Review For reprint orders, please contact
[email protected]
Recent advances in the biology and medicinal chemistry of TRPA1 The transient receptor potential cation channel, subfamily A, member 1 (TRPA1) is a nonselective cation channel that is highly expressed in small-diameter sensory neurons, where it functions as a polymodal receptor, responsible for detecting potentially harmful chemicals, mechanical forces and temperatures. TRPA1 is also activated and/or sensitized by multiple endogenous inflammatory mediators. As such, TRPA1 likely mediates the pain and neurogenic inflammation caused by exposure to reactive chemicals. In addition, it is also possible that this channel may mediate some of the symptoms of chronic inflammatory conditions such as asthma. We review recent advances in the biology of TRPA1 and summarize the evidence for TRPA1 as a therapeutic drug target. In addition, we provide an update on TRPA1 medicinal chemistry and the progress in the search for novel TRPA1 antagonists. The transient receptor potential cation channel, subfamily A, member 1 (TRPA1; formerly ANKTM1) is the latest member of the transient receptor potential (TRP) superfamily and is the only member of the TRPA subfamily in mammals. TRPA1 is so named due to the presence of multiple ankyrin domains in the N-terminus. The TRPA1 gene encodes a protein with six putative transmembrane domains with a proposed pore region between transmembrane domains five and six and with cytoplasmic Nand C-termini. Functional TRPA1 channels are probably homotetrameric structures that form nonselective cation channels in mammalian cells. While TRPA1 was originally cloned from cultured human lung fibroblasts (clone p120) [1] , more recent studies suggest that TRPA1 is most highly expressed in sensory neurons of the dorsal root, trigeminal and nodose ganglia and in hair cells of the inner ear [2–6] . In sensory neurons, TRPA1 expression is most prevalent in smalldiameter neurons where it colocalizes with markers of peptidergic nociceptors such as TRPV1, calcitonin gene-related peptide and substance P, but not TRPM8 [4–8] . Recent evidence suggests that TRPA1 may also be expressed in the intestinal myenteric plexus [2] , epithelial/urothelial cells of both the urinary bladder and prostate gland [9–11] and in human epidermal keratinocytes, melanocytes and fibroblasts [2,12] . In sensory nerves, surface expression of TRPA1 is increased following nerve injury and inflammation in rat [13,14] . Nerve growth factor, one of many inflammatory mediators, also appears to upregulate expression of TRPA1 in dorsal root ganglion (DRG) and trigeminal neurons in vitro and in vivo [4,14] .
Considerable evidence suggests that TRPA1 functions as a polymodal receptor, responsible for detecting potentially harmful chemicals as well as mechanical forces and temperatures.
agonists The vast majority of TRPA1 agonists described to date are highly reactive electrophiles. For example, many deterrent chemicals from plants, such as MO (mustard oil and its active component, allyl isothiocyanate [AITC]), cinnamaldehyde, allicin and various sesquiterpene compounds activate TRPA1 [7,15–20] . Likewise, many of the main reactive constituents of smoke and environmental pollutants (e.g., acrolein, crotonaldehyde, formaldehyde and acetaldehyde) directly activate the TRPA1 channel [15,16,21,22] . A growing list of miscellaneous chemicals, including the active ingredients in several forms of tear gas (e.g., dibenz[b,f]���������������������������������� [1,4]����������������������������� -oxazepine, 1-chloroacetophenone, the active ingredient in Mace® and 2-chlorobenzylidene malononitrile), oxidizing agents, ozone and zinc, for example [23–30] , activate TRPA1 (Table 1) . In addition to these exogenous chemicals, numerous reactive endogenous agents have also been shown to activate TRPA1. These compounds include various a,b-unsaturated
10.4155/FMC.10.29 © 2010 Future Science Ltd
Future Med. Chem. (2010) 2(5), 843–858
TRPA1 as a chemosensor The TRPA1 channel is a sensor of a remarkably broad range of chemical stimuli. Chemicals reported to activate TRPA1 have highly variable structure and reactivity, as well as having a variety of origins. Several of these TRPA1 agonists are listed in Table 1, which serves to highlight the diversity of this group of compounds.
Jason C Rech1, William A Eckert III1, Michael P Maher1, Tue Banke1, Anindya Bhattacharya1 & Alan D Wickenden†1 Johnson & Johnson Pharmaceutical Research & Development, LLC, 3210 Merryfield Row, San Diego, CA 92121, USA † Author for correspondence: Tel.: +1 858 320 3447 E-mail:
[email protected] 1
Electrophilic
ISSN 1756-8919
843
Review | Rech, Eckert, Maher, Banke, Bhattacharya & Wickenden Key Term TRPA1: Transient receptor potential cation channel, subfamily A, member 1. A nonselective cation channel belonging to the TRP superfamily of ion channels
Table 1. Transient receptor potential cation channel A1 agonists. Agonist
EC50 (µM)
Structure
Ref.
Exogenous electrophilic Allyl isothiocyanate (mustard oil)
2–35
Benzyl isothiocyanate
0.3
Mustard oil alkyne
18.4
Toluene diisocyanate
10
[7,20,21,38,47]
NCS
[25]
NCS
[38] [95]
NCO OCN
Methyl isocyanate
25
[23]
Hexamethyl-diisocyanate
2.6
[23]
Formalin
~200
[21]
Cinnamaldehyde
19
Acetaldehyde
76.5
Acrolein
1–5
Pentenal
5
[38]
Crotonaldehyde
16
[85]
[38]
O
[22] [16,38,85]
O
[38]
(2-aminoethyl)methanethiosulphonate 1.6 mM Allicin
7.5
Diallyl disulphide
192
S
[7]
O S
[7]
Morphanthridine
3 nM
N
[25]
Dibenz[b,f][1,4]-oxazepine (tear gas)
0.3 nM
N
[25]
O
1-chloroacetophenone (tear gas)
30 nM
2-cholobenzylidene malonitrile (tear gas) Iodoacetamide
0.9 nM
[25]
[25]
357
Exogenous nonelectrophilic Isoflurane (general anesthetic)
180
[46]
Cl F3 C
O
CHF 2
Nifedipine
400 nM
[108]
Flufenamic acid (NSAID)
78–147
[45]
Niflumic acid (NSAID)
240–540
[45]
Menthol
28
Thymol
63
Farnesyl thiosalicylic acid
5–7
Farnseyl thioacetic acid
90
[47]
OH
[47]
S
[28,47]
CO2 H [28]
BCTC: N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl) tetrahydropyrazine-1(2H)-carbox-amide; FAAH: Fatty acid amide; NA: Not available; TRPA1: Transient receptor potential cation channel A1; -TRPV1: Transient receptor potential cation channel V1.
844
Future Med. Chem. (2010) 2(5)
future science group
Recent advances in the biology & medicinal chemistry of TRPA1
| Review
Table 1. Transient receptor potential cation channel A1 agonists (cont.). Agonist
EC50 (µM)
D -tetrahydrocannabinol
12
[20]
URB597 (FAAH antagonist)
25
[39]
Icilin
NA
9
Structure
Ref.
[6]
S Et 2N
S
S
NEt2 S
BCTC (TRPV1 antagonist)
4.2
N
N
N
Cl
[44]
HN O
Methyl paraben
4.4 mM
[42]
Isovelleral (sesquiterpene)
0.50
[17]
Clotrimazole
NA
[41]
Endogenous electrophilic 4-hydroxynon-2-enal
27
9-nitrooleic acid
1
4-hydroxyhex-2-enal
5–40
4-oxohex-2-enal
2
15-deoxy-D
5–18
[32]
O OH
12,14
-prostoglandin J2
NO2
[36]
CO2 H
[31,88] [31] [28,31]
BCTC: N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl) tetrahydropyrazine-1(2H)-carbox-amide; FAAH: Fatty acid amide; NA: Not available; TRPA1: Transient receptor potential cation channel A1; -TRPV1: Transient receptor potential cation channel V1.
aldehydes such as 4-hydroxynonenal [31,32] , cyclopentane prostaglandin metabolites [28,31,33,34] and products of nitrative stress, such as nitrooleic acid [35,36] . Electrophilic agonists readily form covalent adducts with competent nucleophiles, including the reactive side chains of amino acid residues such as cysteine and lysine. Despite this indiscriminant reactivity, studies have shown electrophilic agonists activate the TRPA1 channel through the modification of three cysteine (Cys-619, Cys-639 and Cys-663) and one lysine (Lys-708) residue located on the intracellular N-terminal ankyrin repeat sequence (Figure 1) [37,38] . These amino acids were identified through a series of point mutations that focused on residues shown to form covalent adducts with electrophiles. It remains to be determined how modification of cysteines in TRPA1 by reactive chemicals translates into channel activation. Nonelectrophilic
agonists Nonelectrophilic agonists of the TRPA1 channel have also been reported including URB597 [39] , menthol [40] , clotrimazole [41] , methyl p-hyd roxybenzoate and related compounds [42] , future science group
WIN 55212-2 [43] , D9-tetrahydrocannabinol [18] , farnesyl thiosalicylic acid [28] , N-(4-tertiary butylphenyl)-4-(3-cholorphyridin-2-yl) tetra hydropyrazine-1(2H)-carboxa mide [44] and several nonelectrophilic NSAIDs, such as flufenamic, niflumic and mefenamic acid, as well as flurbiprofen, ketoprofen, diclofenac and indo methacin [29,45] . TRPA1 can also be activated by certain general anesthetics, including isoflurane, desflurane and propofol, at physiologically relevant concentrations [46] . Since these agonists are not sufficiently reactive to form covalent adducts with cysteine or lysine it is likely they activate TRPA1 by an alternative mechanism. Patapoutian and co-workers demonstrated that the transmembrane domain 5 region plays a significant role in the activation of the TRPA1 channel by nonelectrophilic agonists menthol, thymol and farnesyl thiosalicylic acid and that mutations to key amino acids in this region of the channel eliminated all sensitivity to menthol without compromising activation by the electrophilic agonist allyl isothiocyanate. Based on molecular modeling, these researchers proposed that transmembrane domain 5 region forms a binding pocket for menthol and related www.future-science.com
845
Review | Rech, Eckert, Maher, Banke, Bhattacharya & Wickenden TRPA1 activators (cigarette smoke, ozone, acrolein, crotonaldehyde, nicotine, oxidative stress, chlorinated irritants, isocyanates and tear gas)
TM1 TM1
TM3 TM4 TM4 TM2 TM2 TM3
TM5 TM5
TRPA1 ion channel
TM6 TM6
Ca2+ K710
• Activation • Inflammatory signals leading to sensitization • G protein-coupled receptor/second messenger-mediated phosphorylation
Na+
C665 C641 C621
TRPA1: pulmonary physiology Airway epithelial cells
To spinal and supra-spinal centers
Vagal afferent
• AIRWAY CONSTRICTION • MUCUS SECRETION • SNEEZING • COUGH
Efferent Smooth muscle cells
TRPA1: sensory physiology Efferent Mast cell
Skin
To spinal and supra-spinal centers Blood vessel
Sensory afferent (C and Aδ)
• HYPERALGESIA • ALLODYNIA • RESPONSE TO NOXIOUS COLD • MECHANOSENSATION
Efferent Future Med. Chem. © Future Science Group (2010)
Figure 1. Role of TRPA1 in pulmonary and sensory physiology. Activation of the transient receptor potential cation channel, subfamily A, member 1 (TRPA1) channels located on sensory nerve endings innervating the airway epithelia or skin induces action potential firing in sensory afferents, leading to a variety of responses, including sensory neuropeptide release, neurogenic inflammation, blood vessel dilation, allodynia and hyperalgesia and sensory reflexes such as cough, mucus secretion and airway constriction (refer to the text for further details).
agonists [47] . Further work is required to determine the importance of this region for other noncovalent agonists. Indirect
activators/sensitizers In addition to extracellularly applied modulators, TRPA1 is activated by numerous intracellular pathways. Elevated intracellular calcium alone is sufficient to activate TRPA1 directly via an EF-hand-like domain [18,48,49] . Since 846
Future Med. Chem. (2010) 2(5)
TRPA1 itself raises intracellular calcium, this potentially generates a positive feedback loop that further enhances neuronal excitability. Prolonged activation, however, desensitizes TRPA1. Desensitization may be agonist [50] or calcium dependent [5,51] . In addition to activation through calcium, TRPA1 is activated or sensitized by increases in diacylglycerol (DAG) and/or depletion of phosphatidylinositol-4,5bisphosphate (PIP2) [15,18,52] . Other reports future science group
Recent advances in the biology & medicinal chemistry of TRPA1 suggest that PIP2 sensitizes rather than desensitizes the channel [50,53] ; a biphasic relationship of sensitization/desensitization by PIP2 has been noted in other TRP channels [54] . TRPA1 also appears to be activated by an increase in intracellular pH [55] . Activation via phospholipase C (PLC) and intracellular calcium couples a wide variety of signaling molecules to TRPA1 activation. Nociceptive neurons often express receptors for components of the inflammatory soup, which contains bradykinin, prostaglandins, serotonin and histamine, for example. Theoretically, any of these could activate or sensitize TRPA1 if their respective receptors are coexpressed with the channel. For example, bradykinin has been observed to activate TRPA1 via G protein coupled bradykinin B2 receptors [15,16,18] . Protease activated receptor 2, activated by proteases present in the inflammatory soup, can also couple to TRPA1 through PLC [56] . Recent evidence suggests that activation of second messenger cascades may also regulate TRPA1 signaling via the modulation of cell surface expression levels. Thus, activation of the protein kinase A/PLC pathways can lead to increased surface expression of TRPA1, possibly through incorporation of a ready pool of vesicles [57] . Similar increases in TRPA1 expression occur in response to elevations in intracellular calcium following exposure to TRPA1 agonists or activation of coexpressed calcium permeable channels, such as TRPV1. Dynamic regulation of TRPA1 membrane insertion by second messengers and nociceptor activation likely contributes to the regulation of nociceptor sensitivity to TRPA1 agonists in inflamed tissues [57] . TRPA1 as a sensor for noxious cold TRPA1 may also be activated by noxious cold (
