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
TL;DR
This study introduces a new TRPV1 antagonist, IMTU, which effectively reduces inflammatory pain in mice without causing hyperthermia.
Contribution
The paper presents IMTU, a novel TRPV1 antagonist with analgesic effects and no hyperthermic side effects.
Findings
IMTU is a potent TRPV1 antagonist with an IC50 of 128.34 ± 9.49 nM.
IMTU reduces TRPV1 channel open probability significantly in the presence of capsaicin.
Oral IMTU alleviates inflammatory pain in mice without inducing hyperthermia.
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…
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Fig 7- —Shandong Provincial Natural Science Foundation of China
- —the Affiliated Hospital of Qingdao University Clinical Medical Research Program
- —Medical Health Research Project of Zibo
- —the QLMU research fund
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Taxonomy
TopicsIon Channels and Receptors · Pain Mechanisms and Treatments · Ion channel regulation and function
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.
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..
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 Na_2_SO_4_, 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 NiCl_2_ (2 mmol). Under argon atmosphere, the NaBH_4_ (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 Na_2_SO_4_, 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 (Et_3_N) (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 Na_2_SO_4_, filtered, and concentrated under vacuum. The residue was purified by column chromatography to obtain target compound IMTU. Yield, 88%; white solid*.* ^1^H 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). ^13^C 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 C_21_H_23_F_3_N_6_O, [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 CO_2_ 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% CO_2_/95% O_2_ 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. Ca^2+^ 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 ([Ca^2+^]) was detected by incubating with Ca^2+^-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 NaHCO_3_, 1.3 CaCl_2_, 0.4 KH_2_PO4, 0.1 Na_2_HPO4, 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 MgCl_2_, 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 CaCl_2_, 1.2 Mg Cl_2_, 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 MgCl_2_, 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 MgCl_2_, 1.8 CaCl_2_, 10 glucose, and 10 HEPES, with pH adjusted to 7.4 by NaOH. The standard pipette solution contained (in mM): 140 CsF, 2.5 MgCl_2_, 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 Ca^2+^ 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 IC_50_ 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.
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.
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.
*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 POpen 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.
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.
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.
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 (T1/2 = 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.
Table 1: Pharmacokinetic properties of IMTU in mice.
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.
*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.
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.
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).
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).
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).
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 FigPlasma concentration curve of IMTU in mice via i.v. (10 mg/kg, left) and p.o. (30 mg/kg, right) (n = 3).(TIF)
S2 Fig^1^H NMR spectrum of IMTU.(TIF)
S3 Fig^13^C NMR spectrum of IMTU.(TIF)
S4 FigMass spectrum of IMTU.(TIF)
S1 SchemeSynthetic route of IMTU.a. 4-methylpiperidine, 50 °C, 1 h, yield: 90%; b. NiCl_2_, NaBH_4_, methanol, argon atmosphere, rt, 1 h, yield: 87%; c. Pd/C, H_2_, MeOH, rt, overnight, yield: 94%; d. pyridine, phenyl carbonochloridate, THF, 0-rt, 8 h, yield: 50%; e. GFP, DMF, 50 °C, yield: 88%.(TIF)
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