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

What lessons can we learn
from 20 years of chemokine
receptor
t d
drug di
discovery?
?
John G. Cumming, PhD
5th RSC / SCI symposium on GPCRs in Medicinal Chemistry
15th-17
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
Site of CCL2
release
Recruited
monocyte
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 p
phenotype
yp in disease models
Katschke et al., 2001 Arthritis Rheum, 44, 1022
• Established role in HIV infection
- 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 byy binding/functional
g
assays
y
Allosteric ligands
Early compounds:
TAK-779
CCR5/CCR2
Takeda
N H2
HN
O
O
O
F
N
Cl
N
BX-471
CCR1
Berlex
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
TMI III & TMIII
TMIII-VII
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
potent at CXCR2 than at CXCR1
• 100 times more p
• 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
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-140
PF-4136309
JNJ-17166864
AZD-2423
INCB-003284
BMS-741672
MK-0812
PF-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-3778
GW-766994
CCR4
GSK-2239633
CCR5
NIBR-6465
HGS-004a
TBR-220
SCH-532706
CCR9
CCX-507
CXCR1/2
AZD-4721
navarixin
ladarixin
CXCR2
SB-332235
danirixin
elubrixin
PS-291822
AZD-5069
CXCR3
AMG-487
T-487
CXCR4
MDX-1338a
CTCE 9908d
CTCE-9908
AMD-070
aNeutralizing
asthma, allergic rhinitis
asthma
UK-127,857
INCB-9471
PF-232798
DAPTAd
burixafor
POL 6326
POL-6326
BKT-140d
vicriviroc
cenicriviroc
PRO-140a
AZD-5672
SB-728c
aplaviroc
RA, HIV
vercirnon
IBD, Crohn’s
reparixin
COPD, pancreatic islet
transplantation, bullous
pemphigoid
COPD, cystic fibrosis
psoriasis
ALX40-4Cd
AR 177b
AR-177
olaptesedb
stem cell transplant, multiple
myeloma non-Hodgkins
myeloma,
non Hodgkins
lymphona, HIV
monoclonal antibodies. bAntisense oligonucletide. cZinc finger nuclease. dPeptide
Under Active Development (TR Integrity)
J. Med. Chem. 2012, 55, 9363.
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
hematopoietic stem cells for treatment of adults in
autologous transplantation
whom CCR5-tropic HIV-1
in patients with lymphoma
virus is detectable
p myeloma
y
and multiple
relapsed
p
or refractory
y
CCR4-positive adult Tcell leukaemialymphoma
Advanced clinical C(X)CR antagonists
Na
O
–
O
N
+
O
N
–S
O
Not disclosed
+
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
Crohn’s disease
Rights returned by GSK
i 2013
in
Phase 3 for prevention
of graft dysfunction
after islet
t
transplantation
l t ti in
i type
t
1 diabetes patients
Phase 2 for the
treatment of type 2
diabetes and overt
nephropathy,
h
th and
d
diabetic macular
edoema
Evidence of clinical
efficacy in RA in a
Phase 2 trial*
Ri ht returned
Rights
t
db
by
GSK in 2013
*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
• failed to show efficacy in PoP (Phase 2)
study in asthma patients
• dose limited by safety concerns
• high protein binding and short half-life
• unclear whether adequate receptor
exposure had been achieved
90%
80%
70%
60%
Strategy
50%
PK/PD
40%
Efficacy
Safety
30%
20%
10%
0%
Preclinical (9)
*Projects active 2005-2010
Phase 1 (4)
Phase 2 (3)
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
CCR5 membranes)
Lead optimisation
Main issues: hERG,, PK
Improve LLE
AZD5672
Sub-nanomolar CCR5 antagonist
LLE = 8.1
Failed in pre-clinical safety - insufficient
margin to QT prolongation
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 >8
8h
Dissociation t½ at 37°C 68 min.
• IC50 MIP-1β
β 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
*Cannot measure A2 values in these
assays due to slow off-rate - pseudo
non-competitive behaviour
% max respo
onse
120
control
1nM
2nM
5nM
10nM
100
80
60
40
20
0
-10
-9
-8
-7
log MIP-1 conc(M)
-6
-5
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
SCH-X
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 = 1.7 µM
Chemotaxis IC50 = 2.5 µM
CCR2 binding IC50 = 0.23
0 23 µM
AZD6942
CCR2 binding IC50 = 0.029 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
N
H
N
Cl
O
N
H
Cl
N
N
F
N
O
singleton HTS hit
CCR2 binding IC50 = 170 nM
CCR2 Ca2+ flux IC50 = 965 nM
hERG IC50 = 1.6 µM
N
O
AZD2423
CCR2 binding IC50 = 2.6 nM
CCR2 Ca2+ flux IC50 = 1.2 nM
hERG IC50 = 90 µM
Rat CCR2 Ca2+ flux IC50 = 607 nM
~70-fold selective over CCR5
Cl
F
F
N
N
N
N
AZ889
CCR2 0
0.46
46 nM
hERG 16 µM
Rat CCR2 1.3 0.2 nM
Rat tool compound
N
H
HN
HN
F
O
O
S
N
N
H
N
N
O
HN
N
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
anti CCR2 antibody MLN1202
(Millennium) in RA patients
• MK-0812 is in fact a dual CCR2-CCR5 antagonist
• Alternative disease: neuropathic pain
• Extensive pre-clinical studies with AZ889
• AZD2423 Phase 2a trials in post-traumatic neuralgia
(PTN) and painful diabetic neuropathy (PDN)
MK-0812
Pre-clinical rat neuropathic pain models
Chronic constriction injury (CCI)-induced mechanical hypersensitivity
AZD2423
AZ889
AZD2423
in Chung
g heat hyperalgesia
yp
g
model
AZD2423 in Chung heat hyperalgesia model
125
% Anti-hypera
algesia
mean ± SEM (n
n=6-15)
Emax >100%
EC50 plasma = 191 nM (total), 33 nM (free)
EC50 brain = 63 nM (total), 3.8 nM (free)
original DRC
100
75
50
25
EC50 = 1500nM
(95% CL = 655-3470nM)
Serrano, A. et al. Molecular Pain 2010, 6, 90
0
-7.5
-7.0
-6.5
-6.0
-5.5
-5.0
Log plasma concentration (mol/L)
AZD2423 Ph2a trials in neuropathic pain patients
• Randomized, double-blind,
double blind, placebo
placebo-controlled
controlled multi-centre
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
Rat pain model
Peripheral
Central
Estimated based on IC50 and exposure
80%
17%
Inhibition of CCL2 clearance in vivo
96%
-
Binding ex vivo in peripheral blood monocytes
93 %
-
Calculated based on KB and exposure
97%
90%
(80% reversal of
hyperalgesia in CCI)
PDN and PTN
Phase 2a
studies
(Css,avg 150 mg)
•
•
Both exposure
p
and receptor
p
occupancy
p
y 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
CCR2 Ca2+ flux IC50 = 530 nM
CCR2 Ca2+ flux IC50 = 16 nM
Drop-off to CCR2 binding
Some activity
y 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
- Eff
Effectt off inhibiting
i hibiti T regulatory
l t
cells
ll in
i 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
X ray crystal structure with maraviroc bound3
CCR5 + maraviroc
CXCR4 + IT1t
H
N
N
S
N
S
N
1Wu,
Wu
B.
B et al
al. Science 2010,
2010 330,
330 1066-1071
1066 1071
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
2Park,
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
CXCL8 induced receptor
internalisation
• Effect on CXCL8-induced Ca2+ flux only at high concentrations
yp
binding
g site in CXCR1 does not overlap
p with endogenous
g
• Hypothesised
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
RT = 2.4 min
Ki = 3.6 nM
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
through
g the other receptor
p in cells coexpressing
p
g 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
• Small
S ll molecules
l
l actt via
i allosteric
ll t i modulation
d l ti
• 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
• Is
I 20 years enough
h time
i
to ffully
ll exploit
l i a new target class?
l
?
Acknowledgements
• All AZ colleagues who worked on chemokine receptor projects
• All workers in the chemokine field whose publications I have cited