Conotoxin polypeptide with single disulfide bond and mutant and application of conotoxin polypeptide

Abstract

The invention belongs to the technical field of biological medicines, and relates to a spiro polypeptide with a single disulfide bond and a mutant and application thereof. The amino acid sequence of the conotoxin polypeptide is shown as SEQ ID NO.1 in a sequence table. One or more amino acid site-directed mutagenesis is carried out on the amino acid sequence of the conotoxin polypeptide to obtain a series of mutants. A plurality of mutants have better inhibitory activity and selectivity on an alpha9alpha10 acetylcholine receptor; wherein the IC50 of the M16R is 28.2 nM, and the IC50 of the F19R is 17.5 nM. In addition, M16R and F19R show in-vivo analgesic activity in a chemotherapeutic drug oxaliplatin induced rat crymodynia model, and have the potential of being developed into a neuropathic pain treatment drug targeting alpha9alpha10nAChR.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically a spiropolypeptide with a single disulfide bond, its mutants, and their applications. Background Technology

[0002] Nicotinic acetylcholine receptors (nAChRs) are ligand-gated pentamer ion channels widely distributed in the central and peripheral nervous systems and various non-neuronal tissues, mediating rapid synaptic signal transmission. Among the 17 known nAChR subunits (α1-α10, β1-β4, γ, δ, and ε), the α7, α9, and α10 subunits are unique, as they can assemble into homopentamers or form heteropentamers with other subunits. The homopentamer α7 nAChR is mainly distributed in the central nervous system and is crucial for learning and memory; its dysfunction is closely related to neurodegenerative diseases such as Alzheimer's and Parkinson's. The α9 and α10 nAChRs were initially identified as functional receptors in rat cochlear hair cells and subsequently heterologously expressed in Xenopus oocytes. This receptor has a unique structure within the nAChR family, being the only known heteropentamer composed of two different α subunits.

[0003] Early models considered the α9 subunit to be the functional core because it could form a homologous receptor, while the α10 subunit was considered a helper subunit without independent channel function. However, recent studies based on receptor mutagenesis and molecular chaperones have shown that the α10 subunit can also form a functional homopentamer. The stoichiometry of the α9α10 nAChR subunits is quite complex; in heterologous expression systems, specific assembly forms dominated by (α9)2(α10)3 and (α9)3(α10)2 can be achieved by regulating the injection ratio of the corresponding cRNAs.

[0004] Besides their physiological role in synaptic transmission in the auditory system, the widespread distribution of α9 and α10 nAChR makes them potential targets for treating neuropathic pain. In rat models, antagonism of this receptor subtype produces potent analgesic and anti-inflammatory effects, which may be related to its high expression in dorsal root ganglion (DRG) neurons and immune cells such as macrophages. Furthermore, α7 nAChR is overexpressed in macrophages and tumor cells, significantly regulating cholinergic anti-inflammatory pathways and tumorigenesis. Therefore, developing novel ligands targeting α7 and α9 / α10 nAChR is of great significance for advancing therapeutic strategies for related diseases.

[0005] Alpha-conotoxins, as a class of peptides highly selective for nAChRs, possess significant pharmacological research value. Various α- or α / O-conotoxins and their analogues (such as Vc1.1, RgIA, PeIA, Mr1.1, GeXVIA, ​​and Gex-2) exhibit analgesic activity by acting on the stereobinding sites of α9 and α10 nAChRs. Specifically, Vc1.1 preferentially binds to the α9(+)α9(-) interface in (α9)3(α10)2, while Gex-2 tends to bind to the α10(+)α10(-) interface in (α9)2(α10)3. In contrast, RgIA and PeIA exhibit inhibitory activity against both stoichiometric forms of the receptor because their binding α10(+)α9(-) interface is present in both assemblies. Mr1.1 interacts with both the α10(+)α9(-) and α9(+)α9(-) interfaces simultaneously via hydrogen bonds and salt bridges, stabilizing the channel in a closed state. GeXVIA not only binds to the α9(+)α10(-) interface, but its central pore edge region may also serve as a potential allosteric binding site.

[0006] These studies also raise a key paradox: since the actual stoichiometry of α9α10 nAChR in vivo is not yet clear, peptide drugs that depend on specific stoichiometric interfaces may face efficacy challenges in clinical applications. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides novel conodont polypeptides and their derivatives to resolve the issues of unclear mechanisms and poor selectivity in current α9α10 nAChR antagonists. This conodont polypeptide is a 20-amino acid polypeptide containing a single disulfide bond, capable of potently inhibiting α9α10 nAChR. Furthermore, this invention provides a series of site-directed mutants, among which preferred mutants achieve simultaneous enhancement of both α9α10 nAChR inhibitory activity and selectivity. These molecules, as non-stoichiometric channel blockers, show clear application prospects in the development of targeted drugs for neuropathic pain.

[0008] The present invention first provides a cone snail polypeptide with a single disulfide bond, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] To further enhance the inhibitory effect and selectivity of cone snail polypeptides against α9α10 nAChR, this invention also provides mutants of the cone snail polypeptides modified with single disulfide bonds. These mutants are obtained by performing one or more site-directed mutations on the amino acid sequence shown in SEQ ID NO.1 of the sequence listing. The mutation sites are: G1A, Y2A, L3A, R6A, P7A, R8A, R9A, M10A, R11A, H12A, R13A, K14A, R15A, M16A, L17A, F19A, C5AC20A, G1R, Y2R, L3R, A4R, P7R, M10R, H12R, K14R, M16R, L17R, A18R, and F19R. For mutants containing two cysteine ​​residues, a disulfide bond is formed between these two cysteine ​​residues; for mutants without cysteine ​​residues, the polypeptide is linear.

[0010] Furthermore, in addition to the above-mentioned site-directed amino acid mutations, the mutant is obtained by deleting two cysteine ​​residues or deleting four amino acids from the N-terminus of the amino acid sequence shown in SEQ ID NO.1 of the sequence listing; its amino acid sequences are shown in SEQ ID NO.19 and SEQ ID NO.20 of the sequence listing, respectively.

[0011] Furthermore, the present invention provides the use of the conopod polypeptide or its mutant pair in the preparation of formulations that inhibit acetylcholine receptors α7nAChR or α9α10 nAChR.

[0012] Furthermore, the present invention provides a formulation for inhibiting acetylcholine receptors α7 nAChR or α9 α10 nAChR, the formulation comprising an effective dose of the conopod polypeptide.

[0013] Furthermore, the present invention also provides the use of the formulation in the preparation of pain-relieving compositions, wherein the formulation relieves pain by inhibiting acetylcholine receptors α7 nAChR or α9 α10 nAChR.

[0014] Furthermore, the present invention also provides a pain-relieving composition comprising an effective dose of the conopod polypeptide, the mutant, or the formulation that inhibits acetylcholine receptors α7 nAChR or α9 α10 nAChR.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The conodont polypeptides provided in this invention act as use-dependent channel blockers for α9α10 and α7 nAChRs, a mechanism supported by their non-competitive kinetics and stoichiometric inhibition. Mutational and computational models reveal that their positively charged surfaces bind to the negatively charged pores of both receptors, with subtype selectivity (α9α10>α7) controlled by different electrostatic barriers in the receptor vestibular system. Utilizing this mechanistic insight, this invention has designed analogs with enhanced properties. Notably, [M16R] and [F19R] not only exhibit increased potency against α9α10 but also significantly improved selectivity against α7, making them valuable pharmacological probes and lead compounds. Their efficacy in an oxaliplatin-induced neuropathic pain model, characterized by a favorable duration of action, highlights their therapeutic potential. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the research and design route for an embodiment of the present invention; Figure 2 This invention presents a study on the mechanism of action of conopod polypeptide Qc-037 on the inhibitory effect of α9α10 nAChR, including competitive ligand experiments, receptor mutation studies, and chemometric studies. Specifically, A) the concentration-response relationship induced by Qc-037 co-treatment in α9α10 nAChR; B) a schematic diagram (red) of the high-affinity sites and mutant amino acid positions of the agonist ACh in the α10(+) α9(-) pockets predicted by AlphaFold3; C) the concentration-response relationship of ACh-induced current amplitude in wild-type and mutant hα9α10 nAChRs; and D) the concentration-response relationship of Qc-037 inhibiting the ACh-induced current amplitude in wild-type and mutant hα9α10 nAChRs with an α9 to α10 ratio of 3:1 or 3:1. Figure 3 This diagram illustrates the binding pattern of conodont peptide Qc-037 to the human α9α10 nAChR complex and the inhibition rate of Qc-037 and its analogues against human α9α10 nAChR at a concentration of 50 nM (n≥5). A represents the binding pattern of Qc-037 to the pore domain of α9α10 nAChR; α9 and α10 subunits are shown in magenta and green, respectively; Qc-037 is shown in orange. B represents the percentage inhibition of ACh-induced peak current amplitude by Ala-substituted Qc-037 analogues. C represents the percentage inhibition of ACh-induced peak current by Arg-substituted Qc-037 analogues. D represents the concentration-response relationship of Qc-037 and its analogues on ACh-induced current amplitude at α9α10 nAChR. 50The values ​​were 31.7±3.4, 17.1±4.3, 28.2±5.0, and 17.5±2.4 nM, respectively; values ​​are expressed as mean ± SEM, n=3-8. *P<0.05, **P<0.01, ****P<0.0001 compared with Qc-037 (one-way ANOVA); Figure 4 This diagram illustrates the binding pattern of the conodont peptide Qc-037 to the human α7 nAChR complex and the inhibition rate of Qc-037 and its analogues on human α7 nAChR at a concentration of 50 nM (n≥5). A) shows the binding pattern of Qc-037 and the pore domain binding sites of α7 nAChR; α7 nAChR is shown in cyan; Qc-037 is shown in pink. B) shows the percentage inhibition of ACh-induced peak current amplitude by Ala-substituted Qc-037 analogues. C) shows the percentage inhibition of ACh-induced peak current by Arg-substituted Qc-037 analogues. D) shows the concentration-response relationship of Qc-037 and its analogues on ACh-induced current amplitude at α7 nAChR, IC50. 50 The values ​​were 337.8±69.9, 130.2±16.1, 165.4±6.4, 289.0±65.2, 2753.8±148.3 and 1502.1±191.1 nM, respectively; values ​​are expressed as mean ± SEM, n=3-8; *P<0.05, ****P<0.0001 compared with Qc-037 (one-way ANOVA); Figure 5 The analgesic effects of [M16R] and [F19R] on oxaliplatin-induced cold hyperalgesia were evaluated. Specifically, A) the time course and area under the curve (AUC) of pain relief in acetone assays of [M16R] and [F19R] within 24 hours after intramuscular injection (IM); B) the latency of pain onset and AUC of pain relief in cold plate tests of [M16R] and [F19R] within 24 hours after IM injection. Values ​​are expressed as mean ± SEM, n = 6–8; **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to normal saline (one-way ANOVA). Detailed Implementation

[0017] 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.

[0018] 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.

[0019] Example 1: Design, synthesis and structure-activity relationship study of conopod polypeptide Qc-037 and its analogues like Figure 1 As shown, this invention uses a two-electrode voltage clamp to screen and report a monodisulfide-bridged cone snail toxin discovered from a transcriptome database. This toxin acts as a potent pore blocker of α9α10 nAChR. Structure-activity relationship studies yielded a toxin with enhanced inhibitory activity (IC50). 50 =17 nM) and peptide analogs with significantly improved subtype selectivity (approximately 100-fold). In an oxaliplatin-induced cold abnormal pain rat model, the optimized analogs M16R and F19R exhibited significant in vivo analgesic effects.

[0020] This invention provides a novel cone snail polypeptide sequence that can inhibit the activity of human α7 nAChR and α9α10 nAChR. The sequence is GYLACRPRRMRHRKRMLAFC* (SEQ ID NO.1), where * represents terminal amidation and a disulfide bond is formed between the two cysteine ​​residues. To investigate the structure-activity relationship between Qc-037 and human α7 nAChR and α9α10 nAChR, a complex model of Qc-037-hα7 nAChR and Qc-037-hα9α10 nAChR was first constructed using AlphaFold3, as follows: Figure 2 As shown. Subsequently, Pymol was used to modify the peptide to achieve C-terminal amidation; the specific interactions are as follows. Figure 3 , Figure 4 As shown.

[0021] In this invention, the rationality of the binding mode of the present invention was verified through competitive ligand experiments, receptor mutation and chemometric studies, and electrophysiological data of Qc-037 alanine and arginine scanning mutant analogs (SEQ ID NO.2-17 and SEQ ID NO.21-32): the conodont polypeptide Qc-037 binds to the pore regions of α7 nAChR and α9α10 nAChR. Structural analysis and mutation scanning results show that the conodont polypeptide Qc-037 acts on both α9α10 and α7 nAChR isoforms through a pore-blocking mechanism. To investigate the inhibitory mechanism of Qc-037 on α9α10 nAChR, this invention tested the concentration response curves of ACh at ten different concentrations (…). Figure 2 (A). IC Qc-037 50The values ​​remained almost constant, at 31.7 nM at 6 μM ACh and 33.1 nM at 60 μM ACh, indicating that its inhibitory effect was independent of agonist concentration. This agonist insensitivity suggests a non-competitive mechanism. To provide direct evidence that Qc-037 functions outside the ortho-binding site, this invention introduces triple mutations (α9-I61T, α10-W151T, α10-192T) at the high-affinity ACh binding interface α10(+)α9(-). Figure 2 (Middle B). For example Figure 2 As shown in Figure C, triple mutations in the α9α10 receptor shift the ACh concentration response curve to the right, reducing the half-maximum effective ACh concentration (EC50). 50 The concentration of ACh increased approximately twofold. This confirms that the mutation specifically disrupts the binding ability of ACh at the orthotopic site, while ACh can still activate the receptor through the remaining interface. Crucially, the inhibitory potency of Qc-037 remains unaffected, with both wild-type (WT) and mutant receptors maintaining their half-maximal inhibitory concentrations (IC50). 50 The values ​​are almost the same. Figure 2 (D). This result is consistent with the agonist competition data of the present invention, further confirming that Qc-037 does not compete with ACh for binding at the ortho-binding site. By controlling the injection ratio of cRNA, the present invention can obtain receptors with different stoichiometric compositions in oocytes. Specifically, the stoichiometric ratios of α9:α10 nAChRs expressed at α9:α10 cRNA ratios of 3:1 and 1:3 are (α9)3(α10)2 and (α9)2(α10)3, respectively. Comparison of concentration-response relationships obtained from oocytes with different α9:α10 nAChR subunit ratios shows that this stoichiometric conversion does not change the maximum inhibition of Qc-037 or other curve parameters (D). Figure 2 (Middle D). For WT receptor ratios of 3:1 and 1:3, Qc-037 inhibits the IC50 of ACh-induced current. 50 and Hill slope (n H The IC50 values ​​were 28.1 (1.2) and 30.8 nM (1.2), respectively. For the triple-mutant receptor, the IC50 values ​​were 3:1 and 1:3. 50 and n H The effective values ​​were 27.1 (1.5) and 33.4 nM (1.8), respectively. This stoichiometric efficacy, combined with previous findings of this invention, provides evidence of convergence, suggesting that Qc-037 functions at sites different from the ACh binding interface.

[0022] Its cyclic conformation allows it to interact extensively with residues in the pore lining, including key hydrogen bonds, van der Waals forces, and cation-π interactions. Further site-directed mutagenesis revealed several residues that contribute significantly to binding affinity and inhibitory activity, including arginine sites such as R6, R8, R9, R11, and R15, as well as P7 and H12, which maintain the conformation. Mutations at these sites (such as R6A and Y2A) lead to a significant decrease in activity, while mutations at other non-critical sites have a smaller impact on activity.

[0023] Table 1. Qc-037 and its series of mutants acting on α7 nAChR and α9α10 nAChR, along with their numbers, names, and amino acid sequences. ; * indicates terminal amidation.

[0024] Example 2: Synthesis method of cone snail toxin Qc-037 and its analogues 1. Synthesis of cone snail polypeptide Qc-037 and its mutant linear peptide All peptides involved in this invention were synthesized using solid-phase peptide synthesis (SPPS) and a Fmoc chemistry strategy. The peptides were synthesized using Rink-Amid resin (Loading: 0.637 mmol / g). The resin (0.1 mmol) was swollen in N,N-dimethylformamide (DMF) for 30 min. The Fmoc protecting groups on the resin were removed by treatment with 20% piperidine for 30 min. During coupling, 4 times the amount of Fmoc-protected amino acid, 4 times the amount of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 8 times the amount of N,N-diisopropylethylamine (DIPEA) were added, and the reaction was carried out at room temperature for 60 min. The peptides were washed three times repeatedly with DMF and dichloromethane (DCM). The deprotection-coupling cycle was repeated until the protecting group of the last amino acid was removed. The linear peptide was released from the resin by treatment with TFA cleavage buffer (TFA / H2O / TIPS, 90:5:5) at room temperature for 3 h. Then, an excess of cold ether was added, and a large amount of precipitate immediately formed. After centrifugation at 5000 rpm for 3 minutes, the supernatant was discarded to obtain the crude peptide.

[0025] 2. The disulfide bond oxidation process of cone snail polypeptide Qc-037 and its mutants: Conopod polypeptide Qc-037 and its mutants contain at most one pair of disulfide bonds. During the synthesis process, the protecting group on cysteine ​​is generally the Trt protecting group, and the disulfide bond can be constructed by air oxidation / iodine oxidation.

[0026] Air oxidation method: Dissolve the crude product in a 10% acetonitrile aqueous solution, sonicate to fully dissolve the peptides, adjust the pH to 8-10 with ammonium bicarbonate, add a magnetic stir bar, and expose to air and stir for 48 hours.

[0027] Iodine oxidation method: Dissolve the crude product in a 10% acetonitrile aqueous solution, sonicate to fully dissolve the peptide, slowly add 5 mg / mL iodine solution dropwise until the solution turns light yellow, then stop adding the solution and react in a shaker at room temperature for 20 min.

[0028] The above oxidation process requires real-time monitoring of the oxidation endpoint by LC-MS. After the reaction is complete, L-ascorbic acid aqueous solution is added to reduce the excess iodine.

[0029] 3. Purification and isolation of cone snail polypeptide Qc-037 and its mutants: The obtained crude peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The HPLC mobile phase composition was as follows: Phase A was 10% acetonitrile aqueous solution with 0.05% trifluoroacetic acid (TFA) added; Phase B was 10% aqueous acetonitrile solution with 0.05% TFA added. The mobile phase gradient was 100% mobile phase A uniformly changed to 50% mobile phase and 50% mobile phase B over 40 min. The semi-preparative column was 20*250 mm, 10 μM, and the analytical column was 4.6*250 mm, 5 μM.

[0030] The product peaks were collected and freeze-dried. The identity and purity of the peptides were confirmed by ESI-MS and HPLC, requiring the molecular weight to be consistent with the expected value and the purity to be ≥95%.

[0031] 4. In vitro activity test of cone snail polypeptide Qc-037 and its mutants Mature African clawed frogs in good condition were cryo-anesthetized by burying them deep in crushed ice. Mature oocytes were surgically obtained and placed in OR2 solution. The cell clusters were carefully mechanically and bluntly separated using ophthalmic forceps and digested with 2 mg / mL Clostridium collagenase. After the oocytes were fully dispersed, individual frog eggs were selected. Then, α7 or α9 and α10 cRNA were injected into the frog eggs in proportion using microinjection. The eggs were cultured at 18°C ​​for 2 days in ND96 solution containing antibiotics to express the corresponding receptors. After the dual-electrode voltage clamp device was calibrated and the corresponding receptor expression was confirmed to be successful, the recording electrode was inserted into the oocytes to activate the channel opening with the corresponding concentration of ACh. When the excitation current reached the corresponding amplitude and stabilized, the cells were incubated with the corresponding concentration of test solution for 5 min, and the current change was observed. The experiment was repeated three times and the results were recorded.

[0032] 5. In vivo analgesic activity test of cone snail polypeptide Qc-037 mutant All experiments were conducted using adult male Sprague-Dawley (SD) rats weighing 180–200 g, obtained from Jinan Pengyue Medical Experimental Animal Center (License SCXK 2019−0003). All experiments were performed in accordance with the guidelines of the Animal Ethics Committee of Ocean University of China (EMBWM-2024-14). Oxaliplatin-induced cold hyperalgesia was established by intraperitoneal injection of oxaliplatin (4 mg / kg) for two consecutive days. On day 3, behavioral tests validated the successful establishment of the model. Oxaliplatin was dissolved in 5% glucose, and the control group received the same amount of 5% glucose. Thirty model rats were randomly assigned to five groups: control group, saline group, RgIA4 group, and Qc-037 group. The peptide dissolved in saline was administered via subcutaneous injection in the back. Acetone scoring was used to assess cold hyperalgesia. After acclimatizing to their environment for 30 minutes on a metal frame with a transparent organic box, rats were rapidly sprayed with 100 μL of acetone using an insulin needle onto their hind paws. The responses of 60 rats within a 5-second timeframe were observed and scored: 0 - no response; 0.5 - scanning the paw; 1 - withdrawing or raising the paw; 1.5 - scratching, licking, or bending the paw; 2 - rapid withdrawal of the paw; 3 - prolonged grasping of the paw; 4 - shaking the paw; 5 - extending and licking the paw. Each hind paw was measured three times, with an interval of at least 10 minutes between measurements. Cold plate assay. The cold plate assay was performed. Initially, the baseline latency was measured within 180 seconds at 15°C before oxaliplatin injection. Subsequently, the ability of the tested peptide to react on a cold plate was assessed at 4, 8, 12, and 24 hours post-injection.

[0033] Test results are as follows Figure 5 As shown, in a rat model of oxaliplatin-induced cold abnormal pain, the optimized analogues M16R and F19R exhibited significant in vivo analgesic effects. This study not only identified a new class of α9α10 nAChR inhibitors but also provides promising lead compounds for the treatment of chemotherapy-induced neuropathic pain.

[0034] The above description represents the preferred embodiment 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 described in the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A conopod polypeptide with a single disulfide bond, characterized in that: Its amino acid sequence is shown in SEQ ID NO.1 of the sequence listing.

2. A cone snail polypeptide mutant, characterized in that, The mutant is obtained by performing one or more site-directed mutations on the amino acid sequence shown in SEQ ID NO.1 of the sequence listing; the mutation sites are selected from: G1A, Y2A, L3A, R6A, P7A, R8A, R9A, M10A, R11A, H12A, R13A, K14A, R15A, M16A, L17A, F19A, C5AC20A, G1R, Y2R, L3R, A4R, P7R, M10R, H12R, K14R, M16R, L17R, A18R, F19R.

3. The cone snail polypeptide mutant according to claim 2, characterized in that, For mutants containing two cysteine ​​residues, these two cysteine ​​residues form a disulfide bond; for mutants without cysteine ​​residues, they are linear polypeptides.

4. A cone snail polypeptide mutant, characterized in that, The mutant is obtained by deleting two cysteine ​​residues or four amino acids from the N-terminus of the amino acid sequence shown in SEQ ID NO.1 of the sequence listing; the amino acid sequences are shown in SEQ ID NO.19 and SEQ ID NO.20 of the sequence listing, respectively.

5. The use of the cone snail polypeptide of claim 1 or the mutant of any one of claims 2-4 in the preparation of formulations that inhibit acetylcholine receptors α7 nAChR or α9 α10 nAChR.

6. A formulation for inhibiting acetylcholine receptors α7 nAChR or α9 α10 nAChR, characterized in that, The formulation contains an effective dose of the cone snail polypeptide of claim 1 or the mutant of any one of claims 2-4.

7. The use of the formulation of claim 6 in the preparation of a pain-relieving composition, characterized in that: The formulation relieves pain by inhibiting acetylcholine receptors α7 nAChR or α9 α10 nAChR.

8. A composition for relieving pain, characterized in that, The composition comprises an effective dose of the cone snail polypeptide of claim 1, the mutant of any one of claims 2-4, or the formulation of claim 6 that inhibits acetylcholine receptor α7nAChR or α9α10 nAChR.

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