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. 2025 Aug 21;10(34):39192–39202. doi: 10.1021/acsomega.5c06063

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

Priyanka Yadav †,§, Ashutosh Sharma †,§, Aditya Singh †,§, Mansi Sharma ∥,⊥, Appu Kumar Singh ∥,⊥, Asif Ali ‡,§,*, Aravind Singh Kshatri †,§,*
PMCID: PMC12409571  PMID: 40918390

Abstract

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.

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Introduction

Transient receptor potential (TRP) channels comprise a superfamily of nonselective cation channels that detect and integrate a range of diverse chemical and physical stimuli. Six receptors from this family (TRP vanilloid V1–V4, TRPV1–V4; TRP melastatin 8, TRPM8; and TRP ankyrin A1, TRPA1) are classified as thermosensitive channels due to their intrinsic ability to sense noxious cold to warm temperatures. Even though this subset of TRP members is characterized by their sensitivity to specific ranges of temperatures, they can be activated by different types of stimuli, such as mechanical forces generated by osmolarity or pressure, and endogenous or exogenous chemicals. Upon their activation, Ca2+ primarily enters the cells and generates cell depolarization, leading to the transduction of sensory signals. TRPA1 is the sole constituent of the TRPA subfamily and is characterized as a voltage-dependent, ligand-gated channel that allows conduction of both monovalent and divalent cations. , The TRPA1 channel is predominantly expressed in sensory neurons, which are responsible for the detection of various noxious stimuli. Upon activation by inflammatory mediators and harmful environmental substances, it preferentially conducts Na+ and Ca2+ ions, thus generating a potential that depolarizes the membrane potential toward the action potential threshold. Consequently, the TRPA1 channel has been posited as a chemosensor of nociception as well as a sensor for mechanical and cold stimuli under physiological conditions. ,, The identification of human TRPA1 gain-of-function data (N855S) associated with extreme pain conditions furnishes compelling evidence that the selective and specific blockade of TRPA1 may represent an effective strategy for pain management.

A number of small-molecule potent TRPA1 antagonists, such as HC-030031, A-967079, AMG0902, and GRC-17536, have been reported to provide targeted pain relief. , However, none progressed to clinical development due to poor selectivity, complex pharmacokinetic characteristics, and limited bioavailability. Natural products, characterized by their diverse chemical structures, structural complexity, and historical success, are often used as starting points for optimization into novel drug candidates. Diverse classes of molecules from plants or spices are known to modulate the activity of TRPA1 channels. Some of the best-known natural activators of TRPA1 include allyl isothiocyanate (AITC) from wasabi, allicin from garlic, and cinnamaldehyde from cinnamon. Likewise, monoterpenes such as menthol, camphor, and 1,8-cineole are reported to possess analgesic effects through the inhibition of TRPA1 channels. A natural TRPA1 inhibitor with an ideal safety profile would be desirable for the development of pain therapeutics.

In this article, we report the identification of an unsaturated fatty acid, PHL-B, derived from an endophytic fungus, as a novel natural antagonist of TRPA1 channels. Using calcium imaging and electrophysiological experiments, we have validated the direct inhibition of TRPA1 channels by PHL-B. Furthermore, we demonstrated the specificity of PHL-B on three other typical targets of the TRP family: TRPV1, TRPV4, and TRPM8. Finally, the binding site and the residues involved in PHL-B modulation were identified by using molecular docking and mutagenesis experiments. The data presented here provide a first indication that PHL-B is a new natural product targeting the TRPA1 channel, which can be further used as a scaffold for rational drug design of potent and selective TRPA1 channel inhibitors for antinociception.

Methods

Cell Culture, Transfection, cDNA Constructs, and Mutagenesis

HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated FBS, 1% penicillin/streptomycin, and kept in a humidified incubator at 37 °C with 5% CO2. Cells were plated on 96-well clear-bottom black plates for primary screening assays and Ca2+ imaging experiments, and on 12 mm polylysine-treated glass coverslips for electrophysiology experiments. Transfections were performed 24 h later with the Turbofect reagent (Thermo Scientific). The TRP constructs used in this study were mammalian expression plasmids procured from the DNASU plasmid repository: human TRPA1 (HsCD00080227), human TRPV1 (HsCD00081472), and human TRPV4 (HsCD00830910). The rat TRPM8 plasmid was obtained from Addgene (#64879). The pGP-CMV-GCaMP6s plasmid was a gift from Douglas Kim and the GENIE Project (Addgene plasmid #40753). Mutations were introduced into the TRPA1 construct using a QuikChange site-directed mutagenesis kit (Agilent Genomics) and confirmed by sequencing.

Chemicals and Reagents

Compounds 1–3 were isolated from Murraya koenigii. Compounds 4 and 5 were new synthetic derivatives of mahanimbine. Compound 6 was isolated from Coleus forskohlii, while compound 7 was isolated from Erythrina subrosa. Compound 8 was isolated from Myrica esculenta, compound 9 was isolated from Cholchicum leuteum, and compound 10 was isolated from the leaves of Cannabis sativa. Compounds 11 and 12 were synthetic analogs of THC. Compounds 13–15 were isolated from Artemisia santolinifolia. Compound 16, designated as PHL-B, was isolated from an endophytic fungus, Phialophora mustea, found in the corm of Crocus sativus. Compounds 17–27 were purchased from MedChemExpress (Compound 17, Cat. No. HY-N1464A; Compound 18, Cat. No. HY-101445; Compound 19, Cat. No. HY-16558; Compound 20, Cat. No. HY-N0763; Compound 21, Cat. No. HY-N0228; Compound 22, Cat. No. HY-17387; Compound 23, Cat. No. HY-W015828; Compound 24, Cat. No. HY-N6018; Compound 25, Cat. No. HY-N6607; Compound 26, Cat. No. HY-113303; Compound 27, Cat. No. HY-N0141). The activators and inhibitors were also purchased from MedChemExpress (HC-067047, Cat. No. HY-100208; capsaicin, Cat. No. HY-10448; capsazepine, Cat. No. HY-15640; A-967079, Cat. No. HY-108463). AITC (Cat. No. 377430) and menthol (Cat. No. W266523) were obtained from Sigma-Aldrich. Sesamin was isolated from Piper mullesua. All compound solutions were prepared on the day of the experiment in 100% DMSO and used only for approximately 6 h. The stock solutions of all compounds were kept at a concentration of 25 mM in DMSO. The desired concentrations of compounds were obtained by appropriate dilution in the external SBS solution. DMSO was present at 0.04% (v/v) in the concentration of the compound typically used in this study (10 μM) and at 0.12% (v/v) in the highest dose tested (30 μM).

Primary Screening Assay

HEK293T cells were cotransfected with TRPA1/GCaMP6s for 48 h and seeded at a density of 2 × 104 per well into 96-well clear-bottom black plates, incubated in 5% CO2 at 37 °C overnight. On the day of the assay, the cell culture media was replaced with 100 μL of standard buffer solution (SBS): 160 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES; pH 7.4. The plate was then incubated with the test compounds at a final concentration of 10 μM at room temperature for 10 min. DMSO at these concentrations had no significant effect on the data. Cells were then transferred to an Infinite 200 PRO multimode plate reader (Tecan) for experimentation. Basal fluorescence (F0) was recorded for 80 s before the application of 100 μM AITC. Fluorescence intensity measurements (F) were read by the plate reader at room temperature (22–24 °C) with an excitation wavelength of 497 nm and an emission wavelength of 512 nm. The fluorescence intensity was recorded every 20 s for 600 s.

Compound Selectivity Assay

Selectivity studies of PHL-B were performed on TRPV1, TRPV4, and TRPM8 channels using the fluorescence-based Ca2+ assay. Individual gene-containing vectors were cotransfected with GCaMP6s in HEK293T cells, and the assay was performed after 48 h. For TRPV1 and TRPM8 channels, the SBS solution was used. 10 μM capsaicin was used for the stimulation of TRPV1 channels, and 1 μM capsazepine was used as a reference inhibitor compound. TRPM8 channels were activated with 30 μM menthol and were inhibited with 20 μM sesamin. For TRPV4 channels, we used an isotonic solution containing 105 mM NaCl, 6 mM CsCl, 5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 90 mM d-mannitol, and 10 mM glucose, pH 7.4 with NaOH (osmolarity: 320 mOsm) for recording basal fluorescence values (F0). To activate TRPV4 channels via cell swelling, mannitol was omitted from this solution to yield a hypotonic solution, HTS (osmolarity: 230 mOsm). 1 μM HC-067047 served as a positive inhibitor for TRPV4 channels.

Ca2+ Imaging

TRPA1 transiently transfected or blank HEK293T cells were seeded on 12 × 12 mm glass coverslips for 24 h before being loaded with 5 μM Fluo-4 AM (HY-101896, MedChemExpress) in standard buffer solution (SBS) for 30 min in 5% CO2 at 37 °C. To prevent dye compartmentalization during loading, Pluronic F127 (0.2%) (Cat. No. 786–1536; G-Biosciences) was used to dissolve the Fluo-4 AM dye. The tested compounds were prepared in SBS solution. Cells were then washed twice with SBS solution and subsequently treated with PHL-B and A967079 for 10 min. Later, the basal fluorescence intensity (F0) in each condition was imaged, and then AITC (100 μM) or ionomycin (10 μM) was added individually to induce Ca2+ influx. Fluorescent signals were acquired on a fluorescent microscope (DMI-6000, Leica) using an FITC filter with a 20× objective lens and analyzed using ImageJ software. All experiments were performed at room temperature, and data were expressed as the mean ± SEM.

Cell Viability Assay

Cell viability of HEK293T cells exposed to different concentrations of PHL-B was evaluated using an MTT assay. Briefly, 1 × 104 cells were seeded in 96-well plates and after 24 h the cells were treated with increasing concentrations of PHL-B (from 0.1 μM to 30 μM) in serum-free medium without phenol red and incubated for 24h. Afterward, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well and incubated for 4 h. Finally, 100 μL of solution was added to dissolve formazan, and absorbance was measured at 570 nm, using 620 nm as the wavelength reference.

Patch Clamp Electrophysiology

Whole-cell current recordings and cell-attached single-channel current recordings were performed on HEK293T cells expressing TRPA1 channels. Patch pipettes were fabricated from thick-wall borosilicate glass (1.5 mm O.D. × 0.86 mm I.D.) using a Sutter p-1000 puller and fire-polished. The obtained pipettes had a resistance of 3–5 MΩ when filled with recording solutions. For whole-cell recordings, the extracellular recording solution contained (in mM): 150 NaCl, 5 CsCl, 1.5 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, buffered at pH 7.4 with NaOH. The intracellular solution contained (in mM): 140 CsCl, 4 MgCl2, 5 EGTA, and 10 HEPES, buffered at pH 7.2 with CsOH. For cell-attached recordings, the extracellular recodring solution was used in the patch pipette. The osmolarity of these solutions was adjusted to 290–300 mOsm. No liquid junction potential correction was applied, and all experiments were performed at room temperature (20–25 °C). Gravity perfusion was used to perfuse the compounds, and the perfusion rate was set to 0.5 mL/min. For whole-cell recordings, the membrane potential was clamped at −60 mV, and voltage ramp pulses from −100 to +50 mV were applied every 5 s. For cell-attached recordings, the membrane potential was clamped either at −60 or +60 mV. Currents were recorded using the Multiclamp 700A amplifier (Axon Instruments, Molecular Devices) with low-pass filtering at 2 kHz for ramp currents and 5 kHz for single-channel currents, and digitized with the Axon Digidata 1440A at 1 ms. Clampex and Clampfit software (pClamp10; Axon Instruments) were used for stimulus generation and data acquisition. The current density (pA/pF) was calculated by dividing the whole-cell current (I m) by the cell capacitance (C m). The TRPA1 open channel probability (PO) was determined by single-channel analysis using a 50% amplitude threshold criterion in the Clampfit software.

Molecular Docking

Molecular docking was performed to predict the binding interactions between human TRPA1 and the small molecule PHL-B using the SWISS-DOCK web server, which employs the EADock DSS engine for blind docking across the entire protein surface. The cryo-EM structure of human TRPA1 (PDB ID: 7OR1) was obtained from the Protein Data Bank and processed using UCSF Chimera. All non-protein entities, including ligands and metal ions, were removed, missing hydrogen atoms were added and the structure was subsequently energy-minimized using the CHARMM force field package provided with the SwissDock server. The 3D structure of PHL-B was retrieved from PubChem in SDF format and was then converted to the MOL2 format using Open Babel for docking purposes. The blind docking was performed around the transmembrane domain using default parameters, and output clusters were ranked based on the FullFitness score and estimated binding free energy (ΔG). The top-ranked clusters, particularly those exhibiting the most favorable ΔG values, were visualized using PyMOL and UCSF Chimera to evaluate ligand orientation and key interactions, including hydrogen bonds, hydrophobic contacts, and electrostatics. Top-ranked models were selected for further interpretation to explore how PHL-B may modulate TRPA1 function.

Statistical Analysis

Data are expressed as mean ± SEM and were analyzed using an unpaired Student’s t-test or Ordinary one-way ANOVA, with Dunnett’s or Tukey’s multiple comparisons test applied as appropriate to determine statistical significance (*p < 0.05, **p < 0.01, and ***p < 0.001 vs ) Vehicle/AITC/WT.

Results

TRPA1 Antagonist Primary Screening

In the search for a promising novel TRPA1 channel inhibitor, we evaluated the activity of 27 natural compounds (Figure ) at a concentration of 10 μM in TRPA1-overexpressing cells using a multimode microplate reader fluorescence assay.

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A library of natural products was used for the primary screening assay on TRPA1 channels.

The cells were preincubated with the tested compounds for 10 min and treated with AITC (100 μM) to activate the channels (Figure A). Among the compounds tested, only compound number 16 exhibited a notable reduction in the fluorescence intensity (F/F0) of GCaMP6s to values close to the reference compound (1 μM A967079). Intriguingly, compound 9 displayed a significant enhancement in the F/F0 values, suggesting that it could function as an activator of TRPA1 channels. The remaining compounds had modest inhibitory or activating effects or no effect on TRPA1 channels. After the treatment with compounds, 5 μM ionomycin, a Ca2+ ionophore, was added to the cells to confirm the maximum fluorescence intensity. The percentage inhibition for compound 16 was above the hit threshold (>50% reduction in F/F0), implying that it could be a positive hit (Figure B). This compound is an unsaturated fatty acid known as Phialomustin B (PHL-B), obtained from the endophytic fungus Phialophora mustea of the corms of Crocus sativus. Using the same assay, we determined the concentration-dependent antagonistic effect (IC50) of PHL-B on TRPA1 channels, which was found to be 1.35 ± 0.3 μM (Figure C).

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Identification of PHL-B as an antagonist of TRPA1 channels. (A) Relative fluorescence intensity (F/F0) of GCaMP6s induced by 100 μM AITC after treatment with Vehicle (0.1% DMSO), A967079 (1 μM), and 27 natural compounds at a concentration of 10 μM. (B) A scatter plot showing the percentage inhibition for all the compounds tested. A967079 (red circle) and Vehicle (blue circle) were used as the positive and negative controls, respectively. The cutoff value for a hit molecule was set at 50%, and only PHL-B (green circle) was identified. (C) Normalized fluorescence (F/F0) was plotted against drug concentration and fitted with the Hill equation, yielding an IC50 of 1.35 ± 0.3 μM. All data are expressed as means ± SEM, N = 3–8 independent experiments.

Furthermore, we confirmed TRPA1 inhibition by PHL-B by monitoring [Ca2+]i changes following channel activation by AITC (Figure ). Consistent with the primary screening data, the TRPA1-mediated [Ca2+]i rise in Fluo-4 AM-loaded cells after AITC addition was blocked by pretreatment with PHL-B as well as with A967079. Ionomycin was used as a positive control, resulting in a drastic elevation of intracellular calcium in both TRPA1-overexpressing and nontransfected HEK293T cells. Lastly, we determined the maximal concentration of PHL-B that the cells could tolerate without observable cell death using an MTT assay. A concentration of up to 3 μM PHL-B had no significant toxic effect on HEK293T cells after 24h of treatment (Figure S1). Together, these results indicate that PHL-B is an antagonist of TRPA1 channels with low cytotoxicity.

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Inhibition of AITC-induced intracellular calcium in TRPA1-transfected HEK293T cells by PHL-B. (A) Calcium imaging of untransfected and TRPA1 transiently transfected cells after treatment with Vehicle (0.1% DMSO), PHL-B (10 μM), A967079 (1 μM), and ionomycin (10 μM). Scale bar: 100 μm. (B) The relative fluorescence intensity (ΔF/F0) was calculated based on 25 cells in a glass coverslip (n = 25). The fluorescence intensity was normalized to the value obtained with AITC (10 μM). All data are expressed as means ± SEM and were analyzed using ordinary one-way ANOVA followed by Tukey’s post hoc test. N = 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Vehicle.

Inhibition of TRPA1 Channel Currents by PHL-B

To evaluate the effect of PHL-B on TRPA1 channel inhibition directly, we performed whole-cell patch-clamp experiments on HEK293T cells overexpressing TRPA1 channels. Using a ramp protocol, in which the membrane potential was stepped from a −60 mV holding potential for 100 ms and then ramped from −100 to +50 mV over 2 s, TRPA1 currents were recorded (Figure A). Stimulation with 100 μM AITC elicited a predominantly inward current, which exhibited outward rectification. Application of 5 μM PHL-B (green trace) resulted in a rapid and considerable inhibition of the AITC-elicited TRPA1 currents, which recovered completely following washout (orange trace). The blue trace displays the effect of 1 μM A967079, which produced a near-complete inhibition of TRPA1 channels. The magnitude of inward and outward current densities at −100 mV and +50 mV, respectively, were compared in Figure B. The inhibitory effect of PHL-B was found to be maximal at negative voltages compared to positive voltages (at −100 mV the current density was significantly reduced from −91.73 ± 11.47 pA/pF in AITC to −24.96 ± 6.1 pA/pF in PHL-B compared to 30.5 ± 2.9 pA/pF in AITC and 11.8 ± 1.2 pA/pF in PHL-B). The kinetics of onset and recovery from PHL-B inhibition of TRPA1 channels are shown in Figure C. Interestingly, a faster time course of recovery from PHL-B inhibition (τoff = 28.6 ± 3.4 ms in AITC and 7.3 ± 2.1 ms in AITC + PHL-B) was observed.

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PHL-B inhibited TRPA1 channels in whole-cell recordings. (A) Representative current recordings from HEK293T cells transiently expressing TRPA1 in response to slow voltage ramps from −100 mV to +50 mV. Cells were stimulated with 100 μM AITC and then exposed to AITC + 5 μM PHL-B before washout. A967079 in the presence of AITC was applied at the end of the experiment. (B) Comparison of mean current density (pA/pF) recorded at −100 mV and +50 mV under the same conditions. (C) Time course of recovery of PHL-B inhibition, estimated at a single potential of −60 mV applied in 5-s intervals. Dashed lines represent single exponential fits to the data. Data are expressed as means ± SEM and were analyzed using ordinary one-way ANOVA followed by Dunnett’s post hoc test. N = 4 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to AITC.

Next, we explored whether PHL-B can inhibit TRPA1 single-channel activity when activated by AITC. Figure A illustrates a typical recording of TRPA1 single-channel currents from cell-attached patches. The basal single-channel activity was low, and after the addition of AITC to the bath solution, the channel activity was clearly elevated (orange traces). Application of PHL-B in the presence of AITC significantly reduced the number of open channels (green traces). For example, the channel open probability (PO) at −60 mV increased markedly by approximately 150-fold in AITC, whereas in the presence of PHL-B, PO was increased only 17-fold. The corresponding amplitude histograms of single-channel currents measured at +60 and −60 mV are shown in Figure B where the inhibitory effects of PHL-B can be visualized. Thus, these data support the idea that under physiological conditions, exposure of activated TRPA1-expressing cells to PHL-B would lead to a decreased excitability of these cells.

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TRPA1 single channels were inhibited by PHL-B. (A) Basal single-channel activity (black traces) in the cell-attached configuration at −60 and +60 mV was low under vehicle conditions. After the addition of 100 μM AITC to the bath solution (orange traces), channel activity was clearly elevated. Exposure of the cells to 5 μM PHL-B in the presence of AITC (green traces) substantially reduced the open probability of TRPA1 channels. (B) Histogram plots of single-channel counts vs current amplitude, where the inhibitory effect of PHL-B can be clearly visualized.

Selectivity Evaluation of PHL-B on TRPV1, TRPV4, and TRPM8 Channels

TRPA1 channels share high structural similarity with othe TRP family members, such as TRPV1, TRPV4, and TRPM8 , channels, and they are also found to be coexpressed in nociceptive sensory neurons to participate in pain perception. , To confirm whether PHL-B modulates any activity on these channels, we performed the Ca2+ influx assay using their established agonists: capsaicin (TRPV1), HTS (TRPV4), and menthol (TRPM8). Application of 10 μM capsaicin activated the TRPV1 channels, and pretreatment with different concentrations of PHL-B, ranging from 0.1 μM to 10 μM, did not alter the capsaicin-induced normalized fluorescence values (Figure A,B). However, a reference TRPV1 inhibitor, Capsazepine, at a 1 μM concentration fully abolished the TRPV1-mediated responses. Similarly, the TRPV4 cells were activated with HTS solution to induce Ca2+ mobilization, and 1 μM HC-067047 was used as a positive control. No differences in the fluorescence signals (F/F0) were observed after the PHL-B treatment (Figure C,D). Lastly, the activating effect of menthol on TRPM8-expressing cells also remained unchanged after treatment with different concentrations of PHL-B, whereas 20 μM sesamin practically inhibited all the TRPM8-mediated Ca2+ influx (Figure E,F). These observations suggest that PHL-B does not interact with TRPV1, TRPV4, or TRPM8 channels.

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Lack of inhibitory effect of PHL-B on TRPV1, TRPV4, and TRPM8 channels. (A, C, E) Concentration-dependent relationships of PHL-B on TRPV1, TRPV4, and TRPM8 channels upon stimulation with capsaicin, HTS, and menthol, respectively. Capsazepine, HC-067047, and sesamin were used as reference inhibitors for TRPV1, TRPV4, and TRPM8 channels, respectively. (B, D, F) Plots of normalized fluorescence vs drug concentration at different time points for each channel, where the lack of effect of PHL-B can be clearly visualized. These data could not be fitted with a Hill equation; hence, the IC50 values were not determined. Data are expressed as means ± SEM, N = 3–5 independent experiments.

Identification of the Residues Responsible for PHL-B Inhibition of TRPA1 Channels

We further uncovered the binding region of PHL-B with the TRPA1 channel by using molecular docking and site-directed mutagenesis studies. PHL-B was docked onto the Cryo-EM structure of human TRPA1 (PDB ID: 7OR1), and multiple binding clusters were identified based on spatial convergence and docking scores, which ranged from −7.95 to −3.45 kcal/mol. The highest docking score (−7.95 kcal/mol) corresponded to a site located near a well-characterized ligand-binding pocket, analogous to the vanilloid-binding pocket in the TRPV1 channel. A second site, with a lower docking score (−6.15 kcal/mol), was identified in the extracellular half of the channel, near residue I906 of the pore helix (Figure S2). The remaining sites were either in close proximity to site 1 or at the periphery of the TM region and, therefore, were not considered for analysis. We focused our analysis on the first site, as the pore helix is a structurally rigid element whose modulation could alter channel selectivity, and this extracellular site has not previously been recognized as a druggable region in TRP channels. Docking of PHL-B at the selected site revealed interactions with residues on the S4–S5 linker (Q851, R852) and the distal region of S5 (I860, K868), supporting its functional relevance (Figure A). At this site, the negatively charged headgroup of PHL-B forms a hydrogen bond with residues Q851 and R852, while its hydrophobic tail engages in van der Waals interactions with I860 and K868. To validate the binding site, we individually mutated each of these residues in the WT TRPA1 channel to alanine and examined the concentration-dependent effects of PHL-B using the fluorescence-based Ca2+ assay. The Q851A and R852A mutants did not affect the inhibitory response of PHL-B, whereas the I860A and K868A mutants reduced the potency of PHL-B by 6-fold (p = 0.91; ns) and 29-fold (p < 0.01), respectively (Figure B,C). Intriguingly, the double mutant I860A+K868A further reduced the potency from 1.47 ± 0.6 μM in WT to 179.45 ± 11 μM (122-fold reduction; p < 0.001). We also tested the effects of these mutations on our reference inhibitor, A967079. None of these mutants produced any significant differences in the inhibitory effects of this compound, suggesting that the binding sites and the mechanism of action of PHL-B are different from those of A967079 (Figure S3).

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Identification of residues critical for TRPA1 inhibition by PHL-B. (A) Cryo-EM structure of hTRPA1 showing the location of the ligand PHL-B and surrounding key residues between the S4–S5 linker and distal S5 region (PDB: 7OR1). PHL-B is shown in yellow, and the interacting amino acids (Q851, R852, I860, and K868) are shown as sticks. (B) Concentration–response curves of PHL-B determined through a Ca2+ influx assay in wild-type (WT; white circles), Q851A (blue circles), R852A (pink circles), I860A (green circles), K868A (red circles), and I860A+K868A (cyan circles) mutant channels. (C) A comparison of IC50 values for all the mutant channels is shown. Note the additive reduction in the potency of PHL-B in response to the double mutation. All data are expressed as means ± SEM and were analyzed using ordinary one-way ANOVA followed by Dunnett’s post hoc test. N = 3–6 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to WT.

Discussion

Despite great interest in targeting the TRPA1 channel as a potential analgesic target, the development of its inhibitors has been challenging owing to the poor safety profile, pharmacokinetics, and pharmacodynamics properties of existing synthetic antagonists. Natural products (e.g., morphine and salicylate) have been considered some of the most powerful tools for identifying and manipulating pain pathways since ancient times. Furthermore, these natural sources have also played a role in the ligand deorphanization of many TRP channels, aiding in the understanding of their biology and facilitating their establishment as targets for drug discovery. Recent studies have reported that cardamonin specifically blocked TRPA1 channels (IC50 ∼0.5 μM) and carnosol activated TRPA1 channels (EC50 ∼12 μM) without eliciting any considerable effects on other TRP family members. Inspired by these findings, we aimed to discover natural products with high potency as TRPA1 channel inhibitors for the development of novel analgesics. Our results clearly indicate that the TRPA1 channel can also be modulated by an unsaturated secondary metabolite from the endophytic fungus Phialophora mustea, PHL-B. The secondary metabolites derived from these endophytic fungi have previously been found to possess diverse therapeutic properties. PHL-B was initially shown to exhibit remarkable cytotoxic activity against the human breast cancer cell line, T47D (IC50 = 1 μM). However, our data showed a nontoxic effect of PHL-B at these concentrations on HEK293T cells. A possible speculation of this finding is that TRPA1 channels are known to be upregulated in cancer cells to promote their survival and acquire chemoresistance. In this scenario, PHL-B may have inhibited TRPA1 channels to prevent Ca2+ influx, thereby leading to oxidative stress and cellular toxicity. Recently, PHL-B has also been shown to be a modulator of superoxide dismutase 1 (SOD1) aggregation, which has therapeutic potential in treating Amyotrophic Lateral Sclerosis (ALS). Here, we clearly showed that PHL-B effectively inhibited TRPA1 activation in a dose-dependent manner in the fluorescence-based Ca2+ mobilization assay. These results were supported by a reduction in TRPA1-mediated Ca2+ influx in the presence of PHL-B. Furthermore, in whole-cell patch-clamp recordings, the blocking effect of PHL-B was found to be completely reversible at a 5 μM concentration. Our mutagenesis experiments revealed that the I860A and K868A mutations hampered the inhibitory effects of PHL-B, and the double mutation resulted in an additive loss of function of PHL-B.

Among the TRP channel family, the heat-sensitive TRPV1, the cold-responsive TRPA1 and TRPM8, and the mechanical stimuli-activated TRPV4 are the most studied receptors with respect to physiological and pathological conditions. Moreover, these four members share many structural similarities, such as the presence of ankyrin repeat domains (ARD) in their N-terminal regions, six transmembrane domains (S1–S6), a conserved TRP domain located C-terminal to S6, pore-forming loops, and more. ,, To rule out the possibility of nonselectivity of PHL-B on these thermo TRP channels, we studied its concentration-dependent effects. Our data indicated that PHL-B, at the highest concentration of 10 μM, had no obvious effect on TRPV1, TRPV4, and TRPM8 channels. It is worth noting that some TRP channels are often coexpressed in natural settings for efficient transmission of noxious stimuli. For example, TRPA1-TRPV1 channels form complexes in nociceptive primary sensory neurons to regulate pain transduction. , The effects of PHL-B on these multimeric complexes remain to be elucidated.

The cryo-EM structure of the human TRPA1 ion channel solved with AITC has provided a framework for predicting PHL-B binding sites within the channel. The allosteric agonist AITC binds to the cysteine residues of the cytoplasm adjacent to the regulatory TRP-like domain, which is essential for channel activation. Multiple studies have also proposed that AITC binding triggers S4–S5 linker movements to repack the pore-lining TM helices of S5 and S6, contributing to channel activation by AITC and stabilization of its activated conformation. , Furthermore, analysis of the gain-of-function mutation N855S biophysical properties also highlighted that the linker region and the arrangement of helices S5 and S6 are critical for the TRPA1 channel’s intrinsic gating activity. , Based on our molecular docking and mutagenesis results presented here, we hypothesize that PHL-B may likely participate in direct or indirect interactions with K868 that could suppress the S5 domain motion during AITC-induced channel activation, resulting in channel closure. It is plausible that the Q851A and R852A mutations did not affect TRPA1 activity in part because the predicted binding pose of PHL-B may not be optimal and could adopt an alternative orientation. A967079 is one of the most potent mammalian TRPA1 synthetic antagonists, which inhibits neuropathic and inflammatory pain in vitro and in vivo. Previous work proposed that A967079 interacts with S873, Thr874, and F909 of the TRPA1 channel to exert its inhibitory effects. , In agreement with these studies, we did not observe any loss of the inhibitory effect of A967079 in Q851, R852, I860, and K868 mutant channels, indicating that the binding sites and perhaps the mechanism of action of PHL-B are distinct from those of A967079. Future work involving computational molecular dynamics simulations and cryo-EM structures in apo- and PHL-B-bound states will assist us in understanding the molecular mechanisms by which PHL-B binding instigates TRPA1 inhibition. Nonetheless, our TRPA1/PHL-B docked model helped us identify a pocket that could potentially accommodate PHL-B, supporting its relevance as a candidate binding site. Similarly, detailed structure–activity relationships involving the substitution of the core moiety containing the acidic group of PHL-B with aryl and heteroaryl moieties may also enhance the potency of PHL-B to be better suited as a drug candidate. These studies will provide a great opportunity for identifying the molecular determinants of PHL-B as well as for developing novel classes of natural product-derived potent and specific analgesics targeting TRPA1 channels.

Supplementary Material

ao5c06063_si_001.pdf (467.2KB, pdf)

Acknowledgments

We sincerely acknowledge the generous support of all the members of the Neuroscience and Aging Biology Division, CSIR-CDRI. Additionally, we extend our gratitude to Dr. Prem Yadav for critically reading the manuscript. The institutional (CSIR-CDRI) communication number for this article is 11027.

The data supporting this article has been included as part of the Supporting Information.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c06063.

  • Effects of PHL-B on HEK293T cell viability (Figure S1), the precited alternate binding site of PHL-B near the pore helix of the TRPA1 channel (Figure S2), and data displaying the inhibitory effects of A967079 on PHL-B binding residues (Figure S3) (PDF)

Priyanka Yadav performed investigation and formal analysis. Ashutosh Sharma performed investigation and formal analysis. Aditya Singh performed investigation and formal analysis. Mansi Sharma performed investigation and formal analysis. Appu Singh performed investigation and formal analysis. Appu Singh performed investigation, visualization, and writing-original draft. Asif Ali. performed the synthesis, characterization of compounds, and writing-original draft. Aravind Kshatri provided conceptualization, formal analysis, writingoriginal draft, writingreview and editing, funding acquisition, supervision, visualization, project administration, and validation.

This work was funded by the CSIR-CDRI Institutional Fund (In-house Project No: IHP002 to A. Kshatri and A. Ali) and fellowship grants from AcSIR, DBT, and UGC. This work, in part, was supported by funding and support from ICMR and IIT Kanpur (to A. K. Singh). P.Y., A.S., and M.S. acknowledge CSIR, India, for the fellowship.

The authors declare no competing financial interest.

References

  1. Clapham D. E.. TRP Channels as Cellular Sensors. Nature. 2003;426(6966):517–524. doi: 10.1038/nature02196. [DOI] [PubMed] [Google Scholar]
  2. Tominaga M., Caterina M. J.. Thermosensation and Pain. J. Neurobiol. 2004;61(1):3–12. doi: 10.1002/neu.20079. [DOI] [PubMed] [Google Scholar]
  3. Nagata K., Duggan A., Kumar G., García-Añoveros J.. Nociceptor and Hair Cell Transducer Properties of TRPA1, a Channel for Pain and Hearing. J. Neurosci. 2005;25(16):4052–4061. doi: 10.1523/JNEUROSCI.0013-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ramsey I. S., Delling M., Clapham D. E.. An Introduction to TRP Channels. Annu. Rev. Physiol. 2006;68:619–647. doi: 10.1146/annurev.physiol.68.040204.100431. [DOI] [PubMed] [Google Scholar]
  5. Bautista D. M., Pellegrino M., Tsunozaki M.. TRPA1: A Gatekeeper for Inflammation. Annu. Rev. Physiol. 2013;75:181–200. doi: 10.1146/annurev-physiol-030212-183811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kwan K. Y., Allchorne A. J., Vollrath M. A., Christensen A. P., Zhang D.-S., Woolf C. J., Corey D. P.. TRPA1 Contributes to Cold, Mechanical, and Chemical Nociception but Is Not Essential for Hair-Cell Transduction. Neuron. 2006;50(2):277–289. doi: 10.1016/j.neuron.2006.03.042. [DOI] [PubMed] [Google Scholar]
  7. Brierley S. M., Castro J., Harrington A. M., Hughes P. A., Page A. J., Rychkov G. Y., Blackshaw L. A.. TRPA1 Contributes to Specific Mechanically Activated Currents and Sensory Neuron Mechanical Hypersensitivity. J. Physiol. 2011;589(14):3575–3593. doi: 10.1113/jphysiol.2011.206789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Karashima Y., Talavera K., Everaerts W., Janssens A., Kwan K. Y., Vennekens R., Nilius B., Voets T.. TRPA1 Acts as a Cold Sensor in Vitro and in Vivo. Proc. Natl. Acad. Sci. U. S. A. 2009;106(4):1273–1278. doi: 10.1073/pnas.0808487106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kremeyer B., Lopera F., Cox J. J., Momin A., Rugiero F., Marsh S., Woods C. G., Jones N. G., Paterson K. J., Fricker F. R., Villegas A., Acosta N., Pineda-Trujillo N. G., Ramírez J. D., Zea J., Burley M.-W., Bedoya G., Bennett D. L. H., Wood J. N., Ruiz-Linares A.. A Gain-of-Function Mutation in TRPA1 Causes Familial Episodic Pain Syndrome. Neuron. 2010;66(5):671–680. doi: 10.1016/j.neuron.2010.04.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Achanta S., Jordt S.-E.. Transient Receptor Potential Channels in Pulmonary Chemical Injuries and as Countermeasure Targets. Ann. N. Y. Acad. Sci. 2020;1480(1):73–103. doi: 10.1111/nyas.14472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Koivisto A.-P., Belvisi M. G., Gaudet R., Szallasi A.. Advances in TRP Channel Drug Discovery: From Target Validation to Clinical Studies. Nat. Rev. Drug Discovery. 2022;21(1):41–59. doi: 10.1038/s41573-021-00268-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hinman A., Chuang H., Bautista D. M., Julius D.. TRP Channel Activation by Reversible Covalent Modification. Proc. Natl. Acad. Sci. U. S. A. 2006;103(51):19564–19568. doi: 10.1073/pnas.0609598103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Macpherson L. J., Geierstanger B. H., Viswanath V., Bandell M., Eid S. R., Hwang S., Patapoutian A.. The Pungency of Garlic: Activation of TRPA1 and TRPV1 in Response to Allicin. Curr. Biol. 2005;15(10):929–934. doi: 10.1016/j.cub.2005.04.018. [DOI] [PubMed] [Google Scholar]
  14. Tamura Y., Iwasaki Y., Narukawa M., Watanabe T.. Ingestion of Cinnamaldehyde, a TRPA1 Agonist, Reduces Visceral Fats in Mice Fed a High-Fat and High-Sucrose Diet. J. Nutr. Sci. Vitaminol. 2012;58(1):9–13. doi: 10.3177/jnsv.58.9. [DOI] [PubMed] [Google Scholar]
  15. Xiao B., Dubin A. E., Bursulaya B., Viswanath V., Jegla T. J., Patapoutian A.. Identification of Transmembrane Domain 5 as a Critical Molecular Determinant of Menthol Sensitivity in Mammalian TRPA1 Channels. J. Neurosci. 2008;28(39):9640–9651. doi: 10.1523/JNEUROSCI.2772-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Xu H., Blair N. T., Clapham D. E.. Camphor Activates and Strongly Desensitizes the Transient Receptor Potential Vanilloid Subtype 1 Channel in a Vanilloid-Independent Mechanism. J. Neurosci. 2005;25(39):8924–8937. doi: 10.1523/JNEUROSCI.2574-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Takaishi M., Fujita F., Uchida K., Yamamoto S., Sawada Shimizu M., Hatai Uotsu C., Shimizu M., Tominaga M.. 1,8-Cineole, a TRPM8 Agonist, Is a Novel Natural Antagonist of Human TRPA1. Mol. Pain. 2012;8:1744–8069. doi: 10.1186/1744-8069-8-86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nalli Y., Khajuria V., Gupta S., Arora P., Riyaz-Ul-Hassan S., Ahmed Z., Ali A.. Four New Carbazole Alkaloids from Murraya Koenigii That Display Anti-Inflammatory and Anti-Microbial Activities. Org. Biomol. Chem. 2016;14(12):3322–3332. doi: 10.1039/C6OB00267F. [DOI] [PubMed] [Google Scholar]
  19. Nalli Y., Thakur V., Mohmmed A., Gupta V. K., Ali A.. POCl3-Mediated Cyclization of (+)-S-Mahanimbine Led to the Divergent Synthesis of Natural Product Derivatives with Antiplasmodial Activity. New J. Chem. 2017;41(12):4923–4930. doi: 10.1039/C7NJ00487G. [DOI] [Google Scholar]
  20. Thapa S., Nalli Y., Singh A., Singh S. K., Ali A.. Neuroprotective Effects of Cannabispirenone A against NMDA-Induced Excitotoxicity in Differentiated N2a Cells. Oxid. Med. Cell. Longev. 2024;2024:3530499. doi: 10.1155/2024/3530499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nalli Y., Bashir Mir K., Amin T., Gannedi V., Jameel E., Goswami A., Ali A.. Divergent Synthesis of Fractionated Cannabis Sativa Extract Led to Multiple Cannabinoids C-&O-Glycosides with Anti-Proliferative/Anti-Metastatic Properties. Bioorganic Chem. 2024;143:107030. doi: 10.1016/j.bioorg.2023.107030. [DOI] [PubMed] [Google Scholar]
  22. Nalli Y., Mirza D. N., Wani Z. A., Wadhwa B., Mallik F. A., Raina C., Chaubey A., Riyaz-Ul-Hassan S., Ali A. P. A.. New Antimicrobial and Cytotoxic Metabolites from an Endophytic Fungus, Phialophora Mustea. RSC Adv. 2015;5(115):95307–95312. doi: 10.1039/C5RA18121F. [DOI] [Google Scholar]
  23. Bevan S., Hothi S., Hughes G., James I. F., Rang H. P., Shah K., Walpole C. S., Yeats J. C.. Capsazepine: A Competitive Antagonist of the Sensory Neurone Excitant Capsaicin. Br. J. Pharmacol. 1992;107(2):544–552. doi: 10.1111/j.1476-5381.1992.tb12781.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sui Y., Li S., Zhao Y., Liu Q., Qiao Y., Feng L., Li S.. Identification of a Natural Compound, Sesamin, as a Novel TRPM8 Antagonist with Inhibitory Effects on Prostate Adenocarcinoma. Fitoterapia. 2020;145:104631. doi: 10.1016/j.fitote.2020.104631. [DOI] [PubMed] [Google Scholar]
  25. Everaerts W., Zhen X., Ghosh D., Vriens J., Gevaert T., Gilbert J. P., Hayward N. J., McNamara C. R., Xue F., Moran M. M., Strassmaier T., Uykal E., Owsianik G., Vennekens R., De Ridder D., Nilius B., Fanger C. M., Voets T.. Inhibition of the Cation Channel TRPV4 Improves Bladder Function in Mice and Rats with Cyclophosphamide-Induced Cystitis. Proc. Natl. Acad. Sci. U. S. A. 2010;107(44):19084–19089. doi: 10.1073/pnas.1005333107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Grosdidier A., Zoete V., Michielin O.. SwissDock, a Protein-Small Molecule Docking Web Service Based on EADock DSS. Nucleic Acids Res. 2011;39:W270–W277. doi: 10.1093/nar/gkr366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pettersen E. F., Goddard T. D., Huang C. C., Couch G. S., Greenblatt D. M., Meng E. C., Ferrin T. E.. UCSF Chimera–a Visualization System for Exploratory Research and Analysis. J. Comput. Chem. 2004;25(13):1605–1612. doi: 10.1002/jcc.20084. [DOI] [PubMed] [Google Scholar]
  28. Fischer M. J. M., Balasuriya D., Jeggle P., Goetze T. A., McNaughton P. A., Reeh P. W., Edwardson J. M.. Direct Evidence for Functional TRPV1/TRPA1 Heteromers. Pflugers Arch. 2014;466(12):2229–2241. doi: 10.1007/s00424-014-1497-z. [DOI] [PubMed] [Google Scholar]
  29. Kanju P., Chen Y., Lee W., Yeo M., Lee S. H., Romac J., Shahid R., Fan P., Gooden D. M., Simon S. A., Spasojevic I., Mook R. A., Liddle R. A., Guilak F., Liedtke W. B.. Small Molecule Dual-Inhibitors of TRPV4 and TRPA1 for Attenuation of Inflammation and Pain. Sci. Rep. 2016;6(1):26894. doi: 10.1038/srep26894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Staruschenko A., Jeske N. A., Akopian A. N.. Contribution of TRPV1-TRPA1 Interaction to the Single Channel Properties of the TRPA1 Channel. J. Biol. Chem. 2010;285(20):15167–15177. doi: 10.1074/jbc.M110.106153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Izquierdo C., Martín-Martínez M., Gómez-Monterrey I., González-Muñiz R.. TRPM8 Channels: Advances in Structural Studies and Pharmacological Modulation. Int. J. Mol. Sci. 2021;22(16):8502. doi: 10.3390/ijms22168502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Gouin O., L’Herondelle K., Lebonvallet N., Le gall-Ianotto C., Sakka M., Buhé V., Plée-Gautier E., Carré J.-L., Lefeuvre L., Misery L., Le Garrec R.. TRPV1 and TRPA1 in Cutaneous Neurogenic and Chronic Inflammation: Pro-Inflammatory Response Induced by Their Activation and Their Sensitization. Protein Cell. 2017;8(9):644–661. doi: 10.1007/s13238-017-0395-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Xing R., Wang P., Zhao L., Xu B., Zhang N., Li X.. Mechanism of TRPA1 and TRPV4 Participating in Mechanical Hyperalgesia of Rat Experimental Knee Osteoarthritis. Arch. Rheumatol. 2017;32(2):96–104. doi: 10.5606/ArchRheumatol.2017.6061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Souza Monteiro de Araujo D., Nassini R., Geppetti P., De Logu F.. TRPA1 as a Therapeutic Target for Nociceptive Pain. Expert Opin. Ther. Targets. 2020;24(10):997–1008. doi: 10.1080/14728222.2020.1815191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Calixto J. B., Kassuya C. A. L., André E., Ferreira J.. Contribution of Natural Products to the Discovery of the Transient Receptor Potential (TRP) Channels Family and Their Functions. Pharmacol. Ther. 2005;106(2):179–208. doi: 10.1016/j.pharmthera.2004.11.008. [DOI] [PubMed] [Google Scholar]
  36. Wang S., Zhai C., Zhang Y., Yu Y., Zhang Y., Ma L., Li S., Qiao Y.. Cardamonin, a Novel Antagonist of hTRPA1 Cation Channel, Reveals Therapeutic Mechanism of Pathological Pain. Molecules. 2016;21(9):1145. doi: 10.3390/molecules21091145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zhai C., Liu Q., Zhang Y., Wang S., Zhang Y., Li S., Qiao Y.. Identification of Natural Compound Carnosol as a Novel TRPA1 Receptor Agonist. Molecules. 2014;19(11):18733–18746. doi: 10.3390/molecules191118733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Jha P., Kaur T., Chhabra I., Panja A., Paul S., Kumar V., Malik T.. Endophytic Fungi: Hidden Treasure Chest of Antimicrobial Metabolites Interrelationship of Endophytes and Metabolites. Front. Microbiol. 2023;14:1227830. doi: 10.3389/fmicb.2023.1227830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Takahashi N., Chen H.-Y., Harris I. S., Stover D. G., Selfors L. M., Bronson R. T., Deraedt T., Cichowski K., Welm A. L., Mori Y.. et al. Cancer Cells Co-Opt the Neuronal Redox-Sensing Channel TRPA1 to Promote Oxidative-Stress Tolerance. Cancer Cell. 2018;33(6):985–1003.e7. doi: 10.1016/j.ccell.2018.05.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Unni S., Kommu P., Aouti S., Nalli Y., Bharath M. M. S., Ali A., Padmanabhan B.. Structural Insights into the Modulation Of SOD1 Aggregation By a Fungal Metabolite Phialomustin-B: Therapeutic Potential in ALS. PLoS One. 2024;19(3):e0298196. doi: 10.1371/journal.pone.0298196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Moore C., Gupta R., Jordt S.-E., Chen Y., Liedtke W. B.. Regulation of Pain and Itch by TRP Channels. Neurosci. Bull. 2018;34(1):120–142. doi: 10.1007/s12264-017-0200-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zheng J.. Molecular Mechanism of TRP Channels. Compr. Physiol. 2013;3(1):221–242. doi: 10.1002/cphy.c120001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pumroy R. A., Fluck E. C., Ahmed T., Moiseenkova-Bell V. Y.. Structural Insights into the Gating Mechanisms of TRPV Channels. Cell Calcium. 2020;87:102168. doi: 10.1016/j.ceca.2020.102168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Weng H.-J., Patel K. N., Jeske N. A., Bierbower S. M., Zou W., Tiwari V., Zheng Q., Tang Z., Mo G. C. H., Wang Y., Geng Y., Zhang J., Guan Y., Akopian A. N., Dong X.. Tmem100 Is a Regulator of TRPA1-TRPV1 Complex and Contributes to Persistent Pain. Neuron. 2015;85(4):833–846. doi: 10.1016/j.neuron.2014.12.065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Koldsø H., Jensen M. Ø., Jogini V., Shaw D. E.. Functional Dynamics and Allosteric Modulation of TRPA1. Structure. 2023;31(12):1556–1566.e3. doi: 10.1016/j.str.2023.08.018. [DOI] [PubMed] [Google Scholar]
  46. Benedikt J., Samad A., Ettrich R., Teisinger J., Vlachova V.. Essential Role for the Putative S6 Inner Pore Region in the Activation Gating of the Human TRPA1 Channel. Biochim. Biophys. Acta, Mol. Cell Res. 2009;1793(7):1279–1288. doi: 10.1016/j.bbamcr.2009.04.014. [DOI] [PubMed] [Google Scholar]
  47. Chen J., Joshi S. K., DiDomenico S., Perner R. J., Mikusa J. P., Gauvin D. M., Segreti J. A., Han P., Zhang X.-F., Niforatos W., Bianchi B. R., Baker S. J., Zhong C., Simler G. H., McDonald H. A., Schmidt R. G., McGaraughty S. P., Chu K. L., Faltynek C. R., Kort M. E., Reilly R. M., Kym P. R.. Selective Blockade of TRPA1 Channel Attenuates Pathological Pain without Altering Noxious Cold Sensation or Body Temperature Regulation. Pain. 2011;152(5):1165–1172. doi: 10.1016/j.pain.2011.01.049. [DOI] [PubMed] [Google Scholar]
  48. Paulsen C. E., Armache J.-P., Gao Y., Cheng Y., Julius D.. Structure of the TRPA1 Ion Channel Suggests Regulatory Mechanisms. Nature. 2015;520(7548):511–517. doi: 10.1038/nature14367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Chernov-Rogan T., Gianti E., Liu C., Villemure E., Cridland A. P., Hu X., Ballini E., Lange W., Deisemann H., Li T., Ward S. I., Hackos D. H., Magnuson S., Safina B., Klein M. L., Volgraf M., Carnevale V., Chen J.. TRPA1Modulation by Piperidine Carboxamides Suggests an Evolutionarily Conserved Binding Site and Gating Mechanism. Proc. Natl. Acad. Sci. U. S. A. 2019;116(51):26008–26019. doi: 10.1073/pnas.1913929116. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

ao5c06063_si_001.pdf (467.2KB, pdf)

Data Availability Statement

The data supporting this article has been included as part of the Supporting Information.

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