α-conotoxin peptide lvid mutant, pharmaceutical composition comprising same, and use thereof

Abstract

Provided are an α-conotoxin peptide LvID mutant, a pharmaceutical composition comprising same, and a use thereof. Specifically, provided is an isolated polypeptide, which is a mutant of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, and comprises the following mutations: E5A and Q11A. The isolated polypeptide can specifically block α7 nicotinic acetylcholine receptors (nAChRs), has high selectivity and strong blocking activity for the α7 nAChRs, and has the potential for preparing a drug for treating or preventing diseases related to the α7 nAChRs.

Description

Alpha-conotoxin peptide Lv ID mutant, pharmaceutical composition thereof and use thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biology and medicine, and relates to a mutant of an alpha-conotoxin peptide Lv ID, a pharmaceutical composition thereof and use thereof. BACKGROUND

[0002] Nicotinic acetylcholine receptors (nAChRs) belong to ligand-gated ion channels, are pentameric transmembrane allosteric proteins located on cell membranes, and are the first class of receptors discovered in humans. nAChRs mediate the physiological functions of the central and peripheral nervous systems, the immune system, and the like, including learning, memory, response, analgesia, sensation, hearing, vision, emotion, inflammation, and movement control 1-3 . nAChRs activate the release of dopamine, norepinephrine, serotonin, gamma-aminobutyric acid, and a variety of neurotransmitters, and have important physiological functions and clinical significance 4,5 . They are assembled into many subtypes by different alpha and beta subunits, and are divided into two categories of muscle type and nerve type. The muscle type acetylcholine receptors have only two subtypes of fetal type and adult type, both of which are composed of 5 subunits, contain 2 alpha1 subunits, 1 beta1 subunit, 1 delta subunit, and 1 fetal gamma or adult epsilon subunit. The subtypes of mammalian nerve type acetylcholine receptors are much more complex than the muscle type 6,7 , at least 8 alpha subunits and 3 beta subunits, respectively, alpha2-alpha7, alpha9, alpha10 (alpha8 exists in chicks), and beta2-beta4 (Figure 10). Among them, alpha2, alpha3 and alpha4 can be combined with beta2 or beta4 to form functional receptors, such as alpha2beta2, alpha3beta2, alpha4beta4, and the like. In addition, alpha7 and alpha9 can form homopentamers 8 .

[0003] Each subtype of nAChRs has completely different pharmacological characteristics, but the structures of the subtypes are very similar and difficult to distinguish 9-11 . The abnormal structures and functions of these subtypes are the root causes of many difficult and complex diseases, including pain, addiction, inflammation, depression, dementia (AD), schizophrenia, Parkinson's disease, epilepsy, attention deficit hyperactivity disorder (hyperactivity, ADHD), AIDS (HIV), Rett syndrome, autism, aggressive psychosis, anxiety, phobia, mental retardation, cancer, and the like 12-22 . Therefore, each subtype of nAChRs has become a key target for screening, diagnosis, and treatment of the above-mentioned important diseases 23So far, the fine structure and function of various subtypes of acetylcholine receptors are poorly understood, and there is no specific drug for symptomatic treatment for many diseases related to each receptor subtype, and the pathogenesis is also unclear. Therefore, it is urgently needed to find molecular probes or tools that can distinguish each subtype, which will help to study the structure and function of each subtype, reveal the pathogenesis of diseases related to each subtype, and develop new drugs for treatment. However, due to the lack of highly selective ligand compounds for various subtypes, that is, new molecular lead drugs, it is challenging to study and clarify the fine structure and function of various nAChRs subtypes, and it also limits the development of new drugs for the treatment of many diseases related to them.

[0004] Among them, the α7 acetylcholine receptor subtype (abbreviated as α7 nAChR, or α7 nAChRs, referred to as α7 receptor) is a homologous pentamer transmembrane ion channel composed of 5 identical α7 subunits (Figure 10) 8 Compared with other subtypes, α7 nAChRs have the highest calcium ion (Ca 2+ ) permeability and can rapidly activate and gradually desensitize 24 . α7 nAChRs are widely distributed in the central nervous system, peripheral nervous system and immune system of mammals, including neural cells and non-neural cells, such as the presynaptic region of most neural cells, the postsynaptic region, astrocytes, microglia, macrophages, endothelial cells, etc. 25,26 In particular, α7 nAChRs are highly expressed in the brain and throughout the hippocampus, are closely related to many neuropsychiatric and neurodegenerative diseases, are important targets for the development of new drugs for the treatment of related diseases, and have attracted widespread interest and high attention from scientists around the world 1,27 The affinity of α7 nAChRs for its endogenous neurotransmitter ligand acetylcholine (Ach) is lower than that of other subtypes, and the half-maximum effective concentration (EC 50 ) of α7 nAChRs for Ach is about 200 μM, while the EC 50 of other neuronal subtypes is mostly about 100 μM. The timing of α7 nAChRs activation is related to excitatory signal input, thereby controlling postsynaptic plasticity and affecting theta oscillations. A single basal forebrain cholinergic neuron can dominate all layers of the hippocampus, coordinating the activity of the hippocampal digit 28,29To treat diseases related to dysfunction of α7 nAChRs, it is necessary to ensure the normal function of cholinergic projection neurons, or to use modulators (ligands) of α7 nAChRs, i.e. specific agonists or inhibitors (antagonists), to enhance or weaken the function thereof, so as to maintain the response kinetics in a normal and healthy stable state.

[0005] α7 nAChRs are the second most prevalent subtype in the nervous system next to α4β2 nAChRs, and play an important role in memory and attention in the prefrontal cortex 30 . Meanwhile, α7 nAChRs are also key subtypes in the cholinergic anti-inflammatory pathway, responsible for linking the brain and the immune system, and are distributed in T cells, B cells and other cells involved in the anti-inflammation of tissues and organs 31 . Non-neuronal α7 nAChRs are closely related to neuroinflammation, chronic pain (neuralgia), sepsis, cancer, mental disorders, inflammation, etc. 5,32,33 The decrease or lack of endogenous neurotransmitter acetylcholine (ACh) secretion caused by aging is likely to be caused by the silencing of α7 nAChRs gene by microRNA-6775 (miR-6775). After the silencing of the α7 subunit gene, the expression of α7 receptors is reduced, which promotes the occurrence of diseases such as inflammation, neuroinflammation (neuralgia), mental disorders, etc. in the elderly; and causes the expression of corresponding genes and receptors in lymphocytes and T-cells, and promotes various diseases such as cancer and sepsis caused by immunosuppression and low immunity.

[0006] α7 nAChRs are important for thought processes, and are closely related to cognitive and memory functions due to their high expression in the hippocampus and cortical regions of the human brain 34 . Clinical studies have shown that α7 nAChRs are closely related to cognitive impairment, Alzheimer's disease, Parkinson's disease, epilepsy, schizophrenia and other neurological diseases. Current drug research focuses on the treatment of Alzheimer's disease and schizophrenia by α7 nAChR agonists 35,36 . It has been observed from animal and human studies that α7 nAChR agonists can improve the cognitive ability of schizophrenia to varying degrees, further supporting the involvement of this receptor subtype in the multiple deficits of schizophrenia. Therefore, specific activation of α7 nAChRs has the potential to develop new methods for treating schizophrenia 13,37,38 . Homologous α7 nAChRs are of great significance to the maintenance of cognitive abilities such as memory and attention, and a large number of studies in recent years have shown that this receptor can be used as a therapeutic target for cognitive impairment, and some α7 nAChR agonists have entered phase III clinical trials 39 . Selective α7 nAChR agonist PNU28298 can effectively assist in the treatment of schizophrenia 40 .

[0007] α7 nAChRs not only closely related to many neuropsychiatric and neurodegenerative diseases, but also plays a key role in the development of various malignant tumors. Cholangiocarcinoma is one of the most deadly malignant tumors. Studies have shown that α7 nAChRs is highly expressed in cholangiocarcinoma tissue, which is closely related to the shortening of patient survival. Knockout of α7 nAChRs reduces cell proliferation, increases early apoptosis, and weakens cell migration and invasion. When α7 nAChRs is knocked out, the apoptosis-related proteins and components in the process of epithelial-mesenchymal transition change 41 . The mortality rate of pancreatic ductal adenocarcinoma (PDAC) is almost 100%, because it is already in the advanced stage when it is found, and existing anticancer drugs are ineffective. Studies have found that α7 nAChRs is a major regulator of pancreatic cancer, which promotes the occurrence of PDAC by activating β-adrenergic signaling mediated by neurotransmitters 42 . Pulmonary neuroendocrine cells (PNECs) play an important role in the occurrence of small cell lung cancer (SCLC) and pediatric asthma, and the up-regulation of α7 nAChRs expression promotes the occurrence and deterioration of these two diseases, and smoking is a risk factor for the occurrence of these two diseases 43 . Similarly, smoking is also a risk factor for non-small cell lung cancer (NSCLC), and the up-regulation of α7 nAChRs expression promotes the occurrence of non-small cell lung cancer under the stimulation of nicotine 44 . Nicotine activates α7 nAChRs to increase the population of tumor stem cells, promote their rapid proliferation and self-renewal, and make breast cancer worse and more difficult to treat by resisting anticancer drug treatment in the occurrence, development, angiogenesis, diffusion, metastasis, drug resistance, etc. of breast cancer 45 . Therefore, using specific blockers of α7 nAChRs can effectively inhibit the activity of α7 nAChRs in cholangiocarcinoma, pancreatic cancer, small cell lung cancer, pediatric asthma, non-small cell lung cancer, breast cancer, etc., and down-regulate its expression, which may treat these major diseases.

[0008] α7 nAChRs is widely distributed in the mammalian body, and its physiological and pathological functions are complex and dependent on various factors such as cell type, organ site, development stage, time period, cell microenvironment, other associated molecular chaperones, receptors or ion channels, etc 46-48The fine functions of a7 nAChRs in different cell types, different tissues and organs, and the corresponding physiological environment are still a mystery. Abnormal inactivation and overexpression of the receptor can lead to serious physiological dysfunction and the occurrence of various diseases, the pathogenesis and etiology of which are unclear, and there is a lack of effective treatment drugs in clinical practice. Despite this, research on the physiological function of a7 nAChRs continues to grow, which provides more theoretical basis for the development of new drugs targeting a7 nAChRs. Recent studies have found that during the thrombosis caused by COVID-19, the activation of a7 nAChRs on platelets can increase the level of nitric oxide (NO) and inhibit the over-activated platelets, thereby reducing thrombosis and reducing the damage of inflammation to COVID-19 patients 49 .

[0009] It is particularly noteworthy that the maintenance of fear and anxiety memory requires the activation of a7 nAChRs on a specific subpopulation of astrocytes in the cerebral cortex 50 The results of this study show that in a mouse noise and shock fear aversion animal model, aversive sensory stimuli activate the opening of a7 nAChRs on a subpopulation of astrocytes in the auditory cortex of the mouse brain. Fear memory induces these astrocytes to produce new calcium ion transient currents, which can last for many days with fear memory, and this special current does not exist in the brains of mice without fear memory. Conditional knockout of a7 nAChRs on these astrocytes significantly weakens the persistence of fear memory, and the formation of long-term memory of fear requires the specific activation of a7 nAChRs on this specific subpopulation of astrocytes to produce calcium ion transient influx 50 Thus, this study suggests that if the activation of a7 nAChRs on this population of astrocytes in the auditory cortex of the brain can be specifically blocked, such as the discovery of a7 nAChRs specific blockers, the inhibition of a7 nAChRs opening on these cells, it is expected to treat refractory neuropsychiatric diseases or complications related to excessive long-term memory of fear and aversion, including phobia, anxiety, depression, obsessive-compulsive disorder, mental trauma, schizophrenia, affective disorder, etc.

[0010] A series of conotoxin peptides targeting different subtypes of nAChRs have been found 51 However, there is a severe lack of conotoxin peptides with strong blocking activity specific to a7 nAChRs. The a7 nAChRs blockers found so far either have weak activity; or have blocking effects on other subtypes in addition to the a7 subtype, with poor selectivity; or have a large difference in blocking activity between murine and human a7 nAChRs, with species distinction. It is difficult to use them as molecular probes for studying the corresponding diseases and for the development of new drugs for treatment.

[0011] Therefore, there is an urgent need to find specific strong blockers that have the same activity on rat and human α7 nAChRs, so as to share the experimental results between the rodent experimental animal models and the human disease mechanism research, and the development of new drugs for treatment. Otherwise, the efficacy in experimental animals will not be reproduced in humans, resulting in the failure of new drug development. Because experiments cannot be directly conducted on humans, it is also not conducive to pharmaceutical research on experimental animal models if it only has blocking activity on human α7 nAChRs and has no activity on murine receptors. In view of the important physiological and pathological functions of α7 nAChRs, it is of great significance to find new strong and selective α7 nAChRs specific blockers (inhibitors, antagonists), especially blockers that can distinguish between structurally similar and distributionally overlapping nAChRs subtypes, for the research of disease mechanisms related to α7 nAChRs, the screening of new drugs, and the development of new drugs for treatment.

[0012] SUMMARY

[0013] The present inventors have made intensive studies and creative efforts, and found a new α-conotoxin peptide LvID mutant peptide. The present inventors have surprisingly found that the LvID mutant peptide can specifically block rat and human α7 acetylcholine receptors, has high selectivity and strong blocking activity, and has potential for use in the preparation or screening of drugs for treating or preventing diseases related to α7 acetylcholine receptors. Thus, the following invention is provided:

[0014] One aspect of the present invention relates to an isolated polypeptide which is a mutant of a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1, and comprises the following mutations:

[0015] E5A and Q11A.

[0016] In some embodiments of the present invention, the isolated polypeptide further comprises the following mutations:

[0017] D1G and / or I9R.

[0018] In some embodiments of the present invention, the isolated polypeptide further comprises the following mutations:

[0019] D14H and / or I15D.

[0020] In some embodiments of the present invention, the isolated polypeptide comprises the following mutations:

[0021] E5A, Q11A and D1G.

[0022] In some embodiments of the present invention, the isolated polypeptide comprises the following mutations:

[0023] E5A, Q11A, and I9R.

[0024] In some embodiments of the application, the isolated polypeptide comprises the following mutations:

[0025] E5A, Q11A, D1G, and I9R.

[0026] In some embodiments of the application, the isolated polypeptide comprises the following mutations:

[0027] E5A, Q11A, and D14H.

[0028] In some embodiments of the application, the isolated polypeptide comprises the following mutations:

[0029] E5A, Q11A, and I15D.

[0030] In some embodiments of the application, the isolated polypeptide comprises the following mutations:

[0031] E5A, Q11A, D14H, and I15D.

[0032] In some embodiments of the application, the isolated polypeptide comprises the following mutations:

[0033] E5A, Q11A, D1G, and D14H.

[0034] In some embodiments of the application, the isolated polypeptide comprises the following mutations:

[0035] E5A, Q11A, D1G, and I15D.

[0036] In some embodiments of the application, the isolated polypeptide comprises the following mutations:

[0037] E5A, Q11A, D1G, D14H, and I15D.

[0038] In some embodiments of the application, the isolated polypeptide comprises the following mutations:

[0039] E5A, Q11A, I9R, and D14H.

[0040] In some embodiments of the application, the isolated polypeptide comprises the following mutations:

[0041] E5A, Q11A, I9R, and I15D.

[0042] In some embodiments of the application, the isolated polypeptide comprises the following mutations:

[0043] E5A, Q11A, I9R, D14H, and I15D.

[0044] In some embodiments of the present application, the isolated polypeptide comprises the following mutations:

[0045] E5A, Q11A, D1G, I9R, and D14H.

[0046] In some embodiments of the present application, the isolated polypeptide comprises the following mutations:

[0047] E5A, Q11A, D1G, I9R, and I15D.

[0048] In some embodiments of the present application, the isolated polypeptide comprises the following mutations:

[0049] E5A, Q11A, D1G, I9R, D14H, and I15D.

[0050] In some embodiments of the present application, the isolated polypeptide wherein,

[0051] the first cysteine and the third cysteine from the N terminus of the isolated polypeptide form a disulfide bond, and the second cysteine and the fourth cysteine form a disulfide bond; or the first cysteine and the fourth cysteine from the N terminus of the isolated polypeptide form a disulfide bond, and the second cysteine and the third cysteine form a disulfide bond; or the first cysteine and the second cysteine from the N terminus of the isolated polypeptide form a disulfide bond, and the third cysteine and the fourth cysteine form a disulfide bond.

[0052] Preferably, the carboxyl terminus of the isolated polypeptide is amidated.

[0053] In some embodiments of the present application, the isolated polypeptide is a mutant of a polypeptide having an amino acid sequence shown in SEQ ID NO: 1, and the mutation is selected from any one of (1) to (16) below:

[0054] (1) E5A and Q11A;

[0055] (2) E5A, Q11A, and D1G;

[0056] (3) E5A, Q11A, and I9R;

[0057] (4) E5A, Q11A, D1G, and I9R;

[0058] (5) E5A, Q11A, and D14H;

[0059] (6) E5A, Q11A, and I15D;

[0060] (7) E5A, Q11A, D14H and I15D;

[0061] (8) E5A, Q11A, D1G and D14H;

[0062] (9) E5A, Q11A, D1G and I15D;

[0063] (10) E5A, Q11A, D1G, D14H and I15D;

[0064] (11) E5A, Q11A, I9R and D14H;

[0065] (12) E5A, Q11A, I9R and I15D;

[0066] (13) E5A, Q11A, I9R, D14H and I15D;

[0067] (14) E5A, Q11A, D1G, I9R and D14H;

[0068] (15) E5A, Q11A, D1G, I9R and I15D; and

[0069] (16) E5A, Q11A, D1G, I9R, D14H and I15D;

[0070] Preferably, the first cysteine from the N-terminus of the isolated polypeptide forms a disulfide bond with the third cysteine, and the second cysteine forms a disulfide bond with the fourth cysteine; or the first cysteine from the N-terminus of the isolated polypeptide forms a disulfide bond with the fourth cysteine, and the second cysteine forms a disulfide bond with the third cysteine; or the first cysteine from the N-terminus of the isolated polypeptide forms a disulfide bond with the second cysteine, and the third cysteine forms a disulfide bond with the fourth cysteine.

[0071] Preferably, the carboxyl terminus of the isolated polypeptide is amidated.

[0072] In some embodiments of the application, the isolated polypeptide, wherein,

[0073] The isolated polypeptide has the same or improved activity of blocking human alpha7 nAChR or rat alpha7 nAChR as compared to a polypeptide having an amino acid sequence set forth in SEQ ID NO: 1.

[0074] In some embodiments of the application, the isolated polypeptide has an amino acid sequence set forth in any one of SEQ ID NOs: 7, 10-15 and 18-19.

[0075] Preferably, the first cysteine from the N-terminus of the isolated polypeptide forms a disulfide bond with the third cysteine, and the second cysteine forms a disulfide bond with the fourth cysteine; or the first cysteine from the N-terminus of the isolated polypeptide forms a disulfide bond with the fourth cysteine, and the second cysteine forms a disulfide bond with the third cysteine; or the first cysteine from the N-terminus of the isolated polypeptide forms a disulfide bond with the second cysteine, and the third cysteine forms a disulfide bond with the fourth cysteine.

[0076] Preferably, the carboxyl terminus of the isolated polypeptide is amidated.

[0077] In some embodiments of the present application, the isolated polypeptide is any one of the mutant peptides shown in Table 1.

[0078] Another aspect of the present application relates to an isolated fusion protein comprising at least one polypeptide of any one of the present application.

[0079] Still another aspect of the present application relates to an isolated polynucleotide encoding the isolated polypeptide of any one of the present application or the isolated fusion protein of the present application.

[0080] Still another aspect of the present application relates to a nucleic acid construct comprising the isolated polynucleotide of the present application; preferably, the nucleic acid construct is a recombinant vector; preferably, the nucleic acid construct is a recombinant expression vector.

[0081] Still another aspect of the present application relates to a recombinant host cell comprising the isolated polynucleotide of the present application or comprising the nucleic acid construct of the present application.

[0082] Still another aspect of the present application relates to a pharmaceutical composition comprising at least one isolated polypeptide of any one of the present application, the isolated fusion protein of the present application, the isolated polynucleotide of the present application, the nucleic acid construct of the present application or the recombinant host cell of the present application; preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients; preferably, the pharmaceutical composition is a lyophilized agent or an injection solution.

[0083] Still another aspect of the present application relates to the use of the isolated polypeptide of any one of the present application or the isolated fusion protein of the present application in the preparation of a medicament for blocking or inhibiting α7 nAChR of a mammal; preferably, the mammal is a human or a rat.

[0084] Still another aspect of the present application relates to the use of the isolated polypeptide of any one of the present application or the isolated fusion protein of the present application in the manufacture of a medicament for treating or preventing cancer, asthma, Alzheimer's disease or intractable neuropsychiatric diseases associated with excessive long-term memory of fear and / or aversion or complications thereof;

[0085] Preferably, the cancer is one or more selected from the group consisting of cholangiocarcinoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer and breast cancer;

[0086] Preferably, the asthma is pediatric asthma.

[0087] Preferably, the intractable neuropsychiatric disease associated with excessive long-term memory of fear and / or aversion or complications thereof is one or more selected from the group consisting of phobia, anxiety, depression, obsessive-compulsive disorder, post-traumatic stress disorder, schizophrenia and affective disorder.

[0088] The conotoxin peptides of the present application can be applied to research, diagnosis, screening and treatment of cancer, dementia, schizophrenia, neuropathic pain, Parkinson's disease, depression, fear, anxiety and other nervous system diseases, and as useful molecular probes for research, etc. Different α-conotoxins have different affinities for vertebrate receptors, sometimes by several orders of magnitude. This interspecific difference enables α-conotoxins to be used as useful probes for studying the phylogeny of vertebrate nAChRs and as molecular probes to determine different subtypes of nAchRs. They are candidate drugs, lead drugs and therapeutic drugs for new drug development.

[0089] The isolated polypeptide according to any one of the present application or the isolated fusion protein of the present application is used for blocking or inhibiting α7 nAChR of a mammal; preferably, the mammal is a human or a rat.

[0090] The isolated polypeptide according to any one of the present application or the isolated fusion protein of the present application is used for treating or preventing cancer, asthma, Alzheimer's disease or intractable neuropsychiatric diseases associated with excessive long-term memory of fear and / or aversion or complications thereof;

[0091] Preferably, the cancer is one or more selected from the group consisting of cholangiocarcinoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer and breast cancer;

[0092] Preferably, the asthma is pediatric asthma.

[0093] Preferably, the intractable neuropsychiatric disease associated with excessive long-term memory of fear and / or aversion or complications thereof is one or more selected from the group consisting of phobia, anxiety, depression, obsessive-compulsive disorder, post-traumatic stress disorder, schizophrenia and affective disorder.

[0094] Yet another aspect of the present application relates to a method of blocking or inhibiting α7 nAChR in a subject, comprising the step of administering to a subject in need thereof an effective amount of the isolated polypeptide of any one of the present application or the isolated fusion protein of the present application; the subject is a mammal; preferably, the mammal is a human or a rat.

[0095] Yet another aspect of the present application relates to a method of treating or preventing a disease, comprising the step of administering to a subject in need thereof an effective amount of the isolated polypeptide of any one of the present application or the isolated fusion protein of the present application; wherein the subject is a human; and the disease is one or more selected from the group consisting of cancer, asthma, Alzheimer's disease, and intractable neuropsychiatric diseases associated with excessive long-term memory of fear and / or aversion or complications thereof.

[0096] Preferably, the cancer is one or more selected from the group consisting of cholangiocarcinoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, and breast cancer.

[0097] Preferably, the asthma is pediatric asthma.

[0098] Preferably, the intractable neuropsychiatric diseases associated with excessive long-term memory of fear and / or aversion or complications thereof is one or more selected from the group consisting of phobia, anxiety, depression, obsessive-compulsive disorder, mental trauma, schizophrenia, and affective disorder.

[0099] The research results of Example 6 and Example 7 of the present application show that the α-conotoxin LvID mutant of the present application, particularly [D1G, E5A, I9R, Q11A]LvID, can specifically block α7 nAChRs in the brain of Alzheimer's disease (AD) transgenic model mice, significantly improve the spatial memory impairment of AD mice, and has good application value in the treatment or prevention of AD and learning and memory related diseases.

[0100] In some embodiments, the pharmaceutical composition containing a therapeutically effective amount of the isolated polypeptide of the present application is formulated and administered in a manner that is physiologically compatible with the individual patient, taking into account the individual patient's clinical condition, delivery site, method of administration, schedule of administration, and other factors known to the physician. Thus, an "effective amount" for purposes herein is determined by consideration of these aspects.

[0101] The dosage administered will depend on a number of factors, such as the severity of the condition being treated, the gender, age, weight, and individual response of the patient or animal, and the condition and prior medical history of the patient to be treated. It is common practice in the art to start with a lower level than that required to achieve the desired therapeutic effect, and to increase the dosage until the desired effect is achieved.

[0102] Yet another aspect of the present application relates to a method of producing the isolated polypeptide of any one of the present application, comprising the steps of:

[0103] 1) Synthesis of linear polypeptide on an ABI Prism 433a polypeptide synthesizer, or other polypeptide synthesizer, or by manual method, with the side chain protecting groups of Fmoc amino acids being: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp), Boc (Lys); Cysteine is protected with Trt or Acm protecting group, respectively, to form disulfide bond between the corresponding cysteines;

[0104] 2) Cleavage of the linear polypeptide from the resin obtained in step 1) and precipitation and washing of the linear polypeptide crude product with ice-ethanol, and purification by preparative reverse HPLC C18 column (Vydac);

[0105] 3) Two-step oxidative folding of the product obtained in step 2).

[0106] The following terms used in the present application are explained.

[0107] The term "nucleic acid construct" is defined herein as a single- or double- stranded nucleic acid molecule, preferably an artificially constructed nucleic acid molecule. Optionally, the nucleic acid construct further comprises one or more regulatory sequences operably linked.

[0108] In the present application, the term "operably linked" refers to a functional spatial arrangement of two or more nucleotide regions or nucleic acid sequences. The "operably linked" can be achieved by means of genetic recombination.

[0109] In the present application, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide inhibiting a certain protein is inserted. For example, the vector includes: a plasmid; a phagemid; a cosmid; an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a P1 -derived artificial chromosome (PAC); a bacteriophage such as a lambda phage or a M13 phage; and an animal virus, etc. The animal virus used as a vector includes a retrovirus (including a lentivirus), an adenovirus, an adeno-associated virus, a herpes virus (e.g., a herpes simplex virus), a poxvirus, a baculovirus, a papillomavirus, a papovavirus (e.g., SV40), etc. A vector can contain various elements controlling expression.

[0110] In the present application, the term "host cell" refers to a cell into which a vector is introduced, including a wide variety of cell types such as prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0111] The term "effective amount" refers to a dose that can achieve treatment, prevention, reduction, and / or alleviation of the disease or disorder described in the present application in a subject.

[0112] The term "disease and / or disorder" refers to a physical state of the subject that is associated with the disease and / or disorder described in the present application.

[0113] The term "subject" can refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives a pharmaceutical composition of the present application to treat, prevent, reduce, and / or alleviate the disease or disorder described in the present application.

[0114] In the present application, if not otherwise specified, the concentration unit μM indicates μmol / L, mM indicates mmol / L, and nM indicates nmol / L.

[0115] In the present application, when referring to the amount of drug added to a cell, if not otherwise specified, it generally refers to the final concentration of the drug after addition.

[0116] In the present application, when referring to the term "amino acid" or a specific amino acid name, if not otherwise specified, it refers to the L-form of the amino acid.

[0117] Advantages of the Invention

[0118] The present application achieves one or more of the following technical effects (1) to (3):

[0119] (1) The conotoxin peptide (LvID mutant) of the present application can effectively block α7 nAChRs.

[0120] (2) The conotoxin peptide (LvID mutant) of the present application can specifically block α7 nAChRs.

[0121] (3) The conotoxin peptide of the present application can exert an effect by binding to α7 acetylcholine receptors (nAChR), and has an anticancer and anti-Alzheimer's disease effect. BRIEF DESCRIPTION OF DRAWINGS

[0122] Figure 1: Sequences of a-conotoxin LvID and its mutant peptides, in which cysteine (Cys, C) is shown in red bold font. These polypeptides all contain two pairs of disulfide bonds, with the disulfide bond connection pattern being Cys(I-III, II-IV). Their C-termini are all amidated, indicated by “#”. In the mutant sequences, the amino acid mutation sites that are different from the parent peptide LvID are shown in blue or green font. The mutant is named as “[original site amino acid name + order number of the site from N-terminus to C-terminus + mutated amino acid type, …, …]-LvID”.

[0123] Figure 2: Substituting glutamic acid at position 5 with alanine (A) and isoleucine at position 9 with arginine (R) and glutamine at position 11 with alanine (A) in the sequence of a-conotoxin LvID produces mutant peptide [E5A, I9R, Q11A]LvID (SEQ ID NO: 18). Substituting aspartic acid at position 1 with glycine (G), glutamic acid at position 5 with alanine (A), isoleucine at position 9 with arginine (R), and glutamine at position 11 with alanine (A) in the sequence of a-conotoxin LvID produces mutant peptide [D1G, E5A, I9R, Q11A]LvID (SEQ ID NO: 19). The disulfide bond connection pattern of these two mutant peptides is Cys(I-III, II-IV); “#” indicates C-terminal amidation.

[0124] Figures 3A-3B: HPLC (Figure 3A) and ESI-MS (Figure 3B) chromatograms of [E5A, I9R, Q11A]LvID (SEQ ID NO: 18).

[0125] In Figure 3A, the HPLC analysis conditions are as follows:

[0126] C18 column (Vydac), elution linear gradient is 10-40% B60 (containing 60% acetonitrile aqueous solution) within 0-40 min, monitoring wavelength is 214 nm. Solvent B60 is 90% acetonitrile (ACN, acetonitrile), 0.05% TFA (trifluoroacetic acid) aqueous solution; solvent A is 0.05% TFA aqueous solution; the number of minutes in the HPLC chromatogram represents the retention time of the chromatographic peak; the abscissa is the elution time, in minutes (min); the ordinate is the ultraviolet absorption value (UV 214 ) at a wavelength of 214 nm.

[0127] Figure 4A-B: HPLC (Figure 4A) and ESI-MS (Figure 4B) of [D1G, E5A, I9R, Q11A] LvID (SEQ ID NO: 19).

[0128] Wherein the HPLC analysis conditions in Figure 4A are the same as in Figure 3A.

[0129] Figure 5A-B: Effect of [E5A, I9R, Q11A] LvID (SEQ ID NO: 18, Figure 5A) and [D1G, E5A, I9R, Q11A] LvID (SEQ ID NO: 19, Figure 5B) on the current of each nAChR subtypes expressed in Xenopus oocytes at high concentration of 10 μΜ. All data represent Mean ± S.E.M, n = 4-6. The abscissa is the current response in percentage %, calculated as: the current of each nAChR subtypes at 10 μΜ concentration of [E5A, I9R, Q11A] LvID divided by the current of the respective Control (Control ND96). Here Control means the current produced by Ach stimulation after incubation of the same volume of ND96 buffer as the drugs [E5A, I9R, Q11A] LvID and [D1G, E5A, I9R, Q11A] LvID in the cell chamber for 5 min. The h in the figure stands for human, r for rat, M for mouse, and so on.

[0130] Figure 6A-I: Current traces of [E5A, I9R, Q11A] LvID (SEQ ID NO: 18, Figure 6A-C) and [D1G, E5A, I9R, Q11A] LvID (SEQ ID NO: 19, Figure 6D-I) blocking α7 acetylcholine receptors and other similar subtypes. Both mutants are highly selective and strong blockers of rat and human α7 subtypes. The "C" in the figure means the Control (ND96) current, followed by the concentration of the polypeptide. The arrow points to the current trace of the first Ach pulse after incubation for 5 min, blocked by the polypeptide for the corresponding receptor subtype. Each receptor subtype was expressed in Xenopus oocytes, and the clamped voltage was -70 mV during electrophysiological recording, with 1 s Ach pulse every 1 min according to the experimental procedure.

[0131] [E5A, I9R, Q11A] LvID (SEQ ID NO: 18) can completely block the current of human and rat α7 (Figure 6A) (Figure 6B); but has weak blocking activity on human α6 / α3β4 (Figure 6C) at high concentration (10 μΜ).

[0132] [D1G, E5A, I9R, Q11A] LvID (SEQ ID NO: 19) completely blocked the current of human and rat α7 (Fig. 6D-E); but at high concentration (10 μM), it had higher blocking activity on human α6 / α3β4 (Fig. 6F) and weak blocking activity on rat α3β4 (Fig. 6G), human α3β4 (Fig. 6H), rat α3β2 nAChRs (Fig. 61). Neither of the two mutants had blocking activity on other subtypes.

[0133] wherein,

[0134] Fig. 6A: Effect of 100 nM [E5A, I9R, Q11A] LvID on the current of human α7 nAChR;

[0135] Fig. 6B: Effect of 100 nM [E5A, I9R, Q11A] LvID on the current of rat α7 nAChR;

[0136] Fig. 6C: Effect of 10 μM [E5A, I9R, Q11A] LvID on the current of rat α6 / α3β4 nAChR;

[0137] Fig. 6D: Effect of 100 nM [D1G, E5A, I9R, Q11A] LvID on the current of human α7 nAChR;

[0138] Fig. 6E: Effect of 100 nM [D1G, E5A, I9R, Q11A] LvID on the current of rat α7 nAChR;

[0139] Fig. 6F: Effect of 10 μM [D1G, E5A, I9R, Q11A] LvID on the current of human α6 / α3β4 nAChR;

[0140] Fig. 6G: Effect of 10 μM [D1G, E5A, I9R, Q11A] LvID on the current of rat α3β4 nAChR;

[0141] Fig. 6H: Effect of 10 μM [D1G, E5A, I9R, Q11A] LvID on the current of human α3β4 nAChR;

[0142] Fig. 61: Effect of 10 μM [D1G, E5A, I9R, Q11A] LvID on the current of rat α3β2 nAChR.

[0143] Figure 7A-7B: Concentration dose response curves of [E5A, I9R, Q11A]LvID (SEQ ID NO: 18, Figure 7A) and [D1G, E5A, I9R, Q11A]LvID (SEQ ID NO: 19, Figure 7B) on various nAChRs subtypes. The horizontal axis represents the logarithm of the molar concentration (Log[Peptide]M) of the [E5A, I9R, Q11A]LvID (Figure 7A) and [D1G, E5A, I9R, Q11A]LvID (Figure 7B) polypeptides used; the vertical axis represents the response percentage (%), which is the percentage of the acetylcholine receptor current at the corresponding concentration of the polypeptide relative to the control current. The values in the graph are the average of the current from 4-6 Xenopus oocytes, i.e. Mean ± S.E.M, n = 4-6.

[0144] Figure 8A-8D: α-conotoxin LvID effectively improves the learning and memory abilities of Alzheimer's disease (AD) transgenic mice. Among them:

[0145] Figure 8A: LvID administration time chart; NOR: new object recognition experiment; Platform visibility: water maze platform visibility period; Training: water maze training period; Probe Test: water maze platform-free exploration period (remove the platform, detect the time of the mouse latent swimming in the quadrant where the platform was originally located); Sacrificed: mice were anesthetized and sacrificed, and the mouse brain tissue was removed for subsequent experimental research.

[0146] Figure 8B: Comparison of new object recognition indicators before and after administration, 8 in each group, new object recognition index = exploration new object time / (exploration new object time + exploration old object time); horizontal axis: mouse grouping; vertical axis: new object recognition index.

[0147] WT: wild-type mice,

[0148] WT+saline: wild-type mice injected with saline,

[0149] WT+LvID: wild-type mice injected with LvID,

[0150] 5xFAD: Alzheimer's disease model mice,

[0151] 5xFAD+saline: Alzheimer's disease model mice injected with saline,

[0152] 5xFAD+LvID: Alzheimer's disease model mice injected with LvID,

[0153] ns: no significant difference; *: P < 0.05; **: P < 0.01.

[0154] Figure 8C: Morris water maze (MWM) test results describing escape latency, defined as the time taken to find the hidden platform during the 5-day training phase, 8 mice per group; abscissa: time in days (unit: d) used for water maze training period; ordinate: time in seconds (unit: s) taken by the mice to mount the platform. The different colors in the curves of the figure represent the following groups:

[0155] WT (blue): wild-type mice,

[0156] WT + saline (black): wild-type mice injected with saline,

[0157] WT + LvID (purple): wild-type mice injected with LvID,

[0158] 5xFAD (red): Alzheimer's model mice,

[0159] 5xFAD + saline (gray): Alzheimer's model mice injected with saline,

[0160] 5xFAD + LvID (green): Alzheimer's model mice injected with LvID,

[0161] **: P < 0.01.

[0162] Figure 8D: Time spent in the target quadrant during the probe trial, 8 mice per group; abscissa: mouse group; ordinate: time spent in the target quadrant by the mice (time spent in the quadrant where the platform was located / time spent by the mice in all quadrants).

[0163] WT: wild-type mice,

[0164] WT + saline: wild-type mice injected with saline,

[0165] WT + LvID: wild-type mice injected with LvID,

[0166] 5xFAD: Alzheimer's model mice,

[0167] 5xFAD + saline: Alzheimer's model mice injected with saline,

[0168] 5xFAD + LvID: Alzheimer's model mice injected with LvID,

[0169] ns: no significant difference; *: P < 0.05.

[0170] Figure 9: Mutant [D1G, E5A, I9R, Q11A] LvID (abbreviated as LvIDm, SEQ ID NO: 19) significantly improves learning and memory in Alzheimer's disease (AD) transgenic mice.

[0171] Figure 9A: LvID m Figure 8A: Time chart of drug administration; LvID m : [D1G, E5A, I9R, Q11A] LvID;

[0172] NOR: novel object recognition experiment; Platform visibility: platform visibility period of Morris water maze; Training: training period of Morris water maze; Probe Test: platform-free exploration period of Morris water maze (remove the platform, detect the time of mice latent swimming in the quadrant where the platform was originally located); Sacrificed: mice were anesthetized and sacrificed, and the mouse brain tissue was removed for subsequent experimental study.

[0173] Figure 9B: Comparison of the difference in novel object recognition index before and after administration, 8 mice in each group, novel object recognition index = exploration time of new object / (exploration time of new object + exploration time of old object); abscissa: mouse grouping; ordinate: novel object recognition index.

[0174] WT: wild-type mice,

[0175] WT+saline: wild-type mice injected with saline,

[0176] WT+LvIDm: wild-type mice injected with [D1G, E5A, I9R, Q11A] LvID,

[0177] 5xFAD: Alzheimer's disease model mice,

[0178] 5xFAD+saline: Alzheimer's disease model mice injected with saline,

[0179] 5xFAD+LvIDm: Alzheimer's disease model mice injected with [D1G, E5A, I9R, Q11A] LvID,

[0180] ns: no significant difference;

[0181] *: P < 0.05; **: P < 0.01.

[0182] Figure 9C: Morris water maze (MWM) test results describe the escape latency, defined as the time spent searching for the hidden platform during the 5-day training period, 8 mice in each group; abscissa: time (unit: d) used in the training period of Morris water maze; ordinate: time (unit: s) required for mice to climb onto the platform. The groups represented by different colors in the figure are:

[0183] WT (blue): Wild-type mouse

[0184] WT+saline (black): Wild-type mice injected with physiological saline,

[0185] WT+LvIDm (purple): Wild-type mice injected with [D1G,E5A,I9R,Q11A]LvIDm

[0186] 5×FAD (red): Alzheimer's disease model mice,

[0187] 5×FAD+saline (gray): Alzheimer's disease model mice injected with physiological saline.

[0188] 5×FAD+LvIDm (orange): Alzheimer's disease model mice were injected with [D1G,E5A,I9R,Q11A]LvID.

[0189] **: P < 0.01.

[0190] Figure 9D: Time spent in the target quadrant during the platform-free exploration period, 8 animals per group;

[0191] x-axis: mouse grouping; y-axis: mouse latency time in the target quadrant (mouse latency time in the platform quadrant / mouse time in all quadrants).

[0192] WT: Wild-type mouse,

[0193] WT+saline: Wild-type mice were injected with physiological saline.

[0194] WT+LvIDm: Wild-type mice were injected with [D1G,E5A,I9R,Q11A]LvID.

[0195] 5×FAD: Alzheimer's disease model mice,

[0196] 5×FAD+saline: Alzheimer's disease model mice were injected with physiological saline.

[0197] 5×FAD+LvIDm: Alzheimer's disease model mice were injected with [D1G,E5A,I9R,Q11A]LvID.

[0198] ns: No significant difference

[0199] *: P < 0.05.

[0200] Figure 10: Subunit composition of the neural subtype of nicotinic acetylcholine receptors (nAChRs) and a schematic diagram of the structure of the α7 nAChRs subtype. Detailed Implementation

[0201] Embodiments of the present application will be described in detail below with reference to Examples, but those skilled in the art will understand that the following Examples are for illustrative purposes only and should not be construed as limiting the scope of the present application. Where specific conditions are not specified in the Examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. Where the manufacturer of the reagent or instrument used is not specified, it is a conventional product that can be obtained commercially.

[0202] Example 1: Design of α-conotoxin LvID mutants

[0203] In this Example, the amino acid sequence of the parent peptide α-conotoxin LvID is: DCCSEPPCILQNPDIC#, designated as SEQ ID NO: 1. A series of mutants of α-conotoxin LvID were designed (Table 1, Figure 1), designated as SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, respectively. For example, SEQ ID NO: 2 corresponds to [E5R]LvID, which is obtained by replacing the glutamic acid (E) at the 5th position in the LvID sequence with alanine (A); SEQ ID NO: 3 corresponds to [E5K]LvID, which is obtained by replacing the glutamic acid (E) at the 5th position in the LvID sequence with lysine (K); SEQ ID NO: 4 corresponds to [Q11L]LvID, which is obtained by replacing the glutamine (Q) at the 11th position in the LvID sequence with leucine (L); SEQ ID NO: 5 corresponds to [Q11I]LvID, which is obtained by replacing the glutamine (Q) at the 11th position in the LvID sequence with isoleucine (I).

[0204] Similarly, SEQ ID NO: 6 corresponds to [D1A, Q11A]LvID, in which the aspartic acid (D) at position 1 and the glutamine (Q) at position 11 of the LvID sequence are replaced with alanine (A); SEQ ID NO: 7 corresponds to [E5A, Q11A]LvID, in which the glutamic acid (E) at position 5 and the glutamine (Q) at position 11 of the LvID sequence are replaced with alanine (A); SEQ ID NO: 8 corresponds to [E5A, Q11N]LvID, in which the glutamic acid (E) at position 5 is replaced with alanine (A) and the glutamine (Q) at position 11 is replaced with asparagine (N); SEQ ID NO: 9 corresponds to [E5N, P7N, Q11A]LvID, in which the glutamic acid (E) at position 5, the proline (P) at position 7, and the glutamine (Q) at position 11 of the LvID sequence are replaced with asparagine (N), asparagine (N), and alanine (A); and SEQ ID NO: 10 corresponds to [D1S, 2G, E5A, Q11A]LvID, in which the aspartic acid (D) at position 1 is replaced with serine (S), the amino acid at position 2 is increased by glycine (G), the glutamic acid (E) at position 5, and the glutamine (Q) at position 11 are replaced with alanine (A).

[0205] SEQ ID NO: 11 corresponds to [D1S, 2E, E5A, Q11A]LvID, in which the aspartic acid (D) at position 1, the glutamic acid (E) at position 2, the glutamic acid (E) at position 5 and the glutamine (Q) at position 11 of the sequence of LvID are replaced by a serine (S), an additional glutamic acid (E), an alanine (A) and an alanine (A), respectively; SEQ ID NO: 12 corresponds to [D1G, E5A, Q11A]LvID, in which the aspartic acid (D) at position 1, the glutamic acid (E) at position 5 and the glutamine (Q) at position 11 of the sequence of LvID are replaced by a glycine (G), an alanine (A) and an alanine (A), respectively; SEQ ID NO: 13 corresponds to [E5A, Q11A, D14H, I15D]LvID, in which the glutamic acid (E) at position 5, the glutamine (Q) at position 11, the aspartic acid (D) at position 14 and the isoleucine (I) at position 15 of the sequence of LvID are replaced by an alanine (A), an alanine (A), a histidine (H) and an aspartic acid (D), respectively; SEQ ID NO: 14 corresponds to [E5A, I9R, Q11A, D14H, I15D]LvID, in which the glutamic acid (E) at position 5, the isoleucine (I) at position 9, the glutamine (Q) at position 11, the aspartic acid (D) at position 14 and the isoleucine (I) at position 15 of the sequence of LvID are replaced by an alanine (A), an arginine (R), an alanine (A), a histidine (H) and an aspartic acid (D), respectively; SEQ ID NO: 15 corresponds to [D1G, E5A, I9R, Q11A, D14H, I15D]LvID, in which the aspartic acid (D) at position 1, the glutamic acid (E) at position 5, the isoleucine (I) at position 9, the glutamine (Q) at position 11, the aspartic acid (D) at position 14 and the isoleucine (I) at position 15 of the sequence of LvID are replaced by a glycine (G), an alanine (A), an arginine (R), an alanine (A), a histidine (H) and an aspartic acid (D), respectively.

[0206] SEQ ID NO: 16 corresponds to [E5N, P7A]LvID, in which the glutamic acid (E) at position 5 and the proline (P) at position 7 in the sequence of LvID are replaced by asparagine (N) and alanine (A), respectively; SEQ ID NO: 17 corresponds to [D1G, E5N, P7A, I9R]LvID, in which the aspartic acid (D) at position 1, the glutamic acid (E) at position 5, the proline (P) at position 7 and the isoleucine (I) at position 9 in the sequence of LvID are replaced by glycine (G), asparagine (N), alanine (A) and arginine (R), respectively; SEQ ID NO: 18 corresponds to [E5A, I9R, Q11A]LvID, in which the glutamic acid (E) at position 5, the isoleucine (I) at position 9 and the glutamine (Q) at position 11 in the sequence of LvID are replaced by alanine (A), arginine (R) and alanine (A), respectively; SEQ ID NO: 19 corresponds to [D1G, E5A, I9R, Q11A]LvID, in which the aspartic acid (D) at position 1, the glutamic acid (E) at position 5, the isoleucine (I) at position 9 and the glutamine (Q) at position 11 in the sequence of LvID are replaced by glycine (G), alanine (A), arginine (R) and alanine (A), respectively. These mutation designs are aimed to find new mutants with excellent selectivity and strong blocking activity for both rat and human α7 nAChR subtypes, while reducing the interaction with other nAChR subtypes.

[0207] Table 1: Sequences of LvID and its mutants

[0208] Note: In Table 1, the C-terminus of the sequences of LvID and its mutants are all amidated, as shown in Figure 1.

[0209] Example 2: Artificial synthesis of α-conotoxin LvID mutants

[0210] According to the amino acid sequences of α-conotoxin LvID and its mutants, linear resin peptides are artificially synthesized by Fmoc method (Figure 1, Table 1), and after cleavage, the linear peptides are oxidized and folded to form active Cys (I-III, II-IV) disulfide bond connection mode. Resin peptides are artificially synthesized by Fmoc chemical method, which can be synthesized by polypeptide synthesizer or manual synthesis method. Except for cysteine, the rest of the amino acids are protected by standard side chain protection groups. The -SH of the first and third cysteines (Cys) of LvID and its mutants is protected by Trt (S-trityl), and the -SH of the second and fourth cysteines is protected by Acm (S-acetamidomethyl).

[0211] Specific synthesis procedure: Linear peptides in Figure 1 and Table 1 were synthesized on an ABI Prism 433a peptide synthesizer using Fmoc and FastMoc methods in solid phase synthesis. The side chain protecting groups of Fmoc amino acids were: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp), Boc (Lys). Fmoc HOBT DCC method, Rink amide resin and Fmoc amino acids were used. The synthesis procedure was according to the manual of the instrument. To make the reaction complete, the deprotection time of piperidine and coupling time were prolonged respectively. Double coupling was used for difficult amino acids. The resin peptide was obtained. Linear peptide was cleaved 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 precipitated and washed with ice-ethanol to recover the crude linear peptide. The crude linear peptide was purified by preparative reverse phase HPLC C18 column (Vydac) with a linear gradient of 10-40% B90 in 0-40 min. The monitoring wavelength was 214 nm. Solvent B90 was 90% acetonitrile (ACN, acetonitrile), 0.05% TFA (trifluoroacetic acid) in water; solvent A was 0.05% TFA in water.

[0212] The purified linear peptide was analyzed by analytical HPLC C18 column (Vydac) with the same elution condition as above at a flow rate of 1 mL / min. The purity was more than 95% and was used for oxidative folding.

[0213] The linear peptides of LvID and its mutants were subjected to two-step oxidative folding according to the literature (Dowell, C.; Olivera, B. M.; Garrett, J. E.; Staheli, S. T.; Watkins, M.; Kuryatov, A.; Yoshikami, D.; Lindstrom, J. M.; McIntosh, J. M., Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors. The Journal of neuroscience 2003, 23 (24), 8445-52.).

[0214] The first disulfide bridge was formed between the two cysteines with a Trt protecting group by potassium ferricyanide oxidation (20 mM potassium ferricyanide, 0.1 M Tris, pH 7.5, 45 min). After purification of the monocyclic peptide by reverse phase HPLC C18 column (Vydac), the second disulfide bridge was formed between the other two cysteines by iodine oxidation (10 mM iodine in H2O:trifluoroacetic acid:acetonitrile (78:2:20 by volume, 10 min). The bicyclic peptide was purified by reverse phase HPLC C18 column (Vydac) to obtain the α-conotoxin with the disulfide bonds oriented between the corresponding cysteines in the order from N-terminus to C-terminus, and identified by electrospray-mass spectroscopy (ESI-MS).

[0215] The synthesized polypeptide α-conotoxin LvID mutants have correct molecular weight and high purity. For example, the HPLC chromatogram and ESI-MS mass spectrum of [E5A, I9R, Q11A]LvID (SEQ ID NO: 18) and [D1G, E5A, I9R, Q11A]LvID (SEQ ID NO: 19) after oxidative folding are shown in Figures 3A, 3B, 4A and 4B. The purity of both is above 95%. The measured molecular weight of [E5A, I9R, Q11A]LvID (1674.88 Da) is consistent with the theoretical molecular weight (1672.93 Da), and the measured molecular weight of [D1G, E5A, I9R, Q11A]LvID (1616.94 Da) is consistent with the theoretical molecular weight (1614.9 Da), indicating that the synthesized polypeptide has correct molecular weight and high purity. The concentration of the polypeptide was determined by colorimetry at a wavelength of 280 nm, and the concentration and mass of the polypeptide were calculated according to the Beer-Lambert equation. These quantified and folded polypeptides were used for subsequent activity tests.

[0216] Example 3: Activity of α-conotoxin LvID mutants on human α7 nAChR, rat α7 nAChR, and other nAChRs subtypes

[0217] Methods in reference (Azam L, Yoshikami D, McIntosh JM. Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII [S4A, E11A, L15A]. J Biol Chem. 2008; 283(17): 11625-32.) and the mMessage mMachine in vitro transcription kit (Ambion, Austin, TX) instructions were used to prepare cRNA for various human / rat neuronal nAChR subtypes (a3b2, a6 / a3b2b3, a6 / a3b4, a9a10, a4b2, a4b4, a3b4, a2b2, a2b4, a7), and mouse muscle nAChRs (alblde). The concentration of cRNA was estimated by OD at 260 nm using UV. Oocyte (frog egg) from Xenopus laveis were dissected and cRNA was injected into the frog eggs at 5 ng cRNA per subunit. 0.5-2.5 ng DNA per subunit was injected for rat muscle nAChRs. Frog eggs were cultured in ND-96. cRNA was injected within 1-2 days after oocyte collection and voltage clamp recording of nAChRs was performed within 1-4 days after injection.

[0218] One cRNA-injected frog oocyte was placed in a 50-μL Sylgard recording chamber (4 mm diameter x 2 mm deep) and perfused with ND96 containing 0.1 mg / ml BSA (bovine serum albumin) (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5 mM HEPES, pH 7.1-7.5) or ND96 containing 1 mM atropine (ND96A) at a flow rate of 1 ml / min. All conotoxin solutions also contained 0.1 mg / ml BSA to reduce nonspecific adsorption of the toxins. A switching valve (SmartValve, Cavro Scientific Instruments, Sunnyvale, CA) was used to switch freely between perfusing toxin or acetylcholine (ACh), and a series of three-way solenoid valves (model 161TO31, Neptune Research, Northboro, MA) were used to switch freely between perfusing ND96 and ACh. ACh-gated currents were recorded on-line with a dual-electrode voltage-clamp amplifier (model OC-725B, Warner Instrument Corp., Hamden, CT) set to "slow" clamping, and with the clamp gain at the maximum (x2000) position. Glass electrodes were pulled from 1 mm outer diameter x 0.75 mm inner diameter fiber-filled borosilicate capillaries (WPI Inc., Sarasota, FL) and filled with 3 M KCl for both voltage and current electrodes. Membrane voltage was clamped at -70 mV. The entire system was computer controlled and data were recorded. ACh pulses were automatically perfused for 1 s every 5 min. The concentrations of ACh were 10 μM for rat muscle and neuronal α9α10 nAChRs, 200 μM for rat neuronal α7 nAChR, and 100 μM for all other subtypes. At least four oocytes expressing a given subtype were recorded for current responses to different concentrations of toxin, and current traces were recorded.

[0219] Current data were analyzed using GraphPad Prism software (San Diego, CA) to generate dose-response curves and calculate the half-blocking concentration (IC50) of conotoxin. 50 Various parameters related to the blocking of nAChRs by polypeptides were determined.

[0220] The results are shown in Figures 5A-5B, 6A-6D, and Tables 2-6.

[0221] Table 2: IC50values of [E5A, Q11A] LvID (SEQ ID NO: 7) at rat and human nicotinic acetylcholine receptor homologous subtypes, respectively 50 Hillslope values

[0222] Table 3: IC50values of [D1G, E5A, Q11A] LvID (SEQ ID NO: 12) at rat and human nicotinic acetylcholine receptor homologous subtypes, respectively 50 Hillslope values

[0223] Table 4: IC50values of [D1G, E5A, I9R, Q11A, D14H, I15D] LvID (SEQ ID NO: 15) at rat and human nicotinic acetylcholine receptor homologous subtypes, respectively 50 Hillslope values

[0224] Table 5: Half-blocking dose (IC50) of [E5A, I9R, Q11A] LvID (SEQ ID NO: 18) at all subtypes of human (h, human) and rat (r, rat) nicotinic acetylcholine receptors (nAChRs) and the slope (Hillslope) values of concentration-response curves 50

[0225] a represents less than 50% inhibition at a concentration of 10 μΜ. b m represents mouse. The same below.

[0226] Table 6: IC50values of [D1G, E5A, I9R, Q11A] LvID (SEQ ID NO: 19) at all subtypes of human and rat nicotinic acetylcholine receptors 50 Hillslope values

[0227] a represents less than 50% inhibition at a concentration of 10 μΜ.

[0228] The results show that:

[0229] [E5A, Q11A] LvID (SEQ ID NO: 7) has significant blocking activity at rat a7 nAChR and human a7 nAChR, but has blocking activity at rat a3b2, rat a6 / a3b2b3, human a3b4 and human a6 / a3b4 (Table 2);​

[0230] [D1G, E5A, Q11A] LvID (SEQ ID NO: 12) has significant blocking activity against rat and human α7 nAChRs, but also against rat α3β2, rat α3β4, rat α6 / α3β4, human α3β4 and human α6 / α3β4 (Table 3);

[0231] [D1G, E5A, I9R, Q11A, D14H, I15D] LvID (SEQ ID NO: 15) has extremely significant blocking activity against rat and human α7 nAChRs, but also against rat α3β2, rat α3β4, human α3β4 and human α6 / α3β4 (Table 4);

[0232] [E5A, I9R, Q11A] LvID (SEQ ID NO: 18) has strong blocking activity against human and rat α7 nAChRs, with half-blocking doses (IC 50 ) of 10.6 nM and 2 nM, respectively, and weaker blocking activity against human α6 / α3β4 nAChR, with a half-blocking dose (IC 50 ) of 6190 nM. The blocking activity of [E5A, I9R, Q11A] LvID against human and rat α7 nAChRs is 583-fold and 3095-fold stronger, respectively, than its blocking activity against the human α6 / α3β4 subtype; [E5A, I9R, Q11A] LvID has almost no blocking effect against all other subtypes of receptors at a high concentration of 10 μM (Figures 5A, 6A-6C, Table 5), and 100 nM of this mutant peptide almost completely blocks the current generated by the opening of human and rat α7 nAChRs gated by Ach. In addition, [E5A, I9R, Q11A] LvID (SEQ ID NO: 18) has significant blocking activity against human α7 nAChR, but only weak blocking activity against human α6 / α3β4, with an IC 50 value much lower than its blocking activity against other nAChR subtypes (Table 5), indicating that this mutant has high selectivity;

[0233] [D1G, E5A, I9R, Q11A] LvID (SEQ ID NO: 19) has the strongest blocking activity against human and rat α7 nAChRs, with half-blocking doses (IC 50 ) of 2.2 nM and 0.41 nM, respectively, a half-blocking dose (IC 50 ) of 324 nM against human α6 / α3β4 nAChR, and a half-blocking dose (IC50 ) is 2709 nM, the half-blocking dose (IC50) for human α3β4 50 ) is 6245 nM, the half-blocking dose (IC50) for rat α3β2 50 ) is 7580 nM, and has almost no blocking effect on all other subtypes of receptors (Fig. 5B, Fig. 6D to Fig. 61, Table 6), and the mutant peptide 100 nM almost completely blocks the current generated by the opening of human α7 nAChR and rat α7 nAChR gated by Ach.

[0234] Example 4: Comparison of the activity of preferred mutants of α-conotoxin LvID on rat α7 nAChR subtypes and human α7 nAChR subtypes

[0235] In this example, the activity of LvID mutants on rat and human α7 nAChR subtypes was compared. By a similar method as in the previous Example 3, the blocking effect of the mutants on α7 nAChR from two different species was evaluated (Fig. 7A and Fig. 7B).

[0236] The experimental data show that:

[0237] The [E5A, I9R, Q11A] LvID mutant (SEQ ID NO: 18) has comparable blocking activity on human α7 nAChR and rat α7 nAChR, but slightly stronger blocking activity on human α7 nAChR (Fig. 7A);

[0238] The [D1G, E5A, I9R, Q11A] LvID mutant (SEQ ID NO: 19) has significantly improved blocking activity on human α7 nAChR and rat α7 nAChR compared to [E5A, I9R, Q11A] LvID, although it has weak blocking effect on a few other nAChR subtypes (Fig. 7B).

[0239] Example 5: α-conotoxin LvID effectively improves the learning and memory ability of Alzheimer's disease (AD) transgenic mice

[0240] First, more LvID peptide was prepared according to the method of Example 2, and after being dissolved in 0.9% sterile normal saline, it was used for animal experiments. The body weight of mice was weighed, and after the dissolved LvID was injected into the brain of Alzheimer's disease (AD) model mice (purchased from Jackson Laboratory, USA) and homozygous wild type (WT) mice (from the litter control of AD mice breeding) at a dose of 5 nmol of peptide per 25 g of body weight (5 nmol / 25 g) by brain stereotactic injection, it was continuously injected for 7 days (Figure 8A), and after seven days, the Novel Object Recognition (NOR) test was performed, and the novel object index was significantly increased, indicating that LvID can improve the memory ability of AD mice (Figure 8B). At the same time, the mice were continuously subjected to the Morris Water Maze (MWM) experiment, and after the experiment, the time for the mice to climb onto the platform was counted. The time for the AD mice injected with LvID to climb onto the platform was significantly reduced (Figure 8C), and in addition, the AD mice injected with LvID had a significantly increased latency time in the quadrant where the platform was located (Figure 8D), indicating that LvID can improve the spatial memory impairment of AD mice.

[0241] Example 6: Mutant [D1G, E5A, I9R, Q11A] LvID significantly improves the learning and memory ability of Alzheimer's disease (AD) transgenic mice

[0242] First, more amounts of [D1G, E5A, I9R, Q11A] LvID (SEQ ID NO: 19) peptide were prepared according to the method of Example 2, and were dissolved with 0.9% sterile physiological saline for use. Then the mouse body weight was weighed, and the dissolved [D1G, E5A, I9R, Q11A] LvID was injected into the brain of Alzheimer's disease (AD) model mice (available from Jackson Laboratory, USA) and homozygous wild type (WT) mice (from the same litter as the AD mice) by brain stereotactic injection at a dose of 5 nmol / 25 g, and was injected continuously for 7 days (Figure 9A). After seven days, the Novel Object Recognition (NOR) test was performed, and the novel object index was significantly increased, indicating that [D1G, E5A, I9R, Q11A] LvID can improve the memory ability of AD mice (Figure 9B). At the same time, the mice were continuously subjected to the Morris Water Maze (MWM) test, and after the experiment, the time for the mice to climb onto the platform was counted. The time for the AD mice injected with [D1G, E5A, I9R, Q11A] LvID to climb onto the platform was significantly reduced (Figure 9C), and in addition, the AD mice injected with [D1G, E5A, I9R, Q11A] LvID had a significantly increased latency time in the quadrant where the platform was located (Figure 9D), indicating that [D1G, E5A, I9R, Q11A] LvID can significantly improve the spatial memory impairment of AD mice, and has good new drug research and development value in the field of prevention and treatment of learning and memory related diseases.

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[0295] While the specific embodiments of the application have been described in detail, those skilled in the art will appreciate that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure. The foregoing is intended to cover all modifications and alternatives within the scope of the present application. It is also to be understood that the written form of the specification is to be used as in conjunction with the appended drawings and that the written description is to be taken together with the drawings as a complete disclosure of the application.

Claims

1. An isolated polypeptide which is a mutant of a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1 and which comprises the following mutations: E5A and Q11A.

2. The isolated polypeptide according to claim 1, which further comprises the following mutations: D1G and / or I9R.

3. The isolated polypeptide according to claim 2, which further comprises the following mutations: D14H and / or I15D.

4. The isolated polypeptide according to claim 1, which is a mutant of a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1 and the mutations are selected from any one of (1) to (16) as follows: (1) E5A and Q11A; (2) E5A, Q11A and D1G; (3) E5A, Q11A and I9R; (4) E5A, Q11A, D1G and I9R; (5) E5A, Q11A and D14H; (6) E5A, Q11A and I15D; (7) E5A, Q11A, D14H and I15D; (8) E5A, Q11A, D1G and D14H; (9) E5A, Q11A, D1G and I15D; (10) E5A, Q11A, D1G, D14H and I15D; (11) E5A, Q11A, I9R and D14H; (12) E5A, Q11A, I9R and I15D; (13) E5A, Q11A, I9R, D14H and I15D; (14) E5A, Q11A, D1G, I9R and D14H; (15) E5A, Q11A, D1G, I9R and I15D; and (16) E5A, Q11A, D1G, I9R, D14H and I15D.

5. The isolated polypeptide according to claim 1, which has an amino acid sequence as set forth in any one of SEQ ID NOs: 7, 10 to 15 and 18 to 19.

6. The isolated polypeptide according to any one of claims 1 to 5, wherein, the isolated polypeptide has the same or improved activity of blocking human α7 nAChR or rat α7 nAChR as compared to a polypeptide having an amino acid sequence as set forth in SEQ ID NO:

1.

7. The isolated polypeptide according to any one of claims 1 to 6, wherein, the first and third cysteines from the N-terminus of the isolated polypeptide form a disulfide bond, and the second and fourth cysteines form a disulfide bond; or the first and fourth cysteines from the N-terminus of the isolated polypeptide form a disulfide bond, and the second and third cysteines form a disulfide bond; or the first and second cysteines from the N-terminus of the isolated polypeptide form a disulfide bond, and the third and fourth cysteines form a disulfide bond; preferably, the carboxyl terminus of the isolated polypeptide is amidated.

8. An isolated fusion protein comprising at least one polypeptide according to any one of claims 1 to 7. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 9. An isolated polynucleotide encoding the isolated polypeptide of any one of claims 1 to 7 or the isolated fusion protein of claim 8.

10. A nucleic acid construct comprising the isolated polynucleotide of claim 9; preferably, the nucleic acid construct is a recombinant vector; preferably, the nucleic acid construct is a recombinant expression vector.

11. A recombinant host cell comprising the isolated polynucleotide of claim 9 or comprising the nucleic acid construct of claim 10.

12. A pharmaceutical composition comprising at least one of the isolated polypeptide of any one of claims 1 to 7, the isolated fusion protein of claim 8, the isolated polynucleotide of claim 9, the nucleic acid construct of claim 10 or the recombinant host cell of claim 11; preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients; preferably, the pharmaceutical composition is a lyophilized agent or an injection solution.

13. Use of the isolated polypeptide of any one of claims 1 to 7 or the isolated fusion protein of claim 8 in the manufacture of a medicament for blocking or inhibiting a7 nAChR of a mammal; preferably, the mammal is a human or a rat.

14. Use of the isolated polypeptide of any one of claims 1 to 7 or the isolated fusion protein of claim 8 in the manufacture of a medicament for treating or preventing cancer, asthma, Alzheimer's disease or intractable neuropsychiatric disorders or complications thereof associated with excessive long-term memory of fear and / or aversion; preferably, the cancer is one or more selected from the group consisting of cholangiocarcinoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer and breast cancer; preferably, the asthma is pediatric asthma; preferably, the intractable neuropsychiatric disorders or complications thereof associated with excessive long-term memory of fear and / or aversion is one or more selected from the group consisting of phobia, anxiety, depression, obsessive-compulsive disorder, post-traumatic stress disorder, schizophrenia and affective disorder.

15. The isolated polypeptide of any one of claims 1 to 7 or the isolated fusion protein of claim 8 for use in blocking or inhibiting a7 nAChR of a mammal; preferably, the mammal is a human or a rat.

16. The isolated polypeptide of any one of claims 1 to 7 or the isolated fusion protein of claim 8 for use in treating or preventing cancer, asthma, Alzheimer's disease or intractable neuropsychiatric disorders or complications thereof associated with excessive long-term memory of fear and / or aversion; preferably, the cancer is one or more selected from the group consisting of cholangiocarcinoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer and breast cancer; preferably, the asthma is pediatric asthma; preferably, the intractable neuropsychiatric disorders or complications thereof associated with excessive long-term memory of fear and / or aversion is one or more selected from the group consisting of phobia, anxiety, depression, obsessive-compulsive disorder, post-traumatic stress disorder, schizophrenia and affective disorder.

17. A method of blocking or inhibiting a7 nAChR in a subject, comprising the step of administering to a subject in need thereof an effective amount of the isolated polypeptide of any one of claims 1 to 7 or the isolated fusion protein of claim 8; the subject is a mammal; preferably, the mammal is a human or a rat.

18. A method of treating or preventing a disease comprising the step of administering to a subject in need thereof an effective amount of the isolated polypeptide of any one of claims 1 to 7 or the isolated fusion protein of claim 8; wherein, the subject is a human; and the disease is one or more selected from the group consisting of cancer, asthma, Alzheimer's disease, and intractable neuropsychiatric diseases or complications thereof associated with excessive long-term memory of fear and / or aversion; preferably, the cancer is one or more selected from the group consisting of cholangiocarcinoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, and breast cancer; preferably, the asthma is pediatric asthma; preferably, the intractable neuropsychiatric disease or complication thereof associated with excessive long-term memory of fear and / or aversion is one or more selected from the group consisting of phobia, anxiety, depression, obsessive-compulsive disorder, mental trauma, schizophrenia, and affective disorder.

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