What lessons can we learn from 20 years of chemokine

t d di ?receptor drug discovery?

John G. Cumming, PhD5th RSC / SCI symposium on GPCRs in Medicinal Chemistry15th-17th September 2014, Actelion, Allschwil, Basel,15 17 September 2014, Actelion, Allschwil, Basel, Switzerland

Outline

Background: chemokines and their receptors

Chemokine receptor drug discovery and development

Emerging opportunities for chemokine drug discoveryEmerging opportunities for chemokine drug discovery

Conclusions and learning

Chemokines and chemokine receptorsCXC(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 monocytesHuman monocytes+ CCL2 (red)

Volpe et al. PLoS ONE 2012, 7(5), e37208

CCR2 antagonists inhibit chemotaxis and infiltration

Vasculature

CCL2 release

Spinal or Recruited monocyte

Site of CCL2 release

Peripheral Tissue

CCR2 antagonists inhibit chemotaxis and infiltration

CCR2 antagonist

Circulating monocytemonocyte

CCL2 release from peripheral injury

site or central PAF terminals

CCL2 release

terminals

Role of chemokine system in pathophysiology

• Potential role in inflammatory and autoimmune diseases: Multiple sclerosis, Rheumatoid arthritis, COPD, allergic asthma, IBD, psoriasisasthma, IBD, psoriasis

- Expression levels of chemokines and receptors in relevant tissues and organs of patients and animal disease models

- Mouse knockout phenotype in disease modelsp yp

• 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 antagonistsChemical tractability

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

• Small molecule antagonists and agonists • Identified by binding/functional assaysy g y• Allosteric ligands• Early compounds:

TAK-779CCR5/CCR2Takeda

NH2

ONH O BX-471CCR1

Hopkins et al. NRDD 2014,13, 105–121Cl

ON F

N

CCR1Berlex

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-VIITwo 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 CXCR1p• 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 antagonistsReceptor Phase 1 Phase 2 Phase 3 IndicationsCCR1 AVE-1701

BI-638683 BX-471

MLN-3897 CP-481,715

AZD-4818 CCX-354 C-6448

MS, psoriasis, endometriosis, RA, COPD, multiple myeloma

CCR2 INCB-8696 CCX-915 CCX-872

MLN-1202a

CCX-140PF-4136309

JNJ-17166864AZD-2423

INCB-003284 BMS-741672

MK-0812PF-04634817

RA, atherosclerosis, MS, lupus, type II diabetes, diabetic neuropathy, pain, allergic rhinitis, bone metastases

CCR3 DPC-168 MS-639623 QAP-642

ASM-8b

AZD-3778GW-766994 asthma, allergic rhinitis

CCR4 GSK-2239633 asthmaCCR5 NIBR-6465

HGS-004a

TBR-220 SCH-532706

UK-127,857INCB-9471PF-232798

DAPTAd

vicriviroccenicrivirocPRO-140a

AZD-5672SB-728c

aplaviroc RA, HIV

CCR9 CCX-507 vercirnon IBD, Crohn’sCXCR1/2 AZD-4721 navarixin ladarixin reparixin COPD, pancreatic islet

transplantation, bullouspemphigoid

CXCR2 SB-332235 danirixin

elubrixinPS-291822

AZD-5069 COPD, cystic fibrosis

CXCR3 AMG-487 T-487 psoriasis CXCR4 MDX-1338a

CTCE 9908dburixafor

POL 6326ALX40-4Cd

AR 177bstem cell transplant, multiple myeloma non HodgkinsCTCE-9908d

AMD-070 POL-6326 BKT-140d

AR-177b

olaptesedbmyeloma, non-Hodgkinslymphona, HIV

aNeutralizing monoclonal antibodies. bAntisense oligonucletide. cZinc finger nuclease. dPeptideUnder Active Development (TR Integrity)

J. Med. Chem. 2012, 55, 9363.

Approved C(X)CR antagonists

Anti-CCR4 humanised monoclonal antibody

Drug Plerixafor Maraviroc MogamulizumabBrand name Mozobil Celsentri / Selzentry Poteligeo

Code name AMD-3100 UK-427857 KW-0761

Originator AnorMED Pfizer Kyowa Hakko Kirin Approved 2003 2007 2012Receptor CXCR4 CCR5 CCR4Indications to enhance mobilization of combination antiretroviral relapsed or refractory

hematopoietic stem cells for autologous transplantation in patients with lymphoma and multiple myeloma

treatment of adults in whom CCR5-tropic HIV-1 virus is detectable

p yCCR4-positive adult T-cell leukaemia-lymphoma

p y

Advanced clinical C(X)CR antagonists

O

N+

SOO

N–Na +

Not disclosed

Drug Vercirnon Reparixin - -

NO–

Cl

Brand name Traficet-EN - - -Code name CCX-282 DF-1681 PF-04634817 CCX354-COriginator ChemoCentryx Dompé Pfizer/Incyte ChemoCentryxReceptor CCR9 CXCR1 CCR2 CCR1Status Phase 3 for IBD and

Crohn’s diseaseRights returned by GSK i 2013

Phase 3 for prevention of graft dysfunction after islet t l t ti i t

Phase 2 for the treatment of type 2 diabetes and overt

h th d

Evidence of clinical efficacy in RA in a Phase 2 trial*Ri ht t d bin 2013 transplantation in type

1 diabetes patientsnephropathy, and diabetic macular edoema

Rights returned by GSK in 2013

*Tak et al. Ann. Rheum. Dis. 2013, 72, 337-344

Chemokine receptor projects at AstraZenecaOverview

• Identified small molecule antagonist candidate drugs for almost all chemokine receptor targets prosecuted

- CCR1, CCR2, CCR3, CCR4, CCR5, CX3CR1, CXCR2CCR1, CCR2, CCR3, CCR4, CCR5, CX3CR1, CXCR2• Reasons for candidate drug failure (from the 5 ‘R’s publication*):

90%

100%

Example: AZD3778• failed to show efficacy in PoP (Phase 2) study in asthma patients

• dose limited by safety concerns 60%

70%

80%

90%

Strategy• high protein binding and short half-life• unclear whether adequate receptor exposure had been achieved

20%

30%

40%

50% PK/PD

Efficacy

Safety

0%

10%

20%

Preclinical (9) Phase 1 (4) Phase 2 (3)

Cook et al. NRDD 2014,13, 419-431*Projects active 2005-2010

Chemokine receptor projects at AstraZenecaCCR5 antagonists for the treatment of RA

Hit-to-Lead

HTS hit (SPA binding to CCR5 membranes)

Lead optimisationMain issues: hERG, PK,

Improve LLE

Failed in pre-clinical safety - insufficient margin to QT prolongation

AZD5672Sub-nanomolar CCR5 antagonistLLE = 8.1

Cumming, J. G.* et al. Bioorg. Med. Chem. Lett. 2012, 22, 1655-1659

Chemokine receptor projects at AstraZenecaBinding 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 >8 hDissociation t½ at room temperature 8 h• Dissociation t½ at 37°C 68 min.

• IC50 MIP-1β induced calcium flux 0.16 nM*50 β• 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)*

80

100

120control1nM2nMon

se

Receptor internalisation

*Cannot measure A2 values in these

20

40

60

805nM10nM

% m

ax re

spo2

assays due to slow off-rate - pseudo non-competitive behaviour

-10 -9 -8 -7 -6 -50

log MIP-1 conc(M)

Chemokine receptor projects at AstraZenecaClinical 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 AZSignificant effects on clinical arthritis score reported with SCH X in a rhesus- 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- 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 AstraZenecaCCR2 antagonists for the treatment of RA

singleton HTS hit AZD6942CCR2 binding IC = 0 23 µMsingleton HTS hitCCR2 binding IC50 = 1.7 µMChemotaxis IC50 = 2.5 µM

AZD6942CCR2 binding IC50 = 0.029 µMChemotaxis IC50 = 0.06 µMNo rodent activity

CCR2 binding IC50 = 0.23 µM

AZD6942 discontinued in Phase 1• shorter than predicted half-life• 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 AstraZeneca2nd Generation CCR2 antagonist project

Cl Cl Cl

Cl

Cl

NNH

O

NO

Cl

Cl

NNH

O

N F

Cl O

ONNN

H

singleton HTS hit

N NNH

NNH

AZD2423singleton HTS hitCCR2 binding IC50 = 170 nMCCR2 Ca2+ flux IC50 = 965 nMhERG IC50 = 1.6 µM

AZD2423CCR2 binding IC50 = 2.6 nMCCR2 Ca2+ flux IC50 = 1.2 nMhERG IC50 = 90 µMRat CCR2 Ca2+ flux IC = 607 nMRat CCR2 Ca flux IC50 = 607 nM~70-fold selective over CCR5

AZ889CCR2 0 46 nM

FF

FCl

N

O

ONNN

H

S

CCR2 0.46 nMhERG 16 µMRat CCR2 1.3 0.2 nMRat tool compound

NNH

N

Cumming, J. G. et al. Bioorg. Med. Chem. Lett. 2012, 22, 3895-3899.

Chemokine receptor projects at AstraZenecaSelection of disease indication for AZD2423

• Original target disease was RA• Negative clinical trial outcomes from MK-0812

(Merck) and anti-CCR2 antibody MLN1202(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 modelsChronic constriction injury (CCI)-induced mechanical hypersensitivity

AZ889 AZD2423

E >100% AZD2423 in Chung heat hyperalgesia modelAZD2423 in Chung heat hyperalgesia modelEmax >100% EC50 plasma = 191 nM (total), 33 nM (free)EC50 brain = 63 nM (total), 3.8 nM (free)

AZD2423 in Chung heat hyperalgesia model

75

100

125 original DRC

alge

sia

n=6-

15)

g yp g

25

50

75

EC50 = 1500nM

% A

nti-h

yper

am

ean

± SE

M (n

Serrano, A. et al. Molecular Pain 2010, 6, 90-7.5 -7.0 -6.5 -6.0 -5.5 -5.00

(95% CL = 655-3470nM)

Log plasma concentration (mol/L)

AZD2423 Ph2a trials in neuropathic pain patients

• Randomized, double-blind, placebo-controlled multi-centre trials

PDN 134 patientsPTN 133 patients

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

Kalliomäki, J. et al. Pain 2013, 154, 761-767Kalliomäki, J. et al. Scandinavian Journal of Pain 2013, 4, 77-83

Summary of AZD2423 receptor occupancy

Peripheral Central

AZD2423 is a non-competitive negative allosteric modulator

Peripheral Central

Rat pain model(80% reversal of hyperalgesia in CCI)

Estimated based on IC50 and exposure 80% 17%

PDN and PTN Phase 2a

Inhibition of CCL2 clearance in vivo 96% -

studies(Css,avg 150 mg)

Binding ex vivo in peripheral blood monocytes 93 % -

Calculated based on KB and exposure 97% 90%

• Both exposure and receptor occupancy exceed efficacious levels in rat p p p ypain models

• Therefore conclude that the hypothesis that AZD2423 is analgesic in PDN and/or PTN patients has been tested

Chemokine receptor projects at AstraZeneca3rd Generation CCR2 antagonist projectScreen for new hits using FLIPR (Ca2+ flux) functional assayScreen for new hits using FLIPR (Ca flux) functional assay

CCR2 Ca2+ flux IC50 = 16 nM

Drop-off to CCR2 bindingSome activity at CCR1, CXCR2

CCR2 Ca2+ flux IC50 = 530 nM

y ,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 biologyEff t f i hibiti T l t ll i RA- Effect of inhibiting T regulatory cells in RA

- Effect of blocking chemokine scavenging role of chemokine receptors

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

Future prospects for chemokine receptorsStructure-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 bound3CCR5 X ray crystal structure with maraviroc bound

CCR5 + maraviroc

CXCR4 + IT1t

NH

N

S

1Wu B et al Science 2010 330 1066 1071

N

NS

1Wu, B. et al. Science 2010, 330, 1066-10712Park, S. H. et al. Nature 2012, 491, 779-7833Tan, Q. et al. Science 2013, 341, 1387-1390Fricker, S.P., Metz, M. Future Med. Chem. 2014, 6, 91-114

Future prospects for chemokine receptorsAllosteric 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 receptorDo 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 endogenous yp g p g

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 receptorsRational 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. = KRI50 min. and 240 min. KRI

**

Ki = 6.8 nMRT = 2.4 min

Ki = 3.6 nMRT = 135 min

Vilums, M. et al. J. Med. Chem. 2013, 56, 7706-7714Struthers, M.; Pasternak, A. Curr. Top. Med. Chem. 2010, 10, 1278-98

Future prospects for chemokine receptorsOligomerisation of chemokine receptors

• CCR2, CCR5 and CXCR4 form homo and heterodimers, and oligomers

• Heterodimers shown to exhibit negative agonist bindingHeterodimers shown to exhibit negative agonist binding cooperativity as a result of allosteric modulation

• Selective antagonists show partial inhibition of signalling through the other receptor in cells coexpressing both CCR5 g p p gand 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

ConclusionsKey learning from 20 yrs of chemokine receptor drug discovery

• Chemokine receptors are druggable targets• Chemokine biology is very complex

S ll l l t i ll t i d l ti• Small molecules act via allosteric modulation• 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 appropriateOpportunity for specific intervention in signalling requires appropriate

assays and compound characterisation

I 20 h i f ll l i l ?• Is 20 years enough time to fully exploit a new target class?

Acknowledgements

• All AZ colleagues who worked on chemokine receptor projects

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