WO2024099103A1

WO2024099103A1 - Polypeptide having analgesic effect and use thereof

Info

Publication number: WO2024099103A1
Application number: PCT/CN2023/127050
Authority: WO
Inventors: 杨帆; 张恒
Original assignee: 浙江大学
Priority date: 2022-11-09
Filing date: 2023-10-27
Publication date: 2024-05-16
Also published as: CN118005744A

Abstract

A polypeptide having an analgesic effect and a use thereof. The polypeptide or a construct thereof can be used as a positive allosteric modulator and used in combination with a TRPV1 receptor agonist to enhance an agonistic effect on TRPV1. Administration of the polypeptide or the construct thereof induces agonism of TRPV1 in the presence of a trace amount of the TRPV1 receptor agonist.

Description

Peptide with analgesic effect and its application

Cross-reference to related applications

This patent application claims priority to the Chinese patent application filed on November 9, 2022, with application number 2022113985434 and invention name “Polypeptides with analgesic effect and their applications”. The full text of the above application is incorporated herein by reference.

Technical Field

The present invention relates to the field of biomedicine, and in particular to a polypeptide with analgesic effect and application thereof.

Background technique

Chronic pain not only greatly reduces the quality of life of patients, but also brings huge socioeconomic costs. For example, more than 100 million adults suffer from chronic pain, and the annual economic cost in the United States alone is as high as 600 billion US dollars. Although traditional analgesics, such as opioids and nonsteroidal anti-inflammatory drugs (NSAIDs), have been widely used to treat chronic pain, they either show strong side effects or relatively low efficacy. Therefore, new analgesics with alternative mechanisms of action are needed.

Transient receptor potential vanilloid receptor type 1 (TRPV1) is a multimodally activated, calcium-permeable, non-selective cation channel that is widely expressed in neurons involved in pain perception and plays an important role in pain perception. As a classic analgesic target, TRPV1 antagonists have been the first choice for developing TRPV1-targeted analgesics in the past few decades, although pharmacological inhibition of TRPV1 can effectively relieve oral pain, rectal pain, and thermal pain. However, since TRPV1 also plays an important role in thermoregulation, TRPV1 inhibitors have failed in preclinical studies because of the side effects of hyperthermia and burns in patients. TRPV1 agonists are also used as analgesics: its agonist resiniferatoxin (RTX) induces calcium overload and causes cell death of neurons expressing TRPV1, which helps treat intractable cancer pain, but causes irreversible damage to neurons, which limits its application. In addition, RTX is extracted from resin euphorbia, and currently resin euphorbia is an endangered species, so the source of RTX becomes a limiting factor. Therefore, it is necessary to develop new TRPV1 modulators to better treat pain.

Summary of the invention

The object of the present invention is to provide a polypeptide or a construct thereof.

Another object of the present invention is a pharmaceutical composition comprising the polypeptide or a construct thereof.

Another object of the present invention is the use of the polypeptide or its construct or pharmaceutical composition.

In order to solve the above technical problems, the present invention provides a polypeptide or a construct thereof in the first aspect, wherein the polypeptide has a structure shown in the following formula I:
X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇ -X ₈ -X ₉ -X ₁₀ -X11 -X ₁₂ -X ₁₃ -X ₁₄ -X ₁₅ -X ₁₆ -X ₁₇ -X ₁₈ -X ₁₉ -X ₂₀ -X ₂₁ --X ₂₂ -X ₂₃ ,
Formula I;

wherein X ₂ , X ₇ , X ₁₃ and X ₂₀ are independently C;

X ₁₂ , X ₁₈ and X ₂₂ are all basic amino acids or Q;

X ₁ , X ₂₁ and X ₂₃ are independently none, any amino acid or any peptide segment;

_X3 , _X4 , _X5 , _X6 , X8, _X9 , _X10 , _X11 , _X14 , _X15 , _X16 _, _X17 and _X19 are independently any amino acid or any peptide segment.

In some preferred embodiments, the length of the peptide segment is 1-10 aa, more preferably 1-5 aa, more preferably 1-3 aa, such as 1 aa.

In some preferred embodiments, X ₁₂ , X ₁₈ and X ₂₂ are all basic amino acids;

In some preferred embodiments, X ₁₂ , X ₁₈ and X ₂₂ are independently selected from R, H, K or Q.

In some preferred embodiments, the polypeptide has a structure shown in the following formula II;
_X1 - _X2 -NGVT-X7 _- PSGY- _X12 - _X13- SIVD- _X18 - _QX20 - _X21 - _X22 - _X23 , Formula II;

wherein X ₂ , X ₇ , X ₁₃ and X ₂₀ are independently C;

_X1 is none, any amino acid or any peptide;

X ₂₁ is none, any amino acid or any peptide;

X ₂₃ is none, any amino acid or any peptide;

X ₁₂ , X ₁₈ and X ₂₂ are independently selected from R, H and K, respectively.

In some preferred embodiments, _X12 is R, _X18 is K, and _X22 is K.

In some preferred embodiments, _X1 is nothing or any amino acid.

In some preferred embodiments, _X21 is none or any amino acid.

In some preferred embodiments, _X23 is none or any peptide segment.

In some preferred embodiments, _X1 is P.

In some preferred embodiments, _X21 is 1.

In some preferred embodiments, _X23 is KE.

In some preferred embodiments, the polypeptide has a structure shown in the following formula III:
PX2 _- NGVT- _X7- PSGY- _X12 - _X13- SIVD- _X18 - _QX20 - _IX22 -KE, Formula III;

wherein X ₂ , X ₇ , X ₁₃ and X ₂₀ are independently C;

_X12 is R, _X18 is K, and _X22 is K.

In some preferred embodiments, in the polypeptide, _X2 and _X13 form a disulfide bond with each other.

In some preferred embodiments, in the polypeptide, _X7 and _X20 form a disulfide bond with each other.

In some preferred embodiments, the polypeptide is a linear peptide.

In some preferred embodiments, the linear peptide forms two pairs of disulfide bonds.

In some preferred embodiments, the polypeptide is a cyclic peptide.

In some preferred embodiments, the polypeptide is selected from any one of the following groups:

(a) having the amino acid sequence shown in SEQ ID NO.1;

(b) having an amino acid sequence with greater than 95% homology to that shown in SEQ ID NO.1; and

(c) having an amino acid sequence complementary to the amino acid sequence described in (a) or (b).

In some preferred embodiments, the polypeptide is a chemically synthesized polypeptide.

In some preferred embodiments, the polypeptide is a recombinantly expressed polypeptide.

The second aspect of the present invention provides a polynucleotide comprising a polypeptide or polypeptide construct encoding the polypeptide or polypeptide construct as described in the first aspect of the present invention.

In some preferred aspects, the polynucleotide is an isolated polynucleotide.

In some preferred embodiments, the polynucleotide is optimized for synonymous codon preference.

In some preferred embodiments, the polynucleotide has the sequence shown below:

(a) a polynucleotide sequence having a sequence as shown in SEQ ID NO. 33 or 34;

(b) having a polynucleotide sequence having a homology greater than 80% (preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, more preferably greater than 97%) to the sequence shown in SEQ ID NO. 33 or 34; and

(c) a polynucleotide sequence having a complementary amino acid sequence to the amino acid sequence described in (a) or (b).

The third aspect of the present invention provides a vector, wherein the vector comprises the polynucleotide as described in the second aspect of the present invention.

In some preferred embodiments, the vector is an expression vector.

In some preferred embodiments, the expression vector is a prokaryotic expression vector, such as an Escherichia coli expression vector.

In some preferred embodiments, the expression vector is a eukaryotic expression vector, such as a yeast expression vector, an insect expression vector or mammalian cell expression vector.

In some preferred embodiments, the vector is a plasmid.

The fourth aspect of the present invention provides a host cell, wherein the host cell comprises the vector as described in the third aspect of the present invention or the polynucleotide as described in the second aspect of the present invention is integrated into the chromosome of the host cell.

In some preferred embodiments, the host cell is Escherichia coli, yeast cell or mammalian cell. In some preferred embodiments, the host cell is Escherichia coli, CHO cell or the like.

A fifth aspect of the present invention provides a pharmaceutical composition, comprising:

The polypeptide or polypeptide construct according to the first aspect of the present invention; and a pharmaceutically acceptable carrier or excipient.

In some preferred embodiments, the composition is administered by a mode of administration selected from the group consisting of intravenous, intratumoral, intracavitary, subcutaneous or hepatic arterial administration (such as injection, drip, etc.).

In some preferred embodiments, the pharmaceutical composition is in the form of an oral preparation, an injection preparation or an external preparation.

In some preferred embodiments, the dosage form of the pharmaceutical composition is a solid preparation, a liquid preparation or a semisolid preparation.

In some preferred embodiments, the preparation of the pharmaceutical composition is selected from the following group: tablets, capsules, injections, granules, sprays, and lyophilized agents.

In some preferred embodiments, the pharmaceutical composition is in the form of an injection.

In some preferred embodiments, the injection is an intravenous injection, an intramuscular injection or a subcutaneous injection.

In some preferred embodiments, the polypeptide is administered to a mammal at a dose of 0.01-100 mg/kg body weight (once or every day).

The sixth aspect of the present invention provides a use of the polypeptide or polypeptide construct according to the first aspect of the present invention, or the pharmaceutical composition according to the fifth aspect of the present invention, for at least one of the following uses:

(i) for relieving pain; preferably, for long-term analgesia;

(ii) for the preparation of a medicament for the relief of pain;

(iii) for enhancing the agonistic effect of TRPV1 receptor agonists on TRPV1;

(iv) acts as a positive allosteric modulator of TRPV1 channels;

(v) for specifically binding to the E649 site and/or the E652 site of the TRPV1 protein;

(vi) for inducing death of nerve terminal cells expressing TRPV1; and

(vii)) is used to prolong the desensitization process of TRPV1 channels.

In some preferred embodiments, the pain is pain associated with the TRPV1 channel.

In some preferred embodiments, the pain is neuropathic pain. In some embodiments, the neuropathic pain is diabetic peripheral neuropathy pain. In some embodiments, the pain is post-herpetic neuralgia. In some embodiments, the pain is trigeminal neuralgia.

In some preferred embodiments, the subject suffers from pain associated with or caused by a strain, sprain, arthritis or other joint pain, bruise, back pain, fibromyalgia, endometriosis, surgery, migraine, cluster headaches, psoriasis, irritable bowel syndrome, chronic interstitial cystitis, vulvodynia, trauma, musculoskeletal disease, herpes zoster, sickle cell disease, heart disease, cancer, stroke, or oral ulcers or ulcers caused by chemotherapy or radiation therapy.

A seventh aspect of the present invention provides a method for relieving pain, the method comprising the steps of:

A therapeutically effective amount of the polypeptide or construct thereof according to the first aspect of the present invention, or the pharmaceutical composition according to the fifth aspect of the present invention is administered to the subject.

In an eighth aspect of the present invention, there is provided a method for screening a drug for relieving pain, the method comprising the steps of:

Screening for drugs that can specifically bind to the E649 site and/or the E652 site of the TRPV1 protein.

In some preferred embodiments, the drug forms hydrogen bonds with site E649 and/or E652.

In some preferred embodiments, the method comprises the steps of:

Screening for drugs that can specifically bind to the E649 site and/or the E652 site of the TRPV1 protein and do not specifically bind to the L461 site and the T634 site of the TRPV1 protein.

In some preferred embodiments, the method comprises the steps of:

Screen for drugs that can specifically bind to the E649 site and/or E652 site of the TRPV1 protein, and do not specifically bind to the L461 site, T634 site, K604 site, and K657 site of the TRPV1 protein.

In some preferred embodiments, the drug is a RhTx derivative.

In some preferred embodiments, the drug is at least one, preferably 1 to 10, preferably 1-5, and more preferably 1-3 amino acids in the RhTx sequence that are substituted or deleted, or at least one, or at least two, or at least three identical or different amino acids are inserted into any two amino acid sequences of the RhTx protein to form a polypeptide.

Compared with the prior art, the present invention has at least the following advantages:

(1) The polypeptide or its construct provided in the present invention can be used as a positive allosteric modulator, and can be used in combination with a TRPV1 receptor agonist to enhance the agonism of TRPV1; preferably, the polypeptide or its construct provided in the present invention is administered in the presence of a trace amount of a TRPV1 receptor agonist to induce the agonism of TRPV1;

(2) The polypeptide or its construct provided in the present invention does not directly activate or inhibit the TRPV1 target, but plays the role of enhancing the TRPV1 target agonist. On the basis of having a significant analgesic effect, it has little toxicity and side effects on biological tissues and is safe. high;

(3) The polypeptide or its construct provided in the present invention can target TRPV1 to exert a long-lasting analgesic effect;

(4) The polypeptide of the present invention and its derivative polypeptides have a small molecular weight and are easy to prepare;

(5) The polypeptide of the present invention and its derivative polypeptides have good stability and the polypeptide of the present invention has high specificity.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

FIG1 is a diagram showing the sequences of RhTx and s-RhTx according to an embodiment of the present invention, with the s-RhTx sequence marked in cyan; the lower diagram showing the structural comparison of RhTx (PDB ID: 2MVA, gray, the first three amino acids at the N-terminus are marked in blue) and s-RhTx (marked in cyan, disulfide bonds are marked in orange);

FIG2 is a diagram showing the electrostatic potential energy (kcal/mol/e) of s-RhTx according to an embodiment of the present invention, where positive charges are in blue and negative charges are in red;

FIG3 is a diagram of the reverse phase chromatography purification of s-RhTx according to an embodiment of the present invention, and gradient elution with acetonitrile, where the polypeptide peak corresponds to 31% acetonitrile;

FIG4 is a graph showing the molecular weights of linear s-RhTx (left) and oxidative folded s-RhTx (right) by mass spectrometry according to an embodiment of the present invention;

FIG5 shows that s-RhTx cannot directly activate TRPV1 according to an embodiment of the present invention;

FIG6 shows that s-RhTx dose-dependently enhances the activation effect of 10 nM capsaicin on TRPV1 channels according to an embodiment of the present invention;

FIG. 7 is a concentration effect curve of s-RhTx on TRPV1 channel in the presence of 10 nM capsaicin according to an embodiment of the present invention, fitted by the Hill equation (n=4).

FIG8 is a calcium imaging measurement of HEK293T cells expressing TRPV1 according to an embodiment of the present invention, and the scale bar is 100 μm;

FIG9 is a graph showing the calcium fluorescence variation of the cells in FIG7 according to an embodiment of the present invention (n=6);

FIG. 10 shows that s-RhTx dose-dependently enhances the activation of TRPV1 by protons according to an embodiment of the present invention;

FIG. 11 is a graph showing the relationship between s-RhTx and TRPV1 in the presence of protons (pH 6.5) according to an embodiment of the present invention, which is fitted by the Hill equation. Concentration effect curves of the channels (n=4).

FIG12 is a thermal activation curve of TRPV1 in the presence of s-RhTx or RhTx according to an embodiment of the present invention;

FIG13 is a box plot of the thermal activation threshold of TRPV1 in the presence of s-RhTx or RhTx according to an embodiment of the present invention (n=4-11, **, p<0.01; N.S., no statistical difference);

FIG14 is a graph showing the effect of s-RhTx on TRPV2 in the presence of 500 μM 2-APB according to an embodiment of the present invention. The left graph shows the statistics of the current intensity in the left graph.

FIG15 is a graph showing the effect of s-RhTx on TRPV3 in the presence of 30 μM 2-APB according to an embodiment of the present invention; the right graph shows the statistics of the current intensity in the left graph;

FIG16 is a graph showing the effect of s-RhTx on TRPA1 in the presence of 30 μM AITC in accordance with an embodiment of the present invention; the graph on the right shows the statistics of the current intensity in the graph on the left;

FIG. 17 is a statistical diagram of the enhancement factor of s-RhTx on TRPV1, TRPV2, TRPV3 and TRPA1 channel agonists according to an embodiment of the present invention;

FIG18 is a diagram showing the desensitization process of TRPV1 channels induced by 10 μM capsaicin or 10 μM capsaicin and 50 μM s-RhTx at +80 mV and -80 mV according to an embodiment of the present invention;

FIG. 19 is a statistical diagram of the time constant τ of the TRPV1 desensitization process in FIG. 18 according to an embodiment of the present invention (n=9-10; *, p<0.05; ***, p<0.001);

FIG20 is a statistic of the area under the curve in FIG18 according to an embodiment of the present invention (n=9-10; *, p<0.05; ***, p<0.001);

21 is a photograph of Hoechst-PI staining of HEK293T cells transfected with TRPV1 channels and stimulated with s-RhTx or capsaicin for 18 hours according to an embodiment of the present invention, wherein Hoechst marks the nuclei of all cells and PI marks the nuclei of dead cells;

FIG22 is a statistical diagram of FIG21 according to an embodiment of the present invention;

FIG23 shows that s-RhTx acts on the extracellular region of TRPV1 channels in inside-out (left) and outside-out recording modes according to an embodiment of the present invention;

FIG. 24 is a molecular docking of s-RhTx and TRPV1 channel (PDB ID: 3j5p) according to an embodiment of the present invention. s-RhTx is colored with the electrostatic potential (red represents negative charge, blue represents positive charge). The skeleton of TRPV1 is marked with cyan, and the electron density map of TRPV1 is superimposed on the TRPV1 skeleton. The left and right figures are the top view and side view of the binding of s-RhTx and TRPV1, respectively;

FIG25 is a detailed diagram showing the interaction between s-RhTx and the outer side of the TRPV1 pore region according to an embodiment of the present invention, wherein hydrogen bonds are indicated by red dashed lines;

FIG26 shows the responses of the TRPV1 point mutation to 10 nM capsaicin (a), a mixture of 10 nM capsaicin and 10 μM s-RhTx (b), and 10 μM capsaicin (c) when the clamping potential is +80 mV according to an embodiment of the present invention;

FIG27 shows the ratio of the current induced by a mixture of 10 nM capsaicin and 10 μM s-RhTx to the current induced by 10 nM capsaicin on a TRPV1 point mutation according to an embodiment of the present invention (n=6-10);

FIG28 is a statistical diagram of the desensitization time constant of s-RhTx to TRPV1 point mutations according to an embodiment of the present invention (n=4-6; *, p<0.05; **, p<0.01);

FIG29 is a calcium imaging measurement of HEK293T cells expressing TRPV1 E649A, E652K and E652D point mutations under different stimuli according to an embodiment of the present invention, and the scale bar is 100 μm;

FIG30 is a statistical diagram of changes in calcium fluorescence signals in the calcium imaging assay of FIG29 according to an embodiment of the present invention (n=13-45);

FIG31 is a concentration effect curve of s-RhTx on TRPV1 E652D mutation in the presence of 10 nM capsaicin according to an embodiment of the present invention;

Figure 32 is a concentration effect curve of s-RhTx and s-RhTx R12Q mutation in WT TRPV1 and TRPV1 E652D mutation respectively according to an embodiment of the present invention;

FIG33 is a diagram showing the ligand binding constant represented by Kd during the gating process of ion channels according to an embodiment of the present invention. The Kd value can be calculated from the concentration effect curve of FIG32 (two-tailed t-test, *, p<0.05; **, p<0.01; n=5-8);

FIG34 is a diagram showing the equilibrium constant of the channel from the closed state to the open state after ligand binding in the gating process of the ion channel according to an embodiment of the present invention, represented by L. The L value can be calculated from the concentration effect curve of FIG32 (two-tailed t test, *, p<0.05; **, p<0.01; n=5-8);

Figure 35 is a concentration effect curve of s-RhTx and s-RhTx R12K mutation in WT TRPV1 and TRPV1 E652D mutation respectively according to an embodiment of the present invention;

FIG36 is a Kd value statistic calculated from the concentration effect curve of FIG33 according to an embodiment of the present invention (two-tailed t-test, *, p<0.05; **, p<0.01; ***, p<0.001; n=5-8);

FIG37 is a graph showing L value statistics calculated from the concentration effect curve of FIG34 according to an embodiment of the present invention (two-tailed t-test, *, p<0.05; **, p<0.01; ***, p<0.001; n=5-8);

Figure 38 is a concentration effect curve of s-RhTx and s-RhTx K22R mutation in WT TRPV1 and TRPV1 E652D mutation respectively according to an embodiment of the present invention;

FIG39 is a graph showing Kd value statistics calculated from the concentration effect curve of FIG38 according to an embodiment of the present invention (two-tailed t-test, *, p<0.05; **, p<0.01; N.S., no statistical difference; n=5-8);

FIG40 is a graph showing L value statistics calculated from the concentration effect curve of FIG38 according to an embodiment of the present invention (two-tailed t-test, *, p<0.05; **, p<0.01; NS, no statistical difference; n=5-8);

Figure 41 is a concentration effect curve of s-RhTx and s-RhTx K22Q mutation in WT TRPV1 and TRPV1 E652D mutation respectively according to an embodiment of the present invention;

FIG42 is a graph showing Kd value statistics calculated from the concentration effect curve of FIG41 according to an embodiment of the present invention (two-tailed t-test, **, p<0.01; ***, p<0.001; ****, p<0.0001; n=5-10);

FIG43 is a graph showing L value statistics calculated from the concentration effect curve of FIG41 according to an embodiment of the present invention (two-tailed t-test, **, p<0.01; ***, p<0.001; ****, p<0.0001; n=5-10);

FIG44 is a summary of binding energies calculated according to an embodiment of the present invention;

FIG45 is an enlarged view of the interaction between R12 and E652 (left) and K22 and E652 (right) according to an embodiment of the present invention, wherein hydrogen bonds are indicated by black dashed lines;

Figure 46 is an example of the present invention; the effect of s-RhTx on spontaneous pain in mice induced by capsaicin. Within 10 minutes after the injection of saline (20 μl) containing 200 ng capsaicin and 2 μg oxidative folded s-RhTx or linear s-RhTx, the statistics of the time of licking, lifting and withdrawing the paw of mice per minute were performed (n=6-11, *, p<0.05, two-way ANOVA);

FIG47 is a statistical diagram of the total time of mouse licking paws, lifting paws and withdrawing paws within 10 minutes after injection of the above solution according to an embodiment of the present invention (n=6-11, *, p<0.05; N.S., no statistical difference; two-way ANOVA);

Figure 48 shows the effect of s-RhTx on thermal pain in mice according to an embodiment of the present invention. After injection of saline containing 200ng and 2μg of folded s-RhTx or linear s-RhTx, the paw withdrawal reaction time of mice to thermal stimulation (n=5-6, *, p<0.05, two-way ANOVA);

Figure 49 is an immunohistochemical image of the distribution of TRPV1-positive (TRPV1 ⁺ ) nerve endings after injection of physiological saline containing 200 ng and 2 μg of folded s-RhTx or linear s-RhTx according to an embodiment of the present invention, the scale bar is 100 μm, wherein the mice are Trpv1-Ai32 transgenic mice, and their TRPV1 is labeled with EYFP. Each star-shaped mark represents a nerve ending;

FIG50 is a statistical diagram of the number of nerve endings in FIGd according to an embodiment of the present invention (one-way ANOVA, *, p<0.05; n=3-4);

FIG51 is a schematic diagram showing that s-RhTx enhances TRPV1 channel activity and promotes degeneration of intradermal nerve endings to exert a long-term analgesic effect according to an embodiment of the present invention;

FIG52 is a schematic diagram of the timeline of the establishment of a CFA-induced mouse chronic inflammatory pain model and experimental operation according to an embodiment of the present invention. First, 20 μl CFA was injected to induce chronic inflammatory pain. One day after the CFA injection, physiological saline containing 200 ng and 2 μg of folded s-RhTx or linear s-RhTx was injected;

FIG53 is the effect of oxidative folded s-RhTx on thermal hyperalgesia according to an embodiment of the present invention. BL, basal value. n=6-9, *or#, p<0.05 (two-way ANOVA), compared with mice injected with capsaicin and oxidative folded s-RhTx, respectively;

FIG54 is the effect of oxidative folded s-RhTx on mechanical allodynia according to an embodiment of the present invention. BL, basal value. n=6-9, *or#, p<0.05 (two-way ANOVA), compared with mice injected with capsaicin and oxidative folded s-RhTx, respectively;

FIG55 is the effect of s-RhTx on the body temperature of mice according to an embodiment of the present invention. The changes in the rectal temperature of mice over time after injection of 200 ng capsaicin or 200 ng capsaicin and 2 μg oxidative folded s-RhTx or linear s-RhTx in normal saline. Two-way ANOVA showed no statistically significant difference;

FIG. 56 shows that 100 μM linear s-RhTx cannot enhance the activation effect of low concentration capsaicin on TRPV1 according to an embodiment of the present invention;

FIG57 is a comparison of the enhancing effect of 100 μM linear s-RhTx and 10 μM folded s-RhTx on low concentration capsaicin according to an embodiment of the present invention;

FIG. 58 is a graph showing the effect of linear s-RhTx on changes in intracellular calcium fluorescence signals of cells expressing TRPV1 channels for calcium imaging experiments according to an embodiment of the present invention;

FIG. 59 shows the change in intracellular calcium fluorescence signal intensity over time in calcium imaging assay according to an embodiment of the present invention.

Detailed ways

In the prior art, inhibitors or agonists targeting TRPV1 have been studied as analgesic drugs, but all have serious side effects. In the present invention, a modulator targeting the outside of the TRPV1 pore region is designed and developed, while previously reported TRPV1-activating toxins, such as DkTx, BmP01, RhTx and RhTx2, all bind to the outside of the pore region of the channel and do not directly activate or inhibit the TRPV1 target, but can enhance the role of TRPV1 target agonists, thereby achieving significant analgesic effects and avoiding the side effects of TRPV1 inhibitors or agonists.

In the present invention, the inventors first rationally designed a polypeptide s-RhTx based on RhTx, which targets the outside of the TRPV1 pore area and can serve as a positive allosteric regulator of TRPV1. s-RhTx no longer activates TRPV1. It selectively enhances the TRPV1 current induced by the TRPV1 agonists capsaicin and protons in a concentration-dependent manner, slows down the desensitization process of TRPV1, but does not change the thermal activation threshold of TRPV1. At the molecular level, the inventors proved that R12 and K22 of s-RhTx specifically bind to the E652 residue of different subunits of TRPV1 through thermodynamic double mutation cycle analysis. In in vivo experiments, the inventors found that a mixture of low concentrations of capsaicin and s-RhTx has a significant and lasting analgesic effect in normal and inflammatory model mice in vivo by promoting the degeneration of TRPV1-positive intraepidermal nerve fibers (IENF). Based on this, the present invention is completed.

In addition, the inventors studied the effect of the polypeptide s-RhTx on TRPV1 and developed s-RhTx derivative polypeptides having similar effects to the s-RhTx of the present invention.

Polypeptide or its construct

In the present invention, "polypeptide" refers to a polypeptide having the structure shown in the following formula I:
X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇ -X ₈ -X ₉ -X ₁₀ -X11 -X ₁₂ -X ₁₃ -X ₁₄ -X ₁₅ -X ₁₆ -X ₁₇ -X ₁₈ -X ₁₉ -X ₂₀ -X ₂₁ --X ₂₂ -X ₂₃ ,
Formula I;

wherein X ₂ , X ₇ , X ₁₃ and X ₂₀ are independently C;

X ₁₂ , X ₁₈ and X ₂₂ are all basic amino acids or Q;

In the present invention, the polypeptide spontaneously forms disulfide bonds at _X2 , _X7 , _X13 and _X20 , thereby coordinating with the TRPV1 channel in terms of stereostructure, and the amino acids at the _X12 , _X18 and _X22 sites are positively charged, that is, the amino acids at the _X12 , _X18 and _X22 sites are all basic amino acids, which can bind to the key sites 649 and 652 of the TRPV1 channel; alternatively, the amino acids at the _X12 , _X18 and _X22 sites are hydrophilic amino acids (such as Q), which can hydrogen bond to the key sites 649 and 652 of the TRPV1 channel.

In the present invention, _X1 is not LNNP.

In some embodiments, the peptide segment has a length of 1-10 aa, more preferably 1-5 aa, more preferably 1-3 aa, such as 1 aa.

When _X3 , _X4 , _X5 , _X6 , X8, _X9 , _X10 , _X11 , _X14 , _X15 , _X16 , _X17 and _X19 are all peptides with a length greater than 1 aa, the length of the polypeptide chain structure is relatively high, which affects the binding strength. In a preferred embodiment, at least one of _X3 , _X4 , X5, _X6 , _X8 , _X9 , _X10 , _X11 , _X14 , _X15 , _X16 , _X17 and _X19 is any amino acid, more preferably at least two are any amino acids; more preferably, at least _five are any amino acids; more preferably, at least seven are any amino acids; more preferably, at least nine _are any amino acids; more preferably, at least ten are any amino acids; more preferably, at least eleven are any amino acids; more preferably, at least twelve are any amino acids; more preferably, _X3 , X4, X5, X6, X8, X9, X10, X11, X14, X15, _X16 , X17 and X19 are any amino acids. _X5 , _X6 , _X8 , _X9 , _X10 , _X11 , _X14 , _X15 , _X16 , _X17 and _X19 are all arbitrary amino acids.

In some embodiments, X ₁₂ , X ₁₈ and X ₂₂ are each independently selected from R, H, K or Q.

In some preferred embodiments, the polypeptide has the structure shown in the following formula II;
_X1 - _X2 -NGVT-X7 _- PSGY- _X12 - _X13- SIVD- _X18 - _QX20 - _X21 - _X22 - _X23 , Formula II;

wherein X ₂ , X ₇ , X ₁₃ and X ₂₀ are independently C;

_X1 is none, any amino acid or any peptide;

X ₂₁ is none, any amino acid or any peptide;

X ₂₃ is none, any amino acid or any peptide;

Preferably, _X12 is R, _X22 is K and _X18 is K.

In the present invention, polypeptides should also include polypeptides obtained by mutation based on the structure shown in Formula I or Formula II, for example, based on the structure shown in Formula I or Formula II, any 1 to 5, preferably 1 to 4, preferably 1 to 3, more preferably 1 to 2 amino acids are deleted or replaced by other amino acids, or 1 to 5, preferably 1 to 4, preferably 1 to 3, more preferably 1 to 2, more preferably 1 amino acid is added. It should be noted that the amino acids obtained by mutation of the above-mentioned formula I and formula II are not mutated at the sites of _X2 , _X7 , _X13 , _X18 , _X20 , _X12 and _X22 . It should be noted that the polypeptides obtained by mutation of the above-mentioned formula I and formula II do not change the function of the original protein.

In some embodiments, _Xi is nothing or any amino acid.

In some embodiments, _X21 is nothing or any peptide segment.

In some embodiments, _X23 is nothing or any peptide segment.

In some embodiments, _X1 is any peptide segment, the length of which is 1-10 aa, more preferably 1-5 aa, more preferably 1-3 aa, such as 1 aa. In some embodiments, _X1 is P.

In some embodiments, X ₂₁ is any peptide segment, the length of the peptide segment is 1-10aa, more preferably 1-5aa, more preferably 1-3aa, such as 1aa. In some embodiments, X ₂₁ is 1.

In some embodiments, X ₂₃ is any peptide segment, the length of the peptide segment is 1-10 aa, more preferably 1-5 aa, more preferably 1-2 aa, such as 2 aa. In some embodiments, X ₂₃ is KE.

In a preferred embodiment, the polypeptide has the structure shown in the following formula III:
PX2 _- NGVT- _X7- PSGY- _X12 - _X13- SIVD- _X18 - _QX20 - _IX22 -KE, Formula III;

wherein X ₂ , X ₇ , X ₁₃ and X ₂₀ are independently C;

_X12 is R, _X18 is K, and _X22 is K.

In the present invention, the polypeptides represented by Formula I, Formula II and Formula III can be linear peptides or cyclic peptides.

In a preferred embodiment, _X2 and _X13 form a disulfide bond with each other, and _X7 and _X20 form a disulfide bond with each other. It should be understood that two pairs of disulfide bonds can be formed spontaneously under conditions suitable for preserving proteins.

In a preferred embodiment, the above-mentioned conditions suitable for storing proteins are solutions that can renature proteins.

In a preferred embodiment, the sequence of the polypeptide is selected from any one of the following groups:

(a) having an amino acid sequence as shown in SEQ ID NO.1;

(b) having an amino acid sequence having a homology greater than 80% (preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, more preferably greater than 97%) with the sequence shown in SEQ ID NO.1; and

The sequence described in (b) or (c) above has comparable biological activity to the sequence shown in SEQ ID NO.1.

In the present invention, polypeptides should also include polymers of the structure shown in Formula I, such as dimers, trimers, etc.

In the present invention, the polypeptide can also be modified or labeled with other proteins, compounds or polymers to obtain fusion proteins or modified polypeptides. In some embodiments, alkyl (e.g., methyl, ethyl), alkenyl (e.g., allyl), phenyl or phenyl derivatives (e.g., phenylpropyl), PEG, phosphoric acid are used to modify the polypeptide of the present invention. In other embodiments, fluorescent molecules (e.g., FAM, FITC, Cy5, Cy7) are used to modify the polypeptide of the present invention.

In the present invention, the term "fusion protein" refers to a molecule in the present disclosure that is connected by covalent bonds to two or more proteins or fragments thereof and contained by the main chain of each peptide. The fusion protein is preferably produced by genetic expression of polynucleotide molecules encoding these proteins. In a preferred embodiment, the fusion protein contains an immunoglobulin. In a preferred embodiment, the fusion protein is an Fc-fusion protein. In a preferred embodiment, the polypeptide of the present invention and an Fc fragment, IgG, a lytic tag (e.g., a His tag) and the like constitute a fusion protein.

As used herein, the term "polypeptide" refers to a protein occurring in nature or produced or altered by recombinant chemical or other means, which essentially envisions the three-dimensional structure of the protein being post-translationally processed in the same manner as the native protein.

Polynucleotide

The present invention also relates to a polynucleotide encoding the polypeptide of the present invention or a construct thereof.

"Nucleic acid sequence" or "polynucleotide sequence" in the present invention refers to a sequence of nucleotides or nucleotide monomers formed by naturally occurring bases, sugars and sugar (hubs). The term also includes a limited or alternative sequence containing monomers or a portion thereof that do not exist in nature. The nucleic acid sequence of the present invention can be a deoxyribonucleic acid sequence (DNA) or a ribonucleic acid sequence (RNA), and can contain natural bases of adenine, guanine, cytosine and uracil.

Carrier

The present invention also provides a vector comprising the polynucleotide of the present invention.

The "vector" in the present invention refers to the delivery vector of the polynucleotide in the present invention. In some embodiments, in genetic engineering recombinant technology, the vector includes a polynucleotide sequence encoding a specific protein that can be operably inserted to achieve the expression of the protein. The vector is used to transform, transduce or transfect host cells, and the genetic material elements delivered by the vector can be expressed in the host cells. The "vector" in the present invention can be any suitable vector, including chromosomes, non-chromosomal and synthetic nucleic acid vectors (including a series of nucleic acid sequences of appropriate expression control elements). For example, the vector can be a recombinant plasmid vector, a recombinant eukaryotic virus vector, a recombinant bacterial phage vector, a recombinant yeast mini-chromosome vector, a recombinant bacterial artificial chromosome vector or a recombinant yeast plasmid vector. Exemplarily, the vectors in the present invention include those from SV40 derivatives, bacterial plasmids, Phage DNA, baculovirus, yeast plasmid, a combination of plasmid and phage DNA vectors, and viral nucleic acid (RNA or DNA) vectors. In some embodiments, the vector is an E. coli expression vector. In some embodiments, the vector is a plasmid.

Host cells

The present invention also relates to a host cell comprising the vector of the present invention.

In the present invention, "host cell" is a cell into which exogenous polynucleotides and/or vectors are introduced. The host cell is a eukaryotic host cell or a prokaryotic host cell. Wherein, the eukaryotic host cell can be a mammalian host cell, an insect host cell, a plant host cell, a fungal host cell, a eukaryotic algae host cell, a nematode host cell, a protozoan host cell and a fish host cell. Exemplarily, the host cell in the present invention is a eukaryotic host cell, and the eukaryotic host cell is a mammalian host cell. Wherein, the mammalian host cell is a Chinese hamster ovary cell (CHO cell), COS cell, Vero cell, SP2/0 cell, NS/O myeloid cell, human fetal kidney cell, immature hamster kidney cell, HeLa cell, human B cell, cv-1/EBNA cell, L cell, 3T3 cell, HEPG2 cell, PerC6 cell, and in some embodiments, the mammalian host cell in the present invention is a CHO cell.

The host cell in the present invention may also be a prokaryotic host cell, such as Escherichia coli.

Pharmaceutical composition

The present invention also relates to a pharmaceutical composition comprising the polypeptide of the present invention or a construct thereof.

In the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. The term refers to pharmaceutical carriers that do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not excessively toxic after administration. These carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such carriers include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.

The pharmaceutically acceptable carrier in the therapeutic composition may contain a liquid, such as water, saline, glycerol and ethanol. In addition, auxiliary substances may be present in these carriers, such as wetting agents or emulsifiers, pH buffer substances, etc. Generally, the therapeutic composition can be prepared as an injectable, such as a liquid solution or suspension; it can also be prepared in a solid form suitable for being mixed with a solution or suspension, a liquid carrier before injection. Once the composition of the present invention is prepared, it can be administered by conventional routes, including (but not limited to): intramuscular, intravenous, subcutaneous, intradermal, or topical administration. The subject to be prevented or treated can be an animal; in particular, a human.

In a preferred embodiment, the pharmaceutical composition is in the form of an oral preparation, an injection preparation or an external preparation.

In a preferred embodiment, the dosage form of the pharmaceutical composition is a solid preparation, a liquid preparation or a semisolid preparation.

In a preferred embodiment, the preparation of the pharmaceutical composition is selected from the following group: tablets, capsules, injections, granules, sprays, and lyophilized agents.

Typically, the injection is an intravenous injection, an intramuscular injection or a subcutaneous injection. When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the use situation. Preferably, it is an intravenous preparation or an intratumoral injection.

These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and appropriate pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers are occasionally added, and the formulation process can be carried out in a conventional manner according to the dosage form.

The pharmaceutical composition of the present invention can also be administered in the form of a sustained release agent. For example, the polypeptide of the present invention or a pharmaceutically acceptable salt thereof can be incorporated into a pill or microcapsule with a sustained release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated. As examples of sustained release polymers, ethylene-vinyl acetate copolymers, polyhydroxymethylacrylate (polyhydrometaacrylate), polyacrylamide, polyvinyl pyrrolidone, methylcellulose, lactic acid polymers, lactic acid-glycolic acid copolymers, etc. can be exemplified, and preferably biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers can be exemplified.

The pharmaceutical preparation should be matched with the mode of administration. The medicament of the present invention can also be used with other synergistic therapeutic agents (including before, during or after use). When using the pharmaceutical composition, a safe and effective amount of the drug is applied to the desired object (such as a human or non-human mammal), and the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases does not exceed about 8 milligrams/kg body weight, preferably the dosage is about 10 micrograms/kg body weight-about 1 milligram/kg body weight. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

Indications

The present invention also relates to the use of the polypeptide and its construct, or pharmaceutical composition, for:

(i) for pain relief;

(ii) for the preparation of a medicament for the relief of pain;

(iii) for enhancing the agonistic effect of TRPV1 receptor agonists on TRPV1;

(iv) acts as a positive allosteric modulator of TRPV1 channels;

(vi) for inducing death of nerve terminal cells expressing TRPV1; and

(vii) It is used to prolong the desensitization process of TRPV1 channels.

In some embodiments, the pain is pain associated with the TRPV1 channel.

In some embodiments, the pain is neuropathic pain. In some embodiments, the neuropathic pain is diabetic peripheral neuropathic pain. In some embodiments, the pain is post-herpetic neuralgia. In some embodiments, the pain is trigeminal neuralgia.

In some embodiments, the subject has pain associated with or caused by a strain, sprain, arthritis or other joint pain, bruise, back pain, fibromyalgia, endometriosis, surgery, migraine, cluster headaches, psoriasis, irritable bowel syndrome, chronic interstitial cystitis, vulvodynia, trauma, musculoskeletal disease, herpes zoster, sickle cell disease, heart disease, cancer, stroke, or mouth ulcers or ulcers caused by chemotherapy or radiation therapy.

In the present invention, the term "allosteric regulation" is also called allosteric regulation or allosteric regulation. Receptor ligand molecules can be mainly divided into two categories: orthosteric ligands and allosteric modulators: orthosteric ligands bind to the orthosteric site in the receptor domain, and activate or inhibit receptor-related activities by closing or opening the orthosteric site, such as natural ligands or agonists and competitive antagonists; while the action site of allosteric modulators is topologically different from the binding site of orthosteric ligands. They can induce changes in the conformation and structure of the receptor, thereby affecting the potency and efficacy of orthosteric agonists and playing an allosteric regulatory role on the related functions of the receptor. According to the different characteristics of these allosteric modulator compounds in regulating receptor activity, they can usually be divided into two categories: positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs). PAMs can increase the related effects caused by orthosteric ligands activating receptors; on the contrary, NAMs will reduce the effects induced by orthosteric ligands.

Instructions

The present invention also relates to a method for using the polypeptide, its construct, or pharmaceutical composition, comprising the step of administering a therapeutically effective amount of the polypeptide, its construct, or pharmaceutical composition to a subject.

In the present invention, the term "patient" or "subject" is used throughout the specification to describe an animal, preferably a human or a domesticated animal, to which a polypeptide or a pharmaceutical composition according to the present invention is provided for treatment, including prophylactic treatment. For treatment of those infections, conditions or disease states specific to a particular animal (e.g., a human patient), the term patient refers to the particular animal, including domesticated animals such as dogs or cats or farm animals such as horses, cattle, sheep, etc. In general, in the present invention, unless otherwise specified or implied by the context in which the term is used, the term patient refers to a human patient.

In the present invention, the term "therapeutically effective amount" means that the "therapeutically effective amount" of a compound as used herein refers to the amount of the compound sufficient to provide a therapeutic effect in the treatment or management of a disease or disorder, or sufficient to delay or minimize one or more symptoms associated with the disease or disorder. The therapeutically effective amount of a compound refers to the amount of a therapeutic agent that can provide a therapeutic effect in the treatment or management of a disease or disorder when used alone or in combination with other therapies. The term "therapeutically effective amount" may include an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or disorder, or enhances the effect of another therapeutic agent. The amount of therapeutic efficacy.

In some embodiments, the polypeptide and its constructs, or the pharmaceutical composition are used alone.

In some embodiments, the polypeptide and its construct, or pharmaceutical composition is used in combination with a TRPV1 receptor agonist.

In some embodiments, the TRPV1 receptor agonist is capsaicin.

In some embodiments, the polypeptide or its construct, or the pharmaceutical composition is administered at least once a day for at least 3 days. In some embodiments, it is administered at least once a day for at least 5 days. In some embodiments, it is administered at least once a day for at least 7 days. In some embodiments, the pharmaceutical composition is administered at least once a day for more than 7 days.

In some embodiments, the polypeptide or its construct, or pharmaceutical composition is administered to a mammal at a dose of 0.01-100 mg/kg body weight of the active ingredient (each time or every day). The aforementioned active ingredient refers to the dosage of the polypeptide or its construct.

As used herein, the terms "include", "comprises", and "comprising" are used interchangeably, and include not only open definitions, but also semi-closed and closed definitions. In other words, the terms include "consisting of", "consisting essentially of".

In the present invention, each amino acid and its abbreviation are shown in the following Table 1:

Table 1 Amino acids and their abbreviations

The term neutral amino acid in the present invention refers to an amino acid having an equal number of amino groups and carboxyl groups. However, since the ionization of carboxyl groups is greater than that of amino groups, in aqueous solution, the number of negative ions generated by the ionization of carboxyl groups is greater than the number of positive ions formed after the amino groups combine with H ions. Therefore, neutral amino acid aqueous solutions are acidic, with a pH of <7. If the isoelectric point is to be reached, the number of positive ions must be equal to the number of negative ions. Therefore, in this case, efforts should be made to reduce the number of negative ions in the solution, and the measure taken is to add acid so that the carboxylate negative ions combine with H ions, thereby consuming the negative ions. In order for neutral amino acids to reach the isoelectric point, acid must be added to the system, so its PI < pH < 7.

The term acidic amino acid in the present invention refers to an amino acid with fewer amino groups than carboxyl groups. The solution is acidic, i.e., pH < 7. Therefore, similar to neutral amino acids, the number of negative ions in the solution must be greater than the number of positive ions. In order to reach the isoelectric point, the number of negative ions must be reduced, and acid and H ions must be added to the system. Therefore, its PI < pH < 7. It is usually negatively charged in a solution of 5 < pH < 7. For example, glutamic acid and aspartic acid.

The term basic amino acid in the present invention refers to an amino acid in which the number of amino groups is greater than the number of carboxyl groups. The solution is alkaline, pH>7. Due to the large number of amino groups, the number of positive ions in the solution is greater than the number of negative ions. In order to reach the isoelectric point, the number of positive ions must be reduced, which can be achieved by adding alkali. Adding ^OH- to the solution will consume the H ions bound to the amino groups, thereby reducing the number of positive ions. Therefore, the relationship between the isoelectric point and pH of basic amino acids is: PI>pH>7. Usually, they are positively charged in solutions of 5<pH<7. For example, lysine, histidine and arginine.

To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. In the following examples, capsaicin and ionomycin were purchased from MCE, 2-APB, AITC and complete Freund's adjuvant (CFA) were purchased from Sigma-Aldrich, and other experimental materials and reagents used were all available from commercial sources unless otherwise specified.

Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

In this article, adult C57BL/6 mice (male, 8-10 weeks) were used for behavioral studies. Trpv1cre*Ai32 transgenic mice (Jax Strain#:017769) were used to study the distribution of TRPV1 ⁺ nerve endings. Mice were housed in a 12-hour day and night reversed environment, with room temperature of 22-24 degrees Celsius and free access to food and water. All animal experiments were approved by the Animal Care and Use Committee of Zhejiang University.

experimental method

Cell culture

HEK293T cells were cultured in DMEM (containing 10% fetal bovine serum (FBS)) medium, placed in a 37°C, 5% CO ₂ incubator, and passaged every two days. Mouse TRPV1, TRPV2, TRPV3 and human TRPA1 plasmids all carry GFP or YFP fluorescent tags for subsequent electrophysiological experiments. Lipofectamine 2000 was used for transient transfection of the above plasmids, and electrophysiological recordings were performed 18-24 hours after transfection. TRPV1 point mutations were generated using Takara's rapid point mutation kit, and the point mutation results were confirmed by sequencing.

Synthesis and purification of toxin peptides

Linear s-RhTx was synthesized by Gill Biochemicals with a purity of more than 98%. Linear s-RhTx and s-RhTx point mutations (R12Q, R12K, K22Q, K22R) were dissolved in a solution containing 0.1M sodium chloride, 0.1M Tris-HCl, 5mM glutathione and 0.5mM oxidized glutathione, the pH was adjusted to 5.6-5.8, and the linear peptide was placed at 28 degrees overnight for oxidative folding. The oxidatively folded peptide was purified by reverse phase chromatography using the NGC system, and the peak was detected at 280nm. The concentration of s-RhTx was determined by the absorbance value of nanodrop at 280nm and corrected by the extinction coefficient, which is 0.662mM ^-1 cm ^-1 (calculated by https://web.expasy.org/protparam).

Electrophysiological recordings

Patch clamp recordings were performed using a HEKA EPC10 amplifier and PatchMaster software. Borosilicate glass electrodes were pulled to a resistance of 3-8 MΩ using a P-97 electrode puller. The intracellular and extracellular solutions contained 130 mM NaCl, 0.2 mM EDTA, 10 mM glucose, 3 mM HEPES, and were adjusted to pH = 7.2-7.4 with NaOH. MES was used instead of HEPES in the cell perfusion solution at pH 6.5. Whole-cell recordings were voltage-clamped at ±80 mV and the current sampling frequency was 10 kHz. A gravity perfusion system with a freely rotatable perfusion tube was used for perfusion of peptides and ligands. After the cell membrane was broken by the electrode, the electrode lifted the cell off the bottom of the culture dish and placed it in front of the perfusion tube for subsequent perfusion. All experiments were performed at room temperature.

For the desensitization experiments of TRPV1 channels, the intracellular and extracellular solutions contained 130 mM NaCl, 5 mM KCl, 2 mM MgCl ₂ , 2 mM CaCl ₂ , 10 mM glucose, 10 mM HEPES, and were adjusted to pH 7.2-7.4 with NaOH.

Calcium imaging

HEK293T cells were transiently transfected with TRPV1 for 12-18 hours and then incubated in dark for 1 hour with calcium imaging solution (140mM NaCl, 5mM KCl, 2mM MgCl ₂ , 2mM CaCl ₂ ,10mM glucose, 10mM HEPES, pH 7.4) containing 2μM Fluo-4AM. Fluorescence images of HEK293T cells were taken with an optiMOS CCD camera and recorded with Ocular software. Fluo4-AM was excited at 500nm and fluorescence emission was detected at 535nm. Fluorescence images were analyzed with Fiji software.

Cell death assay

In this experiment, Hoechst and PI double staining method was used to measure cell death, in which Hoechst marked the nuclei of all cells and showed blue fluorescence, while PI marked the nuclei of dead cells and showed red fluorescence. After the cells were treated with peptides or capsaicin for 12 hours, they were trypsinized and centrifuged, resuspended once in PBS, and incubated with Hoechst and PI working solution (Hoechst was 2-10μg/μl, PI was 2μg/μl) at room temperature for 15 minutes. Then, the CCD of a fluorescence microscope (Nikon Eclipse Ti2) was used to take pictures, and 5 fields of view were randomly photographed for each treatment group. The mortality rate is expressed as the ratio of the number of red fluorescent cells to the number of blue fluorescent cells (the number of cells is obtained by the counting function of Fiji), and the mortality rate of each group of cells is the average value of the cell mortality rates of 5 fields of view.

Molecular docking of s-RhTx and TRPV1 channels

Molecular docking of s-RhTx and TRPV1 channel was completed in the protein docking component of Rosetta software version 2019. The structure of s-RhTx was generated from the structure of RhTx NMR (PDB ID: 2MVA) and was first relaxed using the relax component of Rosetta. s-RhTx was placed in the outer pore region of TRPV1 for docking. After generating 30,000 docking results, the 10 models with the highest binding energy between s-RhTx and TRPV1 were selected as the final models for subsequent analysis.

data processing

The data of whole-cell patch clamp recordings were processed in IgorPro version 5.05. The concentration-effect curve was fitted with the Hill equation and the half effective concentration EC50 was obtained. The desensitization time constant tau during TRPV1 desensitization was fitted with an exponential equation.

In the double mutant cycle analysis, the Kd values between the four pairs of peptide-channels were defined as: WT channel, s-RhTx: Kd_1; channel mutant, s-RhTx: Kd_2; WT channel, peptide mutant: Kd_3; channel mutant, peptide mutant: Kd_4. The binding energy was calculated as:

Where, LnΩ is in kT, k is the Boltzmann constant, and T is the Kelvin temperature.

Intradermal injection in mice

Linear or folded s-RhTx was dissolved in saline, and 2 μg of peptide and 200 ng of capsaicin (20 μl) were injected intradermally into the sole of the mouse foot. 20 μl of saline or 200 ng of capsaicin was used as a control. 20 μl of complete Freund's adjuvant was injected to produce a mouse chronic inflammatory pain model.

Mouse behavioral testing

In the spontaneous pain behavior of mice, the time of licking, lifting and withdrawing the paw of mice was recorded within 10 minutes after the peptide or capsaicin was injected into the plantar skin of mice. In the temperature sensitivity test, the threshold of the basic withdrawal time of mice was 4-6s, and the maximum time of heat radiation was set to 10s to avoid tissue damage.

When testing the mechanosensitive pain and thermal pain behavior of mice, the mice were placed in a plexiglass box above a metal mesh for 1 hour in advance. The mechanosensitive threshold of the mouse plantar was measured using von Frey filaments of different specifications (0.02-2.56g). The temperature sensitivity of mice was measured using a Hargreaves thermal radiation device. For the thermal pain model of CFA chronic inflammatory pain mice, the basic value of the mouse paw withdrawal time caused by thermal radiation was 9-12s, and the maximum time of thermal radiation was set to 20s to avoid tissue damage. The paw withdrawal time was tested three times in a row and the average value was taken, with an interval of 5 minutes between each test.

Example 1: Rational design of s-RhTx

In order to develop a regulator targeting the outside of the TRPV1 pore region, in this example, the inventors modified the red-headed centipede toxin RhTx that binds to the outside of the TRPV1 pore region, hoping to obtain a polypeptide with altered activity.

The inventors first synthesized a linear peptide (N-terminus to C-terminus) with a sequence of PCNGVTCPSGYRCSIVDKQCIKKE (s-RhTx, SEQ ID NO: 1), and purified it by HPLC to obtain a linear peptide with a purity of more than 98%. In order to ensure that the polypeptide is correctly folded to form two pairs of disulfide bonds, the polypeptide is placed in a solution containing 0.1M NaCl, 0.1M Tris-HCl, 5mM reduced glutathione and 0.5mM oxidized glutathione, the pH is adjusted to 5.6-5.8, and incubated at 28°C overnight. Purification was performed by reverse phase chromatography, and gradient elution was performed with acetonitrile as the mobile phase. The peak was obtained when the acetonitrile concentration was 31% (Figure 3), and this peak was collected and freeze-dried. The freeze-dried powder was detected by mass spectrometry, and the molecular weight was 2624.125, while the molecular weight of the linear peptide was 2628.211. Compared with the linear peptide, the molecular weight of the oxidized folded polypeptide was reduced by 4, indicating the formation of two pairs of disulfide bonds (Figure 4).

In addition, the inventors synthesized the polypeptides shown in Table 1 below using the same method and oxidatively folded them in the same manner.

Table 1

Example 2: Electrophysiological experiments confirm that s-RhTx is a positive allosteric modulator (PAM) of TRPV1

After obtaining the oxidatively folded polypeptide based on SEQ ID NO: 1, the inventors first evaluated electrophysiology. Perfusing 30 μM s-RhTx into cells expressing TRPV1 failed to induce TRPV1 current (Figure 5). However, s-RhTx can enhance the agonistic effect of low concentration agonist (10 nM capsaicin) on TRPV1 in a dose-dependent manner, with an EC50 of 0.89 ± 0.45 μM (Figures 6 and 7). In addition, calcium imaging results showed that 10 nM capsaicin could hardly induce changes in intracellular calcium fluorescence signals, while 10 nM capsaicin plus 30 μM s-RhTx could induce significant calcium ion influx (Figures 8 and 9). .

TRPV1 is a multimodal receptor that can be activated by a variety of stimuli, such as protons and temperature, so the inventors tested Effect of s-RhTx on acid activation and temperature activation of TRPV1. In acid activation, the TRPV1 current induced by weak acid (pH 6.5) can be enhanced by s-RhTx in a dose-dependent manner, with an EC50 of 1.58±0.50μM (Figures 10 and 11). On the other hand, in the thermal activation of TRPV1, the thermal activation threshold of TRPV1 was 37.9±1.1℃; in the presence of s-RhTx, the thermal activation threshold of TRPV1 was 38.6±0.98℃. Since RhTx can significantly reduce the thermal activation threshold of TRPV1, using RhTx as a control, it was shown that RhTx reduced the thermal activation threshold of TRPV1 to 31.0±1.8℃ (Figures 12 and 13). These data indicate that s-RhTx is a positive allosteric regulator of the capsaicin activation and acid activation processes of TRPV1, but does not affect the thermal activation process of TRPV1.

To determine the selectivity of s-RhTx, the inventors evaluated other members of the TRP family involved in the pain process (TRPV2, TRPV3, TRPA1). For TRPV2, 500 μM 2-APB (agonist of TRPV2 channels) triggered a small TRPV2 current of about 7.06% ± 2.25%, and s-RhTx was unable to enhance the agonistic effect of 2-APB on TRPV2. The solution of s-RhTx plus 2-APB triggered a TRPV2 current of 9.40% ± 3.64% (Figure 14). Similarly, s-RhTx also failed to enhance the activation of TRPV3 by 2-APB and the activation of TRPA1 channels by AITC (Figures 15, 16 and 17). Therefore, s-RhTx is a selective positive allosteric modulator of TRPV1.

Example 3: s-RhTx slows down the desensitization process of TRPV1, thereby inducing calcium overload and inducing cell death

In the presence of calcium ions, TRPV1 undergoes rapid desensitization when activated by capsaicin. Previous literature reports that TRPV1 PAM can greatly prolong the desensitization process of TRPV1 channels, thereby inducing death of nerve endings expressing TRPV1 channels due to calcium overload, thereby achieving analgesic effects. In order to verify the effect of s-RhTx on the desensitization process of TRPV1, the inventors in this example determined the desensitization time constant τ of the TRPV1 channel through electrophysiological experiments. The voltage was clamped at ±80mV for whole-cell recording. At +80mV, the desensitization time constant of TRPV1 channel induced by 10μM capsaicin was 47.37±6.14s, while the mixed solution of s-RhTx and 10μM capsaicin delayed the desensitization time of TRPV1 to 179.3±30.37s; at -80mV voltage, the desensitization time constant of TRPV1 channel induced by 10μM capsaicin was 27.87±3.97s, while the mixed solution of s-RhTx and 10μM capsaicin delayed the desensitization time of TRPV1 to 89.4±28.21s (Figures 18 and 19).

In addition, the amount of calcium ions entering the cell was estimated by calculating the area under the curve (AUC) of the TRPV1 desensitization process. Consistent with the results of the desensitization time constant: at +80 mV, the AUC of 10 μM capsaicin-induced TRPV1 channel desensitization was 72.39 ± 6.61, while the AUC of s-RhTx and 10 μM capsaicin-induced TRPV1 channel desensitization was 164.7 ± 25.37; at -80 mV, the AUC of 10 μM capsaicin-induced TRPV1 channel desensitization was 39.25 ± 5.01, while the AUC of s-RhTx and 10 μM capsaicin-induced TRPV1 channel desensitization was 127.5 ± 33.66 (Figure 20). These results indicate that s-RhTx can indeed significantly increase the amount of calcium ions entering the cell by delaying the desensitization process of TRPV1.

A large influx of calcium ions can induce calcium overload and thus cause cell death. In order to further verify whether s-RhTx channels induce calcium overload and induce cell death, the inventors performed Hoechst and PI (propidium iodide) staining, where Hoechst marks the nuclei of all cells, showing blue fluorescence; PI marks the nuclei of dead cells, showing red fluorescence. TRPV1 channels were transiently transfected in HEK 293T cells, and cell death was measured after 18 hours of treatment with compounds or peptides. The cell death rate of HEK293T cells transfected with TRPV1 was 3.91% ± 0.40%, indicating that transfection with TRPV1 had little effect on cell death. The cell death rate of the 10nM capsaicin treatment group was 9.54% ± 1.21%, while s-RhTx could increase the cell death rate in a dose-dependent manner: in the presence of 10nM capsaicin, the cell death rates induced by 1μM, 10μM, and 100μM s-RhTx were 12.63% ± 2.18, 19.14% ± 2.80%, and 18.58 ± 1.21%, respectively. Among them, the cell death rates induced by 10μM and 100μM s-RhTx were comparable to those of 10μM capsaicin (19.93% ± 1.77%) (Figures 21, 22). These results suggest that s-RhTx induces calcium overload and thus causes cell death expressing TRPV1 by slowing down the desensitization process of TRPV1.

Example 4: E649 and E652 of TRPV1 are essential for the activity of s-RhTx

Given the similarity in sequence and structure, the inventors hypothesized that s-RhTx and RhTx also bind to the outside of the pore region of TRPV1. To verify this hypothesis, the inventors evaluated the effect of s-RhTx using inside-out and outside-out recording modes. In the inside-out mode, 300 μM s-RhTx combined with 10 nM capsaicin also failed to activate the current of TRPV1; however, in the outside-out mode, 30 μM s-RhTx combined with 10 nM capsaicin induced significant current, indicating that s-RhTx binds to the extracellular region of TRPV1 (Figures 23-24).

In order to further study the binding mode of s-RhTx and TRPV1, the inventors performed molecular docking of s-RhTx and the outside of the TRPV1 pore region in Rosetta software and designed some point mutations based on the docking results to verify the docking results. The docking results showed that R12 and K22 of s-RhTx formed hydrogen bond interactions with E652 of two adjacent subunits of TRPV1, respectively, and K18 of s-RhTx formed hydrogen bond interactions with E649 of TRPV1 (Figure 25). Alanine scanning results showed that E649 and E652 of TRPV1 were crucial to the activity of s-RhTx. The E649A point mutation significantly reduced the enhancing effect of s-RhTx on the activity of 10nM capsaicin, but could not completely eliminate the activity of s-RhTx (Figures 26, 29, 30). However, mutation of E652 to A or K with opposite charge completely eliminates the effect of s-RhTx, while E652D can complement the effect of s-RhTx, which suggests that the negative charge of E652 is critical for the activity of s-RhTx, and also suggests that the positive charge of amino acids at positions 12/18/22 on s-RhTx and the binding of the corresponding amino acids at positions 649 and 652 on TRPV1 are also crucial. The inventor's previous studies showed that RhTx interacts with four sites of TRPV1: L461, D602, Y632 and T634, while L461G and T634A do not affect the activity of s-RhTx, which indicates that after removing the three amino acids at the N-terminus of RhTx, the binding mode of the peptide to TRPV1 has changed.

To further confirm the above results, the inventors also evaluated the effect of s-RhTx on TRPV1 point mutations in a capsaicin-induced TRPV1 desensitization experiment. s-RhTx can slow down the desensitization process of TRPV1. If E652 and E649 are indeed very important for the activity of s-RhTx, then in the E652A/K and E649A point mutations, the TRPV1 desensitization time constants induced by perfusion of capsaicin alone and perfusion of a mixture of capsaicin and s-RhTx should be comparable. Consistent with the above results, E652A, E652K and E649A significantly eliminated the effect of s-RhTx on the extension of TRPV1 desensitization time, while E652D complemented the effect of s-RhTx on the extension of TRPV1 desensitization time. Other point mutations such as L461G, T634A, K604E, and K657E had an effect on s-RhTx. There is no effect on delaying the desensitization process of TRPV1.

In addition, the inventors also confirmed these results in calcium imaging experiments. Consistent with the electrophysiological results, the mixture of 30μM s-RhTx and 10nM capsaicin can only increase the intracellular calcium fluorescence intensity of HEK293T cells expressing the E649A point mutation by 23.6%±0.1%, which is lower than the intracellular calcium fluorescence change of cells expressing WT TRPV1 (56.09%±8.62%) (Figure 30, Figure 8-9). Similarly, cells expressing the E652K point mutation only responded to s-RhTx by 7.8%±0.02%, while s-RhTx increased the intracellular calcium fluorescence intensity of cells expressing the E652D mutation by 47.5%±0.05%, which is close to that of cells expressing WT TRPV1. Therefore, the above data indicate that E649 and E652 of TRPV1 are essential for the activity of s-RhTx.

Example 5: Double mutation cycle reveals that R12 and K22 of s-RhTx play important roles in its activity

The above results have confirmed the key role of TRPV1 E652. In order to further reveal whether there is a specific interaction between s-RhTx and E652 (such as the possible formation of hydrogen bonds between R12 and K22 of s-RhTx and E652 of TRPV1 mentioned above), we used double mutation cycles for the next step of verification: that is, to make concentration effect curves of peptides on channels in the following four pairs of channel-peptide combinations: WT TRPV1 and WT s-RhTx; WT TRPV1 and s-RhTx point mutation; TRPV1 E652D and s-RhTx; TRPV1 E652D and s-RhTx point mutation. We synthesized four mutant peptides: R12Q, R12K, K22Q, K22R. Similar to s-RhTx, they were purified by reverse phase chromatography after oxidative folding and renaturation, and then freeze-dried for subsequent experiments.

In the process of verifying the existence of specific interactions by double mutation cycles, two important parameters are: Kd represents the binding ability of ligand and channel; L represents the ability of ligand to bind to the channel and cause conformational changes in the channel, leading to channel opening. Kd and L can be calculated by the following formulas: EC50 = Kd/(1+L), Po max = L/(1+L), where EC50 and Po max (maximum opening probability) can be obtained experimentally. We first evaluated the activity of s-RhTx in the TRPV1 E652D point mutation. The EC50 of the concentration effect curve fitting was 7.16±1.42μM, which was much larger than the EC50 of WT TRPV1: s-RhTx (0.89±0.45μM); Po max was 0.66±0.06, which was smaller than the Po max of WT TRPV1: s-RhTx (0.94±0.03) (Figures 31, 32). Then we evaluated the activity of s-RhTx R12Q point mutation in WT TRPV1 and TRPV1 E652D. The concentration effect curve of R12Q on WT TRPV1 and TRPV1 E652D shifted greatly to the right, which led to a significant increase in Kd value. The Kd value of WT TRPV1:R12Q was 390.6±47.12μM, while the Kd value of WT TRPV1:s-RhTx was 32.06±16.14μM (Figure 33); and the maximum open probability was significantly reduced, resulting in a significant decrease in L value. The L value of WT TRPV1:R12Q was 3.37±1.19 (Figure 34), while the L value of WT TRPV1:s-RhTx was 12.55±5.34. This indicates that the binding ability of R12Q to TRPV1 channel is weakened, and it is more difficult for R12Q to open the channel after binding to the channel. In addition, we also observed similar phenomena in the other three polypeptide point mutations: the Kd values obtained from the concentration-effect curves of R12K, K22Q, and K22R acting on WT TRPV1 and E652D increased, while the L values decreased (Figures 35-43).

Previous studies have shown that when the binding energy is greater than 1.5 kT (or greater than 0.89 kcal/mol at 24°C), a specific interaction is considered to be formed between the ligand and the protein (Figure 44). Therefore, the inventors calculated the R12 and K22 mutants and The binding energy between the channels was found to be greater than 1.5 kT, indicating that R12 and K22 did form specific interactions with the E652 residue of TRPV1 (Figures 44-45).

Example 6: s-RhTx has a long-lasting analgesic effect in in vivo studies

After revealing the binding mode of s-RhTx and TRPV1, the inventors further tested whether s-RhTx has analgesic effect in in vivo experiments. The inventors speculated that the combination of s-RhTx and low-dose capsaicin may cause pain in the initial stage, but in the subsequent stage, it exerts analgesic effect by inducing calcium overload in nerve endings expressing TRPV1.

The inventors first tested the spontaneous pain model induced by capsaicin, and recorded the time of licking, lifting and withdrawing the paws of mice within 10 minutes of injection of capsaicin and s-RhTx. Mice injected with normal saline did not show obvious spontaneous pain behavior, mice injected with low doses of capsaicin (200ng, 20μl) and linear s-RhTx (2μg, very weak enhancing activity) showed mild pain behavior, while mice injected with low doses of capsaicin (200ng, 20μl) and oxidative folded s-RhTx (2μg) showed extremely strong pain behavior. In addition, TRPV1 knockout mice injected with low doses of capsaicin (200ng, 20μl) and oxidative folded s-RhTx (2μg) had no pain behavior (Figures 46 and 47).

The above experimental results show that within 10 minutes of injection, s-RhTx induces significant spontaneous pain behavior in mice by significantly enhancing the activity of TRPV1.

In order to explore whether s-RhTx can show analgesic effect for a long time after injection, the inventors evaluated the paw withdrawal time of mice using noxious radiant heat stimulation. As shown in the figure, the paw withdrawal time of mice injected with low-dose capsaicin and saline and co-injected with capsaicin and linear s-RhTx did not change significantly during the evaluation period (2 weeks), while the paw withdrawal time of mice injected with low-dose capsaicin and folded s-RhTx was significantly prolonged, and this effect can last until the 14th day (Figure 48). Linear s-RhTx does not have the above analgesic effect.

As mentioned above, it is assumed that the co-application of s-RhTx and capsaicin can induce the degeneration of endothelial nerve endings expressing TRPV1, thereby exerting an analgesic effect. In order to verify the above hypothesis, the inventors detected the distribution of TRPV1-positive (TRPV1 ⁺ ) nerve endings after injecting s-RhTx and capsaicin into transgenic mice. Among them, TRPV1 and EYFP in the nerve endings of transgenic mice form a fusion protein, so the nerve endings with positive TPV1 expression carry EGFP. As shown in the figure, there was no significant change in the number of TRPV1 ⁺ nerve endings in mice injected with low doses of capsaicin (200ng) and linear s-RhTx (2μg), while the number of TRPV1 ⁺ endothelial nerve endings in mice injected with low doses of capsaicin (200ng) and folded s-RhTx (2μg) was significantly reduced (Figures 49, 50 and 51).

The above experimental results show that co-injection of s-RhTx and capsaicin exerts a long-lasting analgesic effect by promoting the degeneration of TRPV1 ⁺ endothelial nerve endings.

Example 7: s-RhTx exerts a long-lasting analgesic effect in a chronic inflammatory pain model

Topical application of capsaicin is a common treatment strategy for chronic pain, so we hypothesized that the combination of capsaicin and s-RhTx may have a better effect in chronic pain. In order to test the effect of s-RhTx on chronic pain, the inventors used a chronic inflammatory pain model induced by complete Freund's adjuvant (CFA) for evaluation (Figure 52). After the CFA model was successfully induced, the Hargreaves test was used to test thermal sensitivity and the von Frey test was used to test mechanical sensitivity (Figures 53-54).

In the thermal pain test, CFA-induced chronic inflammatory pain mice showed a slow recovery of thermal pain sensitivity after injection of low-dose capsaicin plus saline or low-dose capsaicin plus linear s-RhTx (Figure 52). After injection of low-dose capsaicin and folded s-RhTx, the thermal pain sensitivity of mice disappeared rapidly and showed insensitivity to thermal pain, and this analgesic effect could last up to 22 days (Figures 53-54). Similarly, in the mechanical pain test, CFA mice injected with capsaicin + saline and capsaicin + linear s-RhTx showed obvious and persistent mechanical pain sensitivity, while the mechanical pain sensitivity of mice injected with capsaicin + folded s-RhTx disappeared rapidly (Figure 54), indicating that the combination of s-RhTx and capsaicin has a lasting analgesic effect in the chronic inflammatory pain model. In addition, the inventors also tested the body temperature of mice and found that there was no significant change in the body temperature of mice after local injection of capsaicin and capsaicin + s-RhTx (Figure 55).

The above experimental results show that the combined use of s-RhTx and capsaicin can increase the thermal and mechanical pain thresholds of mice without affecting body temperature, thereby alleviating chronic inflammatory pain.

Example 8: Evaluation of linear s-RhTx on TRPV1 channel activity

Compared with the folded s-RhTx that forms disulfide bonds, linear s-RhTx has almost no PAM activity on the TRPV1 channel. The results of electrophysiological recordings showed that 100 μM linear s-RhTx had almost no enhancing effect on the activity of 10 nM capsaicin (Figure 56), and the enhancement percentage of 100 μM linear s-RhTx on 10 nM capsaicin was 7.10 ± 3.30%, while in comparison, the enhancing effect of 10 μM folded s-RhTx on 10 nM capsaicin was 78.11 ± 2.88% (Figure 57). In addition, the calcium imaging recording results further verified the above results (Figures 58-59). Linear s-RhTx cannot induce the current of TRPV1, nor can it induce changes in the intracellular calcium fluorescence signal of cells expressing TRPV1. These data indicate that s-RhTx is a positive allosteric modulator of TRPV1, and the two pairs of disulfide bonds of the polypeptide can significantly enhance the positive allosteric activity.

Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

A polypeptide or a construct thereof, characterized in that the polypeptide has a structure shown in the following formula II;
X1 - X2 -NGVT-X7 - PSGY- X12 - X13- SIVD- X18 - QX20 - X21 - X22 - X23 , Formula II;

wherein X 2 , X 7 , X 13 and X 20 are independently C;

X1 is none, any amino acid or any peptide;

X 21 is none, any amino acid or any peptide;

X 23 is none, any amino acid or any peptide;

X 12 , X 18 and X 22 are each independently selected from R, H, K or Q.
The polypeptide according to claim 1, characterized in that the sequence of the polypeptide is selected from any one of the following groups:

(a) having an amino acid sequence as shown in SEQ ID NO.1;

(b) having an amino acid sequence having a homology of greater than 80% with the sequence shown in SEQ ID NO.1; and

(c) having an amino acid sequence complementary to the amino acid sequence described in (a) or (b).
A polynucleotide, characterized in that the polynucleotide encodes the polypeptide or its construct according to claim 1.
An expression vector, characterized in that the vector comprises the polynucleotide as claimed in claim 3.
A host cell, characterized in that the host cell comprises the vector as claimed in claim 4 or the polynucleotide as claimed in claim 3 is integrated into the chromosome of the host cell.
A pharmaceutical composition, characterized in that it comprises the polypeptide according to claim 1, and a pharmaceutically acceptable carrier or excipient.
Use of the polynucleotide according to claim 1 and the pharmaceutical composition according to claim 6 for:

(i) for pain relief;

(ii) for the preparation of a medicament for the relief of pain;

(iii) for enhancing the agonistic effect of TRPV1 receptor agonists on TRPV1;

(iv) acts as a positive allosteric modulator of TRPV1 channels;

(v) for specifically binding to the E649 site and/or the E652 site of the TRPV1 protein;

(vi) for inducing death of nerve terminal cells expressing TRPV1; and

(vii)) is used to prolong the desensitization process of TRPV1 channels.
A method for relieving pain, characterized in that the method comprises the step of administering a therapeutically effective amount of the polypeptide or its construct according to claim 1, or the pharmaceutical composition according to claim 6 to a subject.
The method according to claim 8, characterized in that the pain is pain associated with the TRPV1 channel.
A method for screening a positive allosteric modulator of TRPV1 or a method for screening a drug for relieving pain, characterized in that the method comprises the steps of:

Screening for drugs that can specifically bind to the E649 site and/or the E652 site of the TRPV1 protein.