Discussion
In this study, we identified an irritant chemical nitrobenzene DNFB that is a well known to cause allergic contact dermatitis and can selectively activate TRPA1 channels. DNFB is about 7.5 times more potent than AITC that is currently available agonist of TRPA1. At the single-channel level, DNFB increases the channel open probability through binding to three key residues C621, Y658 and E625 in the channel coupling domain that functions to sense electrophile irritants (Suo et al., 2020). Our identification of chemical DNFB as a selective TRPA1 agonist not only provides a powerful tool for further understanding of the channel pharmacology and pathology, but also demonstrates the importance of TRPA1 as a potential therapeutic target for allergic contact dermatitis (Oh et al., 2013).
Previous studies have shown that several TRP channels such as TRPA1, TRPV1, TRPV3 and TRPV4 are implicated in skin physiology and pathology including skin inflammation (Luo et al., 2018; Nilius et al., 2007; Southall et al., 2003; Toth et al., 2014; Vay et al., 2012; Yoshioka et al., 2009). Our selectivity evaluations indicate that DNFB specifically actives TRPA1 that is featured of a unique electrophilic sensing pocket located in the channel C-terminal coupling domain (Suo et al., 2020). The electrophilic sensing pocket is highly rich in reactive cysteines such as C621 and C655 (Hinman et al., 2006; Macpherson et al., 2007; Suo et al., 2020; Zhao et al., 2020), and the electrophile sensing region is surrounded by a number of nucleophilic aromatic amino acids for facilitating the entry of electrophiles (Suo et al., 2020). In contrast, the cryo-electron microscopy structures of TRPV1 (Cao et al., 2013), TRPV3 (Singh et al., 2018) and TRPV4 (Deng et al., 2018) reveal that these three channels are lack of the electrophile sensing coupling domain that serves to sense electrophilic irritants (Paulsen et al., 2015), which explains the selective activation of TRPA1 by chemical DNFB (Saarnilehto et al., 2014).
Chemical DNFB as an electrophilic reagent binds to TRPA1 through non-covalent hydrogen bonds with C621 and Y658 residues and a unique halogen bond between the fluorine of DNFB and Y684 residue (Fig. 5), consistent with the observation that a non-covalent ligand binding confers a biased agonism of TRPA1 channels (Liu et al., 2020). The non-covalent binding agonist DNFB that activates TRPA1 without causing the channel desensitization is widely used for the model establishment of persistent dermatitis (Kaplan et al., 2012), which is unlike agonist AITC that covalently binds to and induces TRPA1 channel desensitization (Dai et al., 2007; Liu et al., 2020). Other non-covalent TRPA1 agonists such as peptide scorpion toxin (WaTx) or small molecule GNE551 can activate TRPA1 in a slow kinetics fashion without inducing channel desensitization and cause persistent pain (Lin King et al., 2019; Liu et al., 2020). Non-covalent agents are non-reactive to cytosolic abundant nucleophiles (such as glutathione) and are expected to sustain their concentration for longer time, thus leading to more persistent activation of TRPA1 (Liu et al., 2020). In contrast, covalent agents are highly reactive and are not stable in a cytosolic environment containing high concentrations of nucleophiles (Liu et al., 2020). Covalent TRPA1 agonists such as benzoquinone, JT010 and AITC, covalently binding to the cysteine residues of the electrophilic sensing domain of TRPA1, can make TRPA1 desensitization and deactivation, and only cause acute pain (Heber et al., 2019; Ibarra and Blair, 2013; Suo et al., 2020). We also made an effort in docking of DNCB, a derivative of DNFB, into the same electrophilic sensing pocket, which reveals a similar non-covalent binding through hydrogen bonds without the halogen bond to Y684 (data not shown) and DNCB also activates TRPA1, but the skin sensitization mediated by DNCB is much milder than that of DNFB (Hsieh et al., 1996; Tingle et al., 1990).
In summary, we identify the skin sensitizer DNFB that selectively activates TRPA1 channel through binding to the channel irritant sensing domain. DNFB can serve as molecular tool for better understanding of TRPA1 pharmacology and pathology. In addition, pharmacological inhibition of TRPA1 channel may hold a promise for therapy of dermatitis.