combinatorial and fragment-based ligand design...hugo kubinyi, combinatorial and fragment-based...
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Hugo Kubinyi, www.kubinyi.de
Combinatorial andFragment-basedLigand Design
Hugo Kubinyi
Germany
E-Mail [email protected] www.kubinyi.de
Hugo Kubinyi, www.kubinyi.de
„Classical“ and „Combinatorial“ Drug Design
HTS, Docking
Decoration
Linking
Fusion,Assembly
Hugo Kubinyi, www.kubinyi.de
SHAPES Strategy: NMR Screening of Ligands
Kd p38 MAPK = 2 mM
N
NH
N
N
SR2
R1N
NNH
N N
N
NN
F
Me
R R = COOH or S(=O)Me
Kd p38 MAPK = 10-200 nM
Kd p38 MAPK = 200-300 µM
or
J. Fejzo et al., Chem. Biol. 6, 755-769 (1999)
Hugo Kubinyi, www.kubinyi.de
Fragment-Based Approach (Astex)
T. L. Blundell et al., Nature Rev. Drug Discov. 1, 45-54 (2002)
a) complex binding siteb) fragments in different pocketsc) assembly of different fragmentsd) growing out from a fragment
Hugo Kubinyi, www.kubinyi.de
Fragment-Based Approach With Central Scaffold
N N
NHR2
NH
O
R3R1
N N
NHMe
NH
O
MeR1
N N
NHR2
NH
O
MeMe
N N
NHMe
NH
O
R3Me
A library with
100 x R1,100 x R2, and100 x R3 yields
1 mio compounds
100 + 100 + 100 variations yield a library of only300 compounds
R. Carr and M. Hann, ModernDrug Discov., April 2002, 45-48
Hugo Kubinyi, www.kubinyi.de
Enthalpic and Entropic Contributions to Binding:3D Structure of the Biotin-Streptavidin Complex
NH
HO
OH
HN
H
O
OO
O
O
NN
S
O
H H OH
HO
Ser 45
Ser 27
Tyr 43Asn 23
Asp 128
Ser 88
Asn 49
-
-
Hugo Kubinyi, www.kubinyi.de
„Entropic Stabilisation“ of a Ligand-Receptor Complex
Hugo Kubinyi, www.kubinyi.de
Binding Constants of Biotin and Analogs(N. M. Green, Adv. Protein Chem. 29, 85-133 (1975))
-O
O
NN
S
O
H H
-O
O
NN
CH3
O
H H
-O
OCH3
NN
CH3
O
H H
Biotin, Ki = 1,3 x 10-15 M Desthiobiotin, Ki = 5 x 10-13 M
Ki = 3,4 x 10-5 M Ki = 3 x 10-3 M
Hugo Kubinyi, www.kubinyi.de
streptavidin plus two biotin fragments;intermolecular NOEs indicate the „correct“ linkage of the fragments (A. Kline et al., The NMR Newsletter 472, 13 (1997))
NOESY experiment
„Re-discovering“ Biotin by NMR Fragment-Based Screening
Hugo Kubinyi, www.kubinyi.de
Design of a Bisubstrate Inhibitor of Adenylosuccinate Synthase (AdSS)
O NHNH
O
OOH OH
OH
Hydantocidin Hadacidinare natural product inhibitors of AdSS
H N
OOH
COOHN
NN
NO
OH OH
OP
NH
COOH
COOHAdenylosuccinatemonophosphate(AdS-P)
H N
OOH
COOHO NH
N
O
OOH OH
OP
bisubstrate inhibitor
IC50 in µMol, AdSS from E. coli wheat
hyd-P 0.675 1.35hadacidin 3.5 12bisubstrate inhibitor 0.043 0.20S. Hanessian et al., Angew. Chem.
Int. Ed. 38, 3159-3162 (1999)
Hugo Kubinyi, www.kubinyi.de
Michelangelo, The Creation of Adam
(fresco, detail from the Sistine Chapel ceiling, Vaticane)
Hugo Kubinyi, www.kubinyi.de
SAR by NMR(P. J. Hajduk et al., J. Am. Chem. Soc. 119, 5818-5827 (1997))
Hugo Kubinyi, www.kubinyi.de
SAR by NMR: FKBP Ligands
ON
OO
O Me
MeOOMe
OMe
+ ONH
O
OH
Kd = 2 µM
Kd = 100 µM
Kd = 19 nM
ONH
O
OH
ON
OO
O
MeOOMe
OMe
fromFK506
S. B. Shuker, P. J. Hajduk, R. P. Meadows and S. W. Fesik, Science 274, 1531-1534 (1996).
Hugo Kubinyi, www.kubinyi.de
CrystaLEAD - Crystallographic Screening
N
Br
NH2
N
NH2
N
NH2
NH
NH2
N
NH2
OH
N
NHN
N
NH2
Crystallographic hits (Urokinase inhibitors)
optimized structure:
Ki > 500 µM Ki = 200 µM
Ki = 71 µM Ki = 56 µM
Ki = 370 nM(38% bio-availabilityin the rat)
V. L. Nienaber et al., Nat. Biotechnol. 18, 1105-1108 (2000)
Hugo Kubinyi, www.kubinyi.de
HTX in Drug Design: The Astex Pyramid ApproachVirtual screening (AutoDock, up to 1014compounds), soaking (AutoSolve software) and linking of building block libraries
Hugo Kubinyi, www.kubinyi.de
Ibis Pharmaceuticals, www.ibisrna.com/darpa_ii/chemistry.html E. E. Swayze et al., J. Med. Chem. 45, 3816-3819 (2002)
identify binderspreparederivatives
identify interactions between derivativeslink to high-affinity ligand
MS-based screening (MASS)
The SAR by MS Approach for RNA Targets
Hugo Kubinyi, www.kubinyi.de
Combinatorial Libraries of Linked Low-Affinity Binders
low-affinity ligands high-affinity ligand
D. J. Maly, I. C. Choong and J. A. Ellman, PNAS 97, 2419-2424 (2000)
Hugo Kubinyi, www.kubinyi.de
Ki = 40 µµµµM
Hugo Kubinyi, www.kubinyi.de
Ki = 41 µµµµM
Hugo Kubinyi, www.kubinyi.de
D. J. Maly, I. C. Choong and J. A. Ellman, PNAS 97, 2419-2424 (2000)
Fragment-Based Design: Protein Kinase Inhibition
Hugo Kubinyi, www.kubinyi.de
SUNESIS Approach(Thymidilate Synthase)
www.sunesis.com
Hugo Kubinyi, www.kubinyi.de
selected notselected
Design of a Submicromolar TS Inhibitor
D. A. Erlanson et al., PNAS 97, 9367-9372 (2000)
Hugo Kubinyi, www.kubinyi.de
„Needle Screening“: Gyrase Inhibitors
OH OH OH
NOH
NNH
SNHO O
N
NH2
N
NH2
N
NHONH2
NHO
NS
NH2
NNH
O
SO
O„needles“ from LUDI search
optimized structureH.-J. Boehm et al., J. Med. Chem. 43, 2664-2674 (2000)
Hugo Kubinyi, www.kubinyi.de
Cdk4 Inhibitors: Virtual Screening and Library Design
T. Honma et al., J. Med. Chem. 44, 4615-4627 (2001)
NO N
HNH
O
N
Cdk4 inhibitorIC50 = 42 nM
1. Construction of a Cdk4 homology model (starting from the 3D structure of activated Cdk2)2. de novo design of ligands in the binding site (programs LEGEND and SEEDS)3. 382 hits acquired and tested4. design of a diarylurea library5. further chemical modifi- cations
Hugo Kubinyi, www.kubinyi.de
Combinatorial Design of Carbonic Anhydrase Inhibitors
start structure
SNH2
O O
O
NH2
Kd = 120 nM
optimized structure
SNH2
O O
O
NH
NCH3
R enantiomer, Kd = 30 pM(S enantiomer: Kd = 230 pM)
Program CombiSMoG, „best“ N-substituents from 100,000 candidates (20 scored by knowledge-based potentials)B. A. Grzybowski et al., Acc. Chem. Res. 35, 261-269 (2002);B. A. Grzybowski et al., Proc. Natl. Acad. Sci. USA 99, 1270-1273 (2002)
Hugo Kubinyi, www.kubinyi.de
Scaffold-Linker-Functional Group Approach
M. Krier et al., J. Med. Chem. 48, 3816-3822 (2005)
N NMeO O
(CH2)6 NH2MeO
N NMeO O
(CH2)5MeO
N NH
MeO O
F2HCO Design of a structure-based 320-member virtual library with four different scaffolds or ring connections, five linkers and 16 different functional groups; best docking results with FlexX
Zardaverine IC50 PDE4 = 800 nM
N-substituted dihydro-pyridazinone analogs
IC50 PDE4 = 20 nM
IC50 PDE4 = 0.9 nM
Hugo Kubinyi, www.kubinyi.de
Scaffold-Linker-Functional Group Approach
M. Krier et al., J. Med. Chem. 48, 3816-3822 (2005)
Dockingof a 320-memberlibrary into PDE4pocket
subsite Afavors aphenyl ring
subsite Bfavors abasic group(amine)
scaffold
Hugo Kubinyi, www.kubinyi.de
Dynamic Self-Assembly of Ligands and Receptors
Therascope, Heidelberg
Hugo Kubinyi, www.kubinyi.de
O. Ramström and J. M. Lehn, Nature Rev. Drug Discov. 1, 26-36 (2002)
Dynamic Ligand Assembly in a Binding Site
Hugo Kubinyi, www.kubinyi.de
Products
Amines
Alde-hydes
SideProducts
Ligand Assembly in Carbonic Anhydrase
I. Huc and J.-M. Lehn, Proc. Natl. Acad. Sci. USA 94, 2106-2110 (1997)
Hugo Kubinyi, www.kubinyi.de
Ligand Assembly in Neuraminidase, I
NHAcNH2O
COOH
NHAcNH2NH2
COOH
NHAcNH2N
H
COOH
NHAcNH2N
H
COOH
Oseltamivir(Tamiflu, Roche)
Ki = 2.16 µM
Ki = 31.3 µMKi = 1.64 µM
>100-foldamplification
IC50 = 1 nM
M. Hochgürtel et al., Proc. Nat. Acad. Sci. USA 99, 3382-3387 (2002)
Hugo Kubinyi, www.kubinyi.de
Ligand Assembly in Neuraminidase, II
NHAc
OHOH
COOH
OHNH2
NH
NH
NHAcNH
NH2
COOH
NH2
NH
Zanamivir(Relenza, GSK)
Ki = 16 nM
Ki = 352 nM Ki = 52 nM
NHAcNH
NH
COOH
NH2
NH
NHAcNH
NH
COOH
NH2
NH
>100-foldamplification
Ki = 0.1-0.2 nM
M. Hochgürtel et al., Proc. Nat. Acad. Sci. USA 99, 3382-3387 (2002)
Hugo Kubinyi, www.kubinyi.de
Four soakingexperimentsof differentisatins and arylhydrazines in the presenceof Cdk2.
all sulfonamido-arylhydrazonesIC50 = 30 nM
M. S. Congreveet al., Angew.Chem. Int. Ed.42, 4479-4482(2003)
Ligand Assembly in Cdk 2
-Cl-NO2
-CF3
-OCF3
Hugo Kubinyi, www.kubinyi.de
peptidelibrary Dynamic
Selection ofPeptides byAntibodies
O. Ramström and J. M. Lehn, Nature Rev. Drug Discov. 1, 26-36 (2002)
Hugo Kubinyi, www.kubinyi.de
Dynamic Self-Assembly of DNA-Coded Fragments
a) DNA duplex, pairing DNA sequence and two coding DNA sequencesb) DNA triplex, same as a)c) DNA duplex with lead fragment (orange, without coding sequence)
COOH
SO2NH2
SO2NH2
CONH(CH2)8NHO
(CH3)3N
SO2NH2
CONH(CH2)8NHO
NN
N N
O
O
MeMe
+
Ki vs. Carbonic anhydrase
1 µM
90 nM
25 nM
S. Melkko et al., Nat. Biotechnol. 22, 568-574 (2004)
Hugo Kubinyi, www.kubinyi.de
„Click Chemistry“ (K. Barry Sharpless)
The reaction of azides with acetylenes to triazoles is significantly accelerated in the binding site of acetylcholinesterase.W. G. Lewis, L. G. Green, F. Grynszpan, Z. Radic, P. R. Carlier, P. Taylor, M. G. Finn and K. B. Sharpless, Angew. Chem. Int. Ed. Engl. 41, 1053-1057 (2002).
R N N N+ -
C C COOEtEtOOCN
NNR
EtOOC COOEt
H2O, 70o C
C N R N N N+ -
H2O, ZnN
NNN
R
triazoles
tetrazoles
Hugo Kubinyi, www.kubinyi.de
Click Chemistry (B. Sharpless): AChE InhibitorsN
NH2
MeNH O
Me
NH2
N
Et
Cl
NH2
N
NH2 NH2
N
+
+N
NHO
NHO
N
Cl
Cl
EtEt
EtEt
+
+
Ki = 18 nM
Ki = 120 pMKi = 26 pM
Ki = 1100 nM
Ki = 5.2 nM
W. G. Lewis et al., Angew. Chem. 114, 1095-1099 (2002);Angew. Chem. Int. Ed. Engl. 41, 1053-1057(2002).
Hugo Kubinyi, www.kubinyi.de
„Click Chemistry“A home-made Trojan Horse
N
NH
NNN
+
N3
+
N
NH2
NH2
Kd AChEsyn: 77-410 fManti: 0.7-14 pM (different species)
Hugo Kubinyi, www.kubinyi.de
The Future: Combinatorial Drug Design
Hugo Kubinyi, www.kubinyi.de
X
R1
Z
R R2
XX
XY
Combinatorial Ligand Construction
Hugo Kubinyi, www.kubinyi.de
Further Progress in Computer-AidedLigand Design
Ligand flexibilityProtein flexibilityFlexibility of the ligand-protein complexGeometry and strength of hydrogen bondsSolvation and desolvation effects (entropy)Inserted (conserved) water moleculesSynthetic accessibility of ligandsCombinatorial docking of ligands„Last Problem“ - The Scoring Function
Hugo Kubinyi, www.kubinyi.de
Fragment-Based Drug Discoverya) Reviews: D. A. Erlanson et al., Fragment-based drug discovery, J. Med. Chem. 47, 3462- 3482 (2004); M. J. Hartshorn et al., J. Med. Chem. 48, 403-413 (2005)b) Fragment-based de novo design: SKELGEN: M. Stahl et al., JCAMD 16, 459-478 (2002) COREGEN: A. M. Aronov and G. W. Bemis, Proteins 57, 36-50 (2004)c) Fragment space for de novo design: Modified RECAP procedure for Feature tree-based 2D design and 3D design by SkelGen M. Stahl, WATOC January 2005.d) SeeDs libraries for NMR screening, selected by kinase pharmacophore features, MWs about 160-240. N. Baurin et al., JCICS 44, 2157-2166 (2004)