WO2020238692A1 - NOVEL MUTANT OF αO-CONOTOXIN PEPTIDE GEXIVA, PHARMACEUTICAL COMPOSITION THEREOF AND USE THEREOF - Google Patents

NOVEL MUTANT OF αO-CONOTOXIN PEPTIDE GEXIVA, PHARMACEUTICAL COMPOSITION THEREOF AND USE THEREOF Download PDF

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WO2020238692A1
WO2020238692A1 PCT/CN2020/090978 CN2020090978W WO2020238692A1 WO 2020238692 A1 WO2020238692 A1 WO 2020238692A1 CN 2020090978 W CN2020090978 W CN 2020090978W WO 2020238692 A1 WO2020238692 A1 WO 2020238692A1
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seq
polypeptide
gexiva
cysteine
nachr
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罗素兰
长孙东亭
朱晓鹏
吴勇
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海南大学
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Definitions

  • Conotoxin The various polypeptide toxins produced in the venom of the carnivorous mollusk Cono snail living in the tropical ocean are called Conotoxin or Conopeptide.
  • Conotoxin has the special function of specifically binding various ion channels and receptors in animals [1] . It has become the first in animal toxin research and is known as the "treasury of marine drugs" and "undeveloped treasury.” Conotoxin has become a hot spot in neuroscience research and new drug development.
  • Conotoxin has a diversified three-dimensional structure and is considered to be a tiny protein. It has many types, strong activity and high selectivity. It has become a new source of new drug development [2-3] . They are used in analgesia, smoking cessation and anti-cancer , And the treatment of Parkinson's disease, dementia, epilepsy, psychosis and other intractable diseases have excellent application prospects. Its curative effect is definite, not addictive, and it can replace morphine and dulentin to treat neuralgia [4-6]
  • the omega-conotoxin MVIIA which acts on the N-type calcium channel, has been approved by the US FDA as a new therapeutic drug [1, 7-8] .
  • Conotoxins are mostly cysteine (Cys)-rich neuropeptide toxins composed of 7-50 amino acid residues. Conotoxins (peptides) can be divided into ⁇ , ⁇ , ⁇ , ⁇ and other pharmacological families according to their receptor targets. Among them, the ⁇ -family ( ⁇ *-) conotoxin has the special function of specifically blocking different subtypes of nicotinic acetylcholine receptors (nAChRs).
  • nAChRs nicotinic acetylcholine receptors
  • Nicotinic acetylcholine receptors are ubiquitous cell membrane proteins with important physiological functions and clinical research significance in the animal kingdom. They mediate many physiological functions of the central and peripheral nervous system, including learning, memory, response, analgesia and exercise. Control etc. [9-10] . nAChRs activates the release of many neurotransmitters such as dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid.
  • nAChRs are assembled into many subtypes by different ⁇ and ⁇ subunits, each of which has distinct pharmacological characteristics. Due to the lack of highly selective ligand compounds for various subtypes, there are many challenges to study and clarify the fine structure and function of various subtypes of nAChRs. Studies have shown that the ⁇ 9 ⁇ 10 nAChR that exists in the peripheral nervous system is a new target for the treatment of neuralgia drugs, which can be injected into the muscle to exert its efficacy [14-15] . The ⁇ 9 ⁇ 10nAChR blocker has the function of treating neuralgia and accelerating the recovery of injured nerves, which may function through immune mechanisms [16-17] .
  • the polypeptide has substantially the same or improved selectivity or specificity for ⁇ 9 ⁇ 10 nAChR;
  • the polypeptide has a prolonged half-life in the organism (for example, in the digestive tract such as the intestine, or in the blood); preferably, the organism is a mammal such as a human or a rat.
  • the polypeptide is characterized by items 1) and 2).
  • the polypeptide is characterized by items 1) and 3).
  • the polypeptide, wherein the multiple amino acids refer to 2-9 amino acids, such as 2, 3, 4, 5, 6, 7, 8, or 9 amino acids.
  • the polypeptide is characterized by items (6) and (4).
  • the polypeptide is characterized by items (6) and (5).
  • the polypeptide is characterized by items (1), (6), (4) and (5).
  • the polypeptide is characterized by items (1), (2), (6) and (5).
  • the polypeptide wherein the polypeptide contains 0, 1, or 2 disulfide bonds;
  • the polypeptide, wherein the carboxyl terminal of the polypeptide is amidated.
  • the present invention also relates to an isolated polypeptide whose amino acid sequence is as shown in any one of SEQ ID NOs: 2-24 and 26-117;
  • amino acid sequence of the polypeptide is shown in SEQ ID NO:75.
  • any one of SEQ ID NOs: 2-24 and 26-117 represents a linear amino acid sequence.
  • any one of SEQ ID NOs: 2-24 and 26-117 represents a linear amino acid sequence and modifications to the protein shown, such as disulfide bond, carboxyl terminal amidation and/or D Type amino acid substitutions, etc.
  • the polypeptide, wherein the carboxyl terminal of the polypeptide is amidated.
  • Another aspect of the present invention relates to an isolated polynucleotide, which encodes the polypeptide of any one of the present invention.
  • Another aspect of the present invention relates to a transformed cell, which contains the polynucleotide of the present invention, or the nucleic acid construct of the present invention.
  • Another aspect of the present invention relates to the use of the polypeptide of any one of the present invention in the preparation of a medicine for blocking or inhibiting ⁇ 9 ⁇ 10 nAChR.
  • the neuralgia is selected from at least one of the following: sciatica, trigeminal neuralgia, lymphatic neuralgia, multipoint motor neuralgia, acute severe spontaneous neuralgia, crush neuralgia and compound neuralgia;
  • the neuralgia is caused by at least one of the following factors: cancer, cancer chemotherapy, alcoholism, diabetes, sclerosis, herpes zoster, mechanical injury, surgical injury, AIDS, head nerve paralysis, drug poisoning, Industrial pollution poisoning, myeloma, chronic congenital sensory neuropathy, vasculitis, vasculitis, ischemia, uremia, childhood bile liver disease, chronic respiratory disorders, multiple organ failure, sepsis/sepsemia, hepatitis, Porphyria, vitamin deficiency, chronic liver disease, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, perineuritis or allergies;
  • the neuralgia is selected from at least one of the following: sciatica, trigeminal neuralgia, lymphatic neuralgia, multipoint motor neuralgia, acute severe spontaneous neuralgia, crush neuralgia, and compound nerve pain;
  • the neuralgia is caused by at least one of the following factors: cancer, cancer chemotherapy, alcoholism, diabetes, sclerosis, herpes zoster, mechanical injury, surgical injury, AIDS, head nerve paralysis, drug poisoning, Industrial pollution poisoning, myeloma, chronic congenital sensory neuropathy, vasculitis, vasculitis, ischemia, uremia, childhood bile liver disease, chronic respiratory disorders, multiple organ failure, sepsis/sepsemia, hepatitis, Porphyria, vitamin deficiency, chronic liver disease, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, perineuritis or allergies;
  • the cancer is at least one selected from breast cancer, lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, leukemia, neurocytoma and other cancers caused by epithelial cell carcinogenesis.
  • the dosage depends on many factors, such as the severity of the condition to be treated, the gender, age, weight and individual response of the patient or animal, as well as the condition and past medical history of the patient to be treated.
  • the usual practice in the art is to start the dosage from a level lower than the level required to obtain the desired therapeutic effect, and gradually increase the dosage until the desired effect is obtained.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide that inhibits a certain protein can be inserted.
  • the vector includes: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or artificial chromosome (PAC) derived from P1; bacteriophage such as lambda phage or M13 phage And animal viruses.
  • disease and/or disorder refers to a physical state of the subject, which is related to the disease and/or disorder described in the present invention.
  • the concentration unit ⁇ M means ⁇ mol/L
  • mM means mmol/L
  • nM means nmol/L.
  • Figure 2A-2D ⁇ O-conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO: 1) and mutant peptide 16 (Peptide 16, SEQ ID NO: 16, that is, the 17th arginine is alanine
  • UPLC Ultra-high pressure liquid chromatogram
  • ESI-MS electrospray mass spectrometry
  • Figure 7A-7J ⁇ O-conotoxin GeXIVA[1,4] (Peptide 25, SEQ ID NO: 25, represented by a dashed line) and its mutant with alanine at position 23 [I23A]GeXIVA[1, 2] (Peptide 45, SEQ ID NO: 45)
  • the concentration response curve of rat ⁇ 9 ⁇ 10, ⁇ 7, ⁇ 3 ⁇ 2, ⁇ 3 ⁇ 4, ⁇ 6/ ⁇ 3 ⁇ 4, ⁇ 2 ⁇ 2, ⁇ 4 ⁇ 2, ⁇ 2 ⁇ 4, ⁇ 4 ⁇ 2 and mouse ⁇ 1 ⁇ 1 ⁇ nAChRs. Each point on the curve is the mean ⁇ error (mean ⁇ S.E.) of 3-5 frog eggs (n 3-10).
  • FIG 10 ⁇ 10-19 GeXIVA (SEQ ID NO: 63) peptide and alanine scanning mutagenesis (SEQ ID NOs: 64-73; Table 4) Effect of current ⁇ 9 ⁇ 10 nAChR rats.
  • the histogram shows the percentage of each peptide responding to rat ⁇ 9 ⁇ 10 nAChR current induced by Ach (acetylcholine) at a concentration of 10 ⁇ M.
  • the mean ⁇ error of the current (mean ⁇ SE).
  • the current of each peptide was compared with the current of ⁇ 10-19 GeXIVA (SEQ ID NO: 63), and the difference was analyzed statistically. **** indicates the probability p ⁇ 0.0001, which is extremely significant.
  • Figure 14A-14D ⁇ O-conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO: 1) and mutant peptide [C2A, C9A, C20S, C27S] GeXIVA (SEQ ID NO: 86, Table 5) Ultra high pressure liquid chromatography (UPLC) (14A, 14C) and electrospray mass spectrometry (ESI-MS) (14B, 14D).
  • UPLC Ultra high pressure liquid chromatography
  • ESI-MS electrospray mass spectrometry
  • 14A UPLC chromatogram of Peptide 1.
  • 14B The ESI-MS mass spectrum of Peptide 1, the actual molecular weight is 3,452.70 Da.
  • 14C UPLC chromatogram of SEQ ID NO: 86.
  • the ESI-MS mass spectrum of SEQ ID NO: 86, the actual molecular weight is 3360.96 Da.
  • Figure 18A-18H ⁇ O-conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO: 1, 18A) and its D-type amino acid mutant (Table 6), namely 1t-GeXIVA (SEQ ID NO: 88, 18B), 28v-GeXIVA (SEQ ID NO: 89, 18C), 1t, 28v-GeXIVA (SEQ ID NO: 90, 18D), Mt-GeXIVA (SEQ ID NO: 91, 18E), Mf-GeXIVA ( SEQ ID NO: 92, 18F), GeArg-GeXIVA (SEQ ID NO: 93, 18G), GeFlex-GeXIVA (SEQ ID NO: 94, 18H), at a concentration of 1 ⁇ M or 100 nM, for rat ⁇ 9 ⁇ 10(r ⁇ 9 ⁇ 10)nAChR The current influence diagram.
  • the resin peptide is artificially synthesized by Fmoc chemical method, and the resin peptide can be synthesized by a peptide synthesizer or manual synthesis method. Except for cysteine, standard side chain protecting groups are used for other amino acids.
  • each linear peptide (peptide that does not form disulfide bonds) are: using the Fmoc and FastMoc methods in the solid phase synthesis method, the linear peptide is synthesized on the ABI Prism 433a peptide synthesizer.
  • the side chain protecting groups of Fmoc amino acids are: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp), Boc (Lys).
  • Fmoc HOBT DCC method Rink amidation resin and Fmoc For amino acids, refer to the instrument synthesis manual for the synthesis steps.
  • HPLC elution linear gradient is 10%-35% buffer B (B90) within 0-60min.
  • Buffer A is 0.1% TFA (trifluoroacetic acid) aqueous solution
  • Buffer B is 0.05% TFA
  • 90% CAN acetonitrile
  • each subunit was 5-10ng cRNA.
  • Frog eggs are cultured in ND-96.
  • CRNA was injected within 1 to 2 days after the frog eggs were collected, and used for voltage clamp recording of nAChRs within 1 to 4 days after injection.
  • the 23 mutants shown in SEQ ID NOs: 2-24 (prepared in Example 1) have very low nanomolar blocking effects on rat ⁇ 9 ⁇ 10 nAChR, and their IC 50 is below 80 nM, and their activity is comparable to Compared with wild-type GeXIVA[1,2] (SEQ ID NO:1), there is little difference.
  • the activity of these mutants on rat ⁇ 9 ⁇ 10 nAChR varied from 0.7 to 6.2 times, and none of them changed more than 10 times. This indicates that each single amino acid substituted by alanine in the GeXIVA[1,2] sequence has little effect on the blocking activity of ⁇ 9 ⁇ 10 nAChR and can be replaced without significantly affecting their receptor binding activity (Table 8).
  • the activity of the 5 mutants on ⁇ 7 nAChR is similar to that of the wild-type, and the activity of the other mutants on ⁇ 7 nAChR is reduced by more than 2 times, that is to say Compared with the ⁇ 7 nAChR subtype, most mutants have improved selectivity for ⁇ 9 ⁇ 10 nAChR, which is a rare advantage.
  • the half-blocking dose (IC 50 ) is greater than 10 ⁇ M, which means that at a high concentration of 10 ⁇ M, the current blocking of a certain polypeptide for this subtype is less than 50%.
  • the 23 mutants shown in SEQ ID NO: 26-48 have very weak blocking activity against ⁇ 7 nAChR, and their IC 50 is above 1360 nM. Some are even completely inactivated (IC 50 >10000nM), including the 6 mutants of SEQ ID NOs: 31, 39, 40, 41, 43, 44.
  • the activity of these mutants on ⁇ 7 nAChR is not much different from that of wild-type GeXIVA[1,4] (SEQ ID NO:25). Compared with the wild type, their activity on rat ⁇ 7 nAChR has a fold change of 0.5-3.2 times.
  • the measured molecular weight of SEQ ID NO: 25 is 3453.95 Da, and its theoretical molecular weight is 3453.96 Da.
  • the measured molecular weight of SEQ ID NO: 45 is 3410.46 Da, and its theoretical molecular weight is 3410.86 Da.
  • the measured molecular weights of the two are consistent with their theoretical molecular weights ( Figure 4B, 4D), indicating that the synthesized two polypeptides are completely correct.
  • SEQ ID NO:45 has the strongest activity against ⁇ 9 ⁇ 10 nAChR, with an IC 50 of only 1.4 nM, and very weak activity against all other subtypes, and their IC 50 is between 930-8240 nM (Table 10, Figures 7A-7J) .
  • the IC 50 of SEQ ID NO: 25 for ⁇ 9 ⁇ 10 nAChR is 16 nM, and its activity against all other subtypes is weak, and their IC 50 is between 440-4290 nM (Table 10, Figures 7A-7J).
  • the activity and selectivity of SEQ ID NO:45 for ⁇ 9 ⁇ 10 nAChR relative to all other nAChRs subtypes are significantly higher than those of wild-type SEQ ID NO:25.
  • Table 12 ⁇ O-conotoxin GeXIVA[1,2] (SEQ ID NO:1) and its multiple arginine substitution mutants (SEQ ID NOs:49-61) on rat neurotype ⁇ 9 ⁇ 10 nAChR subtype Blocking activity
  • IC 50 half-blocking dose
  • Hill slope the slope of the concentration response curve
  • the unit of IC 50 is nanomole (nM); the data in the brackets are the IC 50 or 95% confidence interval.
  • the range of Hill slope The range of Hill slope.
  • Example 6 ⁇ O-Conotoxin GeXIVA[1,2] and its truncated mutants block ⁇ 9 ⁇ 10 nAChR etc. active
  • brackets refer to the half-blocking dose (IC 50 ) or the slope of the concentration response curve (Hill slope), and the unit of IC 50 is nanomole (nM); the data in brackets are the IC 50 or the 95% confidence interval The range of Hill slope.
  • ⁇ 10-19 GeXIVA (SEQ ID NO: 63) has an IC 50 of 1110 nM for rat ⁇ 9 ⁇ 10, which is relatively weak.
  • ⁇ 10-19 [D5A] GeXIVA (SEQ ID NO: 68) and ⁇ 10-19 GeXIVA# (SEQ ID NO: 74) have an IC 50 of 120 nM and 130 nM for rat ⁇ 9 ⁇ 10, respectively, with similar activities.
  • the activity of SEQ ID NOs: 63, 68, and 74 on ⁇ 9 ⁇ 10 nAChR decreased.
  • the IC 50 of ⁇ 10-19[D5A]GeXIVA#(SEQ ID NO:75) to rat ⁇ 9 ⁇ 10 was 17nM, which kept the activity equivalent to GeXIVA[1,2](SEQ ID NO:1) (Table 13, Figure 12).
  • SEQ ID NO:75 The selectivity of SEQ ID NO:75 to ⁇ 9 ⁇ 10 relative to muscle-type nAChRs was significantly improved compared with wild-type GeXIVA [1,2], which was increased by 3.4 times (Table 14). Therefore, SEQ ID NO: 75 not only maintains strong blocking activity against ⁇ 9 ⁇ 10 nAChR, but also significantly reduces its activity on muscle-type receptors, thereby improving its selectivity. In addition, its synthesis cost is very low, which is a valuable optimization. mutant.
  • ⁇ O-Conotoxin GeXIVA (SEQ ID NOs:1 or 25) in the sequence of cysteine (Cys, C) paired by alanine (A) or serine (S) replaced by a mutant (SEQ ID NOs: 76-87) as shown in Table 5.
  • These mutants were successfully synthesized artificially and confirmed by chromatography and mass spectrometry. They contained a pair of disulfide bonds or no disulfide bonds.
  • the data in this column refers to the ratio between the half-blocking dose (IC 50 ) of polypeptide No. 86 against human ⁇ 9 ⁇ 10 nAChR subtype and the half-blocking dose (IC 50 ) of polypeptide No. 1.
  • SEQ ID NO: 92 The blocking activity of SEQ ID NO: 92 (GeMF, Mf-GeXIVA) on rat and human ⁇ 9 ⁇ 10nAChRs is 2.6 times and 2.8 times higher than that of GeXIVA [1,2] (Table 19-20).
  • the concentration response curve of GeXIVA[1,4] (SEQ ID NO: 25) and its partial aspartic acid scanning mutants (Table 7; SEQ ID NOs: 95, 100, 110-115) to rat ⁇ 9 ⁇ 10 nAChR is shown in the figure As shown in 24, their IC 50 is listed in Table 22, and their IC 50 ranges from 27 to 590 nM. Some mutants have similar activity to wild-type GeXIVA[1,4] (Table 22), such as SEQ ID NO: 95 ( Figure 24A) and SEQ ID NO: 115 ( Figure 24H).

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Abstract

Provided are a novel mutant of αO-conotoxin peptide GeXIVA, a pharmaceutical composition thereof and a use thereof. The amino acid sequence of the novel mutant is obtained by removing one or more amino acids in a parent sequence SEQ ID NO: 1, 62 or 63 or by replacing said acids with the same number of L-type or D-type amino acids. The novel mutant has the activity of blocking α9α10 nAChR, and may be used to prepare drugs such as analgesics, psychiatric disease and cancer-related treatment drugs, and neuroscience tool drugs.

Description

αO-芋螺毒素肽GeXIVA新突变体、其药物组合物及用途αO-conotoxin peptide GeXIVA new mutant, its pharmaceutical composition and use 技术领域Technical field
本发明属于药学、生物化学与分子生物学和神经科学领域，涉及αO-芋螺毒素肽GeXIVA及其一系列新突变体、其药物组合物、其制备方法及用途。本发明还涉及一种阻断烟碱乙酰胆碱受体(nAChRs)的方法、以及所述芋螺毒素肽的制药用途。The invention belongs to the fields of pharmacy, biochemistry, molecular biology and neuroscience, and relates to αO-conotoxin peptide GeXIVA and a series of new mutants thereof, its pharmaceutical composition, its preparation method and application. The invention also relates to a method for blocking nicotinic acetylcholine receptors (nAChRs) and the pharmaceutical use of the conotoxin peptide.
背景技术Background technique
生活在热带海洋中的肉食性软体动物芋螺毒液中产生的各种各样的多肽毒素，称为芋螺毒素或芋螺肽(Conotoxin或Conopeptide)。芋螺毒素具有特异结合动物体内各种离子通道和受体等的特殊功能 [1]，已跃居动物毒素研究的首位，被誉为“海洋药物宝库”与“尚未开发的金库”。芋螺毒素已成为当今神经科学研究和新药研发的热点。 The various polypeptide toxins produced in the venom of the carnivorous mollusk Cono snail living in the tropical ocean are called Conotoxin or Conopeptide. Conotoxin has the special function of specifically binding various ion channels and receptors in animals [1] . It has become the first in animal toxin research and is known as the "treasury of marine drugs" and "undeveloped treasury." Conotoxin has become a hot spot in neuroscience research and new drug development.
芋螺毒素具有多样化的三维结构而被认为是微小蛋白质，其种类多、活性强、选择性高，已成为新药开发的新来源 [2-3]，它们在镇痛、戒烟戒毒、抗癌，以及治疗帕金森症、痴呆、癫痫、精神病等多种疑难杂症方面具有极好的应用前景。其疗效确切、不成瘾，可代替吗啡、杜冷丁等治疗神经痛 [4-6]。作用于N-型钙离子通道的ω-芋螺毒素MVIIA已被美国FDA批准为治疗新药 [1，7-8]。 Conotoxin has a diversified three-dimensional structure and is considered to be a tiny protein. It has many types, strong activity and high selectivity. It has become a new source of new drug development [2-3] . They are used in analgesia, smoking cessation and anti-cancer , And the treatment of Parkinson's disease, dementia, epilepsy, psychosis and other intractable diseases have excellent application prospects. Its curative effect is definite, not addictive, and it can replace morphine and dulentin to treat neuralgia [4-6] The omega-conotoxin MVIIA, which acts on the N-type calcium channel, has been approved by the US FDA as a new therapeutic drug [1, 7-8] .
在我国，芋螺是海南特有而宝贵的药用海洋生物资源，至少有15万种海南产芋螺毒素潜在药源急待开发。我国还没有芋螺毒素药物进入临床试验。芋螺毒素大多是由7－50个氨基酸残基组成的、富含半胱氨酸(Cys)的神经肽毒素。芋螺毒素(肽)按其受体靶位可分为α、ω、μ、δ等多种药理学家族。其中的α-家族(α*-)芋螺毒素具有特异阻断烟碱型乙酰胆碱受体(nAChRs)不同亚型的特殊功能。In our country, cono snails are unique and valuable medical marine biological resources in Hainan, and there are at least 150,000 potential sources of cono toxin produced in Hainan to be developed urgently. my country has not yet entered clinical trials of conotoxin drugs. Conotoxins are mostly cysteine (Cys)-rich neuropeptide toxins composed of 7-50 amino acid residues. Conotoxins (peptides) can be divided into α, ω, μ, δ and other pharmacological families according to their receptor targets. Among them, the α-family (α*-) conotoxin has the special function of specifically blocking different subtypes of nicotinic acetylcholine receptors (nAChRs).
烟碱型乙酰胆碱受体(nAChRs)是动物界普遍存在的具有重要生理作用和临床研究意义的细胞膜蛋白，介导众多中枢和外周神经系统的生理功能，包括学习、记忆、应答、镇痛和运动控制等 [9-10]。nAChRs激活多巴胺、去甲肾上腺素、五羟色胺、γ-氨基丁酸等多种神经递质的释放。已证实nAChRs是筛选诊断和治疗一大类重要疾病药物的关键靶点，这些疾病包括成瘾、疼痛、癌症、智障、帕金森症、精神病、抑郁、重症肌无力等疑难杂症。常用的非选择性的nAChR激动剂如烟碱，虽然可以缓解上述神经疾病的症状，但它们对心脏和胃肠道产生强烈的副作用，且有成瘾性。因此， 开发针对nAChRs各种亚型具有选择性的配体药物是治疗上述疾病的关键所在 [11-13]。 Nicotinic acetylcholine receptors (nAChRs) are ubiquitous cell membrane proteins with important physiological functions and clinical research significance in the animal kingdom. They mediate many physiological functions of the central and peripheral nervous system, including learning, memory, response, analgesia and exercise. Control etc. [9-10] . nAChRs activates the release of many neurotransmitters such as dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid. It has been confirmed that nAChRs is a key target for screening, diagnosis and treatment of a large number of important diseases, such as addiction, pain, cancer, intellectual disability, Parkinson's disease, psychosis, depression, myasthenia gravis and other intractable diseases. Commonly used non-selective nAChR agonists such as nicotine, although they can alleviate the symptoms of the above neurological diseases, they have strong side effects on the heart and gastrointestinal tract and are addictive. Therefore, the development of selective ligand drugs for various subtypes of nAChRs is the key to the treatment of the above-mentioned diseases [11-13] .
然而，开发这样的药物的前提是，要获得可以特异结合nAChRs各种亚型的选择性化合物，作为工具药来研究和鉴定各种亚型的精细组成和生理功能，或直接作为相关疾病的治疗药物。另外，在乳腺癌与小细胞肺癌中，肿瘤细胞膜上烟碱乙酰胆碱受体的激活促进肿瘤细胞增殖，用药物阻断这些受体的激活，或可治疗这些灾难性癌症。目前，芋螺毒素备受关注，已被用于系统地研究与开发nAChRs各种亚型的特异阻断剂。However, the prerequisite for the development of such drugs is to obtain selective compounds that can specifically bind to various subtypes of nAChRs, as tool drugs to study and identify the fine composition and physiological functions of various subtypes, or directly as the treatment of related diseases drug. In addition, in breast cancer and small cell lung cancer, the activation of nicotinic acetylcholine receptors on the tumor cell membrane promotes tumor cell proliferation, and blocking the activation of these receptors with drugs may treat these catastrophic cancers. At present, conotoxin has attracted much attention and has been used to systematically research and develop specific blockers of various subtypes of nAChRs.
nAChRs由不同的α和β亚基组装成很多种亚型，每种亚型都有截然不同的药理学特征。由于缺乏针对各种亚型的高选择性配体化合物，要研究和阐明各种各样的nAChRs亚型的精细结构和功能面临诸多挑战。研究表明，存在于外周神经系统的α9α10 nAChR是治疗神经痛药物的新靶点，可通过肌肉注射发挥药效 [14-15]。α9α10nAChR阻断剂具有治疗神经痛和加速受伤神经恢复的功能，可能是通过免疫机制发挥作用 [16-17]。角化细胞上的α9α10 nAChR在伤口愈合的病理生理学过程中起着很重要的作用 [18]。α9nAChR亚基在乳腺癌组织中过表达，α9亚基变体影响支气管细胞的转化与增殖，该亚基在肺癌的治疗中具有非常重要的意义 [19]。在大鼠动物模型上，α9α10nAChR的阻断剂还可预防和逆转由于癌症化疗引起的神经痛，从而可加大抗癌药物的剂量，减少抗癌药的副作用，达到癌症治疗的目的 [20]。因而，α9α10 nAChR被认为是治疗神经痛 [21-22]、癌症化疗、乳腺癌、肺癌等的新靶点。 nAChRs are assembled into many subtypes by different α and β subunits, each of which has distinct pharmacological characteristics. Due to the lack of highly selective ligand compounds for various subtypes, there are many challenges to study and clarify the fine structure and function of various subtypes of nAChRs. Studies have shown that the α9α10 nAChR that exists in the peripheral nervous system is a new target for the treatment of neuralgia drugs, which can be injected into the muscle to exert its efficacy [14-15] . The α9α10nAChR blocker has the function of treating neuralgia and accelerating the recovery of injured nerves, which may function through immune mechanisms [16-17] . The α9α10 nAChR on keratinocytes plays an important role in the pathophysiological process of wound healing [18] . The α9nAChR subunit is overexpressed in breast cancer tissues, and α9 subunit variants affect the transformation and proliferation of bronchial cells. This subunit has a very important significance in the treatment of lung cancer [19] . In rat animal models, α9α10nAChR blockers can also prevent and reverse neuralgia caused by cancer chemotherapy, which can increase the dose of anticancer drugs, reduce the side effects of anticancer drugs, and achieve the purpose of cancer treatment [20] . Therefore, α9α10 nAChR is considered as a new target for the treatment of neuralgia [21-22] , cancer chemotherapy, breast cancer, lung cancer and so on.
镇痛药市场潜力非常巨大。据流行病学调查研究结果显示，神经痛(慢性痛)影响约20％的人群，已成为世界性公认的健康难题 [23]。人类社会为此付出了沉重的经济负担，例如，美国约有1亿成年人遭受神经痛(慢性痛)的折磨，所花费的医疗费用每年高达约6350－6500美金 [24]。神经痛造成的医疗损失比心脏病、癌症和糖尿病三大类疾病的总和还要多 [25]。广大的发展中国家人口数量众多，神经痛所造成的医疗损失更加巨大。神经痛往往表现为顽固性疼痛，很难治疗，现有治疗神经痛的方法，主要是局部麻醉用药，来阻断由外周神经、神经丛、背根神经、交感神经系统等产生的疼痛信号，但这些治疗只能短时间有镇痛效果，并不能根治神经痛，反复使用很容易引起成瘾。很多疾病都会引起神经痛，包括癌症与癌症化疗、坐骨神经痛、糖尿病、带状疱疹、机械伤和手术伤、艾滋病等等。随着世界人口数量逐渐增加，人口加速老年化，65岁以上的人群中，一半以上(>50％)的老年人都有神经痛的困扰。全球镇 痛药市场年均复合增长率达在25％以上。目前离实现WHO提出的“让癌症患者不痛”的战略还相差甚远，中、重度疼痛的患者尚未得到充分止痛治疗，无成瘾性的镇痛药市场拥有巨大的上升空间。 The market potential of analgesics is huge. According to the results of epidemiological investigations, neuralgia (chronic pain) affects about 20% of the population, and has become a universally recognized health problem [23] . Human society has paid a heavy economic burden for this. For example, about 100 million adults in the United States suffer from neuralgia (chronic pain), and the annual medical expenses are as high as 6350-6500 US dollars [24] . The medical loss caused by neuralgia is more than the sum of the three major diseases of heart disease, cancer and diabetes [25] . The vast developing countries have a large population, and the medical losses caused by neuralgia are even greater. Neuralgia is often manifested as intractable pain, which is difficult to treat. The existing methods of treating neuralgia are mainly local anesthetics to block the pain signals generated by the peripheral nerve, nerve plexus, dorsal root nerve, and sympathetic nervous system. However, these treatments can only have a short-term analgesic effect, and cannot cure neuralgia. Repeated use can easily cause addiction. Many diseases can cause neuralgia, including cancer and cancer chemotherapy, sciatica, diabetes, shingles, mechanical and surgical injuries, AIDS, etc. With the gradual increase in the number of the world's population and the accelerated aging of the population, more than half (>50%) of the elderly over the age of 65 suffer from neuralgia. The compound annual growth rate of the global analgesic market is more than 25%. At present, it is far from realizing the strategy of “make cancer patients painless” proposed by the WHO. Patients with moderate and severe pain have not yet received adequate pain relief treatment, and the non-addictive analgesics market has huge room for growth.
2004年12月，美国FDA批准了世界上第一个海洋芋螺毒素新药Ziconotide(中文译名为奇考诺肽)，不成瘾，被认为是第一个真正针对神经痛的镇痛药，目前美国允许鞘内给药的镇痛药只有2种(芋螺毒素Prialt与吗啡)，该新药仅专利转让费就高达8亿美元，产生了巨大的社会效益和经济效益。奇考诺肽是通过特异阻断N-型钙离子通道发挥镇痛效应，其作用靶点N-型钙离子通道仅存于中枢神经系统，所以需要通过编程泵内置于人体内给药至脊髓，给药途径很麻烦，该给药泵非常昂贵，目前仅限于美欧等发达国家应用，很难在广大的发展中国家使用 [26]。以α9α10 nAChR为靶点的神经痛治疗药物可通过肌肉注射发挥镇痛效应 [22,27]，比目前商业化的芋螺毒素镇痛药物Ziconotide给药途径更简便，市场更广阔。α9α10 nAChR阻断剂还可作为分子探针来研究该亚型的结构与功能，作为工具药探讨与之相关的神经痛、癌症化疗、乳腺癌、肺癌等重大疾病的发病机理，为更好地了解、预防和治疗这些疾病提供最佳途径。 In December 2004, the U.S. FDA approved the world’s first marine conotoxin drug, Ziconotide (Chinese translation for chiconotide), which is not addictive and is considered the first real analgesic against neuralgia. There are only two analgesics that allow intrathecal administration in the United States (conotoxin Prialt and morphine). The patent transfer fee for this new drug alone is as high as US$800 million, which has produced huge social and economic benefits. Chiconotide exerts analgesic effect by specifically blocking N-type calcium ion channels, and its target of action N-type calcium ion channels only exists in the central nervous system, so it needs to be built into the human body through a programmed pump to be administered to the spinal cord , The route of administration is very troublesome, the pump is very expensive, and it is currently only used in developed countries such as the United States and Europe, and it is difficult to use in developing countries [26] . Neuralgia therapeutic drugs targeting α9α10 nAChR can exert analgesic effects through intramuscular injection [22,27] . Compared with the current commercialized conotoxin analgesic drug Ziconotide, the administration route is simpler and the market is broader. α9α10 nAChR blockers can also be used as molecular probes to study the structure and function of this subtype, and as a tool to explore the pathogenesis of major diseases such as neuralgia, cancer chemotherapy, breast cancer, and lung cancer. Provide the best way to understand, prevent and treat these diseases.
本发明人经过多年的努力，筛选鉴定出1个强阻断α9α10 nAChR的αO-新家族芋螺毒素GeXIVA，有3种可能的二硫键异构体，均已人工合成成功，分别命名为GeXIVA[1,2]、GeXIVA[1,3]和GeXIVA[1,4]。其中GeXIVA[1,2]异构体对α9α10 nAChR的半阻断剂量(IC 50)仅为3.8nM/4.6nM [27]，是α9α10 nAChR亚型活性极强的特异阻断剂。GeXIVA的作用靶点清楚，蕴涵着巨大的经济价值和社会效益，并已申请了国家发明专利 [28]和国际PCT专利 [29]。后续研究发现，GeXIVA的3种二硫键异构体，保持了对大鼠和人类α9α10 nAChRs相似的阻断活性和选择性 [30]，这预示着GeXIVA在人类神经痛等的治疗新药研发领域具有广阔的应用前景。而之前发现的α-芋螺毒素RgIA和Vc1.1，对大鼠α9α10 nAChRs的阻断活性很强，而对人类α9α10 nAChRs的阻断活性很微弱，二者相比下降了300－400倍 [30]。这也是导致α-芋螺毒素Vc1.1在II期临床试验中被终止的直接原因。 After years of hard work, the inventors screened and identified a new family of αO-conotoxin GeXIVA that strongly blocks α9α10 nAChR. There are three possible disulfide bond isomers, all of which have been artificially synthesized and named GeXIVA. [1,2], GeXIVA[1,3] and GeXIVA[1,4]. Among them, the half-blocking dose (IC 50 ) of GeXIVA[1,2] isomer for α9α10 nAChR is only 3.8nM/4.6nM [27] , which is a specific blocker with extremely strong activity of α9α10 nAChR subtype. GeXIVA has a clear target point of action, which contains huge economic value and social benefits, and has applied for national invention patents [28] and international PCT patents [29] . Subsequent studies found that the three disulfide bond isomers of GeXIVA maintain similar blocking activity and selectivity to rat and human α9α10 nAChRs [30] , which indicates that GeXIVA can be used in the research and development of new drugs for human neuralgia. have a broad vision of application. The previously discovered α-conotoxins RgIA and Vc1.1 have strong blocking activity against rat α9α10 nAChRs, while their blocking activity against human α9α10 nAChRs is very weak. Compared with the two, they are reduced by 300-400 times [ 30] . This is also the direct cause of the termination of α-conotoxin Vc1.1 in phase II clinical trials.
目前，尚需要开发更多有效的α9α10 nAChRs特异阻断剂。At present, there is still a need to develop more effective α9α10 nAChRs specific blockers.
发明内容Summary of the invention
本发明人经过深入的研究和创造性地劳动，发现了一个αO-芋螺毒素GeXIVA的一系列新突变体，其能够特异性地阻断α9α10乙酰胆碱受体，该受体是神经痛、癌症化疗、乳腺癌、肺癌等的药物作用靶点。其在制备镇痛药物，有关乳腺癌、肺癌、神经精神疾病等治疗药物的研发，以及工具药等方面具有极好的应用前景。由此提供了下述发明：After in-depth research and creative work, the inventors discovered a series of new mutants of αO-conotoxin GeXIVA, which can specifically block α9α10 acetylcholine receptors, which are neuralgia, cancer chemotherapy, Drug targets for breast cancer and lung cancer. It has excellent application prospects in the preparation of analgesics, the research and development of therapeutic drugs such as breast cancer, lung cancer, neuropsychiatric diseases, and tool drugs. This provides the following inventions:
本发明的一个方面涉及一种分离的多肽，其氨基酸序列为将母序列中的一个或多个氨基酸去掉，或者将母序列中的一个或多个氨基酸替换为相同数目的L型氨基酸或D型氨基酸，其中，所述母序列为SEQ ID NO:1、SEQ ID NO:62或SEQ ID NO:63；并且所述多肽具有阻断或抑制α9α10 nAChR的活性；One aspect of the present invention relates to an isolated polypeptide, the amino acid sequence of which is that one or more amino acids in the parent sequence are removed, or one or more amino acids in the parent sequence are replaced with the same number of L-type amino acids or D-type amino acids. Amino acid, wherein the parent sequence is SEQ ID NO: 1, SEQ ID NO: 62 or SEQ ID NO: 63; and the polypeptide has the activity of blocking or inhibiting α9α10 nAChR;
优选地，所述L型氨基酸或D型氨基酸选自：丙氨酸、丝氨酸、2-氨基丁酸、组氨酸、精氨酸、酪氨酸、苏氨酸、赖氨酸、亮氨酸、苯丙氨酸、D-精氨酸、D-丝氨酸、D-苏氨酸、D-缬氨酸、D-天冬氨酸、D-谷氨酸、谷氨酸和天冬氨酸；优选为丙氨酸、D-苏氨酸或D-天冬氨酸；Preferably, the L-type amino acid or D-type amino acid is selected from: alanine, serine, 2-aminobutyric acid, histidine, arginine, tyrosine, threonine, lysine, leucine , Phenylalanine, D-arginine, D-serine, D-threonine, D-valine, D-aspartic acid, D-glutamic acid, glutamic acid and aspartic acid; Preferably it is alanine, D-threonine or D-aspartic acid;
优选地，所述的多肽，其特征在于如下的1)－3)项中的任意1项、2项或3项：Preferably, the polypeptide is characterized by any one, two or three of the following items 1) to 3):
1)与母序列相比，所述多肽具有基本相同或者提高的阻断α9α10 nAChR的活性；1) Compared with the parent sequence, the polypeptide has substantially the same or improved α9α10 nAChR blocking activity;
2)与母序列相比，相对于α7 nAChR和/或α1β1δε nAChR，所述多肽具有基本相同或提高的对α9α10 nAChR的选择性或特异性；2) Compared with the parent sequence, relative to α7 nAChR and/or α1β1δε nAChR, the polypeptide has substantially the same or improved selectivity or specificity for α9α10 nAChR;
3)与母序列相比，所述多肽具有在生物体内(例如在消化道如肠道内，或在血液中)延长的半衰期；优选地，所述生物体为哺乳动物例如人或大鼠。3) Compared with the parent sequence, the polypeptide has a prolonged half-life in the organism (for example, in the digestive tract such as the intestine, or in the blood); preferably, the organism is a mammal such as a human or a rat.
在本发明的一些实施方式中，所述的多肽，其特征在于第1)项。In some embodiments of the present invention, the polypeptide is characterized in item 1).
在本发明的一些实施方式中，所述的多肽，其特征在于第2)项。In some embodiments of the present invention, the polypeptide is characterized by item 2).
在本发明的一些实施方式中，所述的多肽，其特征在于第3)项。In some embodiments of the present invention, the polypeptide is characterized by item 3).
在本发明的一些实施方式中，所述的多肽，其特征在于第1)项和第2)项。In some embodiments of the present invention, the polypeptide is characterized by items 1) and 2).
在本发明的一些实施方式中，所述的多肽，其特征在于第1)项和第3)项。In some embodiments of the present invention, the polypeptide is characterized by items 1) and 3).
在本发明的一些实施方式中，所述的多肽，其特征在于第2)项和第3)项。In some embodiments of the present invention, the polypeptide is characterized by items 2) and 3).
在本发明的一些实施方式中，所述的多肽，其特征在于第1)项至第3)项。In some embodiments of the present invention, the polypeptide is characterized by items 1) to 3).
在本发明的一些实施方式中，所述的多肽，其中，被去掉或被替换的一个或多个氨基酸不是精氨酸。In some embodiments of the present invention, in the polypeptide, one or more amino acids removed or replaced are not arginine.
在本发明的一些实施方式中，所述的多肽，其中，所述多个氨基酸是指2－9个氨基酸，例如2、3、4、5、6、7、8或9个氨基酸。In some embodiments of the present invention, the polypeptide, wherein the multiple amino acids refer to 2-9 amino acids, such as 2, 3, 4, 5, 6, 7, 8, or 9 amino acids.
本发明中，所述“基本相同”是指与母序列相比，阻断α9α10 nAChR的活性或者对α9α10 nAChR的选择性或特异性没有显著的变化(提高或者降低)，例如变化在±10％以内、±20％以内、±30％以内、±40％以内或±50％以内。In the present invention, the "substantially the same" means that compared with the parent sequence, it blocks the activity of α9α10 nAChR or does not significantly change (increase or decrease) the selectivity or specificity of α9α10 nAChR, for example within ±10% Within, within ±20%, within ±30%, within ±40%, or within ±50%.
在本发明的一个或多个实施方式中，所述的分离的多肽，其特征在于如下的(1)－(6)项中的任意的1项、2项、3项、4项或5项：In one or more embodiments of the present invention, the isolated polypeptide is characterized by any of the following (1) to (6) items 1, 2, 3, 4 or 5 :
(1)将SEQ ID NO:1或SEQ ID NO:62中的除了半胱氨酸和丙氨酸之外的任意一个氨基酸替换为一个丙氨酸；(1) Replace any amino acid in SEQ ID NO: 1 or SEQ ID NO: 62 except cysteine and alanine with an alanine;
(2)将SEQ ID NO:1中的9个精氨酸中的任意2个、3个、4个、5个、6个、7个、8个或9个替换为相同数目的丙氨酸和/或丝氨酸；(2) Replace any 2, 3, 4, 5, 6, 7, 8, or 9 of the 9 arginines in SEQ ID NO:1 with the same number of alanines And/or serine;
(3)将SEQ ID NO:63中的任意一个氨基酸替换为一个丙氨酸；(3) Replace any amino acid in SEQ ID NO: 63 with an alanine;
(4)将SEQ ID NO:1中的4个半胱氨酸中的任意1个、2个、3个或4个替换为相同数目的丙氨酸和/丝氨酸；(4) Replace any one, two, three or four of the four cysteines in SEQ ID NO:1 with the same number of alanine and/serine;
(5)将SEQ ID NO:1中的氮末端的苏氨酸替换为D-苏氨酸，碳末端的缬氨酸替换为D-缬氨酸，和/或将SEQ ID NO:1中的9个精氨酸中的任意2个、3个、4个、5个、6个、7个、8个或9个替换为相同数目的D-精氨酸；(5) Replace the nitrogen-terminal threonine in SEQ ID NO:1 with D-threonine, and replace the carbon-terminal valine with D-valine, and/or replace SEQ ID NO:1 Replace any 2, 3, 4, 5, 6, 7, 8, or 9 of the 9 arginines with the same number of D-arginine;
(6)将SEQ ID NO:1、SEQ ID NO:62或SEQ ID NO:63中的除了半胱氨酸和天冬氨酸之外的任意一个氨基酸替换为一个天冬氨酸。(6) Replace any amino acid in SEQ ID NO: 1, SEQ ID NO: 62 or SEQ ID NO: 63 except cysteine and aspartic acid with an aspartic acid.
在本发明的一些实施方式中，所述的多肽，其特征在于第(1)项和第(4)项。In some embodiments of the present invention, the polypeptide is characterized by items (1) and (4).
在本发明的一些实施方式中，所述的多肽，其特征在于第(1)项和第(5)项。In some embodiments of the present invention, the polypeptide is characterized by items (1) and (5).
在本发明的一些实施方式中，所述的多肽，其特征在于第(4)项和第(6)项。In some embodiments of the present invention, the polypeptide is characterized by items (4) and (6).
在本发明的一些实施方式中，所述的多肽，其特征在于第(3)项和第(6)项。In some embodiments of the present invention, the polypeptide is characterized by items (3) and (6).
在本发明的一些实施方式中，所述的多肽，其特征在于第(1)项、第(4)项和第(5)项。In some embodiments of the present invention, the polypeptide is characterized by items (1), (4) and (5).
在本发明的一些实施方式中，所述的多肽，其特征在于第(2)项和第(4)项。In some embodiments of the present invention, the polypeptide is characterized by items (2) and (4).
在本发明的一些实施方式中，所述的多肽，其特征在于第(2)项和第(5)项。In some embodiments of the present invention, the polypeptide is characterized by items (2) and (5).
在本发明的一些实施方式中，所述的多肽，其特征在于第(4)项和第(6)项。In some embodiments of the present invention, the polypeptide is characterized by items (4) and (6).
在本发明的一些实施方式中，所述的多肽，其特征在于第(2)项、第(4)项和 第(5)项。In some embodiments of the present invention, the polypeptide is characterized by items (2), (4) and (5).
在本发明的一些实施方式中，所述的多肽，其特征在于第(6)项和第(4)项。In some embodiments of the present invention, the polypeptide is characterized by items (6) and (4).
在本发明的一些实施方式中，所述的多肽，其特征在于第(6)项和第(5)项。In some embodiments of the present invention, the polypeptide is characterized by items (6) and (5).
在本发明的一些实施方式中，所述的多肽，其特征在于第(4)项和第(6)项。In some embodiments of the present invention, the polypeptide is characterized by items (4) and (6).
在本发明的一些实施方式中，所述的多肽，其特征在于第(6)项、第(4)项和第(5)项。In some embodiments of the present invention, the polypeptide is characterized by items (6), (4) and (5).
在本发明的一些实施方式中，所述的多肽，其特征在于第(1)项、第(6)项、第(4)项和第(5)项。In some embodiments of the present invention, the polypeptide is characterized by items (1), (6), (4) and (5).
在本发明的一些实施方式中，所述的多肽，其特征在于第(2)项、第(6)项、第(4)项和第(5)项。In some embodiments of the present invention, the polypeptide is characterized by items (2), (6), (4) and (5).
在本发明的一些实施方式中，所述的多肽，其特征在于第(1)项、第(2)项、第(4)项和第(5)项。In some embodiments of the present invention, the polypeptide is characterized by items (1), (2), (4) and (5).
在本发明的一些实施方式中，所述的多肽，其特征在于第(1)项、第(2)项、第(6)项和第(5)项。In some embodiments of the present invention, the polypeptide is characterized by items (1), (2), (6) and (5).
在本发明的一些实施方式中，所述的多肽，其特征在于第(1)项、第(2)项、第(4)项和第(6)项。In some embodiments of the present invention, the polypeptide is characterized by items (1), (2), (4) and (6).
在本发明的一些实施方式中，所述的多肽，其特征在于第(1)项、第(2)项、第(6)项、第(4)项和第(5)项。In some embodiments of the present invention, the polypeptide is characterized by items (1), (2), (6), (4) and (5).
在本发明的一个或多个实施方式中，所述的多肽，其中，所述多肽含有0个、1个或2个二硫键；In one or more embodiments of the present invention, the polypeptide, wherein the polypeptide contains 0, 1, or 2 disulfide bonds;
优选地，当母序列为SEQ ID NO:1且所述多肽含有1个或二个二硫键时，所述二硫键选自：Preferably, when the parent sequence is SEQ ID NO: 1 and the polypeptide contains one or two disulfide bonds, the disulfide bonds are selected from:
所述多肽的N末端起的第一个半胱氨酸与第二个半胱氨酸形成的二硫键，以及第三个半胱氨酸与第四个半胱氨酸形成的二硫键；The disulfide bond formed by the first cysteine and the second cysteine from the N-terminus of the polypeptide, and the disulfide bond formed by the third cysteine and the fourth cysteine ；
所述多肽的N末端起的第一个半胱氨酸与第四个半胱氨酸形成的二硫键，以及第二个半胱氨酸与第三个半胱氨酸形成二硫键；和The disulfide bond formed by the first cysteine and the fourth cysteine from the N-terminus of the polypeptide, and the disulfide bond formed by the second cysteine and the third cysteine; with
所述多肽的N末端起的第一个半胱氨酸与第三个半胱氨酸形成的二硫键，以及第二个半胱氨酸与第四个半胱氨酸形成的二硫键。The disulfide bond formed by the first cysteine and the third cysteine from the N-terminus of the polypeptide, and the disulfide bond formed by the second cysteine and the fourth cysteine .
在本发明的一个或多个实施方式中，所述的多肽，其中，所述多肽的羧基末端是酰胺化的。In one or more embodiments of the present invention, the polypeptide, wherein the carboxyl terminal of the polypeptide is amidated.
本发明还涉及一种分离的多肽，其氨基酸序列分别如SEQ ID NOs:2－24、26－117中的任一序列所示；The present invention also relates to an isolated polypeptide whose amino acid sequence is as shown in any one of SEQ ID NOs: 2-24 and 26-117;
优选地，所述多肽的氨基酸序列如SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:23、SEQ ID NO:37、SEQ ID NO:45、SEQ ID NO:62、SEQ ID NO:75、SEQ ID NO:86或SEQ ID NO:94所示；Preferably, the amino acid sequence of the polypeptide is as SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 37, SEQ ID NO: 45, SEQ ID NO: 62, SEQ ID NO: 75, SEQ ID NO: 86 or SEQ ID NO: 94;
更优选地，所述多肽的氨基酸序列如SEQ ID NO:45、SEQ ID NO:75、SEQ ID NO:86或SEQ ID NO:94所示；More preferably, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 45, SEQ ID NO: 75, SEQ ID NO: 86 or SEQ ID NO: 94;
特别优选地，所述多肽的氨基酸序列如SEQ ID NO:75所示。Especially preferably, the amino acid sequence of the polypeptide is shown in SEQ ID NO:75.
在本发明的一个实施方案中，SEQ ID NOs:2－24、26－117中的任一序列表示氨基酸线性序列。In one embodiment of the present invention, any one of SEQ ID NOs: 2-24 and 26-117 represents a linear amino acid sequence.
在本发明的一个实施方案中，SEQ ID NOs:2－24、26－117中的任一序列表示氨基酸线性序列以及对所示蛋白的修饰，例如二硫键、羧基末端酰胺化和/或D型氨基酸替换等。In one embodiment of the present invention, any one of SEQ ID NOs: 2-24 and 26-117 represents a linear amino acid sequence and modifications to the protein shown, such as disulfide bond, carboxyl terminal amidation and/or D Type amino acid substitutions, etc.
在本发明的一个或多个实施方式中，所述的多肽，其中，所述多肽含有0个、1个或2个二硫键；In one or more embodiments of the present invention, the polypeptide, wherein the polypeptide contains 0, 1, or 2 disulfide bonds;
优选地，当母序列为SEQ ID NO:1且所述多肽含有1个或二个二硫键时，所述二硫键选自：Preferably, when the parent sequence is SEQ ID NO: 1 and the polypeptide contains one or two disulfide bonds, the disulfide bonds are selected from:
所述多肽的N末端起的第一个半胱氨酸与第二个半胱氨酸形成的二硫键，以及第三个半胱氨酸与第四个半胱氨酸形成的二硫键；The disulfide bond formed by the first cysteine and the second cysteine from the N-terminus of the polypeptide, and the disulfide bond formed by the third cysteine and the fourth cysteine ；
所述多肽的N末端起的第一个半胱氨酸与第四个半胱氨酸形成的二硫键，以及第二个半胱氨酸与第三个半胱氨酸形成二硫键；和The disulfide bond formed by the first cysteine and the fourth cysteine from the N-terminus of the polypeptide, and the disulfide bond formed by the second cysteine and the third cysteine; with
所述多肽的N末端起的第一个半胱氨酸与第三个半胱氨酸形成的二硫键，以及第二个半胱氨酸与第四个半胱氨酸形成的二硫键。The disulfide bond formed by the first cysteine and the third cysteine from the N-terminus of the polypeptide, and the disulfide bond formed by the second cysteine and the fourth cysteine .
在本发明的一个或多个实施方式中，所述的多肽，其中，所述多肽的羧基末端是酰胺化的。In one or more embodiments of the present invention, the polypeptide, wherein the carboxyl terminal of the polypeptide is amidated.
虽然野生型αO-芋螺毒素GeXIVA已经在本发明人之前的专利和文章中公开，但 很多研究表明，对于芋螺毒素肽，序列上一个氨基酸的差异就可能产生新的受体特异性、活性或选择性的变化，意味着在受体研究中提供了一种新的工具，在新药开发上提供了一种新的候选药物和先导药物 [31-35]。本发明的研究结果(表8－表22)更加证实了这个事实。 Although the wild-type αO-conotoxin GeXIVA has been disclosed in the inventors’ previous patents and articles, many studies have shown that for the conotoxin peptide, a difference in the sequence of one amino acid may produce a new receptor specificity and activity. Or selective changes mean that a new tool is provided in receptor research and a new drug candidate and lead drug in the development of new drugs [31-35] . The research results of the present invention (Table 8-Table 22) further confirm this fact.
本发明鉴定出了GeXIVA及其突变体与α9α10 nAChR之间相互作用的关键氨基酸(如表3，表12)，序列中的多个精氨酸对于与受体相互结合起着至关重要的作用，无论哪两个或两个以上的精氨酸同时被丙氨酸(Ala，A)取代，它们对α9α10 nAChR的阻断活性显著下降。大多数突变肽(SEQ ID NOs:52－61)对α9α10 nAChR的阻断活性至少下降10倍以上，其半阻断剂量(IC 50)均在130nM以上；有的突变肽(SEQ ID NOs:49－51)的阻断活性甚至全部丧失，其半阻断剂量(IC 50)均在10000nM以上，即在10μM高浓度下，这些突变体对α9α10 nAChR的电流阻断超不过50％，或者完全没有阻断作用。详见表12。 The present invention has identified the key amino acids that interact between GeXIVA and its mutants and α9α10 nAChR (such as Table 3, Table 12), and multiple arginines in the sequence play a crucial role in binding to the receptor. No matter which two or more arginines are replaced by alanine (Ala, A) at the same time, their blocking activity on α9α10 nAChR is significantly reduced. The blocking activity of most mutant peptides (SEQ ID NOs: 52-61) on α9α10 nAChR decreased by at least 10 times, and their half-blocking dose (IC 50 ) was above 130 nM; some mutant peptides (SEQ ID NOs: 49 The blocking activity of -51) is even completely lost, and the half-blocking dose (IC 50 ) is above 10000 nM, that is, at a high concentration of 10 μM, the current blocking of α9α10 nAChR by these mutants is not more than 50%, or none at all Blocking effect. See Table 12 for details.
GeXIVA[1,2](SEQ ID NO:1)和GeXIVA[1,4](SEQ ID NO:25)的丙氨酸扫描突变体(表1－2，SEQ ID NOs:2－24，26－48)，以及半胱氨酸替换突变体(表5，SEQ ID NOs:76－87)，除了[R22A]GeXIVA[1,4](SEQ ID NO:44)外(表9)，均保持了对α9α10 nAChR的强阻断活性，其半阻断剂量(IC 50)均在90nM以下，它们的活性变化不大，都在8倍以内(表8－9、表15)。[R22A]GeXIVA[1,4](SEQ ID NO:44)与野生型的GeXIVA[1,4](SEQ ID NO:25)相比，活性下降了约12倍，其IC 50为185nM(表8－9、表15)。其余各种各样的突变体对α9α10 nAChR的阻断活性，详见表13－14与表19－22。 GeXIVA[1,2] (SEQ ID NO:1) and GeXIVA[1,4] (SEQ ID NO:25) alanine scanning mutants (Table 1-2, SEQ ID NOs: 2-24, 26- 48), and cysteine substitution mutants (Table 5, SEQ ID NOs: 76-87), except for [R22A] GeXIVA[1, 4] (SEQ ID NO: 44) (Table 9), all maintained For the strong blocking activity of α9α10 nAChR, the half-blocking dose (IC 50 ) is below 90 nM, and their activity changes little, all within 8 times (Table 8-9, Table 15). [R22A] GeXIVA [1,4] ( SEQ ID NO: 44) and wild type GeXIVA [1,4] (SEQ ID NO : 25) compared to the activity decreased about 12-fold, an IC 50 of 185 nm (Table 8-9, Table 15). The blocking activities of the other various mutants on α9α10 nAChR are shown in Table 13-14 and Table 19-22.
特别是，发现了活性显著增强的([I23A]GeXIVA[1,4]，SEQ ID NO:45)、选择性明显提高的(SEQ ID NO:6，8，15，16，18，23)、稳定性大幅度提高的(Mf-GeXIVA，SEQ ID NO:92；GeFlex-GeXIVA，SEQ ID NO:94)、活性保持序列变短的合成成本更低的( △6-22GeXIVA，SEQ ID NO:62， △10-19[D5A]GeXIVA#，SEQ ID NO:75)，以及活性保持无二硫键的合成成本更低的([C2A,C9A,C20S,C27S]GeXIVA，SEQ ID NO:86)优化突变体等。 In particular, it was found that the activity was significantly enhanced ([I23A]GeXIVA[1,4], SEQ ID NO: 45), and the selectivity was significantly increased (SEQ ID NO: 6, 8, 15, 16, 18, 23), The stability is greatly improved (Mf-GeXIVA, SEQ ID NO: 92; GeFlex-GeXIVA, SEQ ID NO: 94), the activity-maintaining sequence is shortened and the synthesis cost is lower ( △6-22 GeXIVA, SEQ ID NO: 62, △10-19 [D5A]GeXIVA#, SEQ ID NO: 75), and a lower synthesis cost without disulfide bonds ([C2A, C9A, C20S, C27S] GeXIVA, SEQ ID NO: 86) Optimize mutants, etc.
所有这些研究结果，对于设计和研发针对α9α10 nAChR结构与功能研究的分子探针、以及新药研发都具有极高的应用价值。All these research results have extremely high application value for the design and development of molecular probes for the structure and function of α9α10 nAChR, and the development of new drugs.
本发明的再一方面涉及一种分离的融合蛋白，其包含至少一种本发明的多肽。Another aspect of the present invention relates to an isolated fusion protein comprising at least one polypeptide of the present invention.
本发明还包括在本发明αO-芋螺毒素突变肽的N-末端和/或C末端融合了其它肽/多肽的融合多肽或可裂解的融合多肽。产生融合多肽的技术为本领域内已知，包括连接编码本发明肽的编码序列与编码所述其它肽/多肽的编码序列，使它们在同一读框中，并且融合多肽的表达受控于相同的启动子和终止子。The present invention also includes fusion polypeptides or cleavable fusion polypeptides in which other peptides/polypeptides are fused to the N-terminus and/or C-terminus of the αO-conotoxin mutant peptide of the present invention. The technology for producing fusion polypeptides is known in the art, including linking the coding sequence of the peptide of the present invention with the coding sequence of the other peptide/polypeptide, so that they are in the same reading frame, and the expression of the fusion polypeptide is controlled by the same The promoter and terminator.
本发明的再一方面涉及一种分离的多核苷酸，其编码本发明中任一项所述的多肽。Another aspect of the present invention relates to an isolated polynucleotide, which encodes the polypeptide of any one of the present invention.
本发明的再一方面涉及一种核酸构建体，其含有本发明的多核苷酸；优选地，所述核酸构建体为重组载体；优选地，所述核酸构建体为重组表达载体。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.
本发明的再一方面涉及一种转化的细胞，其含有本发明的多核苷酸，或者本发明的核酸构建体。Another aspect of the present invention relates to a transformed cell, which contains the polynucleotide of the present invention, or the nucleic acid construct of the present invention.
本发明的再一方面涉及一种药物组合物，其含有至少一种本发明的多肽；可选地，其还包含药学上可接受的辅料。Another aspect of the present invention relates to a pharmaceutical composition, which contains at least one polypeptide of the present invention; optionally, it also contains pharmaceutically acceptable excipients.
所述药物组合物可用于研究、诊断、缓解或治疗与神经痛、癌症、智障、疼痛、帕金森症、精神病、抑郁、重症肌无力等有关的疾病或病症。在一个实施方案中，含有治疗有效量的本发明肽的药物组合物以利于药用的方式配制和给药，并需考虑到个体病人的临床状况、运送位点、给药方法、给药日程安排和医生已知的其它因素。因此用于本文目的的“有效量”由这些方面的考虑决定。The pharmaceutical composition can be used for research, diagnosis, alleviation or treatment of diseases or disorders related to neuralgia, cancer, intellectual disability, pain, Parkinson's disease, psychosis, depression, myasthenia gravis and the like. In one embodiment, the pharmaceutical composition containing a therapeutically effective amount of the peptide of the present invention is formulated and administered in a medicinal manner, taking into account the clinical condition, delivery site, administration method, and administration schedule of the individual patient. Arrangement and other factors known to the doctor. Therefore, the "effective amount" used for the purposes herein is determined by these considerations.
含治疗有效量的本发明多肽的药物组合物非肠道给药、口服、脑池内给药、鞘内给药等。“药学可接受载体”指无毒的固体、半固体或液体填充物、稀释液、胶囊材料或任何类型的配方辅助物。本文所用术语“非肠道的”表示的给药方式包括静脉内、肌肉内、腹膜内、胸骨内、皮下、鞘内和关节内注射和输注。本发明多肽还可通过缓释系统恰当地给药。The pharmaceutical composition containing a therapeutically effective amount of the polypeptide of the present invention is administered parenterally, orally, intracisternally, intrathecally, and the like. "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filling, diluent, capsule material or any type of formulation aid. The term "parenteral" as used herein refers to administration modes including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intrathecal and intraarticular injections and infusions. The polypeptide of the present invention can also be appropriately administered through a sustained-release system.
本发明的再一方面涉及本发明中任一项所述的多肽在制备阻断或抑制α9α10nAChR的药物中的用途。Another aspect of the present invention relates to the use of the polypeptide of any one of the present invention in the preparation of a medicine for blocking or inhibiting α9α10 nAChR.
现有技术研究表明，α9α10 nAChR是治疗神经痛(慢性痛)、乳腺癌、肺癌、宫颈癌、卵巢癌、癌症化疗、伤口愈合等的药物作用靶点。因此，本发明的新的αO-芋螺毒素GeXIVA突变体在上述疾病的机理研究、诊断、治疗方面具有极高的应用价值。Prior art studies have shown that α9α10 nAChR is a drug target for the treatment of neuralgia (chronic pain), breast cancer, lung cancer, cervical cancer, ovarian cancer, cancer chemotherapy, wound healing, etc. Therefore, the new αO-conotoxin GeXIVA mutant of the present invention has extremely high application value in the mechanism research, diagnosis, and treatment of the above-mentioned diseases.
本发明的再一方面涉及本发明中任一项所述的多肽在制备治疗和/或预防神经系统疾病或癌症的药物中的用途，在制备促进伤口愈合的药物中的用途，或者在制备杀灭害虫、镇痛、抗癌的药物中的用途；Another aspect of the present invention relates to the use of the polypeptide of any one of the present invention in the preparation of drugs for the treatment and/or prevention of neurological diseases or cancer, the use of the preparation of drugs for promoting wound healing, or the preparation of drugs for killing Use in insecticide, analgesic and anti-cancer drugs;
优选地，所述神经系统疾病为选自神经痛(慢性痛)、帕金森症、痴呆、精神分裂症和抑郁中的至少一种；Preferably, the neurological disease is at least one selected from neuralgia (chronic pain), Parkinson's disease, dementia, schizophrenia and depression;
优选地，所述神经痛选自如下的至少一种：坐骨神经痛、三叉神经痛、淋巴神经痛、多点运动神经痛、急性剧烈自发性神经痛、挤压神经痛以及复合神经痛；Preferably, the neuralgia is selected from at least one of the following: sciatica, trigeminal neuralgia, lymphatic neuralgia, multipoint motor neuralgia, acute severe spontaneous neuralgia, crush neuralgia and compound neuralgia;
优选地，所述神经痛由如下因素中的至少一种导致：癌症、癌症化疗、酒精中毒、糖尿病、硬化症、带状疱疹、机械伤、手术伤、艾滋病、头部神经瘫痪、药物中毒、工业污染中毒、骨髓瘤、慢性先天性感觉神经病、脉管炎、血管炎、局部缺血、尿毒症、儿童胆汁肝脏疾病、慢性呼吸障碍、多器官衰竭、脓毒病/脓血症、肝炎、卟啉症、维生素缺乏、慢性肝脏病、原生胆汁硬化、高血脂症、麻疯病、莱姆关节炎、感觉神经束膜炎或过敏症；Preferably, the neuralgia is caused by at least one of the following factors: cancer, cancer chemotherapy, alcoholism, diabetes, sclerosis, herpes zoster, mechanical injury, surgical injury, AIDS, head nerve paralysis, drug poisoning, Industrial pollution poisoning, myeloma, chronic congenital sensory neuropathy, vasculitis, vasculitis, ischemia, uremia, childhood bile liver disease, chronic respiratory disorders, multiple organ failure, sepsis/sepsemia, hepatitis, Porphyria, vitamin deficiency, chronic liver disease, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, perineuritis or allergies;
优选地，所述癌症为选自乳腺癌、肺癌、宫颈癌、卵巢癌、胰腺癌、白血病、神经细胞瘤和其它上皮细胞癌变引起的癌症中的至少一种。Preferably, the cancer is at least one selected from breast cancer, lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, leukemia, neurocytoma, and other cancers caused by epithelial cell carcinoma.
本发明的再一方面涉及一种在体内或体外阻断或抑制α9α10 nAChR或者调节乙酰胆碱水平的方法，包括给予受试者或者施加给细胞有效量的本发明中任一项所述的多肽的步骤。Another aspect of the present invention relates to a method for blocking or inhibiting α9α10 nAChR or regulating the level of acetylcholine in vivo or in vitro, comprising the step of administering to a subject or administering to a cell an effective amount of the polypeptide of any one of the present invention .
根据本发明中任一项所述的多肽，其用于阻断或抑制α9α10 nAChR。The polypeptide according to any one of the present invention is used to block or inhibit α9α10 nAChR.
根据本发明中任一项所述的多肽，其用于治疗和/或预防神经系统疾病或癌症，用于促进伤口愈合，或者用于杀灭害虫、镇痛、抗癌等；The polypeptide according to any one of the present invention is used to treat and/or prevent neurological diseases or cancer, to promote wound healing, or to kill pests, analgesia, anticancer, etc.;
优选地，所述神经系统疾病为选自神经痛、帕金森症、痴呆、精神分裂症和抑郁中的至少一种；Preferably, the nervous system disease is at least one selected from neuralgia, Parkinson's disease, dementia, schizophrenia and depression;
优选地，所述神经痛(慢性痛)选自如下的至少一种：坐骨神经痛、三叉神经痛、淋巴神经痛、多点运动神经痛、急性剧烈自发性神经痛、挤压神经痛以及复合神经痛；Preferably, the neuralgia (chronic pain) is selected from at least one of the following: sciatica, trigeminal neuralgia, lymphatic neuralgia, multipoint motor neuralgia, acute severe spontaneous neuralgia, crush neuralgia, and compound nerve pain;
优选地，所述神经痛由如下因素中的至少一种导致：癌症、癌症化疗、酒精中毒、糖尿病、硬化症、带状疱疹、机械伤、手术伤、艾滋病、头部神经瘫痪、药物中毒、工业污染中毒、骨髓瘤、慢性先天性感觉神经病、脉管炎、血管炎、局部缺血、尿毒 症、儿童胆汁肝脏疾病、慢性呼吸障碍、多器官衰竭、脓毒病/脓血症、肝炎、卟啉症、维生素缺乏、慢性肝脏病、原生胆汁硬化、高血脂症、麻疯病、莱姆关节炎、感觉神经束膜炎或过敏症；Preferably, the neuralgia is caused by at least one of the following factors: cancer, cancer chemotherapy, alcoholism, diabetes, sclerosis, herpes zoster, mechanical injury, surgical injury, AIDS, head nerve paralysis, drug poisoning, Industrial pollution poisoning, myeloma, chronic congenital sensory neuropathy, vasculitis, vasculitis, ischemia, uremia, childhood bile liver disease, chronic respiratory disorders, multiple organ failure, sepsis/sepsemia, hepatitis, Porphyria, vitamin deficiency, chronic liver disease, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, perineuritis or allergies;
优选地，所述癌症为选自乳腺癌、肺癌、宫颈癌、卵巢癌、胰腺癌、白血病、神经细胞瘤和其它上皮细胞癌变引起的癌症中的至少一种。Preferably, the cancer is at least one selected from breast cancer, lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, leukemia, neurocytoma, and other cancers caused by epithelial cell carcinoma.
本发明的再一方面涉及一种治疗和/或预防神经系统疾病或癌症的方法，一种促进伤口愈合的方法，或者一种杀灭害虫、镇痛、抗癌的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的多肽的步骤；Another aspect of the present invention relates to a method of treating and/or preventing neurological diseases or cancer, a method of promoting wound healing, or a method of killing pests, analgesic, and anti-cancer, including administering The step of subjecting an effective amount of the polypeptide of any one of the present invention;
优选地，所述神经系统疾病为选自神经痛、帕金森症、痴呆、精神分裂症和抑郁中的至少一种；Preferably, the neurological disease is at least one selected from neuralgia, Parkinson's disease, dementia, schizophrenia and depression;
优选地，所述神经痛(慢性痛)选自如下的至少一种：坐骨神经痛、三叉神经痛、淋巴神经痛、多点运动神经痛、急性剧烈自发性神经痛、挤压神经痛以及复合神经痛；Preferably, the neuralgia (chronic pain) is selected from at least one of the following: sciatica, trigeminal neuralgia, lymphatic neuralgia, multipoint motor neuralgia, acute severe spontaneous neuralgia, crush neuralgia, and compound nerve pain;
优选地，所述神经痛由如下因素中的至少一种导致：癌症、癌症化疗、酒精中毒、糖尿病、硬化症、带状疱疹、机械伤、手术伤、艾滋病、头部神经瘫痪、药物中毒、工业污染中毒、骨髓瘤、慢性先天性感觉神经病、脉管炎、血管炎、局部缺血、尿毒症、儿童胆汁肝脏疾病、慢性呼吸障碍、多器官衰竭、脓毒病/脓血症、肝炎、卟啉症、维生素缺乏、慢性肝脏病、原生胆汁硬化、高血脂症、麻疯病、莱姆关节炎、感觉神经束膜炎或过敏症；Preferably, the neuralgia is caused by at least one of the following factors: cancer, cancer chemotherapy, alcoholism, diabetes, sclerosis, shingles, mechanical injury, surgical injury, AIDS, cranial nerve paralysis, drug poisoning, Industrial pollution poisoning, myeloma, chronic congenital sensory neuropathy, vasculitis, vasculitis, ischemia, uremia, childhood bile liver disease, chronic respiratory disorders, multiple organ failure, sepsis/sepsemia, hepatitis, Porphyria, vitamin deficiency, chronic liver disease, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, perineuritis or allergies;
优选地，所述癌症为选自乳腺癌、肺癌、宫颈癌、卵巢癌、胰腺癌、白血病、神经细胞瘤和其它上皮细胞癌变引起的癌症等中的至少一种。Preferably, the cancer is at least one selected from breast cancer, lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, leukemia, neurocytoma and other cancers caused by epithelial cell carcinogenesis.
给药剂量取决于许多因素，例如所治疗病况的严重程度，患者或动物的性别、年龄、体重及个体反应，以及待治疗患者的病况和既往病史来选定。本领域通常的做法是，剂量从低于为得到所需治疗效果而要求的水平开始，逐渐增加剂量，直到得到所需的效果。The dosage depends on many factors, such as the severity of the condition to be treated, the gender, age, weight and individual response of the patient or animal, as well as the condition and past medical history of the patient to be treated. The usual practice in the art is to start the dosage from a level lower than the level required to obtain the desired therapeutic effect, and gradually increase the dosage until the desired effect is obtained.
本发明的多肽的制备方法，包括下述步骤：The preparation method of the polypeptide of the present invention includes the following steps:
1)通过多肽合成仪或者手工方法合成线性多肽，Fmoc氨基酸的侧链保护基为：Pmc(Arg)、But(Thr、Ser、Tyr)、OBut(Asp)、Boc(Lys)；半胱氨酸用Trt或Acm 保护基团；1) Synthesize linear peptides by peptide synthesizer or manual method. The side chain protecting groups of Fmoc amino acid are: Pmc (Arg), But (Thr, Ser, Tyr), OBut (Asp), Boc (Lys); Cysteine Use Trt or Acm protecting group;
2)将步骤1)中得到的线性多肽从树脂上切割下来，并用冰乙醚沉淀和洗涤回收线性多肽粗品，用制备型反向HPLC C18柱(Vydac)纯化；2) The linear polypeptide obtained in step 1) is cut from the resin, and the crude linear polypeptide is recovered by precipitation and washing with ice ether, and purified by a preparative reverse HPLC C18 column (Vydac);
3)将步骤2)中得到的产物进行纯化，或进行一步或两步氧化折叠后再纯化。3) Purify the product obtained in step 2), or perform one or two steps of oxidative folding before purification.
本发明中，In the present invention,
术语“核酸构建体”，在文中定义为单链或双链核酸分子，优选是指人工构建的核酸分子。可选地，所述核酸构建体还包含有可操作地连接的1个或多个调控序列。The term "nucleic acid construct" is defined herein as a single-stranded or double-stranded nucleic acid molecule, and preferably refers to an artificially constructed nucleic acid molecule. Optionally, the nucleic acid construct further contains one or more regulatory sequences that are operably linked.
在本发明中，术语“可操作地连接”是指两个或多个核苷酸区域或核酸序列的功能性的空间排列。所述“可操作地连接”可以通过基因重组的手段实现。In the present invention, the term "operably linked" refers to the functional spatial arrangement of two or more nucleotide regions or nucleic acid sequences. The "operably linked" can be achieved by means of gene recombination.
在本发明中，术语“载体”指的是，可将抑制某蛋白的多核苷酸插入其中的一种核酸运载工具。举例来说，载体包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件。In the present invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide that inhibits a certain protein can be inserted. For example, the vector includes: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or artificial chromosome (PAC) derived from P1; bacteriophage such as lambda phage or M13 phage And animal viruses. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papillary polyoma vacuoles Virus (such as SV40). A vector may contain multiple elements that control expression.
在本发明中，术语“宿主细胞”指的是导入载体的细胞，包括如下许多细胞类型，如大肠杆菌或枯草菌等原核细胞，如酵母细胞或曲霉菌等真菌细胞，如S2果蝇细胞或Sf9等昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞的动物细胞。In the present invention, the term "host cell" refers to a cell into which a 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, such as S2 fruit fly cells or Insect cells such as Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
术语“有效量”是指可在受试者中实现治疗、预防、减轻和/或缓解本发明所述疾病或病症的剂量。The term "effective amount" refers to a dose that can treat, prevent, alleviate and/or alleviate the disease or condition described in the present invention in a subject.
术语“疾病和/或病症”是指所述受试者的一种身体状态，该身体状态与本发明所述疾病和/或病症有关。The term "disease and/or disorder" refers to a physical state of the subject, which is related to the disease and/or disorder described in the present invention.
术语“受试者”可以指患者或者其它接受本发明药物组合物以治疗、预防、减轻和/或缓解本发明所述疾病或病症的动物，特别是哺乳动物，例如人、狗、猴、牛、马等。The term "subject" may refer to patients or other animals receiving the pharmaceutical composition of the present invention to treat, prevent, alleviate and/or alleviate the diseases or conditions of the present invention, especially mammals, such as humans, dogs, monkeys, cattle. , Horse, etc.
本发明中，如果没有特别说明，浓度单位μM表示μmol/L，mM表示mmol/L，nM表示nmol/L。In the present invention, unless otherwise specified, the concentration unit μM means μmol/L, mM means mmol/L, and nM means nmol/L.
本发明中，提到细胞中的加药量时，如果没有特别说明，一般是指加药后药物的终浓度。In the present invention, when the amount of drug added in the cell is mentioned, unless otherwise specified, it generally refers to the final concentration of the drug after drug addition.
本发明中提到术语“氨基酸”或者具体的氨基酸名称时，如果没有特别说明，是指L型的氨基酸。When referring to the term "amino acid" or specific amino acid name in the present invention, unless otherwise specified, it refers to an L-shaped amino acid.
发明的有益效果The beneficial effects of the invention
本发明对GeXIVA进行了各种各样的结构优化与改造，研究它们的构效关系，揭示它们与受体相互作用的分子机理，发现了一系列对α9α10 nAChRs保持了阻断活性的新型突变体。与野生型肽相比，有的突变体活性增强、有的突变体的选择性进一步提高，有的突变体氨基酸残基数更少，有的突变体合成成本更低等，这为新型工具药和相关疾病治疗药物的研发提供了更多的候选新药。The present invention carried out various structural optimizations and modifications to GeXIVA, studied their structure-activity relationship, revealed the molecular mechanism of their interaction with the receptor, and discovered a series of novel mutants that maintained blocking activity against α9α10 nAChRs . Compared with wild-type peptides, some mutants have enhanced activity, some mutants have further improved selectivity, some mutants have fewer amino acid residues, and some mutants have lower synthesis costs. This is a new tool drug And the research and development of drugs for the treatment of related diseases provides more new drug candidates.
附图说明Description of the drawings
图1：αO-芋螺毒素GeXIVA的氨基酸序列及其3种可能的二硫键异构体(isomer)，分别命名为GeXIVA[1,2](珠状异构体，bead isomer)、GeXIVA[1,4](带状异构体，ribbon isomer)、GeXIVA[1,3](球状异构体，globular isomer)。3种异构体的二硫键连接方式分别为：GeXIVA[1,2]为[C1-C2，C3-C4]；GeXIVA[1,4]为[C1-C4，C2-C3]；GeXIVA[1,3]为[C1-C3，C2-C4] [27]。 Figure 1: The amino acid sequence of αO-conotoxin GeXIVA and its three possible disulfide bond isomers, named GeXIVA[1,2] (bead isomer) and GeXIVA[ 1,4] (ribbon isomer), GeXIVA[1,3] (globular isomer). The disulfide bond connection modes of the three isomers are: GeXIVA[1,2] is [C1-C2, C3-C4]; GeXIVA[1,4] is [C1-C4, C2-C3]; GeXIVA[ 1,3] is [C1-C3, C2-C4] [27] .
图2A-2D：αO-芋螺毒素GeXIVA[1,2](Peptide 1，SEQ ID NO:1)及突变肽16(Peptide 16，SEQ ID NO:16，即第17位精氨酸被丙氨酸替换的突变体[R17A]GeXIVA[1,2])的超高压液相色谱图(UPLC)(2A、2C)与电喷雾质谱图(ESI-MS)(2B、2D)。(2A)Peptide 1的UPLC色谱图。(2B)Peptide 1的ESI-MS质谱图。(2C)Peptide 16的UPLC色谱图。(2D)Peptide 16的ESI-MS质谱图。Figure 2A-2D: αO-conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO: 1) and mutant peptide 16 (Peptide 16, SEQ ID NO: 16, that is, the 17th arginine is alanine Ultra-high pressure liquid chromatogram (UPLC) (2A, 2C) and electrospray mass spectrometry (ESI-MS) (2B, 2D) of the acid-replaced mutant [R17A]GeXIVA[1,2]). (2A) UPLC chromatogram of Peptide 1. (2B) ESI-MS mass spectrum of Peptide 1. (2C) UPLC chromatogram of Peptide 16. (2D) ESI-MS mass spectrum of Peptide 16.
UPLC分析的条件为：反向C18 Vydac分析柱；所用缓冲液A(Buffer A)为0.075％TFA的水溶液，缓冲液B(Buffer B)组成为0.05％TFA+90％乙腈(ACN)+10％水；线性洗脱梯度为0－3.5min内，buffer B从10％增加到40％。UPLC色谱图中的RT表示该色谱峰的保留时间。The conditions for UPLC analysis are: reverse C18 Vydac analytical column; buffer A (Buffer A) used is 0.075% TFA aqueous solution, and the composition of buffer B (Buffer B) is 0.05% TFA + 90% acetonitrile (ACN) + 10% Water: The linear elution gradient is 0-3.5min, and the buffer B increases from 10% to 40%. RT in the UPLC chromatogram represents the retention time of the chromatographic peak.
图3A-3B：αO-芋螺毒素GeXIVA[1,2](Peptide 1，SEQ ID NO:1)(3A)及其第17位精氨酸被丙氨酸替换的突变体[R17A]GeXIVA[1,2](Peptide 16，SEQ ID NO:16) 3(B)对大鼠α9α10，α7与小鼠肌肉型α1β1δε nAChRs的浓度反应曲线。曲线上每个点是3－10个蛙卵(非洲爪蟾卵母细胞，n＝3－10)的平均值±误差(mean±S.E.)。这两个肽对α9α10:α7和α9α10:α1β1δε的活性选择性分别为207倍和232倍。Figure 3A-3B: αO-conotoxin GeXIVA[1,2](Peptide 1, SEQ ID NO:1)(3A) and its mutant whose 17th arginine was replaced by alanine [R17A]GeXIVA[ 1,2](Peptide 16, SEQ ID NO: 16) 3(B) Concentration response curve of rat α9α10, α7 and mouse muscle α1β1δε nAChRs. Each point on the curve is the mean±error (mean±S.E.) of 3-10 frog eggs (Xenopus oocytes, n=3-10). The activity selectivity of these two peptides to α9α10:α7 and α9α10:α1β1δε were 207 times and 232 times, respectively.
图4A-4D：αO-芋螺毒素GeXIVA[1,4](Peptide 25，SEQ ID NO:25)及突变肽45(Peptide 45，SEQ ID NO:45，即第23位的异亮氨酸被丙氨酸替换的突变体[I23A]GeXIVA[1,2])的超高压液相色谱图(UPLC)(4A、4C)与电喷雾质谱图(ESI-MS)(4B、4D)。(4A)Peptide 25的UPLC色谱图。(4B)Peptide 25的ESI-MS质谱图。(4C)Peptide 45的UPLC色谱图。(4D)Peptide 45的ESI-MS质谱图。Figure 4A-4D: αO-conotoxin GeXIVA[1,4] (Peptide 25, SEQ ID NO: 25) and mutant peptide 45 (Peptide 45, SEQ ID NO: 45, that is, the isoleucine at position 23) Ultra-high pressure liquid chromatogram (UPLC) (4A, 4C) and electrospray mass spectrometry (ESI-MS) (4B, 4D) of the mutant [I23A]GeXIVA[1,2]) substituted with alanine (4A) UPLC chromatogram of Peptide 25. (4B) ESI-MS mass spectrum of Peptide 25. (4C) UPLC chromatogram of Peptide 45. (4D) ESI-MS mass spectrum of Peptide 45.
UPLC分析的条件为：反向C18 Vydac分析柱；所用缓冲液A(Buffer A)为0.075％TFA的水溶液，缓冲液B(Buffer B)组成为0.05％TFA+90％乙腈(ACN)+10％水；线性洗脱梯度为0-3.5min内，buffer B从10％增加到40％。UPLC色谱图中的RT表示该色谱峰的保留时间。The conditions for UPLC analysis are: reverse C18 Vydac analytical column; the buffer A (Buffer A) used is a 0.075% TFA aqueous solution, and the composition of the buffer B (Buffer B) is 0.05% TFA+90% acetonitrile (ACN)+10% Water: The linear elution gradient is 0-3.5min, and the buffer B increases from 10% to 40%. RT in the UPLC chromatogram represents the retention time of the chromatographic peak.
图5A-5B：αO-芋螺毒素GeXIVA[1,4](Peptide 25，SEQ ID NO:25)(5A)与突变肽45(Peptide 45，SEQ ID NO:45)(5B)对大鼠α9α10(rα9α10)nAChR的电流影响图。Figure 5A-5B: αO-conotoxin GeXIVA[1,4] (Peptide 25, SEQ ID NO: 25) (5A) and mutant peptide 45 (Peptide 45, SEQ ID NO: 45) (5B) for rat α9α10 (rα9α10) Current influence diagram of nAChR.
在非洲爪蟾卵母细胞中表达大鼠α9α10 nAChR，细胞膜的钳制电压为-70mV，每隔1分钟给1s(秒)的乙酰胆碱(Ach)脉冲。每个图中显示的是1个多肽在1个卵母细胞上的代表性电流轨迹。获得对照电流后，加入10nM的多肽，温育5min之后的第一个Ach脉冲电流即是该多肽对受体产生影响的电流轨迹，图上用箭头标示。然后对多肽进行洗脱，洗脱过程中对Ach脉冲产生的电流大小及其轨迹也被同时测定。图中的“C”表示ACh激发产生的对照电流。后面的图8A-8C、9A-9C、11A-11F、13A-13F、15A-15B、17A-17E、18A-18H以及19A-19H中的电流轨迹图中的标识，与此说明相同。Rat α9α10 nAChR is expressed in Xenopus oocytes, the clamping voltage of the cell membrane is -70mV, and acetylcholine (Ach) pulses are given for 1s (second) every 1 minute. Each figure shows a representative current trace of a polypeptide on an oocyte. After obtaining the control current, add 10 nM polypeptide, and the first Ach pulse current after 5 min of incubation is the current trajectory of the polypeptide's effect on the receptor, which is marked with an arrow on the figure. Then the polypeptide is eluted, and the magnitude and trajectory of the current generated by the Ach pulse during the elution process are also measured. The "C" in the figure represents the control current generated by ACh excitation. The markings in the current trace diagrams in the following Figures 8A-8C, 9A-9C, 11A-11F, 13A-13F, 15A-15B, 17A-17E, 18A-18H, and 19A-19H are the same as this description.
图6A-6B：αO-芋螺毒素GeXIVA[1,4](Peptide 25，SEQ ID NO:25)(6A)及其第23位被丙氨酸替换的突变体[I23A]GeXIVA[1,2](Peptide 45，SEQ ID NO:45)(6B)对大鼠α9α10，α7与小鼠α1β1δε nAChRs的浓度反应曲线。曲线上每个点是3-10个蛙卵(n＝3-10)的平均值±误差(mean±S.E.)。Figure 6A-6B: αO-conotoxin GeXIVA[1,4](Peptide 25, SEQ ID NO: 25)(6A) and its mutant with alanine at position 23 [I23A]GeXIVA[1,2 ](Peptide 45, SEQ ID NO: 45) (6B) Concentration response curve of rat α9α10, α7 and mouse α1β1δε nAChRs. Each point on the curve is the mean±error (mean±S.E.) of 3-10 frog eggs (n=3-10).
图7A-7J：αO-芋螺毒素GeXIVA[1,4](Peptide 25，SEQ ID NO:25，用虚线表示)及其第23位被丙氨酸替换的突变体[I23A]GeXIVA[1,2](Peptide 45，SEQ ID NO:45) 对大鼠α9α10，α7，α3β2，α3β4，α6/α3β4，α2β2，α4β2，α2β4，α4β2与小鼠α1β1δεnAChRs的浓度反应曲线。曲线上每个点是3-5个蛙卵(n＝3-10)的平均值±误差(mean±S.E.)。Figure 7A-7J: αO-conotoxin GeXIVA[1,4] (Peptide 25, SEQ ID NO: 25, represented by a dashed line) and its mutant with alanine at position 23 [I23A]GeXIVA[1, 2] (Peptide 45, SEQ ID NO: 45) The concentration response curve of rat α9α10, α7, α3β2, α3β4, α6/α3β4, α2β2, α4β2, α2β4, α4β2 and mouse α1β1δεnAChRs. Each point on the curve is the mean±error (mean±S.E.) of 3-5 frog eggs (n=3-10).
图8A-8C：αO-芋螺毒素GeXIVA[1,4](Peptide 25，SEQ ID NO:25)(8A)及其突变体[I23A]GeXIVA[1,2](Peptide 45，SEQ ID NO:45)(8B)对人类α9α10 nAChR的电流影响图；以及对人类α9α10 nAChR的浓度反应曲线(8C),曲线上每个点是4-6个蛙卵(n＝4-6)的平均值±误差(mean±S.E.)。Figure 8A-8C: αO-conotoxin GeXIVA[1,4] (Peptide 25, SEQ ID NO: 25) (8A) and its mutant [I23A] GeXIVA[1,2] (Peptide 45, SEQ ID NO: 45) (8B) Current influence graph on human α9α10 nAChR; and the concentration response curve (8C) of human α9α10 nAChR. Each point on the curve is the average value of 4-6 frog eggs (n=4-6) ± Error (mean±SE).
图9A-9C：αO-芋螺毒素GeXIVA[1,2](Peptide 1，SEQ ID NO:1)的部分精氨酸突变体，即SEQ ID NO:49、50、51对大鼠α9α10(rα9α10)nAChR的电流影响图。Figure 9A-9C: Part of arginine mutants of αO-conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO: 1), namely SEQ ID NO: 49, 50, 51 pair rat α9α10(rα9α10) ) Current influence diagram of nAChR.
在非洲爪蟾卵母细胞中表达大鼠α9α10 nAChR，细胞膜的钳制电压为-70mV，每隔1分钟给1s(秒)的乙酰胆碱(Ach)脉冲。每个图中显示的是1个多肽在1个卵母细胞上的代表性电流轨迹。获得对照电流后，加入10μM的多肽，温育5min之后的第一个Ach脉冲电流即是该多肽对受体产生影响的电流轨迹，图上用箭头标示。然后对多肽进行洗脱，洗脱过程中对Ach脉冲产生的电流大小及其轨迹也被同时测定。图中的“C”表示ACh激发产生的对照电流。Rat α9α10 nAChR is expressed in Xenopus oocytes, the clamping voltage of the cell membrane is -70mV, and acetylcholine (Ach) pulses are given for 1s (second) every 1 minute. Each figure shows a representative current trace of a polypeptide on an oocyte. After obtaining the control current, add 10μM polypeptide, and the first Ach pulse current after 5min incubation is the current trace of the polypeptide's effect on the receptor, which is marked with an arrow on the figure. Then the polypeptide is eluted, and the magnitude and trajectory of the current generated by the Ach pulse during the elution process are also measured. The "C" in the figure represents the control current generated by ACh excitation.
图10： △10-19GeXIVA(SEQ ID NO:63)及其丙氨酸扫描突变肽(SEQ ID NOs:64-73；表4)对大鼠α9α10 nAChR电流的影响。该柱状图显示各个多肽在10μM浓度下，对由Ach(乙酰胆碱)诱发的大鼠α9α10 nAChR电流反应百分数，每个柱子的长度代表3-8个(n＝3-8)非洲爪蟾卵母细胞电流的平均值±误差(mean±S.E.)。各个肽的电流与 △10-19GeXIVA(SEQ ID NO:63)的电流相比，进行了差异统计学分析，****表示概率p<0.0001，具有极显著差异。 FIG 10: △ 10-19 GeXIVA (SEQ ID NO: 63) peptide and alanine scanning mutagenesis (SEQ ID NOs: 64-73; Table 4) Effect of current α9α10 nAChR rats. The histogram shows the percentage of each peptide responding to rat α9α10 nAChR current induced by Ach (acetylcholine) at a concentration of 10μM. The length of each column represents 3-8 (n=3-8) Xenopus oocytes The mean ± error of the current (mean ± SE). The current of each peptide was compared with the current of △10-19 GeXIVA (SEQ ID NO: 63), and the difference was analyzed statistically. **** indicates the probability p<0.0001, which is extremely significant.
图11A-11F：αO-芋螺毒素GeXIVA[1,2](Peptide 1，SEQ ID NO:1)及其截短突变体 △6-22GeXIVA(SEQ ID NO:62)对大鼠(r)和人类(h)α9α10的电流影响(11A-11D)和浓度反应曲线(11E-11F)。 Figure 11A-11F: αO-conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO:1) and its truncated mutant △6-22 GeXIVA (SEQ ID NO:62) against rats (r) And human (h) α9α10 current influence (11A-11D) and concentration response curve (11E-11F).
(11A)GeXIVA[1,2]在10nM浓度下对rα9α10 nAChR的电流影响图。(11B) △6-22GeXIVA在10nM浓度下对rα9α10 nAChR的电流影响图。(11C)GeXIVA[1,2]在50nM浓度下对hα9α10 nAChR的电流影响图。(11D) △6-22GeXIVA在50nM浓度下对hα9α10 nAChR的电流影响图。(11E)GeXIVA[1,2]与 △6-22GeXIVA对大鼠α9α10的浓度反应曲线。(11F)GeXIVA[1,2]与 △6-22GeXIVA对人类α9α10的浓度反应 曲线。E-F曲线上每个点是4-12个蛙卵(n＝4-12)的平均值±误差(mean±S.E.)。 (11A) The graph of the influence of GeXIVA[1,2] on the current of rα9α10 nAChR at a concentration of 10 nM. (11B) △6-22 GeXIVA influences the current of rα9α10 nAChR at a concentration of 10 nM. (11C) GeXIVA[1,2] current influence graph of hα9α10 nAChR at a concentration of 50 nM. (11D) △6-22 GeXIVA influences the current of hα9α10 nAChR at a concentration of 50nM. (11E) Concentration response curve of GeXIVA[1,2] and △6-22 GeXIVA to rat α9α10. (11F) Concentration response curve of GeXIVA[1,2] and △6-22 GeXIVA to human α9α10. Each point on the EF curve is the mean±error (mean±SE) of 4-12 frog eggs (n=4-12).
图12：αO-芋螺毒素GeXIVA[1,2](Peptide 1，SEQ ID NO:1)(红色曲线)及其截短突变体SEQ ID NOs:63、74、68(黑色曲线)与SEQ ID NO:75(蓝色曲线)对大鼠α9α10 nAChR的浓度反应曲线。曲线上每个点是6-10个蛙卵(n＝6-10)的平均值±误差(mean±S.E.)。Figure 12: αO-Conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO:1) (red curve) and its truncated mutant SEQ ID NOs: 63, 74, 68 (black curve) and SEQ ID NO:75 (blue curve) concentration response curve of rat α9α10 nAChR. Each point on the curve is the mean±error (mean±S.E.) of 6-10 frog eggs (n=6-10).
图13A-13F：αO-芋螺毒素GeXIVA[1,2](Peptide 1，SEQ ID NO:1)及其截短突变体 △10-19[D5A]GeXIVA#(SEQ ID NO:75)对大鼠(r)和人类(h)α9α10的电流影响(13A-13D)和浓度反应曲线(13E-13F)。 Figure 13A-13F: αO-conotoxin GeXIVA [1,2] (Peptide 1, SEQ ID NO: 1) and its truncated mutant △10-19 [D5A] GeXIVA# (SEQ ID NO: 75) Mouse (r) and human (h) α9α10 current influence (13A-13D) and concentration response curve (13E-13F).
(13A)GeXIVA[1,2]在10nM浓度下对rα9α10 nAChR的电流影响图。(13B) △10-19[D5A]GeXIVA#在10nM浓度下对rα9α10 nAChR的电流影响图。(13C)GeXIVA[1,2]在50nM浓度下对hα9α10 nAChR的电流影响图。(13D) △10-19[D5A]GeXIVA#在50nM浓度下对hα9α10 nAChR的电流影响图。(13E)GeXIVA[1,2]与 △10-19[D5A]GeXIVA#对大鼠α9α10的浓度反应曲线。(13F)GeXIVA[1,2]与 △10-19[D5A]GeXIVA#对人类α9α10的浓度反应曲线。E-F曲线上每个点是4-6个蛙卵(n＝4-6)的平均值±误差(mean±S.E.)。 (13A) GeXIVA[1,2] current influence graph of rα9α10 nAChR at a concentration of 10 nM. (13B) △10-19 [D5A] GeXIVA# current influence graph of rα9α10 nAChR at a concentration of 10nM. (13C) GeXIVA[1,2] current influence graph of hα9α10 nAChR at a concentration of 50nM. (13D) △10-19 [D5A] GeXIVA# affects the current of hα9α10 nAChR at a concentration of 50nM. (13E) Concentration response curve of GeXIVA[1,2] and △10-19 [D5A]GeXIVA# to rat α9α10. (13F) Concentration response curve of GeXIVA[1,2] and △10-19 [D5A]GeXIVA# to human α9α10. Each point on the EF curve is the mean±error (mean±SE) of 4-6 frog eggs (n=4-6).
图14A-14D：αO-芋螺毒素GeXIVA[1,2](Peptide 1，SEQ ID NO:1)及突变肽[C2A,C9A,C20S,C27S]GeXIVA(SEQ ID NO:86，表5)的超高压液相色谱图(UPLC)(14A、14C)与电喷雾质谱图(ESI-MS)(14B、14D)。(14A)Peptide 1的UPLC色谱图。(14B)Peptide 1的ESI-MS质谱图，其实测分子量为3452.70Da。(14C)SEQ ID NO:86的UPLC色谱图。(14D)SEQ ID NO:86的ESI-MS质谱图，其实测分子量为3360.96Da。Figure 14A-14D: αO-conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO: 1) and mutant peptide [C2A, C9A, C20S, C27S] GeXIVA (SEQ ID NO: 86, Table 5) Ultra high pressure liquid chromatography (UPLC) (14A, 14C) and electrospray mass spectrometry (ESI-MS) (14B, 14D). (14A) UPLC chromatogram of Peptide 1. (14B) The ESI-MS mass spectrum of Peptide 1, the actual molecular weight is 3,452.70 Da. (14C) UPLC chromatogram of SEQ ID NO: 86. (14D) The ESI-MS mass spectrum of SEQ ID NO: 86, the actual molecular weight is 3360.96 Da.
UPLC分析的条件为：反向C18 Vydac分析柱；所用缓冲液A(Buffer A)为0.075％TFA的水溶液，缓冲液B(Buffer B)组成为0.05％TFA+90％乙腈(ACN)+10％水；线性洗脱梯度为0-3.5min内，buffer B从10％增加到40％。UPLC色谱图中的RT表示该色谱峰的保留时间。The conditions for UPLC analysis are: reverse C18 Vydac analytical column; the buffer A (Buffer A) used is a 0.075% TFA aqueous solution, and the composition of the buffer B (Buffer B) is 0.05% TFA+90% acetonitrile (ACN)+10% Water: The linear elution gradient is 0-3.5min, and the buffer B increases from 10% to 40%. RT in the UPLC chromatogram represents the retention time of the chromatographic peak.
图15A-15B：αO-芋螺毒素GeXIVA[1,2](Peptide 1，SEQ ID NO:1)及突变肽[C2A,C9A,C20S,C27S]GeXIVA(SEQ ID NO:86，表5)在10nM的浓度下，对大鼠α9α10 nAChR的电流影响图。Figure 15A-15B: αO-conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO: 1) and mutant peptide [C2A, C9A, C20S, C27S] GeXIVA (SEQ ID NO: 86, Table 5) The graph of the influence on the current of rat α9α10 nAChR at a concentration of 10 nM.
图16A-16H：αO-芋螺毒素GeXIVA[1,2](Peptide 1，SEQ ID NO:1，用虚线表示) 及突变肽[C2A,C9A,C20S,C27S]GeXIVA(SEQ ID NO:86，表5)对大鼠各个神经型与小鼠肌肉型nAChRs的浓度反应曲线。曲线上每个点是4-12个蛙卵(n＝4-12)的平均值±误差(mean±S.E.)。Figure 16A-16H: αO-conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO: 1, represented by a dashed line) and mutant peptide [C2A, C9A, C20S, C27S] GeXIVA (SEQ ID NO: 86, Table 5) Concentration response curves of nAChRs for each neurotype in rats and muscle type in mice. Each point on the curve is the mean±error (mean±S.E.) of 4-12 frog eggs (n=4-12).
图17A-17E：αO-芋螺毒素GeXIVA[1,2](Peptide 1，SEQ ID NO:1)与突变肽[C2A,C9A,C20S,C27S]GeXIVA(SEQ ID NO:86，表5)对人类α9α10 nAChR的电流影响图(17A-D)，以及浓度反应曲线(17E)。(17A)50nM的GeXIVA[1,2]对人类α9α10 nAChR的电流影响图。(17B)50nM的[C2A,C9A,C20S,C27S]GeXIVA对人类α9α10 nAChR的电流影响图。(17C)100nM的GeXIVA[1,2]对人类α9α10 nAChR的电流影响图.(17D)100nM的[C2A,C9A,C20S,C27S]GeXIVA对人类α9α10 nAChR的电流影响图。(17E)浓度反应曲线上每个点是4-6个蛙卵(n＝4-6)的平均值±误差(mean±S.E.)。Figure 17A-17E: αO-conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO: 1) and mutant peptide [C2A, C9A, C20S, C27S] GeXIVA (SEQ ID NO: 86, Table 5) pair Human α9α10 nAChR current effect diagram (17A-D), and concentration response curve (17E). (17A) 50nM GeXIVA[1,2] current effect graph on human α9α10 nAChR. (17B) 50nM [C2A, C9A, C20S, C27S] GeXIVA current effect graph on human α9α10 nAChR. (17C) Graph of the influence of 100nM GeXIVA[1,2] on the current of human α9α10 nAChR. (17D) Graph of the influence of 100nM GeXIVA[C2A,C9A,C20S,C27S] on the current of human α9α10 nAChR. (17E) Each point on the concentration response curve is the mean±error (mean±S.E.) of 4-6 frog eggs (n=4-6).
图18A-18H：αO-芋螺毒素GeXIVA[1,2](Peptide 1，SEQ ID NO:1，18A)及其D-型氨基酸突变体(表6)，即1t-GeXIVA(SEQ ID NO:88，18B)、28v-GeXIVA(SEQ ID NO:89，18C)、1t,28v-GeXIVA(SEQ ID NO:90，18D)、Mt-GeXIVA(SEQ ID NO:91，18E)、Mf-GeXIVA(SEQ ID NO:92，18F)、GeArg-GeXIVA(SEQ ID NO:93，18G)、GeFlex-GeXIVA(SEQ ID NO:94，18H)，在1μM或100nM浓度下，对大鼠α9α10(rα9α10)nAChR的电流影响图。Figure 18A-18H: αO-conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO: 1, 18A) and its D-type amino acid mutant (Table 6), namely 1t-GeXIVA (SEQ ID NO: 88, 18B), 28v-GeXIVA (SEQ ID NO: 89, 18C), 1t, 28v-GeXIVA (SEQ ID NO: 90, 18D), Mt-GeXIVA (SEQ ID NO: 91, 18E), Mf-GeXIVA ( SEQ ID NO: 92, 18F), GeArg-GeXIVA (SEQ ID NO: 93, 18G), GeFlex-GeXIVA (SEQ ID NO: 94, 18H), at a concentration of 1 μM or 100 nM, for rat α9α10(rα9α10)nAChR The current influence diagram.
图19A-19H：αO-芋螺毒素GeXIVA[1,2](Peptide 1，SEQ ID NO:1，19A)及其D-型氨基酸突变体(表6)，即1t-GeXIVA(SEQ ID NO:88，19B)、28v-GeXIVA(SEQ ID NO:89，19C)、1t,28v-GeXIVA(SEQ ID NO:90,19D)、Mt-GeXIVA(SEQ ID NO:91，19E)、Mf-GeXIVA(SEQ ID NO:92，19F)、GeArg-GeXIVA(SEQ ID NO:93，19G)、GeFlex-GeXIVA(SEQ ID NO:94，19H)，在1μM或100nM浓度下，对人类α9α10(hα9α10)nAChR的电流影响图。Figure 19A-19H: αO-Conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO: 1, 19A) and its D-type amino acid mutant (Table 6), namely 1t-GeXIVA (SEQ ID NO: 88, 19B), 28v-GeXIVA (SEQ ID NO: 89, 19C), 1t, 28v-GeXIVA (SEQ ID NO: 90, 19D), Mt-GeXIVA (SEQ ID NO: 91, 19E), Mf-GeXIVA ( SEQ ID NO: 92, 19F), GeArg-GeXIVA (SEQ ID NO: 93, 19G), GeFlex-GeXIVA (SEQ ID NO: 94, 19H), at a concentration of 1 μM or 100 nM, it is effective against human α9α10(hα9α10) nAChR Current influence diagram.
图20A-20B：αO-芋螺毒素GeXIVA[1,2](Peptide 1，SEQ ID NO:1)及其D-型氨基酸突变体(表6，SEQ ID NO:88-94)对大鼠α9α10 nAChRs的浓度反应曲线。曲线上每个点是6-16个蛙卵(n＝6-16)的平均值±误差(mean±S.E.)。Figure 20A-20B: αO-conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO: 1) and its D-type amino acid mutant (Table 6, SEQ ID NO: 88-94) to rat α9α10 Concentration response curve of nAChRs. Each point on the curve is the mean±error (mean±S.E.) of 6-16 frog eggs (n=6-16).
图21A-21E：αO-芋螺毒素GeXIVA[1,2](Peptide 1，SEQ ID NO:1)及其D-型氨基酸取代突变体(SEQ ID NO:93 GeArg-GeXIVA，94 GeFlex-GeXIVA，91 GeMT＝Mt-GeXIVA，92 GeMF＝Mf-GeXIVA；表6)在100％人类血清中的稳定性。 图中横坐标表示该多肽在血清中温育的时间(分钟或小时)；纵坐标表示该多肽在某个时间点血清中剩余的百分数。其中Geflex的半衰期从母体肽GeXIVA[1,2]的40分钟增加到8小时。Figure 21A-21E: αO-conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO: 1) and its D-type amino acid substitution mutant (SEQ ID NO: 93 GeArg-GeXIVA, 94 GeFlex-GeXIVA, 91 GeMT=Mt-GeXIVA, 92 GeMF=Mf-GeXIVA; Table 6) Stability in 100% human serum. The abscissa in the figure represents the time (minutes or hours) of the polypeptide incubation in the serum; the ordinate represents the percentage of the polypeptide remaining in the serum at a certain time point. The half-life of Geflex increases from 40 minutes of the parent peptide GeXIVA [1,2] to 8 hours.
图22：αO-芋螺毒素GeXIVA[1,2](Peptide 1，SEQ ID NO:1，黑色曲线)及其D-型氨基酸取代突变体GeFlex-GeXIVA(SEQ ID NO:94，红色曲线)在人工模拟肠液中的稳定性。图中横坐标表示该多肽在人工模拟肠液中温育的时间(分钟)；纵坐标表示该多肽在某个时间点剩余的百分数。Figure 22: αO-conotoxin GeXIVA[1,2] (Peptide 1, SEQ ID NO: 1, black curve) and its D-type amino acid substitution mutant GeFlex-GeXIVA (SEQ ID NO: 94, red curve) Artificially simulate the stability in intestinal juice. The abscissa in the figure represents the time (minutes) the polypeptide is incubated in artificial intestinal fluid; the ordinate represents the percentage of the polypeptide remaining at a certain time point.
图23A-23B：αO-芋螺毒素GeXIVA[1,4](SEQ ID NO:25)及其部分天冬氨酸扫描突变体(表7；SEQ ID NOs:95-101，110-115)在10μM(23A)或1μM(23B)的浓度下，对大鼠α9α10 nAChR的电流反应百分数柱状图(n＝3)。图中ND96是空白对照，其电流的大小定义为100％，其余多肽产生的电流与对照电流的百分比，即为电流反应百分数(％Response)。Figure 23A-23B: αO-conotoxin GeXIVA[1,4] (SEQ ID NO: 25) and some of its aspartic acid scanning mutants (Table 7; SEQ ID NOs: 95-101, 110-115) Bar graph (n=3) of the current response percentage to rat α9α10 nAChR at a concentration of 10μM (23A) or 1μM (23B). In the figure, ND96 is a blank control, and its current is defined as 100%. The percentage of the current generated by the remaining polypeptides to the control current is the current response percentage (%Response).
图24A-24H：αO-芋螺毒素GeXIVA[1,4](SEQ ID NO:25)及其部分天冬氨酸扫描突变体(表7；SEQ ID NOs:95，100，110-115)对大鼠α9α10 nAChR的浓度反应曲线图。曲线上每个点是3-4个蛙卵(n＝3-4)的平均值±误差(mean±S.E.)。Figure 24A-24H: αO-conotoxin GeXIVA[1,4] (SEQ ID NO: 25) and some of its aspartic acid scanning mutants (Table 7; SEQ ID NOs: 95, 100, 110-115) pair Concentration response curve of rat α9α10 nAChR. Each point on the curve is the mean±error (mean±S.E.) of 3-4 frog eggs (n=3-4).
具体实施方式Detailed ways
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The embodiments of the present invention will be described in detail below in conjunction with examples. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the examples, follow the techniques or conditions described in the literature in the field (for example, refer to the "Molecular Cloning Experiment Guide" translated by J. Sambrook et al., Huang Peitang et al., third edition, Science Press) or follow the product manual. The reagents or instruments used without the manufacturer's indication are all conventional products that are commercially available.
实施例1：αO-芋螺毒素GeXIVA新突变体的序列设计及其人工合成Example 1: Sequence design and artificial synthesis of a new mutant of αO-conotoxin GeXIVA
在αO-芋螺毒素GeXIVA氨基酸序列的基础上(图1，表1-2中的SEQ ID NOs:1与25)，本发明人创造性地设计了一系列新的多肽突变体，其氨基酸序列如表1-7中的SEQ ID NOs:2-24，26-117所示。On the basis of the amino acid sequence of αO-conotoxin GeXIVA (Figure 1, SEQ ID NOs: 1 and 25 in Table 1-2), the inventors creatively designed a series of new polypeptide mutants whose amino acid sequence is as follows: SEQ ID NOs: 2-24, 26-117 in Table 1-7 are shown.
采用Fmoc方法人工合成表1-7中所列多肽的线性肽。具体方法如下：The Fmoc method was used to artificially synthesize the linear peptides of the polypeptides listed in Table 1-7. The specific method is as follows:
树脂肽采用Fmoc化学方法进行人工合成，可用多肽合成仪或手工合成法合成树 脂肽。除了半胱氨酸外，其余氨基酸用标准的侧链保护基团。对于含有4个半胱氨酸(Cys)的多肽，若其二硫键连接方式为[C1-C4，C2-C3]，则其第2和第3个半胱氨酸(Cys)的-SH用Trt(S-trityl)保护，第1和第4个半胱氨酸的-SH用Acm(S-acetamidomethyl)成对保护；若其二硫键连接方式为[C1-C2，C3-C4]，则其第1和第2个半胱氨酸的-SH用Trt(S-trityl)保护，第2和第4个半胱氨酸的-SH用Acm(S-acetamidomethyl)成对保护；以此类推。对于含有2个半胱氨酸(Cys)的多肽，则其两个半胱氨酸的-SH均用Trt(S-trityl)保护。The resin peptide is artificially synthesized by Fmoc chemical method, and the resin peptide can be synthesized by a peptide synthesizer or manual synthesis method. Except for cysteine, standard side chain protecting groups are used for other amino acids. For polypeptides containing 4 cysteines (Cys), if the disulfide bond connection mode is [C1-C4, C2-C3], the second and third cysteine (Cys) -SH Use Trt(S-trityl) to protect, the -SH of the first and fourth cysteine is protected by Acm(S-acetamidomethyl) in pairs; if the disulfide bond connection mode is [C1-C2, C3-C4] , The -SH of the first and second cysteines are protected by Trt (S-trityl), and the -SH of the second and fourth cysteines are protected by Acm (S-acetamidomethyl) in pairs; And so on. For polypeptides containing two cysteines (Cys), the -SH of the two cysteines are protected by Trt (S-trityl).
各个线性肽(没形成二硫键的肽)的合成步骤为：采用固相合成法中的Fmoc与FastMoc方法，在ABI Prism 433a多肽合成仪上合成了线性肽。Fmoc氨基酸的侧链保护基为:Pmc(Arg)、Trt(Cys)、But(Thr、Ser、Tyr)、OBut(Asp)、Boc(Lys).采用Fmoc HOBT DCC方法，Rink酰胺化树脂及Fmoc氨基酸，合成步骤参考仪器合成手册进行。为反应完全，在哌啶脱保护及偶合时间上分别适当延长，对难接氨基酸采用双偶合，获得树脂肽。用reagent K(trifluoroacetic acid/water/ethanedithiol/phenol/thioanisole；90:5:2.5:7.5:5，v/v/v/v/v)将线性肽从树脂上切割下来，并用冰乙醚沉淀和洗涤回收线性肽粗品，The synthesis steps of each linear peptide (peptide that does not form disulfide bonds) are: using the Fmoc and FastMoc methods in the solid phase synthesis method, the linear peptide is synthesized on the ABI Prism 433a peptide synthesizer. The side chain protecting groups of Fmoc amino acids are: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp), Boc (Lys). Using Fmoc HOBT DCC method, Rink amidation resin and Fmoc For amino acids, refer to the instrument synthesis manual for the synthesis steps. In order to complete the reaction, the piperidine deprotection and coupling time were appropriately extended, and the hard-to-connect amino acids were double-coupled to obtain the resin peptide. Cut the linear peptide from the resin with reagent K (trifluoroacetic acid/water/ethanedithiol/phenol/thioanisole; 90:5:2.5:7.5:5, v/v/v/v/v), and precipitate and wash with ice ether Recover crude linear peptides,
用制备型反向HPLC C18柱(Vydac)纯化每个线性肽，其纯度达95％以上。对含有二硫键的多肽继续用于氧化折叠。Each linear peptide was purified with a preparative reverse HPLC C18 column (Vydac) with a purity of more than 95%. Continue to use oxidative folding for polypeptides containing disulfide bonds.
参照文献 [27，36]，对上述线性肽进行一步或两步氧化折叠反应，过程简述如下: With reference to the literature [27 , 36] , one or two-step oxidative folding reaction is performed on the above linear peptide. The process is briefly described as follows:
首先通过铁氰化钾氧化法(20mM potassium ferricyanide，0.1M Tris，pH 7.5，30min)在Trt保护基团的两个半胱氨酸之间形成第一对二硫键；或用0.1M NH 4HCO 3，pH 8.0-8.2的缓冲液进行空气氧化，搅拌过夜，形成第一对二硫键。含有一对二硫键的多肽称为单环肽，纯化后可直接使用或根据需要进行第二步碘氧化，形成第二对二硫键。 Firstly, the first pair of disulfide bonds are formed between the two cysteines of the Trt protecting group by potassium ferricyanide (20mM potassium ferricyanide, 0.1M Tris, pH 7.5, 30min); or 0.1M NH 4 HCO 3 , pH 8.0-8.2 buffer solution is air-oxidized and stirred overnight to form the first pair of disulfide bonds. Polypeptides containing a pair of disulfide bonds are called monocyclic peptides. After purification, they can be used directly or subjected to the second step of iodine oxidation as needed to form a second pair of disulfide bonds.
对于含有两对二硫键的多肽，经过上述第一步氧化形成的单环肽，经反相HPLC C18柱(Vydac)纯化后，进行碘氧化(10mM iodine in H 2O:trifluoroacetic acid:acetonitrile(75:3:25 by volume，15min)，移去另外2个半胱氨酸上的Acm，同时在这2个半胱氨酸之间形成第二对二硫键。二环肽再经反相HPLC C18柱(Vydac)纯化。HPLC洗脱线性梯度为在0－60min内10％-35％buffer B(B90)。Buffer A是0.1％的TFA(三氟乙酸)水溶液，Buffer B是0.05％TFA，90％CAN(乙腈)。 紫外吸收值分析在214nm波长下进行。即获得按照从N端至C端的顺序在相应的半胱氨酸之间定向形成二硫键的芋螺毒素GeXIVA突变体。 For polypeptides containing two pairs of disulfide bonds, the monocyclic peptides formed by the above-mentioned first step oxidation are purified by reversed-phase HPLC C18 column (Vydac) and then subjected to iodine oxidation (10mM iodine in H 2 O:trifluoroacetic acid:acetonitrile( 75:3:25 by volume, 15min), remove the Acm on the other two cysteines, and at the same time form a second pair of disulfide bonds between the two cysteines. The bicyclic peptide is reversed again HPLC C18 column (Vydac) purification. HPLC elution linear gradient is 10%-35% buffer B (B90) within 0-60min. Buffer A is 0.1% TFA (trifluoroacetic acid) aqueous solution, Buffer B is 0.05% TFA , 90% CAN (acetonitrile). Ultraviolet absorption value analysis was carried out at a wavelength of 214nm. That is, the conotoxin GeXIVA mutant was obtained which formed disulfide bonds between the corresponding cysteines in the order from N-terminal to C-terminal.
经氧化折叠且纯化后的多肽，用超高压液相色谱图(UPLC)进行纯度鉴定，用质谱(ESI-MS)鉴定其分子量。UPLC分析的条件为：反向C18 Vydac分析柱；所用缓冲液A(Buffer A)为0.075％TFA的水溶液，缓冲液B(Buffer B)组成为0.05％TFA+90％乙腈(ACN)+10％水；线性洗脱梯度为0-3.5min内，buffer B从10％增加到40％。UPLC色谱图中的RT表示该色谱峰的保留时间。The oxidatively folded and purified polypeptide was identified by ultra high pressure liquid chromatography (UPLC) for purity identification, and mass spectrometry (ESI-MS) was used for identification of its molecular weight. The conditions for UPLC analysis are: reverse C18 Vydac analytical column; the buffer A (Buffer A) used is a 0.075% TFA aqueous solution, and the composition of the buffer B (Buffer B) is 0.05% TFA+90% acetonitrile (ACN)+10% Water: The linear elution gradient is 0-3.5min, and the buffer B increases from 10% to 40%. RT in the UPLC chromatogram represents the retention time of the chromatographic peak.
纯度达到95％以上，质谱测定分子量与其理论分子量一致的多肽，即为合成成功。例如，图2A-2D显示了αO-芋螺毒素GeXIVA[1,2](SEQ ID NO:1)及突变肽16(SEQ ID NO:16，即第17位精氨酸被丙氨酸替换的突变体[R17A]GeXIVA[1,2])的超高压液相色谱图(UPLC)(图2A、2C)与电喷雾质谱图(ESI-MS)(图2B、2D)。这两个肽氧化折叠后的理论分子量与它们的测定分子量完全一致，说明所合成的肽是正确的。The purity reaches more than 95%, and the peptide whose molecular weight is consistent with its theoretical molecular weight determined by mass spectrometry is considered to be successfully synthesized. For example, Figures 2A-2D show αO-conotoxin GeXIVA[1,2] (SEQ ID NO:1) and mutant peptide 16 (SEQ ID NO:16, that is, the 17th arginine is replaced by alanine Ultra-high pressure liquid chromatogram (UPLC) (Figure 2A, 2C) and electrospray mass spectrometry (ESI-MS) (Figure 2B, 2D) of the mutant [R17A]GeXIVA[1,2]). The theoretical molecular weights of these two peptides after oxidative folding are completely consistent with their measured molecular weights, indicating that the synthesized peptides are correct.
经过上述步骤，表1-7中所列的117个多肽全部正确合成，并形成了二硫键连接方式正确的氧化折叠肽。均可用于后续的活性研究。多肽浓度用280nm波长下比色测定，根据Beer-Lambert方程(equation)计算多肽浓度和质量，用于下面实施例中的各项实验。After the above steps, all the 117 polypeptides listed in Table 1-7 were synthesized correctly, and an oxidatively folded peptide with the correct disulfide bond connection mode was formed. Both can be used for subsequent activity studies. The polypeptide concentration was measured colorimetrically at a wavelength of 280 nm, and the polypeptide concentration and mass were calculated according to the Beer-Lambert equation (equation), which was used in the experiments in the following examples.
表1－表7中， a表示各个突变体的突变位点，用下划线标出。 In Table 1-7, a represents the mutation site of each mutant, which is marked with an underline.
表1：αO-芋螺毒素GeXIVA[1,2]及其丙氨酸扫描突变体的序列，其二硫键连接方式为[C1-C2，C3-C4]Table 1: The sequence of αO-conotoxin GeXIVA[1,2] and its alanine scanning mutant, the disulfide bond connection mode is [C1-C2, C3-C4]
SEQ ID NO:SEQ ID NO: 多肽名称Peptide name 氨基酸序列 a Amino acid sequence a
11 GeXIVA[1,2]GeXIVA[1,2] TCRSSGRYCRSPYDRRRRYCRRITDACVTCRSSGRYCRSPYDRRRRYCRRITDACV
22 [T1A]GeXIVA[1,2][T1A]GeXIVA[1,2] ACRSSGRYCRSPYDRRRRYCRRITDACV A CRSSGRYCRSPYDRRRRYCRRITDACV
33 [R3A]GeXIVA[1,2][R3A]GeXIVA[1,2] TC ASSGRYCRSPYDRRRRYCRRITDACV TC A SSGRYCRSPYDRRRRYCRRITDACV
44 [S4A]GeXIVA[1,2][S4A]GeXIVA[1,2] TCR ASGRYCRSPYDRRRRYCRRITDACV TCR A SGRYCRSPYDRRRRYCRRITDACV
55 [S5A]GeXIVA[1,2][S5A]GeXIVA[1,2] TCRS AGRYCRSPYDRRRRYCRRITDACV TCRS A GRYCRSPYDRRRRYCRRITDACV
66 [G6A]GeXIVA[1,2][G6A]GeXIVA[1,2] TCRSS ARYCRSPYDRRRRYCRRITDACV TCRSS A RYCRSPYDRRRRYCRRITDACV
77 [R7A]GeXIVA[1,2][R7A]GeXIVA[1,2] TCRSSG AYCRSPYDRRRRYCRRITDACV TCRSSG A YCRSPYDRRRRYCRRITDACV
88 [Y8A]GeXIVA[1,2][Y8A]GeXIVA[1,2] TCRSSGR ACRSPYDRRRRYCRRITDACV TCRSSGR A CRSPYDRRRRYCRRITDACV
99 [R10A]GeXIVA[1,2][R10A]GeXIVA[1,2] TCRSSGRYC ASPYDRRRRYCRRITDACV TCRSSGRYC A SPYDRRRRYCRRITDACV
1010 [S11A]GeXIVA[1,2][S11A]GeXIVA[1,2] TCRSSGRYCR APYDRRRRYCRRITDACV TCRSSGRYCR A PYDRRRRYCRRITDACV
1111 [P12A]GeXIVA[1,2][P12A]GeXIVA[1,2] TCRSSGRYCRS AYDRRRRYCRRITDACV TCRSSGRYCRS A YDRRRRYCRRITDACV
1212 [Y13A]GeXIVA[1,2][Y13A]GeXIVA[1,2] TCRSSGRYCRSP ADRRRRYCRRITDACV TCRSSGRYCRSP A DRRRRYCRRITDACV
1313 [D14A]GeXIVA[1,2][D14A]GeXIVA[1,2] TCRSSGRYCRSPY ARRRRYCRRITDACV TCRSSGRYCRSPY A RRRRYCRRITDACV
1414 [R15A]GeXIVA[1,2][R15A]GeXIVA[1,2] TCRSSGRYCRSPYD ARRRYCRRITDACV TCRSSGRYCRSPYD A RRRYCRRITDACV
1515 [R16A]GeXIVA[1,2][R16A]GeXIVA[1,2] TCRSSGRYCRSPYDR ARRYCRRITDACV TCRSSGRYCRSPYDR A RRYCRRITDACV
1616 [R17A]GeXIVA[1,2][R17A]GeXIVA[1,2] TCRSSGRYCRSPYDRR ARYCRRITDACV TCRSSGRYCRSPYDRR A RYCRRITDACV
1717 [R18A]GeXIVA[1,2][R18A]GeXIVA[1,2] TCRSSGRYCRSPYDRRR AYCRRITDACV TCRSSGRYCRSPYDRRR A YCRRITDACV
1818 [Y19A]GeXIVA[1,2][Y19A]GeXIVA[1,2] TCRSSGRYCRSPYDRRRR ACRRITDACV TCRSSGRYCRSPYDRRRR A CRRITDACV
1919 [R21A]GeXIVA[1,2][R21A]GeXIVA[1,2] TCRSSGRYCRSPYDRRRRYC ARITDACV TCRSSGRYCRSPYDRRRRYC A RITDACV
2020 [R22A]GeXIVA[1,2][R22A]GeXIVA[1,2] TCRSSGRYCRSPYDRRRRYCR AITDACV TCRSSGRYCRSPYDRRRRYCR A ITDACV
21twenty one [I23A]GeXIVA[1,2][I23A]GeXIVA[1,2] TCRSSGRYCRSPYDRRRRYCRR ATDACV TCRSSGRYCRSPYDRRRRYCRR A TDACV
22twenty two [T24A]GeXIVA[1,2][T24A]GeXIVA[1,2] TCRSSGRYCRSPYDRRRRYCRRI ADACV TCRSSGRYCRSPYDRRRRYCRRI A DACV
23twenty three [D25A]GeXIVA[1,2][D25A]GeXIVA[1,2] TCRSSGRYCRSPYDRRRRYCRRIT AACV TCRSSGRYCRSPYDRRRRYCRRIT A ACV
24twenty four [V28A]GeXIVA[1,2][V28A]GeXIVA[1,2] TCRSSGRYCRSPYDRRRRYCRRITDAC A TCRSSGRYCRSPYDRRRRYCRRITDAC A
表2：αO-芋螺毒素GeXIVA[1,4]及其丙氨酸扫描突变体的序列，其二硫键连接方式为[C1-C4，C2-C3]Table 2: The sequence of αO-conotoxin GeXIVA[1,4] and its alanine scanning mutants, the disulfide bond connection mode is [C1-C4, C2-C3]
Figure PCTCN2020090978-appb-000001
Figure PCTCN2020090978-appb-000001
Figure PCTCN2020090978-appb-000002
Figure PCTCN2020090978-appb-000002
表3：αO-芋螺毒GeXIVA[1,2]的2个或2个以上的精氨酸被替换的突变体序列，其二硫键连接方式为[C1-C2，C3-C4]Table 3: Mutant sequence of αO-conotoxin GeXIVA[1,2] with 2 or more arginines replaced, the disulfide bond connection mode is [C1-C2, C3-C4]
Figure PCTCN2020090978-appb-000003
Figure PCTCN2020090978-appb-000003
Figure PCTCN2020090978-appb-000004
Figure PCTCN2020090978-appb-000004
表4：αO-芋螺毒GeXIVA的截短突变体序列，含有1对二硫键或无二硫键Table 4: Sequences of truncated mutants of αO-conotoxin GeXIVA, containing a pair of disulfide bonds or no disulfide bonds
SEQ ID NO:SEQ ID NO: 多肽名称Peptide name 氨基酸序列 a Amino acid sequence a
6262 △6-22GeXIVA △6-22 GeXIVA GRYCRSPYDRRRRYCRR G RYCRSPYDRRRRYCRR
6363 △10-19GeXIVA △10-19 GeXIVA RSPYDRRRRYRSPYDRRRRY
6464 △10-19[R1A]GeXIVA △10-19 [R1A]GeXIVA ASPYDRRRRY A SPYDRRRRY
6565 △10-19[S2A]GeXIVA △10-19 [S2A]GeXIVA R APYDRRRRY R A PYDRRRRY
6666 △10-19[P3A]GeXIVA △10-19 [P3A]GeXIVA RS AYDRRRRY RS A YDRRRRY
6767 △10-19[Y4A]GeXIVA △10-19 [Y4A]GeXIVA RSP ADRRRRY RSP A DRRRRY
6868 △10-19[D5A]GeXIVA △10-19 [D5A]GeXIVA RSPY ARRRRY RSPY A RRRRY
6969 △10-19[R6A]GeXIVA △10-19 [R6A]GeXIVA RSPYD ARRRY RSPYD A RRRY
7070 △10-19[R7A]GeXIVA △10-19 [R7A]GeXIVA RSPYDR ARRY RSPYDR A RRY
7171 △10-19[R8A]GeXIVA △10-19 [R8A]GeXIVA RSPYDRR ARY RSPYDRR A RY
7272 △10-19[R9A]GeXIVA △10-19 [R9A]GeXIVA RSPYDRRR AY RSPYDRRR A Y
7373 △10-19[Y10A]GeXIVA △10-19 [Y10A]GeXIVA RSPYDRRRR A RSPYDRRRR A
7474 △10-19GeXIVA# △10-19 GeXIVA# RSPYDRRRRY#RSPYDRRRRY#
7575 △10-19[D5A]GeXIVA# △10-19 [D5A]GeXIVA# RSPY ARRRRY# RSPY A RRRRY#
117117 △7-22GeXIVA △7-22 GeXIVA RYCRSPYDRRRRYCRRRYCRSPYDRRRRYCRR
#indicates a C-terminal amide。#，表示C-末端酰胺化。#indicates a C-terminal amide. # Indicates the C-terminal amidation.
△后面的数字依次表示在母序列(SEQ ID NO:1或SEQ ID NO:25)中的起始位点和终止位点。The numbers after △ indicate the start and stop positions in the parent sequence (SEQ ID NO: 1 or SEQ ID NO: 25) in turn.
表5：αO-芋螺毒GeXIVA的半胱氨酸(Cys，C)替换突变体序列，含有1对二硫键或无二硫键Table 5: Cys (C) substitution mutant sequence of αO-conotoxin GeXIVA, containing a pair of disulfide bonds or no disulfide bonds
Figure PCTCN2020090978-appb-000005
Figure PCTCN2020090978-appb-000005
Figure PCTCN2020090978-appb-000006
Figure PCTCN2020090978-appb-000006
表6：αO-芋螺毒素GeXIVA[1,2]的D-型氨基酸突变体序列，其二硫键连接方式为[C1-C2，C3-C4]。相应位置上的L-型氨基酸被D-型氨基酸替换后，用小写字母和下划线显示Table 6: The sequence of the D-type amino acid mutant of αO-conotoxin GeXIVA[1,2], and its disulfide bond connection mode is [C1-C2, C3-C4]. After the L-type amino acid at the corresponding position is replaced by the D-type amino acid, it is displayed with lowercase letters and underscores
Figure PCTCN2020090978-appb-000007
Figure PCTCN2020090978-appb-000007
Figure PCTCN2020090978-appb-000008
Figure PCTCN2020090978-appb-000008
表7：αO-芋螺毒素GeXIVA[1,4](SEQ ID NO:25)的天冬氨酸(Asp，D)扫描突变体的序列，突变氨基酸用下划线显示，其二硫键连接方式为[C1-C4，C2-C3]Table 7: Aspartic acid (Asp, D) of αO-conotoxin GeXIVA[1,4] (SEQ ID NO: 25) scans the sequence of the mutants, the mutant amino acids are underlined, and the disulfide bond connection method is [C1-C4, C2-C3]
Figure PCTCN2020090978-appb-000009
Figure PCTCN2020090978-appb-000009
Figure PCTCN2020090978-appb-000010
Figure PCTCN2020090978-appb-000010
实施例2：检测αO-芋螺毒素GeXIVA及其突变体的受体结合活性的方法Example 2: Method for detecting the receptor binding activity of αO-conotoxin GeXIVA and its mutants
参照文献 [27，37]中的方法，以及体外转录试剂盒(mMessage mMachine in vitro transcription kit(Ambion，Austin，TX))说明书，制备各种大鼠神经型nAChRs亚型(α3β4，α6/α3β4，α9α10，α4β2，α4β4，α3β4，α2β2，α2β4，α7)、人类α9α10，以及小鼠肌肉型nAChRs(α1β1δε)的cRNA，其浓度用UV 260nm下的OD值进行测算。解剖收集非洲爪蟾(Xenopus laveis)卵母细胞(蛙卵)，将cRNA注射入蛙卵中，每个亚基的注射量为5－10ng cRNA。蛙卵在ND-96中培养。蛙卵收集后的1－2天内注射cRNA，注射后1－4天内用于nAChRs的电压钳记录。 According to the method in the literature [ 27, 37 ] and the instructions of the mMessage mMachine in vitro transcription kit (Ambion, Austin, TX)), various rat neuronal nAChRs subtypes (α3β4, α6/α3β4, α9α10, α4β2, α4β4, α3β4, α2β2, α2β4, α7), human α9α10, and mouse muscle-type nAChRs (α1β1δε) cRNA, the concentration of which is measured by the OD value under UV 260nm. The Xenopus laveis oocytes (frog eggs) were dissected and collected, and cRNA was injected into the frog eggs. The injection volume of each subunit was 5-10ng cRNA. Frog eggs are cultured in ND-96. CRNA was injected within 1 to 2 days after the frog eggs were collected, and used for voltage clamp recording of nAChRs within 1 to 4 days after injection.
将1个注射过cRNA的蛙卵置于30μL的Sylgard记录槽中(直径4mm×深度2mm)，重力灌注含有0.1mg/ml BSA(bovine serum albumin)的ND96灌流液(96.0mM NaCl，2.0mM KCl，1.8mM CaCl 2，1.0mM MgCl 2，5mM HEPES，pH 7.1－7.5)或含有1mM atropine的ND96(ND96A)，流速为1ml/min。所有的芋螺毒素溶液也含有0.1mg/ml BSA以减少毒素的非特异性吸附，用转换阀(SmartValve，Cavro Scientific Instruments，Sunnyvale，CA)可以在灌注毒素或乙酰胆碱(ACh)之间进行自由切换，以及一系列三通螺线阀(solenoid valves，model 161TO31，Neptune Research， Northboro，MA)使灌注ND96与ACh等之间进行自由切换。Ach门控的电流由双电极电压箝放大器(model OC-725B，Warner Instrument Corp.，Hamden，CT)设置在“慢”箝，以及clamp gain在最大值(×2000)位置时进行在线记录。用1mm外径×0.75内径mm的玻璃毛细管(fiber-filled borosilicate capillaries，WPI Inc.，Sarasota，FL)拉制玻璃电极，并充满3M KCl作为电压和电流电极。膜电压箝制在-70mV.整个系统均由电脑控制和记录数据。ACh脉冲为每隔5min自动灌注1s的ACh。ACh的浓度分别为，表达肌肉型的nAChRs和神经型α9α10 nAChRs卵为10μM；表达神经型的nAChRs之α7为200μM，其它的亚型都为100μM。至少记录3个卵表达某个亚型对不同多肽浓度的电流反应情况，以及电流轨迹。 Place 1 cRNA-injected frog egg in a 30μL Sylgard recording trough (diameter 4mm×depth 2mm), gravity infuse ND96 perfusate (96.0mM NaCl, 2.0mM KCl) containing 0.1mg/ml BSA (bovine serum albumin) , 1.8mM CaCl 2 , 1.0mM MgCl 2 , 5mM HEPES, pH 7.1-7.5) or ND96 (ND96A) containing 1mM atropine at a flow rate of 1ml/min. All conotoxin solutions also contain 0.1mg/ml BSA to reduce non-specific adsorption of toxins. The switch valve (SmartValve, Cavro Scientific Instruments, Sunnyvale, CA) can be used to switch freely between toxin perfusion or acetylcholine (ACh). And a series of three-way solenoid valves (solenoid valves, model 161TO31, Neptune Research, Northboro, MA) enable free switching between perfusion ND96 and ACh. The current gated by Ach is recorded online when the two-electrode voltage clamp amplifier (model OC-725B, Warner Instrument Corp., Hamden, CT) is set at the "slow" clamp, and the clamp gain is at the maximum (×2000) position. The glass electrode was drawn with a glass capillary (fiber-filled borosilicate capillaries, WPI Inc., Sarasota, FL) with 1 mm outer diameter×0.75 inner diameter mm, and filled with 3M KCl as voltage and current electrodes. The membrane voltage is clamped at -70mV. The entire system is controlled and recorded by a computer. ACh pulse is ACh that is automatically perfused for 1s every 5min. The concentration of ACh was 10μM for nAChRs expressing muscle type and α9α10 nAChRs for neural type; α7 for nAChRs expressing neural type was 200μM, and all other subtypes were 100μM. Record the current response of at least 3 eggs expressing a certain subtype to different peptide concentrations and the current trajectory.
测试的电流数据用GraphPad Prism软件(San Diego，CA)进行统计分析，绘制剂量反应曲线，计算芋螺毒素的半阻滞浓度IC 50等多种参数。 Current test data is performed using GraphPad Prism software (San Diego, CA) statistical analysis, generate a dose response curve 50 is calculated parameters and other semi-conotoxins block concentration IC.
表1-7中所有多肽(SEQ ID NOs:1-117)的受体结合活性均使用上述方法进行检测。The receptor binding activity of all polypeptides (SEQ ID NOs: 1-117) in Table 1-7 were tested using the above method.
实施例3：αO-芋螺毒素GeXIVA[1,2]及其丙氨酸扫描突变体对nAChRs不同亚型Example 3: αO-conotoxin GeXIVA[1,2] and its alanine scanning mutants for different subtypes of nAChRs 的阻断活性Blocking activity
αO-芋螺毒素GeXIVA[1,2](SEQ ID NO:1，图1)及其丙氨酸扫描突变体(SEQ ID NOs:2-24)(表1)对大鼠神经型α9α10、α7、以及小鼠肌肉型α1β1δε nAChRs这三种极其相近的受体亚型的阻断活性，即半阻断剂量(IC 50)的结果，总结在表8中。这些突变体对这3种亚型的阻断活性之间的比值也总结在表8中。 αO-conotoxin GeXIVA[1,2] (SEQ ID NO:1, Figure 1) and its alanine scanning mutants (SEQ ID NOs: 2-24) (Table 1) have an effect on rat neurotype α9α10, α7 , And the blocking activity of three very similar receptor subtypes of mouse muscle α1β1δε nAChRs, that is, the results of the half-blocking dose (IC 50 ), are summarized in Table 8. The ratios of the blocking activities of these mutants to the three subtypes are also summarized in Table 8.
表8：αO-芋螺毒素GeXIVA[1,2](SEQ ID NO:1)及其丙氨酸扫描突变体(SEQ ID NOs:2-24)对大鼠神经型α9α10、α7、以及小鼠肌肉型α1β1δε nAChRs的阻断活性Table 8: αO-Conotoxin GeXIVA[1,2] (SEQ ID NO:1) and its alanine scanning mutants (SEQ ID NOs: 2-24) affect rat neurotype α9α10, α7, and mice The blocking activity of muscle type α1β1δε nAChRs
Figure PCTCN2020090978-appb-000011
Figure PCTCN2020090978-appb-000011
Figure PCTCN2020090978-appb-000012
Figure PCTCN2020090978-appb-000012
Figure PCTCN2020090978-appb-000013
Figure PCTCN2020090978-appb-000013
a括号外的数字是指半阻断剂量(IC 50)，单位是纳摩尔(nM)；括号中的数据是95％置信区间的半阻断剂量(IC 50)的范围。 a The numbers outside the brackets refer to the half-blocking dose (IC 50 ), and the unit is nanomole (nM); the data in parentheses are the range of the half-blocking dose (IC 50 ) in the 95% confidence interval.
b该列数据是指各个多肽针对某个受体亚型的半阻断剂量(IC 50)与1号多肽的半阻断剂量(IC 50)之间的比值。 b The data in this column refers to the ratio between the half-blocking dose (IC 50 ) of each polypeptide for a certain receptor subtype and the half-blocking dose (IC 50 ) of the No. 1 polypeptide.
c各个多肽对α7与α9α10两个亚型的半阻断剂量(IC 50)之间的比值(α7:α9α10) 。 c The ratio of the half-blocking dose (IC 50 ) of each polypeptide to the two subtypes of α7 and α9α10 (α7:α9α10) .
d各个多肽对α1β1δε与α9α10两个亚型的半阻断剂量(IC 50)之间的比值(α1β1δε:α9α10) 。 d The ratio of the half-blocking dose (IC 50 ) of each polypeptide to the two subtypes of α1β1δε and α9α10 (α1β1δε:α9α10) .
结果显示：The results show that:
SEQ ID NOs:2－24所示的23个突变体(实施例1制备)对大鼠α9α10 nAChR均有很低的纳摩尔级的阻断作用，它们的IC 50均在80nM以下，其活性与野生型的GeXIVA[1,2](SEQ ID NO:1)相比差异不大。这些突变体对大鼠α9α10 nAChR的活性与野生型相比，其变化倍数在0.7－6.2倍，无一超过10倍的变化。这表明，GeXIVA[1,2]序列中的各个被丙氨酸替换的单个氨基酸，对α9α10 nAChR的阻断活性影响很小，可以被替换，而不会明显影响它们的受体结合活性(表8)。 The 23 mutants shown in SEQ ID NOs: 2-24 (prepared in Example 1) have very low nanomolar blocking effects on rat α9α10 nAChR, and their IC 50 is below 80 nM, and their activity is comparable to Compared with wild-type GeXIVA[1,2] (SEQ ID NO:1), there is little difference. Compared with the wild type, the activity of these mutants on rat α9α10 nAChR varied from 0.7 to 6.2 times, and none of them changed more than 10 times. This indicates that each single amino acid substituted by alanine in the GeXIVA[1,2] sequence has little effect on the blocking activity of α9α10 nAChR and can be replaced without significantly affecting their receptor binding activity (Table 8).
SEQ ID NOs:2－24所示的23个突变体(表8)，对大鼠α7 nAChR表现出较微弱的阻断活性，它们的IC 50在640－6690nM之间。这些突变体对α7 nAChR的活性与野生型的GeXIVA[1,2](SEQ ID NO:1)相比差异也不大。它们对大鼠α7 nAChR的活性与野生型相比，其变化倍数在0.9－10.3倍，除了SEQ ID NO:17(10.3倍)以外，其变化在8.6倍以内。除了SEQ ID NOs:6、11、13、19和24这5个突变体对α7 nAChR的活性与野生型的活性相似外，其余突变体对α7 nAChR的活性都下降了2倍以上，也就是说，相对于α7 nAChR亚型，大部分突变体对α9α10 nAChR的选择性提高了，这是一个很难得的优点。 The 23 mutants shown in SEQ ID NOs: 2-24 (Table 8) showed weak blocking activity against rat α7 nAChR, and their IC 50 was between 640-6690 nM. The activity of these mutants on α7 nAChR is not much different from that of wild-type GeXIVA[1,2] (SEQ ID NO:1). Compared with the wild type, their activity on rat α7 nAChR has a fold change of 0.9-10.3 times, except for SEQ ID NO: 17 (10.3 times), the change is within 8.6 times. Except for SEQ ID NOs: 6, 11, 13, 19 and 24, the activity of the 5 mutants on α7 nAChR is similar to that of the wild-type, and the activity of the other mutants on α7 nAChR is reduced by more than 2 times, that is to say Compared with the α7 nAChR subtype, most mutants have improved selectivity for α9α10 nAChR, which is a rare advantage.
SEQ ID NOs:2－24所示的23个突变体(表8)，对小鼠肌肉型α1β1δε nAChR也表现出微弱的阻断活性，它们的IC 50在370－6290nM之间。这些突变体对α1β1δε nAChR的活性与野生型的GeXIVA[1,2](SEQ ID NO:1)相比差异也不是特别大。 它们对α1β1δε nAChR的活性与野生型相比，其变化倍数在0.7－12.1倍，除了SEQ ID NOs:16，17，20(>10倍)以外，其变化在9.3倍以内。除了SEQ ID NOs:2，4，5，11，12，13，这6个突变体对α1β1δε nAChR的活性与野生型的活性相似外，其余突变体对α1β1δε nAChR的活性都下降了2倍以上，也就是说，相对于α1β1δε nAChR亚型，大部分突变体对α9α10 nAChR的选择性提高了，这也是一个很好的优点。 The 23 mutants shown in SEQ ID NOs: 2-24 (Table 8) also showed weak blocking activity against mouse muscle α1β1δε nAChR, and their IC 50 was between 370-6290 nM. The activity of these mutants on α1β1δε nAChR is not particularly different from that of wild-type GeXIVA[1,2] (SEQ ID NO:1). Compared with the wild type, their activity on α1β1δε nAChR has a fold change of 0.7-12.1 times, except for SEQ ID NOs: 16, 17, 20 (>10 times), and the change is within 9.3 times. Except for SEQ ID NOs: 2, 4, 5, 11, 12, 13, the activity of these 6 mutants on α1β1δε nAChR is similar to that of the wild type, and the activity of the other mutants on α1β1δε nAChR decreased by more than 2 times. That is to say, compared with the α1β1δε nAChR subtype, most of the mutants have improved selectivity for α9α10 nAChR, which is also a good advantage.
α9α10、α7、α1β1δε nAChRs这三种亚型极其相似，一般情况下很难区分。本发明人发现的野生型GeXIVA[1,2](SEQ ID NO:1)对它们有较好的区分度，对α9α10 nAChR相对于α7与α1β1δε nAChRs有约50倍的区分度。但在高浓度下(>500nM)，GeXIVA[1,2]对α7与α1β1δε nAChRs有一定的阻断活性。从表8可以看出，有6个突变体，即SEQ ID NOs:6，8，15，16，18，23，对α9α10 nAChR相对于α7与α1β1δε nAChRs的选择性明显提高了，其区分度均在100倍以上。也就是说，SEQ ID NOs:6，8，15，16，18，23保持了对α9α10 nAChR的阻断活性，对α7与α1β1δε nAChRs的活性都降低了，是选择性显著提高的优化突变体。The three subtypes of α9α10, α7, and α1β1δε nAChRs are very similar, and it is generally difficult to distinguish them. The wild-type GeXIVA[1,2] (SEQ ID NO:1) discovered by the present inventors has a good degree of discrimination between them, and has about 50 times the degree of discrimination between α9α10 nAChR and α7 and α1β1δε nAChRs. But at high concentrations (>500nM), GeXIVA[1,2] has a certain blocking activity against α7 and α1β1δε nAChRs. It can be seen from Table 8 that there are 6 mutants, namely SEQ ID NOs: 6, 8, 15, 16, 18, 23. The selectivity of α9α10 nAChR over α7 and α1β1δε nAChRs is significantly improved, and their discrimination is all More than 100 times. That is to say, SEQ ID NOs: 6, 8, 15, 16, 18, 23 maintain the blocking activity of α9α10 nAChR, and reduce the activity of α7 and α1β1δε nAChRs, and are optimized mutants with significantly improved selectivity.
譬如，SEQ ID NO:16(表1，8)是将野生型GeXIVA[1,2]的第17位精氨酸用丙氨酸替换后的突变体，即[R17A]GeXIVA[1,2]，其对α9α10、α7、α1β1δε nAChRs这三种亚型的浓度剂量反应曲线如图3A-3B所示，其选择性显著提高。SEQ ID NO:16对α9α10 nAChR的IC 50为27nM，对α7 nAChRs的IC 50为5610nM，对α1β1δε nAChRs的IC 50为6290nM(表8，图3A-3B)。SEQ ID NO:16对α9α10 nAChR相对于α7 nAChRs的选择性高达207.8倍，比GeXIVA[1,2]提高了约4倍。SEQ ID NO:16对α9α10 nAChR相对于α1β1δε nAChRs的选择性高达232.9倍，比GeXIVA[1,2]提高了约5.4倍。与此类似，SEQ ID NOs:6，8，15，18，23的选择性比GeXIVA[1,2]也显著提高(表8)。 For example, SEQ ID NO: 16 (Table 1, 8) is a mutant obtained by replacing the 17th arginine of wild-type GeXIVA[1,2] with alanine, namely [R17A]GeXIVA[1,2] The concentration-dose response curves for the three subtypes of α9α10, α7, and α1β1δε nAChRs are shown in Figure 3A-3B, and the selectivity is significantly improved. SEQ ID NO: α9α10 nAChR IC 16 of 27 nM to 50, for the α7 nAChRs IC 50 of 5610nM, α1β1δε nAChRs of the IC 50 of 6290nM (Table 8, FIG. 3A-3B). The selectivity of SEQ ID NO: 16 to α9α10 nAChR over α7 nAChRs is as high as 207.8 times, which is about 4 times higher than GeXIVA[1,2]. The selectivity of SEQ ID NO:16 to α9α10 nAChR over α1β1δε nAChRs is as high as 232.9 times, which is about 5.4 times higher than GeXIVA[1,2]. Similarly, the selectivity of SEQ ID NOs: 6, 8, 15, 18, 23 is also significantly higher than that of GeXIVA [1,2] (Table 8).
实施例4：αO-芋螺毒素GeXIVA[1,4]及其丙氨酸扫描突变体对nAChRs不同亚型Example 4: αO-conotoxin GeXIVA[1,4] and its alanine scanning mutants for different subtypes of nAChRs 的阻断活性Blocking activity
αO-芋螺毒素GeXIVA[1,4](SEQ ID NO:25，图1)及其丙氨酸扫描突变体(SEQ ID NOs:26-48)(表2)对α9α10、α7、α1β1δε nAChRs这三种极其相近的受体亚型的阻断活性结果，以及对这3种亚型的阻断活性之间的比值总结在表9中。αO-conotoxin GeXIVA[1,4] (SEQ ID NO: 25, Figure 1) and its alanine scanning mutants (SEQ ID NOs: 26-48) (Table 2) are compared with α9α10, α7, α1β1δε nAChRs. The results of the blocking activity of three very similar receptor subtypes and the ratio of the blocking activities of these three subtypes are summarized in Table 9.
表9：αO-芋螺毒素GeXIVA[1,4](SEQ ID NO:25)及其丙氨酸扫描突变体(SEQ ID  NOs:26-48)对大鼠神经型α9α10、α7、以及小鼠肌肉型α1β1δε nAChRs的阻断活性Table 9: αO-conotoxin GeXIVA[1,4] (SEQ ID NO: 25) and its alanine scanning mutants (SEQ ID NOs: 26-48) affect rat neurotype α9α10, α7, and mice The blocking activity of muscle type α1β1δε nAChRs
Figure PCTCN2020090978-appb-000014
Figure PCTCN2020090978-appb-000014
Figure PCTCN2020090978-appb-000015
Figure PCTCN2020090978-appb-000015
a括号外的数字是指半阻断剂量(IC 50)，单位是纳摩尔(nM)；括号中的数据是95％置信区间的半阻断剂量(IC 50)的范围。 a The numbers outside the brackets refer to the half-blocking dose (IC 50 ), and the unit is nanomole (nM); the data in parentheses are the range of the half-blocking dose (IC 50 ) in the 95% confidence interval.
b该列数据是指各个多肽针对某个受体亚型的半阻断剂量(IC 50)与25号多肽的半阻断剂量(IC 50)之间的比值。 b The data in this column refers to the ratio between the half-blocking dose (IC 50 ) of each polypeptide for a certain receptor subtype and the half-blocking dose (IC 50 ) of polypeptide No. 25.
c各个多肽对α7与α9α10两个亚型的半阻断剂量(IC 50)之间的比值(α7:α9α10) 。 c The ratio of the half-blocking dose (IC 50 ) of each polypeptide to the two subtypes of α7 and α9α10 (α7:α9α10) .
d各个多肽对α1β1δε与α9α10两个亚型的半阻断剂量(IC 50)之间的比值(α1β1δε:α9α10)。 d The ratio of the half-blocking dose (IC 50 ) of each polypeptide to the two subtypes of α1β1δε and α9α10 (α1β1δε: α9α10).
e其半阻断剂量(IC 50)大于10μM，是指在10μM高浓度下，某个多肽对该亚型的电流阻断小于50％。 e The half-blocking dose (IC 50 ) is greater than 10 μM, which means that at a high concentration of 10 μM, the current blocking of a certain polypeptide for this subtype is less than 50%.
结果显示：The results show that:
SEQ ID NOs:26-48所示的23个突变体(实施例1制备)对α9α10 nAChR均有很强的阻断活性，它们的IC 50在1.4－120nM之间，其活性与野生型的GeXIVA[1,4](SEQ ID NO:25)相比，绝大部分的差异不大。这些突变体对α9α10 nAChR的活性与野生型相比，其变化倍数在0.1－11.6倍(表9)。其中，SEQ ID NO:44([R22A]GeXIVA[1,4])对α9α10 nAChR的活性比野生型GeXIVA[1,4]下降了11.6倍；而SEQ ID NO:37([D14A]GeXIVA[1,4])，SEQ ID NO:45([I23A]GeXIVA[1,4]) 对α9α10 nAChR的活性比野生型GeXIVA[1,4]却分别增强了3.4倍与11.4倍。除了这三个突变肽之外，其余的20个突变体的活性与野生型GeXIVA[1,4]的活性相比，其变化在0.6－7.5倍的范围内。这说明GeXIVA[1,4]序列中，除了第22位的精氨酸(R)、第14位的天冬氨酸(D)、以及第23位的异亮氨酸(I)被丙氨酸替换后，活性变化较大外，其余位置的单个氨基酸被丙氨酸替换后，对α9α10 nAChR的阻断活性影响很小，且不会明显影响它们的受体结合活性(表9)。 The 23 mutants (prepared in Example 1) shown in SEQ ID NOs: 26-48 have strong blocking activity against α9α10 nAChR, and their IC 50 is between 1.4-120 nM, and their activity is comparable to that of wild-type GeXIVA Compared with [1,4] (SEQ ID NO: 25), most of the differences are not big. Compared with the wild type, the activity of these mutants on α9α10 nAChR varied from 0.1 to 11.6 times (Table 9). Among them, the activity of SEQ ID NO: 44 ([R22A] GeXIVA[1,4]) on α9α10 nAChR is 11.6 times lower than that of wild-type GeXIVA[1,4]; and SEQ ID NO: 37 ([D14A]GeXIVA[1 ,4]), SEQ ID NO:45 ([I23A]GeXIVA[1,4]) has a 3.4-fold and 11.4-fold increase in the activity of α9α10 nAChR compared to wild-type GeXIVA[1,4]. Except for these three mutant peptides, the activity of the remaining 20 mutants was in the range of 0.6-7.5 times compared with the activity of wild-type GeXIVA[1,4]. This shows that in the GeXIVA[1,4] sequence, except for arginine (R) at position 22, aspartic acid at position 14 (D), and isoleucine (I) at position 23 by alanine After acid replacement, the activity changes greatly, and the replacement of a single amino acid in the remaining positions with alanine has little effect on the blocking activity of α9α10 nAChR, and will not significantly affect their receptor binding activity (Table 9).
SEQ ID NO:26-48所示的23个突变体(表9)，对α7 nAChR的阻断活性极弱，它们的IC 50都在1360nM以上。有的甚至完全失活(IC 50>10000nM)，包括SEQ ID NOs:31，39，40，41，43，44这6个突变体。这些突变体对α7 nAChR的活性与野生型的GeXIVA[1,4](SEQ ID NO:25)相比差异不大。它们对大鼠α7 nAChR的活性与野生型相比，其变化倍数在0.5－3.2倍。所有突变体对α7 nAChR的活性与野生型的活性和选择性相似，它们对α9α10相对于α7 nAChR亚型的选择性都很高。其中SEQ ID NO:37和SEQ ID NO:45对α9α10的活性分别增强了3.4倍与11.4倍。相对于α7 nAChR亚型，SEQ ID NOs:37、45这两个优化突变体对α9α10 nAChR的选择性分别为1625倍和971.4倍，比野生型的区分度176.9倍分别提高了9.2倍和5.5倍(表9)。 The 23 mutants shown in SEQ ID NO: 26-48 (Table 9) have very weak blocking activity against α7 nAChR, and their IC 50 is above 1360 nM. Some are even completely inactivated (IC 50 >10000nM), including the 6 mutants of SEQ ID NOs: 31, 39, 40, 41, 43, 44. The activity of these mutants on α7 nAChR is not much different from that of wild-type GeXIVA[1,4] (SEQ ID NO:25). Compared with the wild type, their activity on rat α7 nAChR has a fold change of 0.5-3.2 times. The activity and selectivity of all mutants to α7 nAChR are similar to those of the wild type, and their selectivity to α9α10 relative to α7 nAChR subtypes is very high. The activities of SEQ ID NO: 37 and SEQ ID NO: 45 on α9α10 were increased 3.4 times and 11.4 times, respectively. Compared with the α7 nAChR subtype, the selectivity of the two optimized mutants of SEQ ID NOs: 37 and 45 to α9α10 nAChR is 1625 times and 971.4 times, respectively, which is 9.2 times and 5.5 times higher than that of the wild-type by 176.9 times (Table 9).
SEQ ID NOs:26－48所示的23个突变体(表9)，对α1β1δε nAChR有微弱的阻断活性，它们的IC 50在520－3500nM之间。这些突变体对α1β1δε nAChR的活性与野生型GeXIVA[1,4](SEQ ID NO:25)的很接近。它们对α1β1δε nAChR的活性与野生型相比，其变化倍数在0.7－2.8倍，对α1β1δε nAChR的活性变化很小。 The 23 mutants shown in SEQ ID NOs: 26-48 (Table 9) have weak blocking activity on α1β1δε nAChR, and their IC 50 is between 520-3500 nM. The activity of these mutants on α1β1δε nAChR is very close to that of wild-type GeXIVA[1,4] (SEQ ID NO:25). Compared with the wild type, their activity on α1β1δε nAChR has a fold change of 0.7-2.8 times, and their activity on α1β1δε nAChR has little change.
α9α10、α7、α1β1δε nAChRs这三种亚型极其相似，本发明人发现的野生型GeXIVA[1,4](SEQ ID NO:25)对它们有很高的区分度，对α9α10 nAChR相对于α7与α1β1δε nAChRs分别有177倍和52倍的区分度。但在高浓度下(>500nM)，GeXIVA[1,4]对α1β1δε nAChRs有一定的阻断活性。从表9可以看出，有2个优化突变体，即SEQ ID NO:37([D14A]GeXIVA[1,4])，SEQ ID NO:45([I23A]GeXIVA[1,4])，对α9α10 nAChR的阻断活性增加了，相对于α7nAChR和α1β1δε nAChRs的选择性明显提高。SEQ ID NO:37对α9α10 nAChR相对于α7nAChR与α1β1δε nAChRs的区分度分别为1625倍和187倍。SEQ ID NO:45(表9)对α9α10 nAChR相对于α7nAChR与α1β1δε nAChRs的区分度分别为971倍和664倍，它们比野生型的177倍和52倍分别提高了5.5倍和13倍。The three subtypes of α9α10, α7, and α1β1δε nAChRs are very similar. The wild-type GeXIVA[1,4](SEQ ID NO:25) discovered by the present inventors has a high degree of discrimination between them. The difference between α9α10 nAChR and α7 α1β1δε nAChRs have 177 times and 52 times discrimination. But at high concentrations (>500nM), GeXIVA[1,4] has a certain blocking activity against α1β1δε nAChRs. It can be seen from Table 9 that there are 2 optimized mutants, namely SEQ ID NO: 37([D14A]GeXIVA[1,4]), SEQ ID NO:45([I23A]GeXIVA[1,4]), right The blocking activity of α9α10 nAChR is increased, and the selectivity relative to α7nAChR and α1β1δε nAChRs is significantly improved. SEQ ID NO: 37 distinguishes between α9α10 nAChR and α7nAChR and α1β1δε nAChRs by 1625 times and 187 times, respectively. SEQ ID NO: 45 (Table 9) distinguishes α9α10 nAChR from α7nAChR and α1β1δε nAChRs by 971 times and 664 times, respectively, which are 5.5 times and 13 times higher than wild-type 177 times and 52 times, respectively.
对SEQ ID NO:45([I23A]GeXIVA[1,4])与野生型的GeXIVA[1,4](SEQ ID NO:25)进行了更加深入的对比研究(表10-11，图4A-4D、图5A-5B、图6A-6B、图7A-7J、图8A-8C)。它们在超高压液相色谱图(UPLC)上的出峰时间，即保留时间(RT)略有差异，SEQ ID NO:45的出峰时间为2.04分钟，略早于野生型GeXIVA[1,4]的2.32分钟，SEQ ID NO:45的亲水性略微增强(图4A、4C)。SEQ ID NO:25的实测分子量为3453.95Da，其理论分子量为3453.96Da。SEQ ID NO:45的实测分子量为3410.46Da，其理论分子量为3410.86Da。二者的实测分子量与其理论分子量一致(图4B、4D)，说明合成的这两个多肽完全正确。A more in-depth comparative study of SEQ ID NO:45 ([I23A]GeXIVA[1,4]) and wild-type GeXIVA[1,4] (SEQ ID NO:25) (Table 10-11, Figure 4A- 4D, Figure 5A-5B, Figure 6A-6B, Figure 7A-7J, Figure 8A-8C). Their peak time on the UPLC chart (UPLC), that is, retention time (RT) is slightly different. The peak time of SEQ ID NO:45 is 2.04 minutes, slightly earlier than the wild-type GeXIVA[1,4 At 2.32 minutes], the hydrophilicity of SEQ ID NO: 45 was slightly increased (Figure 4A, 4C). The measured molecular weight of SEQ ID NO: 25 is 3453.95 Da, and its theoretical molecular weight is 3453.96 Da. The measured molecular weight of SEQ ID NO: 45 is 3410.46 Da, and its theoretical molecular weight is 3410.86 Da. The measured molecular weights of the two are consistent with their theoretical molecular weights (Figure 4B, 4D), indicating that the synthesized two polypeptides are completely correct.
表10：αO-芋螺毒素GeXIVA[1,4](SEQ ID NO:25)与突变体[I23A]GeXIVA[1,4](SEQ ID NO:45)对大鼠多种神经型nAChRs、以及小鼠肌肉型α1β1δε nAChRs(Mα1β1δε)的阻断活性比较Table 10: αO-conotoxin GeXIVA[1,4] (SEQ ID NO: 25) and mutant [I23A] GeXIVA[1,4] (SEQ ID NO: 45) effects on various neuronal nAChRs, and Comparison of blocking activity of mouse muscle type α1β1δε nAChRs (Mα1β1δε)
Figure PCTCN2020090978-appb-000016
Figure PCTCN2020090978-appb-000016
Figure PCTCN2020090978-appb-000017
Figure PCTCN2020090978-appb-000017
a括号外的数字是指半阻断剂量(IC 50)，单位是纳摩尔(nM)；括号中的数据是95％置信区间的半阻断剂量(IC 50)的范围。 a The numbers outside the brackets refer to the half-blocking dose (IC 50 ), and the unit is nanomole (nM); the data in parentheses are the range of the half-blocking dose (IC 50 ) in the 95% confidence interval.
b该列数据是指第45号多肽针对某个受体亚型的半阻断剂量(IC 50)与25号多肽的半阻断剂量(IC 50)之间的比值。 b The data in this column refers to the ratio between the half-blocking dose (IC 50 ) of polypeptide No. 45 against a certain receptor subtype and the half-blocking dose (IC 50 ) of polypeptide No. 25.
表11.αO-芋螺毒素GeXIVA[1,4](SEQ ID NO:25)与突变体[I23A]GeXIVA[1,4](SEQ ID NO:45)对人类α9α10 nAChR亚型(hα9α10)的阻断活性比较Table 11. αO-conotoxin GeXIVA[1,4](SEQ ID NO:25) and mutant [I23A]GeXIVA[1,4](SEQ ID NO:45) against human α9α10 nAChR subtype (hα9α10) Comparison of blocking activity
多肽Peptides IC 50(nM) a IC 50 (nM) a Hill slope a Hill slope a Ratio b Ratio b
2525 111(83-150)111(83-150) 0.97(0.65-1.28)0.97(0.65-1.28) 11
4545 11(8-14)11(8-14) 0.85(0.64-1.05)0.85(0.64-1.05) 0.10.1
a括号外的数字是指半阻断剂量(IC 50)或浓度反应曲线的斜率(Hill slope)，IC 50的单位是纳摩尔(nM)；括号中的数据是95％置信区间的IC 50或Hill slope的范围。 a The numbers outside the brackets refer to the half-blocking dose (IC 50 ) or the slope of the concentration response curve (Hill slope), and the unit of IC 50 is nanomole (nM); the data in brackets are the IC 50 or the 95% confidence interval The range of Hill slope.
b该列数据是指第45号多肽针对人类α9α10 nAChR亚型的半阻断剂量(IC 50)与25号多肽的半阻断剂量(IC 50)之间的比值。 b The data in this column refers to the ratio between the half-blocking dose (IC 50 ) of polypeptide No. 45 for human α9α10 nAChR subtype and the half-blocking dose (IC 50 ) of polypeptide No. 25.
图5A-5B显示了GeXIVA[1,4](Peptide 25，SEQ ID NO:25)与突变肽45(Peptide 45，SEQ ID NO:45)对大鼠α9α10(rα9α10)nAChR的电流影响图，10nM的SEQ ID NO:45几乎可以完全阻断大鼠α9α10 nAChR的电流，明显比野生型的活性要强很多。而野生型的SEQ ID NO:25只能阻断大鼠α9α10 nAChR约50％的电流。二者对大鼠α9α10、α7、以及小鼠α1β1δε nAChRs这三种亚型的浓度剂量反应曲线如图6A-6B所示。SEQ ID NO:45对α9α10 nAChR的浓度剂量反应曲线与对α7和α1β1δε nAChRs的曲线之间的距离，要比野生型的远很多。这充分证明了相对于α7nAChR和α1β1δε nAChRs，SEQ ID NO:45对α9α10 nAChR的活性和选择性显著提高。Figures 5A-5B show the current effect of GeXIVA[1,4] (Peptide 25, SEQ ID NO: 25) and mutant peptide 45 (Peptide 45, SEQ ID NO: 45) on rat α9α10 (rα9α10) nAChR current, 10nM The SEQ ID NO:45 can almost completely block the current of rat α9α10 nAChR, which is obviously much stronger than the wild-type activity. The wild-type SEQ ID NO: 25 can only block about 50% of the current of rat α9α10 nAChR. The concentration-dose response curves for the three subtypes of rat α9α10, α7, and mouse α1β1δε nAChRs are shown in Figure 6A-6B. The distance between the concentration-dose response curve of SEQ ID NO:45 for α9α10 nAChR and the curve for α7 and α1β1δε nAChRs is much longer than that of the wild type. This fully proves that compared with α7nAChR and α1β1δε nAChRs, SEQ ID NO:45 has a significantly improved activity and selectivity for α9α10 nAChR.
SEQ ID NO:45([I23A]GeXIVA[1,4])与野生型GeXIVA[1,4](SEQ ID NO:25)对nAChRs更多亚型的阻断活性结果如表10和图7A-7J所示。检测过的亚型包括大鼠α9α10、α7、α3β2、α3β4、α6/α3β4、α2β2、α4β2、α2β4和α4β2以及小鼠α1β1δε nAChRs共10个亚型。SEQ ID NO:45对α9α10 nAChR的活性最强，其IC 50仅为1.4nM，对其它所有亚型的活性很微弱，它们的IC 50在930－8240nM之间(表10，图7A-7J)。 SEQ ID NO:25对α9α10 nAChR的IC 50为16nM，对其它所有亚型的活性较弱，它们的IC 50在440－4290nM之间(表10，图7A-7J)。很显然，SEQ ID NO:45对α9α10 nAChR相对于其它所有nAChRs亚型的活性和选择性，均比野生型SEQ ID NO:25的活性和选择性显著提高。 The blocking activity results of SEQ ID NO:45 ([I23A]GeXIVA[1,4]) and wild-type GeXIVA[1,4] (SEQ ID NO:25) against more subtypes of nAChRs are shown in Table 10 and Figure 7A- Shown in 7J. The subtypes tested included rat α9α10, α7, α3β2, α3β4, α6/α3β4, α2β2, α4β2, α2β4, and α4β2, and mouse α1β1δε nAChRs. SEQ ID NO:45 has the strongest activity against α9α10 nAChR, with an IC 50 of only 1.4 nM, and very weak activity against all other subtypes, and their IC 50 is between 930-8240 nM (Table 10, Figures 7A-7J) . The IC 50 of SEQ ID NO: 25 for α9α10 nAChR is 16 nM, and its activity against all other subtypes is weak, and their IC 50 is between 440-4290 nM (Table 10, Figures 7A-7J). Obviously, the activity and selectivity of SEQ ID NO:45 for α9α10 nAChR relative to all other nAChRs subtypes are significantly higher than those of wild-type SEQ ID NO:25.
SEQ ID NO:45([I23A]GeXIVA[1,4])与野生型GeXIVA[1,4](SEQ ID NO:25)对人类α9α10 nAChR亚型的阻断活性结果如表11和图8A-8C所示。10nM的野生型SEQ ID NO:25不能阻断人类α9α10 nAChR的电流(图8A)；而10nM的SEQ ID NO:45可阻断人类α9α10 nAChR的约50％电流(图8B)，明显比野生型的活性要强很多。二者对人类α9α10 nAChR的浓度剂量反应曲线如图8C所示。SEQ ID NO:45对人类α9α10 nAChR的活性很强，其IC 50仅为11nM，SEQ ID NO:25对人类α9α10 nAChR的IC 50为111nM。SEQ ID NO:45对人类α9α10 nAChR的活性比SEQ ID NO:25增强了10倍。本发明中涉及的所有突变体对大鼠和人类α9α10 nAChRs的活性都是相似的，它们并不区分大鼠和人类α9α10 nAChRs。 The results of the blocking activity of SEQ ID NO:45 ([I23A]GeXIVA[1,4]) and wild-type GeXIVA[1,4] (SEQ ID NO:25) against human α9α10 nAChR subtypes are shown in Table 11 and Figure 8A- Shown in 8C. 10nM wild-type SEQ ID NO: 25 cannot block the current of human α9α10 nAChR (Figure 8A); while 10nM SEQ ID NO:45 can block about 50% of the current of human α9α10 nAChR (Figure 8B), which is significantly better than wild-type The activity is much stronger. The concentration-dose response curves of the two to human α9α10 nAChR are shown in Figure 8C. SEQ ID NO:45 is very active against human α9α10 nAChR, with an IC 50 of only 11 nM, and SEQ ID NO: 25 has an IC 50 of 111 nM for human α9α10 nAChR. The activity of SEQ ID NO:45 on human α9α10 nAChR is 10 times stronger than that of SEQ ID NO:25. All the mutants involved in the present invention have similar activities to rat and human α9α10 nAChRs, and they do not distinguish between rat and human α9α10 nAChRs.
实施例5：αO-芋螺毒素GeXIVA[1,2]及其多个精氨酸替换突变体对α9α10 nAChRExample 5: αO-conotoxin GeXIVA[1,2] and its multiple arginine substitution mutants pair α9α10 nAChR 的阻断活性Blocking activity
表3列出了GeXIVA[1,2](SEQ ID NO:1)序列中的2个以上的精氨酸被替换后的突变体序列(SEQ ID NO:49-61)。它们对大鼠α9α10 nAChR亚型的阻断活性急剧下降(表12，图9A-9C)。当GeXIVA[1,2]中的3个精氨酸(R)被丙氨酸(A)替换后(SEQ ID NO:51)，或4个精氨酸(R)被丙氨酸(A)替换后(SEQ ID NO:50)，或其所有的9个精氨酸(R)被丙氨酸(A)替换后(SEQ ID NO:49)，这三个突变肽对α9α10 nAChR的阻断活性完全丧失，其IC 50>10000nM(表3，表12，图9A-9C)。在10μM高浓度下，SEQ ID NOs:49，50，51对大鼠α9α10 nAChR的电流几乎没有阻断作用(图9A-9C)。其它的2个精氨酸(R)被丙氨酸(A)替换后的突变肽(SEQ ID NOs:52-61)对α9α10 nAChR的阻断活性与野生型GeXIVA[1,2](其IC 50为12nM)相比，下降了10－48倍，它们的IC 50在130－590nM之间(表12)。SEQ ID NOs:52-61突变肽对α9α10 nAChR的活性弱了很多，这说明GeXIVA[1,2]中的多个精氨酸(2个以上)协同起效，对α9α10 nAChR的结合起着关键作用，当其中2个以上的精氨酸被替换后其活性丧失严重(表3，表12)。 Table 3 lists the mutant sequences (SEQ ID NOs: 49-61) after two or more arginines in the GeXIVA[1,2] (SEQ ID NO:1) sequence are replaced. Their blocking activity against rat α9α10 nAChR subtypes dropped sharply (Table 12, Figures 9A-9C). When 3 arginines (R) in GeXIVA[1,2] are replaced by alanine (A) (SEQ ID NO: 51), or 4 arginines (R) are replaced by alanine (A) After replacement (SEQ ID NO: 50), or after all 9 arginines (R) are replaced by alanine (A) (SEQ ID NO: 49), these three mutant peptides block α9α10 nAChR The activity is completely lost, and its IC 50 is >10000 nM (Table 3, Table 12, Figure 9A-9C). At a high concentration of 10 μM, SEQ ID NOs: 49, 50, 51 had almost no blocking effect on the current of rat α9α10 nAChR (Figure 9A-9C). The blocking activity of the mutant peptides (SEQ ID NOs: 52-61) with the other two arginine (R) replaced by alanine (A) on α9α10 nAChR is similar to that of wild-type GeXIVA[1,2] (its IC 50 is 12nM), a decrease of 10-48 times, their IC 50 is between 130-590nM (Table 12). SEQ ID NOs:52-61 mutant peptides have much weaker activity on α9α10 nAChR, which indicates that multiple arginines (more than two) in GeXIVA[1,2] work together and play a key role in the binding of α9α10 nAChR Effect, when more than two arginines are replaced, its activity is severely lost (Table 3, Table 12).
表12：αO-芋螺毒素GeXIVA[1,2](SEQ ID NO:1)及其多个精氨酸替换突变体(SEQ ID NOs:49-61)对大鼠神经型α9α10 nAChR亚型的阻断活性Table 12: αO-conotoxin GeXIVA[1,2] (SEQ ID NO:1) and its multiple arginine substitution mutants (SEQ ID NOs:49-61) on rat neurotype α9α10 nAChR subtype Blocking activity
Figure PCTCN2020090978-appb-000018
Figure PCTCN2020090978-appb-000018
a括号外的数字是指半阻断剂量(IC 50)或浓度反应曲线的斜率(Hill slope)，IC 50的单位是纳摩尔(nM)；括号中的数据是95％置信区间的IC 50或Hill slope的范围。 a The numbers outside the brackets refer to the half-blocking dose (IC 50 ) or the slope of the concentration response curve (Hill slope). The unit of IC 50 is nanomole (nM); the data in the brackets are the IC 50 or 95% confidence interval. The range of Hill slope.
b该列数据是指某个多肽针对大鼠α9α10 nAChR亚型的半阻断剂量(IC 50)与1号多肽的半阻断剂量(IC 50)之间的比值。 b The data in this column refers to the ratio between the half-blocking dose (IC 50 ) of a certain polypeptide against rat α9α10 nAChR subtype and the half-blocking dose (IC 50 ) of the No. 1 polypeptide.
c其半阻断剂量(IC 50)大于10μM，是指在10μM高浓度下，某个多肽对该亚型的电流阻断小于50％。 c The half-blocking dose (IC 50 ) is greater than 10 μM, which means that at a high concentration of 10 μM, the current blocking of a certain polypeptide for this subtype is less than 50%.
实施例6：αO-芋螺毒素GeXIVA[1,2]及其截短突变体对α9α10 nAChR等的阻断Example 6: αO-Conotoxin GeXIVA[1,2] and its truncated mutants block α9α10 nAChR etc. 活性active
对GeXIVA[1,2](SEQ ID NO:1)的序列从两头往中间逐步截短，对其中一个截短突变体 △10-19GeXIVA(SEQ ID NO:63)再进行丙氨酸扫描突变，之后再进行C-末端酰胺化修饰突变。这些截短突变体的序列、命名与编号(SEQ ID NOs:62-75，SEQ ID NO:117)如表4所示。检测了这些截短突变体对大鼠α9α10 nAChR的结合活性(图10，表13-14)。 The sequence of GeXIVA[1,2] (SEQ ID NO: 1) was gradually truncated from the two ends to the middle, and one of the truncated mutants △10-19 GeXIVA (SEQ ID NO: 63) was subjected to alanine scanning mutation , And then carry out C-terminal amidation modification mutation. The sequence, naming and numbering of these truncated mutants (SEQ ID NOs: 62-75, SEQ ID NO: 117) are shown in Table 4. The binding activity of these truncated mutants to rat α9α10 nAChR was tested (Figure 10, Table 13-14).
表13.αO-芋螺毒素GeXIVA[1,2](SEQ ID NO:1)及其截短突变体对大鼠神经型 α9α10 nAChR亚型的阻断活性Table 13. Blocking activity of αO-conotoxin GeXIVA[1,2] (SEQ ID NO:1) and its truncated mutants on rat neuronal α9α10 nAChR subtype
Figure PCTCN2020090978-appb-000019
Figure PCTCN2020090978-appb-000019
a括号外的数字是指半阻断剂量(IC 50)或浓度反应曲线的斜率(Hill slope)，IC 50的单位是纳摩尔(nM)；括号中的数据是95％置信区间的IC 50或Hill slope的范围。 a The numbers outside the brackets refer to the half-blocking dose (IC 50 ) or the slope of the concentration response curve (Hill slope), and the unit of IC 50 is nanomole (nM); the data in brackets are the IC 50 or the 95% confidence interval The range of Hill slope.
表14：αO-芋螺毒素GeXIVA[1,2](SEQ ID NO:1)及其截短突变体 △6-22GeXIVA(SEQ ID NO:62)和 △10-19[D5A]GeXIVA#(SEQ ID NO:75)对大鼠α9α10与α7，以及小鼠肌肉型Mα1β1δε nAChRs的阻断活性 Table 14: αO-conotoxin GeXIVA[1,2] (SEQ ID NO:1) and its truncated mutants △ 6-22GeXIVA (SEQ ID NO: 62) and △ 10-19[D5A]GeXIVA# (SEQ ID NO: ID NO:75) blocking activity of rat α9α10 and α7, and mouse muscle type Mα1β1δε nAChRs
Figure PCTCN2020090978-appb-000020
Figure PCTCN2020090978-appb-000020
a括号外的数字是指半阻断剂量(IC 50)，其单位是纳摩尔(nM)；括号中的数据是95％置信区间的IC 50的范围。 a The number outside the brackets refers to the half-blocking dose (IC 50 ), and its unit is nanomole (nM); the data in the brackets is the IC 50 range of the 95% confidence interval.
b该列数据是指某个多肽针对α7与α9α10 nAChR亚型的半阻断剂量(IC 50)之间的比值(α7:α9α10)。 b The data in this column refers to the ratio (α7:α9α10) between the half-blocking dose (IC 50 ) of a certain polypeptide for α7 and α9α10 nAChR subtypes.
c该列数据是指某个多肽针对α1β1δε与α9α10 nAChR亚型的半阻断剂量(IC 50)之间的比值(α1β1δε:α9α10)。 c The data in this column refers to the ratio of the half-blocking dose (IC 50 ) of a certain polypeptide for α1β1δε and α9α10 nAChR subtypes (α1β1δε:α9α10).
结果发现，在10μM浓度下， △10-19GeXIVA(SEQ ID NO:63)与 △10-19[D5A]GeXIVA(SEQ ID NO:68)可阻断大鼠α9α10 nAChR的80％以上的电流，其阻断活性很强，而其它所试丙氨酸扫描突变肽(SEQ ID NOs:64-67，69-73；表4)突变体对大鼠α9α10  nAChR的电流阻断均在25％以下，其影响很小或完全没有作用，它们的电流反应百分数>75％(图10)。 It was found that at a concentration of 10μM, △10-19 GeXIVA (SEQ ID NO:63) and △10-19 [D5A]GeXIVA (SEQ ID NO:68) can block more than 80% of the current of rat α9α10 nAChR. Its blocking activity is very strong, and other tested alanine scanning mutant peptides (SEQ ID NOs: 64-67, 69-73; Table 4) mutants blocked rat α9α10 nAChR currents below 25%, It has little or no effect at all, and their current response percentage is >75% (Figure 10).
与野生型GeXIVA[1,2](SEQ ID NO:1)相比，截短突变体 △6-22GeXIVA(SEQ ID NO:62)保持了对大鼠(r)和人类(h)α9α10的阻断活性，二者的活性相似(图11A-11F，表13-14)。GeXIVA[1,2]和 △6-22GeXIVA在10nM浓度下对大鼠α9α10 nAChR的电流有阻断作用(图11A-11B)。GeXIVA[1,2]和 △6-22GeXIVA在50nM浓度下对人类α9α10 nAChR的电流阻断作用明显(图11C-11D)。二者对大鼠(r)和人类(h)α9α10受体的浓度反应曲线几乎重合或离得很近(图11E-11F)。GeXIVA[1,2](SEQ ID NO:1)与 △6-22GeXIVA(SEQ ID NO:62)对大鼠α9α10的IC 50分别为15nM和14nM，几乎完全相同(表13-14，图11E)。 △7-22GeXIVA(SEQ ID NO:117)对α9α10的活性与 △6-22GeXIVA(SEQ ID NO:62)相近。 Compared with wild-type GeXIVA [1,2] (SEQ ID NO: 1), the truncated mutant △6-22 GeXIVA (SEQ ID NO: 62) maintains the resistance to rat (r) and human (h) α9α10 Blocking activity, the activities of the two are similar (Figures 11A-11F, Table 13-14). GeXIVA[1,2] and △6-22 GeXIVA can block the current of rat α9α10 nAChR at a concentration of 10nM (Figure 11A-11B). GeXIVA[1,2] and △6-22 GeXIVA have significant current blocking effects on human α9α10 nAChR at a concentration of 50nM (Figure 11C-11D). The concentration response curves of the two to rat (r) and human (h) α9α10 receptors almost overlap or are very close (Figure 11E-11F). The IC 50 of GeXIVA[1,2] (SEQ ID NO:1) and △6-22 GeXIVA (SEQ ID NO:62) to rat α9α10 are 15nM and 14nM respectively, which are almost identical (Table 13-14, Figure 11E ). The activity of △7-22 GeXIVA (SEQ ID NO:117) on α9α10 is similar to that of △6-22 GeXIVA (SEQ ID NO:62).
△10-19GeXIVA(SEQ ID NO:63)对大鼠α9α10的IC 50为1110nM，活性较弱。 △10-19[D5A]GeXIVA(SEQ ID NO:68)与 △10-19GeXIVA#(SEQ ID NO:74)对大鼠α9α10的IC 50分别为120nM和130nM，活性相近。SEQ ID NOs:63，68，74对α9α10 nAChR的活性与野生型SEQ ID NO:1相比，活性均下降了。而 △10-19[D5A]GeXIVA#(SEQ ID NO:75)对大鼠α9α10的IC 50为17nM，保持了与GeXIVA[1,2](SEQ ID NO:1)相当的活性(表13，图12)。 △10-19 GeXIVA (SEQ ID NO: 63) has an IC 50 of 1110 nM for rat α9α10, which is relatively weak. △10-19 [D5A] GeXIVA (SEQ ID NO: 68) and △10-19 GeXIVA# (SEQ ID NO: 74) have an IC 50 of 120 nM and 130 nM for rat α9α10, respectively, with similar activities. Compared with the wild-type SEQ ID NO:1, the activity of SEQ ID NOs: 63, 68, and 74 on α9α10 nAChR decreased. The IC 50 of △ 10-19[D5A]GeXIVA#(SEQ ID NO:75) to rat α9α10 was 17nM, which kept the activity equivalent to GeXIVA[1,2](SEQ ID NO:1) (Table 13, Figure 12).
△10-19[D5A]GeXIVA#(SEQ ID NO:75)同样保持了对人类α9α10 nAChR的强阻断活性(图13A-13F)，与GeXIVA[1,2](SEQ ID NO:1)的活性相当。GeXIVA[1,2]和 △10-19[D5A]GeXIVA#(SEQ ID NO:75)在10nM浓度下对大鼠α9α10 nAChR的电流有阻断作用(图13A-13B)。GeXIVA[1,2]和 △10-19[D5A]GeXIVA#在50nM浓度下对人类α9α10 nAChR的电流阻断作用明显(图13C-13D)。GeXIVA[1,2]和 △10-19[D5A]GeXIVA#对大鼠(r)和人类(h)α9α10受体的浓度反应曲线几乎重合或离得很近(图13E-13F)，二者之间的活性和选择性均很相似。这是很难得的优点，因为 △10-19[D5A]GeXIVA#(SEQ ID NO:75)只含有10个氨基酸，与含有28个氨基酸的野生型GeXIVA相比，其线性肽合成成本大幅度下降；且SEQ ID NO:75不含有二硫键，人工合成时无需氧化折叠，更进一步降低了纯化成本，大大缩短了人工合成的时间。 △10-19 [D5A]GeXIVA#(SEQ ID NO:75) also maintains strong blocking activity against human α9α10 nAChR (Figure 13A-13F), and GeXIVA[1,2](SEQ ID NO:1) The activity is comparable. GeXIVA[1,2] and △10-19 [D5A]GeXIVA# (SEQ ID NO: 75) have a blocking effect on the current of rat α9α10 nAChR at a concentration of 10 nM (Figure 13A-13B). GeXIVA[1,2] and △10-19 [D5A] GeXIVA# have significant current blocking effects on human α9α10 nAChR at a concentration of 50nM (Figure 13C-13D). The concentration response curves of GeXIVA[1,2] and △10-19 [D5A]GeXIVA# to rat (r) and human (h)α9α10 receptors almost overlap or are very close (Figure 13E-13F). The activity and selectivity between are very similar. This is a rare advantage, because △10-19 [D5A]GeXIVA#(SEQ ID NO:75) only contains 10 amino acids. Compared with wild-type GeXIVA containing 28 amino acids, its linear peptide synthesis cost is greatly reduced. ; And SEQ ID NO: 75 does not contain disulfide bonds, and does not require oxidative folding during artificial synthesis, which further reduces the purification cost and greatly shortens the artificial synthesis time.
GeXIVA[1,2](SEQ ID NO:1)及其截短突变体 △6-22GeXIVA(SEQ ID NO:62) 和 △10-19[D5A]GeXIVA#(SEQ ID NO:75)对大鼠α9α10 nAChR的阻断活性极其相似(表14)，其IC 50分别为15nM、14nM和17nM。它们对大鼠α7和小鼠肌肉型Mα1β1δε nAChRs的阻断活性较弱，其IC 50都在500nM以上，其对α9α10 nAChR相对于α7 nAChR的选择性与野生型GeXIVA[1,2]差不多。SEQ ID NO:75对α9α10相对于肌肉型nAChRs的选择性与野生型GeXIVA[1,2]相比显著提高，提高了3.4倍(表14)。因此，SEQ ID NO:75不但保持了对α9α10 nAChR的强阻断活性，对肌肉型受体的活性明显下降了，从而提高了其选择性，加之它的合成成本十分低廉，是很宝贵的优化突变体。 GeXIVA [1,2] (SEQ ID NO: 1) and its truncated mutants △ 6-22GeXIVA (SEQ ID NO: 62) and △ 10-19[D5A] GeXIVA# (SEQ ID NO: 75) to rats α9α10 nAChR blocking activity is very similar (table 14), an IC 50 were 15nM, 14nM and 17nM. They have weak blocking activity on rat α7 and mouse muscle Mα1β1δε nAChRs, and their IC 50 is above 500 nM, and their selectivity for α9α10 nAChR over α7 nAChR is similar to that of wild-type GeXIVA[1,2]. The selectivity of SEQ ID NO:75 to α9α10 relative to muscle-type nAChRs was significantly improved compared with wild-type GeXIVA [1,2], which was increased by 3.4 times (Table 14). Therefore, SEQ ID NO: 75 not only maintains strong blocking activity against α9α10 nAChR, but also significantly reduces its activity on muscle-type receptors, thereby improving its selectivity. In addition, its synthesis cost is very low, which is a valuable optimization. mutant.
实施例7：αO-芋螺毒素GeXIVA及其半胱氨酸替换突变体对α9α10 nAChR等的Example 7: Effect of αO-conotoxin GeXIVA and its cysteine substitution mutant on α9α10 nAChR etc. 阻断活性Blocking activity
αO-芋螺毒GeXIVA(SEQ ID NOs:1或25)序列中的半胱氨酸(Cys，C)成对被丙氨酸(A)或丝氨酸(S)替换所获得的突变体(SEQ ID NOs:76-87)如表5所示。这些突变体均人工合成成功，并经过色谱与质谱分析进行了确证，它们含有1对二硫键或无二硫键。例如，GeXIVA[1,2](SEQ ID NO:1)与无二硫键的突变肽[C2A,C9A,C20S,C27S]GeXIVA(SEQ ID NO:86，表5)的超高压液相色谱图(UPLC)(图14A、14C)显示，SEQ ID NO:86的出峰时间为2.08分钟，略早于GeXIVA[1,2]的出峰时间2.36分钟。这说明SEQ ID NO:86的亲水性相对于GeXIVA[1,2]有所增强，合成纯化后的终产物在色谱图上只有一个峰，其纯度至少在95％以上，符合纯度要求。GeXIVA[1,2]的实测分子量为3452.70Da，计算的理论分子量为3452.94Da(图14B)，[C2A,C9A,C20S,C27S]GeXIVA(13)的实测分子量为3360.90Da，计算的理论分子量为3360.70Da(图14D)。二者的电喷雾质谱图(ESI-MS)(图14B、14D)显示它们的实测分子量与理论分子量一致，合成的多肽完全正确。αO-Conotoxin GeXIVA (SEQ ID NOs:1 or 25) in the sequence of cysteine (Cys, C) paired by alanine (A) or serine (S) replaced by a mutant (SEQ ID NOs: 76-87) as shown in Table 5. These mutants were successfully synthesized artificially and confirmed by chromatography and mass spectrometry. They contained a pair of disulfide bonds or no disulfide bonds. For example, the UHP liquid chromatogram of GeXIVA[1,2](SEQ ID NO:1) and the mutant peptide without disulfide bond [C2A,C9A,C20S,C27S]GeXIVA(SEQ ID NO:86, Table 5) (UPLC) (Figure 14A, 14C) shows that the peak time of SEQ ID NO: 86 is 2.08 minutes, which is slightly earlier than the peak time of GeXIVA[1,2] of 2.36 minutes. This shows that the hydrophilicity of SEQ ID NO: 86 is enhanced relative to GeXIVA [1,2]. The final product after synthesis and purification has only one peak on the chromatogram, and its purity is at least 95%, which meets the purity requirements. The measured molecular weight of GeXIVA [1,2] is 3,452.70 Da, and the calculated theoretical molecular weight is 3,452.94 Da (Figure 14B). The measured molecular weight of [C2A, C9A, C20S, C27S] GeXIVA (13) is 3,360.90 Da, and the calculated theoretical molecular weight is 3360.70 Da (Figure 14D). The electrospray mass spectra (ESI-MS) of the two (Figure 14B, 14D) show that their measured molecular weight is consistent with the theoretical molecular weight, and the synthesized polypeptide is completely correct.
SEQ ID NOs:76-87(表5，表15)对大鼠α9α10 nAChR的阻断活性都很强，其IC 50都在6.1－36nM之间，与野生型的SEQ ID NO:1(IC 50，12nM)或SEQ ID NO:25(IC 50，16nM)的差不多，其活性差异都小于3倍。也就是说GeXIVA中的半胱氨酸和所形成的二硫键对维持其受体结合活性不重要，被丙氨酸或丝氨酸取代后其活性变化很小。这意味着不含半胱氨酸的GeXIVA线性肽突变体仍然具有相似的活性，人工合成时无需进行氧化折叠和繁多的纯化步骤，从而合成成本大大降低。 SEQ ID NOs: 76-87 (Table 5, Table 15) have strong blocking activity against rat α9α10 nAChR, and their IC 50 is between 6.1-36 nM, which is comparable to wild-type SEQ ID NO:1 (IC 50 , 12nM) or SEQ ID NO: 25 (IC 50 , 16nM) is similar, and the difference in activity is less than 3 times. That is to say, the cysteine and the formed disulfide bond in GeXIVA are not important for maintaining its receptor binding activity, and its activity changes little after being replaced by alanine or serine. This means that the GeXIVA linear peptide mutants without cysteine still have similar activities, and there is no need for oxidative folding and numerous purification steps during artificial synthesis, thus greatly reducing the synthesis cost.
其中[C2A,C9A,C20S,C27S]GeXIVA(SEQ ID NO:86)对大鼠α9α10 nAChR的阻断活性比野生型SEQ ID NO:1还增强了两倍(表15)，其IC 50仅为6.1nM。SEQ ID NO:86在10nM的低浓度下，对大鼠α9α10 nAChR的电流阻断超过了一半，比GeXIVA[1,2]的活性要强(图15A-15B)。测试了SEQ ID NO:86对其它亚型的活性，包括α1β1δε，α7，α6/α3β4，α3β2，α3β4，α2β2nAChRs，对这些亚型的活性很弱，其IC 50都在520nM以上，甚至没活性(IC 50>10000nM)，大部分突变体的IC 50均在1000nM以上，即微摩尔(μM)级(表16，图16A-16H)。与GeXIVA[1,2]相比，SEQ ID NO:86的活性增强，选择性也提高了。SEQ ID NO:86对人类α9α10 nAChR的阻断活性也很强，与GeXIVA[1,2]对人类α9α10 nAChR的阻断活性有所增强(表17，图1717A-17E)。在50nM和100nM浓度下，SEQ ID NO:86对人类α9α10 nAChR的电流阻断很显然，比GeXIVA[1,2]阻断的电流要多(图17A-17D)。SEQ ID NO:86对人类α9α10 nAChR的浓度反应曲线与GeXIVA[1,2]的挨得很近，说明它们对人类α9α10 nAChR的阻断活性都很强。 The blocking activity of [C2A, C9A, C20S, C27S] GeXIVA (SEQ ID NO: 86) on rat α9α10 nAChR was twice as strong as that of wild-type SEQ ID NO:1 (Table 15), and its IC 50 was only 6.1nM. SEQ ID NO: 86 blocked the current of rat α9α10 nAChR by more than half at a low concentration of 10 nM, which was stronger than GeXIVA[1,2] (Figure 15A-15B). The activity of SEQ ID NO: 86 against other subtypes was tested, including α1β1δε, α7, α6/α3β4, α3β2, α3β4, and α2β2 nAChRs. The activity on these subtypes is very weak, and its IC 50 is above 520 nM, even inactive ( IC 50> 10000nM), IC 50 of most of the mutants were more than 1000nM, i.e. micromolar ([mu] M) level (table 16, FIGS. 16A-16H). Compared with GeXIVA[1,2], SEQ ID NO:86 has enhanced activity and increased selectivity. The blocking activity of SEQ ID NO: 86 on human α9α10 nAChR is also very strong, and the blocking activity of GeXIVA[1,2] on human α9α10 nAChR is enhanced (Table 17, Figures 1717A-17E). At concentrations of 50 nM and 100 nM, the current blocking of human α9α10 nAChR by SEQ ID NO: 86 is obviously higher than that of GeXIVA [1,2] (Figure 17A-17D). The concentration response curve of SEQ ID NO: 86 to human α9α10 nAChR is very close to that of GeXIVA[1,2], indicating that they have strong blocking activity against human α9α10 nAChR.
并不是所有的半胱氨酸取代突变，都不影响多肽的活性(表18)。事实上，半胱氨酸之间形成的二硫键对大多数的多肽和蛋白的结构和生物活性起着很关键的作用，有的芋螺毒素肽，其二硫键连接方式改变都有可能完全失活，这在大多数芋螺毒素中是普遍存在的。表18中总结的替换半胱氨酸减少或去掉二硫键的突变，对大多数多肽的活性影响巨大，突变体的活性显著下降(IC 50比野生型明显增大)或完全失活(IC 50>10000nM)。因此，对于半胱氨酸能否被取代进行突变而保持多肽活性，要依特定的多肽而定，必须通过实验结果才能下结论。 Not all cysteine substitution mutations did not affect the activity of the polypeptide (Table 18). In fact, the disulfide bond formed between cysteines plays a key role in the structure and biological activity of most polypeptides and proteins. For some conotoxin peptides, the disulfide bond connection mode may change. Complete inactivation, which is common in most cono toxins. Table 18 summarizes the cysteine replacement mutations reduce or eliminate the disulfide bonds, the great majority of activity of the polypeptide, active mutant significantly decreased (IC 50 significantly increased compared to the wild-type) or completely inactivated (IC 50 >10000nM). Therefore, whether cysteine can be substituted and mutated to maintain the activity of the polypeptide depends on the specific polypeptide, and conclusions can be drawn only through experimental results.
表15：αO-芋螺毒素GeXIVA的半胱氨酸(Cys，C)替换突变体(SEQ ID NOs:76-87)，对大鼠α9α10 nAChR亚型的阻断活性Table 15: Cys (Cys, C) substitution mutant of αO-conotoxin GeXIVA (SEQ ID NOs: 76-87), blocking activity against rat α9α10 nAChR subtype
SEQ ID NO:SEQ ID NO: IC 50(nM) a IC 50 (nM) a Hill slope a Hill slope a Ratio b Ratio b
11 12(9-14)12(9-14) 1.00(0.82-1.20)1.00 (0.82-1.20) 1.01.0
7676 16(13-20)16(13-20) 1.33(0.99-1.66)1.33(0.99-1.66) 1.31.3
7777 12(9-15)12(9-15) 1.07(0.84-1.29)1.07(0.84-1.29) 0.90.9
7878 14(11-18)14(11-18) 1.02(0.79-1.25)1.02(0.79-1.25) 1.11.1
7979 11(8-14)11(8-14) 1.01(0.75-1.27)1.01(0.75-1.27) 0.90.9
2525 16(13-20)16(13-20) 1.04(0.83-1.24)1.04 (0.83-1.24) 1.31.3
8080 17(12-23)17(12-23) 0.98(0.71-1.26)0.98 (0.71-1.26) 1.41.4
8181 21(17-27)21(17-27) 1.02(0.82-1.22)1.02(0.82-1.22) 1.81.8
8282 36(30-43)36(30-43) 1.34(1.02-1.65)1.34(1.02-1.65) 2.92.9
8383 13(10-17)13(10-17) 0.99(0.76-1.22)0.99 (0.76-1.22) 1.01.0
8484 14(12-17)14(12-17) 1.11(0.88-1.34)1.11(0.88-1.34) 1.21.2
8585 16(11-22)16(11-22) 1.11(0.76-1.47)1.11 (0.76-1.47) 1.31.3
8686 6.1(4.8-7.6)6.1(4.8-7.6) 0.81(0.69-0.94)0.81(0.69-0.94) 0.50.5
8787 13(10-16)13(10-16) 1.02(0.83-1.22)1.02(0.83-1.22) 1.01.0
a括号外的数字是指半阻断剂量(IC 50)或浓度反应曲线的斜率(Hill slope)，IC 50的单位是纳摩尔(nM)；括号中的数据是95％置信区间的IC 50或Hill slope的范围。 a The numbers outside the brackets refer to the half-blocking dose (IC 50 ) or the slope of the concentration response curve (Hill slope), and the unit of IC 50 is nanomole (nM); the data in brackets are the IC 50 or the 95% confidence interval The range of Hill slope.
b该列数据是指各个多肽针对大鼠α9α10 nAChR亚型的半阻断剂量(IC 50)与1号多肽的半阻断剂量(IC 50)之间的比值。 b The data in this column refers to the ratio between the half-blocking dose (IC 50 ) of each polypeptide against rat α9α10 nAChR subtype and the half-blocking dose (IC 50 ) of polypeptide No. 1.
Figure PCTCN2020090978-appb-000021
Figure PCTCN2020090978-appb-000021
Figure PCTCN2020090978-appb-000022
Figure PCTCN2020090978-appb-000022
表17：αO-芋螺毒素GeXIVA[1,2]与半胱氨酸替换突变体SEQ ID NO:86，对人类α9α10 nAChR亚型的阻断活性Table 17: αO-conotoxin GeXIVA[1,2] and cysteine substitution mutant SEQ ID NO: 86, blocking activity against human α9α10 nAChR subtype
SEQ ID NO:SEQ ID NO: IC 50(nM) a IC 50 (nM) a Hill slope a Hill slope a Ratio b Ratio b
11 52(41-67)52(41-67) 1.00(0.66-1.35)1.00 (0.66-1.35) 11
8686 33(25-42)33(25-42) 1.13(0.83-1.43)1.13 (0.83-1.43) 0.630.63
a括号外的数字是指半阻断剂量(IC 50)或浓度反应曲线的斜率(Hill slope)，IC 50的单位是纳摩尔(nM)；括号中的数据是95％置信区间的IC 50或Hill slope的范围。 a The numbers outside the brackets refer to the half-blocking dose (IC 50 ) or the slope of the concentration response curve (Hill slope). The unit of IC 50 is nanomole (nM); the data in the brackets are the IC 50 or 95% confidence interval. The range of Hill slope.
b该列数据是指第86号多肽针对人类α9α10 nAChR亚型的半阻断剂量(IC 50)与1号多肽的半阻断剂量(IC 50)之间的比值。 b The data in this column refers to the ratio between the half-blocking dose (IC 50 ) of polypeptide No. 86 against human α9α10 nAChR subtype and the half-blocking dose (IC 50 ) of polypeptide No. 1.
表18：对已报道的其它芋螺毒素半胱氨酸进行过替换改造的多肽序列及其活性总结Table 18: Summary of the peptide sequences and their activities of other conotoxin cysteines that have been reported
Figure PCTCN2020090978-appb-000023
Figure PCTCN2020090978-appb-000023
Figure PCTCN2020090978-appb-000024
Figure PCTCN2020090978-appb-000024
*表示C-末端酰胺化。nAChR:乙酰胆碱受体的英文缩写.VGSCs:电源门控钠离子通道的英文缩写。* Indicates C-terminal amidation. nAChR: The English abbreviation of Acetylcholine Receptor. VGSCs: The English abbreviation of Power-Gated Sodium Channel.
实施例8：αO-芋螺毒素GeXIVA及其D-型氨酸突变体对α9α10 nAChR等的阻Example 8: Inhibition of αO-conotoxin GeXIVA and its D-amino acid mutants on α9α10 nAChR etc. 断活性Off activity
将GeXIVA[1,2]序列中的首尾氨基酸、中间的2个或更多个精氨酸(R)用相应的D-型氨基酸进行替换，所获得的突变体编号(SEQ ID NOs:88-94)、命名和序列见表6。它们对大鼠和人类的α9α10 nAChRs均有很强的阻断活性(图18A-18H、图19A-19H、图20A-20B，表19-20)。GeXIVA[1,2](SEQ ID NO:1，图18A)及其D-型氨基酸突变体SEQ ID NOs:88-94(表6)，在1μM或100nM浓度下，对大鼠α9α10(rα9α10)nAChR的90％以上甚至100％的电流全都阻断了(图18A-18H)。类似地，这些突变体在1μM或100nM浓度下，对人类α9α10(hα9α10)nAChR的电流几乎全部阻断(图19A-19H)。其浓度反应曲线(图20A-20B)彼此相隔很近，大部分突变体与GeXIVA[1,2]的曲线相互重叠，活性相似的。Replace the first and last amino acids, two or more arginines (R) in the GeXIVA[1,2] sequence with corresponding D-type amino acids, and the obtained mutant number (SEQ ID NOs: 88- 94). See Table 6 for naming and sequence. They have strong blocking activity against α9α10 nAChRs in rats and humans (Figure 18A-18H, Figure 19A-19H, Figure 20A-20B, Table 19-20). GeXIVA[1,2] (SEQ ID NO:1, Figure 18A) and its D-type amino acid mutant SEQ ID NOs:88-94 (Table 6), at a concentration of 1μM or 100nM, it is effective for rat α9α10 (rα9α10) More than 90% or even 100% of the current of nAChR was blocked (Figure 18A-18H). Similarly, these mutants almost completely blocked the current of human α9α10 (hα9α10) nAChR at a concentration of 1 μM or 100 nM (Figure 19A-19H). The concentration response curves (Figures 20A-20B) are very close to each other, most of the mutants and GeXIVA[1,2] curves overlap each other, and the activities are similar.
GeXIVA[1,2](SEQ ID NO:1)与这些D-型氨基酸突变体SEQ ID NOs:88-94对大鼠α9α10(rα9α10)nAChR的IC 50都在11－32nM之间(表19)；对人类α9α10(hα9α10)nAChR的IC 50都在15－49nM之间(表20)。SEQ ID NOs:88-94对大鼠和人类α9α10nAChRs的阻断活性很接近，且与野生型GeXIVA[1,2]的活性相差不大。这说明用D-型氨基酸进行替换对其受体结合活性的影响很小。这些突变体对大鼠其它亚型的阻断活性很微弱，或完全没有作用(表21)，这些亚型包括α3β4、α3β2、α1β1δε、α7、α6/α3β4、α4β2、α2β4、α4β4和α2β2nAChRs，对这些亚型的活性很弱，其IC 50都在470nM以上，甚至没活性(IC 50>10000nM)，大部分突变体的IC 50均在1000nM以上(表21，图20A-20B)。这些突变体对α9α10 nAChRs相对于其它亚型来说，它们的选择性都很高。其中的SEQ ID NO:92(GeMF，Mf-GeXIVA)对大鼠和人类α9α10nAChRs的阻断活性比GeXIVA[1,2]分别提高了2.6倍与2.8倍(表19－20)。 GeXIVA[1,2] (SEQ ID NO:1) and these D-type amino acid mutants SEQ ID NOs: 88-94 have IC 50 of rat α9α10(rα9α10)nAChR between 11-32nM (Table 19) ; The IC 50 for human α9α10 (hα9α10) nAChR is between 15-49nM (Table 20). The blocking activity of SEQ ID NOs: 88-94 on rat and human α9α10 nAChRs is very close, and the activity is not much different from that of wild-type GeXIVA[1,2]. This shows that the substitution of D-type amino acids has little effect on its receptor binding activity. The blocking activity of these mutants on other rat subtypes is weak or has no effect at all (Table 21). These subtypes include α3β4, α3β2, α1β1δε, α7, α6/α3β4, α4β2, α2β4, α4β4, and α2β2 nAChRs. The activity of these subtypes is very weak, and their IC 50 is above 470 nM, or even inactive (IC 50 >10000 nM), and most of the mutants have IC 50 above 1000 nM (Table 21, Figures 20A-20B). These mutants are highly selective for α9α10 nAChRs compared to other subtypes. The blocking activity of SEQ ID NO: 92 (GeMF, Mf-GeXIVA) on rat and human α9α10nAChRs is 2.6 times and 2.8 times higher than that of GeXIVA [1,2] (Table 19-20).
对GeXIVA[1,2]进行D-型氨基酸替换突变的主要目的是想提高其稳定性。因为多 肽本身在生物体内很容易被各种蛋白酶降解导致多肽的稳定性差。GeXIVA[1,2]中含有9个精氨酸，很容易受胰蛋白酶的攻击而水解。为此，设计合成了这一系列D-型氨基酸替换突变体，幸好这些D-型氨基酸替换保持了其受体结合活性，并没有大的改变。对其中活性增强的4个突变体的血清稳定性进行了研究，结果如图21A-21E所示。这4个突变体(SEQ ID NOs:91-93)分别是SEQ ID NO:91(GeMT＝Mt-GeXIVA)，SEQ ID NO:92(GeMF＝Mf-GeXIVA，SEQ ID NO:93(GeArg-GeXIVA)与SEQ ID NO:94(GeFlex-GeXIVA),)(表6，图21A-21E)。SEQ ID NOs:91-93在100％人类血清中的稳定性与GeXIVA[1,2]相比显著增强。在人类血清中，GeXIVA[1,2]在120分钟内已被降解殆尽，所剩多肽不到原来起始量的20％；而SEQ ID NOs:91-93经过24小时后还剩有约20％。其中SEQ ID NO:94(Geflex)的半衰期从野生型GeXIVA[1,2]的40分钟增加到8小时之久(图21A-21E)。进而测定了SEQ ID NO:94(Geflex)在人工模拟肠液中的稳定性(图22)，其降解速率比GeXIVA[1,2]慢了许多。GeXIVA[1,2]在加入人工模拟肠液的一开始，几乎是瞬间即被完全降解，而SEQ ID NO:94(Geflex)在半小时内还有约40％的多肽留存，75分钟后才被降解完全(图22)。所以，一般的多肽药物不能经过口服发挥药效，多肽经胃肠道被消化降解掉，能进入血液循环的多肽微乎其微，达不到治疗效果。该研究结果说明通过D-型氨基酸替换，的确大幅度地提高了GeXIVA的稳定性，且保持了原有的受体结合活性，这是一个提高多肽药物稳定性的有效途径。The main purpose of D-type amino acid substitution mutation on GeXIVA[1,2] is to improve its stability. Because the peptide itself is easily degraded by various proteases in the organism, the stability of the peptide is poor. GeXIVA [1,2] contains 9 arginines, which are easily hydrolyzed by trypsin. For this reason, a series of D-type amino acid substitution mutants were designed and synthesized. Fortunately, these D-type amino acid substitutions maintained their receptor binding activity without major changes. The serum stability of the four mutants with enhanced activity was studied, and the results are shown in Figures 21A-21E. The four mutants (SEQ ID NOs: 91-93) are SEQ ID NO: 91 (GeMT=Mt-GeXIVA), SEQ ID NO: 92 (GeMF=Mf-GeXIVA, SEQ ID NO: 93 (GeArg-GeXIVA) ) And SEQ ID NO: 94 (GeFlex-GeXIVA),) (Table 6, Figure 21A-21E). The stability of SEQ ID NOs: 91-93 in 100% human serum is significantly enhanced compared with GeXIVA [1,2]. In human serum, GeXIVA[1,2] has been degraded and exhausted within 120 minutes, and the remaining polypeptide is less than 20% of the original initial amount; and SEQ ID NOs: 91-93 has about 24 hours left. 20%. The half-life of SEQ ID NO: 94 (Geflex) increased from 40 minutes of wild-type GeXIVA[1,2] to as long as 8 hours (Figure 21A-21E). Furthermore, the stability of SEQ ID NO: 94 (Geflex) in artificial intestinal juice was determined (Figure 22), and its degradation rate was much slower than GeXIVA [1,2]. GeXIVA[1,2] was completely degraded almost instantaneously when the artificial intestinal fluid was added, and SEQ ID NO:94 (Geflex) still had about 40% of the peptides remaining within half an hour, and it was only after 75 minutes. Degradation is complete (Figure 22). Therefore, ordinary peptide drugs cannot exert their efficacy through oral administration. The peptides are digested and degraded through the gastrointestinal tract, and the peptides that can enter the blood circulation are very small, and the therapeutic effect cannot be achieved. The results of this study indicate that the D-type amino acid substitution has indeed greatly improved the stability of GeXIVA and maintained the original receptor binding activity, which is an effective way to improve the stability of peptide drugs.
表19：αO-芋螺毒GeXIVA[1,2]的D-型氨基酸突变体对大鼠α9α10 nAChR亚型的阻断活性Table 19: Blocking activity of αO-conotoxin GeXIVA[1,2] D-type amino acid mutants on rat α9α10 nAChR subtype
SEQ ID NO:SEQ ID NO: 芋螺毒素名称Conotoxin name IC 50(nM) IC 50 (nM) 浓度反应曲线斜率Concentration response curve slope
11 GeXIVA[1,2]GeXIVA[1,2] 28.67(24.19-33.97)28.67(24.19-33.97) 0.86(0.72-0.99)0.86(0.72-0.99)
8888 1t-GeXIVA1t-GeXIVA 24.70(20.91-29.17)24.70 (20.91-29.17) 0.90(0.77-1.02)0.90 (0.77-1.02)
8989 28v-GeXIVA28v-GeXIVA 31.29(26.15-37.43)31.29 (26.15-37.43) 0.91(0.76-1.05)0.91 (0.76-1.05)
9090 1t,28v-GeXIVA1t,28v-GeXIVA 31.94(27.06-3769)31.94(27.06-3769) 1.03(0.86-1.19)1.03(0.86-1.19)
9191 Mt-GeXIVAMt-GeXIVA 17.37(14.44-20.89)17.37(14.44-20.89) 0.97(0.84-1.13)0.97 (0.84-1.13)
9292 Mf-GeXIVAMf-GeXIVA 11.02(9.08-13.39)11.02(9.08-13.39) 0.90(0.75-1.04)0.90(0.75-1.04)
9393 GeArg-GeXIVAGeArg-GeXIVA 21.67(19.72-28.10)21.67 (19.72-28.10) 0.70(0.57-0.84)0.70(0.57-0.84)
9494 GeFlex-GeXIVAGeFlex-GeXIVA 24.53(20.47-29.39)24.53 (20.47-29.39) 0.96(0.80-1.12)0.96 (0.80-1.12)
表20：αO-芋螺毒GeXIVA[1,2]的D-型氨基酸突变体对人类α9α10 nAChR亚型的阻断活性Table 20: Blocking activity of αO-conotoxin GeXIVA[1,2] D-type amino acid mutants against human α9α10 nAChR subtype
SEQ ID NO:SEQ ID NO: 芋螺毒素名称Conotoxin name 半阻断剂量IC 50(nM) Half-blocking dose IC 50 (nM) 浓度反应曲线斜率Concentration response curve slope
11 GeXIVA[1,2]GeXIVA[1,2] 44.19(39.45-49.51)44.19 (39.45-49.51) 1.196(1.366-1.025)1.196(1.366-1.025)
8888 1t-GeXIVA1t-GeXIVA 45.65(39.83-52.34)45.65(39.83-52.34) 1.131(1.327-0.9338)1.131 (1.327-0.9338)
8989 28v-GeXIVA28v-GeXIVA 48.58(43.61-54.12)48.58 (43.61-54.12) 1.143(1.307-0.9799)1.143 (1.307-0.9799)
9090 1t,28v-GeXIVA1t,28v-GeXIVA 47.54(41.06-55.05)47.54(41.06-55.05) 1.130(1.345-0.9154)1.130 (1.345-0.9154)
9191 Mt-GeXIVAMt-GeXIVA 39.39(33.44-46.40)39.39(33.44-46.40) 1.299(1.589-1.009)1.299(1.589-1.009)
9292 Mf-GeXIVAMf-GeXIVA 15.62(13.52-18.05)15.62(13.52-18.05) 1.145(1.309-0.9801)1.145 (1.309-0.9801)
9393 GeArg-GeXIVAGeArg-GeXIVA 36.94(31.16-43.79)36.94(31.16-43.79) 1.161(1.395-0.9271)1.161 (1.395-0.9271)
9494 GeFlex-GeXIVAGeFlex-GeXIVA 34.34(30.19-39.06)34.34(30.19-39.06) 0.9445(1.038-0.8508)0.9445(1.038-0.8508)
表21：αO-芋螺毒素GeXIVA[1,2]的D-型氨基酸突变体对大鼠其它nAChR亚型的阻断活性(半阻断剂量，IC 50nM) Table 21: Blocking activity of D-amino acid mutants of αO-conotoxin GeXIVA[1,2] on other nAChR subtypes in rats (half blocking dose, IC 50 nM)
Figure PCTCN2020090978-appb-000025
Figure PCTCN2020090978-appb-000025
Figure PCTCN2020090978-appb-000026
Figure PCTCN2020090978-appb-000026
实施例9：αO-芋螺毒素GeXIVA及其天冬氨酸(Asp，D)扫描突变体对α9α10Example 9: αO-conotoxin GeXIVA and its aspartic acid (Asp, D) scanning mutant pair α9α10 nAChR的阻断活性nAChR blocking activity
将GeXIVA[1,4](SEQ ID NO:25)序列中的每个非半胱氨酸分别用天冬氨酸(Asp，D)进行替换，获得天冬氨酸扫描突变体。这些突变体的编号(SEQ ID NOs:95-116)、命名和序列见表7，它们对大鼠α9α10 nAChR均有不同程度的阻断活性(图23A-23B、图24A-24H，表22)。譬如，在10μM高浓度下，SEQ ID NOs:110-115对大鼠α9α10 nAChR的约90％以上的电流都阻断了(图23A，表7)；在1μM浓度下，SEQ ID NOs:95-101以及SEQ ID NOs:110-115对大鼠α9α10 nAChR的约60％以上、甚至是90％以上的电流都阻断了(图23B，表7)。Each non-cysteine in the GeXIVA[1,4] (SEQ ID NO: 25) sequence was replaced with aspartic acid (Asp, D) to obtain an aspartic acid scanning mutant. The number (SEQ ID NOs: 95-116), naming and sequence of these mutants are shown in Table 7. They all have varying degrees of blocking activity against rat α9α10 nAChR (Figure 23A-23B, Figure 24A-24H, Table 22) . For example, at a high concentration of 10μM, SEQ ID NOs:110-115 blocked more than 90% of the current of rat α9α10 nAChR (Figure 23A, Table 7); at a concentration of 1μM, SEQ ID NOs:95- 101 and SEQ ID NOs: 110-115 blocked more than 60%, and even more than 90% of the current of rat α9α10 nAChR (Figure 23B, Table 7).
表22：αO-芋螺毒素GeXIVA[1,4](SEQ ID NO:25)的部分天冬氨酸(Asp，D)扫描突变体SEQ ID NOs:95，100，110-115对大鼠α9α10 nAChR亚型的阻断活性(IC 50) Table 22: Partial Aspartic Acid (Asp, D) Scanning Mutants of αO-Conotoxin GeXIVA[1,4] (SEQ ID NO: 25) SEQ ID NOs: 95, 100, 110-115 to rat α9α10 Blocking activity of nAChR subtypes (IC 50 )
Figure PCTCN2020090978-appb-000027
Figure PCTCN2020090978-appb-000027
Figure PCTCN2020090978-appb-000028
Figure PCTCN2020090978-appb-000028
a括号外的数字是指半阻断剂量(IC 50)或浓度反应曲线的斜率(Hill slope)，IC 50的单位是纳摩尔(nM)；括号中的数据是95％置信区间的IC 50或Hill slope的范围。 a The numbers outside the brackets refer to the half-blocking dose (IC 50 ) or the slope of the concentration response curve (Hill slope), and the unit of IC 50 is nanomole (nM); the data in brackets are the IC 50 or the 95% confidence interval The range of Hill slope.
b该列数据是指各个多肽针对大鼠α9α10 nAChR亚型的半阻断剂量(IC 50)与25号多肽的半阻断剂量(IC 50)之间的比值。 b The data in this column refers to the ratio between the half-blocking dose (IC 50 ) of each polypeptide against rat α9α10 nAChR subtype and the half-blocking dose (IC 50 ) of polypeptide No. 25.
GeXIVA[1,4](SEQ ID NO:25)及其部分天冬氨酸扫描突变体(表7；SEQ ID NOs:95，100，110-115)对大鼠α9α10 nAChR的浓度反应曲线如图24所示，它们的IC 50等列于表22中，其IC 50范围在27－590nM之间。有的突变体与野生型GeXIVA[1,4]的活性相似(表22)，如SEQ ID NO:95(图24A)和SEQ ID NO:115(图24H)。有的突变体比野生型GeXIVA[1,4]的活性明显减弱(表22)，如SEQ ID NO:100(图24B)和SEQ ID NOs:111-112(图24D-24E)。有的突变体比野生型GeXIVA[1,4]的活性轻微减弱(表22)，如SEQ ID NO:110(图24C)和SEQ ID NOs:113-114(图24F-24G)。其余突变体对α9α10 nAChR都有活性，与野生型GeXIVA[1,4]的活性相比差异不是特别大。 The concentration response curve of GeXIVA[1,4] (SEQ ID NO: 25) and its partial aspartic acid scanning mutants (Table 7; SEQ ID NOs: 95, 100, 110-115) to rat α9α10 nAChR is shown in the figure As shown in 24, their IC 50 is listed in Table 22, and their IC 50 ranges from 27 to 590 nM. Some mutants have similar activity to wild-type GeXIVA[1,4] (Table 22), such as SEQ ID NO: 95 (Figure 24A) and SEQ ID NO: 115 (Figure 24H). Some mutants have significantly weaker activity than wild-type GeXIVA[1,4] (Table 22), such as SEQ ID NO: 100 (Figure 24B) and SEQ ID NOs: 111-112 (Figure 24D-24E). Some mutants have slightly weakened activity than wild-type GeXIVA[1,4] (Table 22), such as SEQ ID NO: 110 (Figure 24C) and SEQ ID NOs: 113-114 (Figure 24F-24G). The rest of the mutants are active on α9α10 nAChR, which is not particularly different from the activity of wild-type GeXIVA[1,4].
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28 中国发明专利申请，发明人：罗素兰，长孙东亭，吴勇，朱晓鹏，胡远艳，J.Michael McIntosh。申请人：海南大学。专利名称：αO-超家族芋螺毒素肽、其药物组合物及用途。申请号：201210197589.X；申请日：2012-06-15.28 Chinese invention patent application, inventors: Luo Sulan, Changsun Dongting, Wu Yong, Zhu Xiaopeng, Hu Yuanyan, J. Michael McIntosh. Applicant: Hainan University. Patent name: αO-superfamily conotoxin peptide, its pharmaceutical composition and use. Application number: 201210197589.X; application date: 2012-06-15.
29 PCT申请，发明人：罗素兰，长孙东亭，吴勇，朱晓鹏，胡远艳，J.Michael McIntosh。申请人：海南大学。专利名称：αO-超家族芋螺毒素肽、其药物组合物及用途。国际申请号：PCT/CN2013/076967；申请日：2013-6-8.29 PCT application, inventors: Luo Sulan, Sun Dongting, Wu Yong, Zhu Xiaopeng, Hu Yuanyan, J. Michael McIntosh. Applicant: Hainan University. Patent name: αO-superfamily conotoxin peptide, its pharmaceutical composition and use. International application number: PCT/CN2013/076967; filing date: 2013-6-8.
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31 Halai R,Clark RJ,Nevin ST,Jensen JE,Adams DJ,Craik DJ.Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues crucial for activity at the alpha9alpha10 nicotinic acetylcholine receptor.J Biol Chem 2009；284:20275-20284.31 Halai R, Clark RJ, Nevin ST, Jensen JE, Adams DJ, Craik DJ. Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues Crucial for activity at the alpha9alpha10 nicotinic.acetylcholine 275 Receptor 275 .
32 Kompella SN,Hung A,Clark RJ,Mari F,Adams DJ.Alanine scan of alpha-conotoxin RegIIA reveals a selective alpha3beta4 nicotinic acetylcholine receptor antagonist.J Biol Chem 2015；290:1039-1048.32 Kompella SN, Hung A, Clark RJ, Mari F, Adams DJ. Alanine scan of alpha-conotoxin RegIIA reveals a selective alpha3beta4 nicotinic acetylcholine receptor antagonist. J Biol Chem 2015; 290: 1039-1048.
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39 Green BR,Zhang MM,Chhabra S et al.Interactions of disulfide-deficient selenocysteine analogs of mu-conotoxin BuIIIB with the alpha-subunit of the voltage-gated sodium channel subtype 1.3.FEBS J 2014；281:2885-2898.39 Green BR, Zhang MM, Chhabra S et al. Interactions of disulfide-deficient selenocysteine analogs of mu-conotoxin BuIIIB with the alpha-subunit of the voltage-gated sodium channel subtype 1.3.FEBS J 2014; 281: 2885-2898;
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42 Tabassum N,Tae HS,Jia X et al.Role of CysI-CysIII Disulfide Bond on the Structure and Activity of alpha-Conotoxins at Human Neuronal Nicotinic Acetylcholine Receptors.ACS Omega 2017；2:4621-4631.42 Tabassum N,Tae HS, Jia X et al. Role of CysI-CysIII Disulfide Bond on the Structure and Activity of alpha-Conotoxins at Human Neuronal Nicotinic Acetylcholine Receptors. ACSOmega 631. 2462;
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand. According to all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are within the protection scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (18)

  1. 一种分离的多肽，其氨基酸序列为将母序列中的一个或多个氨基酸去掉，或者将母序列中的一个或多个氨基酸替换为相同数目的L型氨基酸或D型氨基酸，其中，所述母序列为SEQ ID NO:1、SEQ ID NO:62或SEQ ID NO:63；并且所述多肽具有阻断或抑制α9α10 nAChR的活性；An isolated polypeptide whose amino acid sequence is one or more amino acids in the parent sequence removed, or one or more amino acids in the parent sequence are replaced with the same number of L-amino acids or D-amino acids, wherein The parent sequence is SEQ ID NO: 1, SEQ ID NO: 62 or SEQ ID NO: 63; and the polypeptide has the activity of blocking or inhibiting α9α10 nAChR;
    优选地，所述L型氨基酸或D型氨基酸选自：丙氨酸、丝氨酸、2-氨基丁酸、组氨酸、精氨酸、酪氨酸、苏氨酸、赖氨酸、亮氨酸、苯丙氨酸、D-精氨酸、D-丝氨酸、D-苏氨酸、D-缬氨酸、D-天冬氨酸、D-谷氨酸、谷氨酸和天冬氨酸；优选为丙氨酸、D-苏氨酸或D-天冬氨酸；Preferably, the L-type amino acid or D-type amino acid is selected from: alanine, serine, 2-aminobutyric acid, histidine, arginine, tyrosine, threonine, lysine, leucine , Phenylalanine, D-arginine, D-serine, D-threonine, D-valine, D-aspartic acid, D-glutamic acid, glutamic acid and aspartic acid; Preferably it is alanine, D-threonine or D-aspartic acid;
    优选地，所述的多肽，其特征在于如下的1)－3)项中的任意1项、2项或3项：Preferably, the polypeptide is characterized by any one, two or three of the following items 1) to 3):
    1)与母序列相比，所述多肽具有基本相同或者提高的阻断α9α10 nAChR的活性；1) Compared with the parent sequence, the polypeptide has substantially the same or improved α9α10 nAChR blocking activity;
    2)与母序列相比，相对于α7 nAChR和/或α1β1δε nAChR，所述多肽具有基本相同或提高的对α9α10 nAChR的选择性或特异性；2) Compared with the parent sequence, relative to α7 nAChR and/or α1β1δε nAChR, the polypeptide has substantially the same or improved selectivity or specificity for α9α10 nAChR;
    3)与母序列相比，所述多肽具有在生物体内(例如在消化道如肠道内，或在血液中)延长的半衰期；优选地，所述生物体为哺乳动物例如人或大鼠。3) Compared with the parent sequence, the polypeptide has a prolonged half-life in the organism (for example, in the digestive tract such as the intestine, or in the blood); preferably, the organism is a mammal such as a human or a rat.
  2. 根据权利要求1所述的分离的多肽，其特征在于如下的(1)－(6)项中的任意的1项、2项、3项、4项或5项：The isolated polypeptide according to claim 1, characterized in that any of the following (1) to (6) items 1, 2, 3, 4 or 5:
    (1)将SEQ ID NO:1或SEQ ID NO:62中的除了半胱氨酸和丙氨酸之外的任意一个氨基酸替换为一个丙氨酸；(1) Replace any amino acid in SEQ ID NO: 1 or SEQ ID NO: 62 except cysteine and alanine with an alanine;
    (2)将SEQ ID NO:1中的9个精氨酸中的任意2个、3个、4个、5个、6个、7个、8个或9个替换为相同数目的丙氨酸和/或丝氨酸；(2) Replace any 2, 3, 4, 5, 6, 7, 8, or 9 of the 9 arginines in SEQ ID NO:1 with the same number of alanines And/or serine;
    (3)将SEQ ID NO:63中的任意一个氨基酸替换为一个丙氨酸；(3) Replace any amino acid in SEQ ID NO: 63 with an alanine;
    (4)将SEQ ID NO:1中的4个半胱氨酸中的任意1个、2个、3个或4个替换为相同数目的丙氨酸和/丝氨酸；(4) Replace any one, two, three or four of the four cysteines in SEQ ID NO:1 with the same number of alanine and/serine;
    (5)将SEQ ID NO:1中的氮末端的苏氨酸替换为D-苏氨酸，碳末端的缬氨酸替换为D-缬氨酸，和/或将SEQ ID NO:1中的9个精氨酸中的任意2个、3个、4个、5个、6个、7个、8个或9个替换为相同数目的D-精氨酸；(5) Replace the nitrogen-terminus threonine in SEQ ID NO:1 with D-threonine, and replace the carbon-terminus valine with D-valine, and/or replace SEQ ID NO:1 Replace any 2, 3, 4, 5, 6, 7, 8, or 9 of the 9 arginines with the same number of D-arginine;
    (6)将SEQ ID NO:1、SEQ ID NO:62或SEQ ID NO:63中的除了半胱氨酸和天冬氨酸之外的任意一个氨基酸替换为一个天冬氨酸。(6) Replace any amino acid in SEQ ID NO: 1, SEQ ID NO: 62 or SEQ ID NO: 63 except cysteine and aspartic acid with an aspartic acid.
  3. 根据权利要求1或2所述的多肽，其中，所述多肽含有0个、1个或2个二硫键；The polypeptide of claim 1 or 2, wherein the polypeptide contains 0, 1, or 2 disulfide bonds;
    优选地，当母序列为SEQ ID NO:1且所述多肽含有1个或二个二硫键时，所述二硫键选自：Preferably, when the parent sequence is SEQ ID NO: 1 and the polypeptide contains one or two disulfide bonds, the disulfide bonds are selected from:
    所述多肽的N末端起的第一个半胱氨酸与第二个半胱氨酸形成的二硫键，以及第三个半胱氨酸与第四个半胱氨酸形成的二硫键；The disulfide bond formed by the first cysteine and the second cysteine from the N-terminus of the polypeptide, and the disulfide bond formed by the third cysteine and the fourth cysteine ；
    所述多肽的N末端起的第一个半胱氨酸与第四个半胱氨酸形成的二硫键，以及第二个半胱氨酸与第三个半胱氨酸形成二硫键；和The disulfide bond formed by the first cysteine and the fourth cysteine from the N-terminus of the polypeptide, and the disulfide bond formed by the second cysteine and the third cysteine; with
    所述多肽的N末端起的第一个半胱氨酸与第三个半胱氨酸形成的二硫键，以及第二个半胱氨酸与第四个半胱氨酸形成的二硫键。The disulfide bond formed by the first cysteine and the third cysteine from the N-terminus of the polypeptide, and the disulfide bond formed by the second cysteine and the fourth cysteine .
  4. 根据权利要求1至3中任一权利要求所述的多肽，其中，所述多肽的羧基末端是酰胺化的。The polypeptide of any one of claims 1 to 3, wherein the carboxy terminus of the polypeptide is amidated.
  5. 一种分离的多肽，其氨基酸序列分别如SEQ ID NOs:2－24、26－117中的任一序列所示；An isolated polypeptide whose amino acid sequence is shown in any one of SEQ ID NOs: 2-24 and 26-117;
    优选地，所述多肽的氨基酸序列如SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:23、SEQ ID NO:37、SEQ ID NO:45、SEQ ID NO:62、SEQ ID NO:75、SEQ ID NO:86或SEQ ID NO:94所示；Preferably, the amino acid sequence of the polypeptide is as SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 37, SEQ ID NO: 45, SEQ ID NO: 62, SEQ ID NO: 75, SEQ ID NO: 86 or SEQ ID NO: 94;
    更优选地，所述多肽的氨基酸序列如SEQ ID NO:45、SEQ ID NO:75、SEQ ID NO:86或SEQ ID NO:94所示；More preferably, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 45, SEQ ID NO: 75, SEQ ID NO: 86 or SEQ ID NO: 94;
    特别优选地，所述多肽的氨基酸序列如SEQ ID NO:75所示。Especially preferably, the amino acid sequence of the polypeptide is shown in SEQ ID NO:75.
  6. 根据权利要求5所述的多肽，其中，所述多肽含有0个、1个或2个二硫键；The polypeptide of claim 5, wherein the polypeptide contains 0, 1, or 2 disulfide bonds;
    优选地，当母序列为SEQ ID NO:1且所述多肽含有1个或2个二硫键时，所述二硫键选自：Preferably, when the parent sequence is SEQ ID NO: 1 and the polypeptide contains 1 or 2 disulfide bonds, the disulfide bonds are selected from:
    所述多肽的N末端起的第一个半胱氨酸与第二个半胱氨酸形成的二硫键，以及第 三个半胱氨酸与第四个半胱氨酸形成的二硫键；The disulfide bond formed by the first cysteine and the second cysteine from the N-terminus of the polypeptide, and the disulfide bond formed by the third cysteine and the fourth cysteine ；
    所述多肽的N末端起的第一个半胱氨酸与第四个半胱氨酸形成的二硫键，以及第二个半胱氨酸与第三个半胱氨酸形成二硫键；和The disulfide bond formed by the first cysteine and the fourth cysteine from the N-terminus of the polypeptide, and the disulfide bond formed by the second cysteine and the third cysteine; with
    所述多肽的N末端起的第一个半胱氨酸与第三个半胱氨酸形成的二硫键，以及第二个半胱氨酸与第四个半胱氨酸形成的二硫键。The disulfide bond formed by the first cysteine and the third cysteine from the N-terminus of the polypeptide, and the disulfide bond formed by the second cysteine and the fourth cysteine .
  7. 根据权利要求5或6所述的多肽，其中，所述多肽的羧基末端是酰胺化的。The polypeptide of claim 5 or 6, wherein the carboxy terminus of the polypeptide is amidated.
  8. 一种分离的融合蛋白，其包含至少一种权利要求1至7中任一权利要求所述的多肽。An isolated fusion protein comprising at least one polypeptide according to any one of claims 1 to 7.
  9. 一种分离的多核苷酸，其编码权利要求1至7中任一权利要求所述的多肽。An isolated polynucleotide encoding the polypeptide of any one of claims 1-7.
  10. 一种核酸构建体，其含有权利要求9所述的多核苷酸；优选地，所述核酸构建体为重组载体；优选地，所述核酸构建体为重组表达载体。A nucleic acid construct containing the polynucleotide of claim 9; preferably, the nucleic acid construct is a recombinant vector; preferably, the nucleic acid construct is a recombinant expression vector.
  11. 一种转化的细胞，其含有权利要求9所述的多核苷酸，或者权利要求10所述的核酸构建体。A transformed cell containing the polynucleotide of claim 9 or the nucleic acid construct of claim 10.
  12. 一种药物组合物，其含有至少一种权利要求1至7中任一权利要求所述的多肽；可选地，其还包含药学上可接受的辅料。A pharmaceutical composition comprising at least one polypeptide according to any one of claims 1 to 7; optionally, it also comprises pharmaceutically acceptable excipients.
  13. 权利要求1至7中任一权利要求所述的多肽在制备阻断或抑制α9α10 nAChR的药物中的用途。Use of the polypeptide according to any one of claims 1 to 7 in the preparation of a drug for blocking or inhibiting α9α10 nAChR.
  14. 权利要求1至7中任一权利要求所述的多肽在制备治疗和/或预防神经系统疾病或癌症的药物中的用途，在制备促进伤口愈合的药物中的用途，或者在制备镇痛的药物中的用途；Use of the polypeptide according to any one of claims 1 to 7 in the preparation of drugs for the treatment and/or prevention of neurological diseases or cancer, in the preparation of drugs for promoting wound healing, or in the preparation of drugs for analgesia Use in
    优选地，所述神经系统疾病为神经痛(慢性痛)、帕金森症、痴呆、精神分裂症 和抑郁中的至少一种；Preferably, the neurological disease is at least one of neuralgia (chronic pain), Parkinson's disease, dementia, schizophrenia and depression;
    优选地，所述神经痛选自如下的至少一种：坐骨神经痛、三叉神经痛、淋巴神经痛、多点运动神经痛、急性剧烈自发性神经痛、挤压神经痛以及复合神经痛；Preferably, the neuralgia is selected from at least one of the following: sciatica, trigeminal neuralgia, lymphatic neuralgia, multipoint motor neuralgia, acute severe spontaneous neuralgia, crush neuralgia and compound neuralgia;
    优选地，所述神经痛由如下因素中的至少一种导致：癌症、癌症化疗、酒精中毒、糖尿病、硬化症、带状疱疹、机械伤、手术伤、艾滋病、头部神经瘫痪、药物中毒、工业污染中毒、骨髓瘤、慢性先天性感觉神经病、脉管炎、血管炎、局部缺血、尿毒症、儿童胆汁肝脏疾病、慢性呼吸障碍、多器官衰竭、脓毒病/脓血症、肝炎、卟啉症、维生素缺乏、慢性肝脏病、原生胆汁硬化、高血脂症、麻疯病、莱姆关节炎、感觉神经束膜炎或过敏症；Preferably, the neuralgia is caused by at least one of the following factors: cancer, cancer chemotherapy, alcoholism, diabetes, sclerosis, herpes zoster, mechanical injury, surgical injury, AIDS, head nerve paralysis, drug poisoning, Industrial pollution poisoning, myeloma, chronic congenital sensory neuropathy, vasculitis, vasculitis, ischemia, uremia, childhood bile liver disease, chronic respiratory disorders, multiple organ failure, sepsis/sepsemia, hepatitis, Porphyria, vitamin deficiency, chronic liver disease, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, perineuritis or allergies;
    优选地，所述癌症为选自乳腺癌、肺癌、宫颈癌、卵巢癌、胰腺癌、白血病和神经细胞瘤中的至少一种。Preferably, the cancer is at least one selected from breast cancer, lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, leukemia, and neurocytoma.
  15. 一种在体内或体外阻断或抑制α9α10 nAChR或者调节乙酰胆碱水平的方法，包括给予受试者或者施加给细胞有效量的权利要求1至7中任一权利要求所述的多肽的步骤。A method for blocking or inhibiting α9α10 nAChR or regulating the level of acetylcholine in vivo or in vitro, comprising the step of administering to a subject or administering to a cell an effective amount of the polypeptide of any one of claims 1 to 7.
  16. 根据权利要求1至7中任一权利要求所述的多肽，其用于阻断或抑制α9α10 nAChR。The polypeptide according to any one of claims 1 to 7, which is used to block or inhibit α9α10 nAChR.
  17. 根据权利要求1至7中任一权利要求所述的多肽，其用于治疗和/或预防神经系统疾病或癌症，用于促进伤口愈合，或者用于镇痛；The polypeptide according to any one of claims 1 to 7, which is used for treating and/or preventing neurological diseases or cancer, for promoting wound healing, or for analgesia;
    优选地，所述神经系统疾病为神经痛、帕金森症、痴呆、精神分裂症和抑郁中的至少一种；Preferably, the neurological disease is at least one of neuralgia, Parkinson's disease, dementia, schizophrenia and depression;
    优选地，所述神经痛(慢性痛)选自如下的至少一种：坐骨神经痛、三叉神经痛、淋巴神经痛、多点运动神经痛、急性剧烈自发性神经痛、挤压神经痛以及复合神经痛；Preferably, the neuralgia (chronic pain) is selected from at least one of the following: sciatica, trigeminal neuralgia, lymphatic neuralgia, multipoint motor neuralgia, acute severe spontaneous neuralgia, crush neuralgia, and compound nerve pain;
    优选地，所述神经痛由如下因素中的至少一种导致：癌症、癌症化疗、酒精中毒、糖尿病、硬化症、带状疱疹、机械伤、手术伤、艾滋病、头部神经瘫痪、药物中毒、工业污染中毒、骨髓瘤、慢性先天性感觉神经病、脉管炎、血管炎、局部缺血、尿毒症、儿童胆汁肝脏疾病、慢性呼吸障碍、多器官衰竭、脓毒病/脓血症、肝炎、卟啉症、 维生素缺乏、慢性肝脏病、原生胆汁硬化、高血脂症、麻疯病、莱姆关节炎、感觉神经束膜炎或过敏症；Preferably, the neuralgia is caused by at least one of the following factors: cancer, cancer chemotherapy, alcoholism, diabetes, sclerosis, shingles, mechanical injury, surgical injury, AIDS, cranial nerve paralysis, drug poisoning, Industrial pollution poisoning, myeloma, chronic congenital sensory neuropathy, vasculitis, vasculitis, ischemia, uremia, childhood bile liver disease, chronic respiratory disorders, multiple organ failure, sepsis/sepsemia, hepatitis, Porphyria, vitamin deficiency, chronic liver disease, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, perineuritis or allergies;
    优选地，所述癌症为选自乳腺癌、肺癌、宫颈癌、卵巢癌、胰腺癌、白血病和神经细胞瘤中的至少一种。Preferably, the cancer is at least one selected from breast cancer, lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, leukemia, and neurocytoma.
  18. 一种治疗和/或预防神经系统疾病或癌症的方法，一种促进伤口愈合的方法，或者一种镇痛的方法，包括给予有需求的受试者以有效量的权利要求1至7中任一权利要求所述的多肽的步骤；A method of treating and/or preventing neurological diseases or cancer, a method of promoting wound healing, or a method of analgesia, comprising administering to a subject in need an effective amount of any of claims 1 to 7 A step of the polypeptide of claim;
    优选地，所述神经系统疾病为选自神经痛、帕金森症、痴呆、精神分裂症和抑郁中的至少一种；Preferably, the neurological disease is at least one selected from neuralgia, Parkinson's disease, dementia, schizophrenia and depression;
    优选地，所述神经痛(慢性痛)选自如下的至少一种：坐骨神经痛、三叉神经痛、淋巴神经痛、多点运动神经痛、急性剧烈自发性神经痛、挤压神经痛以及复合神经痛；Preferably, the neuralgia (chronic pain) is selected from at least one of the following: sciatica, trigeminal neuralgia, lymphatic neuralgia, multipoint motor neuralgia, acute severe spontaneous neuralgia, crush neuralgia, and compound nerve pain;
    优选地，所述神经痛由如下因素中的至少一种导致：癌症、癌症化疗、酒精中毒、糖尿病、硬化症、带状疱疹、机械伤、手术伤、艾滋病、头部神经瘫痪、药物中毒、工业污染中毒、骨髓瘤、慢性先天性感觉神经病、脉管炎、血管炎、局部缺血、尿毒症、儿童胆汁肝脏疾病、慢性呼吸障碍、多器官衰竭、脓毒病/脓血症、肝炎、卟啉症、维生素缺乏、慢性肝脏病、原生胆汁硬化、高血脂症、麻疯病、莱姆关节炎、感觉神经束膜炎或过敏症；Preferably, the neuralgia is caused by at least one of the following factors: cancer, cancer chemotherapy, alcoholism, diabetes, sclerosis, shingles, mechanical injury, surgical injury, AIDS, cranial nerve paralysis, drug poisoning, Industrial pollution poisoning, myeloma, chronic congenital sensory neuropathy, vasculitis, vasculitis, ischemia, uremia, childhood bile liver disease, chronic respiratory disorders, multiple organ failure, sepsis/sepsemia, hepatitis, Porphyria, vitamin deficiency, chronic liver disease, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, perineuritis or allergies;
    优选地，所述癌症为选自乳腺癌、肺癌、宫颈癌、卵巢癌、胰腺癌、白血病和神经细胞瘤中的至少一种。Preferably, the cancer is at least one selected from breast cancer, lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, leukemia, and neurocytoma.
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