A Potent and Site-Selective Agonist of TRPA1

Abstract

TRPA1 is a member of the transient receptor potential (TRP) cation channel family that is expressed primarily on sensory neurons. This chemosensor is activated through covalent modification of multiple cysteine residues with a wide range of reactive compounds including allyl isothiocyanate (AITC), a spicy component of wasabi. The present study reports on potent and selective agonists of TRPA1, discovered through screening 1657 electrophilic molecules. In an effort to validate the mode of action of hit molecules, we noted a new TRPA1-selective agonist, JT010 (molecule 1), which opens the TRPA1 channel by covalently and site-selectively binding to Cys621 (EC50 = 0.65 nM). The results suggest that a single modification of Cys621 is sufficient to open the TRPA1 channel. The TRPA1-selective probe described herein might be useful for further mechanistic studies of TRPA1 activation.

A Human TRPA1-Specific Pain Model

“…Unexpectedly, the EC 50 was not shifted between experiments at 25°C and 37°C. In summary, recording temperature does not explain the observed differences in EC 50 between the present data and those reported by Takaya et al, 2015. Further limitations of calcium-based assays as dye affinity, ceiling effects of the dynamic range, calcium buffering, and calcium elimination should be mentioned and might affect the inference of the concentration-dependent channel open probability.…”

“…The specificity of JT010 1 M for TRPA1 was established by testing a panel of TRP channels involved in nociception, including TRPV1, TRPV3, TRPV4, TRPM2, TRPM8, and TRPC5, using a concentration of 1 M (Takaya et al, 2015). To allow for estimation of the EC 50 , concentrations of JT010 included 3.1 M. Therefore, lack of activation of TRPV1 expressed in HEK293t cells by JT010 3.1 M (and 10 M) has been demonstrated in this manuscript.…”

“…For example, allyl isothiocyanate (AITC) was recently used for a TRPA1-dependent pain model (Andersen et al, 2017); however, AITC can also activate TRPV1 (Gees et al, 2013). According to the reported EC 50 of 0.65 nM, 2-chloro-N-(4-(4methoxyphenyl)thiazol-2-yl)-N-(3-methoxypropyl)-acetamide (JT010) was the most potent commercially available agonist (Takaya et al, 2015). Further, it was shown to be specific regarding other pain-related TRP channels up to a concentration of 1 M. By means of this substance, and the most potent commercially available antagonist (1E,3E)-1-(4-fluorophenyl)-2-methyl-1-penten-3-one oxime (A-967079), we established a human TRPA1-specific pain model.…”

TRP Channels as Drug Targets to Relieve Itch

“…TRPA1 is selectively expressed by a subpopulation of small-diameter neurons in dorsal root, trigeminal, and nodose ganglia and keratinocytes [36,37,38]. TRPA1 is directly activated by a vast array of noxious and electrophilic compounds (such as allyl isothiocyanate, cinnamaldehyde, allicin, and diallyl disulfide) [39,40,41], and numerous endogenous reactive oxygen species (such as hydrogen peroxide and 4-hydroxynonenal (4-HNE) [42,43]. It can also be activated by the transition metal ion Zn 2+ , leading to acute pain [44].…”

Non-Analgesic Symptomatic or Disease—Modifying Potential of TRPA1

“…The tools to activate TRPA1 have been rather poor for a long time, and substances with a limited range of specificity have been used at concentrations exceeding this range. Compared to e.g., allyl isothiocyanate (AITC), cinnamaldehyde, acrolein, or carvacrol, newer substances like JT010 and PF–4840154 allow potent and selective agonism [14,15]. TRPA1 activation reliably generates pain in humans, and models include AITC, cinnamaldehyde, carvacrol, and JT010 [16,17,18,19].…”