Inhibition of transient receptor potential vanilloid 1 (TRPV1) by a novel selective antagonist for anti-nociceptive effect on inflammatory pain without hyperthermia

Jialiang Guan; Chenru Zhao; Qiqi Zhou; Chao Zhu

doi:10.1371/journal.pone.0345127

Abstract

Nowadays, chronic pain remains a major clinical challenge because of the unsatisfactory efficacy and nonnegligible side effects of current treatments. Pharmacological antagonists of the transient receptor potential vanilloid-1 (TRPV1) channel for chronic pain relief have been confirmed in numerous preclinical studies. However, no TRPV1 antagonist has been approved in clinical, indicating an urgent need to develop effective TRPV1 antagonists with analgesic properties. In this study, we reported a TRPV1 antagonist 1-(1H-indazol-4-yl)-3-((2-(4-methylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)methyl)urea named IMTU. According to the whole-cell patch clamp recording assay, we identified that IMTU was a potent and selective TRPV1 antagonist with an IC₅₀ value of 128.34 ± 9.49 nM and exciting no inhibition of cardiotoxicity-related channels. Further single-channel recording assay revealed that IMTU (300 nM) reduced the channel open probability from 80.8 ± 2.1% to 14.9 ± 2.8% with the presence of capsaicin (100 nM). In addition, does-dependent administration of oral IMTU alleviated nociceptive reactions on mouse models of formalin and inflammatory pain induced by complete Freund’s adjuvant (CFA) without hyperthermia. Finally, studies on molecular docking combined with site-directed mutagenesis suggested that residue Thr550 was critical for the TRPV1 antagonist by IMTU. Taken together, we identified a novel and selective TRPV1 antagonist IMTU exhibiting analgesic properties in mice, which provide a useful lead for the development of analgesia in the future.

Citation: Guan J, Zhao C, Zhou Q, Zhu C (2026) Inhibition of transient receptor potential vanilloid 1 (TRPV1) by a novel selective antagonist for anti-nociceptive effect on inflammatory pain without hyperthermia. PLoS One 21(3): e0345127. https://doi.org/10.1371/journal.pone.0345127

Editor: Alexander G. Obukhov, Indiana University School of Medicine, UNITED STATES OF AMERICA

Received: June 16, 2025; Accepted: March 2, 2026; Published: March 25, 2026

Copyright: © 2026 Guan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: The project is supported by Shandong Provincial Natural Science Foundation of China (ZR2021QH192), Medical Health Research Project of Zibo (20230204005), the QLMU research fund and the Affiliated Hospital of Qingdao University Clinical Medical Research Program (QDFYQN2023112). And there was no additional external funding received for this study. The funders mainly provide financial assistance and experimental guidance for this research.

Competing interests: The authors have declared that no competing interests exist.

1. Introduction

The pain sensation is a physiological response that protects our bodies against harmful conditions, such as tissue damage, chemical or physical stimulus, and inflammatory or neuropathic diseases [1]. Nowadays, chronic pain remains the most common reason for patients to seek medical attention worldwide and a major clinical challenge because of the nonnegligible side effects and unsatisfactory efficacy of currently clinical treatments [2–4].

The transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel abundantly expressed in peripheral sensory neurons, especially in primary afferent neurons that could sense noxious stimuli, including low extracellular pH, heat, and inflammatory or painful mediators [5–8]. Accumulated genetic and pharmacological studies have validated TRPV1 as an analgesic target in several mouse models of chronic pain, such as cancer, postoperative, neuropathic, and inflammatory pain [9–13]. Therefore, developing small molecules of selective TRPV1 receptor antagonist and desensitizer presents a promising strategy for pain management.

In recent years, potent and selective TRPV1 antagonists with well analgesic properties are identified in preclinical studies, such as ABT-116 (Abbott) and JNJ-39729209 (Janssen) [14,15]. However, numerous preclinical studies and clinical trials revealed that those TRPV1 antagonists could result in hyperthermia and elevated noxious heat pain threshold [16–18]. The adverse side effect of hyperthermia directly leaded to the termination of several clinical trials [19,20], and it was why there were no TRPV1 antagonists have been approved in clinical [12]. Therefore, it is an urgent clinical need to develop the second generation of TRPV1 antagonists for pain management.

Further insights into the Cryo-electron microscopy (Cryo-EM) structures of the TRPV1 in complex with agonists or antagonists have revealed the transformative mechanism of TRPV1, among which, the residue Thr550 is critical for TRPV1 activation by resiniferatoxin (RTX) and capsaicin, and the residue Glu570 is critical for TRPV1 inhibition by capsazepine [21,22]. In recent works, we have discovered a series of N-indazole-4-aryl piperazine carboxamide analogues as new TRPV1 ligands based on the Cryo-EM structures of the TRPV1 in complex with ligands [23,24]. Among these analogues, 4-isomers were identified as moderate antagonists. However, their weak antagonistic activity on TRPV1 should be improved. Therefore, it is indeed to discover a new potent TRPV1 antagonist based on the optimization of 4-isomers of N-indazole-4-aryl piperazine carboxamide analogues.

In this study, we indentified 1-(1H-indazol-4-yl)-3-((2-(4-methylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-3yl)methyl)urea (IMTU) as a TRPV1 antagonist by hybridizing hydrophobic region of a known TRPV1 antagonist and indazole of our 4-isomers (Fig 1A). Using fluorescence calcium assay and electrophysiological recordings, we confirmed IMTU as a potent and selective TRPV1 antagonist without inhibition on cardiotoxicity-related channels. Importantly, oral administration of IMTU presented a dose-dependently analgesic effect on mouse models of inflammatory pain without hyperthermia. We also performed molecular docking and site-directed mutagenesis to predict the critical residues for the TRPV1 inhibition by IMTU.

Download:

Fig 1. Inhibition of TRPV1 currents by IMTU in TRPV1-expressed HEK293 cells.

(A) The chemical structure of IMTU. (B) Inhibition of capsaicin-induced intracellular Ca²⁺ increase in TRPV1-expressed HEK-293 cells by pretreatment with different concentrations of IMTU and TRPV1 antagonist BCTC in FlexStation3 calcium fluorescence assay. RFU: Relative Fluorescence Unit; Cap: Capsaicin; HBSS: Hank’s Balanced Salt Solution. (C) Representative current traces showed that TRPV1 currents activated by 100 nM capsaicin were significantly inhibited by perfusion of 300 nM IMTU. The TRPV1 currents were recorded in whole-cell configuration and measured at ± 80 mV. (D) The inhibition rate of whole-cell TRPV1 currents evoked by capsaicin (100 nM) in the presence of TRPV1 antagonist IMTU (300 nM) or BCTC (1 μM). (E) Representative whole-cell current traces showed the responses to 100 nM capsaicin without or with increasing concentrations of IMTU from 3 nM to 1 μM. (F) The dose-response curve for inhibition of TRPV1 outward currents by IMTU was fitted to a Hill equation, with an IC₅₀ of 128.34 ± 9.49 nM (n = 6). The data was represented as means ± SEM..

https://doi.org/10.1371/journal.pone.0345127.g001

2. Materials and methods

2.1. Reagents and Chemicals

The synthetic routes of 1-(1H-indazol-4-yl)-3-((2-(4-methylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)methyl)urea (IMTU) were shown as S1 Scheme in Supplementary Materials. All synthetic reagents were purchased from Bide, Energy Chemical, Macklin and Sinopharm Chemical Reagent Company. Capsaicin, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC), 2-aminoethoxydiphenyl borate (2-APB), allyllsothiocyanate (AITC), Complete Freund’s Adjuvant (CFA) and A-967079 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Compounds were dissolved in dimethyl sulfone (DMSO) for stock solutions before further dilutions with cell bath solutions.

2.2. Chemistry

2.2.1. Synthesis of Intermediates 2 (2-(4-methylpiperidin-1-yl)-6-(trifluoromethyl)nicotinonitrile).

A mixture of compound 1 (1 mmol) and 4-methylpiperidine (10 mL) were stirred at 50 °C for 1 h. The mixture was evaporated under reduced pressure, and the residue was diluted with water and extracted three times with ethyl acetate. The organic layers were combined layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue was purified by column chromatography to obtain target compound 2. Yield, 90%; yellow oil.

2.2.2. Synthesis of Intermediates 3 ((2-(4-methylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)methanamine).

To a solution of 2 (1 mmol) in methanol (20 mL) was added NiCl₂ (2 mmol). Under argon atmosphere, the NaBH₄ (4 mmol) was slowly added to the mixture. The reaction was completed in 1 h at room temperature. The reaction mixture was then filtered through diatomaceous earth. After removing the solvent in vacuo, the residue was purified by column chromatography to obtain compound 3. Yield, 87%; yellow oil.

2.2.3. Synthesis of Intermediates 5 (1H-indazol-4-amine).

To a solution of 4 (1 mmol) in MeOH (20 mL) was added palladium on carbon (5% w/w). The mixture was stirred vigorously under a hydrogen atmosphere at room temperature overnight. The reaction mixture was then filtered through diatomaceous earth, and the filtrate was concentrated under a vacuum. The residue was purified by column chromatography to obtain compound 5. Yield, 94%; white solid.

2.2.4. Synthesis of Intermediates 6 (phenyl (1H-indazol-4-yl)carbamate).

To a solution of 5 (1 mmol) in ultra-dry THF (10 mL) was added pyridine (1.4 mmol). Then phenyl carbonochloridate (1 mmol) was slowly added to the mixture at 0°C, and then stirred at room temperature for 8 h. After the reaction was completed, the residue was diluted with water and extracted three times with ethyl acetate. The combined organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue was purified by column chromatography to obtain target compound 6. Yield, 50%; white solid.

2.2.5. Synthesis of Final Compound IMTU (1-(1H-indazol-4-yl)-3-((2-(4-methylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)methyl)urea).

To a solution of 3 (1.05 mmol) and 6 (1 mmol) in dimethylformamide (DMF) (5 mL) was added triethylamine (Et₃N) (2 mmol). Then the reaction was stirred at 50 °C and monitored by thin-layer chromatography (TLC). When compound 6 was consumed completely, the residue was diluted with water and extracted three times with ethyl acetate. The combined organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue was purified by column chromatography to obtain target compound IMTU. Yield, 88%; white solid. ¹H NMR (400 MHz, DMSO- d₆, ppm) δ 13.01 (s, 1H), 8.94 (d, J = 7.7 Hz, 1H), 8.12 (d, J = 7.5 Hz, 1H), 7.85 (t, J = 7.5 Hz, 1H), 7.59 (t, J = 7.7 Hz, 1H), 7.46 (t, J = 7.9 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 7.07 (t, J = 7.8 Hz, 1H), 6.94 (s, 1H), 4.39 (s, 2H), 3.44 (d, J = 11.1 Hz, 2H), 2.79 (t, J = 10.8 Hz, 2H), 1.72 (d, J = 10.6 Hz, 2H), 1.56 (s, 1H), 1.37–1.26 (m, 2H), 0.97 (t, J = 6.8 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆, ppm) δ 160.48, 155.01, 142.39 (q, J = 33.22 Hz), 140.69, 137.24, 132.56, 130.86, 130.81, 126.77, 121.56 (q, J = 273.31 Hz), 114.79, 113.69, 106.95, 102.99, 49.89, 33.70, 30.12, 21.68. HR-MS (ESI+), calcd for C₂₁H₂₃F₃N₆O, [M + H]+ m/z: 433.1958, found: 433.1954. HPLC purity: 96%.

2.3. Animals

8-week-old male C57BL/6J mice (21 ± 2 g) were ordered from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in a 12/12 h light-dark cyclic room with free access to food and water. Besides, the room temperature was controlled at 23 ± 1 °C, and the relative humidity was maintained between 40–60%. Mice were housed in the room for at least one week for adaptation before behavioral assessment. All the experimental procedures were approved by the Animal Ethics Committee of Qilu Medical University (QLMU, Protocol number: YXLL2021D001) and complied with the ethical guidelines of the International Association for the Study of Pain. Animals were sacrificed as a humane endpoint to minimize the risk of moribundity. Primary euthanasia was done with CO₂ and with cervical dislocation as the secondary euthanasia method.

2.4. Cell culture and transfection of cDNAs

Human embryonic kidney 293 (HEK293) cells were incubated in 5% CO₂/95% O₂ at 37 °C and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) with 10% Fetal bovine serum (FBS, PAN-SERATECH), and 100 U/mL streptomycin and penicillin (Gibco). HEK293 cells were cultured onto glass coverslips 24 h prior to transfection. Cells were transiently transfected with 2 μg cDNAs of mouse Trpv1, Trpv2 and Trpv3 using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Ca²⁺ imaging and electrophysiological recordings were carried out 24 h after transfection. HEK293 cells expressing green fluorescent proteins (GFP) fluorescence were chosen for patch-clamp recordings. Chinese hamster ovary (CHO) cells stably expressing human ether-a-go-go-related gene (hERG) or Nav1.5 channels were maintained in the F12 medium supplemented with 600 μg/mL G418, 10% FBS, and 100 U/mL penicillin/streptomycin.

2.5. Intracellular calcium assay using FlexStation3

The real-time intracellular calcium level ([Ca²⁺]) was detected by incubating with Ca²⁺-sensitive fluorescent dyes using FLIPR calcium 5 assay kit (Molecular Devices, USA) in a FlexStation3 Microplate Reader (Molecular Devices, USA) as previously described [25,26]. Briefly, HEK293 cells transiently expressing mouse TRPV1 channels were seeded in a 96-well plate with a density of about 30,000 cells per well 18 h before intracellular calcium assay. Cells were washed with Hanks’ balanced salt solution (HBSS) containing (in mM): 137 NaCl, 5.4 KCl, 4 NaHCO₃, 1.3 CaCl₂, 0.4 KH₂PO4, 0.1 Na₂HPO4, 5.5 glucose, 20 4-(2-Hydroxyethyl)-1-piperazinethanesulfonic acid (HEPES), with pH adjusted to 7.4 by NaOH, and then incubated with fluorescent dyes from the FLIPR calcium 5 assay kit diluted in HBSS containing 2.0 mM probenecid at 37 °C for 1 h. After incubation, cells were washed twice with HBSS before reading the fluorescent intensity in the FlexStation3 Microplate Reader. Different concentrations of TRPV1 antagonists were added into cells at 17 s, and the TRPV1 agonist capsaicin was added at 100 s. The fluorescent dyes were excited at 494 nm, and the fluorescent intensity was measured at 525 nm at intervals of 1.6 s for a total of 180 s.

2.6. Electrophysiology

Whole-cell patch-clamp recordings were performed at room temperature (20–23°C) using a HEKA EPC10 amplifier and PatchMaster Software (HEKA Electronics, Germany). Borosilicate electrodes with a resistance of 3–6 MΩ were pulled using a laser puller (Model P-2000, Sutter Instrument, USA). For TRPV1 current recordings, the standard pipette solution contained (in mM): 140 NaCl, 5 EthyleneGlycol-bis-(2-Aminoethylether)-N,N,N’,N’-Tetraacetic acid (EGTA), and 10 HEPES with pH adjusted to 7.4 by CsOH, and the standard bath solution contained (in mM): 140 NaCl, 5 KCl, 2 MgCl₂, 10 glucose, and 10 HEPES, with pH adjusted to 7.4 by NaOH. The membrane potential of TRPV1-expressed HEK293 cells was held at 0 mV, and the TRPV1 current traces were elicited by a 400-ms voltage ramp from −100 to +100 mV at an interval of 1 s. TRPV1 activators or antagonists were perfused by using the gravity-driven RSC‐200 perfusion system (Bio‐Logic Science Instruments, France) for the quick change of the bath solution. Membrane currents were low pass filtered at 2 kHz and sampled at 10 kHz. The current amplitude was analyzed at ±80 mV. The dose-response curve was fitted to a Hill logistic equation. The electrophysiological data were analyzed and graphed using Origin 8.5 (OriginLab) and Igor Pro (Wave-metrics).

For recording of hERG currents, cell membrane potential was held at −80 mV. The current traces were elicited by a 3 s depolarizing potential of 40 mV following back to −40 mV for 2 s with an interval of 30 s. The standard bath solution contained (in mM): 137 NaCl, 5.0 KCl, 1.8 CaCl₂, 1.2 Mg Cl₂, 10 glucose, and 10 HEPES, with pH adjusted to 7.4 by NaOH. The standard pipette solution contained (in mM): 75 KF, 65 KCl, 2 MgCl₂, 5 EGTA, and 10 HEPES, with pH adjusted to 7.4 by KOH. Tail currents were analyzed at −40 mV.

For recording of Nav1.5 currents, cell membrane potential was held at −130 mV. Current traces were elicited by repolarized potential at 0 mV for 15 ms following back to a hyperpolarized potential of −120 mV with an interval of 1 s. The standard bath solution contained (in mM): 140 NaCl, 3 KCl, 1 MgCl₂, 1.8 CaCl₂, 10 glucose, and 10 HEPES, with pH adjusted to 7.4 by NaOH. The standard pipette solution contained (in mM): 140 CsF, 2.5 MgCl₂, 5 EGTA, and 10 HEPES (pH 7.3, adjusted with CsOH).

2.7. In vivo Pharmacokinetics

Six male C57BL/6J mice (20–25 g) were randomly divided into two groups (n = 3). Mice were administrated with IMTU (i.v., 10 mg/kg and p.o., 30 mg/kg). Blood samples of mice were collected from cheek at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h after drug administration. The samples (10 μL) were transferred into a 96-well plate, and put 200 μL of internal standard methanol: acetonitrile = 1:1 (100 ng/mL) solution was added. After vortex for 5 minutes, the sample was centrifuged at 4000 rpm under 4°C for 10 min. 100 μL of the supernatant was mixed with 100 μL water and then quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS, Triple Quad 5500+).

2.8. Formalin–induced paw licking behavior

Mice were administered (i.p.) 100 μL saline for the control group, IMTU (5, 10, 30 mg/kg, dissolved in saline) for the experimental group, or ibuprofen (2 mg/kg, dissolved in saline) for the positive control group 1 h before intraplantar injection of 20 µL 5% formalin (formalin in vehicle saline) at the right hind paw. Then mice were placed individually in a transparent observation chamber, where the paw licking behaviors were videotaped. The total time each mouse spent licking the right hind paw was quantified during phase I (0–5 min post-injection) and phase II (15–45 min post-injection).

2.9. Establishment of Complete Freund’s Adjuvant (CFA)–induced inflammatory pain model

Mice were anesthetized with isoflurane before establishing the inflammatory pain model by intraplantar injection of 20 µL complete Freund’s adjuvant (CFA) (Sigma, St. Louis, MO, USA) into the right hind paw. Meanwhile, mice in the vehicle control group were injected with 20 µL saline. 24 h after CFA injection, IMTU (5, 10, 30 mg/kg) or ibuprofen (2 mg/kg) was administered (i.p.), while mice in the vehicle control and model groups were administered the same volume of saline. The paw withdrawal mechanical threshold (PWMT) was measured using a von Frey monofilament by an up-and-down method and the paw withdrawal thermal latency (PWTL) was measured using a plantar analgesia meter as previously described [27], and the time points for measurement were at 0, 1, 2 and 3 h after administration of drugs.

2.10. Determination of PWMT

In brief, mice were placed individually in transparent observation chambers with a metal mesh for 1 h. A set of von Frey fibers with a range of forces (0.008, 0.02, 0.04, 0.07, 0.16, 0.60, 1.0, 1.4, and 2 g) was applied to the plantar of right hind paws until appearing a slight S-shape for 5–6 s. During this period, if a rapid paw withdrawal response was observed, the corresponding force was considered positive; otherwise, it was considered negative. Once crossover was determined, the measurements were repeated four more times. The PWMT was calculated by a formula: PWMT (g) = 10^[Xf+Kδ], where the Xf is the log value of the last von Frey fiber force, the K is the value retrieved from the standardized table based on the up-and-down pattern.

2.11. Determination of PWTL

In brief, mice were placed individually on a transparent plexiglass for about 30 min to adapt the new atmosphere. The PWMT was measured by a fully automatic plantar analgesia meter (BME-410C). The right hind paws of mice were lighted and the withdrawal time was recorded as one PWTL. The PWTL was measured for five times with an interval of 5 min to calculate an average value.

2.12. Body temperature measurement

The rectal temperature of mice was measured before administration using a rectal temperature probe as a basic point. Then the rectal temperature was measured at 0, 0.5, 1, 2 and 3 h after the oral administration of IMTU (20 and 50 mg/kg) or BCTC (20 mg/kg).

2.13. Molecular docking

Molecular docking was performed to model the interaction between IMTU and TRPV1 protein using GOLD Suite 5.1 (Cambridge Crystallographic Data Centre, Cambridge, U.K.). The crystal structure of rat TRPV1-capsazepine complex (PDB ID:5IS0) was obtained from the PDB bank (http://www.rcsb.org/). The structure of IMTU was optimized in SYBYL (Tripos, St Louis, MO, USA) using the Tripos force field (Gasteiger–Hückel charges, distance-dependent dielectric constant = 4.0, nonbonded interaction cutoff = 8 Å and termination criterion = energy gradient < 0.05 kcal/ (mol × Å) for 10,000 iterations). The protein was processed by removing the existing ligand, water, and ions, and then the optimized structure of IMTU was docked into a previously reported pocket consisting of S4 and S4–S5 linker [28]. The top 10 docking results were exported and further analyzed. The ligand-TRPV1 complexes were edited and visualized using UCSF Chimera 1.13.1 software.

2.14. Statistical analysis

Data were acquired from at least three independent experiments and expressed as the mean ± standard error of mean (SEM). Statistical significance was determined using a one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test using GraphPad Prism 7.0 software (GraphPad Software Ltd., San Diego, CA, USA). P < 0.05 was considered as a statistically significant difference.

3. Results

3.1. Identification of IMTU as a potent and selective TRPV1 antagonist

We first tested the inhibitory activity of IMTU on TRPV1 channels overexpressed in HEK293 cells by FlexStation3 calcium fluorescence assay. Application of capsaicin (100 nM) alone robustly increased the intracellular calcium signal in TRPV1 transfected HEK293 cells in comparison with vehicle control (Fig 1B). In contrast, pretreatment with IMTU or known antagonist BCTC significantly inhibited the capsaicin-induced calcium influx (Fig 1B). This result shows that IMTU can significantly inhibit TRPV1-mediated Ca²⁺ influx.

Next, the whole-cell patch-clamp recordings were carried out to determine the inhibitory efficiency of IMTU on TRPV1 channels overexpressed in HEK293 cells. Perfusion of IMTU (300 nM) resulted in an almost complete inhibition on TRPV1 currents evoked by capsaicin (100 nM) (Fig 1C). In addition, the dose-dependent inhibition of TRPV1 currents by IMTU was evaluated. Perfusion of increasing concentrations of IMTU (3–1000 nM) resulted in a dose-dependent inhibition of TRPV1 currents, with an IC₅₀ value of 128.34 ± 9.49 nM and a Hill coefficient of 1.15 ± 0.07 (Fig 1E and F). These results demonstrate that IMTU is a potent TRPV1 channel antagonist.

We further evaluated the selectivity of IMTU on other TRP subfamily channels using the whole-cell patch clamp recording assay. As shown in Fig 2A, in comparison with TRPA1 antagonist A-967079, IMTU (1 μM) had an extremely weak inhibitory effect on TRPA1 current evoked by 300 μM AITC. In contrast, IMTU (1 μM) had no effect on TRPV2 or TRPV3 current evoked by non-selective agonist 2-APB (Fig 2B and 2C). These results indicate that IMTU is a selective TRPV1 antagonist.

Download:

Fig 2. Selectivity of IMTU on TRPA1, TRPV2 and TRPV3 using the whole-cell patch clamp assay.

(A) TRPA1 currents in response to AITC (300 μM), co-application of IMTU (1 μM) displaying weak inhibition on TRPA1 and TRPA1 antagonist A-967079 displaying obvious inhibition on TRPA1. (B) TRPV2 currents in response to 2-APB (2 mM), co-application of IMTU (1 μM) displaying no inhibition on TRPV2 current. (C) TRPV3 currents in response to 2-APB (200 μM), co-application of IMTU (1 μM) displaying no inhibition on TRPV3 current.

https://doi.org/10.1371/journal.pone.0345127.g002

3.2. Direct targeting of TRPV1 single channel by IMTU

To further confirm the targeting inhibition of IMTU on TRPV1 channels, we applied the single-channel recordings of TRPV1 currents in TRPV1 overexpressed HEK293 cells using an inside-out patch configuration. As shown in Fig 3A, 3B and 3D, application of TRPV1 agonist capsaicin (100 nM) obviously increased the channel open probability from 0.4 ± 0.2% (vehicle control) to 80.8 ± 2.1%. The addition of antagonist IMTU (300 nM) in the presence of capsaicin (100 nM) caused a significant reduction of the channel open probability to 14.9 ± 2.8% (Fig 3C and 3D). In addition, perfusion of neither capsaicin nor IMTU altered the single channel conductance of TRPV1 (Fig 3E). These results further verify the direct inhibition of single TRPV1 channel by antagonist IMTU.

Download:

Fig 3. Reduction of single TRPV1 channel open probability by antagonist IMTU.

(A)-(C) Left panels, representative traces recorded from an inside-out patch in the condition of (A) vehicle, (B) after addition of capsaicin (100 nM) and (C) co-perfusion of IMTU (300 nM) and capsaicin (100 nM). Right panels, all-points histograms of single-channel current recording data as shown in left panels and their histograms were fitted to a Gauss functions. (D) Summary for calculated single TRPV1 channel mean P_Open values in the presence of antagonist IMTU and agonist capsaicin (n = 4, ^****P < 0.0001, compared with vehicle control). (E) Summary of TRPV1 single channel conductance after exposure to different regulators (n = 4). Data are shown as the mean ± SEM by one-way ANOVA followed by Bonferroni post hoc multiple comparisons test.

https://doi.org/10.1371/journal.pone.0345127.g003

3.3. IMTU does not affect cardiotoxicity-related channels

Nav1.5 and hERG antagonist are involved in cardiac toxicity and may lead to drug development termination or even market withdrawal [29]. Accordingly, we further tested the effect of IMTU on hERG and Nav1.5 channels by whole-cell patch-clamp recordings. Perfusion of 10 μM IMTU didn’t cause obvious inhibition on hERG and Nav1.5 currents (Fig 4A and B), suggesting that IMTU is a mild and safe TRPV1 antagonist with low cardiotoxicity.

Download:

Fig 4. No altering on hERG and Nav1.5 currents by IMTU using whole-cell patch-clamp configuration.

(A) Representative hERG current traces with or without 10 μM IMTU treatment elicited by the depolarized voltage at +40 mV for 3 s before repolarization to −40 mV for 2 s. The dashed lines represent zero-current levels. (B) Representative Nav1.5 current traces with or without application of 10 μM IMTU elicited by the depolarized voltage at 0 mV before repolarization to −120 mV. The dashed lines represent zero-current levels.

https://doi.org/10.1371/journal.pone.0345127.g004

3.4. In vivo pharmacokinetic profile of IMTU in mice

Before testing the analgesic effect of IMTU on inflammatory pain in mice, we determined the pharmacokinetic profile of IMTU after a single intravenous (IV, 10 mg/kg) or oral (PO, 30 mg/kg) administration in mice. As shown in S1 Fig and Table 1, IMTU showed an excellent oral bioavailability (F ≈ 100%) and good stability (T_1/2 = 2.22 h) after the oral administration of IMTU (30 mg/kg) in mice. IMTU reached a maximum plasma concentration (C_max) of 21800 ng/mL at 0.08 h and 81502 ng/mL at 2.00 h after intravenous and oral administration. These results revealed that antagonist IMTU had promising pharmacokinetic parameters, which could be used to evaluate the analgesic effect in vivo.

Download:

Table 1. Pharmacokinetic properties of IMTU in mice.

https://doi.org/10.1371/journal.pone.0345127.t001

3.5. IMTU alleviates nociceptive behaviors on mouse models of inflammatory pain

We tested the analgesic activity of IMTU on two mouse models of inflammatory pain, in which pain was evoked by formalin or Complete Freund’s Adjuvant (CFA). As shown in Fig 5A, pre-administration of IMTU at 5, 10 and 30 mg/kg does-dependently alleviated nociceptive pain behavior (paw licking) of mice induced by intraplantar injection of formalin in phase II but not in phase I, compared with the saline pre-administered group. As a positive control, pre-administration of ibuprofen (2 mg/kg), a nonsteroid anti-inflammatory drug, also markedly reduced the formalin-induced paw licking time in phase II but not in phase I.

Download:

Fig 5. Analgesic efficacy of IMTU in mice.

(A) Summary for the analgesic effects of IMTU on nociceptive behavior (paw licking) induced by intraplantar injection of formalin (5%) during phase I (0–5 min post-injection) and phase II (15–45 min post-injection) (n = 6-8 mice). IMTU (5, 10 and 30 mg/kg) and ibuprofen (positive control, 2 mg/kg) were intraperitoneally injected into mice 1 h before the injection of formalin. (B) Anti-nociceptive effects of IMTU on inflammatory pain model induced by intraplantar injection of 20 µL CFA (n = 6 mice). 24 h after CFA injection, the paw withdrawal mechanical threshold (PWMT) of mice was measured using von Frey fiber at 0, 1, 2, 3, and 4 h after intraperitoneal injection of IMTU (5, 10 and 30 mg/kg) and ibuprofen (positive control, 2 mg/kg). Error bars represent the means ± SEM. **p < 0.01, ***p < 0.001, by one-way ANOVA.

https://doi.org/10.1371/journal.pone.0345127.g005

Next, we evaluated the analgesic activity of IMTU on CFA-induced inflammatory pain. 24 h after intraplantar injection of CFA, the paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) of mice were measured. CFA injection resulted in obvious mechanical allodynia compared with the control group, while intraperitoneal injection of IMTU at 5, 10 and 30 mg/kg dose-dependently elevated the PWMT, especially within the first 1 h post-administration (Fig 5B, left panel). Similarly, intraperitoneal injection of ibuprofen (2 mg/kg) also markedly elevated the PWMT. In addition, IMTU also dose-dependently increased the PWTL within 3 h (Fig 5B, right panel). Ibuprofen, as a positive control, could also relieve CFA-induced inflammatory pain and its analgesic effect was similar to IMTU at 10 mg/kg. These findings suggested that IMTU possesses a good analgesic effect on inflammatory pain.

3.6. Body temperature

Hyperthermia is a common acute side effect after administration of TRPV1 antagonists. Therefore, we further evaluated the effect of IMTU on body temperature of mice. BCTC, a known TRPV1 antagonist, was selected as a positive control to induce hyperthermia [30]. As shown in Fig 6, oral administration of BCTC (20 mg/kg) resulted in a significant increase of body temperature for about 1°C after 0.5 h and reached peak effect at 1h for about 2°C. In contrast, no obvious temperature increase was monitored after the oral administration of IMTU at higher concentration (20 and 50 mg/kg), indicating IMTU might not cause serious hyperthermia in vivo.

Download:

Fig 6. The effects of IMTU on the rectal body temperature in mice.

The body temperature of mice was measured after the oral administration of IMTU (20 and 50 mg/kg) or BCTC (20 mg/kg). The data was expressed as the mean ± SEM (n = 6).

https://doi.org/10.1371/journal.pone.0345127.g006

3.7. Molecular mechanisms underlying the inhibition of TRPV1 by IMTU

To study the molecular mechanisms underlying TRPV1 inhibition by IMTU, we adopted molecular docking of IMTU into the cryo-EM structure of rat TRPV1 in complex with capsazepine (rTRPV1-capsazepine PDB: 5IS0) using GOLD Suite 5.1 to predict the interactions. The best docking conformation revealed that IMTU was confined into a pocket formed by residues between the S4 and S4-S5 linker (Fig 7A and 7B). The overlap of IMTU with cryo-EM structure of TRPV1-capsazepine complex revealed IMTU fits into the same binding pocket of capsazepine (Fig 7C). Further binding mode indicated the molecular interaction between IMTU and TRPV1 which four hydrogen bonds were formed with residues Tyr511, Ser512, Thr550 and Asn551, suggesting that the four residues were critical for the TRPV1 antagonists by IMTU (Fig 7D).

Download:

Fig 7. Interactions between IMTU and TRPV1 determined by molecular docking and site-directed mutagenesis.

(A and B) Overview of the complex of IMTU with TRPV1. (C) Representative binding conformations of IMTU confined into a pocket of rTRPV1-capsazepine complex (PDB: 5IS0) consisting of the S4, and S4–S5 linker. IMTU: cream stick; capsazepine: blue stick. (D) The putative binding mode of IMTU in the pocket of TRPV1. The subunit of rTRPV1 was shown in cyan and orange, and the IMTU (cream) and key residues for the binding were shown in stick. Hydrogen bonds were shown as red lines. (E-H) The effect of IMTU on TRRV1 mutants expressed in HEK293 cells using the whole-cell patch clamp assay. Current trace of WT (E), T550A (F), Y511A (G) and S512A (H) mutants in response to capsaicin (100 nM) and IMTU (300 nM).

https://doi.org/10.1371/journal.pone.0345127.g007

Then we constructed the rTRPV1 mutants Y511A, S512A and T550A to verify the molecular docking results. As shown in Fig 7E-F, the T550A mutant was insensitive to IMTU (300 nM) in HEK293 cells using whole-cell patch clamp recordings. In contrast, the Y511A and S512A mutants were both insensitive to capsaicin (Fig 7G-H), which was consistent with the observation that Tyr511 stabilize the activation of TRPV1 but unable to further verify the effect of IMTU on these TRPV1 mutants [21]. These results revealed that Thr550 played an essential role in the inhibition of IMTU on TRPV1.

4. Discussion

Capsaicin receptor TRPV1 has been identified as novel directions on the mechanism of nociception, due to its important role in primary afferent neurons sensing noxious stimuli. Although TRPV1 antagonists have been widely accepted as a promising strategy for pain, numerous clinical trials of TRPV1 antagonists have been suspended, mainly due to the adverse hyperthermia events because of impaired regulation of body temperature [31,32]. Activation of TRPV1 by different stimuli involves mechanismic differences related to channel structure or loci of molecule. First generation of TRPV1 antagonists potently block all activation modes which called mode-nonselective (or mode-nonspecific). While second-generation (mode-selective) TRPV1 antagonists potently block the channel activation by capsaicin, with exert different effects in the heat mode. The present study aims to develop and identify novel second-generation TRPV1 antagonists for pain treatment, thus providing alternative therapy for chronic pain relief. After our study, we identified IMTU as a novel and selective TRPV1 antagonist possessing analgesic effects but without hyperthermia side effect in mice. This finding may provide constructive insights into the development of second-generation TRPV1 antagonists.

TRPV1 is abundantly present in primary afferent neurons, such as dorsal root ganglia and trigeminal neurons, where it serves as a biosensor in response to noxious heat, low pH, chemical and endogenous stimuli, and thus generating pain [9,33]. TRPV1 expression increases in the dorsal root ganglia neurons under inflammatory conditions, and endogenous mediators, such as bradykinin, eicosanoids and ATP, produced from the inflammatory tissues could further activate or sensitize TRPV1 in unmyelinated C-fibers, leading to the development of inflammatory responses [34–36]. Unlike the traditional analgesic drugs that either block pain transmission (opiates) or inhibit inflammation (NSAIDs), novel TRPV1 antagonists attempt to inactivate nociceptors and prevent the generation of pain at the beginning [37,38]. In this study, administration of IMTU could dose-dependently alleviate nociceptive behaviors of mice in phase II which represents the inflammatory response stage during 15–45 min post formalin injection, whereas the nociceptive behavior was not attenuated in phase I (0–5 min post formalin injection) representing the acute pain stage [39]. Meanwhile, administration of IMTU dose-dependently alleviated mechanical hyperpathia on a mouse model of inflammatory pain induced by CFA injection. These findings highlight the role of nociceptive TRPV1 channels in inflammatory pain and suggest that targeting antagonists of TRPV1 in primary sensory neurons by IMTU represents a logical and promising strategy for pain relief.

The Cryo-EM structures of the TRPV1 in complex with agonists or antagonists have provided the possible binding pocket for IMTU. Our docking results indicated that IMTU was fitted into a pocket consisting of S4 and S4–S5 linker and shared a similar binding pocket with TRPV1 agonists RTX and capsaicin, as well as the TRPV1 antagonist capsazepine [21,28]. Upon binding, IMTU was identified by four critical residues Tyr511, Ser512, Thr550 and Asn551, among which the two residues Tyr511 and Thr550 were critical for TRPV1 activation by RTX, indicating the molecular mechanism of antagonist IMTU by competitively binding to these critical residues for agonists. Moreover, we found Thr550 played an essential role in the inhibition of IMTU on TRPV1 but not capsazepine (a known TRPA1 antagonist), indicating a unique binding site for developing new generation of TRPV1 antagonists without hyperthermia.

In conclusion, we identify a novel and selective TRPV1 antagonist IMTU that exhibits analgesic effects without hyperthermia in mice, and therefore targeting inhibition of TRPV1 in primary sensory neurons by IMTU represents a promised strategy for pain relief.

Supporting information

S1 Fig. Plasma concentration curve of IMTU in mice via i.v. (10 mg/kg, left) and p.o. (30 mg/kg, right) (n = 3).

https://doi.org/10.1371/journal.pone.0345127.s001

(TIF)

S2 Fig. ¹H NMR spectrum of IMTU.

https://doi.org/10.1371/journal.pone.0345127.s002

(TIF)

S3 Fig. ¹³C NMR spectrum of IMTU.

https://doi.org/10.1371/journal.pone.0345127.s003

(TIF)

S4 Fig. Mass spectrum of IMTU.

https://doi.org/10.1371/journal.pone.0345127.s004

(TIF)

S1 Scheme. Synthetic route of IMTU.

a. 4-methylpiperidine, 50 °C, 1 h, yield: 90%; b. NiCl₂, NaBH₄, methanol, argon atmosphere, rt, 1 h, yield: 87%; c. Pd/C, H₂, MeOH, rt, overnight, yield: 94%; d. pyridine, phenyl carbonochloridate, THF, 0-rt, 8 h, yield: 50%; e. GFP, DMF, 50 °C, yield: 88%.

https://doi.org/10.1371/journal.pone.0345127.s005

(TIF)

References

1. Bourinet E, Altier C, Hildebrand ME, Trang T, Salter MW, Zamponi GW. Calcium-permeable ion channels in pain signaling. Physiol Rev. 2014;94(1):81–140. pmid:24382884
- View Article
- PubMed/NCBI
- Google Scholar
2. Jackson T, Thomas S, Stabile V, Shotwell M, Han X, McQueen K. A systematic review and meta-analysis of the global burden of chronic pain without clear etiology in low- and middle-income countries: Trends in heterogeneous data and a proposal for new assessment methods. Anesthesia and Analgesia. 2016;123(3):739–48. pmid:27537761
- View Article
- PubMed/NCBI
- Google Scholar
3. Tsang A, Von Korff M, Lee S, Alonso J, Karam E, Angermeyer MC, et al. Common chronic pain conditions in developed and developing countries: Gender and age differences and comorbidity with depression-anxiety disorders. J Pain. 2008;9(10):883–91. pmid:18602869
- View Article
- PubMed/NCBI
- Google Scholar
4. Cohen SP, Vase L, Hooten WM. Chronic pain: An update on burden, best practices, and new advances. Lancet. 2021;397(10289):2082–97. pmid:34062143
- View Article
- PubMed/NCBI
- Google Scholar
5. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: A heat-activated ion channel in the pain pathway. Nature. 1997;389(6653):816–24. pmid:9349813
- View Article
- PubMed/NCBI
- Google Scholar
6. Yang S, Yang F, Wei N, Hong J, Li B, Luo L, et al. A pain-inducing centipede toxin targets the heat activation machinery of nociceptor TRPV1. Nat Commun. 2015;6:8297. pmid:26420335
- View Article
- PubMed/NCBI
- Google Scholar
7. Cheng W, Yang F, Takanishi CL, Zheng J. Thermosensitive TRPV channel subunits coassemble into heteromeric channels with intermediate conductance and gating properties. J Gen Physiol. 2007;129(3):191–207. pmid:17325193
- View Article
- PubMed/NCBI
- Google Scholar
8. Cuypers E, Yanagihara A, Karlsson E, Tytgat J. Jellyfish and other cnidarian envenomations cause pain by affecting TRPV1 channels. FEBS Lett. 2006;580(24):5728–32. pmid:17010344
- View Article
- PubMed/NCBI
- Google Scholar
9. Abbas MA. Modulation of TRPV1 channel function by natural products in the treatment of pain. Chem Biol Interact. 2020;330:109178. pmid:32738201
- View Article
- PubMed/NCBI
- Google Scholar
10. Alsalem M, Wong A, Millns P, Arya PH, Chan MSL, Bennett A, et al. The contribution of the endogenous TRPV1 ligands 9-HODE and 13-HODE to nociceptive processing and their role in peripheral inflammatory pain mechanisms. Br J Pharmacol. 2013;168(8):1961–74. pmid:23278358
- View Article
- PubMed/NCBI
- Google Scholar
11. Watanabe M, Ueda T, Shibata Y, Kumamoto N, Ugawa S. The role of TRPV1 channels in carrageenan-induced mechanical hyperalgesia in mice. Neuroreport. 2015;26(3):173–8. pmid:25590988
- View Article
- PubMed/NCBI
- Google Scholar
12. Iftinca M, Defaye M, Altier C. TRPV1-targeted drugs in development for human pain conditions. Drugs. 2021;81(1):7–27. pmid:33165872
- View Article
- PubMed/NCBI
- Google Scholar
13. Luo X, Chen O, Wang Z, Bang S, Ji J, Lee SH, et al. IL-23/IL-17A/TRPV1 axis produces mechanical pain via macrophage-sensory neuron crosstalk in female mice. Neuron. 2021;109(17):2691-2706.e5. pmid:34473953
- View Article
- PubMed/NCBI
- Google Scholar
14. Brown BS, Keddy R, Perner RJ, DiDomenico S, Koenig JR, Jinkerson TK. Discovery of TRPV1 antagonist ABT-116. Bioorganic & Medicinal Chemistry Letters. 2010;20(11):3291–4. pmid:20457518
- View Article
- PubMed/NCBI
- Google Scholar
15. Maher MP, Bhattacharya A, Ao H, Swanson N, Wu NT, Freedman J. Characterization of 2-(2,6-dichloro-benzyl)-thiazolo[5,4-d]pyrimidin-7-yl]-(4-trifluoromethyl-phenyl) -amine (JNJ-39729209) as a novel TRPV1 antagonist. Eur J Pharmacol. 2011;663(1–3):40–50. pmid:21575625
- View Article
- PubMed/NCBI
- Google Scholar
16. Gavva NR, Treanor JJS, Garami A, Fang L, Surapaneni S, Akrami A, et al. Pharmacological blockade of the vanilloid receptor TRPV1 elicits marked hyperthermia in humans. Pain. 2008;136(1–2):202–10. pmid:18337008
- View Article
- PubMed/NCBI
- Google Scholar
17. Garami A, Shimansky YP, Rumbus Z, Vizin RCL, Farkas N, Hegyi J, et al. Hyperthermia induced by transient receptor potential vanilloid-1 (TRPV1) antagonists in human clinical trials: Insights from mathematical modeling and meta-analysis. Pharmacol Ther. 2020;208:107474. pmid:31926897
- View Article
- PubMed/NCBI
- Google Scholar
18. Gavva NR, Bannon AW, Hovland DN, Lehto SG, Klionsky L, Surapaneni S, et al. Repeated administration of vanilloid receptor TRPV1 antagonists attenuates hyperthermia elicited by TRPV1 blockade. J Pharmacol Exp Ther. 2007;323(1):128–37. pmid:17652633
- View Article
- PubMed/NCBI
- Google Scholar
19. Krarup AL, Ny L, Astrand M, Bajor A, Hvid-Jensen F, Hansen MB, et al. Randomised clinical trial: The efficacy of a transient receptor potential vanilloid 1 antagonist AZD1386 in human oesophageal pain. Aliment Pharmacol Ther. 2011;33(10):1113–22. pmid:21410733
- View Article
- PubMed/NCBI
- Google Scholar
20. Miller F, Björnsson M, Svensson O, Karlsten R. Experiences with an adaptive design for a dose-finding study in patients with osteoarthritis. Contemp Clin Trials. 2014;37(2):189–99. pmid:24394343
- View Article
- PubMed/NCBI
- Google Scholar
21. Gao Y, Cao E, Julius D, Cheng Y. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature. 2016;534(7607):347–51. pmid:27281200
- View Article
- PubMed/NCBI
- Google Scholar
22. Liao M, Cao E, Julius D, Cheng Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature. 2013;504(7478):107–12. pmid:24305160
- View Article
- PubMed/NCBI
- Google Scholar
23. Dong L, Zhou Q, Liang Q, Qiao Z, Liu Y, Shao L, et al. Identification of a Partial and Selective TRPV1 Agonist CPIPC for Alleviation of Inflammatory Pain. Molecules. 2022;27(17):5428. pmid:36080196
- View Article
- PubMed/NCBI
- Google Scholar
24. Liang Q, Qiao Z, Zhou Q, Xue D, Wang K, Shao L. Discovery of Potent and Selective Transient Receptor Potential Vanilloid 1 (TRPV1) Agonists with analgesic effects in vivo based on the functional conversion induced by altering the orientation of the indazole core. J Med Chem. 2022;65(17):11658–78. pmid:36008373
- View Article
- PubMed/NCBI
- Google Scholar
25. Han Y, Luo A, Kamau PM, Takomthong P, Hu J, Boonyarat C, et al. A plant-derived TRPV3 inhibitor suppresses pain and itch. Br J Pharmacol. 2021;178(7):1669–83. pmid:33501656
- View Article
- PubMed/NCBI
- Google Scholar
26. Luo J, Zhu Y, Zhu MX, Hu H. Cell-based calcium assay for medium to high throughput screening of TRP channel functions using FlexStation 3. J Vis Exp. 2011;54. pmid:21876521
- View Article
- PubMed/NCBI
- Google Scholar
27. Han K, Zhang A, Mo Y, Mao T, Ji B, Li D, et al. Islet-cell autoantigen 69 mediates the antihyperalgesic effects of electroacupuncture on inflammatory pain by regulating spinal glutamate receptor subunit 2 phosphorylation through protein interacting with C-kinase 1 in mice. Pain. 2019;160(3):712–23. pmid:30699097
- View Article
- PubMed/NCBI
- Google Scholar
28. Cao E, Liao M, Cheng Y, Julius D. TRPV1 structures in distinct conformations reveal activation mechanisms. Nature. 2013;504(7478):113–8. pmid:24305161
- View Article
- PubMed/NCBI
- Google Scholar
29. Zhou Y, Wu H-J, Zhang Y-H, Sun H-Y, Wong T-M, Li G-R. Ionic mechanisms underlying cardiac toxicity of the organochloride solvent trichloromethane. Toxicology. 2011;290(2–3):295–304. pmid:22024336
- View Article
- PubMed/NCBI
- Google Scholar
30. Liu C, Wang W, Zhao S, Chen S, Chen H, Wang S, et al. Discovery of first-in-class highly selective TRPV1 antagonists with dual analgesic and hypoglycemic effects. Bioorg Med Chem. 2024;107:117750. pmid:38776567
- View Article
- PubMed/NCBI
- Google Scholar
31. Lee Y, Hong S, Cui M, Sharma PK, Lee J, Choi S. Transient receptor potential vanilloid type 1 antagonists: A patent review (2011 - 2014). Expert Opin Ther Pat. 2015;25(3):291–318. pmid:25666693
- View Article
- PubMed/NCBI
- Google Scholar
32. Sakakibara S, Imamachi N, Sakakihara M, Katsube Y, Hattori M, Saito Y. Effects of an intrathecal TRPV1 antagonist, SB366791, on morphine-induced itch, body temperature, and antinociception in mice. J Pain Res. 2019;12:2629–36. pmid:31695478
- View Article
- PubMed/NCBI
- Google Scholar
33. Brito R, Sheth S, Mukherjea D, Rybak LP, Ramkumar V. TRPV1: A potential drug target for treating various diseases. Cells. 2014;3(2):517–45. pmid:24861977
- View Article
- PubMed/NCBI
- Google Scholar
34. Roosterman D, Goerge T, Schneider SW, Bunnett NW, Steinhoff M. Neuronal control of skin function: The skin as a neuroimmunoendocrine organ. Physiol Rev. 2006;86(4):1309–79. pmid:17015491
- View Article
- PubMed/NCBI
- Google Scholar
35. Gouin O, L’Herondelle K, Lebonvallet N, Le Gall-Ianotto C, Sakka M, Buhé V, et al. TRPV1 and TRPA1 in cutaneous neurogenic and chronic inflammation: Pro-inflammatory response induced by their activation and their sensitization. Protein Cell. 2017;8(9):644–61. pmid:28364279
- View Article
- PubMed/NCBI
- Google Scholar
36. Chuang HH, Prescott ED, Kong H, Shields S, Jordt SE, Basbaum AI, et al. Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition. Nature. 2001;411(6840):957–62. pmid:11418861
- View Article
- PubMed/NCBI
- Google Scholar
37. Levine JD, Alessandri-Haber N. TRP channels: targets for the relief of pain. Biochim Biophys Acta. 2007;1772(8):989–1003. pmid:17321113
- View Article
- PubMed/NCBI
- Google Scholar
38. Julius D. TRP channels and pain. Annu Rev Cell Dev Biol. 2013;29:355–84. pmid:24099085
- View Article
- PubMed/NCBI
- Google Scholar
39. Xu Y, Yu Y, Wang Q, Li W, Zhang S, Liao X, et al. Active components of Bupleurum chinense and Angelica biserrata showed analgesic effects in formalin induced pain by acting on Nav1.7. J Ethnopharmacol. 2021;269:113736. pmid:33359917
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Bourinet E, Altier C, Hildebrand ME, Trang T, Salter MW, Zamponi GW. Calcium-permeable ion channels in pain signaling. Physiol Rev. 2014;94(1):81–140. pmid:24382884
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Jackson T, Thomas S, Stabile V, Shotwell M, Han X, McQueen K. A systematic review and meta-analysis of the global burden of chronic pain without clear etiology in low- and middle-income countries: Trends in heterogeneous data and a proposal for new assessment methods. Anesthesia and Analgesia. 2016;123(3):739–48. pmid:27537761
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Tsang A, Von Korff M, Lee S, Alonso J, Karam E, Angermeyer MC, et al. Common chronic pain conditions in developed and developing countries: Gender and age differences and comorbidity with depression-anxiety disorders. J Pain. 2008;9(10):883–91. pmid:18602869
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Cohen SP, Vase L, Hooten WM. Chronic pain: An update on burden, best practices, and new advances. Lancet. 2021;397(10289):2082–97. pmid:34062143
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: A heat-activated ion channel in the pain pathway. Nature. 1997;389(6653):816–24. pmid:9349813
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Yang S, Yang F, Wei N, Hong J, Li B, Luo L, et al. A pain-inducing centipede toxin targets the heat activation machinery of nociceptor TRPV1. Nat Commun. 2015;6:8297. pmid:26420335
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Cheng W, Yang F, Takanishi CL, Zheng J. Thermosensitive TRPV channel subunits coassemble into heteromeric channels with intermediate conductance and gating properties. J Gen Physiol. 2007;129(3):191–207. pmid:17325193
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Cuypers E, Yanagihara A, Karlsson E, Tytgat J. Jellyfish and other cnidarian envenomations cause pain by affecting TRPV1 channels. FEBS Lett. 2006;580(24):5728–32. pmid:17010344
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Abbas MA. Modulation of TRPV1 channel function by natural products in the treatment of pain. Chem Biol Interact. 2020;330:109178. pmid:32738201
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Alsalem M, Wong A, Millns P, Arya PH, Chan MSL, Bennett A, et al. The contribution of the endogenous TRPV1 ligands 9-HODE and 13-HODE to nociceptive processing and their role in peripheral inflammatory pain mechanisms. Br J Pharmacol. 2013;168(8):1961–74. pmid:23278358
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Watanabe M, Ueda T, Shibata Y, Kumamoto N, Ugawa S. The role of TRPV1 channels in carrageenan-induced mechanical hyperalgesia in mice. Neuroreport. 2015;26(3):173–8. pmid:25590988
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Iftinca M, Defaye M, Altier C. TRPV1-targeted drugs in development for human pain conditions. Drugs. 2021;81(1):7–27. pmid:33165872
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Luo X, Chen O, Wang Z, Bang S, Ji J, Lee SH, et al. IL-23/IL-17A/TRPV1 axis produces mechanical pain via macrophage-sensory neuron crosstalk in female mice. Neuron. 2021;109(17):2691-2706.e5. pmid:34473953
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Brown BS, Keddy R, Perner RJ, DiDomenico S, Koenig JR, Jinkerson TK. Discovery of TRPV1 antagonist ABT-116. Bioorganic & Medicinal Chemistry Letters. 2010;20(11):3291–4. pmid:20457518
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Maher MP, Bhattacharya A, Ao H, Swanson N, Wu NT, Freedman J. Characterization of 2-(2,6-dichloro-benzyl)-thiazolo[5,4-d]pyrimidin-7-yl]-(4-trifluoromethyl-phenyl) -amine (JNJ-39729209) as a novel TRPV1 antagonist. Eur J Pharmacol. 2011;663(1–3):40–50. pmid:21575625
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Gavva NR, Treanor JJS, Garami A, Fang L, Surapaneni S, Akrami A, et al. Pharmacological blockade of the vanilloid receptor TRPV1 elicits marked hyperthermia in humans. Pain. 2008;136(1–2):202–10. pmid:18337008
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. Garami A, Shimansky YP, Rumbus Z, Vizin RCL, Farkas N, Hegyi J, et al. Hyperthermia induced by transient receptor potential vanilloid-1 (TRPV1) antagonists in human clinical trials: Insights from mathematical modeling and meta-analysis. Pharmacol Ther. 2020;208:107474. pmid:31926897
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Gavva NR, Bannon AW, Hovland DN, Lehto SG, Klionsky L, Surapaneni S, et al. Repeated administration of vanilloid receptor TRPV1 antagonists attenuates hyperthermia elicited by TRPV1 blockade. J Pharmacol Exp Ther. 2007;323(1):128–37. pmid:17652633
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. Krarup AL, Ny L, Astrand M, Bajor A, Hvid-Jensen F, Hansen MB, et al. Randomised clinical trial: The efficacy of a transient receptor potential vanilloid 1 antagonist AZD1386 in human oesophageal pain. Aliment Pharmacol Ther. 2011;33(10):1113–22. pmid:21410733
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref20] 20. Miller F, Björnsson M, Svensson O, Karlsten R. Experiences with an adaptive design for a dose-finding study in patients with osteoarthritis. Contemp Clin Trials. 2014;37(2):189–99. pmid:24394343
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref21] 21. Gao Y, Cao E, Julius D, Cheng Y. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature. 2016;534(7607):347–51. pmid:27281200
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref22] 22. Liao M, Cao E, Julius D, Cheng Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature. 2013;504(7478):107–12. pmid:24305160
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref23] 23. Dong L, Zhou Q, Liang Q, Qiao Z, Liu Y, Shao L, et al. Identification of a Partial and Selective TRPV1 Agonist CPIPC for Alleviation of Inflammatory Pain. Molecules. 2022;27(17):5428. pmid:36080196
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref24] 24. Liang Q, Qiao Z, Zhou Q, Xue D, Wang K, Shao L. Discovery of Potent and Selective Transient Receptor Potential Vanilloid 1 (TRPV1) Agonists with analgesic effects in vivo based on the functional conversion induced by altering the orientation of the indazole core. J Med Chem. 2022;65(17):11658–78. pmid:36008373
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref25] 25. Han Y, Luo A, Kamau PM, Takomthong P, Hu J, Boonyarat C, et al. A plant-derived TRPV3 inhibitor suppresses pain and itch. Br J Pharmacol. 2021;178(7):1669–83. pmid:33501656
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref26] 26. Luo J, Zhu Y, Zhu MX, Hu H. Cell-based calcium assay for medium to high throughput screening of TRP channel functions using FlexStation 3. J Vis Exp. 2011;54. pmid:21876521
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref27] 27. Han K, Zhang A, Mo Y, Mao T, Ji B, Li D, et al. Islet-cell autoantigen 69 mediates the antihyperalgesic effects of electroacupuncture on inflammatory pain by regulating spinal glutamate receptor subunit 2 phosphorylation through protein interacting with C-kinase 1 in mice. Pain. 2019;160(3):712–23. pmid:30699097
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref28] 28. Cao E, Liao M, Cheng Y, Julius D. TRPV1 structures in distinct conformations reveal activation mechanisms. Nature. 2013;504(7478):113–8. pmid:24305161
View Article
PubMed/NCBI
Google Scholar

[110] View Article

[111] PubMed/NCBI

[112] Google Scholar

[ref29] 29. Zhou Y, Wu H-J, Zhang Y-H, Sun H-Y, Wong T-M, Li G-R. Ionic mechanisms underlying cardiac toxicity of the organochloride solvent trichloromethane. Toxicology. 2011;290(2–3):295–304. pmid:22024336
View Article
PubMed/NCBI
Google Scholar

[114] View Article

[115] PubMed/NCBI

[116] Google Scholar

[ref30] 30. Liu C, Wang W, Zhao S, Chen S, Chen H, Wang S, et al. Discovery of first-in-class highly selective TRPV1 antagonists with dual analgesic and hypoglycemic effects. Bioorg Med Chem. 2024;107:117750. pmid:38776567
View Article
PubMed/NCBI
Google Scholar

[118] View Article

[119] PubMed/NCBI

[120] Google Scholar

[ref31] 31. Lee Y, Hong S, Cui M, Sharma PK, Lee J, Choi S. Transient receptor potential vanilloid type 1 antagonists: A patent review (2011 - 2014). Expert Opin Ther Pat. 2015;25(3):291–318. pmid:25666693
View Article
PubMed/NCBI
Google Scholar

[122] View Article

[123] PubMed/NCBI

[124] Google Scholar

[ref32] 32. Sakakibara S, Imamachi N, Sakakihara M, Katsube Y, Hattori M, Saito Y. Effects of an intrathecal TRPV1 antagonist, SB366791, on morphine-induced itch, body temperature, and antinociception in mice. J Pain Res. 2019;12:2629–36. pmid:31695478
View Article
PubMed/NCBI
Google Scholar

[126] View Article

[127] PubMed/NCBI

[128] Google Scholar

[ref33] 33. Brito R, Sheth S, Mukherjea D, Rybak LP, Ramkumar V. TRPV1: A potential drug target for treating various diseases. Cells. 2014;3(2):517–45. pmid:24861977
View Article
PubMed/NCBI
Google Scholar

[130] View Article

[131] PubMed/NCBI

[132] Google Scholar

[ref34] 34. Roosterman D, Goerge T, Schneider SW, Bunnett NW, Steinhoff M. Neuronal control of skin function: The skin as a neuroimmunoendocrine organ. Physiol Rev. 2006;86(4):1309–79. pmid:17015491
View Article
PubMed/NCBI
Google Scholar

[134] View Article

[135] PubMed/NCBI

[136] Google Scholar

[ref35] 35. Gouin O, L’Herondelle K, Lebonvallet N, Le Gall-Ianotto C, Sakka M, Buhé V, et al. TRPV1 and TRPA1 in cutaneous neurogenic and chronic inflammation: Pro-inflammatory response induced by their activation and their sensitization. Protein Cell. 2017;8(9):644–61. pmid:28364279
View Article
PubMed/NCBI
Google Scholar

[138] View Article

[139] PubMed/NCBI

[140] Google Scholar

[ref36] 36. Chuang HH, Prescott ED, Kong H, Shields S, Jordt SE, Basbaum AI, et al. Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition. Nature. 2001;411(6840):957–62. pmid:11418861
View Article
PubMed/NCBI
Google Scholar

[142] View Article

[143] PubMed/NCBI

[144] Google Scholar

[ref37] 37. Levine JD, Alessandri-Haber N. TRP channels: targets for the relief of pain. Biochim Biophys Acta. 2007;1772(8):989–1003. pmid:17321113
View Article
PubMed/NCBI
Google Scholar

[146] View Article

[147] PubMed/NCBI

[148] Google Scholar

[ref38] 38. Julius D. TRP channels and pain. Annu Rev Cell Dev Biol. 2013;29:355–84. pmid:24099085
View Article
PubMed/NCBI
Google Scholar

[150] View Article

[151] PubMed/NCBI

[152] Google Scholar

[ref39] 39. Xu Y, Yu Y, Wang Q, Li W, Zhang S, Liao X, et al. Active components of Bupleurum chinense and Angelica biserrata showed analgesic effects in formalin induced pain by acting on Nav1.7. J Ethnopharmacol. 2021;269:113736. pmid:33359917
View Article
PubMed/NCBI
Google Scholar

[154] View Article

[155] PubMed/NCBI

[156] Google Scholar

Figures

Abstract

1. Introduction

2. Materials and methods

2.1. Reagents and Chemicals

2.2. Chemistry

2.2.1. Synthesis of Intermediates 2 (2-(4-methylpiperidin-1-yl)-6-(trifluoromethyl)nicotinonitrile).

2.2.2. Synthesis of Intermediates 3 ((2-(4-methylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)methanamine).

2.2.3. Synthesis of Intermediates 5 (1H-indazol-4-amine).

2.2.4. Synthesis of Intermediates 6 (phenyl (1H-indazol-4-yl)carbamate).

2.2.5. Synthesis of Final Compound IMTU (1-(1H-indazol-4-yl)-3-((2-(4-methylpiperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)methyl)urea).

2.3. Animals

2.4. Cell culture and transfection of cDNAs

2.5. Intracellular calcium assay using FlexStation3

2.6. Electrophysiology

2.7. In vivo Pharmacokinetics

2.8. Formalin–induced paw licking behavior

2.9. Establishment of Complete Freund’s Adjuvant (CFA)–induced inflammatory pain model

2.10. Determination of PWMT

2.11. Determination of PWTL

2.12. Body temperature measurement

2.13. Molecular docking

2.14. Statistical analysis

3. Results

3.1. Identification of IMTU as a potent and selective TRPV1 antagonist

3.2. Direct targeting of TRPV1 single channel by IMTU

3.3. IMTU does not affect cardiotoxicity-related channels

3.4. In vivo pharmacokinetic profile of IMTU in mice

3.5. IMTU alleviates nociceptive behaviors on mouse models of inflammatory pain

3.6. Body temperature

3.7. Molecular mechanisms underlying the inhibition of TRPV1 by IMTU

4. Discussion

Supporting information

S1 Fig. Plasma concentration curve of IMTU in mice via i.v. (10 mg/kg, left) and p.o. (30 mg/kg, right) (n = 3).

S2 Fig. 1H NMR spectrum of IMTU.

S3 Fig. 13C NMR spectrum of IMTU.

S4 Fig. Mass spectrum of IMTU.

S1 Scheme. Synthetic route of IMTU.

References

S2 Fig. ¹H NMR spectrum of IMTU.

S3 Fig. ¹³C NMR spectrum of IMTU.