Novel TRPA1 Antagonists are Multimodal Blockers of Human TRPA1 Channels: Drug Candidates for Treatment of Familial Episodic Pain Syndrome (FEPS)
Authors: Herz Jeffrey M. , Buated Wannipat , Thomsen William , Mori Yasuo 
Published: April 2020

The TRPA1 receptor‐channel is a therapeutic target for the discovery of novel pain drugs. In man, a single gain‐of‐function mutation in TRPA1, N855S, is known to be the cause of a familial episodic pain syndrome (FEPS). Multimodal activation by various stimuli is a fundamental feature of TRPA1 so that the precise manner in which TRPA1 and N855S‐TRPA1 is activated under pathophysiological conditions is not known, but is thought to be due to a combined effect of an array of inflammatory and nociceptive activators. Since the therapeutic potential of this class of drugs may depend on their efficacy to inhibit TRPA1 function by diverse activation mechanisms, we evaluated the activity of a group of novel TRPA1 antagonists, ALGX‐2542, 2567, 2582, 2586 and 2596 by multiple assay methods to determine mechanisms for inhibition and assess the therapeutic potential for this drug series. In the FLIPR‐calcium based pharmacological assays, the compounds demonstrated potent inhibition of either FFA or AITC induced activation of human TRPA1 (hTRPA1) expressed in HEK‐293 cells with IC50s ranging from 15–88 nM. The potent activity of these ALGX antagonists was confirmed in whole‐cell patch clamp electrophysiological assays. To demonstrate open channel block, hTRPA1 currents were first activated by AITC and upon reaching a pseudo‐steady state, each cell was exposed to a single concentration of an ALGX antagonist. All ALGX antagonists rapidly and completely blocked the open channel currents at 10‐fold their respective IC50 values. To compare open channel block and drug interactions with resting state channels, cells were pre‐incubated with ALGX antagonists for 5 min in the presence of external Ca2+, and then challenged with AITC. For this condition, all ALGX antagonists also completely blocked hTRPA1 activation, indicating that these compounds are effective inhibitors of both open and resting state hTRPA1 channels. Calcium imaging studies also showed that ALGX‐2542 potently inhibits activation by AITC, H202, and cold. The pharmacological activity of this series of antagonists was further defined for native human TRPA1 channels endogenously expressed in the WI‐38 human cell line. Utilizing the pre‐incubation FLIPR protocol, the series of ALGX antagonists was also found inhibit agonist‐induced increases in [Cai2+] with nearly equal IC50 values as found in the recombinant hTRPA1‐HEK assay system. The quantitative agreement (r2=0.95) demonstrates that the pharmacological activity defined in the recombinant assay systems directly translates to native hTRPA1 receptor‐channels. Furthermore, these antagonists also completely inhibited the AITC‐activation of the N855S hTRPA1 channels. Remarkably, the inhibitory effects of this series of antagonists is independent of the mode of TRPA1 activation, which includes structurally diverse agonists, H202, and cold temperatures with similar IC50s. This multimodal profile may be an optimal feature for producing maximal clinical efficacy for a pain drug targeting TRPA1. Our results identify a class of drugs that enable a therapeutic approach to treat pain episodes associated with FEPS.Support or Funding InformationThis work was supported by NIDA 1R44DA044900 to JMH and Algomedix.