TRPA1 in Drug Discovery
TRPA1 is one of the few ion channels with human genetic validation for pain. A TRPA1 gain-of-function mutation is linked to familial episodic pain syndrome in humans. This milestone discovery, coupled with a growing preclinical literature implicating TRPA1 in multiple indications, has made TRPA1 an attractive therapeutic target. With extensive investment across the pharmaceutical industry, several novel nonreactive TRPA1 antagonist series have emerged in patents, and two TRPA1 compounds have recently advanced to human clinical trials. A review of the diverse roles for TRPA1 in pain signaling and other indications such as itch and respiratory diseases is presented along with an overview of known small molecule activators and antagonists of the TRPA1 receptor.
Key wordsTRPA1 antagonists Pain Respiratory disease Overactive bladder Itch
TRPA1, also known as ANKTM1 and p120, belongs to the transient receptor potential (1) superfamily, which consists of a large group of cation channels present in species from yeast to mammals (2, 3). In mammals, there are 28 members from six subfamilies (TRPC, TRPV, TRPM, TRPML, TRPP, and TRPA), playing critical roles in physiological processes ranging from vasorelaxation, fertility, cell growth to sensory function. TRPA1 is the only member of the TRPA subfamily, and distinctively different from other TRP channels with an overall low sequence homology (<40% in transmembrane domains), a large N terminus containing many ankyrin repeat domains (ARD), and sensitivity to a variety of stimuli. Activation of TRPA1 leads to membrane depolarization and increase in intracellular Ca2+ which initiate multiple downstream signaling cascades that ultimately control a variety of physiological processes.
Although TRPA1 was originally cloned from cultured human lung fibroblasts, it is most predominantly expressed in primary sensory neurons where it co-localizes with pain markers and peptidergic nociceptors (e.g., TRPV1, CGRP, substance P, and bradykinin receptors) (4, 5, 6, 7). Among acutely dissociated DRG neurons, 30% respond to TRPA1 agonists (8). TRPA1 expression level is increased in rodent models of inflammatory and neuropathic pain, as well as in avulsion-injured human DRG (4, 6, 9, 10). Furthermore, TRPA1 expression is increased by nerve growth factors and nociceptive signals (4, 11). Besides its predominant expression in sensory neurons, TRPA1 has been reported to also be expressed in nonneuronal cells and tissues, including skin (12), inner ear (13), urinary bladder (14, 15), stomach (16), and cerebral arteries (17).
TRPA1 is activated by electrophilic compounds such as allyl isothiocyante (AITC) through covalent modification. Several cysteine and lysine residues in the N terminus have been identified as covalent modification sites, including C415, C422, and Cys622 in mTRPA1 and C621, C641, C665, and K711 in hTRPA1 (NM_007332) (19, 20). The quadruple mutation in hTRPA1 (C621S/C641S/C655S/K711Q) was found to reduce, but did not eliminate AITC sensitivity, indicating contributions from other yet unidentified residues (21). Another observation is that only one residue is in equivalent position of human and mouse channels (e.g., C622 in mTRPA1 and C621 in hTRPA1), suggesting that covalent modification may differ across species.
TRPA1 contains a large number (at least 12) of ARD in its N zterminus. ARDs are responsible for the difference in heat sensitivity between Drosophila TRPA1 and hTRPA1, and these domains are also involved in modulating sensitivity to chemical irritants and cytosolic Ca2+ (22). The complex regulation of TRPA1 channel gating that is mediated by changes in intracellular Ca2+ has been shown to involve several different structural domains. Cytosolic Ca2+ activates the channel by binding to residues of an EF-hand domain in the N terminus (e.g., S468 and T470 in human TRPA1) (23, 24). Permeation Ca2+ ions potentiate, and then desensitize the channel through binding to an acid residue in the putative selectivity filter (D918 in rat TRPA1) (25). Several residues in the S5 domain (S876/T877 in mouse TRPA1) were identified as critical sites for menthol binding (26). In a previous study, we found that thioaminals covalently modified channel proteins but produced species-specific effects: activation of rat TRPA1, but block of human TRPA1. The opposite gating was attributed to residue difference in several S6 residues: A946 and M949 in rat TRPA1 were shown to be responsible for channel activation, and equivalent residues in human TRPA1 (S943 and I946) resulted in channel block (21). Therefore these residues may constitute a part of the gating machinery of TRPA1.
2 TRPA1 Activators
The most striking property of TRPA1 is its activation by a plethora of stimuli, including electrophilic agonists, nonelectrophilic agonists, and physiological stimuli. This property fulfills its role as a broad chemosensor to changes in external and internal environment.
2.1 Electrophilic Agonists
2.2 Nonelectrophilic Agonists
An increasing number of structurally diverse, nonreactive TRPA1 agonists have also been identified, including arachidonic acid, URB597, farnesyl thiosalicylic acid, trinitrophenol, flufenamic acid, d-9-THC, 2-APPB, diclofenac, isoflurane, and PF-4840154 (Fig. 2) (27, 36, 37, 38, 39, 40). The apparent lack of chemical reactivity suggests that they activate TRPA1 through noncovalent interactions, but the exact mechanism has not been elucidated.
2.3 Physiological Stimuli
Physiological stimuli that activate TRPA1 include changes in intracellular Ca2+, hypertonicity, and arguably noxious cold. Ca2+ is an important regulator of TRPA1 function and has complex effects: intracellular Ca2+ directly activate TRPA1 via binding to an N-terminal EF-hand domain (24); while permeating Ca2+ potentiates and desensitizes the channel (25). Hypertonicity activates both recombinant and native TRPA1 in DRG neurons, implicating a possible role of TRPA1 in mechanosensation (41). Bradykinin activates TRPA1 indirectly through the phospholipase C pathway, and its nociceptive effect is reduced in TRPA1-deficient mice (27, 29). TRPA1 can also be activated downstream of Mas-related G protein-coupled receptors, which mediated histamine-independent itch (42). It was reported originally that noxious cold activated TRPA1 (4), but this issue remains controversial (43, 44).
To date, several antagonists (HC-030031, AP18, A-967079, and Chembridge-5861528) have been utilized as tools for probing TRPA1 function and therapeutic utility (45, 46, 47, 48, 49). The broad interest in TRPA1 antagonists is noted by the large number of patents filed in recent years (>20 patents since 2007).
3.1 Electrophilic Antagonists
In contrast to thioaminals, compounds from an oxime series have more consistent effects across species (46, 49, 51). AP18 was the first reported antagonist within this series, with IC50 of 3.1 μM and 4.5 μM on human and rat TRPA1, respectively (46). Abbott independently identified the same chemotype from a high throughput screen (52). Subsequent medicinal chemistry efforts led to the identification of oxime A-967079 (IC50: 67 nM on hTRPA1 and 290 nM on rat TRPA1) that represented the first potent and selective TRPA1 antagonist that demonstrated good oral bioavailability (51). Although the α, β-unsaturated oxime functionality could potentially react with nucleophiles, neither AP18 nor A-967079 was found positive in a battery of reactivity assays including glutathione assay, La antigen-based ALARM NMR or ALARM-MS (51). Furthermore, block of TRPA1 by A-967079 was reversible. Taken together, these data suggest that some oximes may not covalently modify the channel, or that they could covalently modify the channel but in a rapidly reversible manner.
3.2 Nonreactive Antagonists
3.2.1 Naturally Occurring Antagonists
3.2.2 Pharmaceutical Nonelectrophilic Antagonists
4 TRPA1 Involvement in Pain
Evidence for TRPA1 contributions to pain starts with the observation that local application of “natural” TRPA1 agonists trigger spontaneous pain in humans (54) and induce nocifensive behaviors in animals (29). TRPA1 is expressed in small- and medium-sized peptidergic primary afferent somatosensory neurons that express TRPV1 and receptors for calcitonin gene-related peptide, substance P, and bradykinin, which are key mediators/transmitters in nociceptive signaling (5, 7, 29). TRPA1 expression is increased in these sensory neurons in humans with an avulsion injury as well as in animal models of neuropathic and inflammatory pain (7, 10). Recently, a gain-of-function mutation in TRPA1 was linked to familial episodic pain syndrome in humans, providing strong validation of TRPA1 in human pain states (55).
4.1 Chemical Nociception
TRPA1 is a key chemosensor for many pungent natural products and environmental irritants including AITC from horse radish, cinnamaldehyde from cinnamon, allicin from garlic, acrolein from cigarette smoke, and ozone from air pollutants (56, 57). These agents trigger tissue irritation and a burning sensation. TRPA1 is activated indirectly by bradykinin, a mediator of neurogenic inflammation (27). In behavioral experiments, TRPA1−/− mice lost aversion to oral AITC, and had diminished nocifensive responses to hind paw injection of AITC and intraplantar bradykinin (29, 58).
4.2 Mechanical Nociception
Gene ablation (TRPA1−/−) studies have examined TRPA1 contributions to mechanical nociception. In uninjured rats, mechanical thresholds were not different between wild type and knockout animals, nor was there an impairment in the development of mechanical hyperalgesia after an inflammatory injury (46, 59). However, mechanical sensitization typically induced by mustard oil injection was not observed in TRPA1−/− mice (59), and a TRPA1 antagonist (AP18) did not reduce mechanical hyperalgesia in inflamed knockouts despite effectiveness in wild types (46). Mechanically activated currents in small diameter primary afferent neurons were also significantly reduced in amplitude in TRPA1−/− animals compared to wild types (60). Another study demonstrated that TRPA1−/− animals had less sensitivity to low-intensity mechanical stimuli compared to wild-type mice, and responses to high-intensity (noxious) mechanical stimulation were appreciably impaired (58). Hypertonic solutions were also found to activate recombinant and endogenously expressed rat TRPA1 (41); Taken together, a possible role for TRPA1 in mechanotransmission was suggested by these studies.
Using the skin-nerve preparation, it was found that TRPA1 in normal skin are necessary for mechanotransmission in several types of primary afferent fibers including slowly adapting C-fibers, Aδ-fiber mechanonociceptors, and slowly adapting Aβ-fibers (61). HC-030031 did not alter the responses of C- and A-fibers to low-intensity mechanical stimulation of normal skin (62); however, it reduced the responsiveness of C-fibers once the stimulus force was increased into the high-intensity range. Similarly, with an in vivo preparation, systemic administration of A-967079 reduced the responses of spinal wide dynamic range (WDR) and nociceptive specific neurons to high-intensity mechanical stimulation in uninjured rats as well as in rats with an osteoarthritic (OA) or inflammatory injury (49). In contrast, the transmission of low-intensity mechanical stimulation was impeded by A-967079 only following an inflammatory injury and was not altered in uninjured animals. The combined data suggest that TRPA1 factors into high-intensity mechanotransmission under both normal and pathological conditions, but shifts to include lower intensity stimulation following an inflammatory injury. The lowering of mechanical thresholds could be linked to TRPA1 up-regulation and/or sensitization by inflammatory mediators such as bradykinin and prostaglandins (27, 33).
The physiological data underscore effects of TRPA1 antagonists on mechanical sensitivity in behavioral assays. Responses to mechanical stimuli in inflamed and neuropathic rats were decreased by systemic administration of HC-030031 and Chembridge-5861528 (47, 48, 63). Intraplantar injection of AP18 attenuated mechanical hyperalgesia in wild type but not in knockout mice (46). Intrathecal delivery of A-967079 or Chembridge-5861528 attenuated secondary mechanical sensitivity following capsaicin or formalin injection (64). However, these data are contrasted by the observations that A-967079 did not attenuate mechanical hypersensitivity in models of neuropathic and inflammatory pain, and TRPA1-specific antisense oligodeoxynucleotides. Thus, although the majority of data points to a role for TRPA1 in mechanosensation in uninjured and sensitized states, some inconsistencies still need to be explained.
4.3 Cold Nociception
The role of TRPA1 in noxious cold sensation is highly controversial (43, 44). Channel activation by noxious cold has been reported in several studies (4, 65), but has been disputed or attributed to an indirect intracellular Ca2+-mediated mechanism (5, 24). TRPA1-deficient mice were shown to be less responsive to acetone-mediated evaporative cooling, cold plate, and tail immersion than wild-type animals (58, 65); while other studies showed normal cold sensitivity in knockout mice (29, 66). Injection of A-967079 or HC-030031 did not produce deficits in the cold plate test in uninjured mice, but attenuated cold allodynia in models of neuropathic pain (51, 67). HC030031 also reversed paclitaxel-mediated cold hyperalgesia in diabetic rats, and this effect may have been related to the increased expression of TRPA1 that was triggered by the overproduction of mitochondrial H2O2. TRPA1-specific antisense oligodeoxynucleotides were efficacious in attenuating cold allodyia in inflammatory and neuropathic pain models (7). These data suggest that TRPA1 plays a role in cold allodynia under disease states. It has also been suggested that TRPA1 may play a role in cold sensing in visceral sensory neurons, but not in cutaneous nociceptors (68, 69).
4.4 Heat Nociception
TRPA1 is not considered a heat-activated channel and thus there are limited studies in this area. Genetic ablation of TRPA1 or antagonism of the receptor with HC-030031 does not alter responses to noxious heat stimulation in uninjured animals (47, 58). However, HC-030031 was found to decrease thermal hyperalgesia in the paclitaxel model of chemotherapy-induced neuropathic pain (63). Interestingly, HC-030031 also reduced cold and mechanical hypersensitivity in this model, and this effect was likely mediated through proteinase-activated receptor 2 (PAR2)-related sensitization of neurons containing TRP receptors including TRPA1 (63). Additionally, injection of TRPA1 agonists enhances WDR neuronal responses to heat stimulation in naive rats (70, 71). This effect may not be surprising since topical application of AITC or CA can trigger burning pain and thermal hyperalgesia, which could be attributed to indirect sensitization of TRPV1 (54).
4.5 Spontaneous Nociception
In addition to effects on evoked stimulation, evidence suggests that TRPA1 may be involved in nonevoked discomfort or pain. Nonevoked pain is observed in the majority of patients with chronic injury and is a primary reason for seeking medical care. Spontaneous firing of WDR neurons is elevated in injured animals and likely reflects injury-related sensitization and possibly nonevoked pain (72, 73). Injection of A-967079 reduced “heightened” spontaneous firing of WDR neurons in inflamed rats (49). Interestingly, elevated spontaneous firing of WDR neurons in OA rats was not decreased by injection of A-967079. The differential effect of A-967079 on spontaneous firing in inflamed and OA rats was postulated to be related to the degree of inflammation on the days of neuronal recording.
5 TRPA1 Involvement in Other Diseases
5.1 Respiratory Diseases
Respiratory diseases have become a significant public health burden and unmet medical need. TRPA1 has been linked to airway sensitization, chronic cough, asthma, and Chronic Obstruction of Pulmonary Diseases (COPD) (74, 75, 76). The airways are innervated by sensory neurons that express TRPA1 in vagal and trigeminal nerves, which are responsible for transducing noxious stimuli into pain signals, and for mediating protective behaviors (sneezing, cough, and respiratory depression). A plethora of airway irritants activate TRPA1, including acrolein from cigarette smoke, ozone (air pollutants), isocyanate (industrial waste), and tear gas (chemical warfare) (56, 57). Transient TRPA1 activation by exogenous irritants evokes pain and protective reflex (sneezing, cough, and respiratory depression, avoidance), therefore providing a protective alert to eliminate exposure to irritants and limit damage to the body. However, persistent channel activation by endogenous ligands causes or aggravates existing airway irritation. Asthma, COPD, and rhinitis are associated with increased levels of reactive oxygen species (H2O2), lipid peroxidation production (e.g., 4HNE), and pro-inflammatory mediators (e.g., bradykinin and nerve growth factors) (77, 78). These agents activate TRPA1 directly (e.g., H2O2, 4HNE), indirectly (bradykinin), or increase TRPA1 surface expression (nerve growth factors). In turn, the heightened TRPA1 activity leads to the release of neurotransmitters and cytokines, further promoting neurogenic inflammation (56).
In guinea pig, aqueous extracts from cigarette smoke, acrolein, and crotonaldehyde induce neurotransmitter release, tracheal plasma extravasation, and bronchi contraction. These effects are inhibited by TRPA1 antagonist HC-030031 (56). In mice, airway exposure to oxidants (hypochlorite and H2O2) evokes respiratory depression manifested as a reduction in breathing frequency and increased end expiratory pause, which were abolished by genetic deletion (31). In an ovalbumin-induced mouse asthma model, genetic ablation and treatment with HC-030031 significantly reduced the induction of cytokines, chemokines, neurotransmitters, as well as leukocyte infiltration and airway hyperactivity (32). While these results are encouraging, many respiratory diseases are chronic, multifaceted disorders that involve inflammatory pathways. For example, asthma is associated with acute and chronic inflammation of the airway mucosa, as well as airway remodeling. Chronic obstructive pulmonary disease (COPD) is characterized by activated inflammation pathways that lead to destruction of the lung over time. It remains to be seen whether TRPA1 antagonists can alleviate symptoms and reverse disease progression in inflammatory-mediated respiratory diseases, and it is unclear whether a TRPA1 antagonist will offer more benefits than current therapies (e.g., anti-inflammatory drugs and bronchodilators) and other emerging mechanisms. Nonetheless, respiratory diseases represent an exciting frontier to be explored for TRPA1 antagonists.
5.2 Overactive Bladder
The involvement of TRPA1 in bladder activity is implied by its expression pattern and the effect of TRPA1 agonists and antagonists on bladder function. TRPA1 is expressed on C-fiber bladder afferents and urothelial cells (14, 15). The expression of TRPA1 is significantly increased in overactive bladder conditions in both rats and humans (14, 79). AITC and cinnamaldehyde increase basal bladder pressure and reduce voided volume in naive rats (15). Furthermore, injections of either a TRPA1 antisense oligodeoxynucleotide or antagonist (HC-030031) were both shown to attenuate bladder overactivity induced by spinal cord injury (79). Collectively these studies suggest that TRPA1 may be involved in bladder function under normal and pathological conditions.
Itch is a major complaint in patients with skin, kidney, diabetic, and neurological disorders (80). Ironically, many medical treatments evoke itch, limiting their clinical usage. For example, patients treated with opioids and local anesthetics frequently experience itch (81). The antimalaria drug chloroquine causes intolerable itch, which is resistant to current treatment of antihistamines. A recent study found that TRPA1 knockout mice exhibited diminished itch response to chloroquine and the endogenous pruritogen BAM8-22 (42). Since chloroquine and BAM8-22 evoke itch through two separate Mar-related G protein-coupled receptors (MrgprA3 and MrgprC11), TRPA1 may be a downstream integrator of multiple histamine-independent pathways.
Besides expression in pruritic sensory neurons, TRPA1 is detected in nonneuronal cells from human skin (12). RT-PCR and western blot demonstrated robust TRPA1 expression in primary culture of epidermal keratinocytes, melanocytes, and fibroblasts. Immunohistochemisty of human samples localized TRPA1 in the basal layer of the epidermis, dermis, and epithelium of hair follicle. These results suggest that TRPA1 expressed in the skin may directly participate in itch responses.
The recent finding that a gain-of-function mutation of TRPA1 causes familial episodic pain syndrome in humans is a major milestone for TRPA1 research, establishing TRPA1 as one of two ion channels with human genetic validation for pain (55). This result, coupled with a growing preclinical literature implicating TRPA1 in multiple indications, has made TRPA1 an attractive therapeutic target. With extensive investment across the pharmaceutical industry, several novel nonreactive TRPA1 antagonist series have emerged in patents, and two TRPA1 compounds have recently advanced to human clinical trials. In February 2011, Glenmark announced the entry of the first TRPA1 antagonist into Phase I trials. GRC 17536 is reported to be highly potent (IC50 < 10 nM), selective, and orally available. It is claimed to be efficacious in inflammatory and neuropathic pain, and efficacious in airway inflammation from an asthma model. In January 2012, Cubist Pharmaceuticals/Hydra Biosciences announced entry of CB-625 into Phase I trials. CB625 is reported to be a highly potent, selective TRPA1 antagonist with efficacy in surgically induced and inflammatory mediated pain in animal models. Positive outcomes from these and other clinical trials would galvanize additional TRPA1 drug discovery efforts.