Polypeptides targeting trpv1 and uses thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a peptide targeting TRPV1 and its applications. A peptide targeting TRPV1 has the amino acid sequence shown in SEQ ID NO.1. The peptide also includes a mutant obtained by amino acid scanning mutation based on the sequence shown in SEQ ID NO.1. The amino acid sequence of the mutant is shown in any one of SEQ ID NO.2-16. This peptide is used to prepare formulations that block or inhibit TRPV1 activity. This invention provides a peptide targeting TRPV1 and its applications. Through a strategy combining artificial intelligence screening and structural prediction, SEQ ID NO.1 was successfully identified and its mechanism of action was elucidated. Experimental results show that SEQ ID NO.1 and its mutants exhibit excellent analgesic activity and good safety both in vitro and in vivo, demonstrating significant clinical application potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide targeting TRPV1 and its applications. Background Technology

[0002] Pain is a common symptom in the treatment of clinical diseases, and its pathogenesis involves multiple links, including abnormal activation of peripheral nociceptors and central sensitization. While existing analgesics can relieve pain to some extent, their overall efficacy remains significantly limited. Opioids, as first-line drugs for treating moderate to severe acute pain and cancer pain, are effective in treating moderate to severe pain, but they carry significant risks of tolerability and addiction. Gabapentin and pregabalin, used for neuropathic pain, can relieve some symptoms, but they have slow onset of action, varying efficacy, and are accompanied by central inhibitory adverse reactions such as drowsiness and dizziness (Finnerup et al., 2021; Koivisto et al., 2022). Therefore, exploring analgesic molecules with novel mechanisms of action, precise targets, and higher safety has become an important direction in pain drug research.

[0003] Transient receptor potential (TRPV1) subfamily member 1 is a non-selective cation channel that mediates the perception of thermal stimuli, acidic environments, and inflammatory mediators, and is widely distributed in peripheral sensory neurons. Overactivation of TRPV1 leads to intracellular Ca2+. 2+ Elevated levels of TRPV1 trigger neuronal excitation and amplify pain signals, playing a crucial role in the development of neuropathic and inflammatory pain (Caterina et al., 1117; Julius, 2013). Studies have shown that blocking or modulating TRPV1 activity can effectively inhibit various chronic pain responses, thus TRPV1 has become a highly promising target for analgesics (Koivisto et al., 2022; Andrade et al., 2025). Recent breakthroughs in cryo-electron microscopy have enabled the high-resolution structural analysis of TRPV1, systematically revealing its ligand binding sites and conformational change mechanisms, providing a solid theoretical foundation for structure-based ligand screening and the design of novel inhibitory peptides.

[0004] Another important target involved in pain signal modulation, along with TRPV1, is the α9α10 nicotinic acetylcholine receptor (α9α10 nAChR). This receptor is also expressed in peripheral sensory nerves and participates in the neuromodulation and pain transmission processes mediated by inflammatory factors. Various peptide toxins derived from cone snails (Conus spp.), such as αO-conotoxin GeXIVA, Vc1.1, and RgIA, can inhibit α9α10 nAChR with high affinity, thereby producing significant analgesic effects without inducing addiction (Luo et al., 2015; Jin et al., 2011; Zhu et al., 2022). These natural peptides, due to their high selectivity and good tolerability, have become an important molecular source for the development of non-opioid analgesics.

[0005] It is noteworthy that although TRPV1 and α9α10 nAChR belong to different ion channel families, both are involved in peripheral nociceptive sensation and neuroinflammation-related pain regulation, and may share similar mechanisms at the neuronal sensitization / desensitization level. Therefore, it can be inferred that some peptide antagonists acting on α9α10 nAChR may possess structural features that stabilize the closed or desensitized conformations of other cation channels (such as TRPV1). Furthermore, cone snail toxins targeting α9α10 nAChR and TRPV1 inhibitors have exhibited similar analgesic phenotypes in multiple animal pain models, suggesting that they may share some pharmacophore features or similar binding interface chemical environments. Summary of the Invention

[0006] This invention addresses the problems of insufficient activity and low selectivity of existing TRPV1-targeting analgesic peptides by providing a novel TRPV1-targeting peptide obtained through artificial intelligence screening and its application.

[0007] In a first aspect, the present invention provides a polypeptide targeting TRPV1, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] Furthermore, the peptide targeting TRPV1 is a mutant peptide obtained by amino acid scanning mutation based on the sequence shown in SEQ ID NO.1.

[0009] Furthermore, the amino acid scanning mutation includes positively charged amino acid scanning mutation, negatively charged amino acid scanning mutation, neutral amino acid scanning mutation, rare amino acid substitution mutation, or D-type amino acid substitution mutation; the amino acid sequence of the mutant is shown in any one of SEQ ID NO.2-16 in the sequence listing.

[0010] Furthermore, the present invention also provides the application of the peptide targeting TRPV1, which is used to prepare formulations that block or inhibit TRPV1 activity.

[0011] Furthermore, the formulation is used to prepare analgesic drugs.

[0012] Furthermore, the present invention also provides an formulation for blocking or inhibiting TRPV1 activity, the formulation comprising the aforementioned peptide targeting TRPV1.

[0013] Furthermore, the present invention also provides an analgesic drug, wherein the active ingredient of the analgesic drug comprises an effective dose of the peptide targeting TRPV1, or the preparation that blocks or inhibits TRPV1 activity.

[0014] This invention aims to discover and optimize highly active and selective cone snail toxins targeting TRPV1, providing lead compounds for the development of novel non-opioid analgesics. Using a strategy combining artificial intelligence screening and structural prediction, this invention successfully identified SEQ ID NO.1 and elucidated its mechanism of action. Experimental results show that SEQ ID NO.1 and its mutants exhibit excellent analgesic activity and good safety profiles both in vitro and in vivo, demonstrating significant clinical application potential. This invention provides a new structural template and design approach for the further development of peptide analgesics targeting TRPV1. Attached Figure Description

[0015] Figure 1 The IC50 curve for polypeptide SEQ ID NO.1;

[0016] Figure 2 This diagram illustrates the binding mode and structure-activity relationship of peptide SEQ ID NO.1 with TRPV1; A. Front and top views of the SEQ ID NO.1 / TRPV1 complex structure; B. Schematic diagram of residue interactions between SEQ ID NO.1 and TRPV1; CF. Detailed diagram of residue interactions between SEQ ID NO.1 and TRPV1; G. Inhibitory activity of alanine scanning analog and single-point mutation analog against TRPV1 at 1 μM concentration (n=6); H. Concentration-effect curves of selected mutants [E14A]SEQ ID NO.1 and [L15I]SEQ ID NO.1.

[0017] Figure 3 This is a schematic diagram illustrating the analgesic effect of SEQ ID NO.1 on rats with plantar incision pain; where A is the change curve of the paw withdrawal threshold (PWT) of rats with plantar incision pain over time; and B is the area under the curve (AUC) analysis of the plantar incision pain experiment.

[0018] Figure 4 This is a schematic diagram illustrating the analgesic effect of SEQ ID NO.1 on rats with chronic contractile injury; where A is the change curve of paw withdrawal threshold (PWT) over time in rats with chronic contractile injury; and B is the area under the curve (AUC) analysis of chronic contractile injury. Detailed Implementation

[0019] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0020] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0021] Example 1: Synthesis method of TRPV1-targeting peptide

[0022] Peptides targeting TRPV1 were synthesized manually on Rink amide resin using the standard Fmoc solid-phase peptide synthesis method (Table 1). Standard amino acid residues were activated with HCTU in the presence of N,N-diisopropylethylamine, and Fmoc deprotection was achieved by treating the resin with a 20% piperidine N,N-dimethylformamide solution for 30 minutes. The resin was swollen in DMF for 30 minutes prior to synthesis. The first amino acid was coupled to the resin using 4 equivalents of amino acid and 8 equivalents of DIPEA. The peptide chain was then assembled iteratively through cycles of Fmoc deprotection and amino acid coupling until completion. After peptide chain assembly and removal of the terminal Fmoc groups, the peptides were cleaved from the resin using a TFA / TIPS / H2O (10:5:5, v / v / v) mixture for 3 hours. Resin was then removed by filtration, and the peptides were washed with DCM. The filtrates were combined and concentrated, precipitated with cold diethyl ether, and centrifuged to obtain crude peptides, the molecular weight of which was confirmed by LC-MS. Crude peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 column. The mobile phases were buffer A (H₂O / CH₃CN / TFA, 10:10:0.05) and buffer B (CH₃CN / H₂O / TFA, 10:10:0.05). A linear gradient from 0% to 70% buffer B was used for 70 minutes, and peptide elution was monitored by UV absorption at 214 nm and 280 nm. Peptide purity was assessed by RP-HPLC. All final peptides were confirmed to have a purity >15% by mass spectrometry.

[0023] Table 1. Peptide sequences targeting TRPV1

[0024] ;

[0025] Note: * indicates C-terminal amination; in the computer-readable sequence listing, X in SEQ ID NO.15 represents Dab.

[0026] Example 2: Inhibitory activity test of TRPV1-targeting peptide

[0027] Whole-cell patch-clamp recordings were performed on HEK213T cells at room temperature (22–24°C). Extracellular recording solutions contained (mM): 135 NaCl, 5 KCl, 1 MgCl2, 10 HEPES, 10 glucose, 0.33 NaH2PO4, 5 sodium pyruvate, 5 mannitol, and 5 glucose monohydrate (pH adjusted to 7.4 with NaOH). Electrode intraelectrode solutions contained (mM): 130 CsCl, 5 MgCl2, 5 EGTA, 10 HEPES, and 4 Tris-ATP (pH adjusted to 7.2 with KOH). Recordings were performed using an Axopatch 200B amplifier controlled by Clampex 10 software and a Digidata 1550 data acquisition system. Series resistance and cell capacitance were compensated for approximately 80%. Current signals were filtered at 5.0 kHz and digitally acquired at 200 kHz.

[0028] The patch electrodes were drawn using a P-17 stretcher and polished using an MF-830 microforger, achieving a final resistance of 2–4 MΩ. Before recording, cells were equilibrated in a perfusion bath for 5–10 minutes. The solution was applied via a gravity-driven perfusion system at a rate of 2 mL / min, and the room temperature was maintained at 22–25°C. TRPV1 currents were induced by test pulses depolarizing from a holding potential of -80 mV to -10 mV (every 10 seconds). Peak current amplitudes were recorded before peptide application (I0, control solution only) and after peptide application (I_P, peptide dissolved in the perfusion solution). The percentage inhibition was calculated as: Inhibition rate (%) = (I0 - I_P) / I0 × 100%.

[0029] SEQ ID NO.1 exhibited the best inhibitory activity against the TRPV1 receptor, reaching over 80%. Next, this invention tested the inhibitory activity of SEQ ID NO.1 against the TRPV1 receptor under different concentration conditions, as follows: Figure 1 As shown, its IC50 is 73.13 nM.

[0030] Example 3: Study on the binding mode of SEQ ID NO.1 and TRPV1

[0031] To investigate the inhibitory mechanism of SEQ ID NO.1 on the TRPV1 receptor, this invention used AlphaFold3 to predict the structure of the complex of SEQ ID NO.1 and the TRPV1 receptor. Figure 2 The structure predicted by AlphaFold3 was optimized using molecular dynamics simulations. In the molecular dynamics simulations, the small and stable RMSD value of the SEQ ID NO.1-TRPV1 complex indicated that SEQ ID NO.1 and TRPV1 were stably bound throughout the simulation. The MD-optimized structure showed that, unlike the binding sites of hydrophobic small molecules such as capsaicin, cone snail toxin SEQ ID NO.1 stably embedded in the extracellular vestibular region of the TRPV1 channel, located near the pore entrance (…). Figure 2 (A)

[0032] To further characterize the SEQ ID NO.1 / TRPV1 interaction, an alanine scanning mutation analysis was performed. The results showed that the S4A, P6A, N11A, and H12A mutants almost completely lost their activity; the G1A, Y5A, and P13A mutants showed significantly reduced activity; while the Y10A, E14A, and L15A mutants had activity comparable to the wild type. Figure 2 (F). These functional changes are consistent with the structure-binding pattern. Specifically, S4, N11, H12, and Y5 in SEQ ID NO.1 form hydrogen bonds with TRPV1, and alanine substitutions at these sites significantly reduce activity (F). Figure 2 (CE). The positively charged G1 interacts electrostatically with the side chain of TRPV1 B chain D647. Mutation to alanine may disrupt this interaction by introducing steric hindrance, thereby reducing activity. Figure 2 (C). Since Y10 and L15 do not significantly interact with receptor amino acids, mutations at these sites to alanine do not cause a significant decrease in activity. Figure 2 (D, E). Although E14 forms a hydrogen bond with R617 of the TRPV1 C chain, its mutation to alanine may compensate for the loss of hydrogen bonds by potentially forming a hydrophobic interaction with P623 or reducing steric hindrance, thereby maintaining activity. Figure 2 (F). In addition, mutations in P6 and P13 to alanine may alter the secondary structure of SEQ ID NO.1, leading to a significant reduction in activity.

[0033] To elucidate the interactions mediated by large side-chain residues, this invention employs a conserved mutation strategy, replacing Y5, Y10, E14, and L15 with highly similar residues. Their inhibitory activity against TRPV1 was measured at a 1 μM concentration to assess the contribution of each residue's functional group to peptide activity. The mutation of Y5 to phenylalanine resulted in a significant decrease in activity, primarily due to the disruption of the crucial hydrogen bond between the Y5 hydroxyl group and TRPV1. Interestingly, the activity of the Y5F mutant was lower than that of Y5A, possibly because the smaller steric hindrance of alanine could partially compensate for the loss of tyrosine-mediated hydrogen bonds. Figure 2 The Y10 site does not significantly interact with the receptor; therefore, mutating to phenylalanine does not cause a significant decrease in activity. Figure 2 (D). The salt bridge between the E14Q mutant and R617 in the C chain is replaced by a hydrogen bond, and this mutation only leads to a slight decrease in activity. Figure 2 The presence of the negatively charged carboxyl group at this position (E14) highlights its functional importance. However, mutating E14 to diaminobutyric acid (Dab) not only deprives it of its ability to form hydrogen bonds but also results in a significant loss of activity due to unfavorable charge repulsion with R617 in the C chain. As predicted by the structural model, the L15I mutation did not alter the activity, confirming that this residue plays a minor role in binding. Figure 2 (F). Given that E14A and L15I showed minimal differences from SEQ ID NO.1 in the first two rounds of mutations, this invention further determined their IC50. 50 value( Figure 2 (H). The results were 85.27 ± 10.11 nM and 111 ± 10.22 nM, respectively, indicating a slight decrease in activity compared to SEQ ID NO.1. In summary, the alanine scan and site-directed mutagenesis results validated the reliability of the binding model.

[0034] Example 4: Analgesic effect of SEQ ID NO.1 on rat model of plantar incision pain.

[0035] The analgesic activity of SEQ ID NO.1 was determined in a rat model of plantar incision pain. Before the experiment, rats in identical condition were randomly divided into a positive control group, a treatment group, and a negative control group (n=6-8). Matched rats were intramuscularly injected with saline and 25 mg / kg pregabalin as negative and positive controls, respectively. To determine the degree of pain relief, changes in PWT were assessed at 0, 1, 2, 4, 6, 12, 24, and 48 h after injection of different concentrations of SEQ ID NO.1. The saline treatment group had no effect on mechanosensitivity, and PWT values ​​showed almost no significant change at the measurement time points. Compared with the negative control group, animals treated with pregabalin and SEQ ID NO.1 showed a significant increase in PWT (PWT) within 12 h. Figure 3In the treatment group rats, the analgesic effect of pregabalin and different concentrations of SEQ ID NO.1 reached its peak at 2 hours, then gradually weakened, and disappeared at around 12 hours. The high-dose group (250 μg / kg) showed significantly better analgesic effect than the positive control drug pregabalin (25 mg / kg). The high-dose SEQ ID NO.1 differed from pregabalin in molar mass, and the analgesic effect of SEQ ID NO.1 also showed a clear concentration-dependent trend, indicating that SEQ ID NO.1 can significantly increase the mechanical pain threshold in rats and exhibits a clear dose-dependent effect. Figure 3 (B) is of great significance in relieving postoperative pain.

[0036] Example 5: Analgesic effect of SEQ ID NO.1 on rats with chronic contractile injury.

[0037] CCI rats were randomly assigned to a positive control group, a negative control group, and a treatment group (n=6-8). SEQ ID NO.1 was administered intramuscularly to CCI rats at doses of 250 μg / kg, 25 μg / kg, and 2.5 μg / kg, respectively. Matched CCI rats were administered saline and 25 mg / kg pregabalin intramuscularly as negative and positive controls, respectively. To determine the degree of pain relief, changes in PWT were assessed at intervals of 0, 1, 2, 4, 6, 12, 24, and 48 hours post-injection. The saline treatment group had no effect on mechanosensitivity, and PWT values ​​showed almost no significant change at the measurement time points. Compared to the negative control group, animals treated with pregabalin and SEQ ID NO.1 showed significantly increased PWT (…). Figure 4 In CCI rats, the analgesic effects of both SEQ ID NO.1 and pregabalin peaked 2 hours after injection, then gradually diminished, eventually disappearing after 24 hours. SEQ ID NO.1 was more effective than pregabalin in reducing hyperalgesia, and its analgesic effect at an intramuscular dose of 250 μg / kg was significantly higher than that of pregabalin at an intramuscular dose of 25 mg / kg. These results indicate that SEQ ID NO.1 also possesses significant analgesic activity in neuropathic pain.

[0038] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide targeting TRPV1, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO. 1 of the sequence listing.

2. The polypeptide targeting TRPV1 of claim 1, characterized in that, The polypeptide is a mutant obtained by amino acid scanning mutation based on the sequence shown in SEQ ID NO. 1 of the sequence listing; the amino acid sequence of the mutant is shown in any one of SEQ ID NO. 6, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 16 of the sequence listing.

3. Use of a polypeptide targeting TRPVl according to claim 1 or 2, characterized in that: The polypeptide is used for preparing an analgesic drug.

4. An analgesic medicament, characterized by: The active ingredient of the analgesic drug comprises an effective dose of the TRPV1-targeting polypeptide of claim 1 or 2.

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