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.