O

ClCl

O

N

NH

O

OH

O

N

Cl

Computational Medicinal Chemistry Approaches for GPCRStructure-Based Drug Discovery

© 2019 Heptares Therapeutics Limited

Disclaimer: Sosei Heptares is a trading name. Sosei and the logo are Trade Marks of Sosei Group Corporation, Heptares is a Trade Mark of Heptares Therapeutics Limited. StaR® is a Trade Mark of Heptares Therapeutics Limited

Juan Carlos Mobarec, Conor G. Scully, Lydia Siragusa*,Francesca Deflorian, Robert T. Smith, Alicia Higueruelo, Jonathan S. Mason, Miles Congreve, Chris De Graaf

Lemborexant

Suvorexant

EMPA

dual Ox1/OX2

dual Ox1/OX2

OX2 selective

Targeting lipophilic hotspots and unhappy water sites determine

ligand binding

Trapped unhappy water in Ox1

2.8 log units selectivity from trapped water, ligand would appear to dock fine if water energetics not considered

OX2/Ox1 selectivity:

Binding pocket Ox1 (A3x33) > Ox2 (T3x33)

Examples

Pharmacophore?

Unpublished in-house structures Ox1/Ox2, bound to different ligands and with different water networks

WaterFLAP: MIF based water

networks.

WaterMap: MD based hydration site thermodynamics.

C sp3 probe 1 kcal/mol

C sp2 probe -2.8 kcal/mol

Water probe -5 kcal/mol

GRID Probes:

Pseudo apo water G: Energetically very unhappy water in A2A

Ligand perturbated G: Energetically stabilised water in A2A

WaterFLAP water energetics A2A QSAR

Collaboration S. Cross, G. Cruciani

Bortolato, Mason et al. (2018) Methods Mol Biol

J. Christopher et al., Unpublished

Lipophilic hotspots & water networks in OX1,OX2 and A2a SBDD from hit-ID to clinical trials: Case A2a antagonist

Partnered with:

MonotherapyAZD4635

(A2a antagonist)

Combination with oleclumab(anti-CD73)

Combination with durvalumab(anti-PD-L1)

• SBDD used to re-engineer a virtual screening hit series to target a lipophilic hotspot deep in the pocket, leading high LE

and LLE drug candidates

• Atom by atom optimisation: ligand efficiency (LE)

• Design polar contacts: control lipophilicity (LLE)

• Multiple structures: receptor flexibility & selectivity

• Druglike properties: In vivo efficacy & safety

Langmead (2012) J Med Chem; Congreve (2012) J. Med. Chem

FEP+ based binding affinity prediction for ligand optimization

Ligand/protein tautomer/protonation state

In-house structures of GPCR target with different ligands

C

C

C

C

Linker changesc. x3 Oral Bioavailability

RHSLHS

Pose at 0 nsPose at 20 ns

H278Pose at 0 nsPose at 20 ns

N2566.55

H278

4a

4g

4e

GCMC

Pose at 0 nsPose at 20 ns

A2a - 4a

N256

H2787.43

4a

Pose at 0 nsPose at 20 ns

A2a - 4a

N2566.55

H2787.43

Pose at 0 nsPose at 20 ns

A2a – 4e

N2566.55

H278 Pose at 0 nsPose at 20 ns

A2a – 4e

N2566.55

H278

4e

4g

lacking water

network

4a

4g

4e

No GCMC

R2=0.95

In-house GPCR structures with representative ligand LHS and variable RHS

Ring conformation sampling

FEP+ guided GPCR LO example 1

A2a – 4g A2a – 4g

Alternative residue rotamers

FEP+ guided GPCR LO example 2

Binding site solvation F. Deflorian D. Branduardi

J. Vendome

Structural chemogenomics codification of GPCRome

B

H

G

C

major

D

I

E

J

L

K

minor

A

BioGPS: GRID based identification and comparison of GPCR binding sites across structural GPCRome.

C5aR4156

GPR405706

ECFP4 > 0.4MACCS > 0.8

64 Similar Bioactive Ligands

• Shared pharmacophore features to target GPCR-membrane interface.• Design ideas/rules for conserved GPCR PAM/NAM pockets.

ligands

Lipophilic HB donor HB acceptorLigands

L. Sygura, G. CrucianiComputational chemistry

ON

ClCl NH

O

OH

O

CH3 CH3

OCH3

SBDD in allosteric binding sites: Cases GLP-1R and PAR2

C

Hit 1

GLP-1R pKi 5.0

HTL26005

GLP1 pKi 8.3

MW 527, clogP 5.1

LE 0.31, LLE 3.2

MK-0893

GLP-1R pKi 7.3

GCGR pKi 8.8

HTL26119

GLP1 pKi 7.8

MW 574, clogP 6.4

LE 0.3 LLE 1.4

Clinically studied

GCGR antagonist

Simple novel starting

point for SBDD efforts

GCGR pKi 6.9

Less potent, non-selective

in functional assay

Selective vs GCGR in binding

Less potent as a functional

antagonist

Virtual

Screen

In silico

design

SBDD

NH

O

OH

O

NN

OCl

ClO

NH

O

OH

O

Jazayeri (2016) Nature

GCGR

• Collaboration with AZ, fragment and HTS screening

• Small molecule antagonists inhibit peptide and proteaseactivation of the receptor

• Difficult to optimise in the absence of structuralunderstanding

• Binding site identified in PAR2 X-ray structure

• AZ8838 buried in small binding pocket (TM1-3/7, ECL2)

• X-Chem DNA encoded library technology

• Binding hits confirmed as functional PAR2 antagonists

• AZ3451 binds in novel extra-helical site

• Interaction with PAR2 predominately hydrophobic

• Mechanism of action may be to restrict theconformational inter-helical rearrangement required forPAR2 activation

• PeptiDream DELT focuses on peptide display

• Successful hit generation approach for wide array of targets

• Utilising the PAR2 StaR in collaboration with Heptares Peptidream have identified several series of potent cyclic peptide antagonists of PAR2

• Current efforts to improve potency and stability of these very encouraging peptide lead cpds using SBDD

SLIGKV(model)

AZ3451 (PDB: 5NDZ)AZ8838 (PDB: 5NDD)

cyclic peptide antagonist(unreleased crystal structure)

A. O’ Brien and co-workers 2019PAR2

Cheng (2017) Nature

Sosei Heptares, Steinmetz Building, Granta Park, Great Abington, Cambridge, CB21 6DG United Kingdom

*Molecular Discovery Ltd. Middlesex , United Kingdom

A) Major and minor(ancestral/classical TM) binding sites

95% GPCR ligands in PDB target this pocket

B) G protein intracellular binding site (B)

CCR2 (5T1A)CCR9 (5LWE)β2R (5X7D)

C) Sodium pocketBLT1 (5X33)mGlu5 (4OO9; 5CGC; 5CGD; 6FFH; 6FFI)

D) TM3/4/5/EL2

PAF-lipid/OLC*

(5ZKP; 5ZKQ)

E) TM3/4/5/membrane

C5AR (5O9H; 6C1Q; 6C1R)FFAR1 (5KW2, 5TZY)

GCGR (4L6R*; 5EE7; 5XEZ; 5XF1)GLP-1R (5VEX; 5VEW)

F) TM5/6membrane

G) TM1/2/3/IL1/Membrane

H) TM3/4/IL/membrane

FFAR1 (5KW2, 5TZY) EP4 (5YWY; 5YFI; 5YHL)TA2 (6IIU)CCR2 (5T1A; 6GPS, 6GPX)CB1 (5TGZ, 5U09)

I) TM1/7/membrane J) TM1/7/H8/Membrane K) TM1/2/3/MembraneP2Y1 (4XNV)

L) TM3/4/5/membranePAR2 (5NDZ)

An increasing number of cryo-EM and X-ray crystal structures of GPCR-ligand complexes continue to reveal previously unknown ligand binding sites. Furthermore, emerging sets of

GPCR crystal structures of multiple diverse ligands bound to closely related receptors enable a protein-structure based view of how different ligands bind this major drug target class.

From the analysis of GPCR structures we gather several important learnings and repercussions for computational medicinal chemistry design that should be transferable and relevant

for many targets, including: A) The important roles of lipophilic hot spots and water networks as drivers of GPCR druggability, ligand binding, and selectivity. B) Diverse binding modes

of similar ligands across the structural GPCRome. C) Caveats when using pharmacophore-based similarity principles for modeling receptor-ligand complexes with different ligand

chemotypes.

Multiple ligand binding sites on GPCR structures

• GPCR binding sites can be identified with BioGPS, which utilizes GRID probes to locate and characterize protein pockets.• Pockets can be encoded into bitstrings which can be used to compare different pockets and measure similarity (e.g. Tanimoto).• Phylogenetic relationships can be build to compare pockets in the structural GPCRome.

F

Multiple in-house Ox1/Ox2 crystal structures for SBDD:- Multiple ligand binding modes – pharmacophore models- Critical to consider water networks in docking and FEP+- Design opportunities for smaller ligands with improved

properties

GCMC for exhaustive

water sampling in

protein binding pocket

during FEP+ production

Water mediated receptor-ligand interactions in crystal structuresNo direct polar (H-bond) interactions