CN114751959A - Alpha-conotoxin peptide LvIC and mutant thereof, and pharmaceutical composition and application thereof - Google Patents

Abstract

The invention belongs to the fields of biology and medicine, and relates to alpha-conotoxin peptide LvIC, a mutant thereof, a pharmaceutical composition thereof and application thereof. Specifically, the invention relates to an isolated polypeptide, and the amino acid sequence of the isolated polypeptide is shown as any sequence in SEQ ID NOs:1-3, 5 and 8-30. The polypeptide of the invention can specifically block alpha 6 beta 4 acetylcholine receptors (nAChRs), has high selectivity and strong blocking activity on the alpha 6 beta 4 nAChRs, and has the potential for preparing medicaments for preventing and/or treating diseases related to the alpha 6 beta 4 nAChRs.

Description

Alpha-conotoxin peptide LvIC and mutant thereof, and pharmaceutical composition and application thereof

Technical Field

The invention belongs to the field of biology and medicine, and relates to a novel alpha-conotoxin peptide LvIC, a mutant thereof, a pharmaceutical composition thereof and application thereof.

Background

alpha-Conotoxin polypeptide compounds are specific blockers with the best selectivity for various subtypes of nicotinic acetylcholine receptors (nAChRs), have small molecular weights, generally consist of 12-19 amino acid residues, and are rich in disulfide bonds (P.Gopalakrinkrone LJC, Sun Luo.toxins and Drug discovery. Springer Nature (Publisher) 2017; ISBN 978-94-007-. The alpha-conotoxin has various types, diverse activities and complex structure. Alpha-conotoxins can be classified by their highly conserved signal peptide sequence, pharmacological activity and cysteine pattern (Kaas Q, Yu R, Jin AH, Dutertre S and Craik DJ. ConoServer: updated content, knowledge, and discovery tools in the linkage Database. nucleic Acids Research (2012)40(Database issue): D325-30).

The cysteine pattern of alpha-conotoxin is CC-C-C, in which disulfide bonds are C1-C3 and C2-C4, 2 loop loops are formed between disulfide bonds, and alpha-conotoxin can be divided into several subfamilies of alpha 3/5, alpha 4/7, alpha 4/6, alpha 4/4 and alpha 4/3 according to the difference of amino acid number between di-tri-and tri-tetra-cysteine, and the characteristics and residue composition of each loop are different, namely the basis of toxin acting on different receptor subtypes (Ying Fu, Cheng Li, Shuai Dong, Yong Wu, Dong Zhang and Sulan Luo. Discovey method of non Conotoxins from Conom Drugs,2018,16, 417; Mardoi: 10.3390/md 10416117)

Nicotinic Acetylcholine receptors (nAChRs) are pentameric allosteric proteins on cell membranes belonging to ligand-gated ion channels that mediate a wide variety of physiological functions of the central and peripheral nervous systems, as well as the Immune System (Zoli M, Pucci S, Vilella A, Gotti C.Neuro and Extraneous Nicotinic acid neuro metabolism 2018; 16:338-349.Fujii T, Mashimo M, Moriwaki Y, Misawa H, Ono S, Horiguchi K, Kawashima K.expression and Function of the Choline in Immune cells. immunity in immunity 2017; 8: 1085), including learning, memory, response, analgesia, and motor control. nAChRs activate the release of various neurotransmitters, such as dopamine, norepinephrine, serotonin, gamma-aminobutyric acid, etc., and have important physiological functions and clinical significance (Giribaldi J, Dutert S. alpha. -Conotoxins to the molecular, physiological and physiological functions of neurological bacterial receptors. Neurosci Lett.2017Dec 2. pi: S0304-3940(17) 30972-2.).

nAChRs are assembled into many subtypes from different alpha and beta subunits, and the muscle acetylcholine receptor consists of 5 subunits, including 2 alpha 1 subunits, 1 beta subunit, 1 delta subunit, and 1 gamma or epsilon subunit depending on whether it is a fetal or adult acetylcholine receptor. Mammalian neural nAChRs are also composed of 5 subunits, the more complex subset of which (Bertrand D, Terry AV, Jr. the wireless and neural, acetyl choline receptors. biochemical pharmacology 2018; 151: 214. quadrature. 225.), at least 8 alpha subunits, 3 beta subunits, α 2, α 3, α 4, α 5, α 6, α 7, α 9 and α 10, respectively, and β 2, β 3 and β 4. Wherein α 2, α 3 and α 4 can bind to β 2 or β 4, respectively, to form functional receptors, such as α 2 β 2, α 3 β 2, α 2 β 4, and the like. Furthermore, α 6 β 4 and α 9 may form homomultimers.

Each subtype has distinct pharmacological characteristics and is a key target for screening drugs for diagnosing and treating a large class of important diseases, such as pain, addiction, inflammation, Parkinson's disease, depression, dementia, cancer, etc. (Rollema H, Bertrand D, Hurst RS. Nicotinic Agents and Antagonists.2014.Hone AJ, McIntoh JM. Nicotinic acetic Choline receptors in neuropathic and inflimatory pain. FEBS letters 2018; 592: 1045-.

The lack of highly selective ligand compounds for various subtypes presents numerous challenges in studying and elucidating the fine structure and function of the various subtypes of nAChRs. To date, the fine structure and function of various subtypes of acetylcholine receptors are poorly understood, and there is no specific medicine for symptomatic treatment of many diseases related to various subtypes of receptors, and the pathogenesis is unclear.

Therefore, it is urgently required to find a specific ligand compound, a molecular probe or a tool drug capable of distinguishing each subtype, which will contribute to the study of the structure and function of each subtype, the disclosure of the pathogenesis of diseases associated with each subtype, and the development of new therapeutic drugs.

Among them, the α 6/α 3 β 4nAChRs (α 6 β 4nAChRs, indicates α 6/α 3 chimeric subunit or other subunits), and are mainly distributed in the brain, retina (Marritt, a.m., Cox, b.c., Yasuda, r.p., mcinosh, j.m., Xiao, y., wolffe, b.b., and keller, K.J. (2005) Nicotinic chorolic chloride receptors in the said rat rete: simple and mixed cholesterol subunit, mol pharmacological 68,1656-1668), adrenal chromaffin cells (hernanz-vivano, a.2012, Hone, a.j, scanden, m.l., single-hidalsgo, b, endothelial cell, hippocampus, 108, a.42, hippocampus, d, hippocampus, d, d.g. 13, d. alpha.l., hormone, d.l., sea horse, d. alpha.k, d. alpha.d. 13, hippocampus, d. alpha.42, d. hormone, d. c. c. 1. c. 13, d. c. 1. c. alpha. c, d. alpha. c. alpha. c, d. d, d. d, d. d, d. d, d. d, d. d, d. d, d. d, d. d, d, l., Maskos, u., Changeux, j.p., Dowell, c.d., Christensen, s., De Biasi, m., and McIntosh, j.m. (2010) alpha-Conotoxin Buia [ T5A; P6O: a novel tissue and that of a tissue and tissue repair, wherein the tissue of tissue and tissue repair is selected from FASEB J24, 5113-; 96:194-204.). The basic functional studies on the α 6 β 4nAChRs subtype are very lacking, and the fine structure and related physiological and pathological roles and functions are not known, and there is no understanding of them.

It has been shown that α 6 β 4 nAChRs expressed in DRG interact directly with the extracellular nucleotide-binding ligand-gated ion channel P2X2/3 receptor, and cross-inhibition, which causes symptoms of neuropathic pain inversely correlated to the expression level of the α 6 subunit gene (CHRNA6) (mineral, A.J., Talley, T.T., Bobango, J., HuidobRo Melo, C., Hararah, F., Gajewick, J., Christensen, S., Harvey, P.J., Craik, D.J., and McIntosh, J.M. (2018) Molecular determinants of alpha-toxin reactivity for interaction of man and alpha6beta4 organic carboxylic acid receptors. J. Biol. Chem 293,17838-17hone, A.J., and Internally, J.M. Pat. No. 6 beta. 20154 organic carboxylic acid receptors, J. Biotin 293,17838-17hone, A.J., and hormone, J.852, J.M. nitine, and biological genes 20182, and 35, 32, 35, 32, 35, 2, and 5932, 7, three, four, three, four, three, four. α 6 β 4 nAChRs are potential new targets for neuropathic pain therapy.

α 6 β 4 nAChRs on human adrenal chromaffin cells can regulate catecholamine release, and it is speculated that α 6 β 4 nAChRs may also be associated with cardiovascular diseases [ Perez-Alvarez, A., et al, Native alpha6beta4, physiological receptors control exotosis in human cardiovascular cells of the advanced gland. FASEB J,2012.26(1): p.346-54.Hernandez-Vivanco, A., et al, Monkey additional myocardial cells expressage alpha beta 6a 4 physiological acetic acid receptors. PLoS One,2014.9(4 p.94142.). Activation of α 6 nAChRs and Dopamine (DA) neurons was sufficient to cause hyperlocomotion. Mice containing the α 6 gene mutant, α 6(L9S) nAChRs, were extremely active in the cage and were unable to adapt to the new environment. Low doses of nicotine selectively activate mouse α 6 nAChRs by stimulating DA neurons, but not GABA neurons, leading to increased motor capacity and to a high dopaminergic state In vivo [ Drenan, R.M., et al, In vivo activity of polyamide nanoparticles vitamins vitamin sensitive, high-affinity alpha 6nicotinic acetyl choline receptors, neuron,2008.60(1): p.123-36 ]. This effect of α 6(L9S) nAChRs is primarily mediated by α 6 β 4 nAChRs [ Drenan, R.M., et al, Cholanergic modulation of pathology and striatal dopamine release therapy by alpha6alpha 4. bacterial acetyl choline receptors. J Neurosci,2010.30(29): p.9877-89 ].

There was no difference in baseline resting and climbing times between the α 6 Knockout (KO) and corresponding Wild Type (WT) mice. Compared with wild-type mice, bupropion significantly reduced the resting time of α 6KO mice, increasing the climbing time of the mice. Both low (3mg/kg) and high (10mg/kg) doses of bupropion in α 6 receptor-deficient mice increased the time to locomotion in the mice, and only high (10mg/kg) doses of bupropion in wild-type mice increased the time to locomotion in the wild-type mice. This suggests that the absence of these α 6 nAChRs may enhance the antidepressant effect of bupropion [ Bagdas, d., et al., Blockade of a nicotinic acetyl choline receptor enhancer process to the patient for the treated swim test. behavv Brain Res,2019.360: p.262-269 ]. This suggests that blocking α 6 β 4 nAChRs may enhance the efficacy of some antidepressants, i.e. blockers/antagonists/blockers of this receptor may treat or potentiate depression treatment with other drugs.

Levodopa-induced dyskinesia is a serious side effect of dopamine replacement therapy in most parkinson's disease patients. Research on levodopa-induced dyskinesia of alpha6 knockout mice shows that the alpha6 knockout mice can reduce levodopa-induced involuntary movement. This suggests that α 6 nAChRs may be drug targets for the treatment of Parkinson's disease [ Quik, M., et al, Role for alpha6 toxic receptors in l-dopa-induced dyskiniae in parkinsonian mice.Neuropharmacography, 2012.63(3): p.450-9 ]. It was found that in ICSA and nicotine-induced VTA DA neuronal activity, the α 4 subunit is essential, not the α 6 subunit, and that the α 4 and α 6 subunits together regulate DA transmission in NAc. These data indicate that the central firing of α 4 dominated dopamine neurons is dependent on DA transmission in NAc, where both α 4 and α 6 subunits control DA transmission [ Exley, R., et al, Distingction considerations of a nicotinic acetylcholinergic receptor subunit alpha4 and subunit alpha6 to the reinforming effects of a nucleotide. Proc Natl Acad Sci U S A,2011.108(18): p.7577-82 ].

Mice containing β 4 subunit-deficient acetylcholine receptors (β 4. multidot. nAChRs) have nicotine-induced suppression of reward behavior [ Donvito, G., et al, Neuronal nicotinic acetylcholine receptors (9) -THC dependency: Mouse and human students. Addit Biol,2020.25(1): p.e12691 ]. Reward behavior is restored when β 4 gene receptor expression is restored in the medial reins and interpeduncular nuclei of the mouse brain [ Semenova, S., et al, Mice lacing the beta4 distribution of the bacterial acetyl choline receptor morphology definitions, absolute and expression-like Receptors, and minor amino-induced expression of the bacterial receptor library, 2012.14(11): p.1346-55.Husson, M., et al, beta 4-bacterial Receptors arc clinical expression in modified peptides and genes and lf-Regulation of nucleic acids in modification of nucleic acids receptor, J.56 (3465-3477). In addition to improving the withdrawal symptoms of tetrahydrocannabis, β 4 gene deficient mice also have an improved effect on nicotine-induced withdrawal symptoms [ marker, A.K., B.Olivier, and A.Markou, Role of alpha7-and beta 4-coordinating organic acetyl choline receptors in the affective and therapeutic aspects of organic with regard to the wavelength: students in knock out mice, Behav Genet,2012.42(3): p.423-36 ]. Compared with wild-type mice, the beta4 knockout mice had lower levels of 7.5, 15, 30 and 60 ug/kg/infusion intravenous self-administration (IVSA). In vivo microdialysis results showed that β 4KO mice had higher levels of nucleus accumbens extracellular Dopamine (DA) than WT mice and showed different sensitivity to nicotine-induced DA. In addition, electrophysiological recordings of the Ventral Tegmental Area (VTA) showed that β 4KO mouse DA neurons were more sensitive to low doses of nicotine than wild type mice. The re-expression of β 4 nAChRs in IPN neurons completely restored nicotine IVSA and attenuated VTA DA neurons sensitivity to nicotine. These results indicate that β 4 nAChRs play a Role in maintaining Nicotine IVSA [ Harrington, l., et al, Role of beta 4. nicotic acetic acid Receptors in the habenuclo-interpenetrated reactor Pathway in nicotin re-enforcement in rice.neuropsychomatology, 2016.41(7): p.1790-802 ].

In addition, β 4 receptor deficient mice will exhibit memory deficits and β 4 knockout mice exhibit less anxiety-like behavior in the light-black box than normal mice. Meanwhile, the depression-like behavior of β 4 knockout Mice decreases in tail overhang tests and increases in forced swim tests [ Semenova, s., et al, rice lacing the beta4 study of the nutritional acetyl choline receptor show memory deficits, accurate and predicted-like behavor, and differentiated amino-induced amino-expression Tob Res,2012.14(11): p.1346-55 ]. The literature reports that β 4 knockout mice can prevent somatic signs of nicotine withdrawal in mice compared to controls [ Salas, r., f. pieri, and m.de Biasi, secreted signs of nicotine with dry in the mouse null for the beta4nicotinic acetyl choline receptor subunit.j Neurosci,2004.24(45): p.10035-9 ]. Mice deficient in the beta4 receptor gene show a lower withdrawal response after chronic nicotine. Whereas β 4 acetylcholine receptors are most densely expressed in the MHb-IPN pathway, these findings suggest a role for the MHb-IPN loop and for these receptor subtypes in mediating nicotine withdrawal [ Salas, R., et al, Nicotinic receptors in the nicotine system area research for nicotine with dry road in micro. J Neurosci,2009.29(10): p.3014-8 ].

The β 4 subunit knockout males exhibited increased exotic evoked motor activity, lower baseline anxiety, and lack of motivation in the operative conditioning reflex and reward-based go/no-go tasks awarded to savory foods. To further explore loss of reward, studies using the intracranial self-administration (ICSA) method of directly injecting nicotine into the Ventral Tegmental Area (VTA) of mice found that at low nicotine doses, β 4KO self-administration was less than that of wild-type (WT) mice. In contrast, at high nicotine doses, this was reversed, with β 4KO self-administered more than WT mice, while β 4 over-expressing mice avoided nicotine injection. In conclusion, the lack of functional β 4 nAChRs resulted in a loss of reward sensitivity, including an increase in ICSA at high doses of nicotine, whereas ICSA was restored by the re-expression of β 4 nAChRs in MHb-IPN. These data indicate that β 4 subunit-containing acetylcholine Receptors Are a key regulator that regulates Reward-Related behavior [ Husson, M., et al, beta4-nicotin Receptors arc criticality invented in altered-modified carbohydrates and Self-Regulation of nicotin resistance. J Neurosci,2020.40(17): p.3465-3477 ].

The research shows that the beta4 nAChRs have the function of participating in body temperature regulation, the body temperature baseline value of a beta4 gene knockout mouse is lower than that of a normal mouse, and meanwhile, a beta4 deficient mouse can improve a low-temperature response caused by nicotine. Thus, it is presumed that β 4 may be involved in the thermoregulatory center [ Sack, R., et al., Lower core body temperature and expressed nucleic-induced hyperthermic stress in the beta4 neurological viral acetyl kinase subunit Branin resin, 2005.66(1): p.30-6 ]. Thus, blockers of α 6 β 4 nAChRs are likely to be useful in the treatment of disorders associated with body temperature disorders such as fever.

α 6 β 4 nAChRs play such important physiological roles and are closely related to pain, learning, memory, exercise, body temperature, depression, addiction, cardiovascular and cerebrovascular diseases, and the like. Due to the lack of specific ligands or tool drugs for α 6 β 4 nAChRs, the only study on physiological function and pathological mechanism was in iceberg. Therefore, the research and development of new strong blockers of α 6 β 4 nAChRs with good selectivity, especially blockers capable of distinguishing subtypes of nAChRs with similar structure and overlapping distribution, such as α 3 β 4 and α 6 β 2 nAChRs, etc., and the research and development of disease mechanisms, new drug screening, and new drug therapy related to α 6 β 4 nAChRs, have very important scientific significance and great economic value.

Disclosure of Invention

The present inventors have found a novel alpha-conotoxin peptide (named LvIC) through intensive research and creative efforts, and further prepared a mutant of LvIC. The inventors have surprisingly found that LvIC or LvIC mutants are capable of specifically blocking α 6 β 4 acetylcholine receptors, have a highly selective strong blocking activity, and have the potential to be used for preparing or screening drugs for treating or preventing diseases related to α 6 β 4 acetylcholine receptors. The following invention is thus provided:

one aspect of the invention relates to an isolated polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs:1-3, 5 and 8-30.

In some embodiments of the invention, the polypeptide, wherein,

the first cysteine from the N-terminus of the polypeptide forms a disulfide bond with the third cysteine, and the second cysteine forms a disulfide bond with the fourth cysteine; or the first cysteine from the N-terminus of the polypeptide forms a disulfide bond with the fourth cysteine, and the second cysteine forms a disulfide bond with the third cysteine; or the first cysteine from the N-terminus of the polypeptide forms a disulfide bond with the second cysteine, and the third cysteine forms a disulfide bond with the fourth cysteine;

Preferably, the carboxy terminus of the polypeptide is amidated.

Another aspect of the invention relates to an isolated fusion protein comprising at least one polypeptide according to any of the present invention.

Yet another aspect of the invention relates to an isolated polynucleotide encoding a polypeptide of any of the invention or a fusion protein of the invention.

Yet another aspect of the invention relates to a nucleic acid construct comprising a polynucleotide of the invention; preferably, the nucleic acid construct is a recombinant vector; preferably, the nucleic acid construct is a recombinant expression vector.

Yet another aspect of the invention relates to a transformed cell comprising a polynucleotide of the invention, or comprising a nucleic acid construct of the invention.

Yet another aspect of the present invention relates to the use of a polypeptide according to any of the present invention, a fusion protein according to the present invention or a polynucleotide according to the present invention for the preparation of a medicament for blocking an acetylcholine receptor; wherein the acetylcholine receptor is an α 6 β 4 acetylcholine receptor.

The polypeptide of any one of the present invention, the fusion protein of the present invention or the polynucleotide of the present invention, which is used for preparing a medicament for treating and/or preventing depression, Parkinson's disease, anxiety, fever, cardiovascular and cerebrovascular diseases, addiction, pain or learning and memory disorder;

Preferably, the pain is neuropathic pain;

preferably, the depression, parkinson's disease, anxiety, fever, cardiovascular and cerebrovascular disorders, addiction, pain or learning memory disorders are associated with or caused by expression levels of α 6 β 4 acetylcholine receptors (e.g., α 6/α 3 β 4nAChRs) that are higher than those of normal humans or that are higher than those of normal patients.

Yet another aspect of the invention relates to a pharmaceutical composition comprising at least one polypeptide according to any of the invention, a fusion protein according to the invention or a polynucleotide according to the invention; optionally, it further comprises one or more pharmaceutically acceptable excipients.

In some embodiments, pharmaceutical compositions containing a therapeutically effective amount of a polypeptide of the invention are formulated and administered in a manner that facilitates pharmaceutical use, taking into account the clinical condition of the individual patient, the site of delivery, the method of administration, the schedule of administration, and other factors known to physicians. Thus an "effective amount" for the purposes herein is determined by consideration of these aspects.

A further aspect of the invention relates to the use of a polypeptide according to any of the invention, a fusion protein according to the invention or a polynucleotide according to the invention for the preparation of a medicament for blocking acetylcholine receptors; wherein the acetylcholine receptor is an α 6 β 4 acetylcholine receptor.

A further aspect of the present invention relates to the use of a polypeptide according to any of the present invention, a fusion protein according to the present invention or a polynucleotide according to the present invention in the manufacture of a medicament for the treatment and/or prevention of depression, parkinson's disease, anxiety, fever, cardiovascular and cerebrovascular diseases, addiction, pain or learning and memory disorder;

preferably, the pain is neuropathic pain;

preferably, the depression, parkinson's disease, anxiety, fever, cardiovascular and cerebrovascular diseases, addiction, pain or learning memory disorders are associated with or caused by expression levels of α 6 β 4 acetylcholine receptors (e.g., α 6/α 3 β 4nAChRs) that are higher than those of normal humans or higher than those of patients.

Yet another aspect of the present invention relates to a method for the therapeutic and/or prophylactic treatment and/or prevention of depression, parkinson's disease, anxiety, fever, cardiovascular and cerebrovascular diseases, addiction, pain or learning and memory disorders, comprising the step of administering to a subject in need thereof an effective amount of a polypeptide of any one of the present invention, a fusion protein of the present invention or a polynucleotide of the present invention;

preferably, the pain is neuropathic pain;

Preferably, the depression, parkinson's disease, anxiety, fever, cardiovascular and cerebrovascular diseases, addiction, pain or learning memory disorders are associated with or caused by expression levels of α 6 β 4 acetylcholine receptors (e.g., α 6/α 3 β 4nAChRs) that are higher than those of normal humans or higher than those of patients.

The dosage to be administered will depend on a number of factors, such as the severity of the condition being treated, the sex, age, weight and individual response of the patient or animal, and the condition and past medical history of the patient being treated. It is common practice in the art to start doses from a level below that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

Yet another aspect of the present invention relates to a method of blocking acetylcholine receptors or modulating acetylcholine levels in vivo or in vitro comprising the step of administering to a cell or experimental animal an effective amount of a polypeptide of any of the present invention or a fusion protein of the present invention; wherein the acetylcholine receptor is an α 6 β 4 acetylcholine receptor.

A further aspect of the invention relates to the use of a polypeptide according to any of the invention, a fusion protein according to the invention or a polynucleotide according to the invention for the preparation of a drug screening model,

The drug screening model is a cell model or an animal model,

the medicine is used for treating and/or preventing depression, Parkinson's disease, anxiety, fever, cardiovascular and cerebrovascular diseases, addiction, pain or learning and memory disorder;

preferably, the pain is neuropathic pain;

preferably, the depression, parkinson's disease, anxiety, fever, cardiovascular and cerebrovascular diseases, addiction, pain or learning memory disorders are associated with or caused by expression levels of α 6 β 4 acetylcholine receptors (e.g., α 6/α 3 β 4nAChRs) that are higher than those of normal humans or higher than those of patients.

Yet another aspect of the present invention relates to a method for preparing a drug screening model, comprising the step of administering an effective amount of the polypeptide of any of the present invention, the fusion protein of the present invention, or the polynucleotide of the present invention to a target cell or a target animal,

the drug screening model is a cell model or an animal model;

the medicine is used for treating and/or preventing depression, Parkinson's disease, anxiety, fever, cardiovascular and cerebrovascular diseases, addiction, pain or learning and memory disorder;

Preferably, the pain is neuropathic pain;

preferably, the depression, parkinson's disease, anxiety, fever, cardiovascular and cerebrovascular disorders, addiction, pain or learning memory disorders are associated with or caused by expression levels of α 6 β 4 acetylcholine receptors (e.g., α 6/α 3 β 4nAChRs) that are higher than those of normal humans or that are higher than those of normal patients.

In some embodiments of the invention, the cellular model or animal model has a decreased level of α 6 β 4 acetylcholine receptors or a blocked α 6 β 4 acetylcholine receptors. In some embodiments of the invention, the decreased levels of α 6 β 4 acetylcholine receptors or the blockade of α 6 β 4 acetylcholine receptors in a cellular or animal model may be achieved by methods including, but not limited to, one of: administering to a target cell or a target animal an effective amount of a polypeptide of any of the present invention or a fusion protein of the present invention; or by gene transduction, the polynucleotide or nucleic acid construct of the present invention is transferred into a target cell or a target animal to produce an effective amount of the polypeptide of any one of the present invention or the fusion protein of the present invention.

Yet another aspect of the invention relates to a method for preparing a polypeptide according to any one of the invention, comprising the steps of:

1) synthesizing linear polypeptide on ABI Prism 433a polypeptide synthesizer or other polypeptide synthesizers or by manual method, wherein the side chain protecting group of Fmoc amino acid is Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp), Boc (Lys); cysteine uses Trt or Acm protecting group to form disulfide bond between corresponding cysteine;

2) cleaving the linear polypeptide obtained in step 1) from the resin and precipitating and washing with glacial ethyl ether to recover a crude linear polypeptide, which is purified using a preparative reverse phase HPLC C18 column (Vydac);

3) carrying out two-step oxidation folding on the product obtained in the step 2).

In the invention:

the term "nucleic acid construct", defined herein as a single-or double-stranded nucleic acid molecule, preferably refers to an artificially constructed nucleic acid molecule. Optionally, the nucleic acid construct further comprises 1 or more regulatory sequences operably linked.

In the present invention, the term "operably linked" refers to a functional spatial arrangement of two or more nucleotide regions or nucleic acid sequences. The "operably linked" may be achieved by means of genetic recombination.

In the present invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide inhibiting a protein can be inserted. By way of example, the carrier includes: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosome (YAC), Bacterial Artificial Chromosome (BAC), or artificial chromosome (PAC) of P1 origin; bacteriophage such as lambda bacteriophage or M13 bacteriophage, animal virus, etc. Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), pox viruses, baculoviruses, papilloma viruses, papovaviruses (e.g., SV 40). A vector may contain a variety of elements for controlling expression.

In the present invention, the term "host cell" refers to a cell into which a vector is introduced, and includes 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 fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

It is known to those skilled in the art that mammalian (e.g., rodents, primates, such as humans, etc.) acetylcholine receptors (muscle or neuronal) are pentameric transmembrane proteins composed of 5 identical or different subunits, with a wide variety of subtypes. The structures of the individual subtypes are very similar, but their pharmacological characteristics and physiological functions are quite different. For example, α 2 β 2nAChRs represent functional receptors containing both α 2 and β 2 subunits, and the number of α 2 subunits and the number of β 2 subunits add up to 5, i.e., pentameric proteins. And the rest is analogized.

α 6 β 4nAChRs represent chimeric subunits in the receptor (e.g., α 6/α 3) containing the α 6 subunit or containing the extracellular ligand binding region of α 6, as well as the β 4 subunit. In some embodiments of the invention, α 6 further comprises α 6/α 3, wherein α 6/α 3 represents the extracellular region of α 6 and the transmembrane region chimeric subunit of α 3, facilitating expression of α 6 β 4nAChRs in vitro. The α 6/α 3 chimeric subunit is functionally equivalent to the α 6 subunit. In some embodiments of the invention, the α 6 β 4 acetylcholine receptor is α 6 β 4nAChRs or α 6/α 3 β 4 nAChRs.

The term "effective amount" refers to a dose that achieves treatment, prevention, alleviation and/or amelioration of a disease or disorder described herein in a subject.

The term "disease and/or disorder" refers to a physical condition of the subject that is associated with the disease and/or disorder of the present invention.

The term "subject" can refer to a patient or other animal, particularly a mammal, e.g., a human, dog, monkey, cow, horse, etc., that receives a pharmaceutical composition of the invention to treat, prevent, ameliorate, and/or alleviate a disease or disorder described herein.

In the present invention, the concentration unit. mu.M means. mu. mol/L, mM means mmol/L, and nM means nmol/L, unless otherwise specified.

In the present invention, when the amount of a drug to be administered into cells is referred to, the final concentration of the drug after administration is generally referred to unless otherwise specified.

In the present invention, the term "amino acid" or a specific amino acid name refers to an L-form amino acid unless otherwise specified.

Advantageous effects of the invention

The invention achieves one or more of the following technical effects:

(1) the conotoxin peptides (LvIC and mutants thereof) of the present invention are able to effectively block α 6 β 4 nAChRs.

(2) The conotoxin peptides (LvIC and mutants thereof) of the present invention are capable of specifically blocking α 6 β 4 nAChRs.

(3) The conotoxin peptide of the present invention can act by binding to α 6 β 4 acetylcholine receptors (nAChRs), and has activity of treating and/or preventing depression, parkinson's disease, anxiety, fever, cardiovascular and cerebrovascular diseases, addiction, pain, learning and memory disorder, or any disease associated with α 6 β 4 acetylcholine receptors, etc.

(4) The conotoxin peptide can be applied to research, diagnosis, screening and treatment of depression, Parkinson's disease, anxiety, fever, cardiovascular and cerebrovascular diseases, addiction, pain, learning and memory disorder or any diseases related to alpha 6 beta 4 acetylcholine receptors, and can be used as a useful molecular probe for research and other aspects. The affinity of different alpha-conotoxins for vertebrate receptors varies, sometimes by several orders of magnitude. This phylogenetic difference makes alpha-conotoxin useful as a probe for studying phylogeny of vertebrate nAChRs, and as a molecular probe for determining different subtypes of nAChRs. They are candidates, lead drugs and therapeutic drugs for new drug development.

Drawings

FIG. 1: alpha-conotoxin LvIC (polypeptide 1, SEQ ID NO:1) propeptide gene sequence and propeptide generated by coding and mature peptide generated by posttranslational modification. The arrow indicates the processing site for post-translational modification. The putative proteolytic processing site 1(processing site 1) follows the basic amino acid lysine (K); the C-terminal amidation processing site may be at the position of the two glycines indicated by the arrows, i.e., processing site 2. The glycine residue immediately C-terminal to cysteine (Cys) of the mature peptide is often the processing site for amidation post-translational modification, and the mature peptide produced by amidation from processing site 2 is designated LvIC.

FIG. 2: the sequence and disulfide bond connection mode of alpha-conotoxin LvIC and its mutant. Wherein cysteine is italicized; # denotes C-terminal amidation.

FIG. 3: amino acid sequences of [ D1G,. DELTA.Q 14] LvIC (polypeptide 3, SEQ ID NO:3) and mutants thereof (polypeptide 4-26, SEQ ID NOs: 8-30).

FIGS. 4A-4B: HPLC and ESI-MS spectra of [ D1G,. DELTA.Q 14] LvIC, respectively. Conditions for HPLC analysis: c18 column (Vydac), linear gradient 10-40% B90 in 0-40min, detection wavelength 214 nm. Solvent B: an aqueous solution containing 90% Acetonitrile (ACN), 0.05% tfa (trifluoracetic acid); solvent A: 0.075% TFA in water.

FIG. 5 is a schematic view of: LvIC (polypeptide 1, SEQ ID NO:1), [ Δ Q14] LvIC (polypeptide 2, SEQ ID NO:2) and [ D1G, [ Δ Q14] LvIC (polypeptide 3, SEQ ID NO:3) are plotted as a response to α 6/α 3 β 4nAChRs concentration.

FIG. 6: [ D1G,. DELTA.Q 14] LvIC (10. mu.M) effect on the current of various subtypes of nAChRs expressed in Xenopus oocytes. Data represent mean ± SEM. n is 6-8.

FIGS. 7A-7C: [ D1G,. DELTA.Q 14] LvIC blocks the current trace plot of rat alpha 6/alpha 3 beta 4nAChRs, and is a high-selectivity specific blocker of the alpha 6/alpha 3 beta 4 nAChRs. Wherein:

FIG. 7A: the current influence of 1 μ M [ D1G, Δ Q14] LvIC on α 6/α 3 β 4 nAChRs;

FIG. 7B: the current influence of 10 μ M [ D1G, Δ Q14] LvIC on α 3 β 4 nAChRs;

FIG. 7C: current contribution of 10. mu.M [ D1G, Δ Q14] LvIC to α 6/α 3 β 2 β 3 nAChRs.

FIG. 8: [ D1G,. DELTA.Q 14] LvIC alanine scanning mutants (polypeptides 4-14, SEQ ID NOs:8-18) were plotted as a response to α 6/α 3 β 4nAChRs concentration with data representing mean. + -. SEM. n-6-8, wherein:

FIG. 9: effect of [ D1G, Δ Q14] LvIC alanine scanning mutants on currents of α 3 β 4 nAChRs.

FIGS. 10A-10B: amino acid substitution mutants of [ D1G,. DELTA.Q 14] LvIC (polypeptides 14-26, SEQ ID NOs:19-30) are plotted against α 6/α 3 β 4 and α 3 β 4nAChRs concentration, and the data are presented as mean. + -. SEM. n-6-8, wherein:

FIG. 10A: the amino acid substitution mutant of [ D1G,. DELTA.Q 14] LvIC reacts to the concentration of alpha 6/alpha 3 beta 4nAChRs in a curve;

FIG. 10B: amino acid substitution mutants of [ D1G,. DELTA.Q 14] LvIC are shown in response to concentrations of α 3 β 4 nAChRs.

FIGS. 11A-11D: the mutants [ G10A ] + [ D1G,. DELTA.Q 14] LvIC (polypeptide 10, SEQ ID NO:14) and [ N9A, G10A ] + [ D1G,. DELTA.Q 14] LvIC (polypeptide 26, SEQ ID NO:30) block the current trace graphs of rat α 6/α 3 β 4 and α 3 β 4nAChRs, where:

FIG. 11A: the current influence of the 100nM mutant [ G10A ] + [ D1G,. DELTA.Q 14] LvIC on α 6/α 3 β 4 nAChRs;

FIG. 11B: the current influence of 10. mu.M mutant [ G10A ] + [ D1G,. DELTA.Q 14] LvIC on α 3 β 4 nAChRs;

FIG. 11C: the current contribution of 1. mu.M mutant [ N9A, G10A ] + [ D1G,. DELTA.Q 14] LvIC to α 6/α 3 β 2 β 3 nAChRs;

FIG. 11D: the current contribution of 10. mu.M mutant [ N9A, G10A ] + [ D1G,. DELTA.Q 14] LvIC to α 3 β 4 nAChRs.

The partial sequences involved in the present invention are shown in Table A below:

table a: partial sequences to which the invention relates

Detailed Description

Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to J. SammBruk et al, molecular cloning, A laboratory Manual, third edition, science Press, translated by Huang Petang et al), the corresponding references, or the product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: cloning and sequence analysis of alpha-conotoxin LvIC gene

1. Extraction, cloning and sequence analysis of genomic DNA of tridentiger

The method comprises the steps of taking onymous tridentiger (C.lividus) collected in the Hainan sea area as a raw material, obtaining a toxic gland tissue through dissection, extracting genomic DNA of a toxic gland by using a marine animal genomic DNA extraction kit (purchased from Beijing Tiangen Biochemical technology Co., Ltd., China), and specifically referring to kit specifications. According to the intron sequence of the alpha-conotoxin precursor gene and the 3 'untranslated region (3' -UTR) sequence thereof, two alpha-conotoxin specific primers are designed:

the upstream intron primer sequence is 5'-GTGGTTCTGGGTCCAGCA-3' (SEQ ID NO: 6);

the downstream 3 ' -UTR primer sequence is 5'-GTCGTGGTTCAGAGGGTC-3' (SEQ ID NO: 7).

Diluting the extracted genome DNA stock solution to be used as a PCR amplification template, then recovering a PCR specific amplification product, connecting the PCR specific amplification product with a T vector, transforming the PCR specific amplification product into escherichia coli, selecting recombinants by utilizing ampicillin resistance, and finally sequencing, analyzing, extracting and purifying the recombinant plasmids.

The sequence of the obtained PCR specific amplification product is analyzed by DNAStar software, and the sequence of the coding protein and the sequence of the 3' -untranslated region (UTR) are obtained. Prediction of signal, propeptide and mature peptides of the conotoxin precursor protein was analyzed using an on-line ProP 1.0Server (Duckert, P.; Brunak, S.; Blum, N., Prediction of protein converting cleavage sites, design & selection: PEDS 2004,17(1), 107-12.).

Through sequence analysis and comparison, a new alpha-conotoxin LvIC precursor gene is obtained (as shown in figure 1):

TCTAATGGCAGGAATGCTGCAGCCGGCGACAAACCGTCTTACTGGATCACTCTGGCCATCACGGATTGCTGCGCCAATCCCGTCTGTAACGGGAAACACTGTCAGGGAAGACGC(SEQ ID NO:4)

according to the characteristics of precursor genes and conotoxin, the amino acid sequence of the propeptide of the conotoxin LvIC is presumed as follows:

SNGRNAAAGDKPSYWITLAITDCCANPVCNGKHCQGR R(SEQ ID NO:5)

the mature peptide LvIC is further deduced from the propeptide sequence, its amino acid sequence is DCCANPVCNGKHCQ (polypeptide 1, SEQ ID NO:1), and the methods and principles of inference are described in Luo S, Zhang Sun D, Zhang B, Quan Y, Wu Y. novel alpha-toxins identified by gene sequencing from the peptide sequence from natural source to Hainan, and the same sequence diversity. J peptide Sci.2006,12(11): 693-.

LvIC (peptide 1, SEQ ID NO:1) is a novel alpha-conotoxin containing the CC-C-C cysteine pattern linked in the disulfide bond pattern Cys (I-III, II-IV), i.e., two pairs of disulfide bonds are formed between the first and third cysteines, and between the second and fourth cysteines, respectively, and the number of amino acids therebetween is 4. The amino acid sequence of LvIC differs from other known alpha-conotoxins.

Example 2: alpha-conotoxin [ D1G, delta Q14]]Design of LvIC mutants

According to the sequence of mature peptide LvIC, removing the last cysteine and then directly amidating the amino acid to obtain [ delta Q14] LvIC, wherein the sequence is DCCANPVCNGKHC (polypeptide 2, SEQ ID NO: 2); on the basis of polypeptide 2, the first amino acid was replaced with glycine (G) to give a new peptide, [ D1G,. DELTA.Q 14] LvIC, whose sequence was GCCANPVCNGKHC (polypeptide 3, SEQ ID NO:3) (FIG. 2).

On the basis of the sequence structure of [ D1G,. DELTA.Q 14] LvIC (polypeptide 3), alanine scanning substitution mutations were first made for all amino acids, i.e., all amino acids except cysteine (C) and alanine (A) were individually substituted with alanine (A). Subsequently, substitution mutation of various amino acids was performed with respect to glycine (G) at position 10, and finally, as a result of substitution of asparagine (N) at position 9 with alanine (a), double mutants at positions 9 and 10 were formed (fig. 3). The amino acid sequence is shown in SEQ ID NOs:8-30, and is shown in the following table 1.

Table 1: polypeptide sequence of [ D1G, delta Q14] LvIC and mutant thereof

In Table 1, # denotes C-terminal amidation,. DELTA.NH₂It is emphasized that the C-terminus is not amidated. In the sequence 11, O represents L-hydroxyproline.

Example 3: alpha-conotoxin [ D1G, Delta Q14]]Artificial synthesis of LvIC and mutants

Polypeptides 1, 2 and the polypeptides shown in table 1 were synthesized.

The linear peptide was artificially synthesized by Fmoc method. The remaining amino acids, except cysteine, are protected with standard side chain protecting groups. the-SH of 1 st and 3 rd cysteine (Cys) of LvIC and its mutant is protected by Trt (S-trityl), and the-SH of 2 nd and 4 th cysteine is protected by Acm (S-acetamidomethyl) in pairs; the side chain protecting groups of the amino acid are Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp) and Boc (Lys). The Fmoc HOBT DCC method, Rink amidated resin and Fmoc amino acid are adopted, and the synthetic steps are carried out according to an instrument synthesis manual. In order to complete the reaction, the piperidine deprotection and coupling time are respectively and properly prolonged, and the amino acid difficult to be grafted is subjected to double coupling to obtain the resin peptide. The linear peptide was cleaved from the resin with reagent K (trifluoracetic acid/water/ethanoldithiol/phenol/thioanisole; 90:5:2.5:7.5:5, v/v/v/v/v/v), the linear crude peptide was precipitated with glacial ethyl ether and recovered by washing several times, purified with a preparative RP-HPLC C18 column (Vydac) eluting a linear gradient of 10-40% B90 in 0-40min, monitoring wavelength 214 nm. Solvent B90 is an aqueous solution containing 90% Acetonitrile (ACN), 0.05% tfa (trifluoracetic acid); solvent a was 0.075% TFA in water. The purity of the purified linear peptide was checked by analytical RP-HPLC C18 column (Vydac) under the same elution conditions as above and at a flow rate of 1 mL/min. The purity of the product reaches more than 95 percent, and the product is used for oxidation folding.

Reference is made to the literature (Dowlel, C.; Olivera, B.M.; Garrett, J.E.; Stahelli, S.T.; Watkins, M.; Kuryatov, A.; Yoshikami, D.; Lindstrom, J.M.; McIntosh, J.M., Alpha-toxin PIA is selective for Alpha 6. suburbitut-connecting acidic ethylene chloride receptors. the Journal of neuroscience 2003,23(24),8445-52.) for the two-step oxidative folding of LvIC and mutant linear peptides, the process is briefly described below:

first a first pair of disulfide bonds was formed between the two cysteines of the Trt protecting group by potassium ferricyanide oxidation (20mM potassium ferricyanide,0.1M Tris, pH 7.5, 45 min). After purification by RP-HPLC C18 column (Vydac), iodoxidation (10mM iododine in H) was performed₂(ii) trifluoroacetic acid acetonitrile (78:2:20by volume, 10min), remove another 2 cysteinesAcm, while forming a second pair of disulfide bonds between these 2 cysteines. Purifying by RP-HPLC C18 column (Vydac) to obtain alpha-conotoxin oriented to form disulfide bond, and identifying whether the relative molecular mass is consistent with the theoretical value by mass spectrum (ESI-MS).

Taking polypeptide [ D1G,. DELTA.Q 14] LvIC (polypeptide 3) as an example, the purity of the product after oxidative folding was 95% or more, and the molecular weight was found to be consistent with the theoretical molecular weight (FIGS. 4A-4B). The polypeptide concentration was determined colorimetrically at a wavelength of 280nm and the polypeptide concentration and mass were calculated according to the Beer-Lambert equation (equalisation).

These quantified folded polypeptides were used in the following activity tests.

Example 4: alpha-conotoxin LvIC and its mutant for alpha 6/alpha 3 beta 4nAChRs and other nAChRs subtypes Study on Activity

With reference to the literature (Azam L, Yoshikami D, McIntosh JM. amino acid derivatives of high selectivity of the alpha6 nicotinic acetyl choline receptor subunit to alpha-ecoxin MII [ S4A, E11A, L15A ]. J Biol chem.2008; 283(17):11625-32.), and the in vitro transcription kit (Ambion, Austin TX)) specification, various subtypes of rat neural nAChRs (α 3 β 2, α 6/α 3 β 2 β 3, α 6/α 3 β 4, α 9 α 10, α 4 β 2, α 4 β 4, α 3 β 4, α 2 β 2, α 2 β 4, α 7 β 7, and the concentration of small muscle RNA (α 1. delta. 1) at (. alpha.) pH were measured using UV measurement of cRNA at wavelength of α 1 nm. Xenopus laevis (Xenopus laveis) oocytes (frog eggs) were dissected and cRNA was injected into the frog eggs in an amount of 5ng cRNA per subunit. Intramuscular nAChRs were injected with 0.5-2.5ng DNA per subunit. Frog eggs were cultured in ND-96. cRNA was injected 1-2 days after frog egg collection and voltage clamp recordings for nAChRs 1-4 days after injection.

1 frog egg injected with cRNA was placed in a 30. mu.L Sylgard recording chamber (4 mM diameter. times.2 mM depth), and perfusion fluid ND96 (96.0mM NaCl,2.0mM KCl,1.8mM CaCl) containing 0.1mg/ml BSA (bone serum album) was gravity-perfused₂,1.0mM MgCl₂5mM HEPES, pH 7.1-7.5) or containingND96(ND96A) with 1mM atropine at a flow rate of 1 ml/min. All conotoxin solutions also contained 0.1mg/ml BSA TO reduce non-specific adsorption of toxins, free switching between perfusion toxins or acetylcholine (ACh) using a switching valve (SmartValve, Cavro Scientific Instruments, Sunnyvale, CA), and free switching between perfusion ND96 and ACh etc. using a series of three-way solenoid valves (solenoid valves, model 161TO31, Neptune Research, Northboro, MA). The Ach-gated current is set in a "slow" clamp by a two-electrode voltage clamp amplifier (model OC-725B, Warner Instrument core, Hamden, CT), and recorded online with clamp gain at the maximum (x 2000) position. A glass capillary (fiber-filled silicon capsules, WPI Inc., Sarasota, FL) of 1mm OD x 0.75 ID was drawn across the glass electrode and filled with 3M KCl as voltage and current electrodes. The film voltage clamp is-70 mV., and the whole system is controlled and recorded by a computer. The ACh pulse was automatically infused for 1s every 5 min. The concentration of ACh is respectively 10 mu M of eggs expressing muscle type nAChRs and nerve type alpha 9 alpha 10 nAChRs; the α 7 expression of neural nAChRs was 200. mu.M, and the other subtypes were 100. mu.M. At least 4 eggs were recorded expressing the current response of a certain subtype to different toxin concentrations, as well as the current traces.

The current data measured were statistically analyzed using GraphPad Prism software (San Diego, Calif.), dose-response curves were plotted, and the half-blocking concentration IC of conotoxin was calculated₅₀And various parameters related to polypeptide blockade of nAChRs.

The results are shown in fig. 5, 6, 7A to 7C, and table 2.

Table 2: LvIC and its mutant acting on alpha 6/alpha 3 beta 4nAChRs and IC of other nAChRs₅₀Value summary table

Note:^arepresents a 95% confidence interval;^brepresentative IC₅₀The value is greater than 10. mu.M. Values are mean. + -. SEM for 6 to 8 oocytes.

The results show that: LvIC (polypeptide 1, SEQ ID NO:1) has blocking activity on rat alpha 6/alpha 3 beta 4nAChRs, and half-blocking dose (IC)₅₀) 3300 nM; [ Delta Q14]LvIC (polypeptide 2, SEQ ID NO:2) has about 10-fold enhanced blocking activity on rat alpha 6/alpha 3 beta 4nAChRs, the most half-blocking dose (IC)₅₀) Is 310 nM; [ D1G,. DELTA.Q 14]LvIC (polypeptide 3, SEQ ID NO:3) has the strongest blocking activity on rat alpha 6/alpha 3 beta 4nAChRs and half-blocking dose (IC)₅₀) Only 24nM (fig. 5).

The results also show that: [ D1G,. DELTA.Q 14] LvIC (polypeptide 3) showed no blocking effect on all other subtypes of receptors at a high concentration of 10. mu.M, and showed a percent response of 90% or more in comparison to control current, and 1. mu.M [ D1G,. DELTA.Q 14] LvIC almost completely blocked the open-production current of rat α 6/α 3 β 4nAChRs (FIG. 6, FIG. 7A-FIG. 7C, Table 2).

Example 5: alpha-conotoxin [ D1G, Delta Q14]]Activity of respective mutants of LvIC on α 6/α 3 β 4 and α 3 β 4nAChRs Sexual research and structure-activity relationship research

The activity of 11 alanine-scanning mutants of [ D1G,. DELTA.Q 14] LvIC (polypeptide 3, SEQ ID NO:3) (polypeptides 4 to 14, corresponding to SEQ ID NOs:8 to 18, respectively) on each of the α 6/α 3 β 4 and α 3 β 4nAChRs was determined as described in example 4.

The results are shown in fig. 8, 9 and table 3.

The site of activity loss or great reduction is the key amino acid site of [ D1G, delta Q14] LvIC. The results showed that position 9 had little effect on activity and that position 10 exhibited blocking activity of proliferation of α 3 β 4 nAChRs.

Further, the present inventors performed amino acid substitution mutation at position 10, and selected a plurality of different amino acids to replace glycine (G) at position 10, respectively (fig. 3). The activity of 8 amino acid substitution mutants of [ D1G,. DELTA.Q 14] LvIC (polypeptides 15 to 26, corresponding to SEQ ID NOs:19 to 30, respectively) on each of the α 6/α 3 β 4 and α 3 β 4nAChRs subtypes was determined as described in example 4.

The results are shown in FIGS. 10A to 10B, FIGS. 11A to 11D, and Table 4.

Table 4: [ D1G,. DELTA.Q 14]LvIC amino acid substitution mutants act on IC of alpha 6/alpha 3 beta 4 and alpha 3 beta 4nAChRs₅₀Value summary table

Note:^arepresents a 95% confidence interval;^brepresentative IC₅₀The value is greater than 10. mu.M.^cIndicates that the mutant is related to [ D1G,. DELTA.Q 14]IC of LvIC body pair alpha 6/alpha 3 beta 4₅₀The ratio of (a) to (b).^d IC representing alpha 3 beta 4 and alpha 6/alpha 3 beta 4₅₀The ratio of the values. Values are mean. + -. SEM for 6 to 8 oocytes.

The results show that: blockade of α 3 β 4nAChRs occurs following the substitution of glycine (G) at position 10 with a variety of aliphatic hydrocarbon chain amino acids, such as valine (V), leucine (L) and isoleucine (I), similar to the proliferative α 3 β 4nAChRs activity at position 10 with alanine (a). However, the double mutation [ N9A, G10A ] + [ D1G,. DELTA.Q 14] LvIC (polypeptide 26, SEQ ID NO:30) abolished the proliferative α 3 β 4nAChRs activity caused by the replacement of glycine (G) at position 10 with alanine (A) and maintained α 6/α 3 β 4nAChRs blocking activity consistent with the mutants [ N9A ] + [ D1G,. DELTA.Q 14] LvIC (polypeptide 9, SEQ ID NO:13) and the wild-type [ D1G,. DELTA.Q 14] LvIC; the double mutants using acidic amino acid exchange or basic amino acid exchange at position 9 all showed varying degrees of loss of activity.

Therefore, through the above series of mutant experimental results, the following structural-activity relationship conclusion of [ D1G, delta Q14] LvIC is obtained: first, a number of key amino acid positions (5 th, 6 th, 11 th, 12 th) of [ D1G,. DELTA.Q 14] LvIC were found, and alterations at these positions would lose activity; the blocking activity of proliferating alpha 3 beta 4nAChRs occurs after the glycine (G) at the 10 th position is replaced by an aliphatic amino acid; the substitution of asparagine (N) at position 9 with alanine (A) has the additional receptor inhibitory activity of restoring the alanine mutation at position 10, i.e., positions 9 and 10 are critical for the selectivity of α 6/α 3 β 4nAChRs for [ D1G, Δ Q14] LvIC.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Many modifications and variations of those details may be made in light of the overall teachings of the disclosure, and such variations are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110> Guangxi university

<120> alpha-conotoxin peptide LvIC, mutant thereof, pharmaceutical composition thereof and application thereof

<130> IDC200533

<160> 30

<170> PatentIn version 3.5

<210> 1

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> LvIC

<400> 1

Asp Cys Cys Ala Asn Pro Val Cys Asn Gly Lys His Cys Gln

1 5 10

<210> 2

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [ΔQ14]LvIC

<400> 2

Asp Cys Cys Ala Asn Pro Val Cys Asn Gly Lys His Cys

1 5 10

<210> 3

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [D1G,ΔQ14]LvIC

<400> 3

Gly Cys Cys Ala Asn Pro Val Cys Asn Gly Lys His Cys

1 5 10

<210> 4

<211> 114

<212> DNA

<213> Artificial Sequence

<220>

<223> LvIC precursor Gene

<400> 4

tctaatggca ggaatgctgc agccggcgac aaaccgtctt actggatcac tctggccatc 60

acggattgct gcgccaatcc cgtctgtaac gggaaacact gtcagggaag acgc 114

<210> 5

<211> 38

<212> PRT

<213> Artificial Sequence

<220>

<223> propeptide of LvIC

<400> 5

Ser Asn Gly Arg Asn Ala Ala Ala Gly Asp Lys Pro Ser Tyr Trp Ile

1 5 10 15

Thr Leu Ala Ile Thr Asp Cys Cys Ala Asn Pro Val Cys Asn Gly Lys

20 25 30

His Cys Gln Gly Arg Arg

35

<210> 6

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> primer 1

<400> 6

gtggttctgg gtccagca 18

<210> 7

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> GTCGTGGTTCAGAGGGTC

<400> 7

gtcgtggttc agagggtc 18

<210> 8

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [G1A]+[D1G,ΔQ14]LvIC

<400> 8

Ala Cys Cys Ala Asn Pro Val Cys Asn Gly Lys His Cys

1 5 10

<210> 9

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [N5A]+[D1G,ΔQ14]LvIC

<400> 9

Gly Cys Cys Ala Ala Pro Val Cys Asn Gly Lys His Cys

1 5 10

<210> 10

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [P6A]+[D1G,ΔQ14]LvIC

<400> 10

Gly Cys Cys Ala Asn Ala Val Cys Asn Gly Lys His Cys

1 5 10

<210> 11

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [P6O]+[D1G,ΔQ14]LvIC

<220>

<221> MOD_RES

<222> (6)..(6)

<223> X represents L-hydroxyproline

<400> 11

Gly Cys Cys Ala Asn Xaa Val Cys Asn Gly Lys His Cys

1 5 10

<210> 12

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [V7A]+[D1G,ΔQ14]LvIC

<400> 12

Gly Cys Cys Ala Asn Pro Ala Cys Asn Gly Lys His Cys

1 5 10

<210> 13

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [N9A]+[D1G,ΔQ14]LvIC

<400> 13

Gly Cys Cys Ala Asn Pro Val Cys Ala Gly Lys His Cys

1 5 10

<210> 14

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [G10A]+[D1G,ΔQ14]LvIC

<400> 14

Gly Cys Cys Ala Asn Pro Val Cys Asn Ala Lys His Cys

1 5 10

<210> 15

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [K11A]+[D1G,ΔQ14]LvIC

<400> 15

Gly Cys Cys Ala Asn Pro Val Cys Asn Gly Ala His Cys

1 5 10

<210> 16

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [K11R]+[D1G,ΔQ14]LvIC

<400> 16

Gly Cys Cys Ala Asn Pro Val Cys Asn Gly Arg His Cys

1 5 10

<210> 17

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [H12A]+[D1G,ΔQ14]LvIC

<400> 17

Gly Cys Cys Ala Asn Pro Val Cys Asn Gly Lys Ala Cys

1 5 10

<210> 18

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [ΔNH2]+[D1G,ΔQ14]LvIC

<400> 18

Gly Cys Cys Ala Asn Pro Val Cys Asn Gly Lys His Cys

1 5 10

<210> 19

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [G10V]+[D1G,ΔQ14]LvIC

<400> 19

Gly Cys Cys Ala Asn Pro Val Cys Asn Val Lys His Cys

1 5 10

<210> 20

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [G10L]+[D1G,ΔQ14]LvIC

<400> 20

Gly Cys Cys Ala Asn Pro Val Cys Asn Leu Lys His Cys

1 5 10

<210> 21

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [G10I]+[D1G,ΔQ14]LvIC

<400> 21

Gly Cys Cys Ala Asn Pro Val Cys Asn Ile Lys His Cys

1 5 10

<210> 22

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [G10K]+[D1G,ΔQ14]LvIC

<400> 22

Gly Cys Cys Ala Asn Pro Val Cys Asn Lys Lys His Cys

1 5 10

<210> 23

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [G10D]+[D1G,ΔQ14]LvIC

<400> 23

Gly Cys Cys Ala Asn Pro Val Cys Asn Asp Lys His Cys

1 5 10

<210> 24

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [G10N]+[D1G,ΔQ14]LvIC

<400> 24

Gly Cys Cys Ala Asn Pro Val Cys Asn Asn Lys His Cys

1 5 10

<210> 25

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [G10T]+[D1G,ΔQ14]LvIC

<400> 25

Gly Cys Cys Ala Asn Pro Val Cys Asn Thr Lys His Cys

1 5 10

<210> 26

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [G10F]+[D1G,ΔQ14]LvIC

<400> 26

Gly Cys Cys Ala Asn Pro Val Cys Asn Phe Lys His Cys

1 5 10

<210> 27

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [N9D,G10A]+[D1G,ΔQ14]LvIC

<400> 27

Gly Cys Cys Ala Asn Pro Val Cys Asp Ala Lys His Cys

1 5 10

<210> 28

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [N9K,G10A]+[D1G,ΔQ14]LvIC

<400> 28

Gly Cys Cys Ala Asn Pro Val Cys Lys Ala Lys His Cys

1 5 10

<210> 29

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [N9R,G10A]+[D1G,ΔQ14]LvIC

<400> 29

Gly Cys Cys Ala Asn Pro Val Cys Arg Ala Lys His Cys

1 5 10

<210> 30

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> [N9A,G10A]+[D1G,ΔQ14]LvIC

<400> 30

Gly Cys Cys Ala Asn Pro Val Cys Ala Ala Lys His Cys

1 5 10

Claims (11)

1. An isolated polypeptide, the amino acid sequence of which is shown in any one of SEQ ID NOs:1-3, 5 and 8-30.

2. The polypeptide of claim 1, wherein,

the first cysteine from the N-terminus of the polypeptide forms a disulfide bond with the third cysteine, and the second cysteine forms a disulfide bond with the fourth cysteine; or the first cysteine from the N-terminus of the polypeptide forms a disulfide bond with the fourth cysteine, and the second cysteine forms a disulfide bond with the third cysteine; or the first cysteine from the N-terminus of the polypeptide forms a disulfide bond with the second cysteine, and the third cysteine forms a disulfide bond with the fourth cysteine;

Preferably, the carboxy terminus of the polypeptide is amidated.

3. An isolated fusion protein comprising at least one polypeptide of any one of claims 1-2.

4. An isolated polynucleotide encoding the polypeptide of any one of claims 1 to 2 or the fusion protein of claim 3.

5. A nucleic acid construct comprising the polynucleotide of claim 4; preferably, the nucleic acid construct is a recombinant vector; preferably, the nucleic acid construct is a recombinant expression vector.

6. A transformed cell comprising the polynucleotide of claim 4 or the nucleic acid construct of claim 5.

7. A pharmaceutical composition comprising at least one polypeptide according to any one of claims 1 to 2, a fusion protein according to claim 3 or a polynucleotide according to claim 4; optionally, it further comprises one or more pharmaceutically acceptable excipients.

8. Use of the polypeptide of any one of claims 1 to 2, the fusion protein of claim 3, or the polynucleotide of claim 4 in the preparation of a medicament for blocking acetylcholine receptors; wherein the acetylcholine receptor is an α 6 β 4 acetylcholine receptor.

9. Use of a polypeptide according to any one of claims 1 to 2, a fusion protein according to claim 3 or a polynucleotide according to claim 4 for the manufacture of a medicament for the treatment and/or prevention of depression, parkinson's disease, anxiety, fever, cardiovascular and cerebrovascular diseases, addiction, pain or learning and memory disorders;

preferably, the pain is neuropathic pain.

10. A method of blocking acetylcholine receptors or modulating acetylcholine levels in vitro comprising the step of administering to a cell an effective amount of the polypeptide of any of claims 1-2 or the fusion protein of claim 3; wherein the acetylcholine receptor is an α 6 β 4 acetylcholine receptor.

11. Use of the polypeptide of any one of claims 1 to 2, the fusion protein of claim 3, or the polynucleotide of claim 4 in the preparation of a drug screening model,

the drug screening model is a cell model or an animal model,

the medicine is used for treating and/or preventing depression, Parkinson's disease, anxiety, fever, cardiovascular and cerebrovascular diseases, addiction, pain or learning and memory disorder;

preferably, the pain is neuropathic pain.

Images