α-Conotoxin peptides LvIE and LvIF, their pharmaceutical compositions and uses

Abstract

This invention belongs to the field of biomedicine and relates to a novel α-conotoxin peptide LvIE and LvIF, its pharmaceutical composition, and its uses. An isolated polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs:4-5 and SEQ ID NOs:7-8. The α-conotoxin peptide of this invention can effectively and specifically block α3β2 acetylcholine receptors and α6β2*nAChRs acetylcholine receptors, and has the potential to prevent and treat neuralgia, addiction, Parkinson's disease, dementia, schizophrenia, or depression, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a novel α-conotoxin peptide LvIE and LvIF, its pharmaceutical composition, and its uses. This invention also relates to the precursor peptide of the said conotoxin peptide, its nucleic acid construct, its expression vector and transformed cells, and its fusion protein. This invention further relates to a method for blocking acetylcholine receptors and the pharmaceutical use of the said conotoxin peptide. Background Technology

[0002] Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels, pentamer-variant transmembrane proteins on the cell membrane, and are ubiquitous in the animal kingdom. They have very important physiological functions and clinical research significance. nAChRs are the earliest discovered class of receptors in humans, which can be divided into two main categories: muscle type and neuronal type. They are assembled from different α and β subunits into many subtypes, each with distinct pharmacological characteristics (Marcovich I, Moglie MJ, Carpaneto Freixas AE, Trigila AP, Franchini LF, Plazas PV, Lipovsek M, Elgoyhen AB. Distinct Evolutionary Trajectories of Neuronal and Hair Cell Nicotinic Acetylcholine Receptors. Mol Biol Evol. 2020; 37(4):1070-1089.).

[0003] nAChRs are assembled from different α and β subunits into many subtypes. The muscular acetylcholine receptor (NCR) consists of five subunits: two α1 subunits, one β subunit, one δ subunit, and one γ or ε subunit. The γ or ε subunit depends on whether the receptor is from a fetus or an adult. Mammalian neural nAChRs also consist of five subunits, but their subtypes are more complex, involving at least eight α subunits, three β subunits (α2, α3, α4, α5, α6, α7, α9, and α10), and β2, β3, and β4. α2, α3, and α4 can bind to β2 or β4, respectively, to form functional receptors, such as α2β2, α3β2, and α2β4. In addition, α6β4* and α9 can form homopolymers (P. Gopalakrishnakone LJC, Sulan Luo. Toxins and Drug Discovery. Springer Nature (Publisher) 2017; ISBN 978-94-007-6451-4: Conotoxin: p148-187.).

[0004] nAChRs are widely distributed in the central, peripheral, and immune systems, mediating numerous physiological functions of the central and peripheral nervous systems, including learning, memory, response, analgesia, and motor control. nAChRs activate the release of various neurotransmitters such as dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid (GABA). (Zoli M, Pucci S, Vilella A, Gotti C. Neuronal and Extraneuronal Nicotinic Acetylcholine Receptors. Currentneuropharmacology 2018; 16:338-349. Fujii T, Mashimo M, Moriwaki Y, Misawa H, Ono S, Horiguchi K, Kawashima K. Expression and Function of the Cholinergic System in Immune Cells. Frontiers in Immunology 2017; 8:1085.)nAChRs have been confirmed as key targets for screening drugs to diagnose and treat a large class of important diseases, including pain, tobacco and drug addiction, intellectual disability, dementia, schizophrenia, central nervous system disorders, epilepsy, Parkinson's disease, psychosis, neuromuscular blockade, myasthenia gravis, depression, hypertension, arrhythmia, asthma, muscle relaxation, stroke, deafness, obesity, breast cancer, and lung cancer (Bertrand D, Terry AV, Jr. The wonderland of neuronal nicotinic acetylcholine receptors. Biochemical pharmacology 2018; 151:214-225. Gu S, Knowland D, Matta JA, O'Carroll ML, Davini WB, Dhara M, Kweon HJ, Bredt DS. Hair cell α9α10 nicotinic acetylcholine receptor functional expression regulated by ligand binding and deafness gene products. Proc Natl Acad Sci US). A.2020;117(39):24534-24544.HoneAJ,McIntosh JM.Nicotinic acetylcholine receptors in neuropathic and inflammatory pain.FEBS Letters 2018;592:1045-1062.). To date, there is no specific drug for the treatment of the above-mentioned diseases.

[0005] The lack of highly selective ligand compounds targeting various nAChR subtypes presents numerous challenges in studying and elucidating the fine structures and functions of diverse nAChR subtypes. Therefore, developing highly selective ligand drugs targeting various nAChR subtypes is crucial for treating these diseases. Specific ligands (blockers / inhibitors / antagonists or agonists / openers) for a particular nAChR subtype will be invaluable tool drugs and lead compounds for major innovative drugs, possessing immense application value and development potential in the study of various disease mechanisms and the development of new drugs for related diseases.

[0006] Conotoxins, or conopeptides (CTX), secreted by the carnivorous mollusc Conus, which lives in tropical oceans, possess unique functions in regulating various ion channels and have shown significant clinical value, possessing enormous potential for new drug development (Jin AH, Muttenthaler M, Dutertre S, Himaya SWA, Kaas Q, Craik DJ, Lewis RJ, Alewood PF. Conotoxins: Chemistry and Biology. Chemicalreviews 2019; 119:11510-11549. Muttenthaler M, King GF, Adams DJ, Alewood PF. Trends in peptide drug discovery. Nat Rev Drug Discov. 2021 Feb 3. Epub ahead of print.). Conotoxins typically contain 10–46 amino acids, are rich in disulfide bonds, exhibit strong biological activity, and can specifically act on receptors and ion channels on animal cell membranes. In particular, they exhibit excellent selectivity for voltage-gated or ligand-gated ion channels (including a few G-protein-associated receptors). Conozotoxins can be classified into various pharmacological families based on their receptor target sites, such as α, ω, μ, and δ.

[0007] Alpha-conotoxin is a highly selective and specific blocker of various subtypes of nicotinic acetylcholine receptors (nAChRs). It has a relatively small molecular weight, generally composed of 12-19 amino acid residues, and is rich in disulfide bonds (P. Gopalakrishnakone LJC, Sulan Luo. Toxins and Drug Discovery. Springer Nature (Publisher) 2017; ISBN 978-94-007-6451-4: Conotoxin: p148-187.). Alpha-conotoxins are diverse in species, activities, and structures. They can be classified based on their highly conserved signal peptide sequences, pharmacological activities, and cysteine ​​patterns. The cysteine ​​sequence of α-conotoxin is CC-CC, with disulfide bonds linked by C1-C3 and C2-C4, forming two loops. Based on the different amino acid numbers between the di- and tri-cysteine ​​residues and the tri- and tetra-cysteine ​​residues, α-conotoxin can be further divided into several subfamilies, such as α3 / 5, α4 / 7, α4 / 6, α4 / 4, and α4 / 3. The characteristics and residue composition of each loop are the basis for the toxin's action on different receptor subtypes (Kaas Q, Yu R, Jin AH, Dutertre S, Craik DJ. ConoServer: updated content, knowledge, and discovery tools in the conopeptide database. Nucleic acids research 2012; 40: D325-330.). Therefore, α-conotoxins and their target nAChRs are of great value in the study of various disease mechanisms and drug development (Ying Fu, Cheng Li, Shui Dong, Yong Wu, Dongting Zhangsun and Sulan Luo*. Discovery Methodology of Novel Conotoxins from Conus Species. Marine Drugs, 2018, 16, 417; doi:10.3390 / md16110417.).

[0008] The natural peptide of α-conotoxin has disulfide bonds linked by C1-C3 and C2-C4, known as the globular isomer, forming two loop rings between the disulfide bonds. The linear α-conotoxin peptide containing four cysteine ​​residues often produces three isomers upon oxidative folding: besides the natural peptide disulfide bond linkage between C1-C3 and C2-C4 (the globular isomer), the other two isomers are the ribbon isomer and the bead isomer. The ribbon isomer has disulfide bonds linked by C1-C4 and C2-C3; the bead isomer has disulfide bonds linked by C1-C2 and C3-C4. Spherical isomers have full biological activity, while ribbon-like isomers sometimes exert biological activity through different mechanisms of action, and bead-like isomers often have reduced activity (Ulens C, Hogg RC, Celie PH, et al. Structural determinants of selective alpha-conotoxin binding to a nicotinic acetylcholine receptor homolog AChBP[J]. Proc Natl Acad Sci USA 2006;103:3615–20;Terlau, H.;Olivera, BM, Conus venoms: a rich source of novel ion channel-targeted peptides. Physiological reviews 2004,84(1),41-68. Grishin AA, Wang CI, Muttenthaler M, Alewood PF, Lewis RJ, Adams DJ. Alpha-conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity tostoichiometry of alpha3beta4 nicotinic acetylcholine receptors. J BiolChem. 2010, 285(29):22254-63).

[0009] Neuropathic pain (chronic pain) is a pain syndrome caused by damage or dysfunction of the central or peripheral nervous system. Many diseases, including cancer, trauma (nerve injury, compression), metabolic disorders (diabetes), and poisoning (chemotherapy), can cause neuropathic pain. Neuropathic pain is a major disease affecting human quality of life, causing a huge social and economic burden. According to epidemiological and medical cost surveys in the United States (Vadivelu N, Kai AM, Kodumudi G, Babayan K, Fontes M, Burg MM. Pain and Psychology-A Reciprocal Relationship. The Ochsner journal 2017; 17:173-180), neuropathic pain affects more than 20% of the global population; more than 50% of people over 65 years of age suffer from neuropathic pain. In the United States, there are as many as 100 million adult chronic pain patients, with annual medical costs exceeding $600 billion ($635-$650 billion). Neuropathic pain not only severely impacts patients' physical and mental health but also consumes hundreds of billions of dollars in medical resources annually. The medical losses caused by neuropathic pain exceed the combined losses from cancer, heart disease, and diabetes (Gaskin DJ, Richard P. The economic costs of pain in the United States. The Journal of Pain: Official Journal of the American Pain Society 2012; 13:715-724). my country has an even larger number of neuropathic pain patients and has paid a heavy price for it. However, there is currently no specific drug for treating neuropathic pain in clinical practice.

[0010] nAChRs containing α3-subunits, including the α3β2 and α3β4 subtypes, are mainly expressed in the peripheral nervous system and are targets for neuropathic pain drugs. α-conotoxins that block α3β2 or α3β4 nAChRs have shown good analgesic activity in various preclinical models of chronic pain and are not addictive. Intractable pain is a global health problem, and new treatments are urgently needed. (Napier, IA; Klimis, H.; Rycroft, BK; Jin, AH; Alewood, PF; Motin, L.; Adams, DJ; Christie, MJ, Intrathecal α-conotoxins Vc1.1, AuIB and MII acting on distinct nicotinic receptor subtypes reverse signs of neuropathic pain. Neuroopharmacology 2012, 62(7), 2202-2207. Blyth, FM; March, LM; Brnabic, AJ; Jorm, LR; Williamson, M.; Cousins, MJ, Chronic pain in Australia: a prevalence study. PAIN 2001, 89(2-3), 127-34. Cousins, MJ; Brennan, F.; Carr, DB, Pain relief: a universal human right. PAIN) 2004,112(1-2),1-4. Eisenberg, E.; McNicol, ED; Carr, DB. Efficacy and safety of opioid agonists in the treatment of neuropathic pain of nonmalignant origin: systematic review and meta-analysis of randomized controlled trials. JAMA: the journal of the American Medical Association 2005, 293(24), 3043-52.).

[0011] Drug addiction is both a medical challenge and a serious social problem. Smoking addiction is caused by nicotine in tobacco, and its receptors in the body are nicotinic acetylcholine receptors (nAChRs) (Azam L, McIntosh JM. Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors. Acta Pharmacol Sin. 2009; 30(6):771-783). Studies have shown that nAChRs expressed in dopaminergic (DA) neurons are drug targets for the treatment of neuropsychiatric disorders, such as addiction to nicotine, morphine, and cocaine, Parkinson's disease, dementia, schizophrenia, and depression (Larsson, A.; Jerlhag, E.; Svensson, L.; Soderpalm, B.; Engel, JA. Is an alpha-conotoxin MII-sensitive mechanism involved in the neurochemical, stimulatory, and rewarding effects of ethanol? Alcohol 2004, 34(2-3), 239-50. Jerlhag, E.; Egecioglu, E.; Dickson, SL; Svensson, L.; Engel, JA. Alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors are involved in mediating the ghrelin-induced locomotor stimulation and dopamine overflow in nucleus). accumbens.European neuropsychopharmacology, 2008,18(7),508-18).α-conotoxin MII, which blocks α3β2 and α6β2* (* represents other subunits) nAChRs, can partially and differentially block nicotinic dopamine release in the striatum. Presynaptic nAChRs contain at least two subtypes, namely MII-sensitive and MII-insensitive, which regulate DA release from dopamine neurons (Kaiser SA, Soliakov L, Harvey SC, Luetje CW, Wonnacott S. Differential inhibition by α-conotoxin-MII of the nicotinic stimulation of [3H]dopamine release from rat striatal synaptosomes and slices. J Neurochem 1998; 70:1069-76). Recent studies have reported that blocking α6β2-containing nAChRs can effectively prevent the onset of nicotine and morphine addictions and significantly suppress the desire to smoke and take drugs (Brunzell DH, Boschen KE, Hendrick ES, Beardsley PM, McIntosh JM. Alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors in the nucleus accumbens shell regulate progressive ratio responding maintained by nicotine. Neuropsychopharmacology, 2010; 35(3):665-673.).

[0012] Alpha-6β2*-nAChRs subtypes in the striatum of mammalian brains are considered drug targets for treating nicotine, drug, and alcohol addictions (Exley, R.; Clements, MA; Hartung, H.; McIntosh, JM; Cragg, SJ, Alpha6-containing nicotinic acetylcholine receptors dominate the nicotine control of dopamine neurotransmission in nucleus accumbens. Neuropsychopharmacology 2008, 33(9), 2158-66. Gao, F.; Chen, D.; Ma, X.; Sudweeks, S.; Yorkason, JT; Gao, M.; Turner, D.; Eaton, JB; McIntosh, JM; Lukas, RJ; Whiteaker, P.; Chang, Y.; Steffensen, SC; Wu, J. Alpha6-containing nicotinic Acetylcholine receptor (AChR) is a highly sensitive target of alcohol. (Neuropharmacology 2019, 149, 45-54.) The α6 subunit is not widely distributed in the brain, but it is enriched in dopaminergic neurons in the midbrain, an area closely related to pleasure, reward, and mood control. This suggests that α6*nAChRs play a crucial role in the regulation of drug-induced addiction and mood control.

[0013] α3β2 nAChRs play crucial physiological roles and are closely related to pain, learning, memory, movement, body temperature, depression, addiction, and cardiovascular diseases. However, due to the lack of specific ligands or drug targets for α3β2 nAChRs, research on their physiological functions and pathological mechanisms is still insufficient. Therefore, there is an urgent need to discover new, highly specific nAChRs, especially α3β2 nAChR blockers. Developing new, potent α3β2 nAChR blockers, particularly those capable of differentiating other subtypes of nAChRs with similar structures and overlapping distributions, as well as researching disease mechanisms related to α3β2 nAChRs, screening new drugs, and developing new therapeutic agents, has significant scientific importance and enormous economic value. Summary of the Invention

[0014] Through in-depth research and creative labor, the inventors discovered novel α-conotoxin peptides (named LvIE and LvIF, corresponding to SEQ ID NO:5 and SEQ ID NO:8, respectively), and further synthesized these two peptides artificially. The inventors surprisingly found that both LvIE and LvIF can specifically block α3β2 acetylcholine receptors, exhibiting highly selective and strong blocking activity, and possessing the potential for use in the preparation or screening of drugs for the treatment and / or prevention of diseases related to α3β2, α6 / α3β2β3 (α6β2*) acetylcholine receptors. Therefore, the following invention is provided:

[0015] One aspect of the present invention relates to an isolated polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs:4-5 and SEQ ID NOs:7-8.

[0016] In some embodiments of the present invention, the polypeptide, wherein...

[0017] The first cysteine ​​residue at the N-terminus of the polypeptide forms a disulfide bond with the third cysteine ​​residue, and the second cysteine ​​residue forms a disulfide bond with the fourth cysteine ​​residue; or the first cysteine ​​residue at the N-terminus of the polypeptide forms a disulfide bond with the fourth cysteine ​​residue, and the second cysteine ​​residue forms a disulfide bond with the third cysteine ​​residue; or the first cysteine ​​residue at the N-terminus of the polypeptide forms a disulfide bond with the second cysteine ​​residue, and the third cysteine ​​residue forms a disulfide bond with the fourth cysteine ​​residue.

[0018] Preferably, the carboxyl terminus of the polypeptide is amidated.

[0019] Another aspect of the present invention relates to an isolated fusion protein comprising at least one polypeptide as described in any one of the present invention.

[0020] Another aspect of the invention relates to an isolated polynucleotide encoding the polypeptide or fusion protein described in any one of the inventions.

[0021] Another aspect of the present invention relates to a nucleic acid construct containing the polynucleotide of the present invention; preferably, the nucleic acid construct is a recombinant vector; preferably, the nucleic acid construct is a recombinant expression vector.

[0022] Another aspect of the invention relates to a transformed cell containing the polynucleotide of the invention, or containing the nucleic acid construct of the invention.

[0023] The polypeptide, fusion protein, or polynucleotide of any one of the present invention is used to prepare a drug that blocks acetylcholine receptors; wherein the acetylcholine receptor is an α3β2 acetylcholine receptor or an α6β2* acetylcholine receptor.

[0024] The polypeptide, fusion protein, or polynucleotide of any one of the present inventions is used to prepare a medicament for treating and / or preventing neuralgia, addiction, Parkinson's disease, dementia, schizophrenia, or depression.

[0025] Preferably, the neuralgia is caused by one or more of the following factors: cancer and cancer chemotherapy, alcohol poisoning, sciatica, diabetes, trigeminal neuralgia, sclerosis, herpes zoster, mechanical injury and surgical injury, AIDS, cephalopathy, drug poisoning, industrial pollution poisoning, lymphatic neuralgia, myeloma, multipoint motor neuralgia, chronic congenital sensory neuropathy, acute severe spontaneous neuralgia, crush neuralgia, vasculitis, vasculitis, local ischemia, uremia, childhood biliary liver disease, chronic respiratory disorders, complex neuralgia, multiple organ failure, sepsis / septicemia, hepatitis, porphyria, vitamin deficiency, chronic liver disease, primary cholecystitis, hyperlipidemia, leprosy, Lyme arthritis, sensory neuritis, or allergy.

[0026] Preferably, the addiction is a smoking addiction, an alcohol addiction, or a drug addiction;

[0027] Preferably, the addiction is caused by one or more of the following factors: nicotine, opium, heroin, methamphetamine (ice), morphine, cannabis, or cocaine.

[0028] Another aspect of the invention relates to a pharmaceutical composition comprising at least one polypeptide, fusion protein, or polynucleotide as described in any one of the inventions; optionally, it further comprises one or more pharmaceutically acceptable excipients.

[0029] In some embodiments, pharmaceutical compositions containing a therapeutically effective amount of the polypeptide of the present invention are formulated and administered in a manner conducive to pharmaceutical use, taking into account the individual patient's clinical condition, delivery site, method of administration, dosing schedule, and other factors known to the physician. Therefore, the term "effective amount" used for the purposes herein is determined by these considerations.

[0030] Another aspect of the present invention relates to the use of any of the polypeptides, fusion proteins, or polynucleotides described in any one of the present invention in the preparation of a medicament that blocks acetylcholine receptors; wherein the acetylcholine receptor is an α3β2 acetylcholine receptor or an α6β2* acetylcholine receptor.

[0031] Another aspect of the present invention relates to the use of any of the polypeptides, fusion proteins, or polynucleotides described in any one of the present invention in the preparation of medicaments for the treatment and / or prevention of neuralgia, addiction, Parkinson's disease, dementia, schizophrenia, or depression.

[0032] Preferably, the neuralgia is caused by one or more of the following factors: cancer and cancer chemotherapy, alcohol poisoning, sciatica, diabetes, trigeminal neuralgia, sclerosis, herpes zoster, mechanical injury and surgical injury, AIDS, cephalopathy, drug poisoning, industrial pollution poisoning, lymphatic neuralgia, myeloma, multipoint motor neuralgia, chronic congenital sensory neuropathy, acute severe spontaneous neuralgia, crush neuralgia, vasculitis, vasculitis, local ischemia, uremia, childhood biliary liver disease, chronic respiratory disorders, complex neuralgia, multiple organ failure, sepsis / septicemia, hepatitis, porphyria, vitamin deficiency, chronic liver disease, primary cholecystitis, hyperlipidemia, leprosy, Lyme arthritis, sensory neuritis, or allergy.

[0033] Preferably, the addiction is a smoking addiction, an alcohol addiction, or a drug addiction;

[0034] Preferably, the addiction is caused by one or more of the following factors: nicotine, opium, heroin, methamphetamine (ice), morphine, cannabis, or cocaine.

[0035] Another aspect of the invention relates to a method for treating and / or preventing neuralgia, addiction, Parkinson's disease, dementia, schizophrenia, or depression, comprising the step of administering to a subject in need an effective amount of any of the polypeptides, fusion proteins, or polynucleotides of the invention described in any one of the inventions.

[0036] Preferably, the neuralgia is caused by one or more of the following factors: cancer and cancer chemotherapy, alcohol poisoning, sciatica, diabetes, trigeminal neuralgia, sclerosis, herpes zoster, mechanical injury and surgical injury, AIDS, cephalopathy, drug poisoning, industrial pollution poisoning, lymphatic neuralgia, myeloma, multipoint motor neuralgia, chronic congenital sensory neuropathy, acute severe spontaneous neuralgia, crush neuralgia, vasculitis, vasculitis, local ischemia, uremia, childhood biliary liver disease, chronic respiratory disorders, complex neuralgia, multiple organ failure, sepsis / septicemia, hepatitis, porphyria, vitamin deficiency, chronic liver disease, primary cholecystitis, hyperlipidemia, leprosy, Lyme arthritis, sensory neuritis, or allergy.

[0037] Preferably, the addiction is a smoking addiction, an alcohol addiction, or a drug addiction;

[0038] Preferably, the addiction is caused by one or more of the following factors: nicotine, opium, heroin, methamphetamine (ice), morphine, cannabis, or cocaine.

[0039] Dosage is determined by many factors, such as the severity of the condition being treated, the sex, age, weight, and individual response of the patient or animal, as well as the patient's condition and medical history. Common practice in the art is to start with a dose below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0040] Another aspect of the present invention relates to a method for blocking acetylcholine receptors or regulating acetylcholine levels in vitro, comprising the step of applying to cells an effective amount of any of the polypeptides, fusion proteins, or polynucleotides of the present invention as described above; wherein the acetylcholine receptor is an α3β2 acetylcholine receptor or an α6β2* acetylcholine receptor.

[0041] Another aspect of the present invention relates to the use of any of the polypeptides, fusion proteins, or polynucleotides described in any one of the present invention in the preparation of drug screening models, wherein,

[0042] The drug screening model is a cell model or an animal model.

[0043] The drug is used to treat and / or prevent neuralgia, addiction, Parkinson's disease, dementia, schizophrenia, or depression;

[0044] Preferably, the neuralgia is caused by one or more of the following factors: cancer and cancer chemotherapy, alcohol poisoning, sciatica, diabetes, trigeminal neuralgia, sclerosis, herpes zoster, mechanical injury and surgical injury, AIDS, cephalopathy, drug poisoning, industrial pollution poisoning, lymphatic neuralgia, myeloma, multipoint motor neuralgia, chronic congenital sensory neuropathy, acute severe spontaneous neuralgia, crush neuralgia, vasculitis, vasculitis, local ischemia, uremia, childhood biliary liver disease, chronic respiratory disorders, complex neuralgia, multiple organ failure, sepsis / septicemia, hepatitis, porphyria, vitamin deficiency, chronic liver disease, primary cholecystitis, hyperlipidemia, leprosy, Lyme arthritis, sensory neuritis, or allergy.

[0045] Preferably, the addiction is a smoking addiction, an alcohol addiction, or a drug addiction;

[0046] Preferably, the addiction is caused by one or more of the following factors: nicotine, opium, heroin, methamphetamine (ice), morphine, cannabis, or cocaine.

[0047] Another aspect of the present invention relates to a method for preparing a drug screening model, comprising the step of administering to target cells or target animals an effective amount of any of the polypeptides, fusion proteins, or polynucleotides of the present invention, wherein...

[0048] The drug screening model is a cell model or an animal model;

[0049] The drug is used to treat and / or prevent neuralgia, addiction, Parkinson's disease, dementia, schizophrenia, or depression;

[0050] Preferably, the neuralgia is caused by one or more of the following factors: cancer and cancer chemotherapy, alcohol poisoning, sciatica, diabetes, trigeminal neuralgia, sclerosis, herpes zoster, mechanical injury and surgical injury, AIDS, cephalopathy, drug poisoning, industrial pollution poisoning, lymphatic neuralgia, myeloma, multipoint motor neuralgia, chronic congenital sensory neuropathy, acute severe spontaneous neuralgia, crush neuralgia, vasculitis, vasculitis, local ischemia, uremia, childhood biliary liver disease, chronic respiratory disorders, complex neuralgia, multiple organ failure, sepsis / septicemia, hepatitis, porphyria, vitamin deficiency, chronic liver disease, primary cholecystitis, hyperlipidemia, leprosy, Lyme arthritis, sensory neuritis, or allergy.

[0051] Preferably, the addiction is a smoking addiction, an alcohol addiction, or a drug addiction;

[0052] Preferably, the addiction is caused by one or more of the following factors: nicotine, opium, heroin, methamphetamine (ice), morphine, cannabis, or cocaine;

[0053] Preferably, the neuralgia, addiction, Parkinson's disease, dementia, schizophrenia, or depression is related to or caused by α3β2 acetylcholine receptors or α6β2* acetylcholine receptors, or by α3β2 acetylcholine receptors or α6β2* acetylcholine receptors with expression levels higher than normal or higher than normal expression levels in patients.

[0054] In some embodiments of the present invention, the cell model or animal model exhibits reduced or blocked levels of α3β2 acetylcholine receptors or α6β2* acetylcholine receptors. In some embodiments of the present invention, the reduced or blocked levels of α3β2 acetylcholine receptors or α6β2* acetylcholine receptors in the cell model or animal model can be achieved by methods including, but not limited to, one of the following: applying an effective amount of the polypeptide or fusion protein of any one of the present invention to the target cells or target animal; or transferring the polynucleotide or nucleic acid construct of the present invention into the target cells or target animal via gene transduction to produce an effective amount of the polypeptide or fusion protein of any one of the present invention.

[0055] Another aspect of the present invention relates to a method for preparing the polypeptide according to any one of the present invention, comprising the following steps:

[0056] 1) Linear peptides were synthesized on an ABI Prism 433a peptide synthesizer or by manual method. The side chain protecting groups of Fmoc amino acids were: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp), Boc (Lys); cysteine ​​was protected by Trt or Acm groups, and disulfide bonds were formed between the corresponding cysteine ​​residues at specific sites.

[0057] 2) The linear polypeptide obtained in step 1) was cut off from the resin, and the crude linear polypeptide was recovered by precipitation and washing with ice-cold ether, and purified by preparative reverse HPLC C18 column (Vydac).

[0058] 3) Perform a two-step oxidative folding on the product obtained in step 2).

[0059] In this invention:

[0060] The term "nucleic acid construct" is defined herein as a single-stranded or double-stranded nucleic acid molecule, preferably an artificially constructed nucleic acid molecule. Optionally, the nucleic acid construct may further comprise one or more operatively linked regulatory sequences.

[0061] In this invention, the term "operably linked" refers to the functional spatial arrangement of two or more nucleotide regions or nucleic acid sequences. This "operably linked" arrangement can be achieved through gene recombination.

[0062] In this invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide that inhibits a protein can be inserted. For example, vectors include: plasmids; phage particles; Cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression.

[0063] In this invention, the term "host cell" refers to the cell into which the vector is introduced, including many cell types such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0064] Those skilled in the art will understand that acetylcholine receptors (muscular or neural types) in mammals (such as rodents and primates like humans) are pentamer transmembrane proteins composed of five identical or different subunits, and there are many subtypes. The structures of these subtypes are very similar, but their pharmacological characteristics and physiological functions are quite different. For example, α2β2nAChRs indicates a functional receptor containing both α2 and β2 subunits, and the total number of α2 and β2 subunits is 5, i.e., a pentamer protein. The rest can be deduced similarly.

[0065] α6β2*nAChRs represent a chimeric subunit of the receptor containing an α6 subunit or an extracellular ligand-binding region of α6 (e.g., α6 / α3), and a β2 subunit. In some embodiments of the invention, α6 further includes α6 / α3, wherein α6 / α3 represents a chimeric subunit of the extracellular region of α6 and the transmembrane region of α3, which facilitates the in vitro expression of α6β2*nAChRs. The α6 / α3 chimeric subunit is functionally equivalent to the α6 subunit. In some embodiments of the invention, the α6β2*acetylcholine receptor is α6β2 nAChRs or α6 / α3β2β3 nAChRs.

[0066] The term "effective dose" refers to a dose that can achieve therapeutic, preventive, alleviating and / or relieving disease or condition as described in this invention in a subject.

[0067] The term "disease and / or symptom" refers to a physical condition of the subject that is related to the disease and / or symptom described in this invention.

[0068] The term "subject" can refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the pharmaceutical composition of the present invention to treat, prevent, reduce and / or alleviate the disease or condition described in the present invention.

[0069] In this invention, unless otherwise specified, the concentration units μM represent μmol / L, mM represent mmol / L, and nM represent nmol / L.

[0070] In this invention, when referring to the amount of drug added to cells, unless otherwise specified, it generally refers to the final concentration of the drug after addition.

[0071] When the term "amino acid" or a specific amino acid name is mentioned in this invention, unless otherwise specified, it refers to L-type amino acids.

[0072] Beneficial effects of the invention

[0073] This invention achieves one or more of the following technical effects:

[0074] (1) The cone snail toxin peptide (LvIE or LvIF) of the present invention can effectively block α3β2 acetylcholine receptors or α6β2* acetylcholine receptors.

[0075] (2) The cone snail toxin peptide (LvIE or LvIF) of the present invention can specifically block α3β2 acetylcholine receptors or α6β2* acetylcholine receptors.

[0076] (3) The cone snail toxin peptide of the present invention can exert its effects by binding to α6β2* acetylcholine receptors (nAChRs) and has the activity of treating and / or preventing neuralgia, addiction, Parkinson's disease, dementia, schizophrenia or depression or any disease related to α3β2 acetylcholine receptors or α6β2* acetylcholine receptors.

[0077] (4) The cone snail toxin peptides of the present invention can be applied to the research, diagnosis, screening, and treatment of neuralgia, addiction, Parkinson's disease, dementia, schizophrenia, or depression, or any disease related to α3β2 acetylcholine receptors or α6β2* acetylcholine receptors, as well as as useful molecular probes for research. Different α-conotoxins have different affinities for vertebrate receptors, sometimes differing by several orders of magnitude. This interspecies difference makes α-conotoxins useful probes for studying the phylogeny of vertebrate nAChRs and for identifying different subtypes of nAChRs. They are candidate drugs, lead drugs, and therapeutics for new drug development.

[0078] The α-conotoxin peptide of this invention can specifically block acetylcholine receptors (nAChRs) and has strong analgesic and addiction-reducing activities, as well as therapeutic effects on diseases such as Parkinson's disease, dementia, schizophrenia, and depression. Attached Figure Description

[0079] Figure 1A-Figure 1B α-Conotoxin LvIE (Peptide 1) ( Figure 1A ) and LvIF (peptide 2) ( Figure 1B The sequence of ) and its disulfide bond linkage Cys(I-III,II-IV). Each substituted amino acid is underlined. Cysteine ​​is indicated in italics. # indicates C-terminal amidation.

[0080] Figures 2A-2B α-Conotoxin LvIE ( Figure 2A ) and LvIF( Figure 2BThe sequence of the propeptide gene and the propeptide it encodes, along with the mature peptide resulting from post-translational modifications, are shown. Arrows indicate post-translational modification sites. The inferred proteolytic processing site 1 is located after the basic amino acid arginine (R); the C-terminal amidation site is likely located at the two glycine residues indicated by the arrow, i.e., processing site 2. The glycine residue immediately adjacent to cysteine ​​(Cys) at the C-terminus of the mature peptide is often the processing site for amidation post-translational modifications. The mature peptide resulting from amidation at processing site 2 is named LvIE, with the sequence: GCCSHPACAGNNPHIC# (# indicates C-terminal amidation). The propeptide region is shown in italics, the mature peptide is underlined, and cysteine ​​(C) is shown in bold.

[0081] Figure 3A and Figure 3C These are the high-performance liquid chromatograms of LvIE and LvIF, respectively.

[0082] The HPLC analysis conditions were as follows: C18 column (Vydac), linear elution gradient of 10-40% B60 (containing 60% acetonitrile aqueous solution) over 0-40 min, and monitoring wavelength of 214 nm. Solvent B60 was an aqueous solution containing 90% acetonitrile (ACN) and 0.05% trifluoroacetic acid (TFA); solvent A was an aqueous solution of 0.05% TFA.

[0083] The minute count in an HPLC chromatogram indicates the retention time of that chromatographic peak.

[0084] The horizontal axis represents elution time in minutes (min); the vertical axis represents the ultraviolet absorbance (UVA) at a wavelength of 214 nm. 214 ).

[0085] Figure 3B and Figure 3D These are the ESI-MS mass spectra of LvIE and LvIF, respectively.

[0086] Figures 4A-4BThe figures show the effects of α-CTx LvIE and LvIF (10 μM) on the currents of various nAChR isoforms expressed in Xenopus laevis oocytes. All data represent Mean ± SEM, n = 4–6. The x-axis represents the percentage of current response (%), calculated as: the current of each nAChR isoform at a 10 μM LvIE concentration divided by its respective control current (ND96). Here, control refers to the current generated by ACh excitation after incubation for 5 min in the cell culture medium with the same volume of ND96 buffer as LvIE or LvIF; this is the control current.

[0087] Figures 5A-5D The diagram shows the current trajectory of LvIE or LvIF blocking α3β2 in rats, representing highly selective and specific α3β2 inhibitors. In the diagram, "C" represents the control (ND96) current, and the value immediately following "C" is the peptide concentration. The arrows indicate the current trajectory formed by the first Ach pulse of the corresponding receptor subtype after 5 minutes of incubation, where the peptide blocks these impulses.

[0088] Figure 5A Effects of 10 nM LvIE on the current of rat α3β2 nAChR;

[0089] Figure 5B Effects of 10 nM LvIE on the current of rat α6 / α3β2β3 nAChR;

[0090] Figure 5C Effects of 100 nM LvIF on the current of rat α3β2 nAChR;

[0091] Figure 5D Effects of 100 nM LvIF on the current of rat α6 / α3β2β3 nAChR;

[0092] Rat α3β2 and α6 / α3β2β3 nAChRs were expressed in Xenopus laevis oocytes. The clamping voltage during electrophysiological recording was -70 mV, and ACh pulses were administered every 1 second (s) per minute according to the experimental procedure. LvIE and LvIF inhibited the expression of rat α3β2 ( Figure 5A Figure 5C ) and rat α6 / α3β2β3 nAChRs ( Figure 5B Figure 5D It has a good blocking effect on the current.

[0093] Figures 6A-6BThe concentration-dose response curves of LvIE and LvIF for all nAChR subtypes are shown. The horizontal axis represents the logarithm of the molar concentration (M) of the LvIE and LvIF peptides used (Log[Peptide]M); the vertical axis represents the percentage of concentration response (Response%), which is the ratio of the acetylcholine receptor current to the control current at the corresponding peptide concentration. All values ​​in the figure are the average current values ​​taken from 4-6 Xenopus oocytes, i.e., Mean±SEM, n=4-6.

[0094] The sequences involved in this invention are shown in Table 1 below.

[0095] Table 1: SEQ ID NO and sequence number of LvIE and LvIF

[0096]

[0097] Detailed Implementation

[0098] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press), relevant references, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0099] Example 1: Cloning and sequence analysis of the α-conotoxin LvIE gene

[0100] 1. Extraction of genomic DNA from Conophytum fasciatus

[0101] Live cone snails (Conus lividus) collected from coastal areas of Hainan Island and the Xisha Islands were used as materials and stored at -80℃ for later use. First, the venom glands of the cone snails were dissected and weighed. Then, genomic DNA was extracted from the venom glands using a marine animal genomic DNA extraction kit (purchased from Beijing Tiangen Biotech Co., Ltd., China). Specific procedures are detailed in the kit's instructions.

[0102] Total genomic DNA extracted from cone snails was dissolved in 100 μL TE buffer, and 5 μL was used for 1.0% agarose gel electrophoresis. The integrity and size of the obtained DNA were assessed using the λ-EcoT14 I digest DNA marker as a standard. The OD260, OD280 values, and OD260 / OD280 ratio of the DNA solution were measured using a nucleic acid and protein analyzer, and the DNA concentration (μg·ml) was calculated. -1 Purity and DNA yield (μg·g) -1 The extracted intact DNA was used as a template for the next step of PCR amplification to clone the cone snail toxin gene.

[0103] 2. Cloning, sequencing, and sequence analysis of PCR reactions and their products.

[0104] Based on the intron sequence of the α-conotoxin precursor gene and its 3' untranslated region (3'-UTR) sequence, α-conotoxin-specific primers were designed, each primer being an 18-base oligonucleotide fragment.

[0105] The upstream intron primer sequence is 5'-GTGGTTCTGGGTCCAGCA-3' (SEQ ID NO:1);

[0106] The downstream 3'-UTR primer sequence is 5'-GTCGTGGTTCAGAGGGTC-3' (SEQ ID NO:2).

[0107] The extracted genomic DNA stock solution was diluted and used as a template for PCR amplification. The PCR-specific amplification product was recovered, ligated into the T-easy vector (Promega), and transformed into E. coli XL1 strain. Recombinants were selected using blue-white colony and ampicillin resistance methods, and the recombinant plasmids were extracted and purified for sequencing analysis.

[0108] The obtained PCR-specific amplification product sequences were analyzed using DNAStar software to determine their encoded protein sequences and 3'-untranslated region (UTR) sequences. The signal peptide, propeptide, and mature peptide of the cone snail toxin precursor protein were predicted using the online ProP1.0 Server (Duckert, P.; Brunak, S.; Blom, N., Prediction of proprotein converter cleavage sites. Protein engineering, design & selection: PEDS 2004, 17(1), 107-12.).

[0109] Sequence analysis and comparison revealed a novel α-conotoxin (named LvIE) precursor gene. Figure 2A ):

[0110] GTGGTTCTGGGTCCAGCATTTGATGGCAGGAATGCTGCAGCCAGCGACAAAGCGTCTGAGCTGATGGCTCTGGCCGTCAGG GGA TGCTGTTCCCATCCTGCCTGTGCTGGGAATAATCCACATATCTGT GGC AGAAGACGCTGATGCCCCAGGACCCTCTGAACCACGAC(SEQ ID NO:3)

[0111] Based on the precursor gene and characteristics of cone snail toxins, the LvIE cone snail toxin precursor peptide was deduced, and its amino acid sequence is as follows:

[0112] VVLGPAFDGRNAAASDKASELMALAVRGCCSHPACAGNNPH ICGRRR(SEQ ID NO:4)

[0113] Based on the propeptide sequence, the mature peptide LvIE was deduced, with the amino acid sequence GCCSHPACAGNNPHIC (SEQ ID NO: 5). For the deduction method and principle, please refer to Luo S, Zhangsun D, ​​Zhang B, Quan Y, Wu Y. Novel alpha-conotoxins identified by gene sequencing from cone snails native to Hainan, and their sequence diversity. J Pept Sci. 2006, 12(11): 693-704. For detailed derivation results, please refer to [link to relevant documentation]. Figure 1A , Figure 2A .

[0114] LvIE is a novel α-conotoxin with an amidation modification at its C-terminus. LvIE contains a CC-CC cysteine ​​sequence, and its disulfide linkage is Cys(I-III,II-IV). Figure 1A LvIE forms two disulfide bonds between the first and third cysteine ​​residues, and between the second and fourth cysteine ​​residues, respectively. LvIE differs from other known α-conotoxins.

[0115] Example 2: Cloning and sequence analysis of the α-conotoxin LvIF gene

[0116] 1. Extraction of genomic DNA from Conophytum fasciatus

[0117] Same as Example 1 above.

[0118] 2. Cloning, sequencing, and sequence analysis of PCR reactions and their products.

[0119] Same as Example 1 above.

[0120] Sequence analysis and comparison revealed a novel α-conotoxin (named LvIF) precursor gene. Figure 2B ):

[0121] GTGGTTCTGGGTCCAGCATTTGATGGCAGGAATGCTGCAGCCAGCGACAAAGCTTCTGAGCTGATGGCTCTGGCCGTCAGG GGA TGCTGTTCCCATCCTGCCTGTGCCGGGAATAATCAAGACATCTGT GGC TGAAGACGCTGATGCCCCAGGACCCTCTGAACCACGAC(SEQ ID NO:6)

[0122] Based on the precursor gene and characteristics of cone snail toxins, the LvIF cone snail toxin precursor peptide was deduced, and its amino acid sequence is as follows:

[0123] VVLGPAFDGRNAAASDKASELMALAVRGCCSHPACAGNNQDICGRRR(SEQ ID NO:7)

[0124] Based on the propeptide sequence, the mature peptide LvIF was deduced, with the amino acid sequence GCCSHPACAGNNQDIC (SEQ ID NO: 8). For the deduction method and principle, please refer to Luo S, Zhangsun D, ​​Zhang B, Quan Y, Wu Y. Novel alpha-conotoxins identified by gene sequencing from cone snails native to Hainan, and their sequence diversity. J Pept Sci. 2006, 12(11): 693-704. For detailed derivation results, please refer to [link to derivation results]. Figure 1B , Figure 2B .

[0125] LvIF is a novel α-conotoxin with an amidation modification at its C-terminus. LvIF contains a CC-CC cysteine ​​sequence, with disulfide bonds linked in a Cys(I-III,II-IV) configuration. Figure 1B LvIF forms two disulfide bonds between the first and third cysteine ​​residues, and between the second and fourth cysteine ​​residues, respectively. LvIF differs from other known α-conotoxins.

[0126] Example 3: Artificial Synthesis of α-Conotoxins LvIE and LvIF

[0127] Based on the amino acid sequences of the mature α-conotoxin peptides LvIE and LvIF (SEQ ID NO:5 and SEQ ID NO:8), a linear peptide was artificially synthesized using the Fmoc method. Figure 1A and Figure 1B ).

[0128] The specific method is as follows:

[0129] Resin peptides are synthesized artificially using the Fmoc chemical method, which can be performed using a peptide synthesizer or by manual synthesis. Except for cysteine, the remaining amino acids are protected with standard side-chain protection groups. The -SH groups of the first and third cysteine ​​residues (Cys) in LvIE are protected with Trt (S-trityl), and the -SH groups of the second and fourth cysteine ​​residues are paired with Acm (S-acetamidomethyl).

[0130] The specific synthesis steps were as follows: The Fmoc and FastMoc methods from solid-phase synthesis were used to synthesize the peptide on an ABI Prism 433a peptide synthesizer. Figure 1A and Figure 1B The linear peptides were synthesized using Fmoc amino acids. The side-chain protecting groups for Fmoc amino acids were: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp), and Boc (Lys). The Fmoc HOBT DCC method was employed, with Rink amidating the resin and Fmoc amino acids. The synthesis steps were performed according to the instrument synthesis manual. To ensure complete reaction, the deprotection and coupling times with piperidine were appropriately extended. For difficult-to-couple amino acids, double coupling was used to obtain the resin peptide. The linear peptide was cleaved from the resin using reagent K (trifluoroaceticacid / water / ethanedithiol / phenol / thioanisole; 90:5:2.5:7.5:5, v / v / v / v / v). The crude linear peptide was recovered by precipitation and washing with ice-cold diethyl ether. Purification was performed using a preparative reverse-phase HPLC C18 column (Vydac) with an elution linear gradient of 10-40% B90 over 0-40 min at a monitoring wavelength of 214 nm. Solvent B90 is an aqueous solution containing 90% acetonitrile (CAN) and 0.05% trifluoroacetic acid (TFA); Solvent A is an aqueous solution containing 0.05% TFA.

[0131] The purified linear peptide was analyzed for purity using an analytical HPLC C18 column (Vydac) under the same elution conditions as above, at a flow rate of 1 mL / min. Its purity was above 95%, and it was used for oxidative folding.

[0132] Referring to the literature (Dowell, C.; Olivera, BM; Garrett, JE; Staheli, ST; Watkins, M.; Kuryatov, A.; Yoshikami, D.; Lindstrom, JM; McIntosh, JM, Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors. The Journal of Neuroscience 2003, 23(24), 8445-52.), a two-step oxidative folding reaction of the linear peptide of LvIE was performed, and the process is briefly described as follows:

[0133] First, the first disulfide bond was formed between the two cysteine ​​residues of the Trt protecting group by potassium ferricyanide oxidation (20 mM potassium ferricyanide, 0.1 M Tris, pH 7.5, 45 min). After purification by reversed-phase HPLC on a C18 column (Vydac), the monocyclic peptide was oxidized with iodine (10 mM iodine in H2O:trifluoroacetic acid:acetonitrile (78:2:20 by volume, 10 min) to remove the Acm residues from the other two cysteine ​​residues, simultaneously forming a second disulfide bond between these two cysteine ​​residues. The dicyclic peptide was then purified by reversed-phase HPLC on a C18 column (Vydac) to obtain α-conotoxin with disulfide bonds oriented between the corresponding cysteine ​​residues in a sequence from the N-terminus to the C-terminus, and identified by electrospray-mass spectrometry (ESI-MS).

[0134] The HPLC chromatograms and ESI-MS mass spectra of LvIE and LvIF after oxidative folding are shown below. Figure 3A and Figure 3C , Figure 3B and Figure 3D As shown in Table 2, the purity of the synthesized LvIE and LvIF was above 95%. The measured molecular weights of LvIE and LvIF were consistent with the theoretical molecular weights.

[0135] Table 2: Molecular weight of α-CTx LvIE and its mutants

[0136] polypeptide Theoretical molecular weight (Da, average) Measured molecular weight (Da) LvIE 1580.7 1578.8 LvIF 1589.8 1587.8

[0137] The results showed that the synthesized polypeptide had the correct molecular weight and high purity.

[0138] The peptide concentration was determined colorimetrically at a wavelength of 280 nm, and the peptide concentration and mass were calculated according to the Beer-Lambert equation. These quantified, folded peptides were used in Example 4 below.

[0139] Example 4: Effects of α-conotoxins LvIE and LvIF on rat α3β2 and rα6 / α3β2β3nAChRs and all others Activity studies of nAChRs subtypes

[0140] Following the method described in the literature (Azam L, Yoshikami D, McIntosh JM. Amino acid residues that confess high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII [S4A, E11A, L15A]. J Biol Chem. 2008; 283(17): 11625-32.), and the instructions of the in vitro transcription kit (mMessage mMachine in vitro transcription kit (Ambion, Austin, TX)), cRNAs of various rat neural nAChR subtypes (α3β2, α6 / α3β2β3, α6 / α3β4, α9α10, α4β2, α4β4, α3β4, α2β2, α2β4, α7) and rat muscle nAChRs (α1β1δε) were prepared, and their concentrations were calculated using OD values ​​at UV 260nm. African clawed frog (Xenopus laveis) oocytes were dissected and collected. cRNA was injected into the frog eggs at a dose of 5 ng per subunit. Rat muscle-based nAChR was administered via injection of 0.5–2.5 ng DNA per subunit. Frog eggs were cultured in ND-96. cRNA was injected 1–2 days after egg collection, and voltage-clamp recordings of nAChR were performed 1–4 days post-injection.

[0141] One frog egg injected with cRNA was placed in a 50 μL Sylgard recording well (4 mm in diameter × 2 mm in depth) and gravity-perfused with ND96 perfusion medium containing 0.1 mg / ml BSA (bovine serum albumin) (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5 mM HEPES, pH 7.1-7.5) or ND96 containing 1 mM atropine (ND96A) at a flow rate of 1 ml / min. All cone snail venom solutions also contained 0.1 mg / ml BSA to reduce non-specific adsorption of the venom. A switching valve (SmartValve, Cavro Scientific Instruments, Sunnyvale, CA) allowed for free switching between venom perfusion and acetylcholine (ACh), and a series of solenoid valves (model161TO31, Neptune Research, Northboro, MA) allowed for free switching between ND96 perfusion and ACh perfusion. ACh-gated current was set to "slow" clamping by a two-electrode voltage-clamped amplifier (model OC-725B, Warner Instrument Corp., Hamden, CT), and recorded online at the maximum clamp gain (×2000). Glass electrodes were drawn using 1 mm outer diameter × 0.75 mm inner diameter glass capillaries (fiber-filled borosilicate capillaries, WPI Inc., Sarasota, FL) and filled with 3M KCl as voltage and current electrodes. The membrane voltage was clamped at -70mV. The entire system was computer-controlled and data recorded. ACh pulses were performed by automatically injecting ACh for 1 second every 5 minutes. The ACh concentrations were as follows: 10 μM for α9 and α10 nAChRs expressing rat muscle and rat neural types; 200 μM for α7 of nAChRs expressing rat neural type; and 100 μM for other subtypes. The current responses and current trajectories of at least four oocytes expressing a specific subtype to different toxin concentrations were recorded.

[0142] The tested current data were statistically analyzed using GraphPad Prism software (San Diego, CA), and dose-response curves were plotted. Figures 6A to 6B ), calculate the half-blocking concentration (IC50) of cone snail toxin. 50 Various parameters related to peptide blocking nAChRs (Table 3).

[0143] Table 3: The effects of LvIE and LvIF (prepared in Example 3) on IC50 of different nicotinic acetylcholine receptor subtypes. 50 value

[0144]

[0145] a This indicates that at a concentration of 10 μM, the inhibition rate is less than 50%.

[0146] "--" indicates that no relevant experiments were conducted or that the experiment is not applicable.

[0147] The results show that LvIE ( Figure 6A ) and LvIF( Figure 6B It exhibits the strongest blocking activity against rat rα3β2 and rα6 / α3β2β3 nAChRs, with a half-blocking dose (IC50) of 100%. 50 The concentrations were 1.35 nM and 2.91 nM (LvIE), and 9.2 nM and 14.4 nM (LvIF), respectively (Table 2). At high concentrations of 10 μM, LvIE and LvIF showed almost no blocking effect on any other receptor subtypes. Figures 4A to 4B Table 3 shows good specificity. 10 nM LvIE and 100 nM LvIF effectively blocked the current generated by the opening of Ach-gated rat rα3β2 and rα6 / α3β2β3nAChRs. After LvIE and LvIF blocked rat rα3β2 and rα6 / α3β2β3nAChRs, the elution rate was rapid, and the current returned to the control level within 3 minutes. Figures 5A to 5D LvIE and LvIF showed reversible blockade of rat rα3β2 and rα6 / α3β2β3 nAChRs.

[0148] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof. SEQUENCE LISTING <110> Guangxi University <120> α-Conotoxin peptides LvIE and LvIF, their pharmaceutical compositions and uses <130> IDC210176 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 gtggttctgg gtccagca 18 <210> 2 <211> 18 <212> DNA<所 <213> Artificial Sequence <220> <223> Primer <400> 2 gtcgtggttc agagggtc 18 <210> 3 <211> 170 <212> DNA <213> Artificial Sequence <220> <223> LvIE precursor gene <400> 3 gtggttctgg gtccagcatt tgatggcagg aatgctgcag ccagcgacaa agcgtctgag 60 ctgatggctc tggccgtcag gggatgctgt tcccatcctg cctgtgctgg gaataatcca 120 catatctgtg gcagaagacg ctgatgcccc aggaccctct gaaccacgac 170 <210> 4 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> LvIE precursor protein <400> 4 2]Val Val Leu Gly Pro Ala Phe Asp Gly Arg Asn Ala Ala Ala Ser Asp 1 5 10 15 It should be noted that there is an unclear "所" in the translation of . You may need to check and correct it according to the actual situation. Lys Ala Ser Glu Leu Met Ala Leu Ala Val Arg Gly Cys Cys Ser His 20 25 30 Pro Ala Cys Ala Gly Asn Asn Pro His Ile Cys Gly Arg Arg Arg 35 40 45 <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> LvIE mature peptide <400> 5 Gly Cys Cys Ser His Pro Ala Cys Ala Gly Asn Asn Pro His Ile Cys 1 5 10 15 <210> 6 <211> 170 <212> DNA <213> Artificial Sequence <220> <223> LvIF precursor gene <400> 6 gtggttctgg gtccagcatt tgatggcagg aatgctgcag ccagcgacaa agcttctgag 60 ctgatggctc tggccgtcag gggatgctgt tcccatcctg cctgtgccgg gaataatcaa 120 gacatctgtg gctgaagacg ctgatgcccc aggaccctct gaaccacgac 170 <210> 7 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> LvIF precursor protein <400> 7 Val Val Leu Gly Pro Ala Phe Asp Gly Arg Asn Ala Ala Ala Ser Asp 1 5 10 15 Lys Ala Ser Glu Leu Met Ala Leu Ala Val Arg Gly Cys Cys Ser His 20 25 30 Pro Ala Cys Ala Gly Asn Asn Gln Asp Ile Cys Gly Arg Arg Arg 35 40 45 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> LvIF mature peptide <400> 8 Gly Cys Cys Ser His Pro Ala Cys Ala Gly Asn Asn Gln Asp Ile Cys 1 5 10 15

Claims

1. An isolated polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 4-5 and SEQ ID NOs: 7-8, wherein, The first cysteine ​​residue at the N-terminus of the polypeptide forms a disulfide bond with the third cysteine ​​residue, and the second cysteine ​​residue forms a disulfide bond with the fourth cysteine ​​residue.

2. The polypeptide according to claim 1, wherein, The carboxyl terminus of the polypeptide is amidated.

3. An isolated polynucleotide encoding the polypeptide as claimed in any one of claims 1 to 2.

4. A nucleic acid construct comprising the polynucleotide of claim 3.

5. The nucleic acid construct according to claim 4 is a recombinant vector.

6. The nucleic acid construct according to claim 4 is a recombinant expression vector.

7. A transformed cell containing the polynucleotide of claim 3, or containing a nucleic acid construct of any one of claims 4 to 6.

8. A pharmaceutical composition comprising at least one polypeptide as described in any one of claims 1 to 2 or a polynucleotide as described in claim 3.

9. The pharmaceutical composition according to claim 8, wherein, The pharmaceutical composition also includes one or more pharmaceutically acceptable excipients.

10. Use of the polypeptide according to any one of claims 1 to 2 in the preparation of a medicament for treating neuralgia or addiction.

11. The use according to claim 10, wherein, The neuralgia is caused by one or more of the following factors: sciatica, trigeminal neuralgia, lymphatic neuralgia, multipoint motor neuralgia, acute severe spontaneous neuralgia, compression neuralgia, and complex neuralgia.

12. The use according to claim 10, wherein, The addictions mentioned are nicotine addiction, alcohol addiction, or drug addiction.

13. The use according to claim 12, wherein, The addiction is caused by one or more of the following factors: nicotine, opium, heroin, methamphetamine, morphine, marijuana, or cocaine.

14. A method for blocking acetylcholine receptors or regulating acetylcholine levels in vitro, comprising the step of applying an effective amount of the polypeptide according to any one of claims 1 to 2 to cells; wherein, The acetylcholine receptor is either an α3β2 acetylcholine receptor or an α6 / α3β2β3 acetylcholine receptor.

15. Use of the polypeptide according to any one of claims 1 to 2 in the preparation of a drug screening model, wherein, The drug screening model is a cell model or an animal model. The drug in question is used to treat neuralgia or addiction.

16. The use according to claim 15, wherein, The neuralgia is caused by one or more of the following factors: sciatica, trigeminal neuralgia, lymphatic neuralgia, multipoint motor neuralgia, acute severe spontaneous neuralgia, compression neuralgia, and complex neuralgia.

17. The use according to claim 15, wherein, The addictions mentioned are nicotine addiction, alcohol addiction, or drug addiction.

18. The use according to claim 17, wherein, The addiction is caused by one or more of the following factors: nicotine, opium, heroin, methamphetamine, morphine, marijuana, or cocaine.

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