What lessons can we learn from 20 years of chemokine receptdtor drug discovery?

John G. Cumming, PhD 5th RSC / SCI symposium on GPCRs in Medicinal Chemistry 15th-17th September 2014, Actelion, Allschwil, Basel, Switzerland Outline

Background: chemokines and their receptors

Chemokine receptor drug discovery and development

Emerging opportunities for chemokine drug discovery

Conclusions and learning Chemokines and chemokine receptors

CXC(α)

• Chemokines (chemoattractant cytokines) are 70-120 aa proteins • 44 chemokines in 4 major families and 22 chemokine receptors in human genome

• ‘Cell positioning system’ in the body

• Many receptors bind multiple ligands • Many ligands bind multiple receptors Chemotaxis

Human monocytes + CCL2 (red)

Volpe et al. PLoS ONE 2012, 7(5), e37208 CCR2 antagonists inhibit chemotaxis and infiltration

Vasculature

CCL2 release Spinal or Peripheral Tissue Recruited monocyte Site of CCL2 release CCR2 antagonists inhibit chemotaxis and infiltration

CCR2 antagonist

Circulating monocyte

CCL2 release CCL2 release from peripheral injury site or central PAF terminals Role of chemokine system in pathophysiology

• Potential role in inflammatory and autoimmune diseases: Multiple sclerosis, Rheumatoid arthritis, COPD, allergic asthma, IBD, psoriasis - Expression levels of chemokines and receptors in relevant tissues and organs of patients and animal disease models - Mouse knockout ppyphenotype in disease models

• Established role in HIV infection Katschke et al., 2001 Arthritis Rheum, 44, 1022 - CCR5 and CXCR4 act as HIV-1 co-receptors for virus entry to T-cells (M-tropic and T-topic strains respectively) - Individuals with Δ32-CCR5 mutation protected from HIV infection

Garin, Proudfoot Exp. Cell Res. 2011,317, 602-612 Discovery of chemokine receptor antagonists Chemical tractability

• Class A GPCRs • Example of protein-protein interaction • Challenging to find low MWt, high LLE antagonists

• Small molecule antagonists and agonists • Identified byyg binding/functional assa ys • Allosteric ligands • Early compounds:

TAK-779 CCR5/CCR2 Takeda

NH2

NH O O BX-471

O F CCR1 N Berlex N Cl Hopkins et al. NRDD 2014,13, 105–121 CCR5 allosteric ligand binding site hypotheses

• Receptor homology modelling based on rhodopsin structures • Site directed mutagenesis to identify critical residues • Two hydrophobic pockets - extracellular face of TMI-III & TMIII -VII • Glu283 essential for almost all compound interactions (except TAK-779) • Partially overlaps with endogenous ligand binding site 2

Metz, M. et al. J. Am. Chem. Soc. 2011, 133, 16477-16485 Identification of intracellular allosteric site

• Potency measured by inhibiting calcium response • 100 times more potent at CXCR2 than at CXCR1 • Potency increases with increased lipophilicity in cellular Ca2+ flux relative to cell-free radioligand displacement

• CXCR1-2-1 chimera constructs used to probe site • Site directed mutagenesis studies • Receptor modelling based on rhodopsin structure • Key amino acid residues influencing antagonism between 302 and 327 (C-terminal tail), particularly 320 (Asp in CXCR1, Lys in CXCR2)

Nicholls et al. Mol. Pharm. 2008, 74, 1193-1202 Clinical development of C(X)CR antagonists

Receptor Phase 1 Phase 2 Phase 3 Indications CCR1 AVE-1701 BX-471 AZD-4818 MS, psoriasis, endometriosis, RA, BI-638683 MLN-3897 CCX-354 COPD, multiple myeloma CP-481,715 C-6448 CCR2 INCB-8696 MLN-1202a INCB-003284 RA, atherosclerosis, MS, lupus, CCX-915 CCX-140 BMS-741672 type II diabetes, diabetic CCX-872 PF-4136309 MK-0812 neuropathy, pain, allergic rhinitis, JNJ-17166864 PF-04634817 bone metastases AZD-2423 CCR3 DPC-168 ASM-8b GW-766994 asthma, allergic rhinitis MS-639623 AZD-3778 QAP-642 CCR4 GSK-2239633 asthma CCR5 NIBR-6465 UK-127,857 vicriviroc aplaviroc RA, HIV HGS-004a INCB-9471 cenicriviroc TBR-220 PF-232798 PRO-140a SCH-532706 DAPTAd AZD-5672 SB-728c CCR9 CCX-507 vercirnon IBD, Crohn’s CXCR1/2 AZD-4721 navarixin ladarixin reparixin COPD, pancreatic islet transplantation, bullous pemphigoid CXCR2 SB-332235 elubrixin AZD-5069 COPD, cystic fibrosis danirixin PS-291822 CXCR3 AMG-487 T-487 psoriasis CXCR4 MDX-1338a burixafor ALX40-4Cd stem cell transplant, multiple CTCE-9908d POL-6326 AR-177b myeloma, non -Hodgkins AMD-070 BKT-140d olaptesedb lymphona, HIV aNeutralizing monoclonal antibodies. bAntisense oligonucletide. cZinc finger nuclease. dPeptide J. Med. Chem. 2012, 55, 9363. Under Active Development (TR Integrity) Approved C(X)CR antagonists

Anti-CCR4 humanised monoclonal antibody

Drug Plerixafor Maraviroc Mogamulizumab Brand name Mozobil Celsentri / Selzentry Poteligeo Code name AMD-3100 UK-427857 KW-0761 Originator AnorMED Pfizer Kyowa Hakko Kirin Approved 2003 2007 2012 Receptor CXCR4 CCR5 CCR4 Indications to enhance mobilization of combination antiretroviral relappysed or refractory hematopoietic stem cells for treatment of adults in CCR4-positive adult T- autologous transplantation whom CCR5-tropic HIV-1 cell leukaemia- in patients with lymphoma virus is detectable lymphoma and multippyle myeloma Advanced clinical C(X)CR antagonists

Na + O O S O N–

N+ Not disclosed – O Cl

Drug Vercirnon Reparixin - - Brand name Traficet-EN - - - Code name CCX-282 DF-1681 PF-04634817 CCX354-C Originator ChemoCentryx Dompé Pfizer/Incyte ChemoCentryx Receptor CCR9 CXCR1 CCR2 CCR1 Status Phase 3 for IBD and Phase 3 for prevention Phase 2 for the Evidence of clinical Crohn’s disease of graft dysfunction treatment of type 2 efficacy in RA in a Rights returned by GSK after islet diabetes and overt Phase 2 trial* in 2013 tlttiittransplantation in type nephthhropathy, and Rig hts re turne d by 1 diabetes patients diabetic macular GSK in 2013 edoema

*Tak et al. Ann. Rheum. Dis. 2013, 72, 337-344 Chemokine receptor projects at AstraZeneca Overview

• Identified small molecule antagonist candidate drugs for almost all chemokine receptor targets prosecuted - CCR1, CCR2, CCR3, CCR4, CCR5, CX3CR1, CXCR2 • Reasons for candidate drug failure (from the 5 ‘R’s publication*):

100% Example: AZD3778 90% • failed to show efficacy in PoP (Phase 2) 80% study in asthma patients 70% • dose limited by safety concerns 60% Strategy • high protein binding and short half-life 50% PK/PD • unclear whether adequate receptor 40% Efficacy exposure had been achieved Safety 30% 20% 10% 0% Preclinical (9) Phase 1 (4) Phase 2 (3)

*Projects active 2005-2010 Cook et al. NRDD 2014,13, 419-431 Chemokine receptor projects at AstraZeneca CCR5 antagonists for the treatment of RA

Hit-to-Lead

HTS hit (SPA binding to Lead optimisation CCR5 membranes) Main issues: hERG, PK

Improve LLE

AZD5672 Failed in pre-clinical safety - insufficient Sub-nanomolar CCR5 antagonist margin to QT prolongation LLE = 8.1

Cumming, J. G.* et al. Bioorg. Med. Chem. Lett. 2012, 22, 1655-1659 Chemokine receptor projects at AstraZeneca Binding kinetics and potency of AZD5672

• Binding Ki 0.16 nM - displacement of [125I]MIP-1α • Binding Kd 0.10 nM - binding of [3H]-AZD5672 • Dissociation t½ at room temperature >8h8 h • Dissociation t½ at 37°C 68 min.

• IC50 MIP-1β induced calcium flux 0.16 nM* • IC50 MIP-1β induced chemotaxis 0.63 nM* • Pseudo A2 MIP-1β induced receptor internalisation in human whole blood 0.76 nM (0.05 nM free drug)* Receptor internalisation

120 control 100 1nM

*Cannot measure A values in these nse 2 oo 80 2nM assays due to slow off-rate - pseudo 5nM 60 10nM non-competitive behaviour 40 % max resp 20

0 -10 -9 -8 -7 -6 -5 log MIP-1 conc(M) Chemokine receptor projects at AstraZeneca Clinical development of AZD5672

• Target validation - Expression of CCR5 and its ligands significantly increased in RA patient synovial tissue - Some studies link CCR5Δ32 allele to lower incidence of RA - AZD5672 and other CCR5 antagonists show no activity vs rodent CCR5 - no disease model work undertaken at AZ - Significant effects on clinical arthritis score reported with SCH-X in a rhesus monkey collagen-induced arthritis model • Clinical results - AZD5672 well tolerated and showed good PK in Phase 1 - Phase 2b study in 371 RA patients receiving methotrexate - 20, 50, 100, 150 mg qd AZD5672 vs placebo vs etanercept - Increased rate of infection vs placebo - Failed primary endpoint: no significant effect on ACR20 response at week 12 - Ex vivo receptor internalisation assay showed complete inhibition of CCR5 - Negative results also seen with maraviroc and SCH351125 in RA

Gerlag et al. Arthritis Rheum. 2010, 62, 3154-3160 Chemokine receptor projects at AstraZeneca CCR2 antagonists for the treatment of RA

singleton HTS hit CCR2 binding IC50 =023µM= 0.23 µM AZD6942 CCR2 binding IC50 = 1.7 µM CCR2 binding IC50 = 0.029 µM Chemotaxis IC50 = 2.5 µM Chemotaxis IC50 = 0.06 µM No rodent activity

AZD6942 discontinued in Phase 1 • shorter than predicted half-life • poorly tolerated

‘Discovery and Optimisation of Small Molecule CCR2b Antagonists’ Kettle, J. ACS National Meeting 25 August 2004 Chemokine receptor projects at AstraZeneca 2nd Generation CCR2 antagonist project

Cl Cl Cl O O O

Cl N N Cl N N F N N H H H N N O N O

NH NH N N N

singleton HTS hit AZD2423

CCR2 binding IC50 = 170 nM CCR2 binding IC50 = 2.6 nM 2+ 2+ CCR2 Ca flux IC50 = 965 nM CCR2 Ca flux IC50 = 1.2 nM hERG IC50 = 1.6 µM hERG IC50 = 90 µM 2+ Rat CCR2 Ca flux IC50 = 607 nM Cl ~70-fold selective over CCR5 F S O F AZ889 F N N N H CCR2 0. 46 nM N O hERG 16 µM NH Rat CCR2 1.3 0.2 nM N Rat tool compound

Cumming, J. G. et al. Bioorg. Med. Chem. Lett. 2012, 22, 3895-3899. Chemokine receptor projects at AstraZeneca Selection of disease indication for AZD2423

• Original target disease was RA • Negative clinical trial outcomes from MK-0812 (Merck) and anti-CCR2 antibody MLN1202 (Millennium) in RA patients • MK-0812 is in fact a dual CCR2-CCR5 antagonist MK-0812

• Alternative disease: neuropathic pain • Extensive pre-clinical studies with AZ889 • AZD2423 Phase 2a trials in post-traumatic neuralgia (PTN) and painful diabetic neuropathy (PDN) Pre-clinical rat neuropathic pain models Chronic constriction injury (CCI)-induced mechanical hypersensitivity AZ889 AZD2423

AZD2423AZD2423 inin ChungChung Chun heat heatg hyperalgesia heathyperalgesia hyp modeleral model gesia model Emax >100% 125 EC50 plasma = 191 nM (total), 33 nM (free) original DRC EC brain = 63 nM (total), 3.8 nM (free) 50 100 =6-15) lgesia nn aa 75

50 % Anti-hyper% mean ± ( SEM 25 EC50 = 1500nM (95% CL = 655-3470nM) 0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 Serrano, A. et al. Molecular Pain 2010, 6, 90 Log plasma concentration (mol/L) AZD2423 Ph2a trials in neuropathic pain patients

• Randomized, double-blind, placebo -controlled multi-centre trials • 20 mg or 150 mg AZD2423 or placebo, once daily for 28 days

PTN 133 patients PDN 134 patients

Kalliomäki, J. et al. Pain 2013, 154, 761-767 Kalliomäki, J. et al. Scandinavian Journal of Pain 2013, 4, 77-83 Summary of AZD2423 receptor occupancy AZD2423 is a non-competitive negative allosteric modulator

Peripheral Central

Rat pain model Estimated based on IC50 and exposure 80% 17% (80% reversal of hyperalgesia in CCI)

PDN and PTN Inhibition of CCL2 clearance in vivo 96% - Phase 2a studies Binding ex vivo in peripheral blood monocytes 93 % -

(Css,avg 150 mg)

Calculated based on KB and exposure 97% 90%

• Both expppposure and receptor occupancy exceed efficacious levels in rat pain models • Therefore conclude that the hypothesis that AZD2423 is analgesic in PDN and/or PTN patients has been tested Chemokine receptor projects at AstraZeneca 3rd Generation CCR2 antagonist project

Screen for new hits using FLIPR (Ca2+ flux) functional assay

2+ 2+ CCR2 Ca flux IC50 = 530 nM CCR2 Ca flux IC50 = 16 nM

Drop-off to CCR2 binding Some activityy, at CCR1, CXCR2 No activity at CCR5

Different binding site to piperazine ureas?

Bengtsson, B.A. et al. WO 2011114148 Why have CCR antagonists failed in the clinic?

• “Redundancy” in the chemokine system? - Discrete chemokines under temporal and spatial control in vivo - Early leukocyte migration experiments lacked complete cell population phenotyping - Biased agonism - functionally distinct, not redundant, responses - Chemokine/receptor mouse knockout phenotypes do not show redundancy • The wrong choice of targets - Differences between rodent and human chemokine biology - Rodent disease model experiments challenging due to lack of species crossover • Insufficient therapeutic exposure in clinical trials - Assessment of the true potency of antagonists - Need to mount a continuous effective blockade to infiltrating pro-inflammatory cells • Lack of understanding of chemokine biology - Effec t o f i nhibiti ng T regul at ory cell s i n RA - Effect of blocking chemokine scavenging role of chemokine receptors

Proudfoot, A.E.I. et al. Exp. Opin. Invest. Drugs 2010, 19, 345-355 Schall, T.J., Proudfoot, A.E.I. Nat.Rev. Immunol. 2011, 11, 355-363 Lebre, M.C. et al. PLoS ONE 2011, 6(7), e21772 Future prospects for chemokine receptors Structure-based design opportunities

• CXCR4 X-ray crystal structure with IT1t bound1 • CXCR1 solid state NMR structure in phospholipid bilayer2 • CCR5 X-ray crystal structure with maraviroc bound3

CCR5 + maraviroc

CXCR4 + IT1t

H N N

S

N S N

1Wu, B . et al. Science 2010, 330, 1066-1071 2Park, S. H. et al. Nature 2012, 491, 779-783 3Tan, Q. et al. Science 2013, 341, 1387-1390 Fricker, S.P., Metz, M. Future Med. Chem. 2014, 6, 91-114 Future prospects for chemokine receptors Allosteric binding and biased signalling

• Dompé CXCR1/2 inhibitors • Potent inhibitors of CXCL8 (IL-8)-induced migration of PMNs • Do not inhibit CXCL8 binding to the receptors or CXCL8-induced receptor internalisation • Effect on CXCL8-induced Ca2+ flux only at high concentrations • Hypothesised binding site in CXCR1 does not overlap with endog enous agonist binding site and is not involved in G protein binding

reparixin DF 2156A

Allegretti, M. et al. TIPS (2008), 29, 280-286 Future prospects for chemokine receptors Rational design of insurmountable antagonists

• Optimisation of residence time as well as affinity • Competition association assay with [3H]INCB3344 at 25°C, ratio of binding at 50 min. and 240 min. = KRI

*

Ki = 6.8 nM Ki = 3.6 nM RT = 2.4 min RT = 135 min

Vilums, M. et al. J. Med. Chem. 2013, 56, 7706-7714 Struthers, M.; Pasternak, A. Curr. Top. Med. Chem. 2010, 10, 1278-98 Future prospects for chemokine receptors Oligomerisation of chemokine receptors

• CCR2, CCR5 and CXCR4 form homo and heterodimers, and oligomers • Heterodimers shown to exhibit negative agonist binding cooperativity as a result of allosteric modulation • Selective antagonists show partial inhibition of signalling througgph the other receptor in cells coexppgressing both CCR5 and CXCR4 • TAK-779 (CCR2/5 antagonist) inhibits leukocyte migration into mouse air pouch in response to CXCL12-induced stimulation of CXCR4

• Both challenges and opportunities for drug discovery!

Sohy, D. et al. J. Biol. Chem. 2009, 284, 31270-31279 Conclusions Key learning from 20 yrs of chemokine receptor drug discovery

• Chemokine receptors are druggable targets • Chemokine biology is very complex • SlllSmall molecul es act tillti via allosteric mo dltidulation • High quality drug molecules are required with near-complete receptor occupancy throughout dosing period - insurmountable antagonists • Improvements in GPCR structure-based design and understanding of GPCR biology are starting to impact the field • Opportunity for specific intervention in signalling - requires appropriate assays and compound characterisation

• I20Is 20 years enoughih time to fllfully exp liloit a new target class ? Acknowledgements

• All AZ colleagues who worked on chemokine receptor projects

• All workers in the chemokine field whose publications I have cited