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Hugo Kubinyi, www.kubinyi.de
Structure-Based
Ligand Design
Hugo Kubinyi
Germany
E-Mail [email protected]
HomePage www.kubinyi.de
Hugo Kubinyi, www.kubinyi.de
A. Cressy Morrison
Man in a Chemical World
Medicinal
Chemist
The Service of Chemical Industry
Ch. Scribner‘s Sons, NY, 1937
Modeller
„Chemical Industry, Upheld
by Pure Science, Sustains
the Production of Man‘s
Necessities“
Hugo Kubinyi, www.kubinyi.de
Strategies in
Drug Design
no protein
3D structure,
no ligands
combichem, HTS,
virtual screening
no protein 3D
structure, ligands
LBLD
pharmacophores,
(3D) similarity,
(3D) QSAR
Hugo Kubinyi, www.kubinyi.de
Protein Crystallography
protein
3D structure,
no ligands
de novo design
(protein flexibility !)
protein 3D structure,
ligands
structure-based
design
SBLD
Hugo Kubinyi, www.kubinyi.de
Crystallisation Techniques
protein
solution
protein bulk
solution solvens
protein
solution
protein
solution
C°
solvens
vapour diffusion
gel
dialysis
membrane
microdialysis
cocrystallisation
batch
cooling
soaking
precipitant
protein
ligand
(weeks)
mother liquour
with ligand
crystal (days)
Hugo Kubinyi, www.kubinyi.de
Thrombin and Thrombin Inhibitor Complexes
Thrombin-Thrombstop Complex
Hugo Kubinyi, www.kubinyi.de
Number of Protein 3D Structures in the PDB
total number of 3D structures
(including NMR structures)
n = 33,585 (November 05, 2005)
number of protein X-ray
structures: n = 27,797
Hugo Kubinyi, www.kubinyi.de
Protein Domain Superfold Families
α/β
β doubly wound
2FOX - flavodoxin
TIM barrel
7TIM
α/β
β sandwich
1APS - hydrolase
α up-down
256B cytochrome
globin
1THB - hemoglobin
jelly roll 2STV - virus
trefoil
1I1B - IL-1β
β
greek key - Ig
2RHE - Bence
Jones
αβ roll
Hugo Kubinyi, www.kubinyi.de
new folds (% of new 3D structures)
Proportion of New Protein Folds / Year
Hugo Kubinyi, www.kubinyi.de
Binding Site
Recognition
a) coat the surface
with water
b) remove surfaceexposed water
c) accrete new
water molecules
d) filter
e) repeat process
f) identify „active
site points“
(PASS algorithm,
G. P. Brady Jr.,
JCICS 14, 383-401
(2002))
R. C. Willis, Mod.
Drug Discov.,
Sept. 2002, 28-34.
Hugo Kubinyi, www.kubinyi.de
The Problem of Protein 3D Structure Resolution
Hugo Kubinyi, www.kubinyi.de
Problems of PDB Protein 3D Structures
Binding site geometry may be different in free protein and complex
Lacking hydrogen atoms, orientation of hydroxyl groups
can be added automatically
Protonation of acidic and basic side chains
Amino acid pKa values: pKa shifts?
His: protonation
equilibrium
HN
HN
N
+
NH
N
NH
No differentiation between C, O and N
rotamer equilibria of his, thr, asn, gln
Me
N
H
OH
O
HO
N
H
Me
O
O
NH 2
H2N
O
Hugo Kubinyi, www.kubinyi.de
HIV Protease,
without a ligand
HIV Protease,
with a ligand
Hugo Kubinyi, www.kubinyi.de
Flexibility of the Binding Site of Cruzain
a) Glu 205 flexibility, induced by
Phe (gray) and Arg (blue) inhibitors
b) Interactions of an Arg
inhibitor with Glu 205 and
a C-terminal Gly 212‘
S. A. Gillmor et al., Protein Sci. 6, 1603-1611 (1997)
Hugo Kubinyi, www.kubinyi.de
RELIBASE - Comparison of MTX Binding Sites
www.ccdc.cam.ac.uk/prods/relibase/index.html
Hugo Kubinyi, www.kubinyi.de
RELIBASE - Benzamidine / COO- Interactions
www.ccdc.cam.ac.uk/prods/relibase/index.html
Hugo Kubinyi, www.kubinyi.de
RELIBASE
Binding Site
Flexibility
white: e.g. 1BR6
blue: 1IFS and 1APG
red: ligand-free
structures 2AII,
1RTC and 1IFT
yellow: R180H
mutants 1OBS
and 1OBT
J. Günther et al.,
J. Mol. Biol. 326,
621-636 (2003)
Hugo Kubinyi, www.kubinyi.de
Structure-based Design of Protein Ligands
ß2-1
ß2-His 2
ß2-His 143
O
O P O
O
ß1- His 143
2,3-DPG
reduces
the oxygen
affinity of
hemoglobin
O
O P -O
ß1-His 2
O
ß1-1
O - O
ß1- Lys 82
ß2-His 143
H
ß2-1-N
ß2-His 2
SO3-
-O S
3
ß1-His 143
COO
-
N- ß1-1
H
ß1-Lys 82
ß1-His 2
Hugo Kubinyi, www.kubinyi.de
Design of Dihydrofolate
NH
Arg 57
Reductase (DHFR)
H
H
N + N
Ligands
H
H
N
N
H
+
N
H
O
O-
H
O
O
N
H
N
N
H
O
O
O
H
N
CH3
N
α -carboxylate
O
O-
γ -carboxylate
methotrexate (MTX)
Hugo Kubinyi, www.kubinyi.de
Design of Dihydrofolate Reductase (DHFR)
Ligands
R
CH3
O
O
H
N
H
OR
N
H
N
H
+
N
H
O
O-
OMe
OMe
trimethoprim, R = CH3
CH2COOH
(CH2)2COOH
Ki [nM]
1.3
2.6
0.37
(CH2)3COOH
(CH2)4COOH
0.035
(CH2)5COOH
(CH2)6COOH
0.024
0.066
0.050
Hugo Kubinyi, www.kubinyi.de
Design of DHFR Ligands
Hugo Kubinyi, www.kubinyi.de
General Strategies for the Rational Design of
Protease Inhibitors
a) Exchange of the scissile bond in the substrate
(„substrate-like“aspartyl protease inhibitors)
b) Introduction of a group that interacts with a metal
ion („carboxy-terminal“ metalloprotease inhibitors)
c) Introduction of a group that covalently interacts
with the catalytic amino acid („amino-terminal“
serine and cysteine protease inhibitors)
Strong non-covalent interactions with the binding
site pockets (especially hydrophobic interactions)
Hugo Kubinyi, www.kubinyi.de
Relative Potency of Zn++-Complexing Groups
OH
R
Ki [nM]
O
R=H
6200
CH 2COOH
450
CH 2S(=NH)2CH3
250
OP(=O)(OH)2
140
CH 2SH
11
Hugo Kubinyi, www.kubinyi.de
Structure-Based Design of Captopril
Carboxypeptidase
Zn2+
{
Zn2+
O
H
N
N
H
R
O
O
+
NH2
O
H2N
substrate
O
IC50 = 330 µM
HO
+
NH2
O
N
H
inhibitor
H2 N
N
H
CH3
N
HOOC
O
HS
COOH
N
O
Captopril
COOH
IC50 = 23 nM
Hugo Kubinyi, www.kubinyi.de
Captopril Analogs
HS
N
O
H3C
S
N
COOEt
IC50 = 17 µ M
H
N
HS
O
O
COOH
IC50 = 4300 µ M
HS
COOH
COOH
IC50 = 1100 µ M
IC50 = 2.8 µ M
Free thiol and carboxylate groups are required for binding.
Esterification of the carboxylate group reduces the binding
affinity by nearly two orders of magnitude. S-methylation
leads to a 20,000-fold reduction in binding affinities.
Also the central carbonyl group seems to be essential for
high binding affinity (lower right analog).
Hugo Kubinyi, www.kubinyi.de
Structure-Based Design of Dorzolamide
H3C
Ki = 300 nM
O
S
Methazolamide
N
N
NH2
H3C
N
S
O O
H3C
S
NH2
O O
CH3
CH3
NH2
HN
O O
S
HN
CH3
Ki = 100 µM
F3C
S
NH2
S
O O
S
S
NH2
O O
H3C
S
O O
S
S
O O
O O
Ki = 2.0 nM
MK 927
Ki = 0.7 nM
NH2
Dorzolamide
Ki = 0.37 nM
Hugo Kubinyi, www.kubinyi.de
Binding Mode of Carbonic Anhydrase Inhibitors
Thr 199
N
H
CH3
O
H
HN
H
O
S N2+
O
Zn
S
S
O O
Dorzolamide,
anion
Gln 92 H
K i = 0.37 nM
N
H
O
H3 C
CH3SO2NH2, Ki = 100 µM,
CF3SO2NH2, Ki = 2 nM,
pKa = 10.5
pKa = 5.8
Hugo Kubinyi, www.kubinyi.de
Design of HIV Protease Inhibitors
H 2N
H
O
O
N
H
N
N
H
O
Saquinavir
H
OH
H
N
Phe
OH
H
N
HO
CONH-t-Bu
CH3 O
Nelfinavir
OH
H
N
N
N
CONH-t-Bu
H3C
O
Ritonavir
H3C
N
H 3C
S
N
CH3
N
H
OH
Phe
NH2
O
O
Indinavir
O
HN
O S
N
O
Phe
O
CH3
H
N
O
CONH-t-Bu
S
Phe
N
H
N
OH
Phe
N
H
O
OH
S
N
Amprenavir
O
Hugo Kubinyi, www.kubinyi.de
Design of HIV Protease Inhibitors
NH2
H
O
O
N
OH
H
N
N
H
H
N
O
CONH-t-Bu
Phe
Saquinavir
H
OH
H
N
HO
H
N
CH3 O
S
Nelfinavir
CONH-t-Bu
Phe
Hugo Kubinyi, www.kubinyi.de
HIV Protease Inhibitors Against Resistant Strains
H3C
O
H3C
N
H3C
S
N
CH3
H3 C
O
Ritonavir
HN
Lopinavir
N
H
CH3
Phe
O
H
N
O
CH3
OH
Phe
H
N
N
O
OH
Phe
N
H
S
O
N
Phe
O
N
H
O
therapeutically applied in combination: lopinavir is active
against ritonavir-resistant strains; its pharmacokinetics is
improved by the CYP 3A4 inhibitor ritonavir.
Hugo Kubinyi, www.kubinyi.de
Success Stories and Failures of
Structure-based Design
OH
HO
phospholipase
inhibitor
HO
CH3 CH2
OH
N
O
HIV protease
inhibitor
MeO
O
O O
S
N
H
N
N
OH
COOH
O
HO
O
OH
AcNH
N
H
thrombin inhibitor
COOH
HN
N
HN
neuraminidase
inhibitor
NH2
NH2
NH
Hugo Kubinyi, www.kubinyi.de
Rational Design of HIV-Protease Inhibitors
pharmacophore hypothesis
hit from 3D search
8,5-12,0 Å
P1
MeO
P1'
3,5-6,5 Å
HO
OH
HO
H-bond
donor / acceptor
HN
P1'
OH
O
O
O
P1
OMe
P2
NH
P1
P1'
HO
OH
P1
N
HO
N
P2'
OH
P1'
Hugo Kubinyi, www.kubinyi.de
Binding Mode of the DuPont HIV-Protease Inhibitors
Ile B50
Ile A50
Asp A25
Asp B25
Hugo Kubinyi, www.kubinyi.de
DuPont HIV-Protease Inhibitors
O
HN
R
R
NH2
H2N
NH
O
HO
O
OH
N
N
N
DMP-412
N
Ki = 4500 nM
HO
HO
OH
OH
HN N
DMP-323
R = -CH2OH
O
N
HO
N
Ki = 0.3 nM
OH
DMP-450
R = -NH2
Ki = 0.3 nM
Ki = 0.3 nM
NH2
H3C
DMP-850
Ki = 0.02 nM
O
N
HO
N
OH
Hugo Kubinyi, www.kubinyi.de
HIV-Protease Inhibitor vs. Resistant Strains
Gly-48
O
N
H
O
3,1
Ile-50'
3,0
Ile-50
3,5
HO
O
O
3,5
H
N
HN
NH
N
H
N
H
3,0
2,8
H
N
N
H
4,0
OH
NH
3,2
-O
3,1
3,4
O
N
O
Gly-48'
O
H
N
N
O
Asp-30
Asp-30'
3,4
O
H
2,9
2,9 O
O 2,9
Asp-25'
OH O
Asp-25
Ki = 0.018 nM
Hugo Kubinyi, www.kubinyi.de
General Recommendations for Ligand Design
Complementary hydrophobic surfaces contribute to
affinity but lipophilicity increase reduces solubility.
Hydrogen bonds are important for recognition and
orientation; they increase or reduce affinity.
The effect of repulsive interactions is hardly predictable.
The effect of MEP and dipole interactions is most often
neglected.
Replacement of water molecules may reduce affinity.
Small and/or flexible ligands are only weakly active;
they may bind to many different targets.
binding site:
complex ligand
small ligand
+ + - + - - + - + + +
- - + - + + - + - - - + - + -
M. M. Hann et al., J. Chem. Inf. Comput. Sci. 41, 856-864 (2001)
Hugo Kubinyi, www.kubinyi.de
Rational Design of Protease Inhibitors:
Transition State Mimics
O
N
H
OH
O OH
P
X
substrate
X = -CH2-, -NH-, -OOH
CH
OH
CHO , as
O OH
N
H
O
, as
transition state
OH
OH
mimics
HO OH
X
X
OH
B
OH
X = -CF3,
-CF2-R, -Aryl
Hugo Kubinyi, www.kubinyi.de
Rational Design: Transition State Mimics
NH2
N
N
N
N
HO
hypothetical
transition
state
OH
N
N
N
HO
pentostatin
Ki = 2.5 pM
O
inosine HO OH
H
active
agent HN
OH
N
N
N
HO
O
HO OH
N
N
HO
N
N
O
OH
N
N
HN
N
HN
N
sugar
N
adenosine
deaminase
HO OH adenosine
HO
N
HN
O
H
O
H2N OH
nebularine
Ki = 0.3 pM
O
HO OH
Hugo Kubinyi, www.kubinyi.de
Transition State Mimics are Picomolar Inhibitors
O
N
HO
O
N
NH
N N R
+:
O :
..... OPO-
HO
H
N
N
NH
N
R
3
HO OH
transition state
R. W. Miles et al., Biochemistry
37, 8615-8621 (1998)
G. B. Evans et al., J. Med. Chem.
46, 155-160 (2003)
HO OH
transition
state mimic
R = H, Immucillin-H
Ki hPNP = 72 pM
R = NH2, Immucillin-G
Ki hPNP = 29 pM
Hugo Kubinyi, www.kubinyi.de
Design of Selective ERα
α and ERβ
β Ligands
blue: hERα
α
green: hERß
estradiol
hERα
α ! hERß
„upper“ side:
Leu384 ! Met336
„lower“ side:
Met421 ! Ile373
A. Hillisch et al., Ernst Schering Res. Found. Workshop 46, 47-62
(2004); A. Hillisch et al., Mol. Endocrinol. 18, 1599-1609 (2004)
Hugo Kubinyi, www.kubinyi.de
Design of Selective ERα
α and ERβ
β Ligands
Potency ERα : 40 % of E2
Selectivity:
300 fold
Potency ERβ : 50 % of E2
Selectivity:
190 fold
Hugo Kubinyi, www.kubinyi.de
Influenza
In 1918/19, the „Spanish Flu“
killed about 20-40 mio people.
Especially young and very old
people died from influenza.
The heavy death toll of this
pandemic disease has to be
compared to the number of
11 mio victims of World War I.
Egon Schiele prepared this
drawing of his wife, one day
before her death and four days
before he died himself, only
28 years old.
Hugo Kubinyi, www.kubinyi.de
Influenza Virus
schematic view
and electron microscopic picture
Hugo Kubinyi, www.kubinyi.de
Influenza Virus
Hemagglutinine
sialic acid
binding sites
Neuraminidase
sialic acid
cleavage sites
Kansas City, 1918
viral
surface
Hugo Kubinyi, www.kubinyi.de
hemagglutinine + sialic acid (green)
neuraminidase + DANA (red)
Hugo Kubinyi, www.kubinyi.de
Design of Neuraminidase Inhibitors
O
OH OH
HO
AcNH
HO
OH
O
OR
HO
OH OH
AcNH
HO
HO
AcNH
OH
HO
+
Glu-119
Arg-371
O
O
-
O
O
OH
HO 4
sialic acid, R = H
Glu-276
O
Neu5Ac2en
Ki = 1 000 nM
Arg-292
Arg-118
4
result of a GRID search with
a positively charged probe
Glu-227
Hugo Kubinyi, www.kubinyi.de
Design of Neuraminidase Inhibitors
O
OH OH
HO
AcNH
HO
OH
O
OR
HO
OH OH
AcNH
HO
HO
AcNH
Arg-371
O
OH
O
O
4
HN
+
H2N
NH2
Glu-119
Glu-227
O
OH
HO 4
sialic acid, R = H
Glu-276
O
Neu5Ac2en
Ki = 1 000 nM
Arg-292
Arg-118
4-Guanidino-Neu5Ac2en
Ki = 0.1-0.2 nM
Zanamivir (Relenza,
Glaxo-Wellcome)
Hugo Kubinyi, www.kubinyi.de
1a4g
glu
arg
Hugo Kubinyi, www.kubinyi.de
Design of Bioavailable Neuraminidase Inhibitors
OH
O
HO
COOH
R
COOH
AcNH
HO
AcNH
HN
NH2
4-NH2-Neu5Ac2en
Ki = 50 nM
COOH
HO
NH
NH2
a) R = H Ki = 8 µM
b) R = CH(OH)CH(OH)CH2OH
Ki > 100 µM
HO
COOH
AcNH
AcNH
NH2 IC > 200 µM
50
NH2 IC50 = 6.3 µM
Hugo Kubinyi, www.kubinyi.de
Design of Bioavailable Neuraminidase Inhibitors
RO 3
AcNH 4
2
1 COOH
6
5
NH2
GS 4071, R = CH(Et)2
IC50 = 1 nM
(Et)2CHO
COOEt
AcNH
NH2
GS 4104 (ester prodrug
of GS 4071)
Oseltamivir (Tamiflu, Roche)
R=
H
CH3
CH2CH3
CH2CH2CH3
CH2CH2CF3
CH2OCH3
CH2CH=CH2
CH2CH2CH2CH3
CH2CH(CH3)2
CH(CH3)CH2CH3
CH(CH2CH3)2
CH(CH2CH2CH3)2
Cyclopentyl
Cyclohexyl
Phenyl
IC50 (nM)
6 300
3 700
2 000
180
225
2 000
2 200
300
200
10
1
16
22
60
530
Hugo Kubinyi, www.kubinyi.de
2qwk
arg
glu
Hugo Kubinyi, www.kubinyi.de
Binding Mode
of Oseltamivir
C. U. Kim et al.,
J. Am. Chem. Soc.
119, 681-690 (1997)
Hugo Kubinyi, www.kubinyi.de
Other Neuraminidase Inhibitors
NH
H2 N
HN
OH
HN
OH
HN
OH
HN
O
H2 N
O
O
H3C
O
CH3
BANA-113
(BioCryst,
Univ. Alabama)
CH3
OH
CH3
O
BCX-1812 =
RWJ-270201
(BioCryst, J&J)
OH
N
H
IC50 = 0.1-11 nM
N
BANA-206
OH
O
A. F. Abdel-Magid et al., Curr. Opin. Drug. Discov. Dev. 4, 776-791 (2001)
Hugo Kubinyi, www.kubinyi.de
Other Neuraminidase Inhibitors
COOH
COOH
NH2
O
N
O
H3C
CF3
HN
O
HN
O
A-315 675
Ki = 0.02 - 0.31 nM
(different strains)
Drug Discov. Today
7, 1066 (2002)
Me
CH3
COOH
A-192 558
(Abbott Labs)
A. F. Abdel-Magid et al.,
Curr. Opin. Drug. Discov.
Dev. 4, 776-791 (2001)
O
NHAc
Ki = 45 nM
S. Hanessian et
al., Bioorg. Med.
Chem. Lett. 12,
3425-3429 (2002)
Hugo Kubinyi, www.kubinyi.de
The Relevance of Protein Crystal Structures:
Conformational flexibility of PNP in the crystal
favorable
unfavorable
Asn 243
hydrogen
hydrogen
bonds
bonds ?
O
H
H N
H
Thr 242
H
O
O
O
CH3
H
N
HN 6
7
HN 6
N
9
3
favorable
H
N
3
H2N
N
H2N
N
hydrogen
S
bond
H
N
Asn 243
H O
O
H
N
7
9
H
Thr 242
CH3
N
H
unfavorable
polar/nonpolar
and steric
interactions
S
Hugo Kubinyi, www.kubinyi.de
The Relevance of Protein Crystal Structures:
Enzymatic activity of PNP crystals
O
N
HO
N
O
NH
N
HO
NH2
+ H2PO-4
O
OPO3H
PNP
HO OH
HO OH
O
-
N
NH
+
N
H
N
remove crystal
reaction
rate
(% product)
remove
crystal
add crystal
add crystal
time
NH2