Enabling fragment-based lead discovery & structure- based design for GPCRs using stabilized receptor (StaR®) technology

Jonathan S Mason

Overview

ƒ GPCRs the largest drug-target gene family • 50 well validated but poorly tractable current Pharma targets • Instability of isolated GPCRs major obstacle to drug discovery

ƒ Integrated GPCR Drug Discovery Engine based on stabilised receptor (StaR®) technology overcomes this issue

ƒ $33M Series A fund raise completed Feb 2009

ƒ Focus on internal drug discovery pipeline

ƒ $200M deal on single non-pipeline target with Novartis

ƒ Scope for additional, broad-based strategic alliance GPCR Drug Discovery

Pharma HTS success rate only 1:10

• GPCRs once considered highly tractable targets but very slow progress over last decade

• Yet GPCRs still form 30% of current Pharma targets due to compelling biology

• Most recent pipeline compounds large and lipophilic - high-attrition chemotypes

• Need Structure-Based Design approaches to produce atom-efficient NCEs Wenlock, Austin, Barton, Davis and Leeson, J. Med. Chem. 2003, 1250 • But GPCR discovery previously limited to testing in cells - StaR® s are the solution

GPCR Drug Launches

GPCR Drugs Launched compared with all NMEs

ƒ 24% of launched drugs in the last decade hit GPCRs ƒ This is 63 NMEs ƒ The numbers of launched GPCRs has actually increased in 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 the last few years ƒ However only about 1 new GPCR Drugs Launched in 2009 GPCR is drugged per year Nuvigil armodafinil α1‐adrenoceptor agonist ƒ Many drugs are ‘me-too’ or have Saphris asenapine poly‐pharmacology monoamine receptors Firmagon degarelix acetate GnRH antagonist spectrums of activity vs multiple Fanapt Iloperidone D2/D3/α2c/5HT1A/5HT6 previously drugged receptors Onbrez Breezhaler indacaterol β2 agonist Victoza GLP1 agonist ƒ There have been multiple phase Remitch nalfurafine HCl κ‐opioid Mozobil plerixafor CXCR4 3 failures in the last 2 years for Talion bepotastine H1 antagonist new MoAs Effient prasugrel P2Y12 antagonist Nucynta tapentadol MOR agonist (and noradrenaline reuptake inh) Samsca tolvaptan vasopressin V2 antagonist Taltirelin (2000) Ramatroban (2000) (2001) Aprepitant (2003)

TRH receptor agonist / Thromboxane receptor antagonistNK1 antagonist / oral oral DP2 antagonist / oral (ETA / ETB) / oral MWT = 534 MWT = 405 MWT = 416 MWT = 552 cLogP = 4.8 cLogP = -1.4 cLogP = 4.0 cLogP = 4.2 O O O N F O N O S F NH HN O OH O HN N O O N O F H O N N O O O NH O N FF S O N H N N F 2 N NH N F N OH H F

Cinacalcet (2004)Ramelteon (2005) Conivaptan (2005) Rimonabant (2006)

Calcium-sensing receptor MT1/2 agonist /oral V1A/V2 antagonist / IV CB1 inverse agonist / oral allosteric modulator / oral MWT = 259 MWT = 499 Now withdrawn MWT = 357 cLogP = 2.5 cLogP = 5.0 MWT = 464 cLogP = 6.4 cLogP = 6.5

N NH H F N Cl N F NH F O O O N Cl N N HN O O

NH Cl Maraviroc (2007) Plerixafor (2009) CCR5 antagonist / oral CXCR4 antagonist / SC MWT = 514 MWT = 502 Biological Agents cLogP = 3.3 cLogP = -0.2 Atosiban V1A/oxytocin (2000) F F Ganirelix GnRH (2000) N N N N GLP1 (2005) ONH N HN NH NH HN N B2 (2009) N N H H

Intractable GPCR Targets

Marketed Small molecule Marketed biological 57 25 152 105 36 High disease validation

Lack of Selectivity Poor/limited chemistry No drug-like molecules Muscarinic M1. M4 Many Chemokines GLP1 CRF1 PTH Serotonin receptors MC4 Ghrelin Lipid receptors/fatty acid VIP Dopamine D1 TGR5 PARs FSH Complement C3a,C5a CGRP GnRH mGluRs 6 StaRs® are a bridge between established discovery paradigms

StaR® Technology

GPCRs Structure-based Drug Design

Proven Target Superfamily Proven Technology

7 Heptares Therapeutics Ltd

Advantages of SBDD over Empirical Lead Optimisation

SBDD targets out perform GPCR targets in terms of numbers of clinical compounds and smaller numbers of discontinued projects • 3 times the success rate of agents in Phase 1 for SBDD vs GPCR • Higher numbers of agents in P3 and pre-registration (28 vs 12) 2 carefully matched sets of 10 targets, • 70% GPCR projects discontinued SBDD vs GPCRs ƒ +/- Same number of launched vs 43% SBDD drugs for both ƒ Clinically validated MOA ƒ Industry ‘hot’ targets ƒ Large data set (Thomson Pharma

n=1095) What is a StaR®? • A GPCR containing a small number of point mutations that greatly improve its thermostability - Stable in purified, detergent solubilised form - Functional and drug-binding characteristics preserved - Trapped in relevant conformation that matches drug Product Profile - Patent protected technology - Suitable for uHTS, Biacore (kinetics), crystallisation etc. - Transferrable across GPCR superfamily

Heptares StaR® Technology

AR AR* AR*G

Binding Functional assay Assay

RR*R*G • Receptors embedded in cell membrane exist in multiple conformations – Highly unstable when removed – Not suitable for structure based drug discovery methods • Heptares’ technology is used to make a stabilized versions of target GPCRs (StaRs) held in a specific chosen conformation – Stable in functionally-relevant, purified form • Discover Leads using the conformation that fits pharmacology of Target Product Profile – N.B. always follow up with wild type screens StaR® -Based GPCR Drug Discovery

Cell Based Assays Protein-Ligand Fragment Screening High Throughput Screening Complexes SPR / NMR direct binding

Recombinant SPR Kinetics Receptor and Membrane Thermodynamics Assays

In vitro In vitro Pharmacology Pharmacology Isolated Receptor Selectivity Conformations In vivo Modelling by Modelling by Therapeutic Pharmacology SDM with Biophysical Antibodies Efficacy and Radioligand MappingTM Target Validation Receptor Occupancy 11

Rapid Pipeline Progress Since Series A Technology Fully Transferrable Across Superfamily

StaR StaR Drug Discovery generated Structural Information Hits to Lead Candidate leads Optimization Selection

Adenosine A2a programme – Parkinson’s disease

CNS

Cognition

Oncology

>20 StaRs to >12 receptors: • 4 receptors • 2 Family B receptors • Family A receptors •Multiple Conformations

Target Indications: •CNS • Metabolic •Cancer •Inflammation Proprietary Process for Creating StaRs

Select AATCAGCTT Conformation GTCGAA Iteration

Ligand selection Mutagenesis Unstable Assay development Native receptor

100

75

50

25

0 5 10 15 20 25 30 35 40 45 50 Temperature Recombination Thermostability of mutants

New Crystallise GPCR structure

Fragment screening StaR®

Kinetics Purify Agonist or Antag or… protein conformation e.g. SPR/Biacore Pharmacology FSEC TINS/Zobio

TypesTypes ofof StaRsStaRs

Antagonist StaR Inverse Agonist StaR

Agonist state 1 Agonist state 2 StaR StaR

PAM StaR NAM StaR

StaR proteins are locked in the conformation derived from the pharmacology of the ligand used in their creation Types of StaRs

Inverse Antagonist Agonist Agonist/ Agonist G protein

Inverse Antagonist Agonist Agonist/ Agonist G protein Agonist affinity Low Low High High Antagonist affinity High High Low Low

Signalling N/Y (high agonist) Y (high agonist) Partial or N Y Ionic lock Yes Partial Broken Broken

StaRs give a general approach to thermostabilisation Family A – solubilised Family B (normalised data)

27°C 27°C

39°C 43°C

Family A peptide - purified Chemokine receptor (raw data shows 30°C higher yield of functional protein) StaR

43°C WT and early StaRs Pharmacology correlates with the isolated conformation ƒ Inverse agonist StaR shows excellent correlation to wild-type for binding of antagonists / inverse agonists from a range of chemical classes ƒ Indicates antagonist binding site is unaltered ƒ Improved affinity for StaR due to inverse agonist conformational trapping ƒ Conformation specific to pharmacological class not chemotype

WT v StaR2 [3H]-ZM241385 competition

10 CGS15943

9 SCH58261

8 XAC KW6002 7 DPCPX , StaR2 I 6 Theophylline pK 5 CGS21680 NECA 4

4 5 6 7 8 9 10

pKI, WT

StaRs show increasing conformational selection during optimisation

Family A peptide receptor StaR signalling Antagonist StaR Generation Process

Thermostability

Number of mutations

Conformational selection

Shift in equilibrium to ground state Log agonist [peptide]

Agonist potency Isolating R and R* conformations of GPCRs •StaRsof different conformations of the same receptor highlight ability to screen for desired pharmacology using a binding assay •Agonist affinities at Family A agonist and antagonist StaRs •Useful for screening for specific pharmacologies

10 Increased affinity at agonist StaR (R*) Does the difference 9 in affinity at agonist 8 and antagonist StaR

, StaR represent efficacy ? I 7 pK 6

5 Reduced affinity at antagonist StaR (R)

5 6 7 8 9 10

pKI, wild-type

Understanding GPCR Pharmacology

Ä First remove the immense bias and potential “force fitting” we have when only ligand structures are known

Ä A real issue for the key drug class of GPCRs

… Until we could stabilize them in antagonist/agonist/… conformations and do X-ray structures with ligands / fragments & biophysical (fragment) screening & binding site mapping (using stabilized mutant structures) Need to be careful about biases in how we see data – based only on ligands?

Courtesy of Arthur Doweyko, BMS

A2A StaR Crystallography Conventional Detergents/Vapour Diffusion

Wide range of crystals in 5 different detergents

Crystals up to 0.5mm in size

Heptares A2A crystal structure solved from single crystal

8 co-structures, wide range of potency and size (10nM to Visible UV 20uM, 2-600 Da)

Greater stability => better quality protein, reduced flexibility => better crystals Beta-1 Adrenoceptor (β1 AR) StaR X-ray Structure Collaboration with LMB

Entrance to ligand binding site well defined – high resolution

F327A 9 drug co-crystal structures M90V now solved in detergent Agonists and Antagonists F338M Low and High Affinity

Y227A Activation and G-protein binding R68S region retained Multiple loop conformations resolved cf biased agonism A282L

β1 AR agonists & antagonists cluster into different binding modes

• Agonist ligands – green carbons • Antagonists ligands – light carbons

Significant changes in ligand S212 position, hydrogen bonding, backbone and side-chains S215 observed β1 vs β2 Crystal Structure Comparison

Water Probe Grid Map

Hydrophobic Probe Grid Map

β1 AR (2VT4) β2 AR (2RH1)

rhodopsin agonist v’s antagonist 2X72 c.f. 1HZX

top side

P U

IN

NI

I N OUT & TWIST

OUT What causes activation?

TM4 UP TM3 TM2 IN TM5

TM1

NI TWIST I

TM6 TM7 N

β2 agonist v’s antagonist 3POG c.f. 2RH1

top side

P U

IN

NI

I N TWIST

OUT β2 agonist v’s antagonist 3POG c.f. 2RH1

D3.32

S5.42

S5.46 Y7.42 P5.50 I3.40

N7.39

W6.48

Agonists,“agonists”& antagonists

• Binding site surfaces of β2 agonist in β2 agonist structure (3POG), β1 agonist in β1 antagonist StaR structure (2Y03) S5.42 and β1 antagonist in β1 antagonist StaR structure D3.32 (2VT4) showing contraction of the site due S5.46 to agonist binding and receptor activation Heptares Drug Discovery

Biophysical Mapping Hits to Leads Virtual Lead Screening Optimisation SPR Fragment Receptor screening Kinetics StaR Protein Protein-Ligand Complexes

Pharmacokinetics In vivo efficacy Protein Crystallography

Adenosine A2A Antagonist Virtual Screen

• Homology models based on β1 structure built and refined by extensive mutagenesis data. Point mutants that affect ligand binding cluster around active site. – Model adjusted significantly to fit with mutation/ligand-binding data (Modeller, MOE) • Library of 540K compounds (CNS property-filtered etc) screened in silico by docking using Glide/SP. Bias towards compounds which docked into the most buried part of the site, remote form the low confidence region bordered by the ECL2 loop. • 372 compounds were prioritized following post-processing and visualization in the models. 231 compounds were purchased – 20 exhibited activity in binding assay (IC50<55μM) covering 12 chemotypes – Hit rate of 9% • The most potent and ligand efficient molecules were selected – Resulted in 4 hit series • Subsequent comparison with X-ray structure showed good agreement in particular around the binding mode of ZM- 241385. Fragment Screening Cascade

Primary screening validated with • SPR NMR/ • SPR SPR NMR/ Primary • NMR TINS TINS Screening • HCS • CE HCS CE Hits triaged by • SPR kinetics SPR Hit SPR Radioligand Thermal • SPR stoichiometry kinetics / binding shift Confirmation • Binding assays stoichiometry • Thermal shift SAR X-ray BPM Hits validated by Hit • SAR / analogues Validation • X-ray / modelling Structural • BPM / SDM model

33

Fragment Screening: The new possibilities for GPCRs as well as enzymes

Enzymes GPCRs

StaR™ PAM Select NAM conformation Stabilize

StaR proteins are locked in the conformation derived from the pharmacology of the ligand used Immobilized protein in their creation fragment screening Biophysical mapping SPR (Biacore) (SPR), Competition Identify binding TINS (Zobio) studies (SPR & TINS) & position / mode X-ray structures NMR/TINS method for finding hits: fragment screening Ä Immobilized protein – only small amounts needed (~1mg) Ä Very sensitive: higher mM hits identified (not found by SPR)

TINS = Target Immobilized NMR Screening: Zobio

Adenosine A2A Binding Modes: Biophysical Mapping comparison with Crystal Structures

StaR Mutant panel

Biophysical Mapping

• Not possible with native receptors due to instability and lack of sensitivity Data Analysis Protein / Ligand Map • Provides detailed 3D structural data in the absence of X-ray structure

• Structure based approach drives efficient lead optimisation

– Maintain ligand efficiency whilst improving potency/selectivity

– Greatly reduced timelines during LO.

– Knowledge driven rather than empirical chemistry 3D – Greatly improved drug like properties Structural leading to reduced risk and attrition Model Structure based discovery of A2A Antagonists

CH3 for Parkinson’s Disease O O O

N N N Preladenant in Ph III N N N H2N N N (Gold Standard)

• Range of Structure based approaches used to

discover novel series of A2A antagonists • Lead generation from virtual screening and fragment screening • Very rapid lead optimisation phase – 18months to candidate selection phase

• Lead optimisation informed by: Effect of istradefylline, preladenant or HTL (1 mg/kg, PO) on haloperidol-induced catalepsy in rats – Biophysical mapping using SPR – Rapid co-crystallization of lead compounds 100 1h post-dose 2h post-dose • Kinetic profiling by SPR on all compounds 80

– Selection of slow off rate compounds 60

• Heptares candidate 40

– Greatly improved properties compared to other A2A antagonists (eg molecular weight, pharmacokinetics) 20

– Nanomolar affinity and selectivity 0 % 0.8 mg/kghaloperidol response – Very high oral bioavailability (80-100%), low clearance, nt dol ine a TL i n H fyll e low plasma binding (~90%), high solubility per e d lo d la a a H tr – Oral efficacy in vivo ED50 of <1 mg/kg across multiple Is Pre compounds

Family A Chemokine Receptor Antagonist Breakthrough to a Highly Intractable Target

ƒ SBDD and fragment screening ƒ 5% hit rate from Heptares’ 800-member Fragment library ƒ Clinical gold standard is not Rule of 5 compliant ƒ Potent and low molecular weight start-point ƒ Promising low-nanomolar atom efficient lead series

Surface of hit compound bound to chemokine Heptares’ Lead Clinical Gold Standard homology model 38 Summary - Heptares

X Transformational technology for GPCR drug discovery - Structures at last for agonist and antagonist ligands etc + early biophysical screening to identify fragments etc + binding modes etc

X Validated StaR® SBDD platform

Summary

• Transformational technology for GPCR drug discovery • Validated StaR technology platform • Experienced management • Established drug discovery capability – Adenosine Receptor programme (A2a antagonist in PD) in candidate selection • Balanced business model: – 'Platform & Product‘ – Pipeline focussed on difficult/intractable but validated targets – First major deal ($200M) done with Novartis Oct 2009 on a single target

– Discovery Alliance – new drug leads to designated set of targets • Strong cash position to invest in future growth and development – $30M Series A February 2009 – Clarus, MVM, NOF Heptares

Malcolm Weir CEO Fiona Marshall CSO Barry Kenny CBO

Miles Congreve: Head of Chemistry Heptares Therapeutics Ltd Jonathan Mason: Head of Comp Chem BioPark Chris Langmead: Head of Pharmacology Broadwater Road Welwyn Garden City Molecular Biology Herts AL7 3AX Protein Sciences UK Structure Group

LMB [email protected] Richard Henderson Tel: +44 (0)1707 358 649 Gebhard Schertler [email protected] Chris Tate Tel: +44 (0)1707 358 629

www.heptares.com