toluidyl 0.099 1 0.5 32
3-chlorophenyl H 1.6 16 16 16
* E. coli GyrB ATPase assay; SA, S. aureus ATCC 29213; EF, E. faecalis ATCC 29212; SP, S. pyogenes ATCC 51339
N
NH
O
NH
NH
OR1
R2
Initial structural optimisation at C4
Ian Yule, Sarah NarramoreIan Yule, Sarah Narramore
R1 R2 GyrB IC50 (µM)* MIC (µg / mL)
SA EF SP
2-thiazolyl NMe-pip 33.2 >64 >64 >64
3-tolyl NMe-pip 18.0 >64 >64 >64
2-thiazolyl 3-pyridyl 0.56 >64 64 64
3-chlorophenyl 1-imidazolyl 0.55 >64 32 64
3-chlorophenyl 1-pyrazolyl 2.96 8 1 4
3-pyridyl NHCH2(3-py) 0.66 >64 8 8
3-tolyl NHC6H11 2.12 >64 >64 >64
2-thiazolyl 3-toluidyl 0.098 >64 >64 >64
3-tolyl 3-toluidyl 0.099 1 0.5 32
3-chlorophenyl H 1.6 16 16 16
* E. coli GyrB ATPase assay; SA, S. aureus ATCC 29213; EF, E. faecalis ATCC 29212; SP, S. pyogenes ATCC 51339
N
NH
O
NH
NH
OR1
R2
Initial structural optimisation at C4
Ian Yule, Sarah NarramoreIan Yule, Sarah Narramore
Possible role of intramolecular H-bonding?
Ian Yule, Sarah NarramoreIan Yule, Sarah Narramore
N-heterocycles at C-4
R cLogPIC50s (µM) MICs (µg / mL)
GyrB ParE SA EC
0.79 49 105 64 >128
1.56 0.26 0.68 16 >128
-0.02 5.2 1.0 64 >128
0.11 8.9 1.2 >128 >128
1.22 59 5.3 64 >128
NN
NN
Br
NN
N
N
NN
N
N
NH
O
NH
NH
O
RN
Ian Yule, Sarah NarramoreIan Yule, Sarah Narramore
R1 R2 GyrB IC50 (µM)* MIC (µg / mL)
SA SA + HS EF SP
Ph NHPh 0.040 1 nd 0.25 2
3-tolyl NHPh 0.140 1 nd 0.125 2
3-Cl-Phe NHPh 0.150 0.5 nd 0.125 4
3-pyridyl NHPh 0.170 1 4 0.125 1
6-mor-3-py NHPh 0.111 2 16 0.25 1
6-acetam-3-py NHPh 0.039 2 8 0.125 8
6-triaz-3-py NHPh 0.153 >16 >16 0.25 >16
phenyl NH-3-Py 0.430 1 2 2 0.25
3-pyridyl NH-3-Py 0.093 8 nd 0.5 1
phenyl OPh 4.90 >16 >16 >16 >16
* E. coli GyrB ATPase assay; SA, S. aureus ATCC 29213; EF, E. faecalis ATCC 29212; SP, S. pyogenes ATCC 51339; SA + HS, S. aureus + 50% horse serum
• Core structural element of R2 = NHAr was retained and amide varied leading to a series of potent antibacterial compounds with low levels of serum binding
N
NH
O
NH
NH
OR1
R2
R1 =
R1 =
R1 =
N
NO
N
AcHN
N
N
N
N
Amide optimisation
Ian Yule, Sarah NarramoreIan Yule, Sarah Narramore
R1 R2 GyrB IC50 (µM)* MIC (µg / mL)
SA SA + HS EF SP
Ph NHPh 0.040 1 nd 0.25 2
3-tolyl NHPh 0.140 1 nd 0.125 2
3-Cl-Phe NHPh 0.150 0.5 nd 0.125 4
3-pyridyl NHPh 0.170 1 4 0.125 1
6-mor-3-py NHPh 0.111 2 16 0.25 1
6-acetam-3-py NHPh 0.039 2 8 0.125 8
6-triaz-3-py NHPh 0.153 >16 >16 0.25 >16
phenyl NH-3-Py 0.430 1 2 2 0.25
3-pyridyl NH-3-Py 0.093 8 nd 0.5 1
phenyl OPh 4.90 >16 >16 >16 >16
* E. coli GyrB ATPase assay; SA, S. aureus ATCC 29213; EF, E. faecalis ATCC 29212; SP, S. pyogenes ATCC 51339; SA + HS, S. aureus + 50% horse serum
• Core structural element of R2 = NHAr was retained and amide varied leading to a series of potent antibacterial compounds with low levels of serum binding
N
NH
O
NH
NH
OR1
R2
R1 =
R1 =
R1 =
N
NO
N
AcHN
N
N
N
N
Amide optimisation
Ian Yule, Sarah NarramoreIan Yule, Sarah Narramore
• Bacteria have developed β-lactamase enzymes which cleave the amide bond in the β-lactam antibiotics to render the drug molecules ineffective.
• This method of antibiotic resistance is particularly prevalent in Gram-negative bacteria.
• There are four different classes of enzymes: A, B, C and D.
• Class B are made up of the metallo-β-lactamases whereas classes A, C and D are the serine-β-lactamases.
Example 2: -lactamases
• Most important class of β-lactamase enzymes as they can hydrolyse all β-lactams except for the monobactam aztreonam.
• 3 different subclasses: B1, B2 and B3.
• B1 has two zinc ions in the active site but can be active in the mono or di-zinc forms.
• B2 is only active in the mono-zinc form whereas the B3 subclass is active in the di-zinc form.
• A number of structurally related variants of MβLs, including IMP-1, VIM-2 and NDM-1 which all belong to the B1 subclass.
Metallo--lactamases
Example 2: -lactamases
Boronates to mimic tetrahedral intermediate?
Bind to MBL
planar tetrahedral
Mechanism-based inhibition
In vitro screening reveals broad spectrum activity
Brem et al, Nature Commun., 2016
In vitro screening reveals broad spectrum activity
Brem et al, Nature Commun., 2016
2
Structural elucidation
Brem et al, Nature Commun., 2016
Structural elucidation
Brem et al, Nature Commun., 2016
Structural elucidation
Brem et al, Nature Commun., 2016
Conclusions
In silico fragment-based molecular design can be In silico fragment-based molecular design can be used to rapidly produce novel enzyme inhibitors.used to rapidly produce novel enzyme inhibitors.
The initially-designed molecules can act as The initially-designed molecules can act as templates for further rounds of enhancement via templates for further rounds of enhancement via synthesis and de novo design. synthesis and de novo design.
Cellular activity difficult to predict- once Cellular activity difficult to predict- once established can be improved via chemistryestablished can be improved via chemistry
Acknowledgements
Ian YuleIan YuleSarah NarramoreSarah NarramoreRicky CainRicky CainRachel JohnsonRachel JohnsonProf. Peter JohnsonProf. Peter JohnsonDr. Alex O’NeillDr. Alex O’Neill
Prof. Chris SchofieldProf. Chris SchofieldDr. Lloyd CzaplewskiDr. Lloyd CzaplewskiProf. Tony MaxwellProf. Tony MaxwellDr. Clare StevensonDr. Clare StevensonDr. Jim SpencerDr. Jim SpencerDr. Jurgen BremDr. Jurgen Brem
EPSRC, BBSRC, Medical Research Council, Medicines for Malaria Venture, EPSRC, BBSRC, Medical Research Council, Medicines for Malaria Venture, Biota Europe Ltd (UK), Wellcome Trust, NIHBiota Europe Ltd (UK), Wellcome Trust, NIH
R = NH-PNB
R = NH2
R = NHCONHEt
R = OH
R = thiazol-2-yl amino
R = OHR = Cl
R = imidazol-1-yl (34 %)
R = 4-me-piperazin-1-yl
R = imidazol-1-ylR = 4-me-piperazin-1-yl
R = imidazol-1-yl
R = 4-me-piperazin-1-yl (80 %)
m-toluidineHCl
EtOH51%
N
EtO2CHN
Cl N
EtO2CHN
R N
HN
NH
O
R
NH
O
N
EtO2CR
R N
EtO2CR
Cl N
EtO2CR
NH
NH
O
N
R
NH
NH
ONH
OAr
N
HO2CR
NH
NH
O
p-MeOCH2NH2 PhMe, ,62%
62 %
NaOH (aq),
90 %
TFA, Et3SiH98 %
EtNCO, 43 %
POCl3, 79 %
ArNH2, HOBTEDC.MeI, DMF, rt, 35 %
ImH, NaH, DMF or
NMe-pip, TEA
Pd(OAc)2, xantphosN-ethylurea, KOtBu,
79-92 %
NaOH (aq),
56 -97 %
ArNH2, HOBT, EDC.MeI, DMF, rt,
5-51 %
routes based upon dichloropyidine
Ian Yule, Sarah NarramoreIan Yule, Sarah Narramore
• Proving a link between target (enzyme) activity and antibacterial activity is important in early evaluation of a novel antibacterial compound
• Promiscuous cytotoxic activity (e.g. membrane damaging) observed alongside coincidental enzyme activity can be mis-leading
Wild-typeGyrB
Wild-typeParE
dualinhibitor
Wild-typeBacterium
antibacterialactivity
Mode of action studies via mutagenesis
• Proving a link between target (enzyme) activity and antibacterial activity is important in early evaluation of a novel antibacterial compound
• Promiscuous cytotoxic activity (e.g. membrane damaging) observed alongside coincidental enzyme activity can be mis-leading
Wild-typeGyrB
Wild-typeParE
dualinhibitor
Wild-typeBacterium
antibacterialactivity
X X
Mode of action studies via mutagenesis
• Proving a link between target (enzyme) activity and antibacterial activity is important in early evaluation of a novel antibacterial compound
• Promiscuous cytotoxic activity (e.g. membrane damaging) observed alongside coincidental enzyme activity can be mis-leading
Wild-typeGyrB
Wild-typeParE
dualinhibitor
Wild-typeBacterium
antibacterialactivity
X X
Mode of action studies via mutagenesis
• Proving a link between target (enzyme) activity and antibacterial activity is important in early evaluation of a novel antibacterial compound
• Promiscuous cytotoxic activity (e.g. membrane damaging) observed alongside coincidental enzyme activity can be mis-leading
Wild-typeGyrB
Wild-typeParE
dualinhibitor
Wild-typeBacterium
antibacterialactivity
X X
X X
Mode of action studies via mutagenesis
R1 R2MIC (µg / mL)
SA T173N / T167N
(GyrB / ParE)SA SA T173N (GyrB) SP SP A53S (ParE)
Ph NHPh 1 2 2 16 >64
3-Cl-Phe NHPh 0.5 1 4 32 >64
3-pyridyl NHPh 1 4 1 2 >64
SA, S. aureus (ATCC 29213); SA T173N, S. aureus GyrB mutant (ATCC 080519); SA T173N / T167N, S. aureus GyrB / ParE mutant (ATCC 090108); SP, S. pyogenes (ATCC 51339); SP A53S, S. pyogenes ParE mutant (ATCC 071201).
N
NH
O
NH
NH
OR1
R2
Point mutation in GyrB ATPase site
Mode of action studies via mutagenesis
R1 R2MIC (µg / mL)
SA T173N / T167N
(GyrB / ParE)SA SA T173N (GyrB) SP SP A53S (ParE)
Ph NHPh 1 2 2 16 >64
3-Cl-Phe NHPh 0.5 1 4 32 >64
3-pyridyl NHPh 1 4 1 2 >64
SA, S. aureus (ATCC 29213); SA T173N, S. aureus GyrB mutant (ATCC 080519); SA T173N / T167N, S. aureus GyrB / ParE mutant (ATCC 090108); SP, S. pyogenes (ATCC 51339); SP A53S, S. pyogenes ParE mutant (ATCC 071201).
N
NH
O
NH
NH
OR1
R2
Point mutation in ParE ATPase site
Mode of action studies via mutagenesis
R1 R2MIC (µg / mL)
SA T173N / T167N
(GyrB / ParE)SA SA T173N (GyrB) SP SP A53S (ParE)
Ph NHPh 1 2 2 16 >64
3-Cl-Phe NHPh 0.5 1 4 32 >64
3-pyridyl NHPh 1 4 1 2 >64
SA, S. aureus (ATCC 29213); SA T173N, S. aureus GyrB mutant (ATCC 080519); SA T173N / T167N, S. aureus GyrB / ParE mutant (ATCC 090108); SP, S. pyogenes (ATCC 51339); SP A53S, S. pyogenes ParE mutant (ATCC 071201).
N
NH
O
NH
NH
OR1
R2
Dual mutation in GyrB and ParE sites
Mode of action studies via mutagenesis
R1 R2MIC (µg / mL)
SA T173N / T167N
(GyrB / ParE)SA SA T173N (GyrB) SP SP A53S (ParE)
Ph NHPh 1 2 2 16 >64
3-Cl-Phe NHPh 0.5 1 4 32 >64
3-pyridyl NHPh 1 4 1 2 >64
SA, S. aureus (ATCC 29213); SA T173N, S. aureus GyrB mutant (ATCC 080519); SA T173N / T167N, S. aureus GyrB / ParE mutant (ATCC 090108); SP, S. pyogenes (ATCC 51339); SP A53S, S. pyogenes ParE mutant (ATCC 071201).
N
NH
O
NH
NH
OR1
R2
Dual mutation in GyrB and ParE sites
Mode of action studies via mutagenesis
Yule, Narramore, Yule, Narramore, et alet al (2014), (2014), European Journal of Medicinal ChemistryEuropean Journal of Medicinal Chemistry , 86, 31 - 38 , 86, 31 - 38