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TRPA1 as a drug Target

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




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




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


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


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




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


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


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


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 (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 (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




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

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trpa1 as a drug - maggi churchouse eventsmaggichurchouseevents.co.uk/bmcs/downloads/archive/ion...

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