structure-based ligand design - kubinyikubinyi.de/dd-16.pdf · 2005. 12. 25. · a. cressy morrison...

Hugo Kubinyi, www.kubinyi.de

Structure-BasedLigand Design

Hugo Kubinyi

Germany

E-Mail [email protected] www.kubinyi.de


A. Cressy Morrison

Man in a Chemical WorldThe Service of Chemical Industry

Ch. Scribner‘s Sons, NY, 1937

„Chemical Industry, Upheld by Pure Science, Sustains the Production of Man‘s Necessities“

Medicinal Chemist

Modeller


no protein 3D structure, no ligands

combichem, HTS, virtual screening

protein 3D structure,no ligands

de novo design(protein flexibility !)

protein 3D structure,ligands

structure-baseddesign

no protein 3D structure, ligands

pharmacophores, (3D) similarity, (3D) QSAR

Strategies in Drug Design

LBLD SBLD


Protein Crystallo-graphy


Crystallisation Techniquesproteinsolution

solvens

vapour diffusionmicrodialysis

batchcooling

bulksolvens

dialysismembrane

gel

C°

cocrystallisation

(weeks)

precipitant

ligandproteinmother liquourwith ligand

crystal (days)

proteinsolution

proteinsolution

proteinsolution

soaking


Thrombin and Thrombin Inhibitor Complexes

Thrombin-Thrombstop Complex


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


αααα/ββββ doubly wound2FOX - flavodoxin

TIM barrel7TIM

αααα/ββββ sandwich1APS - hydrolase

greek key - Ig2RHE - Bence

Jones

αααα up-down256B -

cytochrome

globin1THB - hemoglobin jelly roll 2STV - virus

trefoil1I1B - IL-1ββββ

αβαβαβαβ roll

Protein Domain Superfold Families


Proportion of New Protein Folds / Year

new

fold

s (%

of n

ew 3

D s

truc

ture

s)


Binding SiteRecognitiona) coat the surface with waterb) remove surface- exposed waterc) accrete new water moleculesd) filtere) repeat processf) 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.


The Problem of Protein 3D Structure Resolution


Problems of PDB Protein 3D StructuresBinding site geometry may be different in free protein and complexLacking hydrogen atoms, orientation of hydroxyl groups

can be added automaticallyProtonation of acidic and basic side chains

Amino acid pKa values: pKa shifts?His: protonation

equilibrium

No differentiation between C, O and N rotamer equilibria of his, thr, asn, gln

NNH NHNH NHN+

OHMe

NH O

MeOH

NH O

NH2O ONH2


HIV Protease,without a ligand

HIV Protease,with a ligand


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)


RELIBASE - Comparison of MTX Binding Sites

www.ccdc.cam.ac.uk/prods/relibase/index.html


RELIBASE - Benzamidine / COO- Interactions

www.ccdc.cam.ac.uk/prods/relibase/index.html


RELIBASEBinding Site Flexibility

white: e.g. 1BR6blue: 1IFS and 1APGred: ligand-free structures 2AII, 1RTC and 1IFTyellow: R180H mutants 1OBS and 1OBT

J. Günther et al., J. Mol. Biol. 326,621-636 (2003)


Structure-based Design of Protein Ligands

2,3-DPGreducesthe oxygenaffinity ofhemoglobin

ß2-1

ß1-1ß2-His 2

ß2-His 143

ß1- His 143

ß1-His 2

ß1- Lys 82

-

O PO

OO

O PO

OO

O O

-

-

--

O3SSO3

COO

-N

H

H

N-

--

-

ß2-His 2ß1-His 2

ß2-His 143

ß1-His 143

ß2-1

ß1-1

ß1-Lys 82


N

N N

NN

N

NCH3

N

OHH

H

H H

OO

O

O

methotrexate (MTX)

O O

NH

H

NH

NH

H

OO

+

+

αααα-carboxylate

γγγγ-carboxylate

Arg 57

H

-

-

-

Design of Dihydrofolate Reductase (DHFR)Ligands


trimethoprim, R = CH3

O

OO

+

-

HO

N

N

N

N

OR

OMe

H

H

H H OMe

R Ki [nM]CH3 1.3CH2COOH 2.6(CH2)2COOH 0.37(CH2)3COOH 0.035(CH2)4COOH 0.066(CH2)5COOH 0.024(CH2)6COOH 0.050

Design of Dihydrofolate Reductase (DHFR) Ligands


Design of DHFR Ligands


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)


RO

OHKi [nM]

R = H 6200 CH2COOH 450

CH2S(=NH)2CH3 250

OP(=O)(OH)2 140

CH2SH 11

Relative Potency of Zn++-Complexing Groups


Structure-Based Design of Captopril

N

ON O

ORNH2

NH2 Nsubstrate

Zn2+

H

H

H

{ +

Carboxy-peptidase

-OH

OO

ONH2

NH2 N

Zn2+

H

+-

inhibitor

N

COOHO

HOOC

IC50 = 330 µM

N

COOHO

SHCH3

IC50 = 23 nMCaptopril


N

COOEtO

SH N

COOHO

SCH3

N

COOHO

SHIC50 = 17 µµµµM IC50 = 4300 µµµµM

IC50 = 2.8 µµµµM IC50 = 1100 µµµµM

SH

COOH

H

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).

Captopril Analogs


SNH2

O O

Ki = 300 nM

SNH2

O O

N

S

N

N

O

CH3

CH3 Methazolamide

SNH2

O O

SS

OO

NH

CH3

CH3

MK 927Ki = 0.7 nM

SNH2

O O

SS

OO

NH

CH3

CH3

DorzolamideKi = 0.37 nM

CH3 SNH2

O O

Ki = 100 µM

F3C SNH2

O O

Ki = 2.0 nM

Structure-Based Design of Dorzolamide


Gln 92

Thr 199

HN

OH

SOO

NH

CH3

CH3S

SO

ON

H

HN

HO

Zn2+

-

Dorzolamide,anion Ki = 0.37 nM

Binding Mode of Carbonic Anhydrase Inhibitors

CH3SO2NH2, Ki = 100 µµµµM, pKa = 10.5CF3SO2NH2, Ki = 2 nM, pKa = 5.8


Design of HIV Protease Inhibitors

HN

OH

O

N

SPhe

CONH-t-Bu

H

H

CH3

OH

NelfinavirSaquinavir

NN

ON

OH

O

N

Phe

NH2O

CONH-t-Bu

H

H

H

H

NN

NCONH-t-Bu

OH

O

NOH

Phe

Indinavir

H

OHN O

O

N

SN

O

CH3CH3

N

O

NCH3

N

SPhe

Phe

CH3

CH3

Ritonavir

H

H

H

NH2

SN

OO

OH

NH O

OO

Amprenavir


Design of HIV Protease Inhibitors

HNH

OH

O

N

SPhe

CONH-t-Bu

H

H

CH3

OH

Nelfinavir

Saquinavir

NNH

O

O

NH2

NH

OHN

PheCONH-t-Bu

H

H

O


OHN O

O

N

SN

O

CH3CH3

N

O

NCH3

N

SPhe

Phe

CH3

CH3

Ritonavir

H

H

H

LopinavirOH

N

ON

O

CH3CH3

N

Phe

Phe

NH

OO

H

H

therapeutically applied in combination: lopinavir is active against ritonavir-resistant strains; its pharmacokinetics is improved by the CYP 3A4 inhibitor ritonavir.

HIV Protease Inhibitors Against Resistant Strains


O COOH

NHAcNH

OH

OHOH

NH

NH2

neuraminidase inhibitor

HIV protease inhibitor

phospholipase inhibitor CH2CH3

MeO

thrombin inhibitor

H

H

SO O

N

O N COOH

O

N

NH NH2

N

NN

O

OHOH

OH OH

Success Stories and Failures of Structure-based Design


OH

OH

OH

OMe

MeO 8,5-12,0 ÅP1 P1'

3,5-6,5 Å

H-bond donor / acceptor

pharmacophore hypothesis hit from 3D search

O

P1 P1'OH

NHNH

O

OH OH

P1 P1'

NN

OP2 P2'

OH OHP1 P1'

Rational Design of HIV-Protease Inhibitors


Asp A25 Asp B25

Ile B50 Ile A50

Binding Mode of the DuPont HIV-Protease Inhibitors


Ki = 4500 nM

Ki = 0.3 nM

NH NH

O

OHOH

N N

O

OHOH

Ki = 0.3 nM

N N

O

OHOH

R R

DMP-323R = -CH2OH

DMP-412 N N

O

OHOH

NH2 NH2

Ki = 0.02 nM N N

O

OHOH

NNH

CH3NH2

DMP-850

Ki = 0.3 nM

DMP-450R = -NH2

DuPont HIV-Protease Inhibitors


N N

O

O

NHNH NH

NOH OHN N

H H

OHO

OO

H

Ile-50 Ile-50'

Asp-25 Asp-25'-

OO

N

O

-O O

N

O

-H H

Asp-30 Asp-30'

NO

HN

OH

H

Gly-48 Gly-48'

Ki = 0.018 nM

3,53,0 3,1

4,0

3,0 3,1

3,2

3,4

3,5

2,8

3,4

2,9 2,9

2,9

HIV-Protease Inhibitor vs. Resistant Strains


General Recommendations for Ligand DesignComplementary 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)


substrate

transition state

N

O

N

O OH

mimics OH

PX

O OH

O

X

X = -CH2-, -NH-, -O-

, as CHO CHOH

OH

X = -CF3, -CF2-R, -ArylX

OH OH

BOH

OH

H

H

OH

OH

, as

Rational Design of Protease Inhibitors: Transition State Mimics


Rational Design: Transition State Mimics

NH

N N

N

sugar

NH2 OH

adenosinedeaminaseadenosine inosine

pentostatinKi = 2.5 pM

hypotheticaltransition state

OOH

OH

N

NNH

N

OHH

OOH

OH

NH

N N

NO

OH

OOH

OH

N

N N

NNH2

OH

OOH

OH

N

N N

N

OHnebularineKi = 0.3 pM

OOH

OH

NH

N N

N

OH

OHHactive agent


Transition State Mimics are Picomolar Inhibitors

N

OHOH

OH N

NHN

N

O

R-+

N

NHN

N

O

R

OPO3

transition state

transition state mimic

R = H, Immucillin-HKi hPNP = 72 pMR = NH2, Immucillin-GKi hPNP = 29 pM

O

OHOH

OH ::.....

H

R. W. Miles et al., Biochemistry37, 8615-8621 (1998) G. B. Evans et al., J. Med. Chem. 46, 155-160 (2003)


estradiol

Design of Selective ERαααα and ERββββ Ligandsblue: hERαααα

green: hERß

hERαααα !!!! 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)


Design of Selective ERαααα and ERββββ Ligands

Potency ERαααα : 40 % of E2Selectivity: 300 fold

Potency ERββββ : 50 % of E2Selectivity: 190 fold


InfluenzaIn 1918/19, the „Spanish Flu“killed about 20-40 mio people.Especially young and very oldpeople died from influenza. The heavy death toll of thispandemic disease has to be compared to the number of 11 mio victims of World War I.

Egon Schiele prepared thisdrawing of his wife, one daybefore her death and four daysbefore he died himself, only28 years old.


Influenza Virusschematic viewand electron micro-scopic picture


Influenza Virus

Neuraminidasesialic acid cleavage sites

viralsurface

Hemagglutininesialic acidbinding sites

Kansas City, 1918


hemagglutinine + sialic acid (green) neuraminidase + DANA (red)


Design of Neuraminidase Inhibitors

OHOH OH

O

OHAcNH

O

O

OHOH OH

O

OH

OH

O

AcNH

Neu5Ac2en

Ki = 1 000 nM

-

Glu-119 Glu-227

Arg-371

Arg-292

Arg-118

Glu-276

4

4

OHOH OH

OH

OH

O

OORAcNH

sialic acid, R = H

+result of a GRID search witha positively charged probe


Design of Neuraminidase Inhibitors

OHOH OH

O

NH

H2N NH2

AcNH

O

O

OHOH OH

O

OH

OH

O

AcNH

Neu5Ac2en

Ki = 1 000 nM

-

+

Glu-119 Glu-227

Arg-371

Arg-292

Arg-118

Glu-276

4

4

OHOH OH

OH

OH

O

OORAcNH

sialic acid, R = H

4-Guanidino-Neu5Ac2en Ki = 0.1-0.2 nMZanamivir (Relenza, Glaxo-Wellcome)


1a4g

argglu


Design of Bioavailable Neuraminidase Inhibitors

a) R = H Ki = 8 µMb) R = CH(OH)CH(OH)CH2OH Ki > 100 µM

AcNH

OH

NH2

COOH

AcNH

OH

NH2

COOH

IC50 = 6.3 µM

R

AcNH

COOH

NH

NH2

NH

O

NH2

COOHOH

OHOH

AcNH

4-NH2-Neu5Ac2enKi = 50 nM

IC50 > 200 µM


AcNH

(Et)2CHO

NH2

COOEt

GS 4104 (ester prodrug of GS 4071)Oseltamivir (Tamiflu, Roche)

AcNH

RO

NH2

COOHIC50 (nM)123

4 5 6

GS 4071, R = CH(Et)2 IC50 = 1 nM

R = H 6 300 CH3 3 700 CH2CH3 2 000 CH2CH2CH3 180 CH2CH2CF3 225 CH2OCH3 2 000 CH2CH=CH2 2 200 CH2CH2CH2CH3 300 CH2CH(CH3)2 200 CH(CH3)CH2CH3 10 CH(CH2CH2CH3)2 16 Cyclopentyl 22 Cyclohexyl 60 Phenyl 530

CH(CH2CH3)2 1

Design of Bioavailable Neuraminidase Inhibitors


2qwk

arg

glu


Binding Modeof OseltamivirC. U. Kim et al., J. Am. Chem. Soc.119, 681-690 (1997)


Other Neuraminidase Inhibitors

OH

O

OHNH

CH3CH3

CH3O

NH

NHNH2

BCX-1812 =RWJ-270201(BioCryst, J&J)

IC50 = 0.1-11 nM

OH

ONH2

NHNH

O CH3

BANA-113(BioCryst, Univ. Alabama)

NH

OHO

NO

OH

OH BANA-206

A. F. Abdel-Magid et al., Curr. Opin. Drug. Discov. Dev. 4, 776-791 (2001)


Other Neuraminidase Inhibitors

NHO

NH O

COOH A-315 675Ki = 0.02 - 0.31 nM(different strains)

N

NH2

CF3

OO

CH3

CH3Me

COOH

A-192 558(Abbott Labs)

COOH

ONHAc

Ki = 45 nM

A. F. Abdel-Magid et al., Curr. Opin. Drug. Discov. Dev. 4, 776-791 (2001)

Drug Discov. Today 7, 1066 (2002)

S. Hanessian et al., Bioorg. Med. Chem. Lett. 12, 3425-3429 (2002)


The Relevance of Protein Crystal Structures:Conformational flexibility of PNP in the crystal

NH

N N

N

O

NH2

S

NH

HO

CH3

H

Asn 243

Thr 242

6 73 9

H NO

H

NH

N

N

O

NH2

S

NH

H

Asn 243

H

NH

O Thr 242

63

79

H

H O

CH3

unfavorablepolar/nonpolarand stericinteractions

favorablehydrogenbonds

favorablehydrogenbond

unfavorablehydrogenbonds ?


The Relevance of Protein Crystal Structures:Enzymatic activity of PNP crystals

O

OHOH

OH N

NHN

N

O

NH2

N

NHN

N

O

NH2-+

+

removecrystal

add crystal

reactionrate(% product)

H2PO4 H

O

OHOH

OH OPO3H-

PNP

remove crystal

timeadd crystal

structure-based ligand design - kubinyikubinyi.de/dd-16.pdf · 2005. 12. 25. · a. cressy morrison...

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