Identification of Phialomustin‑B (PHL-B) as a Novel Natural TRPA1 Channel Inhibitor

The TRPA1 channel has recently emerged as a critical target for pain relief since its antagonists target the beginning of the pain transduction pathway and, thus, are devoid of side effects such as sedation, dizziness, somnolence, or cognitive impairment. Despite this clinical significance, currently, no TRPA1 inhibitors suitable for therapeutic usage exist to target these channels. Since ancient times, natural products have been known to be a rich source of new drugs, useful therapeutic agents, as well as pharmacological tools. To discover novel natural TRPA1 antagonists, we screened a diverse range of natural products belonging to medicinal plants and endophytic microbes. Using a fluorescence-based calcium-influx assay, we identified that an unsaturated fatty acid known as Phialomustin B (PHL-B) exhibited potent TRPA1 inhibitory activity (IC50 = 1.35 ± 0.3 μM). In subsequent whole-cell/cell-attached patch clamp recordings, PHL-B displayed a reversible and voltage-dependent block of the TRPA1 ion channel at submicromolar concentrations. Our off-target profiling data indicated that PHL-B selectively inhibited TRPA1 channels without any considerable effect on other thermo-TRPs such as TRPV1, TRPV4, and TRPM8 channels. Docking of PHL-B on the TRPA1 channel structure revealed a binding pocket in a hotspot region for a gain-of-function mutation, N855S, that results in pain syndromes. Mutagenesis data demonstrated that I860 and K868 residues of the TRPA1 channel participate in PHL-B interactions, and when mutated, the potency of PHL-B is significantly mitigated. Collectively, our data indicate that PHL-B could function as a novel natural antinociceptive agent targeting TRPA1-related diseases with a TRPA1-mediated adverse component.