2
Overview
TRPA1 – an introduction Validation as a target for drug discovery
For migraine For respiratory disorders For inflammatory and neuropathic pain
Medicinal chemistry of TRPA1 antagonists
3
TRP channels play diverse (patho)physiological roles
TRPC3, C5, C6TRPM2, M4, M7, ML1TRPC6, TRPV1, V4,
TRPA1, TRPM8
TRPV1, V3, TRPA1
TRPC6, TRPM6, TRPP2
TRPV1, V2, V4, TRPM8, TRPA1
TRPV1, V3, V4, TRPM3, M8, TRPA1
NeuronalRespiratory
Skin
Pain
Kidney
Gastrointestinal
Also bladder disorders, diabetes
4
TRPA1 introduction
Member of the transient receptor potential (TRP) superfamily of ion channels Characterised by extended chain of repeating ankyrin repeat domains Non-selective cation channel; carries both monovalent and divalent cations
TRPA1 activation can drive both depolarisation and calcium signalling Multiple modes of activation
Low temperatures – still some controversy/species differences? Mechanical pressure – still some controversy? Exogenous mediators such as airborne irritants Endogenous mediators such as reactive oxygen species, inflammatory mediators
Expressed in many different tissue beds and implicated in many disease areas
5
TRPA1: A chemosensitive ion channel
FEPS: familial episodic pain syndrome
Chen, Arch Pharm, 2015Preti, Patent Review, 2015
6
TRPA1 receptors integrate signals from multiple sourcesNociception and neurogenic inflammation
TRPA1 agonists activate channel GPCR dependent activation of PLC reduces TRPA1
‘brake’ by PIP2 PLC elevation triggers Ca2+ release from stores which
may increase TRPA1 Ca2+ flux Ca2+ influx via TRPV1 also increases TRPA1 activity TRPA1 also activated by:
Various migraine – causing irritants Methylglyoxal: implications for diabetic neuropathic pain Formalin: mechanistic basis for a common inflammatory
pain model
Heightened intracellular Ca2+ levels trigger release of pro-inflammatory neuropeptides, such as substance P or CGRP, initiating neurogenic inflammation
Chen, Arch Pharm, 2015
7
TRPA1 receptors integrate signals from multiple sourcesNociception and neurogenic inflammation
TRPA1 agonists activate channel GPCR dependent activation of PLC reduces TRPA1
‘brake’ by PIP2 PLC elevation triggers Ca2+ release from stores which
may increase TRPA1 Ca2+ flux Ca2+ influx via TRPV1 also increases TRPA1 activity TRPA1 also activated by:
Various migraine – causing irritants Methylglyoxal: implications for diabetic neuropathic pain Formalin: mechanistic basis for a common inflammatory
pain model
Heightened intracellular Ca2+ levels trigger release of pro-inflammatory neuropeptides, such as substance P or CGRP, initiating neurogenic inflammation
Bessac. Physiology (2008) PMID: 19074743
8
Overview
TRPA1 – an introduction Validation as a target for drug discovery
For migraine For respiratory disorders For inflammatory and neuropathic pain
Medicinal chemistry of TRPA1 antagonists
9
TRPA1: role in migraineActivators of TRPA1 cause migraine
Migraine is triggered by CGRP release from trigeminal neurons, which express TRPA1 Exogenous or endogenous molecules that act as triggers for migraine activate TRPA1 Californian ‘headache tree’ the bay laurel Umbellularia californica releases the reactive
ketone umbellulone, which activates TRPA1
O
RS
O O O
Umbellulone Verbenone PiperitoneThiol adduct
10
TRPA1: role in migraineActivators of TRPA1 cause migraine
No effect on TRPV1 or TRPM8 Umbellulone activation of TRPA1 evokes currents in
trigeminal ganglion neurones Umbellulone releases CGRP from rat spinal cord and
evokes an increase in meningeal blood flow
Nassini, Brain, 2012
11
TRPA1: role in migraineTRPA1 desensitisers protect against migraine
Feverfew is used to treat pain, headaches and migraine The natural product parthenolide (isolated from feverfew) is a partial agonist at TRPA1 Initial activation of TRPA1 develops into desensitisation and reduced neuropeptide release
Nassini, Pain, 2013
Parthenolide
12
TRPA1: role in migraineTRPA1 desensitisers protect against migraine
Feverfew is used to treat pain, headaches and migraine The natural product parthenolide (isolated from feverfew) is a partial agonist at TRPA1 Initial activation of TRPA1 develops into desensitisation and reduced neuropeptide release
Nassini, Pain, 2013
Parthenolide
13
TRPA1: role in migraine
Role of endogenous and exogenous agents that modulate TRPA1 and play a role in migraine
Benemei, BJP, 2013
14
Overview
TRPA1 – an introduction Validation as a target for drug discovery
For migraine For respiratory disorders For inflammatory and neuropathic pain
Medicinal chemistry of TRPA1 antagonists
15
TRPA1: role in respiratory disorders
TRPA1 is expressed in lung fibroblasts, epithelial cells, and c-type neurons Activated by airborne irritants such as cigarette smoke, acrolein
TRPA1 is implicated in cough following exposure to respiratory irritants, bronchoconstriction/airway hyper-responsiveness leading to asthma, and neurogenic inflammation leading to COPD The receptor is activated by inflammatory mediators present in the lung such as bradykinin,
histamine, prostanoids, as well as reactive oxygen species TRPA1 plays a crucial role in airway inflammation and the late asthmatic response TRPA1 integrates interactions between the immune and nervous systems in the airways
TRPA1 antagonists could be effective in diseases such as asthma, COPD and rhinitis
16
TRPA1: role in respiratory disordersTRPA1 is activated by many airway irritants
Concentration response curves for various irritants against TRPA1
CS = CS tear gasCN = CN tear gasCR = CR tear gasPS = PS tear gasBenzBr = Benzyl BromideBrAc = BromoacetoneBessac. FASEB J 2009
Bandell. Neuron 2004Andrè. J Clin Invest (2008) PMID: 18568077
Isocyanates induce lacrimation, pain, airway irritation, and edema
Similar responses are elicited by chemicals used as tear gases
Other irritants such as acrolein, which is present in cigarette smoke, also activate TRPA1
CSE triggers trachea plasma extravasation
17
TRPA1: role in respiratory disordersTRPA1 is activated by many airway irritants
Bessac. FASEB J 2009Bandell. Neuron 2004Andrè. J Clin Invest (2008) PMID: 18568077
Isocyanates induce lacrimation, pain, airway irritation, and edema
Similar responses are elicited by chemicals used as tear gases
Other irritants such as acrolein, which is present in cigarette smoke, also activate TRPA1
CSE triggers trachea plasma extravasation
CSE promotes neuropeptide release from guinea pig airways
CSE increase tracheal plasma extravasation in TRPA1+/+ mice only
18
TRPA1: role in respiratory disordersNAPQI increases neuropeptide release through TRPA1
Epidemiological evidence has associated use of therapeutic Acetaminophen (APAP) doses with an increased risk of COPD and asthma
The metabolite of APAP (NAPQI) activates TRPA1 and causes the release of pro‐inflammatory neuropeptides from sensory nerve terminals in rodent airways, thereby initiating
neurogenic edema and neutrophilia
Nassini. FASEB Journal (2010) PMID: 20720158
NAPQl promotes neuropeptide release from guinea pig airways
NAPQl increases tracheal plasma extravasation in TRPA1+/+ mice only & is abolished by HC-030031
19
TRPA1: role in respiratory disordersNAPQI increases airway inflammation through TRPA1
Repeated therapeutic APAP doses to mice increased neutrophil numbers, myeloperoxidase activity, and cytokine and chemokine levels in the airways ablated by TRPA1 inhibition (HC‐030031) and knockout of TRPA1
NAPQl induced migration of neutrophils into guinea pig airways is dependent on a TRPA1 mechanism
Nassini. FASEB Journal (2010) PMID: 20720158
20
TRPA1: role in respiratory disordersTRPA1 is activated by reactive oxygen species
Bessac. J Clin Invest, 2008Boldogh. J Clin Invest, 2005
Activation of TRPA1 by hydrogen peroxide Activation of TRPA1 by sodium hypochlorite
During inflammation reactive oxygen species are generated by infiltrating macrophages and neutrophils Hypochlorite is generated through myeloperoxidase mediated catalysis in neutrophlis During inflammation hypochlorite concentrations exceed those required for TRPA1 activation
TRPA1 knockout mice show large reduction in airway responsiveness to hydrogen peroxide administered by aerosol Antigenic pollen proteins induce ROS production in allergic airway inflammation
21
TRPA1: role in respiratory disordersKO of TRPA1 reduces symptoms in a mouse asthma model
Number of inflammatory cells in bronchoalveolar lavage fluid (BALF) from mice challenged with ovalbumin with/without TRPA1
Caceres. PNAS (2009) PMID: 19458046
Comparison of mouse airway resistance (R) produced by i.v. acetylcholine
Genetic silencing of TRPA1 significantly reduced the number of inflammatory cells & cytokines in BALF from mice challenged with ovalbumin
Genetic silencing of TRPA1 significantly reduced airway resistance produced by i.v. acetylcholine in mice challenged with ovalbumin
22
TRPA1: role in respiratory disordersTRPA1 inhibition reduces symptoms in a mouse asthma model
Number of inflammatory cells in bronchoalveolar lavage fluid (BALF) from mice challenged with ovalbumin with/without HC-030031
Caceres. PNAS (2009) PMID: 19458046
Comparison of mouse airway resistance (R) produced by i.v. acetylcholine
Pharmacological inhibition (HC‐030031) of TRPA1 significantly reduced the number of inflammatory cells & cytokines in BALF from mice challenged with ovalbumin
TRPA1 inhibition significantly reduced airway resistance produced by i.v. acetylcholine in mice challenged with ovalbumin
23
TRPA1: role in respiratory disorders TRP receptors are implicated in bradykinin and prostaglandin responses in the airways
Bradykinin and PGE2 trigger depolarisation in GP vagus Signal is inhibited by TRPA1 antagonists such as HC‐030031, as well as TRPV1 antagonists Depolarisation evoked by airway irritants is blocked by TRPA1 antagonists Further roles for TRPM8 (menthol) and TRPV4 (citric acid)
24
TRPA1: role in respiratory disorders TRP receptors are implicated in bradykinin and prostaglandin responses in the airways
acro
AP-365 1
0uM
acro
+AP-36
5
Acro
0.0
0.2
0.4
0.6
0.8
Dep
olar
isat
ion
(mV)
acro
AP-739 1
0uM
acro
+AP-73
9
Acro
0.0
0.2
0.4
0.6
Dep
olar
isat
ion
(mV)
Bradykinin and PGE2 trigger depolarisation in GP vagus Signal is inhibited by TRPA1 antagonists such as HC‐030031, as well as TRPV1 antagonists Depolarisation evoked by airway irritants is blocked by TRPA1 antagonists Further roles for TRPM8 and TRPV4
25
TRPA1: role in respiratory disorders TRP receptors are implicated in bradykinin and prostaglandin responses in the airways
Bradykinin and PGE2 trigger depolarisation in GP vagus Signal is inhibited by TRPA1 antagonists such as HC‐030031, as well as TRPV1 antagonists Depolarisation evoked by airway irritants is blocked by TRPA1 antagonists Further roles for TRPM8 and TRPV4
26
Overview
TRPA1 – an introduction Validation as a target for drug discovery
For migraine For respiratory disorders For inflammatory and neuropathic pain
Medicinal chemistry of TRPA1 antagonists
27
TRPA1: role in pain signallingStrong validation for the therapeutic potential of TRPA1 antagonists
Expression profile High expression in small/medium sized peptidergic fibres (co‐expressed with TRPV1) TRPA1 is upregulated in nerve injury and inflammatory pain models
Genetic rationale Gain of function TRPA1 mutation associated with familial episodic pain syndrome TRPA KO has reduced response to formalin‐induced pain, bradykinin induced mechanical hyperalgesia /
nocifensive behaviours and suprathreshold mechanical stimulation TRPA1 antisense reduced cold allodynia in a neuropathic (SNL) and inflammatory pain model (CFA)
Human and rodent pathophysiology linked to elevated TRPA1 agonists Exogenous mediators: formaldehyde, cinnamaldehyde, AITC, acrolein cause pain in humans and rodents 4‐HNE increased in osteoarthritis; methylglyoxal increased in diabetic peripheral neuropathy
• Human and rodent pharmacological evidence TRPA1 antagonists reverse pain scores in diverse animal models Efficacy of Glenmark GRC17536 in diabetic neuropathy patients
28
TRPA1: expression and function in pain signaling
1. Activated by inflammatory mediators and molecules released during tissue damage2. Elicits generator potential3. Localization at pre-synapse and effects on transmitter release4. Flare – basis for a straightforward clinical biomarker?5. Itch
12
34
5
1 2 3 4 5 6
N
C
TRPA1
29
TRPA1: a rare genetic channelopathy validates the targetFamilial episodic pain syndrome (FEPS)
Kremeyer et al (2010) Neuron 66(5): 671-80
Parameter Mean (17 patients)
Pain severity (0‐10) 9.7
Min duration 34 min.
Max duration 62 min.
Triggers Fasting (100%), Cold (71%), Exercise (71%), Illness (47%)
Location of pain Shoulders/Arms (47%), Thorax (35%), Abdomen (12%), Whole body (12%)
Rare gain of function mutation leading to increased TRPA1 current and a severe pain phenotype
N855S Mutation
30
Reactive pro-algesic mediators – hydroxynonenal4-Hydroxy-2-nonenal (HNE) is produced when ROS peroxidate phospholipids
31
Reactive pro-algesic mediators – hydroxynonenalHydroxynonenal activates TRPA1
HNE activates the tet‐induced TRPA1 channel in HEK293 cells expressing rTRPA1 Effect is blocked by ruthenium red HNE triggers release of neuropeptides substance P and CGRP from central and peripheral nerves
and promotes extravasation HNE induces tactile allodynia in the rat hind paw
32
Reactive pro-algesic mediators – methylglyoxal Methylglyoxal (MG) and diabetic peripheral neuropathy (DPN) in diabetes patients
TRPA1 activation by MG in diabetes Bierhaus et al (2012) Nature Medicine 18(6): 926-934
H
O
O
Plasma levels of MG are correlated with pain in DPN
33
Reactive pro-algesic mediators – methylglyoxal Methylglyoxal induced acute and chronic pain is TRPA1 dependent
Andersson et al (2013) PLoS One 8(10): e77986
MG induces acute nocifensive behaviours
Elevated MG levels induced by GLO1 inhibition induce hyperalgesia
34
TRPA1 antagonists are active in multiple pain modelsAP-00750 inhibits PF-4840154-induced mechanical allodynia
Compounds were administered 1 hour prior to the intraplantar injection PF-4840154 (0.05nmol/50µl/paw),
*, p<0.05 vs respective vehicle-treated group (Student’s t-test).
- 60 0 30 60 120 180 240 min
PHE orStandard
PF
Mechanical allodynia
AP-00750
1214161820222426
0 30 60 120 180 240
* ** *
VEH 2.5% DMSO/30%Solutol, po, n =11
PHE750 30mol/kg, po, n=8 (15mg/kg)
10mol/kg, po, n=9 (5mg/kg)
3mol/kg, po, n=7 (1.5mg/kg)
** * *
**
Time (min)Pa
w W
ithdr
awal
Thr
esho
ld (g
)
AP-00750
3456789100
20
40
60
80
100
Janssen IC50: 45 nMHC-030031 IC50: 12000 nM
PHE750 IC50: 15 nM
-log[Compounds] (M)% A
goni
st (P
F-48
4015
4) r
espo
nse
AP-00750
AP-00750
3456789100
20
40
60
80
100
120Janssen IC50: 48 nMPHE750 IC50: 14 nM
-log[Compounds] (M)% A
goni
st (P
F-48
4015
4) r
espo
nse
Janssen IC50 48nM
AP-00750 IC50 14nM
Human A549 cell line
Rat dorsal ganglia neurons
PF-4840154
35
TRPA1 antagonists are active in multiple pain modelsAP-00750 inhibits Oxaliplatin-induced mechanical allodynia
0
5000
10000
15000
20000
25000
PHE750 30mol/kg
HC-030031 298mol/kg
Janssen 30mol/kg
Del
ta A
UC
(0-3
00)v
s V
ehic
lePa
w W
ithdr
awal
Thr
esho
ld
- 7-days 0 30 60 120 180 240 min
Oxaliplatin
Mechanical allodynia
STD or PHE
Testing was performed 7-day after a single challenge with oxaliplatin (2.5 mg/kg, i.v., dissolved in normal saline)*, p<0.05 vs respective vehicle-treated group (Student’s t-test).
2022242628303234363840
0 30 60 120 180 240 300
VEH 2.5%DMSO/30%Solutol, po, n=4
3mol/kg, po, n=6 (1.5mg/kg)
10mol/kg, po, n=6 (5mg/kg)
PHE750 30mol/kg, po, n=4 (15mg/kg)
* * ** ** *
* **
Time (min)
Paw
With
draw
al T
hres
hold
(g)
AP-00750AP-00750
AP-00750
36
TRPA1 antagonists are active in multiple pain modelsAP-00750 inhibits mechanical allodynia after CCI
- 10-day 0 30 60 120 180 240 min
Mechanical allodynia
CCI STD or AP-00750
Pretest 30 60 120 180 240
202428323640444852
Vehicle (IPSI), n =12Vehicle (CONTRA), n =12Gabap (IPSI) 580mol/kg, n =11Gabap (CONTRA) 580mol/kg, n =11Pregab (IPSI) 188mol/kg, n =7Pregab (CONTRA) 188mol/kg, n =7
* * **
** *
Time (min)Pretest 30 60 120 180 240
202428323640444852
Vehicle (IPSI), n =12
Janssen (IPSI) 100mol/kg, n =6Vehicle (CONTRA), n =12
Janssen (CONTRA) 100mol/kg, n =6
* * *
Time (min)
Experiments were performed 10 days after the lesion. Drugs were administered at the indicated dose by oral gavage.
Standard-of-careTRPA1 antagonist
Pretest 30 60 120 180 240
202428323640444852
Vehicle (IPSI), n=12Vehicle (CONTRA), n =12PHE750 (IPSI) 100mol/kg, n =14PHE750 (CONTRA) 100mol/kg, n =14
* * *
Time (min)
Pres
sure
Thr
esho
ld (g
)
AP-00750
AP750AP750
37
TRPA1 antagonists are active in multiple pain modelsAP-00750 inhibits mechanical allodynia after CFA
- 1-day 0 30 60 120 180 240 min
CFA
Mechanical allodynia
STD or AP-00750
Testing was performed 1-day after a single intraplantar challenge with CFA
Pretest 30 60 120 180 240
10
14
18
22
26
30
34
Vehicle (IPSI), n =7
Vehicle (CONTRA), n =7
Gabap (IPSI) 580mol/kg, n =9
Gabap (CONTRA) 580mol/kg, n =9
** *
*
Time (min)
Pres
sure
Thr
esho
ld (g
)
Pretest 30 60 120 180 240
10
14
18
22
26
30
34
Vehicle (IPSI), n =7
Vehicle (CONTRA), n =7
Indomenthacin (IPSI) 30mol/kg, n =6
Indomethacin (CONTRA) 30mol/kg, n =6
* * **
Time (min)Pretest 30 60 120 180 240
10
14
18
22
26
30
34
Vehicle (IPSI)Vehicle (CONTRA)PHE750 (IPSI) 100mol/kg, n =18PHE750 (CONTRA) 100mol/kg, n =18
* ** * *
Time (min)
Pres
sure
Thr
esho
ld (g
)
*, p<0.05 vs respective vehicle-treated group (Student’s t-test).
AP750AP750
38
Overview
TRPA1 – an introduction Validation as a target for drug discovery
For migraine For respiratory disorders For inflammatory and neuropathic pain
Medicinal chemistry of TRPA1 antagonists
39
TRPA1 antagonists – first generationXanthine derivatives and analogues – generally poorly soluble and poor PK
2007, 5M
2010, 4nM
HC030031
2013, 93nM
2014, <25nM
Hydra Hydra/Cubist
2009, <40nM
2010, <50nM
Glenmark
40
TRPA1 antagonists – first generationXanthine analogues with amide isostere - Amgen
rTRPA1 71nMhTRPA1 131nM
iv Cl = 2.5 L/h/Kgiv Vss = 1.7 L/Kgpo F = 60%po T1/2 = 2.8hr
Assay in AITC-induced flinching in rats
Schenkel, JMC, 2016
41
TRPA1 antagonists – second generationNew generation of smaller TRPA1 antagonists
Janssen, 2010, 7nM Roche, 2014, 12nM
Roche, 2014, 13nM
Hydra/Cubist, 2013, 93nM
Orion, 2012, 15M
42
TRPA1 antagonists – more diverse antagonists?New generation of smaller TRPA1 antagonists
F
FF
N H
N N H
O
Orion, 2014, 100nM Astra Zeneca, 2012, 60nM
H
O H
F
NH
Merck, 2011, 19nM Astra Zeneca, 2014, 110nM
Novartis, 2014F
Cl Cl
O OSF
O H
N
Genentech, 2016hTRPA1 110nMrTRPA1 7.3M
rTRPA1 85nMhTRPA1 15nM
Iv Cl = 26 ml/min/KgIv Vss = 2.39 L/KgPo F = 39%D50 = 5.5mpk in CFA rat
Mustard-induced allodynia in mice
43
Binding Site Hypothesis 1Janssen , Amgen, Glenmark, Hydra antagonists
N
O
S
Large Hydrophobic pocket in N-terminal domainOccupation induces allosteric regulation of TRPA1Single Occupation ACTIVATES
Site prefers lipophilic side chains
NS
OClClCl
N+
O
O
hTRPA1 – C621
1
JACS, 2015, 137 (50) 15859 JT activatorMol Pharm, 2013, 83, 1120 pBQN
44
Binding Site Hypothesis 1Janssen , Amgen, Glenmark, Hydra antagonists
N
O
S
NS
OClClCl
N+
O
O
hTRPA1 – C621
1
Hydrophilic pocket with a number of potential contacts. Xanthine core fits well.
2
JACS, 2015, 137 (50) 15859 JT activatorMol Pharm, 2013, 83, 1120 pBQN
45
Binding Site Hypothesis 2AZ, Abbott, Novartis antagonists
Point mutations identify sites in the pore that play a critical role in AZ-868 and A-967079 bindingBinding of xanthane-like ligands such as HC030031 is not affected by these mutations
Dabrowski, Biophys J, 2013, 104, 798
M956
T877
V879
F880
L906
Five residues alter affinity for compound 31No effects on mustard oil activationSites mapped onto a homology model of the mouse TRPA1 channel based on KcsA
Moldenhauser, PLOS, 2014, 9 (9) 1
46
TRPA1 cryo-EM structureA Platform to Great Achievements
Determined at 4Å as 3J9P, released April 2015
Accurately describes cytoplasmic site of covalent activators and some antagonists (BS1); allows classification of different antagonist classes
Accurately describes pore region antagonist site (BS2) and confirms binding of A-967079 in this region (but not HC-030031)
Julius, Nature, 2015,
HC-030031
A-967079
47
TRPA1 antagonists – hurdles along the way
Poor DMPK properties
Low ligand efficiency
Mixed partial agonist/antagonist properties
Covalent and non-covalent binding
Species differences
48
TRPA1 antagonists – clinical developmentAnd other approaches……
Cubist/Hydra CB-625 enters clinical development in 2012 discontinued for PK reasons
Glenmark GRC17536 enters clinical development 2010 Positive data in patients with PDN announced 2014
Hydra announces progression of HX-100 into Phase 2 for PDN and asthma January 2016
Mouse monoclonal antibodies to TRPA1 act as antagonists of multiple modes of channel activation (Gavva et al, J PET, 2014)
Thanks for your attention