WO2022117116A1 - Α9α10 NACHR INIBITORY PEPTIDE AND USE THEREFOR - Google Patents

Α9α10 NACHR INIBITORY PEPTIDE AND USE THEREFOR Download PDF

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WO2022117116A1
WO2022117116A1 PCT/CN2021/141818 CN2021141818W WO2022117116A1 WO 2022117116 A1 WO2022117116 A1 WO 2022117116A1 CN 2021141818 W CN2021141818 W CN 2021141818W WO 2022117116 A1 WO2022117116 A1 WO 2022117116A1
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amino acid
polypeptide
gex
peptide
conotoxin
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Chinese (zh)
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于日磊
李晓
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中国海洋大学
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Definitions

  • the invention belongs to the field of biopharmaceuticals, in particular to a polypeptide with analgesic activity and application thereof, in particular to a single-chain polypeptide with analgesic activity obtained by design, preparation and screening based on natural ⁇ -conotoxin and its mechanism of action Polypeptides and their derivatives.
  • Nicotinic acetylcholine receptors are ubiquitous ion channel receptors in the animal kingdom, from lower nematodes to higher mammals contain such receptors (Nicke, A. (2004) Learning about structure and function of neuronal nicotinicacetylch oline receptors. Lessons from snails. European journal of biochemistry/FEBS 271, 2293). nAChRs receptors are located in and out of synapses at the nerve-muscle (and/or) nerve-nerve junction, and activate the release of various neurotransmitters such as dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid.
  • nAChRs mediate many physiological functions of the central and peripheral nervous system, including learning, memory, response, analgesia, sensory signal processing and motor control, etc. They have important physiological functions and clinical research significance. Numerous studies have shown that nAChRs are key targets for screening, diagnosing and treating a large class of important diseases, including pain, addiction, cancer, intellectual disability, Parkinson's disease, mental illness, depression, myasthenia gravis and other intractable diseases.
  • nAChRs are pentameric transmembrane proteins composed of 5 subunits, which can be divided into two categories: muscle-type and neuron-type, among which neuron-type nAChRs are very complex, they are composed of different ⁇ and/or ⁇ subunits, heterologous or homologous.
  • the functional receptor subtype of the source type in vertebrates, has at least 12 subunits, namely ⁇ 2– ⁇ 10, ⁇ 2– ⁇ 4.
  • the ⁇ 9 ⁇ 10 nicotinic acetylcholine receptor is a transmembrane pentamer composed of two subunits, ⁇ 9 and ⁇ 10, and is a ligand-gated ion channel. This receptor was first identified in cochlear hair cells and mediates synaptic transmission between oligocochlear cholinergic fibers and efferent nerves, and was later shown to be involved in leukocytes, dorsal root ganglia, pituitary gland, skin keratinocytes, and semen are also distributed.
  • ⁇ 9 ⁇ 10nAChR is a new target for the treatment of neuralgia drugs (McIntosh, J.M.; Absalom, N.; Chebib, M.; Elgoyhen, A.B.; Vincler, M., Alpha9nicotinic acetylcholine receptors and the treatment of pain. Biochemal pharmacology 2009, 78(7), 693-702. Satkunanathan, N.; Livett, B.; Gayler, K.; Sandall, D.; Down, J.; Khalil, Z., Alpha-conotoxin Vc1.1alleviates neuropathic pain andaccelerates function al recovery of injured neurons.
  • Neuropathic pain is a complex and debilitating syndrome caused by damage to the somatosensory nervous system, affecting millions of people worldwide.
  • Epidemiological studies have shown that neuralgia afflicts more than 8% of the global population, not only seriously affecting the physical and mental health and quality of life of patients, but also consuming tens of billions of dollars in medical resources.
  • traditional analgesics are not effective in treating neuropathic pain, and long-term use is addictive, so the research and development of new analgesics is particularly important.
  • ⁇ 9 ⁇ 10nAChR blockers have the functions of treating neuralgia, preventing nerve injury, and accelerating the recovery of injured nerves, possibly through immune mechanisms (Holtman, J.R.; Dwoskin, L.P.; Dowell, C.; Wala, E.P.; Zhang, Z.
  • ⁇ 9 ⁇ 10nAChR is also closely related to wound healing, ear diseases, lung cancer, breast cancer and skin diseases and many other diseases.
  • ⁇ 9 ⁇ 10nAChR on keratinocytes plays an important role in the pathophysiology of wound healing (Chernyavsky, A.I.; Arredondo, J.; Vetter, D.E.; Grando, S.A., Central role of alpha9acetylcholinereceptor in coordinating keratinocyt e adhesion and motility at theinitiation of epithelialization. Experimental cell research 2007, 313(16), 3542-55).
  • the ⁇ 9 subunit variant affects the transformation and proliferation of bronchial cells, and this subunit has a very important significance in the treatment of lung cancer (Chikova, A. Grando, S.A., Naturally occurring variants of human Alpha9nicotinic receptordifferentially affect bronchial cell proliferation and transformation. PloS one 2011, 6(11), e27978.).
  • Conotoxin is a biologically active polypeptide of marine origin, usually composed of 10-50 amino acid residues, rich in secondary structure and several disulfide bonds. Its stable structure and diverse pharmacological effects make it suitable for drug development. A very broad prospect.
  • Conotoxins are divided into different gene families according to the similarity of the endoplasmic reticulum signal peptide sequence of their precursor proteins and the cysteine pattern. So far, all known conotoxins can be divided into 18 superfamilies; Spirotoxins (peptides) can be divided into ⁇ , ⁇ , ⁇ , ⁇ and other pharmacological families according to their receptor targets.
  • ⁇ -Conotoxins have the function of blocking nicotinic acetylcholine receptors (nAChRs); Conantokins, which do not contain cysteine, have the function of blocking N-methyl-D-day The function of aspartic acid receptor (NMDA receptor, N-methyl-D-aspartic acid receptor, NMDAR).
  • NMDA receptor N-methyl-D-aspartic acid receptor
  • ⁇ -conotoxins RgIA, Vc1.1 and GeXIVA which selectively inhibit ⁇ 9 ⁇ 10 acetylcholine receptors, have shown good analgesic effects in animal pain models. Subcutaneous or intramuscular injection can alleviate trauma, inflammation or nerve damage. caused pain. After structural optimization, such polypeptides are expected to form a major breakthrough in the research of analgesic drugs.
  • the ⁇ -conotoxin that can specifically bind to and inhibit mouse ⁇ 9 ⁇ 10nAChR significantly reduces the inhibitory activity of human ⁇ 9 ⁇ 10nAChR (see Molecular basis for the differential sensitivity of rat and human alpha9alpha10nAChRs to alpha- conotoxin RgIA.Azam L,McIntosh JM.J Neurochem.2012Sep;122(6):1137-44.;Determination of the alpha-conotoxin Vc1.1binding site on the alpha9alpha10nicotinic acetylcholine receptor.Yu R, Kompella SN,Adams DJ,Craik DJ, Kaas Q.J Med Chem.
  • natural ⁇ -conotoxin is a mixture of peptide chains containing multiple cysteines, and multiple cysteines on the monomer peptide chain can form different disulfide bonds with each other, resulting in disulfide Bond isomers; monomeric peptide chains may also be connected to each other by intermolecular disulfide bonds.
  • These disulfide bond isomers and multimers have different inhibitory activities on ⁇ 9 ⁇ 10nAChR, which are difficult to synthesize during the production process, require oxidative folding, etc. to form the correct conformation, difficult to purify, and low yield.
  • the present invention provides a single-chain analgesic peptide.
  • a single-chain analgesic peptide According to the mechanism of the interaction between ⁇ -conotoxin and ⁇ 9 ⁇ 10nAChR (ie: ⁇ 9 ⁇ 10 nicotinic acetylcholine receptor), a plurality of ⁇ -conotoxin single peptides are designed. somatic peptide mutants. The monomeric peptide mutant Gex-2 with high inhibitory activity and good analgesic effect was screened.
  • Gex-2 was mutated by means of alanine scanning, aspartic acid scanning, arginine scanning, arginine-citrulline substitution, and D-amino acid substitution to obtain a series of derivatives of Gex-2.
  • the Gex-2 can improve the pain threshold of CCI rats and has a good analgesic effect; compared with Gex-2, some members of the Gex-2 series derivatives have higher inhibitory activity on ⁇ 9 ⁇ 10 acetylcholine receptors.
  • the Gex-2 and its series derivatives can be used as ⁇ 9 ⁇ 10nAChR inhibitors for analgesia, anti-tumor, treatment of nervous system diseases and the like.
  • the present invention provides an active polypeptide characterized by having a deletion or deletion mutation of one or more cysteine residues compared to the ⁇ -conotoxin active monomeric peptide;
  • the ⁇ -conotoxin active monomer peptide has the function of binding nAChR.
  • the active polypeptide of the present invention is characterized in that the ⁇ -conotoxin active monomer peptide comprises at least three consecutive arginine residues, and has 1, 2, 3, 4 or 5 a cysteine residue.
  • the active polypeptide of the present invention is characterized in that the ⁇ -conotoxin active monomer peptide includes ⁇ -conotoxin natural monomer peptide, and on the basis of the amino acid sequence of the ⁇ -conotoxin natural monomer peptide One or several amino acid residues at the C-terminus and/or the N-terminus are deleted.
  • the active polypeptide of the present invention is characterized in that the ⁇ -conotoxin natural monomer peptide is derived from ⁇ -conotoxin selected from the group consisting of GeXIVA, GeXXVIIA, Vc1.1, PeIA, RgIA, B- VxXXIVA, S-GVIIIB, D-GeXXA, O-GeXXVIIA, D--Lt28.1, Mr1.1, BuIA, ImI, or AuIB; the ⁇ -conotoxin natural monomeric peptides include the Mature peptide monomeric peptide, precursor peptide monomeric peptide.
  • the active polypeptide of the present invention is characterized in that the ⁇ -conotoxin natural monomer peptide comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the active polypeptide of the present invention is characterized in that the active polypeptide does not contain cysteine residues, does not form intramolecular disulfide bonds or intermolecular disulfide bonds, and has more than 10 amino acid residues.
  • the active polypeptide of the present invention is characterized in that the active polypeptide comprises the sequence shown in SEQ ID NO: 3 and has 12-20 amino acid residues.
  • any one of the aforementioned active polypeptides of the present invention includes a linear peptide, a cyclic peptide, or a D-type amino acid substitution derivative peptide having an amino acid sequence structure selected from any one of SEQ ID NOs: 4-9.
  • the active polypeptide according to any one of the foregoing in the present invention includes a mutant obtained by performing single-point amino acid scanning mutation on the basis of the amino acid sequence shown in any of SEQ ID NOs: 4-9, and the amino acid scanning mutation includes positive Charged amino acid scanning mutations, negatively charged amino acid scanning mutations, neutral amino acid scanning mutations, rare amino acid substitution mutations, and D-type amino acid substitution mutations.
  • the active polypeptide of the present invention wherein the positively charged amino acid scanning mutation includes arginine scanning mutation; the negatively charged amino acid scanning mutation includes aspartic acid scanning mutation; the uncharged amino acid scanning includes alanine scanning mutation; D Type amino acid scanning includes single point mutants obtained by replacing each amino acid residue with its corresponding D-type amino acid residue on the basis of the sequences shown in SEQ ID NOs: 4-9.
  • the active polypeptides of the present invention include the polypeptide sequences produced by the combination of the above-mentioned several modification strategies.
  • the analogs obtained, for example, were performed simultaneously with two strategies of arginine scanning and D-form amino acid substitution.
  • the active polypeptide of the present invention has the amino acid sequence of any one of SEQ ID NO: 10-73, preferably has SEQ ID NO: 5, 20, 21, 22, 23, 30, 34, 46, 50, The amino acid sequence shown in 51, 53, 66, or 69.
  • the present invention provides a method for preparing an active polypeptide, comprising deleting one or more cysteine residues in the amino acid sequence of an ⁇ -conotoxin active monomer peptide, the ⁇ -conotoxin active Monomeric peptides function to bind nAChRs.
  • the method for preparing the ⁇ -conotoxin active monomer peptide according to the present invention is characterized in that it further comprises truncating the ⁇ -conotoxin natural monomer peptide at the C-terminus and/or the N-terminus.
  • the method for preparing an active polypeptide according to the present invention is characterized in that the ⁇ -conotoxin natural monomer peptide is derived from ⁇ -conotoxin selected from the group consisting of GeXIVA, GeXXVIIA, Vc1.1, PeIA, RgIA , B-VxXXIVA, S-GVIIIB, D-GeXXA, O-GeXXVIIA, D--Lt28.1, Mr1.1, BuIA, ImI, or AuIB; the ⁇ -conotoxin natural monomer peptide includes ⁇ -conotoxin Mature peptide monomer peptide, precursor peptide monomer peptide of spirotoxin.
  • the present invention provides a method for preparing any one of the aforementioned active polypeptides, including recombinant expression method and chemical synthesis method, wherein the chemical synthesis method includes solid-phase synthesis method, liquid-phase synthesis method, solid-phase-liquid synthesis method Combined synthetic method, natural chemical linking method (NCL).
  • the chemical synthesis method includes solid-phase synthesis method, liquid-phase synthesis method, solid-phase-liquid synthesis method Combined synthetic method, natural chemical linking method (NCL).
  • the method for preparing active polypeptides of the present invention is characterized in that the solid-phase synthesis of linear polypeptides includes Fmoc solid-phase synthesis method and Boc solid-phase synthesis method; To the N-terminal synthesis method, from the N-terminal to the C-terminal synthesis method.
  • the method for preparing an active polypeptide according to the present invention wherein the Fomc solid-phase synthesis method from the C-terminal to the N-terminal comprises:
  • the method for preparing an active polypeptide of the present invention is characterized in that the cyclic polypeptide is synthesized by the NCL method, including:
  • the present invention provides a fusion protein or conjugate comprising the active polypeptide sequence described in any one of the foregoing, and the fusion protein or conjugate has the function of binding nAChR.
  • the present invention provides a multimer, which is formed by the polymerization of two or more polypeptide monomers, wherein at least one polypeptide monomer is the active polypeptide described in any one of the foregoing; The polypeptide monomers are covalently linked.
  • the multimer of the present invention is a homodimer or a heterodimer
  • each polypeptide monomer constituting the multimer is connected by a flexible linker or a PEG linker, preferably, each polypeptide monomer is connected by a flexible linker or a PEG linker at the respective amino terminus connected to each other.
  • the multimer of the present invention has a higher targeting activity of inhibiting human ⁇ 9 ⁇ 10 nAChR than any of the aforementioned active polypeptides in the monomeric form.
  • the present invention provides a nucleic acid molecule encoding any of the aforementioned active polypeptides, or the aforementioned fusion proteins or conjugates.
  • the present invention provides a construct comprising the aforementioned nucleic acid molecule.
  • the present invention provides a host cell comprising the aforementioned nucleic acid molecule and/or the aforementioned construct, or the host cell is transformed or transfected with the aforementioned nucleic acid molecule and/or the aforementioned construct.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the active polypeptide described in any of the foregoing, the foregoing fusion protein or conjugate, the foregoing nucleic acid molecule, the foregoing multimer, the foregoing construct, The aforementioned host cells, and optional pharmaceutically acceptable excipients.
  • the present invention provides the active polypeptide, the fusion protein or the conjugate, the nucleic acid molecule, the multimer, the construct, the host cell, and the pharmaceutical composition described in any one of the aforementioned ⁇ 9 ⁇ 10 nAChR inhibitors Use in , wherein the ⁇ 9 ⁇ 10 nAChR includes human ⁇ 9 ⁇ 10 nAChR, murine ⁇ 9 ⁇ 10 nAChR.
  • the present invention provides the active polypeptide, the fusion protein or the conjugate, the nucleic acid molecule, the multimer, the construct, the host cell, and the pharmaceutical composition described in any one of the foregoing in analgesia. , anti-tumor, and/or use in the treatment of neurological diseases.
  • the application of the present invention wherein the active polypeptide, fusion protein or conjugate, nucleic acid molecule, multimer, construct, host cell, and/or pharmaceutical composition, can be combined with optional analgesia, Combination or combination pharmaceuticals of active ingredients for antitumor and/or treatment of nervous system diseases.
  • the diseases include neuralgia, addiction, Parkinson's disease, epilepsy, ischemia, excitatory neuronal cell death, dementia, breast cancer, lung cancer, encephalomyelitis, cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis, herpes zoster, mechanical and surgical injuries, AIDS, paralysis of head nerves, drug poisoning, industrial pollution poisoning, lymphatic neuralgia, myeloma, Multipoint motor neuralgia, chronic congenital sensory neuropathy, acute severe idiopathic neuralgia, crush neuralgia, vasculitis, vasculitis, ischemia, uremia, childhood biliary liver disease, chronic respiratory disorders, complex neuralgia , multiple organ failure, sepsis/sepsis, hepatitis, porphyria, vitamin deficiency, chronic liver disease, native bile sclerosis, hyperlipidemia, lepro
  • polypeptide is intended to encompass a single “polypeptide” as well as a plurality of “polypeptides” and refers to a molecule composed of monomers (amino acids) linked linearly by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain or chains containing two or more amino acids, and does not refer to a product of a particular length.
  • polypeptides peptides, dipeptides, tripeptides, oligopeptides, "proteins," “chains of amino acids,” or any other term used to denote one or more chains of two or more amino acids are included within the term “polypeptide.” definitions, and the term “polypeptide” may be used instead or interchangeably with any of these terms.
  • Polypeptides of the present invention may have about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more amino acids size. Polypeptides can have a defined three-dimensional structure, but they need not have this structure. A polypeptide that has a defined three-dimensional structure is said to be folded, and a polypeptide that does not have a defined three-dimensional structure, but can adopt many different conformations, is said to be unfolded. Polypeptides as described herein may be terminally modified, eg -NH2 at the carboxy terminus.
  • the active polypeptide derived from conotoxin of the present invention can be extended or shortened according to the sequence of natural conotoxin, and cleavage of the sequence of conotoxin usually reduces its activity, but there are exceptions, such as the N-terminal free end of GID conotoxin (4 amino acid free polypeptide fragment), can still retain the activity of targeting ⁇ 4 ⁇ 2 nAChR after being cut (J Biol Chem. 2009 Feb 20; 284(8): 4944-51). Therefore, it is possible to substantially retain the activity of the polypeptide by cleaving the N-terminal 1-4 amino acids.
  • polypeptide is also intended to mean the post-expression modification product of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage or modified with non-naturally occurring amino acids.
  • Polypeptides may be derived from natural biological sources or produced by recombinant techniques, but need not be translated from a given nucleic acid sequence. It can be produced in any way, including by chemical synthesis. In the process of polypeptide structure optimization, non-natural modification or glycosylation strategy is usually used to improve the activity, stability and selectivity of the polypeptide.
  • polypeptides can be improved by replacing natural amino acids in the sequence with unnatural amino acids of similar physicochemical properties (J Biol Chem. 2009 Apr 3; 284(14): 9498-512.; ACS Chem Neurosci. 2019 Oct 16; 10(10 ): 4328-4336).
  • Arg-1 the side chain contains 4 C-length arginine analogs
  • Arg-2 the side chain contains 2 C-length arginine analogs
  • Arg-3 the side chain contains 1 C-length arginine analogs
  • the replacement of the basic amino acid Arg in the Gex-2 sequence can potentially retain or improve its activity, using Pro analogs 4-(R)-hydroxy-L-proline, [4-(R) -OH], 4-(R)-amino-L-proline, [4-(R)-NH2], 4-(S)-amino-L-proline, [4-(S)-NH2], 4- (R)-guanidino-L-proline,[4-(R)-Gn], 4-(R)-betainamidyl-L-proline,[4-(R)-Bet], 4-(R)-fluoro- L-proline,[4-(R)-F], 4-(S)-fluoro-L-proline,[4-(S)-(S
  • glycosylation Terends Biochem. Sci. 2006, 31, 156–163; Carbohydr. Res. 2009, 344, 1508–1514; 155
  • currently glycosylated amino acids mainly include Ser, Thr and Asn, so it can be expected that glycosylation of Ser at position 5 or Thr at position 17 in the Gex-2 sequence can improve the stability, half-life and bioavailability of the polypeptide.
  • Modification of linear polypeptides with long-chain fatty acids containing C12–C20 can improve the binding of polypeptides to serum proteins, thereby significantly improving the stability and half-life of polypeptides in plasma (J Med Chem. (2015) 58:7370–80.) Therefore, modification of Gex-2 with C12–C20 long-chain fatty acids is expected to improve the stability and bioavailability of the peptide.
  • the two ends of the active polypeptide derived from conotoxin of the present invention can be "capped" to improve its stability, especially its stability in plasma.
  • the N-terminus of Gex can be acetylated, methylated, etc. to continue to retain its activity, and its C-terminus can be free end, amination, esterification, etc., which does not affect the basic structure of the polypeptide and retains the activity of the polypeptide, improving the polypeptide. stability.
  • an "isolated" polypeptide or fragment, variant or derivative thereof is intended to be a polypeptide that is not in its natural surroundings. No specific purification level is required.
  • an isolated polypeptide can be removed from its native or natural environment.
  • Recombinantly produced polypeptides and proteins expressed in host cells for the purposes of the present invention are considered isolated, as are native or recombinant polypeptides that are isolated, fractionated, or partially or substantially purified by any suitable technique.
  • polypeptides of the present invention also include fragments, derivatives, analogs or variants of the foregoing polypeptides, and any combination thereof.
  • fragments include retaining at least some biological activity and/or function of the corresponding polypeptide.
  • Variants of the polypeptides of the present invention include fragments of the polypeptides, as well as polypeptides having altered amino acid sequences due to amino acid substitutions, deletions or insertions. Variants may or may not be naturally occurring. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. Variant polypeptides may contain conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of polypeptides are polypeptides that have been altered so as to exhibit additional characteristics not found on the native polypeptide. Examples include fusion proteins.
  • variant polypeptides may also be referred to herein as "polypeptide analogs.”
  • a “derivative" of a polypeptide refers to a subject polypeptide having one or more residues derivatized by reactive chemistry of functional pendant groups. "Derivatives” also include those peptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace serine; and ornithine can replace Substitute lysine.
  • the amino acids used in the present invention may be D- or L-isomers or mixtures thereof.
  • amino acid scanning refers to a site-directed mutagenesis technique used to determine the contribution of particular wild-type residues to the stability or function (eg, binding affinity) of a given protein or polypeptide. This technique involves substituting a wild-type residue in a polypeptide with a specific amino acid, such as an alanine residue, and subsequently assessing the stability or function (eg, binding affinity) of the alanine-substituted derivative or mutant polypeptide and comparing it with the wild-type polypeptide. Compared. Techniques for substituting alanine for wild-type residues in polypeptides are known in the art.
  • nucleic acid refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.
  • An "isolated" nucleic acid or polynucleotide is intended to be a nucleic acid molecule, DNA or RNA, removed from its native environment.
  • a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention.
  • isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partial or substantially) polynucleotides in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the invention.
  • Isolated polynucleotides or nucleic acids according to the present invention also include synthetically produced said molecules.
  • a polynucleotide or nucleic acid can be or can include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.
  • a "coding region” is a portion of a nucleic acid that consists of codons that are translated into amino acids. Although a "stop codon" (TAG, TGA or TAA) is not translated into amino acids, it can be considered part of the coding region, but any flanking sequences such as promoters, ribosome binding sites, transcription terminators, introns Subsidiaries etc. are not part of the coding region. Additionally, a vector, polynucleotide or nucleic acid of the invention may encode a heterologous coding region fused or unfused to a nucleic acid encoding the polypeptide or fragment, variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretion signal peptides or heterologous functional domains.
  • the polynucleotide or nucleic acid is DNA.
  • a polynucleotide comprising a nucleic acid encoding a polypeptide may generally comprise a promoter and/or other transcriptional or translational control elements operably associated with one or more coding regions. Operably associated is for the coding region of a gene product (eg, a polypeptide) to associate with one or more regulatory sequences in such a manner that expression of the gene product is under the influence or control of the regulatory sequences under.
  • a promoter region will be operably associated with a nucleic acid encoding a polypeptide so long as the promoter is capable of effecting transcription of the nucleic acid.
  • the promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in the intended cell.
  • transcriptional control elements such as enhancers, operators, repressors, and transcription termination signals can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein.
  • transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but are not limited to, from cytomegalovirus (immediate early promoter, associated with intron-A), simian virus 40 (early promoter), and reverse Promoter and enhancer segments of videoviruses such as Rous sarcoma virus.
  • Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit beta-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells.
  • RNA eg, in the form of messenger RNA (mRNA).
  • mRNA messenger RNA
  • polynucleotides and nucleic acid coding regions of the invention may have associated additional coding regions encoding secretion or signal peptides that direct secretion of the polypeptides encoded by the polynucleotides of the invention.
  • proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum is initiated.
  • polypeptides secreted by vertebrate cells often have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the intact or "full-length" polypeptide to produce a secreted or "mature” polypeptide. "form of polypeptides.
  • a native signal peptide eg, a conotoxin signal peptide, or a functional derivative of the sequence that retains the ability to direct secretion of a polypeptide with which it is operably associated is used.
  • heterologous mammalian signal peptides or functional derivatives thereof can be used.
  • the wild-type leader sequence can be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse beta-glucuronidase.
  • vector or “construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and an insert.
  • Recombinant expression vectors are typically generated for the purpose of expressing and/or propagating an insert, or for the purpose of constructing other recombinant nucleotide sequences. Inserts may or may not be operably linked to promoter sequences, and may or may not be operably linked to DNA regulatory sequences.
  • fusion protein chimeric protein
  • protein conjugate and the like are meant to include amino acids in addition to the amino acid sequence encoding the original or native full-length protein or a subsequence thereof, including substitutions encoding the original or native full-length protein or a subsequence thereof.
  • the amino acid sequence of the long protein or subsequence thereof comprising less than the amino acid sequence encoding the original or native full length protein or subsequence thereof and/or comprising a different amino acid sequence than that encoding the original or native full length protein or subsequence thereof.
  • More than one additional domain can be added to an active polypeptide as described herein, eg, one epitope tag or purification tag, or multiple epitope tags or purification tags. Additional domains may be attached, eg, which may add additional activity, targeting function, or affect physiological processes (eg, vascular permeability or biofilm integrity). Alternatively, domains can be associated to create physical affinity between different polypeptides, resulting in multi-chain polymer complexes.
  • composition or “pharmaceutical composition” can include a composition comprising an active polypeptide of the present disclosure and, for example, a pharmaceutically acceptable carrier, excipient or diluent, which composition is administered to an individual subject .
  • compositions that, within the scope of sound medical judgment, are suitable for contact with human and animal tissue without undue toxicity or other complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable refers to compositions that, within the scope of sound medical judgment, are suitable for contact with human and animal tissue without undue toxicity or other complications, commensurate with a reasonable benefit/risk ratio.
  • the polypeptides, polynucleotides, compositions and vaccines described herein are pharmaceutically acceptable.
  • an “effective amount” is an amount effective for treatment or prevention when administered to a subject in a single dose or as part of a series of doses.
  • the term "subject” refers to any subject in need of diagnosis, prognosis, immunization or treatment, particularly mammalian subjects.
  • Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals such as bears, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, cows; Primates, such as apes, monkeys, orangutans, and chimpanzees; canids, such as dogs and wolves; felines, such as cats, lions, and tigers; equines, such as horses, donkeys, and zebras; food animals, such as cattle , pigs and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs.
  • the subject is a human.
  • improvement refers to any therapeutically beneficial outcome in the treatment of a disease state, such as cancer, including prevention, reduction in severity or progression of the disease state, alleviation, or cure.
  • ⁇ -conotoxin Based on the spatial structure of ⁇ -conotoxin and its receptor ⁇ 9 ⁇ 10nAChR, a series of active polypeptides were designed and synthesized by computer-aided molecular simulation. On the one hand, an active polypeptide with a small number of amino acid residues, no cysteine, and high specific human ⁇ 9 ⁇ 10 nAChR inhibitory activity was prepared. It is not only easy to chemically synthesize, eliminates the generation of complex disulfide bond isomers, but also avoids the influence of amino acid substitution on biological activity, and maintains the specific inhibitory function of natural ⁇ -conotoxin on human ⁇ 9 ⁇ 10nAChR.
  • the inhibitory activity of ⁇ 9 ⁇ 10nAChR in different species was analyzed, and the results showed that the polypeptide involved in the present invention can not only inhibit mouse ⁇ 9 ⁇ 10nAChR, but also effectively inhibit human ⁇ 9 ⁇ 10nAChR; on the other hand, according to the effect of inhibiting ⁇ 9 ⁇ 10nAChR Mechanism, pain threshold analysis was performed on rat animal model, and good analgesic effect was obtained, and there was a dose-dependent relationship within a certain range.
  • Figure 1 The relative inhibitory intensity of active peptides on the peak currents stimulated by acetylcholine on human ⁇ 9 ⁇ 10 acetylcholine receptors, in which the whole-cell currents were stimulated by 6 ⁇ M acetylcholine, and the peptide concentration was 30 nM.
  • Figure 2A IC50 of Gex-2 for human ⁇ 9 ⁇ 10 nAChR.
  • Figure 2B Comparison of inhibitory activities of Gex-2 and its dimers on human ⁇ 9 ⁇ 10 acetylcholine receptors
  • the X-axis represents time (unit: days); the Y-axis represents mechanical pain threshold (unit: grams)
  • the X-axis represents time (unit: days); the Y-axis represents thermal pain threshold (unit: seconds)
  • Figure 5 The relative inhibitory intensity of the active polypeptide Gex-2 alanine scanning variant on the peak current excited by acetylcholine on the human ⁇ 9 ⁇ 10 acetylcholine receptor, wherein the whole-cell current was stimulated by 6 ⁇ M acetylcholine, and the peptide concentration was 30 nM.
  • Figure 6 The relative inhibitory intensity of the active polypeptide Gex-2 aspartate scanning variant on the peak current stimulated by acetylcholine on the human ⁇ 9 ⁇ 10 acetylcholine receptor, wherein the whole-cell current was stimulated by 6 ⁇ M acetylcholine, and the peptide concentration was 30 nM.
  • Figure 7 The relative inhibitory intensity of the active polypeptide Gex-2 arginine scanning variant on the peak current excited by acetylcholine on the human ⁇ 9 ⁇ 10 acetylcholine receptor, wherein the whole-cell current was stimulated by 6 ⁇ M acetylcholine, and the peptide concentration was 30 nM.
  • Figure 8 The relative inhibitory intensity of the active polypeptide Gex-2 arginine-citrulline substitution variant on the peak current induced by acetylcholine on the human ⁇ 9 ⁇ 10 acetylcholine receptor, wherein the whole-cell current was stimulated by 6 ⁇ M acetylcholine, and the peptide concentration was 30 nM .
  • Figure 9 The relative inhibitory intensity of the D-amino acid substitution variant of the active polypeptide Gex-2 on the peak current stimulated by acetylcholine on the human ⁇ 9 ⁇ 10 acetylcholine receptor, wherein the whole-cell current was stimulated by 6 ⁇ M acetylcholine, and the peptide concentration was 30 nM.
  • Figure 12 PWT of rat plantar mechanical pain measured 1 h after Gex-2 administration
  • Figure 13 PWT of heat pain in rats 1 h after Gex-2 administration
  • Figure 14 PWT of rat plantar mechanical pain 24h after Gex-2 administration
  • Figure 15 PWT of rat plantar heat pain 24h after Gex-2 administration
  • Figure 16 Evaluation of the analgesic activity and toxicity of Gex-2 in an animal model of trigeminal neuralgia.
  • A evaluation of the analgesic activity of Gex-2
  • B evaluation of the effect of Gex-2 on the movement and balance ability of animals.
  • polypeptide in the present invention can be synthesized by Fmoc or Boc solid-phase synthesis method.
  • the peptide sample was purified by preparative reverse HPLC C8 column, solvent A was 90% pure water, 10% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA); solvent B was 60% pure Water, 40% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA), eluting with a linear gradient of solvent B from 0%-60% in 40 min;
  • Cyclic peptides were synthesized by NCL method. Cyclic peptides do not have a head-to-tail distinction. Linear peptides are synthesized first, and then cyclized according to the reaction mechanism of the NCL method. A straight-chain peptide is synthesized from the C-terminal to the N-terminal, and the amino acid residue connected to the cysteine residue-NH2 side of the cyclic peptide is used as the first amino acid at the C-terminal of the linear peptide, and cysteine is used as the last amino acid. residues, which are then cyclized via an acyl transfer reaction.
  • the peptide sample was purified by preparative reverse HPLC C8 column, solvent A was 90% pure water, 10% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA); solvent B was 60% pure Water, 40% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA), eluting with a linear gradient of solvent B from 0%-60% in 40 min;
  • the cRNA of human ⁇ 9 ⁇ 10 acetylcholine receptor was prepared by in vitro transcription kit, and its concentration was measured by OD value under UV260m. Xenopus oocytes were dissected and collected, and the cRNAs of the two subunits were injected into frog eggs on the first and second day, and the injection amount was 5ng, and cultured in ND-96. Oocytes expressing the ⁇ 9 ⁇ 10 acetylcholine receptor were tested for electrophysiological activity 1-4 days after injection.
  • the specific test method is as follows: Place 1 cRNA-injected Xenopus oocyte in a 30uL Sylgard recording tank with a diameter of 4mm and a depth of 2mm, and gravity perfusion with ND96 perfusate containing 0.1mg/ml bovine serum albumin (BSA). (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2 1.0 mM MgCl2 , 5 mM HEPES, pH 7.1-7.5).
  • BSA bovine serum albumin
  • All conotoxin solutions also contained 0.1 mg/mL BSA to reduce non-specific adsorption, a switch valve was used to switch freely between perfusion toxin or acetylcholine, and the ACh-gated current was set at "slow" by a two-electrode voltage clamp amplifier file, and the clamp gain is recorded online when it is at the maximum ( ⁇ 2000) position.
  • Glass electrodes were drawn with glass capillaries with an outer diameter of 1 mm and an inner diameter of 0.75 mm, and filled with 3M KCl as voltage and current electrodes. The membrane voltage was clamped at -70mV, and the entire system was controlled and recorded by a computer.
  • the ACh pulse was automatically perfused with ACh for 1 s every 5 min, and the concentration of ACh was 6 ⁇ M.
  • the current responses of at least 6 oocytes were recorded for each polypeptide, and the tested current data were statistically analyzed with GraphPad Prism software.
  • Example 3 Construction of an animal model of in vivo analgesic activity
  • the modeling process is as follows: after the rats were anesthetized by intraperitoneal injection of 2% pentobarbital sodium, the hair of the right leg was removed, the skin of the right lower limb was incised 1-2 cm under aseptic conditions, the muscles and fascia were bluntly separated, and the trunk of the sciatic nerve was exposed.
  • the mechanical pain pain threshold of rats was measured with IITC electronic pain measuring instrument, and the thermal pain pain threshold of rats was measured with IITC plantar hot spot pain measuring instrument.
  • the conotoxin targeting the ⁇ 9 ⁇ 10 acetylcholine receptor was searched on ConoServer, and GeXIVA and GeXXVIIA were selected for sequence alignment. Based on the overlapping part of the two sequences, combined with peptide docking and molecular dynamics simulation analysis, the overlap between the two was analyzed. The fragments were modified reasonably, and a series of polypeptides were designed and synthesized. The sequences are shown in Table 1.
  • Fmoc solid-phase synthesis method was used to synthesize linear polypeptide, and cyclic peptide Gex-4 was synthesized using NCL synthesis method.
  • NCL synthesis method using Xenopus oocytes expressing ⁇ 9 ⁇ 10 acetylcholine receptors, the inhibitory activities of eight polypeptides on ⁇ 9 ⁇ 10 acetylcholine receptors were tested, and the activity test results are shown in FIG. 1 .
  • Example 2 the Xenopus oocytes expressing the ⁇ 9 ⁇ 10 acetylcholine receptor were used to detect and calculate the IC50 value of Gex-2 for the inhibition of the human ⁇ 9 ⁇ 10 acetylcholine receptor, and the results are shown in FIG. 2A .
  • Figure 2 shows that Gex-2A has an IC50 of ⁇ 25 nM for inhibition of human ⁇ 9 ⁇ 10 nAChR.
  • Gex-2 was coupled to form a dimer by linker (PEG13), and the inhibitory activity of Gex-2 and its dimer on human ⁇ 9 ⁇ 10 acetylcholine receptor was compared, and the results were shown in Figure 2B.
  • the results of Figure 2B show that the inhibitory activity of the dimer on the ⁇ 9 ⁇ 10 acetylcholine receptor is significantly improved compared with the Gex-2 monomer.
  • Gex-2 was selected as the representative of active polypeptide, and its analgesic effect was further studied in animal model. According to the method of Example 3, the analgesic activity test of Gex-2 was carried out using the rat sciatic nerve compression injury (CCI) model,
  • the mechanical pain pain threshold of rats was measured with IITC electronic pain measuring instrument, and the thermal pain pain threshold of rats was measured with IITC plantar hot spot pain measuring instrument.
  • the working light intensity was set. is "025", the standby light intensity is set to "010”, the preset time is set to "20”, and the obtained data is statistically analyzed with GraphPad Prism software.
  • Gex-2 was scanned for alanine, and alanine-scanned variants were designed and synthesized, as shown in Table 3.
  • Example 2 using Xenopus oocytes expressing ⁇ 9 ⁇ 10 acetylcholine receptors, the inhibitory activity of Gex-2 polypeptide alanine scanning variants on ⁇ 9 ⁇ 10 acetylcholine receptors was detected, and the activity test results are shown in FIG. 5 .
  • Figure 5 shows that the relative current intensities of Gex-19, Gex-20, Gex-21, and Gex-22 are lower than 0.4, and the relative current intensities of other variants are all higher than those of Gex-2.
  • the above experimental results show that the four amino acid residues of R11-R14 in Gex-2 have little effect on the activity of Gex-2, and the activity can still be retained or even improved after single mutation. It can retain or even improve its biological activity; while other amino acid residues in the Gex-2 sequence are substituted by Ala, the activity is greatly reduced, indicating that the amino acids at these positions are crucial for maintaining the activity or structure of the polypeptide. Afterwards, the activity of the polypeptide will be greatly reduced.
  • substitution of amino acid residues with similar physicochemical properties such as Arg in the basic amino acid substitution sequence of Lys/Dab/Dap
  • substitution of hydrophobic amino acids such as Leu/Val for Ile
  • substitution of Asp in the acidic amino acid sequence such as Glu
  • Example 6 Gex-2 Aspartate and Arginine Scanning Variants and Inhibitory Activity on Human ⁇ 9 ⁇ 10 nAChR
  • Example 2 using Xenopus oocytes expressing ⁇ 9 ⁇ 10 acetylcholine receptors, the inhibitory activity of Gex-2 polypeptide aspartate scanning variants and arginine scanning variants on ⁇ 9 ⁇ 10 acetylcholine receptors was detected.
  • the test results are shown in Figure 6 and Figure 7, respectively.
  • Figure 6 shows that the replacement of Y3 and P6 of Gex-2 polypeptide with aspartic acid has little effect on the activity, while the introduction of negatively charged amino acids at other positions will reduce the activity of the polypeptide or even inactivate it.
  • Y3 or P6 is crucial for maintaining the activity of the polypeptide.
  • the introduction of Asp acidic amino acids with very different physicochemical properties can preserve the activity of the polypeptide, indicating that the introduction of negatively charged sides at positions 3 and 6
  • the amino acids of the chain (Asp or Glu, etc.) can compensate for the important role played by the side chain in the original amino acid.
  • the substitution of the negatively charged amino acid Asp at the N-terminal 1 residue of Gex-2 can greatly reduce the activity of Gex-2.
  • Figure 7 shows that when a positively charged amino acid Arg is introduced into the S5 or 116 position of the polypeptide, the activity of the polypeptide will be greatly improved, indicating that the introduction of a positively charged amino acid (Lys/Dab/Dap, etc.) at the 5th or 16th position will enhance the polypeptide's activity Activity; when Y13 or D18 is replaced by R, it has little effect on the activity of the polypeptide, indicating that the side chain of R can compensate for the effect of the side chain of Y13 and D18; the introduction of R at other positions will reduce the activity of the polypeptide.
  • Example 7 Gex-2 polypeptide arginine-citrulline substitution variant and its inhibitory activity on human ⁇ 9 ⁇ 10 nAChR
  • Example 2 using Xenopus oocytes expressing ⁇ 9 ⁇ 10 acetylcholine receptors, the inhibitory activity of Gex-2 polypeptide arginine-citrulline substitution variants on ⁇ 9 ⁇ 10 acetylcholine receptors was detected, and the activity test results are shown in the figure 8 shown.
  • Example 8 D-type amino acid substitution variant of Gex-2 polypeptide and its inhibitory activity on human ⁇ 9 ⁇ 10 nAChR
  • Example 2 using Xenopus oocytes expressing ⁇ 9 ⁇ 10 acetylcholine receptors, the inhibitory activity of Gex-2 polypeptide D-type amino acid substitution variants on ⁇ 9 ⁇ 10 acetylcholine receptors was detected, and the activity test results are shown in Figure 9 .
  • Figure 9 shows that the substitution of D-type amino acids can maintain the activity of the polypeptide, and the substitution of R11 or R14 by D-type amino acid can significantly improve the activity of the polypeptide.
  • the inhibition rate of Gex-2 at 30nM concentration is about 50%. It can be seen from the results in the above figure: except for positions 2 and 4, after the amino acid configuration of other positions is changed from L-type to D-type, the activity of the polypeptide is all to a certain extent. improve. Therefore, it is inferred that the combination of multiple D-type amino acid analogs of Gex-2 is beneficial to improve the activity of the polypeptide. Meanwhile, based on previous studies (J Med Chem. 2020 Apr 9; 63(7): 3475-3484; Mar Drugs. 2019 Feb 28; 17(3): 142.), the introduction of D-amino acids into conotoxin can improve its stability , it can be known that the D-amino acid analog of Gex-2 can retain or even improve its activity.
  • the rats were anesthetized by intraperitoneal injection of 65 mg/kg sodium pentobarbital solution, and the rats were completely anesthetized in about 10 minutes, and the muscle reaction disappeared; the surgical area of the right lower limb of the rats was soaked with 75% alcohol, and the rat hair was removed; The prone position is placed on the ultra-clean workbench, and a sterile hole towel is placed on it, and only the surgical site is exposed to avoid rat hair entering the wound and causing foreign body infection.
  • the skin was sutured, and some penicillin powder was sprinkled on the skin wound after suture to prevent postoperative infection of the rats.
  • the rats were placed in an incubator until the rats were awake, and after they regained their mobility, they were reared in a single cage until the wounds healed.
  • the pain threshold of the CCI rats will decrease to the minimum value, and the drug administration experiment can be carried out at this time.
  • the modeling steps of the sham-operated rats were similar to those of the CCI rats, the only difference was that the sciatic nerve was not ligated in the sham-operated rats, only the sciatic nerve was exposed by surgery, and then directly flushed and sutured. Sham-operated rats were used to simulate the effects of skin wounds and internal muscle tears on rat pain thresholds.
  • the single-dose experiment was mainly used to evaluate the analgesic effect of Gex-2 polypeptide after a single dose, the time from onset to peak value and the duration of the analgesic effect.
  • the single-dose experiment we set up the control group, the sham-operated group, the normal saline group, the Gex-2 group and the morphine group, in which the rats in the control group did not receive any treatment, and the pain threshold measured was the whole test process.
  • the baseline of represents the pain threshold of normal rats; the sham-operated group was used to simulate the effect of skin injury and muscle tear on the pain threshold of rats; the Gex-2 group was the experimental group; the normal saline group was used as a negative control; morphine group as a positive control.
  • the mode of administration and dosage are shown in the table:
  • the drugs were respectively dissolved/diluted to the concentration required for the experiment within 0.5h before administration, and administered by in situ intramuscular injection.
  • the administration site was in the muscle area of the middle leg of the operation side, and the dose of each rat was controlled. in 200 ⁇ L.
  • the mechanical pain and thermal pain thresholds were tested at 0.5h, 1h, 2h, 4h, 6h, 12h, 24h and 48h respectively. Each rat was measured three times, and the mean value was taken as the pain threshold.
  • the rats used for the long-term analgesic activity test experiment were divided into six groups: control group, sham operation group, normal saline negative control group, GeXIVA positive control group, Gex-2 low-dose group and Gex-2 high-dose group.
  • the rats in the control group did not receive any treatment and served as a blank control; the rats in the sham-operated group were used to evaluate the effect of skin injury and muscle laceration on the pain threshold of the rats; the normal saline group was used as a negative control; Gex-2 was divided into two groups.
  • the dose per rat was 0.075 ⁇ g/kg, while in the high-dose group the dose per rat was 0.25 ⁇ g/kg; GeXIVA was used as the For the positive control, the dosage of each rat was the same as that of the Gex-2 high-dose group, which was 0.25 ⁇ g/kg.
  • GeXIVA and Gex-2 were prepared to the required concentration with physiological saline 0.5h before administration every day, and the dosage of each rat was controlled at 200 ⁇ L, which were administered by in situ intramuscular injection. After continuous administration for 14 days, the mechanical pain threshold and thermal pain threshold of rats were tested at 1h and 24h after each administration, each rat was measured three times, and the mean value was taken as the measured pain threshold data.
  • the original data unit of the mechanical pain paw withdrawal threshold is g
  • the original data unit of the thermal pain paw withdrawal threshold is s. All data are expressed as mean ⁇ SEM, and GraphPad Prism7.0 software was used for statistical processing and graphing.
  • the mechanical pain PWT measured after a single dose of Gex-2 is shown in Figure 10:
  • the abscissa is time, the unit is hour (h), the ordinate is the rat plantar mechanical pain PWT, the unit is grams (g).
  • Blue is the PWT value of plantar mechanical pain in the control group, red is the sham-operated group, purple is the Gex-2 group, orange is the morphine group, and green is the normal saline group.
  • the purple and orange markers above the broken line represent the significance level of the difference between the Gex-2 group and the morphine group and the saline group, respectively, at that time point.
  • the mechanical pain PWT of the normal rats is about 65g, which is consistent with the results measured in the previous experiments;
  • the PWT of the sham-operated rats is basically the same as that of the control group, indicating that the skin wounds and muscles caused by the surgery are Laceration had no effect on the changes of plantar mechanical pain threshold in CCI rats, probably because the skin and muscle injuries of the rats were recovered within two weeks before the experiment;
  • the thermal pain PWT measured after a single administration of Gex-2 is shown in Figure 11:
  • the horizontal axis is time, the unit is hour (h), the vertical axis is the rat plantar thermal pain PWT, the unit is second(s).
  • the blue is the PWT value of plantar heat pain in the control group, the red is the sham-operated group, the purple is the Gex-2 group, the orange is the morphine group, and the green is the normal saline group.
  • the purple and orange markers above the broken line represent the significance level of the difference between the Gex-2 group and the morphine group and the saline group, respectively, at that time point.
  • 0.25 ⁇ g/kg Gex-2 exhibited stronger and longer-lasting analgesic effects in the CCI model.
  • 0.25 ⁇ g/kg of Gex-2 can effectively relieve sciatica in CCI rats, making the plantar PWT of the affected limb of the rat close to the plantar PWT of the normal rat.
  • Gex-2 takes effect quickly, and the analgesic effect is about 2h Reaching the peak, the drug effect can last for at least 48 hours.
  • the GeXIVA group, the Gex-2 group and the normal saline group were administered intramuscularly for 14 consecutive days, and the pain thresholds of mechanical pain and thermal pain were tested 1 h after each administration, and the graphs were analyzed.
  • Figure 12 shows the results of PWT of mechanical plantar pain in rats measured 1 h after administration: in the figure, the abscissa is the number of days, and the ordinate is the PWT of mechanical plantar pain of rats, and the unit is grams (g).
  • Blue is the PWT value of plantar mechanical pain in the control group
  • red is the sham-operated group
  • orange is the 0.25 ⁇ g/kg Gex-2 group
  • purple is the 0.075 ⁇ g/kg Gex-2 group
  • black is the GeXIVA group
  • green is the physiological group saline group.
  • the orange, purple and black marks above the broken line represent the significance level of the difference between the corresponding group and the normal saline group, respectively.
  • the results of PWT of rat heat pain 1 h after administration are shown in Figure 13: as shown in Figure 13, the abscissa is the number of days, and the rats were given continuous intramuscular injection for 14 days; the ordinate is the heat pain PWT of the rat, the unit is second(s).
  • Blue is the PWT value of plantar mechanical pain in the control group, red is the sham-operated group, orange is the 0.25 ⁇ g/kg Gex-2 group, purple is the 0.075 ⁇ g/kg Gex-2 group, black is the GeXIVA group, and green is the physiological group saline group.
  • the orange, purple and black marks above the broken line represent the significance level of the difference between the corresponding group and the normal saline group, respectively.
  • the pain thresholds of the rats in the control group and the sham-operated group were between 17-20s, while the pain thresholds of the rats in the normal saline group decreased to less than 10s, indicating that skin wounds and muscle injuries did not affect the rats.
  • Thermal pain sensitivity has a significant effect, and the decrease in pain threshold in rats is mainly caused by chronic compression of the sciatic nerve after ligation.
  • CCI rats were injected with 0.3nM Gex-2 or 0.25 ⁇ g/kg GeXIVA, the thermal pain thresholds of the rats were significantly increased, although the pain thresholds of the rats could not be restored to the normal level, and due to individual differences The data fluctuated greatly, but there were significant differences between the two groups and the saline group.
  • Gex-2 has a very good analgesic effect at very low doses, and administration for 14 consecutive days does not induce dependence in rats.
  • the analgesic effect of Gex-2 at a dose of 0.075 ⁇ g/kg was similar to that of GeXIVA at 0.25 ⁇ g/kg, indicating that the analgesic activity of the compound was superior to that of GeXIVA; at a dose of 0.25 ⁇ g/kg, it was almost The mechanical pain PWT of CCI rats was restored to the preoperative threshold, and the claudication and foot valgus of the rats were significantly improved, and the drug did not cause drug dependence in the rats for up to 14 days.
  • Figure 14 shows the PWT results of mechanical pain in the plantar of the rat 24 hours after administration: as shown in Figure 14, the abscissa is the time, the unit is day; the ordinate is the mechanical pain PWT of the rat, the unit is grams (g) .
  • Blue is the PWT value of plantar mechanical pain in the control group, red is the sham-operated group, orange is the 0.25 ⁇ g/kg Gex-2 group, purple is the 0.075 ⁇ g/kg Gex-2 group, black is the GeXIVA group, and green is the physiological group saline group.
  • the orange, purple and black marks above the broken line represent the difference between the corresponding group and the saline group, respectively.
  • Rats The PWT was still significantly different from the normal saline group; and when Gex-2 was continuously administered at a dose of 0.25 ⁇ g/kg, the analgesic effect after 24 hours of each administration could still restore the pain threshold of CCI rats to Compared with the level of normal rats, the analgesic effect was not significantly weakened after administration for 1 h, indicating that 0.25 ⁇ g/kg of Gex-2 has a strong and durable analgesic effect, and the rats in the Gex-2 administration group The mean value of PWT in the second week was higher than that in the first week, indicating that Gex-2 also had a cumulative effect on its efficacy. In addition, during the 14 consecutive days of administration, the gait of the rats gradually tended to be normal, and the phenomenon of lameness and foot eversion were significantly improved, indicating that the pain of the rats was significantly relieved. .
  • Figure 15 shows the PWT results of plantar heat pain in rats 24 hours after administration: as shown in Figure 15, the abscissa is time, the unit is day; the ordinate is the heat pain PWT of the rat, the unit is second (s) .
  • the blue is the PWT value of plantar heat pain in the control group, the red is the sham operation group, the orange is the 0.25 ⁇ g/kg Gex-2 group, the purple is the 0.075 ⁇ g/kg Gex-2 group, the black is the GeXIVA group, and the green is the physiological group saline group.
  • the orange, purple and black marks above the broken line represent the significance level of the difference between the corresponding group and the normal saline group, respectively.
  • mice can be maintained for at least 24h.
  • Example 12 Activity evaluation experiment of Gex-2 polypeptide on trigeminal neuralgia
  • the bone cancer pain evaluation experiment was carried out according to the following table 9, and the results were tested at 1 hour and 24 hours after administration, respectively, as shown in Figures 17-18.
  • the results showed that: (1) Gex-2 played a good analgesic effect. , with statistical significance, but the activity after 24 hours of administration was lower than that after 1 hour of administration;
  • the device used in the conditioned place preference experiment is a three-box CPP system.
  • the inside of the left box is all black, and the inside of the right box is black and white with equally spaced stripes. It is 30cm x 10cm x 30cm.
  • Pretreatment stage On the first day after the rats adapt to the environment, the partition of the CPP box is removed to open the shuttle door, the rats enter from the middle box, and the rats are allowed to shuttle freely in the three boxes for 15 minutes, and no data is recorded at this time. . On days 2-3, record the stay time of the rats in each box within 15 minutes, take the average of the two-day experimental data, and select rats with no obvious preference for follow-up experiments. , the rat's non-preferred box was used as the companion box.
  • the rats were divided into four groups, which were the morphine administration group with the black box as the medicine box, the morphine administration group with the black and white box as the medicine box, and the Gex-2 administration group with the black box as the medicine box group and Gex-2 administration group with black and white box as medicine box, 6 animals in each group.
  • the morphine administration group and the Gex-2 group were injected intramuscularly with the corresponding drugs.
  • the dosage of morphine group was gradually increased from 5 mg/kg to 10 mg/kg, the dosage of Gex-2 group was kept at 1 nM, and the control group was injected with normal saline, and the drug injection volume was 200 ⁇ L per animal.
  • the rats were allowed to move freely for 40 min with the medicine box.
  • the three groups were injected intramuscularly with the same amount of normal saline, and put the rats into the non-accompanied medicine box. 40 minutes of free time.
  • Test phase On the first day after the training is completed, the rat CPP score test is performed.
  • the specific method is as follows: take out the partition of the CPP box to open the shuttle door, put the rat in the middle box to make it shuttle freely between different boxes, record the time the rat stays in the different boxes within 15 minutes, and observe the Changes in the preference of rats; on the second day after training, each rat was injected with 0.01 mg/kg naloxone to induce withdrawal reactions, and then the rats were placed in the middle box to make them in different boxes. Freely shuttle between the bodies, record the time that the rats stay in different boxes within 15 minutes, and observe the changes in the preference of the rats. During the test, the experimenter performed the double-blind principle.
  • the ordinate is the ratio of the time the rat spends in the companion medicine box to the total time. The higher the ratio, the longer the time the rat spends in the medicine companion box, the stronger the preference for the rat. .
  • Natural Preference (white) refers to the natural preference of the rats before the experiment. Since the medicine box we chose is the natural non-preference box of the rats, the data of the Natural Preference group are all less than 50%. This was followed by 8 days of continuous administration training. During this process, the rats in the saline group and Gex-2 group behaved normally, while the rats in the morphine group gradually developed symptoms of drug addiction, such as mania and malaise, and abnormal post-administration. Excited, exaggerated range of motion, etc.

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Abstract

Provided is an α9α10 nicotinic acetylcholine receptor (nAChR) inhibitory peptide and a use thereof. On the basis of the mechanism of interaction between α-conotoxin and α9α10 nicotinic acetylcholine receptors, mutants having a plurality of α-conotoxin monomeric peptides are designed, and a monomeric peptide mutant GEX-2 having high inhibitory activity and a good analgesic effect is screened from among the mutants. Further, a series of derivatives of GEX-2 are obtained by mutating GEX-2 by means such as alanine scanning, aspartic acid scanning, arginine scanning, arginine-citrulline substitution and D-amino acid substitution. The described GEX-2 can improve the pain threshold of a CCI rat, and has a good analgesic effect. In comparison with GEX-2, the inhibitory activity of a portion of members in the GEX-2 series derivatives on α9α10 nAChR is higher. The described GEX-2 and the series derivatives thereof may be used as α9α10 nAChR inhibitors for analgesia, fighting tumors, treating nervous system diseases, etc.

Description

一种α9α10nAChR抑制活性肽及其应用A kind of α9α10nAChR inhibitory active peptide and its application 技术领域technical field
本发明属于生物制药领域，具体涉及一种具有镇痛活性的多肽及其应用，特别涉及基于天然α-芋螺毒素及其作用机理设计、制备、筛选获得的一种具有镇痛活性的单链多肽及其衍生物。The invention belongs to the field of biopharmaceuticals, in particular to a polypeptide with analgesic activity and application thereof, in particular to a single-chain polypeptide with analgesic activity obtained by design, preparation and screening based on natural α-conotoxin and its mechanism of action Polypeptides and their derivatives.
背景技术Background technique
烟碱型乙酰胆碱受体(nAChRs)是动物界普遍存在的离子通道型受体，从低等的线虫到高等的哺乳动物都含有该类受体(Nicke,A.(2004)Learning about structure and function of neuronal nicotinicacetylch oline receptors.Lessons from snails.European journal ofbiochemistry/FEBS 271,2293)。nAChRs受体位于神经-肌肉(和/或)神经-神经接头的突触内和突触外，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。nAChRs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、感觉信号加工和运动控制等，它们具有重要的生理功能和临床研究意义。大量研究显示nAChRs是筛选诊断和治疗一大类重要疾病药物的关键靶点，这些疾病包括疼痛、成瘾、癌症、智障、帕金森症、精神病、抑郁、重症肌无力等疑难杂症。nAChRs是由5个亚基组成的五聚体跨膜蛋白，分为肌肉型和神经型两大类，其中神经型nAChRs异常复杂，它们由不同的α和/或β亚基组成异源或同源型的功能性受体亚型，在脊椎动物中，至少有12个亚基，即α2–α10,β2–β4。发现和开发各个亚型的特异性分子探针，将有利于揭示和阐释它们在生命体内的功能，同时有可能研发出针对上述不同疾病的治疗药物。Nicotinic acetylcholine receptors (nAChRs) are ubiquitous ion channel receptors in the animal kingdom, from lower nematodes to higher mammals contain such receptors (Nicke, A. (2004) Learning about structure and function of neuronal nicotinicacetylch oline receptors. Lessons from snails. European journal of biochemistry/FEBS 271, 2293). nAChRs receptors are located in and out of synapses at the nerve-muscle (and/or) nerve-nerve junction, and activate the release of various neurotransmitters such as dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid. nAChRs mediate many physiological functions of the central and peripheral nervous system, including learning, memory, response, analgesia, sensory signal processing and motor control, etc. They have important physiological functions and clinical research significance. Numerous studies have shown that nAChRs are key targets for screening, diagnosing and treating a large class of important diseases, including pain, addiction, cancer, intellectual disability, Parkinson's disease, mental illness, depression, myasthenia gravis and other intractable diseases. nAChRs are pentameric transmembrane proteins composed of 5 subunits, which can be divided into two categories: muscle-type and neuron-type, among which neuron-type nAChRs are very complex, they are composed of different α and/or β subunits, heterologous or homologous. The functional receptor subtype of the source type, in vertebrates, has at least 12 subunits, namely α2–α10, β2–β4. The discovery and development of specific molecular probes for each subtype will help to reveal and explain their functions in life, and it is possible to develop therapeutic drugs for the above-mentioned different diseases.
在神经型nAChRs的各种亚型中，α9α10亚型在生物医药领域备受关注。α9α10烟碱型乙酰胆碱受体是由α9和α10两种亚基组成的跨膜五聚体，是一种配体门控离子通道。该受体最早被发现存在于耳蜗毛细胞中，介导橄榄耳蜗胆碱能纤维和传出神经之间的突触传递，后被证明在白细胞、背根神经节、垂体、皮肤角质细胞和精液中也有分布。研究表明，α9α10nAChR是治疗神经痛药物的新靶点(McIntosh,J.M.；Absalom,N.；Chebib,M.；Elgoyhen,A.B.；Vincler,M.,Alpha9nicotinic acetylcholine receptors and thetreatment of pain.Bioche mical pharmacology 2009,78(7),693-702.Satkunanathan,N.；Livett,B.；Gayler,K.； Sandall,D.；Down,J.；Khalil,Z.,Alpha-conotoxin Vc1.1alleviates neuropathic pain andaccelerates function al recovery of injured neurones.Brain research2005,1059(2),149-58.)。神经病理性疼痛是由于对体感神经系统的损害导致的一种复杂而衰弱的综合征，影响着全球数百万人。流行病调查研究表明，神经痛折磨着全球范围内超过8％的人口，不仅严重影响患者的身心健康和生活质量，而且消耗了数以百亿美元计的医疗资源。但是，传统镇痛药对于神经疼痛的治疗效果欠佳，且长期使用具有成瘾性，因此新型镇痛药物的研制和开发显得尤为重要。α9α10nAChR阻断剂具有治疗神经痛、预防神经受伤、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(Holtman,J.R.；Dwoskin,L.P.；Dowell,C.；Wala,E.P.；Zhang,Z.；Crooks,P.A.；McIntosh,J.M.,The novel small moleculealpha9alpha10nicotinic acetylcholine recep tor antagonist ZZ-204G isanalgesic.European journal of pharmacology2011,670(2-3),500-8.Zheng,G.；Zhang,Z.；Dowell,C.；Wala,E.；Dwoskin,L.P.；Holtman,J.R.；McIntosh,J.M.；Crooks,P.A.,Discovery of non-peptide,smallmolecule antagonists of alpha9alpha10n icotinic acetylcholinereceptors as novel analgesics for the treatment of neuropathic a ndtonic inflammatory pain.Bioorganic&medicinal chemistry letters2011,21(8),2476-9)。Among the various subtypes of neuronAChRs, the α9α10 subtype has attracted much attention in the field of biomedicine. The α9α10 nicotinic acetylcholine receptor is a transmembrane pentamer composed of two subunits, α9 and α10, and is a ligand-gated ion channel. This receptor was first identified in cochlear hair cells and mediates synaptic transmission between oligocochlear cholinergic fibers and efferent nerves, and was later shown to be involved in leukocytes, dorsal root ganglia, pituitary gland, skin keratinocytes, and semen are also distributed. Studies have shown that α9α10nAChR is a new target for the treatment of neuralgia drugs (McIntosh, J.M.; Absalom, N.; Chebib, M.; Elgoyhen, A.B.; Vincler, M., Alpha9nicotinic acetylcholine receptors and the treatment of pain. Biochemal pharmacology 2009, 78(7), 693-702. Satkunanathan, N.; Livett, B.; Gayler, K.; Sandall, D.; Down, J.; Khalil, Z., Alpha-conotoxin Vc1.1alleviates neuropathic pain andaccelerates function al recovery of injured neurons. Brain research 2005, 1059(2), 149-58.). Neuropathic pain is a complex and debilitating syndrome caused by damage to the somatosensory nervous system, affecting millions of people worldwide. Epidemiological studies have shown that neuralgia afflicts more than 8% of the global population, not only seriously affecting the physical and mental health and quality of life of patients, but also consuming tens of billions of dollars in medical resources. However, traditional analgesics are not effective in treating neuropathic pain, and long-term use is addictive, so the research and development of new analgesics is particularly important. α9α10nAChR blockers have the functions of treating neuralgia, preventing nerve injury, and accelerating the recovery of injured nerves, possibly through immune mechanisms (Holtman, J.R.; Dwoskin, L.P.; Dowell, C.; Wala, E.P.; Zhang, Z. ;Crooks,P.A.;McIntosh,J.M.,The novel small moleculealpha9alpha10nicotinic acetylcholine recep tor antagonist ZZ-204G isanalgesic.European journal of pharmacology2011,670(2-3),500-8.Zheng,G.;Zhang,Z.;Dowell, C.; Wala, E.; Dwoskin, L.P.; Holtman, J.R.; McIntosh, J.M.; Crooks, P.A. chemistry letters 2011, 21(8), 2476-9).
此外，α9α10nAChR还与伤口愈合、耳部疾病、肺癌、乳腺癌以及皮肤病等诸多疾病密切相关。角化细胞上的α9α10nAChR在伤口愈合的病理生理学过程中起着很重要的作用(Chernyavsky,A.I.；Arredondo,J.；Vetter,D.E.；Grando,S.A.,Central role of alpha9acetylcholinereceptor in coordinating keratinocyt e adhesion and motility at theinitiation of epithelialization.Experimental cell research 2007,313(16),3542-55)。新近研究表明，α9亚基在乳腺癌组织中过表达，促进乳腺癌的发生(Chen,C.S.,Lee,C.H.,Hsieh,C.D.,Ho,C.T.,Pan,M.H.,Huang,C.S.,Tu,S.H.,Wang,Y.J.,Chen,L.C.,Chang,Y.J.,Wei,P.L.,Yang,Y.Y.,Wu,C.H.,and Ho,Y.S.(2011)Nicotine-induced human breast cancer cell proliferation attenuated bygarcinol through down-regulation of the nicotinic receptor and cyclinD3proteins.Breast cancer research and treatment 125,73-87.Lee,C.H.,Huang,C.S.,Chen,C.S.,Tu,S.H.,Wang,Y.J.,Chang,Y.J.,Tam,K.W.,Wei,P.L.,Cheng,T.C.,Chu,J.S.,Chen,L.C.,Wu,C.H.,and Ho,Y.S.(2010)Overexpression and acti  vation of thealpha9-nicotinic receptor during tumorigenesis in human breastepithelia l cells.Journal of the National Cancer Institute 102,1322-1335)。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(Chikova,A。Grando,S.A.,Naturally occurring variants of human Alpha9nicotinic receptordifferen tially affect bronchial cell proliferation and transformation.PloS one 2011,6(11),e27978.)。In addition, α9α10nAChR is also closely related to wound healing, ear diseases, lung cancer, breast cancer and skin diseases and many other diseases. α9α10nAChR on keratinocytes plays an important role in the pathophysiology of wound healing (Chernyavsky, A.I.; Arredondo, J.; Vetter, D.E.; Grando, S.A., Central role of alpha9acetylcholinereceptor in coordinating keratinocyt e adhesion and motility at theinitiation of epithelialization. Experimental cell research 2007, 313(16), 3542-55). Recent studies have shown that the α9 subunit is overexpressed in breast cancer tissues and promotes the occurrence of breast cancer (Chen, C.S., Lee, C.H., Hsieh, C.D., Ho, C.T., Pan, M.H., Huang, C.S., Tu, S.H., Wang , Y.J., Chen, L.C., Chang, Y.J., Wei, P.L., Yang, Y.Y., Wu, C.H., and Ho, Y.S. (2011) Nicotine-induced human breast cancer cell proliferation attenuated bygarcinol through down-regulation of the nicotinic receptor and cyclinD3proteins .Breast cancer research and treatment 125,73-87.Lee,C.H.,Huang,C.S.,Chen,C.S.,Tu,S.H.,Wang,Y.J.,Chang,Y.J.,Tam,K.W.,Wei,P.L.,Cheng,T.C.,Chu, J.S., Chen, L.C., Wu, C.H., and Ho, Y.S. (2010) Overexpression and acti vation of thealpha9-nicotinic receptor during tumororigenesis in human breastepithelia l cells. Journal of the National Cancer Institute 102, 1322-1335). The α9 subunit variant affects the transformation and proliferation of bronchial cells, and this subunit has a very important significance in the treatment of lung cancer (Chikova, A. Grando, S.A., Naturally occurring variants of human Alpha9nicotinic receptordifferentially affect bronchial cell proliferation and transformation. PloS one 2011, 6(11), e27978.).
芋螺毒素是海洋来源的生物活性多肽，通常由10-50个氨基酸残基组成，含有丰富的二级结构以及数个二硫键，稳定的结构和多样的药理作用使其在药物开发方面展现出了非常广阔的前景。芋螺毒素按其前体蛋白的内质网信号肽序列的相似性以及半胱氨酸模式，分为不同的基因家族，至今，所有已知的芋螺毒素可分为18个超家族；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类芋螺毒素(α-Conotoxins)具有阻断烟碱乙酰胆碱受体(nAChRs)的功能；不含有半胱氨酸的芋螺毒素肽Conantokins则具有阻断N-甲基-D-天冬氨酸受体(NMDA受体，N-methyl-D-aspartic acid receptor，NMDAR)的功能。其中，选择性抑制α9α10乙酰胆碱受体的α-芋螺毒素RgIA、Vc1.1和GeXIVA在动物疼痛模型中表现出了良好的镇痛效果，通过皮下或肌肉注射可以减轻由外伤、炎症或神经损伤引起的疼痛。此类多肽在经过结构优化改造后，有望在镇痛药物的研究上形成重大突破。Conotoxin is a biologically active polypeptide of marine origin, usually composed of 10-50 amino acid residues, rich in secondary structure and several disulfide bonds. Its stable structure and diverse pharmacological effects make it suitable for drug development. A very broad prospect. Conotoxins are divided into different gene families according to the similarity of the endoplasmic reticulum signal peptide sequence of their precursor proteins and the cysteine pattern. So far, all known conotoxins can be divided into 18 superfamilies; Spirotoxins (peptides) can be divided into α, ω, μ, δ and other pharmacological families according to their receptor targets. Among them, α-Conotoxins have the function of blocking nicotinic acetylcholine receptors (nAChRs); Conantokins, which do not contain cysteine, have the function of blocking N-methyl-D-day The function of aspartic acid receptor (NMDA receptor, N-methyl-D-aspartic acid receptor, NMDAR). Among them, α-conotoxins RgIA, Vc1.1 and GeXIVA, which selectively inhibit α9α10 acetylcholine receptors, have shown good analgesic effects in animal pain models. Subcutaneous or intramuscular injection can alleviate trauma, inflammation or nerve damage. caused pain. After structural optimization, such polypeptides are expected to form a major breakthrough in the research of analgesic drugs.
尽管在前期的研究中发现α-芋螺毒素能够阻断nAChRs，部分α-芋螺毒素还呈现出对α9α10nAChR的特异性阻断和抑制作用。然而仍有三方面问题制约了α-芋螺毒素的临床应用。首先，人和鼠α9α10nAChR存在差异，前期研究中能特异性结合并抑制鼠α9α10nAChR的α-芋螺毒素对人α9α10nAChR的抑制活性显著降低(参见Molecular basis for the differential sensitivity of rat and human alpha9alpha10nAChRs to alpha-conotoxin RgIA.Azam L,McIntosh JM.J Neurochem.2012Sep；122(6):1137-44.；Determination of the alpha-conotoxin Vc1.1binding site on the alpha9alpha10nicotinic acetylcholine receptor.Yu R,Kompella SN,Adams DJ,Craik DJ,Kaas Q.J Med Chem.2013May 9；56(9):3557-67.)。其次，α-芋螺毒素衍生物对α9α10nAChR的特异性不高，天然α-芋螺毒素通常具有多种 活性，对其进行化学结构改造往往削弱甚至丧失了对α9α10nAChR的特异性阻断活性，而特异性抑制肌肉型nAChR等(参见“基于αO-芋螺毒素GeXIVA的突变短肽设计合成与活性研究”，海南大学，硕士学位论文，黄艺)。另外，天然α-芋螺毒素是由含有多个半胱氨酸的肽链形成的混合物，单体肽链上的多个半胱氨酸相互之间可形成不同的二硫键，产生二硫键异构体；单体肽链之间也可能通过分子间二硫键相互连接。这些二硫键异构体、多聚体对α9α10nAChR的抑制活性有所不同，生产过程中合成困难、需要通过氧化折叠等才有可能形成正确的构象、纯化难度大、产率低。Although α-conotoxins were found to be able to block nAChRs in previous studies, some α-conotoxins also showed specific blocking and inhibitory effects on α9α10 nAChRs. However, there are still three problems that restrict the clinical application of α-conotoxin. First, there are differences between human and mouse α9α10nAChR. In previous studies, the α-conotoxin that can specifically bind to and inhibit mouse α9α10nAChR significantly reduces the inhibitory activity of human α9α10nAChR (see Molecular basis for the differential sensitivity of rat and human alpha9alpha10nAChRs to alpha- conotoxin RgIA.Azam L,McIntosh JM.J Neurochem.2012Sep;122(6):1137-44.;Determination of the alpha-conotoxin Vc1.1binding site on the alpha9alpha10nicotinic acetylcholine receptor.Yu R,Kompella SN,Adams DJ,Craik DJ, Kaas Q.J Med Chem. 2013 May 9;56(9):3557-67.). Secondly, the specificity of α-conotoxin derivatives for α9α10nAChR is not high. Natural α-conotoxin usually has multiple activities, and chemical structural modification of it often weakens or even loses the specific blocking activity of α9α10nAChR, while the Specific inhibition of muscle-type nAChR, etc. (see "Study on the Design, Synthesis and Activity of Mutant Short Peptides Based on αO-Conotoxin GeXIVA", Hainan University, Master's Thesis, Huang Yi). In addition, natural α-conotoxin is a mixture of peptide chains containing multiple cysteines, and multiple cysteines on the monomer peptide chain can form different disulfide bonds with each other, resulting in disulfide Bond isomers; monomeric peptide chains may also be connected to each other by intermolecular disulfide bonds. These disulfide bond isomers and multimers have different inhibitory activities on α9α10nAChR, which are difficult to synthesize during the production process, require oxidative folding, etc. to form the correct conformation, difficult to purify, and low yield.
发明内容SUMMARY OF THE INVENTION
为解决上述技术问题，本发明提供一种单链镇痛肽，根据α-芋螺毒素与α9α10nAChR(即：α9α10烟碱型乙酰胆碱受体)相互作用的机理，设计多条α-芋螺毒素单体肽突变体。筛选抑制活性高、镇痛效果好的单体肽突变体Gex-2。进一步通过对Gex-2进行丙氨酸扫描、天冬氨酸扫描、精氨酸扫描、精氨酸-瓜氨酸替代、D型氨基酸替换等手段进行突变获得Gex-2的系列衍生物。所述Gex-2能够提高CCI大鼠的痛阈阈值，具有良好的镇痛作用；与Gex-2相比，Gex-2系列衍生物中部分成员对α9α10乙酰胆碱受体的抑制活性更高。所述Gex-2及其系列衍生物可作为α9α10nAChR抑制剂用于镇痛、抗肿瘤、治疗神经系统疾病等。In order to solve the above-mentioned technical problems, the present invention provides a single-chain analgesic peptide. According to the mechanism of the interaction between α-conotoxin and α9α10nAChR (ie: α9α10 nicotinic acetylcholine receptor), a plurality of α-conotoxin single peptides are designed. somatic peptide mutants. The monomeric peptide mutant Gex-2 with high inhibitory activity and good analgesic effect was screened. Further, Gex-2 was mutated by means of alanine scanning, aspartic acid scanning, arginine scanning, arginine-citrulline substitution, and D-amino acid substitution to obtain a series of derivatives of Gex-2. The Gex-2 can improve the pain threshold of CCI rats and has a good analgesic effect; compared with Gex-2, some members of the Gex-2 series derivatives have higher inhibitory activity on α9α10 acetylcholine receptors. The Gex-2 and its series derivatives can be used as α9α10nAChR inhibitors for analgesia, anti-tumor, treatment of nervous system diseases and the like.
具体而言：in particular:
一方面，本发明提供一种活性多肽，其特征在于与α-芋螺毒素活性单体肽相比具有一个或多个半胱氨酸残基的删除或缺失突变；In one aspect, the present invention provides an active polypeptide characterized by having a deletion or deletion mutation of one or more cysteine residues compared to the α-conotoxin active monomeric peptide;
其中，所述α-芋螺毒素活性单体肽具有结合nAChR的功能。Wherein, the α-conotoxin active monomer peptide has the function of binding nAChR.
进一步，本发明所述活性多肽，其特征在于所述α-芋螺毒素活性单体肽包括至少三个连续的精氨酸残基，并具有1个、2个、3个、4个或5个半胱氨酸残基。Further, the active polypeptide of the present invention is characterized in that the α-conotoxin active monomer peptide comprises at least three consecutive arginine residues, and has 1, 2, 3, 4 or 5 a cysteine residue.
进一步，本发明所述活性多肽，其特征在于所述α-芋螺毒素活性单体肽包括α-芋螺毒素天然单体肽，以及在α-芋螺毒素天然单体肽氨基酸序列的基础上缺失C端和/或N端的一个或数个氨基酸残基。Further, the active polypeptide of the present invention is characterized in that the α-conotoxin active monomer peptide includes α-conotoxin natural monomer peptide, and on the basis of the amino acid sequence of the α-conotoxin natural monomer peptide One or several amino acid residues at the C-terminus and/or the N-terminus are deleted.
进一步，本发明所述活性多肽，其特征在于所述α-芋螺毒素天然单体肽来源于选自下组的α-芋螺毒素，GeXIVA、GeXXVIIA、Vc1.1、PeIA、RgIA、 B-VxXXIVA、S-GVIIIB、D-GeXXA、O-GeXXVIIA、D--Lt28.1、Mr1.1、BuIA、ImI、或AuIB；所述α-芋螺毒素天然单体肽包括α-芋螺毒素的成熟肽单体肽、前体肽单体肽。Further, the active polypeptide of the present invention is characterized in that the α-conotoxin natural monomer peptide is derived from α-conotoxin selected from the group consisting of GeXIVA, GeXXVIIA, Vc1.1, PeIA, RgIA, B- VxXXIVA, S-GVIIIB, D-GeXXA, O-GeXXVIIA, D--Lt28.1, Mr1.1, BuIA, ImI, or AuIB; the α-conotoxin natural monomeric peptides include the Mature peptide monomeric peptide, precursor peptide monomeric peptide.
进一步，本发明所述活性多肽，其特征在于所述α-芋螺毒素天然单体肽包括SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列。Further, the active polypeptide of the present invention is characterized in that the α-conotoxin natural monomer peptide comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.
进一步，本发明所述活性多肽，其特征在于所述活性多肽不含有半胱氨酸残基、不形成分子内二硫键或分子间二硫键，且其氨基酸残基数>10。Further, the active polypeptide of the present invention is characterized in that the active polypeptide does not contain cysteine residues, does not form intramolecular disulfide bonds or intermolecular disulfide bonds, and has more than 10 amino acid residues.
进一步，本发明所述活性多肽，其特征在于所述活性多肽包含SEQ ID NO:3所示的序列，且具有12-20个氨基酸残基。Further, the active polypeptide of the present invention is characterized in that the active polypeptide comprises the sequence shown in SEQ ID NO: 3 and has 12-20 amino acid residues.
进一步，本发明前述任一所述活性多肽，其包括具有选自SEQ ID NO:4-9中任一所示氨基酸序列结构的线性肽、环肽、或D型氨基酸替换衍生肽。Further, any one of the aforementioned active polypeptides of the present invention includes a linear peptide, a cyclic peptide, or a D-type amino acid substitution derivative peptide having an amino acid sequence structure selected from any one of SEQ ID NOs: 4-9.
进一步，本发明前述任一项所述活性多肽，其包括SEQ ID NO:4-9中任一所示氨基酸序列的基础上进行单点氨基酸扫描突变获得的突变体，所述氨基酸扫描突变包括正电荷氨基酸扫描突变、负电荷氨基酸扫描突变、中性氨基酸扫描突变、稀有氨基酸替换突变、以及D-型氨基酸替换突变。Further, the active polypeptide according to any one of the foregoing in the present invention includes a mutant obtained by performing single-point amino acid scanning mutation on the basis of the amino acid sequence shown in any of SEQ ID NOs: 4-9, and the amino acid scanning mutation includes positive Charged amino acid scanning mutations, negatively charged amino acid scanning mutations, neutral amino acid scanning mutations, rare amino acid substitution mutations, and D-type amino acid substitution mutations.
更进一步，本发明所述活性多肽，其中，正电荷氨基酸扫描突变包括精氨酸扫描突变；负电荷氨基酸扫描突变包括天冬氨酸扫描突变；不带电荷氨基酸扫描包括丙氨酸扫描突变；D型氨基酸扫描包括在SEQ ID NO:4-9所示序列的基础上逐一对每个氨基酸残基采用其相应的D型氨基酸残基替换获得的单点突变体。Further, the active polypeptide of the present invention, wherein the positively charged amino acid scanning mutation includes arginine scanning mutation; the negatively charged amino acid scanning mutation includes aspartic acid scanning mutation; the uncharged amino acid scanning includes alanine scanning mutation; D Type amino acid scanning includes single point mutants obtained by replacing each amino acid residue with its corresponding D-type amino acid residue on the basis of the sequences shown in SEQ ID NOs: 4-9.
更进一步，本发明所述活性多肽，包括上述几种修改策略的组合所产生的多肽序列。例如采用精氨酸扫描与D型氨基酸替换两种策略同时进行所获得的类似物。Furthermore, the active polypeptides of the present invention include the polypeptide sequences produced by the combination of the above-mentioned several modification strategies. The analogs obtained, for example, were performed simultaneously with two strategies of arginine scanning and D-form amino acid substitution.
更进一步，本发明所述活性多肽，其具有SEQ ID NO:10-73任一所述氨基酸序列，优选具有SEQ ID NO:5、20、21、22、23、30、34、46、50、51、53、66、或69所示的氨基酸序列。Further, the active polypeptide of the present invention has the amino acid sequence of any one of SEQ ID NO: 10-73, preferably has SEQ ID NO: 5, 20, 21, 22, 23, 30, 34, 46, 50, The amino acid sequence shown in 51, 53, 66, or 69.
第二方面，本发明提供一种制备活性多肽的方法，包括将α-芋螺毒素活性单体肽氨基酸序列中的一个或多个半胱氨酸残基删除，所述α-芋螺毒素活性单体肽具有结合nAChR的功能。In a second aspect, the present invention provides a method for preparing an active polypeptide, comprising deleting one or more cysteine residues in the amino acid sequence of an α-conotoxin active monomer peptide, the α-conotoxin active Monomeric peptides function to bind nAChRs.
进一步，本发明所述制备α-芋螺毒素活性单体肽的方法，其特征在于还包括将α-芋螺毒素天然单体肽在C端和或N端截短。Further, the method for preparing the α-conotoxin active monomer peptide according to the present invention is characterized in that it further comprises truncating the α-conotoxin natural monomer peptide at the C-terminus and/or the N-terminus.
进一步，本发明所述制备活性多肽的方法，其特征在于所述α-芋螺毒素天然单体肽来源于选自下组的α-芋螺毒素，GeXIVA、GeXXVIIA、Vc1.1、PeIA、RgIA、B-VxXXIVA、S-GVIIIB、D-GeXXA、O-GeXXVIIA、D--Lt28.1、Mr1.1、BuIA、ImI、或AuIB；所述α-芋螺毒素天然单体肽包括α-芋螺毒素的成熟肽单体肽、前体肽单体肽。Further, the method for preparing an active polypeptide according to the present invention is characterized in that the α-conotoxin natural monomer peptide is derived from α-conotoxin selected from the group consisting of GeXIVA, GeXXVIIA, Vc1.1, PeIA, RgIA , B-VxXXIVA, S-GVIIIB, D-GeXXA, O-GeXXVIIA, D--Lt28.1, Mr1.1, BuIA, ImI, or AuIB; the α-conotoxin natural monomer peptide includes α-conotoxin Mature peptide monomer peptide, precursor peptide monomer peptide of spirotoxin.
第三方面，本发明提供一种制备前述任一所述活性多肽的方法，包括重组表达法和化学合成法，其中所述化学合成法包括固相合成法、液相合成法、固相-液相联合合成法、自然化学连接法(NCL)。In a third aspect, the present invention provides a method for preparing any one of the aforementioned active polypeptides, including recombinant expression method and chemical synthesis method, wherein the chemical synthesis method includes solid-phase synthesis method, liquid-phase synthesis method, solid-phase-liquid synthesis method Combined synthetic method, natural chemical linking method (NCL).
在一个具体的实施方案中，本发明所述的制备活性多肽的方法，其特征在于线性多肽的固相合成包括Fmoc固相合成法、Boc固相合成法；固相合成的方式包括由C端至N端合成法、由N端至C端合成法。In a specific embodiment, the method for preparing active polypeptides of the present invention is characterized in that the solid-phase synthesis of linear polypeptides includes Fmoc solid-phase synthesis method and Boc solid-phase synthesis method; To the N-terminal synthesis method, from the N-terminal to the C-terminal synthesis method.
进一步，本发明所述制备活性多肽的方法，其中C端至N端的Fomc固相合成法包括：Further, the method for preparing an active polypeptide according to the present invention, wherein the Fomc solid-phase synthesis method from the C-terminal to the N-terminal comprises:
(1)固相树脂的预处理；(1) Pretreatment of solid phase resin;
(2)脱除固相树脂上氨基的Fmoc保护基；(2) remove the Fmoc protecting group of amino group on solid-phase resin;
(3)加入C端第一个氨基酸进行缩合反应，将C端第一个氨基酸连接到固相树脂上；(3) adding the first amino acid at the C-terminal to carry out a condensation reaction, and connecting the first amino acid at the C-terminal to the solid phase resin;
(4)脱除C端第一个氨基酸氨基上的Fmoc保护基，加入C端第二个氨基酸进行缩合反应，将C端第二个氨基酸连接到第一个氨基酸的氨基上；循环往复，从C端至N端将氨基酸逐一连接形成多肽链；(4) removing the Fmoc protecting group on the amino group of the first amino acid at the C-terminal, adding the second amino acid at the C-terminal to carry out a condensation reaction, and connecting the second amino acid at the C-terminal to the amino group of the first amino acid; The amino acids are linked one by one from the C-terminus to the N-terminus to form a polypeptide chain;
(5)将多肽链从固相树脂上切割回收、纯化。(5) The polypeptide chain is cut, recovered and purified from the solid phase resin.
在另一个具体的实施方案中，本发明所述制备活性多肽的方法，其特征在于环形多肽采用NCL法合成，包括：In another specific embodiment, the method for preparing an active polypeptide of the present invention is characterized in that the cyclic polypeptide is synthesized by the NCL method, including:
(1)固相合成直链肽，使N端为半胱氨酸；(1) Solid-phase synthesis of straight-chain peptides, making the N-terminal cysteine;
(2)从固相载体上切下N端为半胱氨酸、C端含硫酯的线性肽；(2) excising a linear peptide whose N-terminal is cysteine and C-terminal contains thioester from the solid phase carrier;
(3)通过酰基转移反应完成线性肽的环合。(3) The cyclization of the linear peptide is accomplished by an acyl transfer reaction.
第四方面，本发明提供一种融合蛋白或缀合物，其包含前述任一项所述的活性多肽序列，所述融合蛋白或缀合物具有结合nAChR的功能。In a fourth aspect, the present invention provides a fusion protein or conjugate comprising the active polypeptide sequence described in any one of the foregoing, and the fusion protein or conjugate has the function of binding nAChR.
第五方面，本发明提供一种多聚体，由两个或更多个多肽单体聚合而成，其中至少一个多肽单体为前述任一项所述的活性多肽；所述多聚体的各多肽单体之 间通过共价连接。In a fifth aspect, the present invention provides a multimer, which is formed by the polymerization of two or more polypeptide monomers, wherein at least one polypeptide monomer is the active polypeptide described in any one of the foregoing; The polypeptide monomers are covalently linked.
进一步，本发明所述多聚体，其为同源二聚体或异源二聚体；Further, the multimer of the present invention is a homodimer or a heterodimer;
进一步，本发明所述多聚体，其中组成多聚体的各多肽单体之间通过柔性linker或PEG连接臂连接，优选的，各多肽单体在各自的氨基端通过柔性linker或PEG连接臂相互连接。Further, in the multimer of the present invention, each polypeptide monomer constituting the multimer is connected by a flexible linker or a PEG linker, preferably, each polypeptide monomer is connected by a flexible linker or a PEG linker at the respective amino terminus connected to each other.
进一步，本发明所述多聚体，其靶向抑制人α9α10nAChR的活性高于单体形式的前述任一项所述活性多肽。Further, the multimer of the present invention has a higher targeting activity of inhibiting human α9α10 nAChR than any of the aforementioned active polypeptides in the monomeric form.
第六方面，本发明提供一种核酸分子，其编码前述任一项所述的活性多肽、或前述融合蛋白或缀合物。In a sixth aspect, the present invention provides a nucleic acid molecule encoding any of the aforementioned active polypeptides, or the aforementioned fusion proteins or conjugates.
第七方面，本发明提供一种构建体，其包含前述的核酸分子。In a seventh aspect, the present invention provides a construct comprising the aforementioned nucleic acid molecule.
第八方面，本发明提供一种宿主细胞，其包含前述的核酸分子和/或前述的构建体，或者所述宿主细胞被前述的核酸分子和/或前述的构建体转化或转染。In an eighth aspect, the present invention provides a host cell comprising the aforementioned nucleic acid molecule and/or the aforementioned construct, or the host cell is transformed or transfected with the aforementioned nucleic acid molecule and/or the aforementioned construct.
第九方面，本发明提供一种药物组合物，所述药物组合物包含前述任一项所述的活性多肽、前述融合蛋白或缀合物、前述核酸分子、前述多聚体、前述构建体、前述宿主细胞，以及可选的药学上可接受的辅料。In a ninth aspect, the present invention provides a pharmaceutical composition comprising the active polypeptide described in any of the foregoing, the foregoing fusion protein or conjugate, the foregoing nucleic acid molecule, the foregoing multimer, the foregoing construct, The aforementioned host cells, and optional pharmaceutically acceptable excipients.
第十方面，本发明提供前述任一项所述的活性多肽、前述融合蛋白或缀合物、前述核酸分子、前述多聚体、前述构建体、前述宿主细胞、前述药物组合物在α9α10nAChR抑制剂中的用途，其中所述α9α10nAChR包括人α9α10nAChR、鼠α9α10nAChR。In a tenth aspect, the present invention provides the active polypeptide, the fusion protein or the conjugate, the nucleic acid molecule, the multimer, the construct, the host cell, and the pharmaceutical composition described in any one of the aforementioned α9α10 nAChR inhibitors Use in , wherein the α9α10 nAChR includes human α9α10 nAChR, murine α9α10 nAChR.
第十一方面，本发明提供前述任一项所述的活性多肽、前述融合蛋白或缀合物、前述核酸分子、前述多聚体、前述构建体、前述宿主细胞、前述药物组合物在镇痛、抗肿瘤、和/或治疗神经系统疾病中的应用。In the eleventh aspect, the present invention provides the active polypeptide, the fusion protein or the conjugate, the nucleic acid molecule, the multimer, the construct, the host cell, and the pharmaceutical composition described in any one of the foregoing in analgesia. , anti-tumor, and/or use in the treatment of neurological diseases.
进一步，本发明所述的应用，其中所述活性多肽、融合蛋白或缀合物、核酸分子、多聚体、构建体、宿主细胞、和/或药物组合物，可与任选的镇痛、抗肿瘤、和/或治疗神经系统疾病的活性成分联用或联合制药。Further, the application of the present invention, wherein the active polypeptide, fusion protein or conjugate, nucleic acid molecule, multimer, construct, host cell, and/or pharmaceutical composition, can be combined with optional analgesia, Combination or combination pharmaceuticals of active ingredients for antitumor and/or treatment of nervous system diseases.
进一步，本发明所述的应用，其中所述疾病包括神经痛、成瘾、帕金森症、癫痫症、局部缺血、兴奋性神经元细胞死亡、痴呆、乳腺癌、肺癌、脑脊髓炎、癌症与癌症化疗、酒精中毒、坐骨神经痛、糖尿病、三叉神经痛、硬化症、带状疱疹、机械伤和手术伤、艾滋病、头部神经瘫痪、药物中毒、工业污染中毒、淋巴神经痛、骨髓瘤、多点运动神经痛、慢性先天性感觉神经病、急性剧烈自发性 神经痛、挤压神经痛、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、复合神经痛、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。Further, the application of the present invention, wherein the diseases include neuralgia, addiction, Parkinson's disease, epilepsy, ischemia, excitatory neuronal cell death, dementia, breast cancer, lung cancer, encephalomyelitis, cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis, herpes zoster, mechanical and surgical injuries, AIDS, paralysis of head nerves, drug poisoning, industrial pollution poisoning, lymphatic neuralgia, myeloma, Multipoint motor neuralgia, chronic congenital sensory neuropathy, acute severe idiopathic neuralgia, crush neuralgia, vasculitis, vasculitis, ischemia, uremia, childhood biliary liver disease, chronic respiratory disorders, complex neuralgia , multiple organ failure, sepsis/sepsis, hepatitis, porphyria, vitamin deficiency, chronic liver disease, native bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, sensory perineuritis, or allergy disease.
为更好理解本发明，首先定义一些术语。其他定义则贯穿具体实施方式部分而列出。For a better understanding of the present invention, some terms are first defined. Other definitions are listed throughout the Detailed Description.
如本文所用，术语“多肽”意图涵盖单个“多肽”以及多个“多肽”，并且是指由通过酰胺键(还被称为肽键)线性连接的单体(氨基酸)构成的分子。术语“多肽”是指含有两个或更多个氨基酸的任何一个或多个链，并且不是指特定长度的产物。因此，肽、二肽、三肽、寡肽、“蛋白质”、“氨基酸链”或用于表示两个或更多个氨基酸的一个或多个链的任何其它术语都被包括在“多肽”的定义内，并且术语“多肽”可以代替或与任何这些术语互换使用。As used herein, the term "polypeptide" is intended to encompass a single "polypeptide" as well as a plurality of "polypeptides" and refers to a molecule composed of monomers (amino acids) linked linearly by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains containing two or more amino acids, and does not refer to a product of a particular length. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "chains of amino acids," or any other term used to denote one or more chains of two or more amino acids are included within the term "polypeptide." definitions, and the term "polypeptide" may be used instead or interchangeably with any of these terms.
本发明的多肽可具有约3个或更多个、5个或更多个、10个或更多个、20个或更多个、25个或更多个、50个或更多个氨基酸的大小。多肽可具有限定的三维结构，但是它们不必具有这种结构。具有限定的三维结构的多肽被称为折叠的，并且不具有限定的三维结构、而是可采用许多不同构象的多肽被称为未折叠的。如本文所述的多肽可以是两端修饰的，例如在羧基端进行-NH 2修饰。 Polypeptides of the present invention may have about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more amino acids size. Polypeptides can have a defined three-dimensional structure, but they need not have this structure. A polypeptide that has a defined three-dimensional structure is said to be folded, and a polypeptide that does not have a defined three-dimensional structure, but can adopt many different conformations, is said to be unfolded. Polypeptides as described herein may be terminally modified, eg -NH2 at the carboxy terminus.
本发明源自芋螺毒素的活性多肽可以根据天然芋螺毒素的序列延长或缩短，剪切芋螺毒素的序列通常会降低其活性，但也存在例外情形，例如GID芋螺毒素N末端自由端(4个氨基酸的自由多肽片段)，被剪切后仍然可以保留靶向α4β2nAChR的活性(J Biol Chem.2009Feb 20；284(8):4944-51)。因此，通过剪切N末端1-4个氨基酸而基本保留多肽的活性是有可能的。The active polypeptide derived from conotoxin of the present invention can be extended or shortened according to the sequence of natural conotoxin, and cleavage of the sequence of conotoxin usually reduces its activity, but there are exceptions, such as the N-terminal free end of GID conotoxin (4 amino acid free polypeptide fragment), can still retain the activity of targeting α4β2 nAChR after being cut (J Biol Chem. 2009 Feb 20; 284(8): 4944-51). Therefore, it is possible to substantially retain the activity of the polypeptide by cleaving the N-terminal 1-4 amino acids.
术语“多肽”还意图表示多肽的表达后修饰产物，所述修饰包括但不限于糖基化、乙酰化、磷酸化、酰胺化、通过已知保护/阻断基团衍生化、蛋白水解性裂解或被非天然存在的氨基酸修饰。多肽可以源自天然生物来源或由重组技术产生，但不必从指定核酸序列翻译。它可以任何方式产生，包括通过化学合成。在多肽结构优化过程中通常采用非天然修饰或者糖基化策略提高多肽的活性、稳定性与选择性。通过采用类似物理化学性质的非天然氨基酸替代序列中的天然氨基酸可以提高多肽的稳定性(J Biol Chem.2009Apr 3；284(14):9498-512.；ACS Chem Neurosci.2019Oct 16；10(10):4328-4336)。例如采用Arg-1(侧链含有4 个C长度的精氨酸类似物)、Arg-2(侧链含有2个C长度的精氨酸类似物)、Arg-3(侧链含有1个C长度的精氨酸类似物)等替代Gex-2序列中碱性氨基酸Arg可以潜在保留或提高其活性，采用Pro的类似物4-(R)-hydroxy-L-proline、[4-(R)-OH]、4-(R)-amino-L-proline,[4-(R)-NH2]、4-(S)-amino-L-proline,[4-(S)-NH2]、4-(R)-guanidino-L-proline,[4-(R)-Gn]、4-(R)-betainamidyl-L-proline,[4-(R)-Bet]、4-(R)-fluoro-L-proline,[4-(R)-F]、4-(S)-fluoro-L-proline,[4-(S)-F]、4-(R)-phenyl-L-proline,[4-(R)-Ph]、4-(S)-phenyl-L-proline,[4-(S)-Ph4-(R)-1-naphthylmethyl-L-proline、4-(R)-Nap]4-()-benzyl-L-proline,[4-(R)-Bzl3-(S)-phenyl-L-proline,[3-(S)-Ph]、5-(R)-phenyl-L-proline,[5-(R)-Ph]等可以潜在保留甚至提高Gex-2的活性。采用糖基化方法可以显著提高多肽的稳定性、半衰期与生物利用度(Trends Biochem.Sci.2006,31,156–163；Carbohydr.Res.2009,344,1508–1514；Front Pharmacol.2013Dec 13；4:155)，目前糖基化的氨基酸主要包括Ser、Thr及Asn，因此可以预期对Gex-2序列中5号位Ser或者17位Thr进行糖基化可以提高多肽的稳定性、半衰期与生物利用度。采用含有C12–C20长链脂肪酸修饰直链多肽，可以提高多肽与血清蛋白的结合，从而可以显著提高多肽在血浆中的稳定性与半衰期(J Med Chem.(2015)58:7370–80.)，因此对Gex-2进行C12–C20长链脂肪酸修饰，有望提高多肽的稳定性与生物利用度。The term "polypeptide" is also intended to mean the post-expression modification product of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage or modified with non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced by recombinant techniques, but need not be translated from a given nucleic acid sequence. It can be produced in any way, including by chemical synthesis. In the process of polypeptide structure optimization, non-natural modification or glycosylation strategy is usually used to improve the activity, stability and selectivity of the polypeptide. The stability of polypeptides can be improved by replacing natural amino acids in the sequence with unnatural amino acids of similar physicochemical properties (J Biol Chem. 2009 Apr 3; 284(14): 9498-512.; ACS Chem Neurosci. 2019 Oct 16; 10(10 ): 4328-4336). For example, Arg-1 (the side chain contains 4 C-length arginine analogs), Arg-2 (the side chain contains 2 C-length arginine analogs), Arg-3 (the side chain contains 1 C-length arginine analogs) The replacement of the basic amino acid Arg in the Gex-2 sequence can potentially retain or improve its activity, using Pro analogs 4-(R)-hydroxy-L-proline, [4-(R) -OH], 4-(R)-amino-L-proline, [4-(R)-NH2], 4-(S)-amino-L-proline, [4-(S)-NH2], 4- (R)-guanidino-L-proline,[4-(R)-Gn], 4-(R)-betainamidyl-L-proline,[4-(R)-Bet], 4-(R)-fluoro- L-proline,[4-(R)-F], 4-(S)-fluoro-L-proline,[4-(S)-F], 4-(R)-phenyl-L-proline,[4 -(R)-Ph], 4-(S)-phenyl-L-proline, [4-(S)-Ph4-(R)-1-naphthylmethyl-L-proline, 4-(R)-Nap]4 -()-benzyl-L-proline,[4-(R)-Bzl3-(S)-phenyl-L-proline,[3-(S)-Ph],5-(R)-phenyl-L-proline , [5-(R)-Ph], etc. can potentially retain or even improve the activity of Gex-2. The stability, half-life and bioavailability of polypeptides can be significantly improved by glycosylation (Trends Biochem. Sci. 2006, 31, 156–163; Carbohydr. Res. 2009, 344, 1508–1514; 155), currently glycosylated amino acids mainly include Ser, Thr and Asn, so it can be expected that glycosylation of Ser at position 5 or Thr at position 17 in the Gex-2 sequence can improve the stability, half-life and bioavailability of the polypeptide. . Modification of linear polypeptides with long-chain fatty acids containing C12–C20 can improve the binding of polypeptides to serum proteins, thereby significantly improving the stability and half-life of polypeptides in plasma (J Med Chem. (2015) 58:7370–80.) Therefore, modification of Gex-2 with C12–C20 long-chain fatty acids is expected to improve the stability and bioavailability of the peptide.
另外，本发明源自芋螺毒素的活性多肽，两端可以通过“封帽”策略，提高其稳定性，尤其提高其在血浆中的稳定性。例如Gex的N末端可以被乙酰化、甲基化等可以继续保留其活性，其C-末端可以为自由端、氨基化、酯化等，不影响多肽的基本结构而保留多肽的活性，提高多肽的稳定性。In addition, the two ends of the active polypeptide derived from conotoxin of the present invention can be "capped" to improve its stability, especially its stability in plasma. For example, the N-terminus of Gex can be acetylated, methylated, etc. to continue to retain its activity, and its C-terminus can be free end, amination, esterification, etc., which does not affect the basic structure of the polypeptide and retains the activity of the polypeptide, improving the polypeptide. stability.
“分离的”多肽或其片段、变体或衍生物意图为不在其天然周围环境中的多肽。不要求特定的纯化水平。例如，分离的多肽可以是从其原生或天然环境中移出的。出于本发明的目的在宿主细胞中表达的重组产生的多肽和蛋白质被认为是分离的，通过任何适合的技术分离、分级或者部分或实质性纯化的天然或重组多肽也被认为是分离的。An "isolated" polypeptide or fragment, variant or derivative thereof is intended to be a polypeptide that is not in its natural surroundings. No specific purification level is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells for the purposes of the present invention are considered isolated, as are native or recombinant polypeptides that are isolated, fractionated, or partially or substantially purified by any suitable technique.
本发明的多肽还包括前述多肽的片段、衍生物、类似物或变体以及其任何组合。在提及本发明的多肽时，术语“片段”、“变体”、“衍生物”以及“类似物” 包括保持相应多肽的至少一些生物活性和/或功能。The polypeptides of the present invention also include fragments, derivatives, analogs or variants of the foregoing polypeptides, and any combination thereof. The terms "fragments," "variants," "derivatives," and "analogs" when referring to polypeptides of the present invention include retaining at least some biological activity and/or function of the corresponding polypeptide.
本发明多肽的变体包括多肽的片段，以及由于氨基酸取代、缺失或插入而具有改变的氨基酸序列的多肽。变体可天然存在或可以是非天然存在的。非天然存在的变体可使用本领域已知的诱变技术来产生。变体多肽可包含保守或非保守氨基酸取代、缺失或添加。多肽的衍生物是已经被改变以便展现在天然多肽上未发现的另外特征的多肽。实例包括融合蛋白。变体多肽在本文中还可被称为“多肽类似物”。如本文所用，多肽的“衍生物”是指具有通过官能侧基的反应化学衍生的一个或多个残基的主题多肽。“衍生物”还包括含有20二十个标准氨基酸的一个或多个天然存在的氨基酸衍生物的那些肽。例如，4-羟基脯氨酸可以取代脯氨酸；5-羟基赖氨酸可以取代赖氨酸；3-甲基组氨酸可以取代组氨酸；高丝氨酸可以取代丝氨酸；并且鸟氨酸可以取代赖氨酸。在本发明中使用的氨基酸可以是D-或L-异构体或它们的混合物。Variants of the polypeptides of the present invention include fragments of the polypeptides, as well as polypeptides having altered amino acid sequences due to amino acid substitutions, deletions or insertions. Variants may or may not be naturally occurring. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. Variant polypeptides may contain conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of polypeptides are polypeptides that have been altered so as to exhibit additional characteristics not found on the native polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogs." As used herein, a "derivative" of a polypeptide refers to a subject polypeptide having one or more residues derivatized by reactive chemistry of functional pendant groups. "Derivatives" also include those peptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace serine; and ornithine can replace Substitute lysine. The amino acids used in the present invention may be D- or L-isomers or mixtures thereof.
术语“氨基酸扫描”是指用于测定特定野生型残基对给定蛋白质或多肽的稳定性或功能(例如结合亲和力)的贡献的一种定点诱变技术。该技术涉及用特定氨基酸，例如丙氨酸残基取代多肽中的野生型残基，随后评估丙氨酸取代的衍生物或突变型多肽的稳定性或功能(例如结合亲和力)并与野生型多肽相比较。用丙氨酸取代多肽中的野生型残基的技术是本领域已知的。The term "amino acid scanning" refers to a site-directed mutagenesis technique used to determine the contribution of particular wild-type residues to the stability or function (eg, binding affinity) of a given protein or polypeptide. This technique involves substituting a wild-type residue in a polypeptide with a specific amino acid, such as an alanine residue, and subsequently assessing the stability or function (eg, binding affinity) of the alanine-substituted derivative or mutant polypeptide and comparing it with the wild-type polypeptide. Compared. Techniques for substituting alanine for wild-type residues in polypeptides are known in the art.
术语“核酸”是指多核苷酸中存在的任何一种或多种核酸区段，例如DNA或RNA片段。“分离的”核酸或多核苷酸意图为从其原生环境中移出的核酸分子、DNA或RNA。例如，出于本发明的目的，载体中所含有的编码多肽的重组多核苷酸被认为是分离的。分离的多核苷酸的其它实例包括维持在异源宿主细胞中的重组多核苷酸或溶液中的纯化(部分或实质性)的多核苷酸。分离的RNA分子包括本发明的多核苷酸的体内或体外RNA转录物。根据本发明的分离的多核苷酸或核酸还包括合成产生的所述分子。另外，多核苷酸或核酸可以是或可以包括调控元件，如启动子、核糖体结合位点或转录终止子。The term "nucleic acid" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide. An "isolated" nucleic acid or polynucleotide is intended to be a nucleic acid molecule, DNA or RNA, removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partial or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the invention. Isolated polynucleotides or nucleic acids according to the present invention also include synthetically produced said molecules. Additionally, a polynucleotide or nucleic acid can be or can include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.
如本文所用，“编码区”是核酸中由翻译成氨基酸的密码子组成的一部分。虽然“终止密码子”(TAG、TGA或TAA)不翻译成氨基酸，但是它可以被认为是编码区的一部分，但任何侧翼序列，例如启动子、核糖体结合位点、转录终止子、内含子等不是编码区的一部分。另外，本发明的载体、多核苷酸或核酸可编码异源编码区，所述异源编码区融合或未融合至编码所述多肽或其片段、变体或衍生 物的核酸。异源编码区包括但不限于特化的元件或基序，如分泌信号肽或异源功能性结构域。As used herein, a "coding region" is a portion of a nucleic acid that consists of codons that are translated into amino acids. Although a "stop codon" (TAG, TGA or TAA) is not translated into amino acids, it can be considered part of the coding region, but any flanking sequences such as promoters, ribosome binding sites, transcription terminators, introns Subsidiaries etc. are not part of the coding region. Additionally, a vector, polynucleotide or nucleic acid of the invention may encode a heterologous coding region fused or unfused to a nucleic acid encoding the polypeptide or fragment, variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretion signal peptides or heterologous functional domains.
在某些实施方案中，多核苷酸或核酸是DNA。在DNA的情况下，包含编码多肽的核酸的多核苷酸通常可包含启动子和/或与一个或多个编码区可操作地缔合的其它转录或翻译控制元件。可操作地缔合是针对基因产物(例如多肽)的编码区按以下这种方式与一个或多个调控序列缔合，所述方式使得所述基因产物的表达处于所述调控序列的影响或控制之下。如果启动子功能的诱导导致编码所需基因产物的mRNA的转录，并且如果两个DNA片段之间的键联的性质不干扰表达调控序列引导基因产物的表达的能力或不干扰DNA模板转录的能力，那么两个DNA片段(如多肽编码区和与其缔合的启动子)是“可操作地缔合”或“可操作地连接的”。因此，启动子区将与编码多肽的核酸可操作地缔合，只要启动子能够实现所述核酸的转录。启动子可以是只在预定细胞中引导DNA的实质性转录的细胞特异性启动子。除了启动子以外，其它转录控制元件例如增强子、操纵子、阻遏子以及转录终止信号可与多核苷酸可操作地缔合以便引导细胞特异性转录。本文公开了适合的启动子和其它转录控制区。In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid encoding a polypeptide may generally comprise a promoter and/or other transcriptional or translational control elements operably associated with one or more coding regions. Operably associated is for the coding region of a gene product (eg, a polypeptide) to associate with one or more regulatory sequences in such a manner that expression of the gene product is under the influence or control of the regulatory sequences under. If the induction of promoter function results in the transcription of the mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression control sequences to direct the expression of the gene product or the ability of the DNA template to be transcribed , then two DNA fragments (eg, a polypeptide coding region and a promoter associated therewith) are "operably associated" or "operably linked." Thus, a promoter region will be operably associated with a nucleic acid encoding a polypeptide so long as the promoter is capable of effecting transcription of the nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in the intended cell. In addition to promoters, other transcriptional control elements such as enhancers, operators, repressors, and transcription termination signals can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein.
多种转录控制区是本领域的技术人员已知的。这些包括但不限于在脊椎动物细胞中起作用的转录控制区，如但不限于来自巨细胞病毒(立即早期启动子，与内含子-A结合)、猿猴病毒40(早期启动子)以及逆转录病毒(如劳斯肉瘤病毒(Rous sarcoma virus))的启动子和增强子区段。其它转录控制区包括来源于脊椎动物基因的那些，如肌动蛋白、热休克蛋白、牛生长激素以及兔β-球蛋白，以及能够在真核细胞中控制基因表达的其它序列。其它适合的转录控制区包括组织特异性启动子和增强子以及淋巴因子可诱导的启动子(例如，可由干扰素或白介素诱导的启动子)。类似地，多种翻译控制元件是本领域的普通技术人员已知的。这些包括但不限于核糖体结合位点、翻译起始和终止密码子以及来源于小核糖核酸病毒的元件(特别是内部核糖体进入位点或IRES，还被称为CITE序列)。在其它实施方案中，本发明的多核苷酸是RNA，例如，呈信使RNA(mRNA)的形式。Various transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but are not limited to, from cytomegalovirus (immediate early promoter, associated with intron-A), simian virus 40 (early promoter), and reverse Promoter and enhancer segments of videoviruses such as Rous sarcoma virus. Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit beta-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (eg, promoters inducible by interferons or interleukins). Similarly, various translation control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and picornavirus-derived elements (especially the internal ribosomal entry site or IRES, also known as CITE sequences). In other embodiments, the polynucleotides of the invention are RNA, eg, in the form of messenger RNA (mRNA).
本发明的多核苷酸和核酸编码区可缔合有另外的编码区，所述另外的编码区编码分泌或信号肽，所述分泌或信号肽指导由本发明的多核苷酸编码的多肽的分泌。根据信号假说，由哺乳动物细胞分泌的蛋白质具有信号肽或分泌前导序 列，一旦生长中的蛋白质链跨过粗面内质网的输出被启动，那么所述序列就从成熟蛋白质裂解。本领域的普通技术人员应意识到由脊椎动物细胞分泌的多肽通常具有融合至多肽的N-末端的信号肽，所述信号肽从完整或“全长”多肽裂解，以产生分泌的或“成熟”形式的多肽。在某些实施方案中，使用原生信号肽，例如芋螺毒素信号肽，或所述序列的保留指导与其可操作地缔合的多肽的分泌的能力的功能性衍生物。或者，可使用异源哺乳动物信号肽或其功能衍生物。例如，野生型前导序列可被人组织纤溶酶原活化因子(TPA)或小鼠β-葡糖苷酸酶的前导序列取代。The polynucleotides and nucleic acid coding regions of the invention may have associated additional coding regions encoding secretion or signal peptides that direct secretion of the polypeptides encoded by the polynucleotides of the invention. According to the signaling hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum is initiated. One of ordinary skill in the art will appreciate that polypeptides secreted by vertebrate cells often have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the intact or "full-length" polypeptide to produce a secreted or "mature" polypeptide. "form of polypeptides. In certain embodiments, a native signal peptide, eg, a conotoxin signal peptide, or a functional derivative of the sequence that retains the ability to direct secretion of a polypeptide with which it is operably associated is used. Alternatively, heterologous mammalian signal peptides or functional derivatives thereof can be used. For example, the wild-type leader sequence can be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse beta-glucuronidase.
术语“载体”或“构建体”在本文中可互换用于指代包含载体和一种插入物的DNA分子。重组表达载体通常出于使插入物表达和/或繁殖的目的，或出于构建其他重组核苷酸序列的目的而产生。插入物可或可不以可操作方式连接于启动子序列，并且可或可不以可操作方式连接于DNA调控序列。The terms "vector" or "construct" are used interchangeably herein to refer to a DNA molecule comprising a vector and an insert. Recombinant expression vectors are typically generated for the purpose of expressing and/or propagating an insert, or for the purpose of constructing other recombinant nucleotide sequences. Inserts may or may not be operably linked to promoter sequences, and may or may not be operably linked to DNA regulatory sequences.
术语“融合蛋白”、“嵌合蛋白”、“蛋白缀合物”等术语是指包含除了编码原始或天然全长蛋白或其子序列的氨基酸序列之外的氨基酸、包含代替编码原始或天然全长蛋白或其子序列的氨基酸序列、包含少于编码原始或天然全长蛋白或其子序列的氨基酸序列和/或包含不同于编码原始或天然全长蛋白或其子序列的氨基酸序列的蛋白。可以将多于一个的另外的结构域添加至如本文中所述的活性多肽，例如一个表位标签或纯化标签、或多个表位标签或纯化标签。可以连接另外的结构域，例如，其可以增加另外的活性、靶向功能，或其影响生理过程(例如血管通透性或生物膜的完整性)。可替代地，可以将结构域缔合以产生不同多肽之间的物理亲和力，从而产生多链聚合物复合物。The terms "fusion protein", "chimeric protein", "protein conjugate" and the like are meant to include amino acids in addition to the amino acid sequence encoding the original or native full-length protein or a subsequence thereof, including substitutions encoding the original or native full-length protein or a subsequence thereof. The amino acid sequence of the long protein or subsequence thereof, comprising less than the amino acid sequence encoding the original or native full length protein or subsequence thereof and/or comprising a different amino acid sequence than that encoding the original or native full length protein or subsequence thereof. More than one additional domain can be added to an active polypeptide as described herein, eg, one epitope tag or purification tag, or multiple epitope tags or purification tags. Additional domains may be attached, eg, which may add additional activity, targeting function, or affect physiological processes (eg, vascular permeability or biofilm integrity). Alternatively, domains can be associated to create physical affinity between different polypeptides, resulting in multi-chain polymer complexes.
除非另外说明，否则术语“病症”和“疾病”在本文中互换使用。Unless otherwise indicated, the terms "disorder" and "disease" are used interchangeably herein.
术语“组合物”或“药物组合物”可包括含有本公开的活性多肽以及例如药学上可接受的载剂、赋形剂或稀释剂的组合物，所述组合物被施用至受试者个体。The term "composition" or "pharmaceutical composition" can include a composition comprising an active polypeptide of the present disclosure and, for example, a pharmaceutically acceptable carrier, excipient or diluent, which composition is administered to an individual subject .
术语“药学上可接受的”是指在合理的医学判断范围内适合于与人类和动物的组织接触而无过度毒性或其它并发症、与合理的利益/风险比相称的组合物。在一些方面，本文所述的多肽、多核苷酸、组合物和疫苗是药学上可接受的。The term "pharmaceutically acceptable" refers to compositions that, within the scope of sound medical judgment, are suitable for contact with human and animal tissue without undue toxicity or other complications, commensurate with a reasonable benefit/risk ratio. In some aspects, the polypeptides, polynucleotides, compositions and vaccines described herein are pharmaceutically acceptable.
“有效量”是以单剂量或作为一系列剂量的一部分向个体施用对于治疗或预防而言有效的量。An "effective amount" is an amount effective for treatment or prevention when administered to a subject in a single dose or as part of a series of doses.
术语“受试者”是指需要诊断、预后、免疫或治疗的任何受试者，特别是哺乳动物受试者。哺乳动物受试者包括但不限于人、家养动物、农场动物、动物园动物如熊、运动动物、宠物动物如狗、猫、豚鼠、兔、大鼠、小鼠、马、牛、熊、奶牛；灵长类动物，如猿、猴、猩猩和黑猩猩；犬科动物，如狗和狼；猫科动物，如猫、狮子和老虎；马科动物，如马、驴和斑马；食用动物，如牛、猪和绵羊；有蹄类动物，如鹿和长颈鹿；啮齿动物，如小鼠、大鼠、仓鼠和豚鼠等。在一个优选的实施例中，受试者是人。The term "subject" refers to any subject in need of diagnosis, prognosis, immunization or treatment, particularly mammalian subjects. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals such as bears, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, cows; Primates, such as apes, monkeys, orangutans, and chimpanzees; canids, such as dogs and wolves; felines, such as cats, lions, and tigers; equines, such as horses, donkeys, and zebras; food animals, such as cattle , pigs and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs. In a preferred embodiment, the subject is a human.
术语“改善”是指在疾病状态，例如癌症的治疗中的任何治疗有益的结果，包括该疾病状态的预防、严重程度或进展的降低、减轻或治愈。The term "improvement" refers to any therapeutically beneficial outcome in the treatment of a disease state, such as cancer, including prevention, reduction in severity or progression of the disease state, alleviation, or cure.
本发明取得了以下技术效果：The present invention has achieved the following technical effects:
在α-芋螺毒素及其受体α9α10nAChR的空间结构基础上通过计算机辅助分子模拟设计合成了系列活性多肽。一方面，制备了氨基酸残基数量较少、不含半胱氨酸、特异性人α9α10nAChR抑制活性高的活性多肽。不仅易于化学合成、消除了复杂二硫键异构体的产生，而且避免了氨基酸替换对生物活性的影响，保持了天然α-芋螺毒素对人α9α10nAChR的特异性抑制功能。另一方面，与氨基酸残基数量更少(≤10)的短肽或基序(motif)相比，则具有更高的人α9α10nAChR特异性和更强的抑制活性，IC 50值为～25nM。而RSPYDRRRRY针对α9α10nAChR的抑制活性为320nM，因此Gex-2的活性大约为RSPYDRRRRY活性的12倍。 Based on the spatial structure of α-conotoxin and its receptor α9α10nAChR, a series of active polypeptides were designed and synthesized by computer-aided molecular simulation. On the one hand, an active polypeptide with a small number of amino acid residues, no cysteine, and high specific human α9α10 nAChR inhibitory activity was prepared. It is not only easy to chemically synthesize, eliminates the generation of complex disulfide bond isomers, but also avoids the influence of amino acid substitution on biological activity, and maintains the specific inhibitory function of natural α-conotoxin on human α9α10nAChR. On the other hand, compared with short peptides or motifs with fewer amino acid residues (≤10), it has higher specificity for human α9α10 nAChR and stronger inhibitory activity with IC50 value of ~25nM. The inhibitory activity of RSPYDRRRRY against α9α10 nAChR was 320 nM, so the activity of Gex-2 was about 12 times that of RSPYDRRRRY.
利用设计获得的系列活性多肽，一方面，针对不同物种α9α10nAChR的抑制活性进行分析，结果表明本发明涉及的多肽不仅能够抑制鼠α9α10nAChR，也能有效抑制人α9α10nAChR；另一方面，根据抑制α9α10nAChR的作用机理，在大鼠动物模型上进行疼痛阈值分析，取得了良好的镇痛效果，并且在一定范围内存在剂量依赖关系。Using the designed series of active polypeptides, on the one hand, the inhibitory activity of α9α10nAChR in different species was analyzed, and the results showed that the polypeptide involved in the present invention can not only inhibit mouse α9α10nAChR, but also effectively inhibit human α9α10nAChR; on the other hand, according to the effect of inhibiting α9α10nAChR Mechanism, pain threshold analysis was performed on rat animal model, and good analgesic effect was obtained, and there was a dose-dependent relationship within a certain range.
对经过功能验证的活性多肽进行定点突变，采用中性氨基酸、酸性氨基酸、碱性氨基酸、稀有氨基酸替换、D型氨基酸替换扫描的方式获得突变体。全景扫描式突变方案，不仅明确了关键的氨基酸残基位点和类型，而且还获得了人α9α10nAChR特异性抑制活性进一步提高的活性多肽。Site-directed mutagenesis of functionally verified active polypeptides was performed, and mutants were obtained by scanning neutral amino acids, acidic amino acids, basic amino acids, rare amino acids, and D-type amino acids. The panorama scanning mutation scheme not only clarified the key amino acid residue sites and types, but also obtained an active polypeptide with further improved specific inhibitory activity of human α9α10nAChR.
附图说明Description of drawings
通过阅读下文优选实施方式的详细描述，各种其他的优点和益处对于本领 域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的，而并不认为是对本发明的限制。而且在整个附图中，用相同的参考符号表示相同的部件。在附图中：Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are for the purpose of illustrating preferred embodiments only and are not to be considered limiting of the invention. Also, the same components are denoted by the same reference numerals throughout the drawings. In the attached image:
图1：活性多肽对人α9α10乙酰胆碱受体上乙酰胆碱激发的峰电流的相对抑制强度，其中，全细胞电流由6μM乙酰胆碱激发，多肽使用浓度为30nM。Figure 1: The relative inhibitory intensity of active peptides on the peak currents stimulated by acetylcholine on human α9α10 acetylcholine receptors, in which the whole-cell currents were stimulated by 6 μM acetylcholine, and the peptide concentration was 30 nM.
图2A：Gex-2对人α9α10nAChR的IC 50。 Figure 2A: IC50 of Gex-2 for human α9α10 nAChR.
图2B：Gex-2与其二聚体对人α9α10乙酰胆碱受体的抑制活性对比Figure 2B: Comparison of inhibitory activities of Gex-2 and its dimers on human α9α10 acetylcholine receptors
图3：大鼠的机械痛痛阈测试结果Figure 3: Mechanical pain threshold test results in rats
X轴表示时间(单位：天)；Y轴表示机械痛痛阈(单位：克)The X-axis represents time (unit: days); the Y-axis represents mechanical pain threshold (unit: grams)
图4：大鼠的热敏痛痛阈测试结果Figure 4: Thermal Pain Threshold Test Results in Rats
X轴表示时间(单位：天)；Y轴表示热痛痛阈(单位：秒)The X-axis represents time (unit: days); the Y-axis represents thermal pain threshold (unit: seconds)
图5：活性多肽Gex-2丙氨酸扫描变体对人α9α10乙酰胆碱受体上乙酰胆碱激发的峰电流的相对抑制强度，其中，全细胞电流由6μM乙酰胆碱激发，多肽使用浓度为30nM。Figure 5: The relative inhibitory intensity of the active polypeptide Gex-2 alanine scanning variant on the peak current excited by acetylcholine on the human α9α10 acetylcholine receptor, wherein the whole-cell current was stimulated by 6 μM acetylcholine, and the peptide concentration was 30 nM.
图6：活性多肽Gex-2天冬氨酸扫描变体对人α9α10乙酰胆碱受体上乙酰胆碱激发的峰电流的相对抑制强度，其中，全细胞电流由6μM乙酰胆碱激发，多肽使用浓度为30nM。Figure 6: The relative inhibitory intensity of the active polypeptide Gex-2 aspartate scanning variant on the peak current stimulated by acetylcholine on the human α9α10 acetylcholine receptor, wherein the whole-cell current was stimulated by 6 μM acetylcholine, and the peptide concentration was 30 nM.
图7：活性多肽Gex-2精氨酸扫描变体对人α9α10乙酰胆碱受体上乙酰胆碱激发的峰电流的相对抑制强度，其中，全细胞电流由6μM乙酰胆碱激发，多肽使用浓度为30nM。Figure 7: The relative inhibitory intensity of the active polypeptide Gex-2 arginine scanning variant on the peak current excited by acetylcholine on the human α9α10 acetylcholine receptor, wherein the whole-cell current was stimulated by 6 μM acetylcholine, and the peptide concentration was 30 nM.
图8：活性多肽Gex-2精氨酸-瓜氨酸替换变体对人α9α10乙酰胆碱受体上乙酰胆碱激发的峰电流的相对抑制强度，其中，全细胞电流由6μM乙酰胆碱激发，多肽使用浓度为30nM。Figure 8: The relative inhibitory intensity of the active polypeptide Gex-2 arginine-citrulline substitution variant on the peak current induced by acetylcholine on the human α9α10 acetylcholine receptor, wherein the whole-cell current was stimulated by 6 μM acetylcholine, and the peptide concentration was 30 nM .
图9：活性多肽Gex-2的D-型氨基酸替换变体对人α9α10乙酰胆碱受体上乙酰胆碱激发的峰电流的相对抑制强度，其中，全细胞电流由6μM乙酰胆碱激发，多肽使用浓度为30nM。Figure 9: The relative inhibitory intensity of the D-amino acid substitution variant of the active polypeptide Gex-2 on the peak current stimulated by acetylcholine on the human α9α10 acetylcholine receptor, wherein the whole-cell current was stimulated by 6 μM acetylcholine, and the peptide concentration was 30 nM.
图10：Gex-2单次给药后测得的机械痛PWTFigure 10: Mechanical pain PWT measured after a single dose of Gex-2
图11：Gex-2单次给药后测得的热痛PWTFigure 11: Thermal Pain PWT Measured After a Single Dosing of Gex-2
图12：Gex-2给药1h后测得的大鼠足底机械痛PWTFigure 12: PWT of rat plantar mechanical pain measured 1 h after Gex-2 administration
图13：Gex-2给药后1h大鼠热痛PWTFigure 13: PWT of heat pain in rats 1 h after Gex-2 administration
图14：Gex-2给药后24h后大鼠足底机械痛PWTFigure 14: PWT of rat plantar mechanical pain 24h after Gex-2 administration
图15：Gex-2给药后24h后大鼠足底热痛PWTFigure 15: PWT of rat plantar heat pain 24h after Gex-2 administration
图16：Gex-2在三叉神经疼痛动物模型上的镇痛活性评价与毒副作用评价。A，Gex-2镇痛活性评价；B，Gex-2对动物运动与平衡能力影响评价。Figure 16: Evaluation of the analgesic activity and toxicity of Gex-2 in an animal model of trigeminal neuralgia. A, evaluation of the analgesic activity of Gex-2; B, evaluation of the effect of Gex-2 on the movement and balance ability of animals.
图17：Gex-2給药1小时后痛阈测试结果Figure 17: Pain threshold test results 1 hour after Gex-2 administration
图18：Gex-2給药24小时后痛阈测试结果Figure 18: Pain threshold test results 24 hours after Gex-2 administration
图19：大鼠CPP实验结果Figure 19: Results of the rat CPP experiment
具体实施方式Detailed ways
下面将参照附图更详细地描述本公开的示例性实施方式。虽然附图中显示了本公开的示例性实施方式，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present disclosure will be more thoroughly understood, and will fully convey the scope of the present disclosure to those skilled in the art.
实施例1：多肽的合成方法Example 1: Synthetic method of polypeptide
本发明中的多肽可以采用Fmoc或者Boc固相合成方法。The polypeptide in the present invention can be synthesized by Fmoc or Boc solid-phase synthesis method.
1.1线性肽的合成方法1.1 Synthesis of linear peptides
以Fmoc固相合成法为例，合成步骤如下：Taking Fmoc solid-phase synthesis as an example, the synthesis steps are as follows:
1.称取0.2mmol RAM树脂，置于固相反应管中，加入等比例的N,N-二甲基甲酰胺(DMF)和二氯甲烷(DCM)过夜溶胀；1. Weigh 0.2 mmol RAM resin, place it in a solid-phase reaction tube, and add equal proportions of N,N-dimethylformamide (DMF) and dichloromethane (DCM) to swell overnight;
2.加入20％的哌啶于37度摇床内反应0.5h，脱除树脂上氨基的Fmoc保护基，反应完成后分别用DMF和DCM清洗树脂三次；2. Add 20% piperidine and react in a shaking table at 37 degrees for 0.5h to remove the Fmoc protecting group of the amino group on the resin. After the reaction is completed, wash the resin three times with DMF and DCM respectively;
3.加入5eq的C端第一个氨基酸、4.5eq HCTU和8eq DIPEA置于37度摇床，进行第一个氨基酸的缩合反应，待1h反应完成后分别用DMF和DCM清洗树脂三次；3. Add 5eq of the C-terminal first amino acid, 4.5eq of HCTU and 8eq of DIPEA and place them on a shaker at 37 degrees to carry out the condensation reaction of the first amino acid. After the 1h reaction is completed, wash the resin three times with DMF and DCM respectively;
4.再加入20％哌啶脱除Fmoc，脱除后再进行第二个氨基酸的缩合反应，如此循环往复，将氨基酸从C端到N端逐一地连接在多肽链上；4. Add 20% piperidine to remove Fmoc, and then carry out the condensation reaction of the second amino acid after the removal, so that the amino acids are connected to the polypeptide chain one by one from the C-terminus to the N-terminus;
5.直链肽合成完毕后，用K试剂(三氟乙酸/水/乙二硫醇/苯酚/硫苯甲醚＝90:5:2.5:7.5:5)将多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；5. After the linear peptide is synthesized, use K reagent (trifluoroacetic acid/water/ethanedithiol/phenol/thioanisole=90:5:2.5:7.5:5) to cut the peptide from the resin, and use The crude linear peptide was recovered by precipitation and washing with ice ether;
6.质谱确定分子量无误后，用制备型反向HPLC C8柱纯化多肽样品，溶剂A为90％纯水、10％乙腈(ACN)和0.1％三氟乙酸(TFA)；溶剂B为60％纯水， 40％乙腈(ACN)和0.1％三氟乙酸(TFA)，洗脱线性梯度为在40min内使溶剂B从0％-60％；6. After the molecular weight was confirmed by mass spectrometry, the peptide sample was purified by preparative reverse HPLC C8 column, solvent A was 90% pure water, 10% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA); solvent B was 60% pure Water, 40% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA), eluting with a linear gradient of solvent B from 0%-60% in 40 min;
7.纯化完成后，对于不含二硫键的多肽，用分析型的HPC C18柱进行纯度检测，保证其纯度达到96％以上即可进行冻干；对于有二硫键的多肽，则进行进一步氧化折叠，将多肽溶液的pH调制7-8，在空气中搅拌氧化48h连接形成二硫键，再进行纯度检测，多肽纯度达到96％，冻干。7. After purification, for peptides without disulfide bonds, use an analytical HPC C18 column for purity testing to ensure that the purity is over 96% and then lyophilize; for peptides with disulfide bonds, further Oxidative folding, adjusting the pH of the polypeptide solution to 7-8, stirring and oxidizing in the air for 48 h to form disulfide bonds, and then performing purity testing, the polypeptide purity reached 96%, and was freeze-dried.
1.2环肽的合成方法1.2 Synthesis of cyclic peptides
环肽的合成采用的是NCL法。环肽是不具有首尾之分的，先合成线性肽，然后根据NCL法的反应机理环合。以C端到N端的方向合成出直链肽，以环肽上半胱氨酸残基-NH2侧连接的氨基酸残基作为线性肽C端第一个氨基酸，使半胱氨酸作为最后一个氨基酸残基，然后通过酰基转移反应环合。Cyclic peptides were synthesized by NCL method. Cyclic peptides do not have a head-to-tail distinction. Linear peptides are synthesized first, and then cyclized according to the reaction mechanism of the NCL method. A straight-chain peptide is synthesized from the C-terminal to the N-terminal, and the amino acid residue connected to the cysteine residue-NH2 side of the cyclic peptide is used as the first amino acid at the C-terminal of the linear peptide, and cysteine is used as the last amino acid. residues, which are then cyclized via an acyl transfer reaction.
具体合成方法为：The specific synthesis method is:
1.称取0.2mmol RAM树脂，置于固相反应管中，加入等比例的N,N-二甲基甲酰胺(DMF)和二氯甲烷(DCM)过夜溶胀；1. Weigh 0.2 mmol RAM resin, place it in a solid-phase reaction tube, and add equal proportions of N,N-dimethylformamide (DMF) and dichloromethane (DCM) to swell overnight;
2.加入20％的哌啶于37度摇床内反应0.5h，脱除树脂上氨基的Fmoc保护基，反应完成后分别用DMF和DCM清洗树脂三次；2. Add 20% piperidine and react in a shaking table at 37 degrees for 0.5h to remove the Fmoc protecting group of the amino group on the resin. After the reaction is completed, wash the resin three times with DMF and DCM respectively;
3.加入3eq 3,4-二氨基苯甲酸(氨基被Fmoc保护)、2.7eq HCTU和0.1mL DIPEA置于37度摇床反应过夜，反应完成后分别用DMF和DCM清洗树脂三次；3. Add 3eq 3,4-diaminobenzoic acid (the amino group is protected by Fmoc), 2.7eq HCTU and 0.1mL DIPEA and place it on a shaker at 37 degrees to react overnight. After the reaction is completed, wash the resin three times with DMF and DCM respectively;
4.加入20％的哌啶于37度摇床内反应0.5h，脱除3,4-二氨基苯甲酸上的Fmoc保护基，反应完成后分别用DMF和DCM清洗树脂三次；4. Add 20% piperidine and react in a shaking table at 37 degrees for 0.5h to remove the Fmoc protecting group on 3,4-diaminobenzoic acid. After the reaction is completed, the resin is washed three times with DMF and DCM respectively;
5.为保证Fmoc脱除完全，重复第四步；5. To ensure complete removal of Fmoc, repeat the fourth step;
6.加入5eq的酪氨酸、4.5eq HCTU和8eq DIPEA置于37度摇床，进行第一个氨基酸的缩合反应，待2h反应完成后分别用DMF和DCM清洗树脂三次；6. Add 5eq of tyrosine, 4.5eq of HCTU and 8eq of DIPEA and place them on a shaker at 37 degrees to carry out the condensation reaction of the first amino acid. After the 2h reaction is completed, wash the resin three times with DMF and DCM respectively;
7.再加入20％哌啶脱除Fmoc，脱除后再进行第二个氨基酸的缩合反应，按照传统的固相多肽合成法，将氨基酸从C端到N端逐一地连接在多肽链上，最后一个氨基酸为Boc-Cys(Trt)-OH；7. Add 20% piperidine to remove Fmoc, and then carry out the condensation reaction of the second amino acid. According to the traditional solid-phase peptide synthesis method, the amino acids are connected to the polypeptide chain one by one from the C-terminus to the N-terminus, The last amino acid is Boc-Cys(Trt)-OH;
8.直链肽合成结束后，将树脂抽干，转移至干燥的茄型瓶中，加入7eq对硝基苯基氯甲酸酯，加入适量DCM作溶剂，反应2h；8. After the synthesis of the linear peptide, drain the resin, transfer it to a dry eggplant-shaped bottle, add 7eq of p-nitrophenyl chloroformate, add an appropriate amount of DCM as a solvent, and react for 2h;
9.反应结束后将树脂转移至固相反应管中，洗涤树脂，分别用DMF和DCM清洗树脂三次，再加入1ml DIPEA和适量DMF溶液反应30min，结束后分别用 DMF和DCM清洗树脂三次；9. After the reaction, transfer the resin to the solid-phase reaction tube, wash the resin, wash the resin three times with DMF and DCM respectively, then add 1ml DIPEA and an appropriate amount of DMF solution to react for 30min, and wash the resin three times with DMF and DCM respectively after finishing;
10.用K试剂(三氟乙酸/水/乙二硫醇/苯酚/硫苯甲醚＝90:5:2.5:7.5:5)将多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，回收后进行冻干；10. Use K reagent (trifluoroacetic acid/water/ethanedithiol/phenol/thioanisole = 90:5:2.5:7.5:5) to cleave the polypeptide from the resin, and recover the linearity by precipitation and washing with glacial ether Crude peptide, lyophilized after recovery;
11.称取20mg粗肽，加入40eq二硫苏糖醇，200eq醋酸铵，加入水/乙腈溶液至溶液澄清，室温反应40min；11. Weigh 20mg crude peptide, add 40eq dithiothreitol, 200eq ammonium acetate, add water/acetonitrile solution until the solution is clear, react at room temperature for 40min;
12.质谱确定反应完成后，用制备型反向HPLC C8柱纯化多肽样品，溶剂A为90％纯水、10％乙腈(ACN)和0.1％三氟乙酸(TFA)；溶剂B为60％纯水，40％乙腈(ACN)和0.1％三氟乙酸(TFA)，洗脱线性梯度为在40min内使溶剂B从0％-60％；12. After the reaction was confirmed by mass spectrometry, the peptide sample was purified by preparative reverse HPLC C8 column, solvent A was 90% pure water, 10% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA); solvent B was 60% pure Water, 40% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA), eluting with a linear gradient of solvent B from 0%-60% in 40 min;
13.纯化完成后，将多肽溶液的pH调制7-8，在空气中搅拌氧化48h氧化折叠形成二硫键，用分析型的HPCC18柱进行纯度检测，若多肽纯度达到96％，则可冻干。13. After the purification is completed, adjust the pH of the peptide solution to 7-8, stir and oxidize in the air for 48h to form disulfide bonds, and use an analytical HPCC18 column for purity detection. If the peptide purity reaches 96%, it can be lyophilized. .
采用Boc固相合成，其过程类似于Fmoc，但每一步的去保护(Boc)反应，需要采用较低浓度的TFA。多肽链合成完后需要采用较高浓度的三氟乙酸进行切割，把多肽从树脂上切割下来。本专利工艺包括采用Boc法合成多肽。Using Boc solid-phase synthesis, the process is similar to Fmoc, but each step of the deprotection (Boc) reaction requires the use of lower concentrations of TFA. After the polypeptide chain is synthesized, a higher concentration of trifluoroacetic acid needs to be used for cleavage, and the polypeptide is cleaved from the resin. The patented process includes the use of the Boc method to synthesize polypeptides.
实施例2：电生理学活性测试方法Example 2: Electrophysiological Activity Test Method
为了验证活性多肽对人α9α10乙酰胆碱受体的抑制活性，检测多肽对人α9α10乙酰胆碱受体上乙酰胆碱激发的峰电流的相对抑制强度。方法如下：In order to verify the inhibitory activity of the active polypeptide on the human α9α10 acetylcholine receptor, the relative inhibitory intensity of the polypeptide on the peak current excited by acetylcholine on the human α9α10 acetylcholine receptor was detected. Methods as below:
使用体外转录试剂盒制备人α9α10乙酰胆碱受体的cRNA,其浓度用UV260m下的OD值进行测算。解剖收集非洲爪蟾卵母细胞,在第一天和第二天将两个亚基的cRNA注射入蛙卵中,注射量均为5ng，在ND-96中培养。注射后1-4天将表达了α9α10乙酰胆碱受体的卵母细胞用于电生理学活性测试。具体测试方法为：将1个注射过cRNA的爪蟾卵母细胞置于直径为4mm深度为2mm的30uL Sylgard记录槽中，重力灌注含有0.1mg/ml牛血清白蛋白(BSA)的ND96灌流液(96.0mM NaC1，2.0mM KC1，1.8mM CaCl 2 1.0mM MgCl 2，5mM HEPES，pH 7.1-7.5)。所有的芋螺毒素溶液也含有0.1mg/mL BSA以减少非特异性吸附,用转换阀可以在灌注毒素或乙酰胆碱之间进行自由切换,ACh门控的电流由双电极电压钳放大器设置在“慢”档,以及clamp gain在最大值(×2000)位置时进行在线记录。用外径1mm内径0.75mm的玻璃毛细管拉制玻璃电极，并充满3M KCl作为电压和电流电极。膜电压钳制在-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的浓度为6μM。每条多肽至少记录6个卵母细胞的电流反应情况，测试的电流数据用GraphPad Prism软件进行统计分析。 The cRNA of human α9α10 acetylcholine receptor was prepared by in vitro transcription kit, and its concentration was measured by OD value under UV260m. Xenopus oocytes were dissected and collected, and the cRNAs of the two subunits were injected into frog eggs on the first and second day, and the injection amount was 5ng, and cultured in ND-96. Oocytes expressing the α9α10 acetylcholine receptor were tested for electrophysiological activity 1-4 days after injection. The specific test method is as follows: Place 1 cRNA-injected Xenopus oocyte in a 30uL Sylgard recording tank with a diameter of 4mm and a depth of 2mm, and gravity perfusion with ND96 perfusate containing 0.1mg/ml bovine serum albumin (BSA). (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2 1.0 mM MgCl2 , 5 mM HEPES, pH 7.1-7.5). All conotoxin solutions also contained 0.1 mg/mL BSA to reduce non-specific adsorption, a switch valve was used to switch freely between perfusion toxin or acetylcholine, and the ACh-gated current was set at "slow" by a two-electrode voltage clamp amplifier file, and the clamp gain is recorded online when it is at the maximum (×2000) position. Glass electrodes were drawn with glass capillaries with an outer diameter of 1 mm and an inner diameter of 0.75 mm, and filled with 3M KCl as voltage and current electrodes. The membrane voltage was clamped at -70mV, and the entire system was controlled and recorded by a computer. The ACh pulse was automatically perfused with ACh for 1 s every 5 min, and the concentration of ACh was 6 μM. The current responses of at least 6 oocytes were recorded for each polypeptide, and the tested current data were statistically analyzed with GraphPad Prism software.
实施例3：体内镇痛活性动物模型的构建Example 3: Construction of an animal model of in vivo analgesic activity
采用动物模型评估活性多肽对痛觉的影响。镇痛活性的测试采用的是大鼠坐骨神经慢性压迫损伤(CCI)模型。Animal models were used to evaluate the effect of active peptides on pain perception. The analgesic activity was tested using the rat sciatic nerve chronic compression injury (CCI) model.
造模过程如下：大鼠经腹腔注射2％戊巴比妥钠麻醉后，剔除右腿鼠毛，无菌条件下切开右下肢皮肤1-2cm，钝性分离肌肉和筋膜，暴露坐骨神经主干，用4-0铬制羊肠线以间距为1mm打四个结，其松紧程度要求至大鼠脚趾微微抽搐为止，以不影响神经外膜的血运为度，结扎完成后用四万单位青霉素钠清洗伤口，最后缝合，术后连续三天在患肢肌肉注射4万单位的青霉素钠防止感染。假手术组，只暴露坐骨神经主干而不进行结扎压迫，模拟伤口对大鼠痛阈的影响。The modeling process is as follows: after the rats were anesthetized by intraperitoneal injection of 2% pentobarbital sodium, the hair of the right leg was removed, the skin of the right lower limb was incised 1-2 cm under aseptic conditions, the muscles and fascia were bluntly separated, and the trunk of the sciatic nerve was exposed. , use 4-0 chrome catgut to tie four knots with a spacing of 1mm, and the tightness is required until the rat's toes twitch slightly, so as not to affect the blood supply of the epineurium, after the ligation is completed, use 40,000 units The wound was cleaned with sodium penicillin and finally sutured, and 40,000 units of sodium penicillin was intramuscularly injected into the affected limb for three consecutive days after the operation to prevent infection. In the sham operation group, only the trunk of the sciatic nerve was exposed without ligation and compression to simulate the effect of wound on the pain threshold of rats.
用IITC电子测痛仪测定大鼠的机械痛疼痛阈值，用IITC足底热点测痛仪测定大鼠的热敏痛疼痛阈值，The mechanical pain pain threshold of rats was measured with IITC electronic pain measuring instrument, and the thermal pain pain threshold of rats was measured with IITC plantar hot spot pain measuring instrument.
实施例4：α9α10nAChR抑制活性多肽的设计及初步验证Example 4: Design and preliminary verification of α9α10nAChR inhibitory active polypeptide
在ConoServer上查找了靶向α9α10乙酰胆碱受体的芋螺毒素，选取GeXIVA和GeXXVIIA进行了序列比对，以两者序列重叠的部分为基础，结合多肽对接和分子动力学模拟分析，对二者重叠片段进行合理的修饰改造，设计合成了系列多肽，序列如表1所示。The conotoxin targeting the α9α10 acetylcholine receptor was searched on ConoServer, and GeXIVA and GeXXVIIA were selected for sequence alignment. Based on the overlapping part of the two sequences, combined with peptide docking and molecular dynamics simulation analysis, the overlap between the two was analyzed. The fragments were modified reasonably, and a series of polypeptides were designed and synthesized. The sequences are shown in Table 1.
表1：α9α10nAChR抑制活性多肽的设计Table 1: Design of polypeptides with α9α10nAChR inhibitory activity
Figure PCTCN2021141818-appb-000001
Figure PCTCN2021141818-appb-000001
根据实施例2的方法，使用Fmoc固相合成法合成直链多肽，环肽Gex-4使用NCL合成法进行合成。按照实施例2的方法，利用表达α9α10乙酰胆碱受体的非洲爪蟾卵母细胞，测试八条多肽对α9α10乙酰胆碱受体的抑制活性，活性测试结果如图1所示。According to the method of Example 2, Fmoc solid-phase synthesis method was used to synthesize linear polypeptide, and cyclic peptide Gex-4 was synthesized using NCL synthesis method. According to the method of Example 2, using Xenopus oocytes expressing α9α10 acetylcholine receptors, the inhibitory activities of eight polypeptides on α9α10 acetylcholine receptors were tested, and the activity test results are shown in FIG. 1 .
图1结果表明，设计的8条短肽对α9α10乙酰胆碱受体的抑制活性均具有抑制活性，其中Gex-2、Gex-3完全不含有半胱氨酸，纯化更容易、收率更高，且二者的活性最高。Gex-3是Gex-2的D-型氨基酸类似物，翻转氨基酸构型可以基本保留多肽的α9α10乙酰胆碱受体抑制活性。前期研究发现，采用D型氨基酸替换L型氨基酸，可以显著提高多肽的稳定性，保留其活性(J Med Chem.2020Apr 9；63(7):3475-3484；Mar Drugs.2019Feb 28；17(3):142.)。因此，尽管与Gex-2相比Gex-3的活性略有降低，Gex-3能获得更高的稳定性。The results in Figure 1 show that the designed 8 short peptides all have inhibitory activity on α9α10 acetylcholine receptors. Among them, Gex-2 and Gex-3 do not contain cysteine at all, the purification is easier, the yield is higher, and Both have the highest activity. Gex-3 is a D-type amino acid analog of Gex-2, and the inversion of the amino acid configuration can basically retain the α9α10 acetylcholine receptor inhibitory activity of the polypeptide. Previous studies have found that replacing L-amino acids with D-amino acids can significantly improve the stability of the polypeptide and retain its activity (J Med Chem.2020Apr 9;63(7):3475-3484;Mar Drugs.2019Feb 28;17(3 ): 142.). Therefore, Gex-3 achieves higher stability despite slightly reduced activity compared to Gex-2.
进一步按照实施例2的方法，利用表达α9α10乙酰胆碱受体的非洲爪蟾卵母细胞检测并计算Gex-2对人α9α10乙酰胆碱受体抑制的IC50值，结果如图2A所示。图2表明Gex-2A对人α9α10nAChR抑制的IC50为～25nM。Further according to the method of Example 2, the Xenopus oocytes expressing the α9α10 acetylcholine receptor were used to detect and calculate the IC50 value of Gex-2 for the inhibition of the human α9α10 acetylcholine receptor, and the results are shown in FIG. 2A . Figure 2 shows that Gex-2A has an IC50 of ~25 nM for inhibition of human α9α10 nAChR.
将Gex-2通过linker(PEG13)偶联形成二聚体，对比Gex-2与其二聚体对人α9α10乙酰胆碱受体的抑制活性，结果如图2B所示。图2B结果表明与Gex-2单体相比，二聚体对α9α10乙酰胆碱受体的抑制活性显著提高。Gex-2 was coupled to form a dimer by linker (PEG13), and the inhibitory activity of Gex-2 and its dimer on human α9α10 acetylcholine receptor was compared, and the results were shown in Figure 2B. The results of Figure 2B show that the inhibitory activity of the dimer on the α9α10 acetylcholine receptor is significantly improved compared with the Gex-2 monomer.
实施例5：Gex-2多肽的镇痛效果Example 5: Analgesic effect of Gex-2 polypeptide
选择Gex-2为活性多肽的代表，进一步在动物模型体内研究其镇痛效果。按 照实施例3的方法，使用大鼠坐骨神经压迫性损伤(CCI)模型进行Gex-2的镇痛活性测试，Gex-2 was selected as the representative of active polypeptide, and its analgesic effect was further studied in animal model. According to the method of Example 3, the analgesic activity test of Gex-2 was carried out using the rat sciatic nerve compression injury (CCI) model,
于济南朋悦动物繁育中心购置30只120-150g的大鼠，分为五组进行适应性训练(表2)，训练一周待大鼠适应环境后，对其中三组进行造模，其余两组中，一组作为空白对照，一组作为假手术组。待一周后三组造模大鼠的疼痛阈值降低到最低值后，进行连续两周的原位肌肉注射给药，第一组每天注射生理盐水，第二组给Gex-2，给药剂量为7.5×10 -5mg/kg，第三组给Gex-2，给药剂量为2.5×10 -4mg/kg。于每天给药后的24h，用IITC电子测痛仪测定大鼠的机械痛疼痛阈值，用IITC足底热点测痛仪测定大鼠的热敏痛疼痛阈值，热痛测试中，工作光强设置为“025”，待机光强设置为“010”，预置时间设置为“20”，获得的数据用GraphPad Prism软件进行统计分析。 Purchased 30 rats of 120-150 g in Jinan Pengyue Animal Breeding Center, and divided them into five groups for adaptive training (Table 2). , one group was used as blank control, and the other group was used as sham operation group. After one week, the pain thresholds of the three groups of model rats were reduced to the lowest value, and they were administered by in situ intramuscular injection for two consecutive weeks. The first group was injected with normal saline every day, and the second group was given Gex-2 at a dose of 7.5×10 -5 mg/kg, the third group was given Gex-2 at a dose of 2.5×10 -4 mg/kg. 24h after daily administration, the mechanical pain pain threshold of rats was measured with IITC electronic pain measuring instrument, and the thermal pain pain threshold of rats was measured with IITC plantar hot spot pain measuring instrument. In the thermal pain test, the working light intensity was set. is "025", the standby light intensity is set to "010", the preset time is set to "20", and the obtained data is statistically analyzed with GraphPad Prism software.
表2：动物模型镇痛实验分组Table 2: Grouping of animal model analgesia experiments
组别group 剥离神经stripped nerves 结扎神经ligated nerve 肌肉注射intramuscular injection
对照组control group -- -- --
假手术组mock surgical group √√ -- --
生理盐水组saline group √√ √√ 生理盐水normal saline
Gex-2组(7.5×10 -5mg/kg) Gex-2 group (7.5×10 -5 mg/kg) √√ √√ Gex-2Gex-2
Gex-2组(2.5×10 -4mg/kg) Gex-2 group (2.5×10 -4 mg/kg) √√ √√ Gex-2Gex-2
连续给药两周，分别在每次给药后的24h测试大鼠的机械痛痛阈和热敏痛痛阈，结果分别如图3和图4所示。After continuous administration for two weeks, the mechanical pain threshold and thermal pain threshold of the rats were tested at 24 h after each administration, and the results were shown in Figure 3 and Figure 4, respectively.
图3的结果表明，正常大鼠的机械痛阈值大都分布在55-70克之间，而生理盐水组的CCI模型大鼠的痛阈普遍低于20克，皮肤伤口和神经剥离(假手术组)并没有对大鼠的痛阈造成明显的影响，2.5×10 -4mg/kg的Gex-2可以使CCI大鼠的痛阈回复到接近于正常大鼠的水平，而7.5×10 -5mg/kg的Gex-2也可以使CCI大鼠的痛阈大幅提高，并与阴性对照组具有显著差异。这说明Gex-2在极低剂量时就具有良好的镇痛作用，而这种镇痛作用的强度在一定范围内与用药剂量相关。 The results in Figure 3 show that the mechanical pain thresholds of normal rats are mostly distributed between 55-70 grams, while the pain thresholds of CCI model rats in the normal saline group are generally lower than 20 grams, with skin wounds and nerve stripping (sham operation group) There was no obvious effect on the pain threshold of rats. 2.5×10 -4 mg/kg of Gex-2 could restore the pain threshold of CCI rats to the level of normal rats, while 7.5×10 -5 mg Gex-2/kg can also significantly improve the pain threshold of CCI rats, and there is a significant difference with the negative control group. This shows that Gex-2 has a good analgesic effect at very low doses, and the intensity of this analgesic effect is related to the dosage within a certain range.
图4的结果表明，正常大鼠在25的光强下，热痛阈值几乎都可以达到极限值20秒，而生理盐水组的CCI模型大鼠的痛阈普遍低于10秒，皮肤伤口和神经剥离(假手术组)并没有对大鼠的痛阈造成明显的影响，2.5×10 -4mg/kg的 Gex-2可以使CCI大鼠的痛阈回复到接近于正常大鼠的水平，而7.5×10 -5mg/kg的Gex-2也可以使CCI大鼠的痛阈大幅上升，与阴性对照组产生显著差异。这说明Gex-2在极低剂量时就具有良好的镇痛作用，但由于时间限制为20秒，镇痛作用的剂量依赖性并没有充分体现。 The results in Figure 4 show that under the light intensity of 25, the thermal pain threshold of normal rats can almost reach the limit value of 20 seconds, while the pain threshold of CCI model rats in the normal saline group is generally lower than 10 seconds. Skin wounds and nerves Stripping (sham-operated group) did not significantly affect the pain threshold of rats. Gex-2 at 2.5×10 -4 mg/kg could restore the pain threshold of CCI rats to a level close to that of normal rats, while 7.5×10 -5 mg/kg of Gex-2 can also significantly increase the pain threshold of CCI rats, which is significantly different from the negative control group. This shows that Gex-2 has a good analgesic effect at very low doses, but the dose-dependent analgesic effect is not fully reflected due to the time limit of 20 seconds.
实施例5：Gex-2丙氨酸扫描变体及对人α9α10nAChR的抑制活性Example 5: Gex-2 Alanine Scanning Variants and Inhibitory Activity on Human α9α10 nAChR
为了表征Gex-2多肽中氨基酸侧链对于活性的影响，阐明其构-效关系，对Gex-2进行了丙氨酸扫描，设计合成了丙氨酸扫描变体，如表3所示。In order to characterize the effect of amino acid side chains in Gex-2 polypeptide on the activity and clarify its structure-activity relationship, Gex-2 was scanned for alanine, and alanine-scanned variants were designed and synthesized, as shown in Table 3.
表3：Gex-2多肽的丙氨酸扫描变体Table 3: Alanine Scanning Variants of Gex-2 Polypeptides
Figure PCTCN2021141818-appb-000002
Figure PCTCN2021141818-appb-000002
按照实施例2的方法，利用表达α9α10乙酰胆碱受体的非洲爪蟾卵母细胞，检测Gex-2多肽丙氨酸扫描变体对α9α10乙酰胆碱受体的抑制活性，活性测试结果如图5所示。According to the method of Example 2, using Xenopus oocytes expressing α9α10 acetylcholine receptors, the inhibitory activity of Gex-2 polypeptide alanine scanning variants on α9α10 acetylcholine receptors was detected, and the activity test results are shown in FIG. 5 .
图5显示Gex-19、Gex-20、Gex-21、Gex-22的相对电流强度低于0.4，其它各变体的相对电流强度则均高于Gex-2。上述实验结果表明，Gex-2中R11-R14四个氨基酸残基对Gex-2的活性影响不大，单突变后仍可保留甚至提高活性，因此采用其它任意氨基酸取代11–14号位置的氨基酸可以保留甚至提高其生物活性；而Gex-2序列中的其他的氨基酸残基被Ala取代后活性大幅度下降，说明这些位置上的氨基酸对于维系多肽的活性或者结构是至关重要的，被突变后会使多肽的活性大幅降低。然而，采用物化性质相似的氨基酸残基取代，例如Lys/Dab/Dap碱性氨基酸取代序列中的Arg，Leu/Val等疏水氨基酸取代Ile，Glu等酸性氨基酸取代序列中的Asp潜在可以保留甚至提高其活性。Figure 5 shows that the relative current intensities of Gex-19, Gex-20, Gex-21, and Gex-22 are lower than 0.4, and the relative current intensities of other variants are all higher than those of Gex-2. The above experimental results show that the four amino acid residues of R11-R14 in Gex-2 have little effect on the activity of Gex-2, and the activity can still be retained or even improved after single mutation. It can retain or even improve its biological activity; while other amino acid residues in the Gex-2 sequence are substituted by Ala, the activity is greatly reduced, indicating that the amino acids at these positions are crucial for maintaining the activity or structure of the polypeptide. Afterwards, the activity of the polypeptide will be greatly reduced. However, the substitution of amino acid residues with similar physicochemical properties, such as Arg in the basic amino acid substitution sequence of Lys/Dab/Dap, substitution of hydrophobic amino acids such as Leu/Val for Ile, and substitution of Asp in the acidic amino acid sequence such as Glu, can potentially preserve or even improve the its activity.
实施例6：Gex-2天冬氨酸和精氨酸扫描变体及对人α9α10nAChR的抑制活性Example 6: Gex-2 Aspartate and Arginine Scanning Variants and Inhibitory Activity on Human α9α10 nAChR
为了分析侧链电荷对Gex-2多肽活性的影响，对Gex-2进行了天冬氨酸(负电荷)和精氨酸(正电荷)扫描，设计合成了天冬氨酸扫描变体、精氨酸扫描变体，如表4所示。In order to analyze the effect of side chain charge on the activity of Gex-2 polypeptide, aspartic acid (negative charge) and arginine (positive charge) scanning were performed on Gex-2, and aspartic acid scanning variants, arginine scanning variants were designed and synthesized. Amino acid scanning variants are shown in Table 4.
表4：Gex-2多肽的天冬氨酸/精氨酸扫描变体Table 4: Aspartate/Arginine Scanning Variants of Gex-2 Polypeptides
Figure PCTCN2021141818-appb-000003
Figure PCTCN2021141818-appb-000003
Figure PCTCN2021141818-appb-000004
Figure PCTCN2021141818-appb-000004
按照实施例2的方法，利用表达α9α10乙酰胆碱受体的非洲爪蟾卵母细胞，检测Gex-2多肽天冬氨酸扫描变体、精氨酸扫描变体对α9α10乙酰胆碱受体的抑制活性，活性测试结果分别如图6、图7所示。According to the method of Example 2, using Xenopus oocytes expressing α9α10 acetylcholine receptors, the inhibitory activity of Gex-2 polypeptide aspartate scanning variants and arginine scanning variants on α9α10 acetylcholine receptors was detected. The test results are shown in Figure 6 and Figure 7, respectively.
图6表明Gex-2多肽的Y3和P6被天冬氨酸替代时对活性影响不大，而在其他位置引入带负电荷氨基酸会使多肽活性降低甚至失活。基于前面的Ala扫描 得知Y3或P6对于维系多肽的活性是至关重要的，然而引入物化性质差别很大的Asp酸性氨基酸可以保留多肽的活性，说明在3号与6号位置引入带负电侧链的氨基酸(Asp或者Glu等)可以补偿原氨基酸中侧链发挥的重要作用。在Gex-2的N末端1号位残基被带负电荷氨基酸Asp取代，可以大幅度降低Gex-2的活性。Figure 6 shows that the replacement of Y3 and P6 of Gex-2 polypeptide with aspartic acid has little effect on the activity, while the introduction of negatively charged amino acids at other positions will reduce the activity of the polypeptide or even inactivate it. Based on the previous Ala scan, it is known that Y3 or P6 is crucial for maintaining the activity of the polypeptide. However, the introduction of Asp acidic amino acids with very different physicochemical properties can preserve the activity of the polypeptide, indicating that the introduction of negatively charged sides at positions 3 and 6 The amino acids of the chain (Asp or Glu, etc.) can compensate for the important role played by the side chain in the original amino acid. The substitution of the negatively charged amino acid Asp at the N-terminal 1 residue of Gex-2 can greatly reduce the activity of Gex-2.
图7表明当多肽中S5或116位引入正电荷氨基酸Arg时，多肽的活性会大幅度提高，表明5号位或者16号位引入带正电荷氨基酸(Lys/Dab/Dap等)会增强多肽的活性；Y13或D18被R替代时对多肽的活性影响不大，说明R的侧链可以补偿Y13与D18侧链的作用；其它位置引入R都会引起多肽活性降低。Figure 7 shows that when a positively charged amino acid Arg is introduced into the S5 or 116 position of the polypeptide, the activity of the polypeptide will be greatly improved, indicating that the introduction of a positively charged amino acid (Lys/Dab/Dap, etc.) at the 5th or 16th position will enhance the polypeptide's activity Activity; when Y13 or D18 is replaced by R, it has little effect on the activity of the polypeptide, indicating that the side chain of R can compensate for the effect of the side chain of Y13 and D18; the introduction of R at other positions will reduce the activity of the polypeptide.
实施例7：Gex-2多肽精氨酸-瓜氨酸替换变体及对人α9α10 nAChR的抑制活性Example 7: Gex-2 polypeptide arginine-citrulline substitution variant and its inhibitory activity on human α9α10 nAChR
为了评估稀有氨基酸替换对Gex-2多肽活性的影响，基于精氨酸与瓜氨酸相似的性质和结构，将多肽链中的精氨酸逐一用瓜氨酸替代，制备Gex-2多肽的精氨酸-瓜氨酸替换变体，序列如表5所示。In order to evaluate the effect of rare amino acid substitutions on the activity of Gex-2 polypeptides, based on the similar properties and structures of arginine and citrulline, the arginines in the polypeptide chain were replaced with citrulline one by one to prepare the Gex-2 polypeptides. Amino-citrulline substitution variants, the sequences are shown in Table 5.
表5：Gex-2多肽的精氨酸-瓜氨酸替换变体Table 5: Arginine-citrulline substitution variants of Gex-2 polypeptides
多肽名称Peptide name 多肽序列polypeptide sequence
Gex-53Gex-53 G(Cit)YRSPYDRRRRYRRITD-NH 2 G(Cit)YRSPYDRRRRYRRITD-NH 2
Gex-54Gex-54 GRY(Cit)SPYDRRRRYRRITD-NH 2 GRY(Cit)SPYDRRRRYRRITD-NH 2
Gex-55Gex-55 GRYRSPYD(Cit)RRRYRRITD-NH 2 GRYRSPYD(Cit)RRRYRRITD-NH 2
Gex-56Gex-56 GRYRSPYDR(Cit)RRYRRITD-NH 2 GRYRSPYDR(Cit)RRYRRITD-NH 2
Gex-57Gex-57 GRYRSPYDRR(Cit)RYRRITD-NH 2 GRYRSPYDRR(Cit)RYRRITD-NH 2
Gex-58Gex-58 GRYRSPYDRRR(Cit)YRRITD-NH 2 GRYRSPYDRRR(Cit)YRRITD-NH 2
Gex-59Gex-59 GRYRSPYDRRRRY(Cit)RITD-NH 2 GRYRSPYDRRRRY(Cit)RITD-NH 2
Gex-60Gex-60 GRYRSPYDRRRRYR(Cit)ITD-NH 2 GRYRSPYDRRRRYR(Cit)ITD-NH 2
按照实施例2的方法，利用表达α9α10乙酰胆碱受体的非洲爪蟾卵母细胞，检测Gex-2多肽精氨酸-瓜氨酸替换变体对α9α10乙酰胆碱受体的抑制活性，活性测试结果如图8所示。According to the method of Example 2, using Xenopus oocytes expressing α9α10 acetylcholine receptors, the inhibitory activity of Gex-2 polypeptide arginine-citrulline substitution variants on α9α10 acetylcholine receptors was detected, and the activity test results are shown in the figure 8 shown.
图8的实验结果表明，精氨酸被瓜氨酸替代后会使多肽活性大幅降低，趋近 于失活。由于Cit的结构类似于Arg，可以认为多肽活性的大幅度降低主要由正电荷的去除所导致的。因此，保留多肽中带正电荷的氨基酸是Gex-2及相关类似物具有活性的前提。The experimental results in Figure 8 show that the replacement of arginine by citrulline will greatly reduce the activity of the polypeptide, approaching inactivation. Since the structure of Cit is similar to Arg, it can be considered that the large reduction in polypeptide activity is mainly caused by the removal of positive charges. Therefore, retention of positively charged amino acids in polypeptides is a prerequisite for the activity of Gex-2 and related analogs.
实施例8：Gex-2多肽D-型氨基酸替换变体及对人α9α10nAChR的抑制活性Example 8: D-type amino acid substitution variant of Gex-2 polypeptide and its inhibitory activity on human α9α10 nAChR
由于无二硫键的多肽稳定性较差，前期研究表明在多肽链中引人D-型氨基酸可以提高多肽链的稳定性，因此对多肽链中不参与二级结构的残基进行了D-型氨基酸替换扫描，序列如表6所示。Due to the poor stability of polypeptides without disulfide bonds, previous studies have shown that the introduction of D-type amino acids into the polypeptide chain can improve the stability of the polypeptide chain. Type amino acid replacement scan, the sequence is shown in Table 6.
表6：Gex-2多肽的D-型氨基酸替换变体Table 6: D-form amino acid substitution variants of Gex-2 polypeptides
多肽名称Peptide name 多肽序列polypeptide sequence
Gex-61Gex-61 GrYRSPYDRRRRYRRITD-NH 2 GrYRSPYDRRRRYRRITD-NH 2
Gex-62Gex-62 GRyRSPYDRRRRYRRITD-NH 2 GRyRSPYDRRRRYRRITD-NH 2
Gex-63Gex-63 GRYrSPYDRRRRYRRITD-NH 2 GRYrSPYDRRRRYRRITD-NH 2
Gex-64Gex-64 GRYRsPYDRRRRYRRITD-NH 2 GRYRsPYDRRRRYRRITD-NH 2
Gex-65Gex-65 GRYRSPYDRRrRYRRITD-NH 2 GRYRSPYDRRrRYRRITD-NH 2
Gex-66Gex-66 GRYRSPYDRRRrYRRITD-NH 2 GRYRSPYDRRRrYRRITD-NH 2
Gex-67Gex-67 GRYRSPYDRRRRyRRITD-NH 2 GRYRSPYDRRRRyRRITD-NH 2
Gex-68Gex-68 GRYRSPYDRRRRYrRITD-NH 2 GRYRSPYDRRRRYrRITD-NH 2
Gex-69Gex-69 GRYRSPYDRRRRYRrITD-NH 2 GRYRSPYDRRRRYRrITD-NH 2
Gex-70Gex-70 GRYRSPYDRRRRYRRiTD-NH 2 GRYRSPYDRRRRYRRiTD-NH 2
Gex-71Gex-71 GRYRSPYDRRRRYRRItD-NH 2 GRYRSPYDRRRRYRRItD-NH 2
Gex-72Gex-72 GRYRSPYDRRRRYRRITd-NH 2 GRYRSPYDRRRRYRRITd-NH 2
按照实施例2的方法，利用表达α9α10乙酰胆碱受体的非洲爪蟾卵母细胞，检测Gex-2多肽D-型氨基酸替换变体对α9α10乙酰胆碱受体的抑制活性，活性测试结果如图9所示。According to the method of Example 2, using Xenopus oocytes expressing α9α10 acetylcholine receptors, the inhibitory activity of Gex-2 polypeptide D-type amino acid substitution variants on α9α10 acetylcholine receptors was detected, and the activity test results are shown in Figure 9 .
图9表明D-型氨基酸的替代大都能保持多肽活性，R11或R14被D-型取代后会使多肽的活性显著提高。Gex-2在30nM浓度的抑制率约为50％，由上图结果得知：除了2、4号位，其它位置氨基酸构型由L-型转变为D-型后，多肽的活性均一定程度提高。由此推断，Gex-2多个D-型氨基酸类似物的组合有利于提高多肽的活性。同时，基于前期研究(J Med Chem.2020Apr 9；63(7):3475-3484；Mar Drugs.2019Feb 28；17(3):142.)，向芋螺毒素中引入D型氨基酸可以提高其稳定，可以得知Gex-2的D型氨基酸类似物可以保留甚至提高其活性。Figure 9 shows that the substitution of D-type amino acids can maintain the activity of the polypeptide, and the substitution of R11 or R14 by D-type amino acid can significantly improve the activity of the polypeptide. The inhibition rate of Gex-2 at 30nM concentration is about 50%. It can be seen from the results in the above figure: except for positions 2 and 4, after the amino acid configuration of other positions is changed from L-type to D-type, the activity of the polypeptide is all to a certain extent. improve. Therefore, it is inferred that the combination of multiple D-type amino acid analogs of Gex-2 is beneficial to improve the activity of the polypeptide. Meanwhile, based on previous studies (J Med Chem. 2020 Apr 9; 63(7): 3475-3484; Mar Drugs. 2019 Feb 28; 17(3): 142.), the introduction of D-amino acids into conotoxin can improve its stability , it can be known that the D-amino acid analog of Gex-2 can retain or even improve its activity.
实施例9：Gex-2多肽对坐骨神经痛的作用Example 9: Effect of Gex-2 polypeptide on sciatica
1、坐骨神经慢性压迫性损伤(CCI)模型的构建1. Construction of the sciatic nerve chronic compression injury (CCI) model
给大鼠腹腔注射65mg/kg的戊巴比妥钠溶液进行麻醉，约10min大鼠会麻醉完全，肌肉反应消失；75％的酒精浸湿大鼠右下肢手术区域，剔除鼠毛；将大鼠俯卧位置于超净工作台上，铺上无菌洞巾，仅暴露手术部位，避免鼠毛进入伤口引发异物感染。局部皮肤涂抹安尔碘消毒，75％酒精脱碘；用剪刀剪开大鼠的皮肤，使表皮露出1cm左右的口子，钝性分离筋膜和肌肉，即可看到一条白色的较粗的坐骨神经贴在肌肉上，用玻璃分针将其轻轻与筋膜分离，使坐骨神经完全暴露。用4-0医用肠线在坐骨神经上进行四道结扎，每一道之间的间隔控制在1mm，结扎松紧度以大鼠脚趾微微抽搐为宜。结扎结束后用玻璃分针轻轻将坐骨神经拨回原位，还原肌肉位置，并用四万单位的青霉素钠溶液重复冲洗伤口。最后缝合外皮，缝合后在外皮伤口处撒一些青霉素粉末防止大鼠术后感染。手术完成后将大鼠放入保温箱直至大鼠苏醒，待其恢复行动能力后单笼饲养直至伤口愈合。The rats were anesthetized by intraperitoneal injection of 65 mg/kg sodium pentobarbital solution, and the rats were completely anesthetized in about 10 minutes, and the muscle reaction disappeared; the surgical area of the right lower limb of the rats was soaked with 75% alcohol, and the rat hair was removed; The prone position is placed on the ultra-clean workbench, and a sterile hole towel is placed on it, and only the surgical site is exposed to avoid rat hair entering the wound and causing foreign body infection. Apply Aner iodine to the local skin for disinfection, and 75% alcohol for deiodination; cut the skin of the rat with scissors to expose an opening of about 1 cm in the epidermis, bluntly separate the fascia and muscles, and a thick white sciatic nerve can be seen Attach to the muscle and use a glass minute needle to gently separate it from the fascia to fully expose the sciatic nerve. Four ligatures were performed on the sciatic nerve with 4-0 medical catgut, and the interval between each ligature was controlled at 1mm. After the ligation, the sciatic nerve was gently pulled back to its original position with a glass minute needle to restore the muscle position, and the wound was repeatedly washed with 40,000 units of penicillin sodium solution. Finally, the skin was sutured, and some penicillin powder was sprinkled on the skin wound after suture to prevent postoperative infection of the rats. After the operation, the rats were placed in an incubator until the rats were awake, and after they regained their mobility, they were reared in a single cage until the wounds healed.
两周后，CCI大鼠的痛阈会降低到最小值，此时即可进行给药实验。After two weeks, the pain threshold of the CCI rats will decrease to the minimum value, and the drug administration experiment can be carried out at this time.
2假手术大鼠构建2 Sham-operated rat construction
假手术大鼠的造模步骤与CCI大鼠相似，唯一的区别就是假手术大鼠不进行坐骨神经的结扎，仅通过手术暴露坐骨神经，之后直接进行冲洗缝合。假手术大鼠用来模拟大鼠皮肤伤口和内部肌肉撕裂对大鼠痛阈的影响。The modeling steps of the sham-operated rats were similar to those of the CCI rats, the only difference was that the sciatic nerve was not ligated in the sham-operated rats, only the sciatic nerve was exposed by surgery, and then directly flushed and sutured. Sham-operated rats were used to simulate the effects of skin wounds and internal muscle tears on rat pain thresholds.
3单次给药实验中大鼠的分组与给药处理3 Grouping and administration of rats in single administration experiment
单次给药实验主要用来评判Gex-2多肽在单次给药后的镇痛效果、起效到达峰值的时间以及镇痛效应的持续时间。在单次给药实验中，我们设置了对照组、假手术组、生理盐水组、Gex-2组和吗啡组，其中对照组的大鼠不进行任何处理，测得的痛阈是整个测试过程的基线，代表正常大鼠的疼痛阈值；假手术组大鼠用 来模拟外皮伤和肌肉撕裂伤对大鼠疼痛阈值的影响；Gex-2组为实验组；生理盐水组作为阴性对照；吗啡组作为阳性对照。给药方式和给药剂量如表所示：The single-dose experiment was mainly used to evaluate the analgesic effect of Gex-2 polypeptide after a single dose, the time from onset to peak value and the duration of the analgesic effect. In the single-dose experiment, we set up the control group, the sham-operated group, the normal saline group, the Gex-2 group and the morphine group, in which the rats in the control group did not receive any treatment, and the pain threshold measured was the whole test process. The baseline of , represents the pain threshold of normal rats; the sham-operated group was used to simulate the effect of skin injury and muscle tear on the pain threshold of rats; the Gex-2 group was the experimental group; the normal saline group was used as a negative control; morphine group as a positive control. The mode of administration and dosage are shown in the table:
表7单次给药实验中大鼠分组Table 7 Grouping of rats in single-dose experiment
Figure PCTCN2021141818-appb-000005
Figure PCTCN2021141818-appb-000005
给药前0.5h内分别将药物分别溶解/稀释至实验所需浓度，通过原位肌肉注射的方式给药，给药部位在手术侧腿中部的肌肉区域，每个大鼠的给药量控制在200μL。给药完成后，分别在给药后0.5h、1h、2h、4h、6h、12h、24h和48h进行机械痛和热痛痛阈测试，每只大鼠测三次，求均值作为痛阈。The drugs were respectively dissolved/diluted to the concentration required for the experiment within 0.5h before administration, and administered by in situ intramuscular injection. The administration site was in the muscle area of the middle leg of the operation side, and the dose of each rat was controlled. in 200 μL. After the administration was completed, the mechanical pain and thermal pain thresholds were tested at 0.5h, 1h, 2h, 4h, 6h, 12h, 24h and 48h respectively. Each rat was measured three times, and the mean value was taken as the pain threshold.
4、连续长期给药实验中大鼠的分组与给药处理4. Grouping and administration of rats in continuous long-term administration experiments
为了评判了Gex-2多肽在连续给药两周的情况下对于神经性疼痛的缓解作用，观察该过程中镇痛效果的变化。In order to evaluate the relieving effect of Gex-2 polypeptide on neuropathic pain under the condition of continuous administration for two weeks, the changes of analgesic effect during the process were observed.
用作长期镇痛活性测试实验的大鼠共分为六组：对照组、假手术组、生理盐水阴性对照组、GeXIVA阳性对照组、Gex-2低剂量组和Gex-2高剂量组。对照组大鼠不做任何处理，作为空白对照；假手术组大鼠用来评判外皮伤和肌肉撕裂伤对大鼠疼痛阈值的影响；生理盐水组作为阴性对照；Gex-2被分为两组，低剂量组和高剂量组，低剂量组中，每只大鼠的给药量为0.075μg/kg，而高剂量组中每只大鼠的给药量为0.25μg/kg；GeXIVA作为阳性对照，每只大鼠的给药量与Gex-2高剂量组相同，为0.25μg/kg。The rats used for the long-term analgesic activity test experiment were divided into six groups: control group, sham operation group, normal saline negative control group, GeXIVA positive control group, Gex-2 low-dose group and Gex-2 high-dose group. The rats in the control group did not receive any treatment and served as a blank control; the rats in the sham-operated group were used to evaluate the effect of skin injury and muscle laceration on the pain threshold of the rats; the normal saline group was used as a negative control; Gex-2 was divided into two groups. group, low-dose group and high-dose group, in the low-dose group, the dose per rat was 0.075 μg/kg, while in the high-dose group the dose per rat was 0.25 μg/kg; GeXIVA was used as the For the positive control, the dosage of each rat was the same as that of the Gex-2 high-dose group, which was 0.25 μg/kg.
表8大鼠的分组与给药处理Table 8 Grouping and administration of rats
Figure PCTCN2021141818-appb-000006
Figure PCTCN2021141818-appb-000006
每天给药0.5h前用生理盐水将GeXIVA和Gex-2配置到所需浓度，每个大鼠的给药量控制在200μL，均采用原位肌肉注射的方式给药。连续给药14天，分别在每次给药后的1h和24h测试大鼠的机械痛阈值和热痛阈值，每只大鼠测三次，求均值作为测得的痛阈数据。GeXIVA and Gex-2 were prepared to the required concentration with physiological saline 0.5h before administration every day, and the dosage of each rat was controlled at 200 μL, which were administered by in situ intramuscular injection. After continuous administration for 14 days, the mechanical pain threshold and thermal pain threshold of rats were tested at 1h and 24h after each administration, each rat was measured three times, and the mean value was taken as the measured pain threshold data.
5大鼠痛阈的测定5 Determination of Pain Threshold in Rats
大鼠机械痛痛阈测定：将大鼠放入可自由活动的亚克力观察格中，底部是带有网眼的金属架，网眼规格为1.8mm×1.8mm。待大鼠放松安静后，用Electric Von Frey电子测痛仪的探头从金属架的网眼中刺激大鼠足底中部，逐渐增加探头压力，待大鼠由于疼痛将脚从探头移开时，记录仪器所示数字，该数字代表大鼠的机械痛足爪缩回阈值(PWT)，共测试3次，每次测试的时间间隔为10min。Determination of mechanical pain threshold in rats: The rats were placed in a freely movable acrylic observation grid with a metal frame with mesh at the bottom, and the mesh size was 1.8mm×1.8mm. After the rat was relaxed and quiet, the probe of the Electric Von Frey electronic pain meter was used to stimulate the middle of the rat's sole from the mesh of the metal frame, and the pressure of the probe was gradually increased. When the rat removed the foot from the probe due to pain, the instrument was recorded. The numbers shown, which represent the paw withdrawal threshold (PWT) of mechanical pain in rats, were tested 3 times, and the time interval of each test was 10 min.
大鼠热敏痛痛阈测定：将大鼠放入可自由活动的亚克力观察格中，底部是玻璃板。仪器的参数设置如下：工作光强设置为“025”，待机光强设置为“010”，预置时间设置为“20”，预置时间设置为“20”的意义是倘若在足底刺激20s时大鼠仍不出现疼痛反应，光照刺激会即刻停止，防止大鼠脚底烫伤影响后续痛阈测定的准确性。实验前打开仪器开关预热30min，待大鼠在观察格中放松安静后，打开光源刺激大鼠足底中部，大鼠出现如抬脚、舔脚等疼痛反应时，仪器所示数字即为大鼠的热敏痛缩爪阈值，共测试3次，每次测试的时间间隔为10min。Determination of thermal pain threshold in rats: The rats were placed in a freely movable acrylic observation grid with a glass plate at the bottom. The parameter settings of the instrument are as follows: the working light intensity is set to "025", the standby light intensity is set to "010", the preset time is set to "20", and the preset time is set to "20". When the rat still does not have a pain response, the light stimulation will stop immediately to prevent the scalding of the sole of the rat's foot from affecting the accuracy of subsequent pain threshold determination. Before the experiment, turn on the switch of the instrument to preheat for 30 minutes. After the rat is relaxed and quiet in the observation box, turn on the light source to stimulate the middle of the sole of the rat. When the rat has pain reactions such as lifting the foot, licking the foot, etc., the number displayed on the instrument is the big one. The thermal pain paw withdrawal threshold of mice was tested 3 times, and the time interval of each test was 10 min.
6、统计分析6. Statistical analysis
机械痛足爪缩回阈值原始数据单位为g，热敏痛足爪缩回阈值的原始数据单位为s，所有数据均用mean±SEM表示，用GraphPad Prism7.0软件进行统计处理和作图。The original data unit of the mechanical pain paw withdrawal threshold is g, and the original data unit of the thermal pain paw withdrawal threshold is s. All data are expressed as mean ± SEM, and GraphPad Prism7.0 software was used for statistical processing and graphing.
实验结果表明：Experimental results show that:
1、CCI手术对大鼠疼痛阈值的影响1. The effect of CCI surgery on the pain threshold of rats
大鼠在经过CCI手术之后，大约在7-14天后痛阈会降低到最低值。分别测定大鼠术前和术后14天的疼痛阈值，发现术前大鼠机械痛的PWT均值为62.1g，术后14天大鼠的机械痛PWT均值降为17.7g；术前大鼠的热痛PWT均值为19.27s，术后大鼠的热敏痛PWT均值降为9.32s。大鼠手术前后的疼痛阈值存在显著性差异，而且我们可以非常明显的观察到CCI手术后的大鼠出现跛行、后肢不落地或者走路时患肢足外翻的现象，以上均表示该模型建立成功。After CCI surgery in rats, pain threshold will be reduced to the lowest value after about 7-14 days. The pain thresholds of rats before and 14 days after operation were measured respectively, and it was found that the mean PWT of mechanical pain in rats before operation was 62.1 g, and the mean PWT of mechanical pain in rats at 14 days after operation was reduced to 17.7 g; The mean PWT of heat pain was 19.27s, and the mean PWT of heat pain in rats after operation was reduced to 9.32s. There are significant differences in the pain thresholds of rats before and after surgery, and we can clearly observe that the rats after CCI surgery appear lameness, the hind limbs do not fall, or the affected limbs are valgus when walking. The above all indicate that the model was successfully established. .
2、Gex-2单次给药后镇痛活性分析2. Analysis of analgesic activity after single administration of Gex-2
Gex-2单次给药后测得的机械痛PWT如图10所示：在图10中，横坐标为时间，单位为小时(h)，纵坐标是大鼠足底机械痛PWT，单位为克(g)。蓝色为对照组大鼠足底机械痛PWT值，红色为假手术组，紫色为Gex-2组，橙色为吗啡组，绿色为生理盐水组。折线上方的紫色和橙色标志分别代表在该时间点Gex-2组和吗啡组与生理盐水组之间的差异显著性水平。The mechanical pain PWT measured after a single dose of Gex-2 is shown in Figure 10: In Figure 10, the abscissa is time, the unit is hour (h), the ordinate is the rat plantar mechanical pain PWT, the unit is grams (g). Blue is the PWT value of plantar mechanical pain in the control group, red is the sham-operated group, purple is the Gex-2 group, orange is the morphine group, and green is the normal saline group. The purple and orange markers above the broken line represent the significance level of the difference between the Gex-2 group and the morphine group and the saline group, respectively, at that time point.
由图10可知，正常大鼠(对照组)的机械痛PWT在65g左右，与前期实验测得的结果一致；假手术组大鼠的PWT与对照组基本持平，说明手术造成的皮肤伤口和肌肉撕裂伤对CCI大鼠的足底机械痛阈变化没有造成影响，可能是因为实验开展前的两周内，大鼠的皮肤伤和肌肉伤得到了恢复；生理盐水组的数据与前期测得的CCI大鼠的痛阈相比没有产生明显变化；而吗啡在CCI大鼠上产生了良好的镇痛作用，自注射起很快起效，到2h时镇痛作用达到峰值，接近正常大鼠的痛阈，2h后开始下降，药效总共可持续约12h，24h时吗啡几乎已失效，不再发挥镇痛作用；而对于我们的多肽Gex-2，则表现出优于吗啡的镇痛效果，起效时间与吗啡相似，且同样在2h时达到峰值，达峰值时的PWT与正 常大鼠的PWT一致，说明它具有很强的镇痛作用，随后药效以极其缓慢的方式降低，直至48h依然可以与正常大鼠的疼痛阈值持平。说明Gex-2可能通过多种在体内发挥作用，在拮抗α9α10nAChR发挥镇痛作用后，可能会通过抗炎作用来发挥持续药效。It can be seen from Figure 10 that the mechanical pain PWT of the normal rats (control group) is about 65g, which is consistent with the results measured in the previous experiments; the PWT of the sham-operated rats is basically the same as that of the control group, indicating that the skin wounds and muscles caused by the surgery are Laceration had no effect on the changes of plantar mechanical pain threshold in CCI rats, probably because the skin and muscle injuries of the rats were recovered within two weeks before the experiment; There was no significant change in the pain threshold of the CCI rats compared with 100%; while morphine produced a good analgesic effect on the CCI rats, and it took effect soon after the injection, and the analgesic effect reached its peak at 2h, which was close to the normal rat. After 2 hours, the pain threshold began to decrease, and the drug effect lasted for about 12 hours. At 24 hours, morphine was almost ineffective and no longer exerted analgesic effect. For our polypeptide Gex-2, it showed better analgesic effect than morphine. , the onset time is similar to that of morphine, and the peak value is also reached at 2h. The PWT at the peak is consistent with the PWT of normal rats, indicating that it has a strong analgesic effect, and then the drug effect decreases in an extremely slow manner until 48h can still be equal to the pain threshold of normal rats. It indicates that Gex-2 may exert various effects in vivo. After antagonizing α9α10nAChR to exert analgesic effect, it may exert a sustained drug effect through anti-inflammatory effect.
Gex-2单次给药后测得的热痛PWT如图11所示：在图11中，横坐标为时间，单位为小时(h)，纵坐标是大鼠足底热痛PWT，单位为秒(s)。蓝色为对照组大鼠足底热痛PWT值，红色为假手术组，紫色为Gex-2组，橙色为吗啡组，绿色为生理盐水组。折线上方的紫色和橙色标志分别代表在该时间点Gex-2组和吗啡组与生理盐水组之间的差异显著性水平。The thermal pain PWT measured after a single administration of Gex-2 is shown in Figure 11: In Figure 11, the horizontal axis is time, the unit is hour (h), the vertical axis is the rat plantar thermal pain PWT, the unit is second(s). The blue is the PWT value of plantar heat pain in the control group, the red is the sham-operated group, the purple is the Gex-2 group, the orange is the morphine group, and the green is the normal saline group. The purple and orange markers above the broken line represent the significance level of the difference between the Gex-2 group and the morphine group and the saline group, respectively, at that time point.
在做动物行为学实验时，由于不同因素的干扰可能会导致数据波动从而使数据误差较大，但是在图11中我们仍可以观察到不同组别之间的显著差异。由图10可知，对照组和假手术组大鼠的热痛PWT都接近20s；生理盐水对照组大鼠的PWT均值不超过10s；吗啡在6h内可以使CCI大鼠的痛阈回复到正常阈值，6h后作用逐渐下降，24h时已几乎失效，48h时完全失去镇痛活性；而Gex-2可以使更有效的缓解CCI大鼠的神经痛，使大鼠48h内的热痛PWT始终与正常大鼠保持一致。When doing animal behavior experiments, the data may fluctuate due to the interference of different factors, resulting in large data errors, but we can still observe significant differences between different groups in Figure 11. It can be seen from Figure 10 that the thermal pain PWT of the rats in the control group and the sham-operated group is close to 20s; the average PWT of the rats in the saline control group does not exceed 10s; morphine can restore the pain threshold of the CCI rats to the normal threshold within 6h , the effect gradually decreased after 6h, almost ineffective at 24h, and completely lost its analgesic activity at 48h; while Gex-2 can more effectively relieve neuralgia in CCI rats, so that the heat pain PWT in rats within 48h is always the same as normal. Rats remain consistent.
综上，与5mg/kg的吗啡相比，0.25μg/kg的Gex-2在CCI模型上表现出了更强更持久的镇痛作用。0.25μg/kg的Gex-2可以有效的缓解CCI大鼠的坐骨神经痛，使大鼠患肢的足底PWT接近正常大鼠足底PWT，Gex-2起效很快，镇痛作用约在2h到达峰值，药效可持续至少48h。In conclusion, compared with 5 mg/kg morphine, 0.25 μg/kg Gex-2 exhibited stronger and longer-lasting analgesic effects in the CCI model. 0.25μg/kg of Gex-2 can effectively relieve sciatica in CCI rats, making the plantar PWT of the affected limb of the rat close to the plantar PWT of the normal rat. Gex-2 takes effect quickly, and the analgesic effect is about 2h Reaching the peak, the drug effect can last for at least 48 hours.
实施例10：Gex-2多肽的急性镇痛活性实验Example 10: Acute analgesic activity test of Gex-2 polypeptide
对GeXIVA组、Gex-2组和生理盐水组进行连续14天的肌肉注射给药，在每次给药后1h测试大鼠的机械痛和热痛疼痛阈值，并进行了作图分析。The GeXIVA group, the Gex-2 group and the normal saline group were administered intramuscularly for 14 consecutive days, and the pain thresholds of mechanical pain and thermal pain were tested 1 h after each administration, and the graphs were analyzed.
给药1h后测得的大鼠足底机械痛PWT的结果如图12所示：图中，横坐标是天数，纵坐标是大鼠足底机械痛PWT，单位是克(g)。蓝色为对照组大鼠足底机械痛PWT值，红色为假手术组，橙色为0.25μg/kg Gex-2组，紫色为0.075μg/kg Gex-2组，黑色为GeXIVA组，绿色为生理盐水组。折线上方的橙色、紫色和黑色标志分别代表对应组别与生理盐水组之间的差异显著性水平。Figure 12 shows the results of PWT of mechanical plantar pain in rats measured 1 h after administration: in the figure, the abscissa is the number of days, and the ordinate is the PWT of mechanical plantar pain of rats, and the unit is grams (g). Blue is the PWT value of plantar mechanical pain in the control group, red is the sham-operated group, orange is the 0.25 μg/kg Gex-2 group, purple is the 0.075 μg/kg Gex-2 group, black is the GeXIVA group, and green is the physiological group saline group. The orange, purple and black marks above the broken line represent the significance level of the difference between the corresponding group and the normal saline group, respectively.
由图12可知，对照组和假手术组大鼠的痛阈均在60g左右，而生理盐水组大鼠的痛阈降低至20g，说明皮肤伤口和肌肉伤并没有对大鼠的痛阈造成明显的影响，大鼠痛阈的降低主要是由于结扎后对坐骨神经的慢性压迫导致的。当给CCI大鼠注射0.075μg/kg的Gex-2时，大鼠的机械痛阈值显著提高，提高幅度与0.25μg/kg的阳性对照GeXIVA类似，说明Gex-2比阳性对照GeXIVA的镇痛作用更强；而当Gex-2的剂量为0.25μg/kg时，大鼠的机械痛阈值几乎可以恢复到与正常大鼠一致的水平。我们还观察到，在连续注射药物的过程中，大鼠跛行和足外翻的行为都得到了明显的缓解。以上结果说明，在极低剂量下，Gex-2就具有非常好的镇痛作用，并且这种作用具有剂量一定的依赖性，以同一剂量连续注射14天，大鼠的痛阈几乎都与第一次给药时一致，并且在14天内大鼠的饮食饮水都正常，行为也没有出现明显变化，Gex-2给药组大鼠的体重也与对照组接近，说明大鼠未对该化合物产生明显依赖性。It can be seen from Figure 12 that the pain thresholds of the rats in the control group and the sham-operated group were both around 60g, while the pain thresholds of the rats in the saline group were reduced to 20g, indicating that skin wounds and muscle injuries did not significantly affect the pain thresholds of the rats. The decrease of pain threshold in rats was mainly caused by chronic compression of sciatic nerve after ligation. When 0.075 μg/kg of Gex-2 was injected into CCI rats, the mechanical pain threshold of the rats was significantly increased, and the increase was similar to that of the positive control GeXIVA at 0.25 μg/kg, indicating that Gex-2 was more analgesic than the positive control GeXIVA. stronger; and when the dose of Gex-2 was 0.25μg/kg, the mechanical pain threshold of rats could almost recover to the level consistent with that of normal rats. We also observed that both lameness and foot eversion behaviors were significantly relieved in the rats during continuous drug injection. The above results show that Gex-2 has a very good analgesic effect at very low doses, and this effect is dose-dependent. With the same dose of continuous injection for 14 days, the pain threshold of rats is almost the same as the first. It was the same at the time of one administration, and within 14 days, the rats' diet and drinking were normal, and there was no obvious change in behavior. The body weight of the rats in the Gex-2 administration group was also similar to that of the control group, indicating that the rats did not produce the compound. obvious dependence.
给药后1h大鼠热痛PWT的结果如图13所示：如图13所示，横坐标是天数，大鼠连续肌肉注射给药14天；纵坐标即为大鼠热痛PWT，单位是秒(s)。蓝色为对照组大鼠足底机械痛PWT值，红色为假手术组，橙色为0.25μg/kg Gex-2组，紫色为0.075μg/kg Gex-2组，黑色为GeXIVA组，绿色为生理盐水组。折线上方的橙色、紫色和黑色标志分别代表对应组别与生理盐水组之间的差异显著性水平。The results of PWT of rat heat pain 1 h after administration are shown in Figure 13: as shown in Figure 13, the abscissa is the number of days, and the rats were given continuous intramuscular injection for 14 days; the ordinate is the heat pain PWT of the rat, the unit is second(s). Blue is the PWT value of plantar mechanical pain in the control group, red is the sham-operated group, orange is the 0.25 μg/kg Gex-2 group, purple is the 0.075 μg/kg Gex-2 group, black is the GeXIVA group, and green is the physiological group saline group. The orange, purple and black marks above the broken line represent the significance level of the difference between the corresponding group and the normal saline group, respectively.
由图13可知，对照组和假手术组大鼠的痛阈均在17-20s之间，而生理盐水组大鼠的痛阈降低至10s以下，说明皮肤伤口和肌肉伤并没有对大鼠的热痛敏感性造成明显的影响，大鼠痛阈的降低主要是由于结扎后对坐骨神经的慢性压迫导致的。当给CCI大鼠注射0.3nM的Gex-2或0.25μg/kg的GeXIVA时，大鼠的热痛阈值均显著提高，虽无法使大鼠的痛阈恢复到正常水平，且由于个体差异造成了数据较大的波动，但两组与生理盐水组都产生了明显差异。而0.25μg/kg的Gex-2产生了更强的镇痛作用，可以把CCI大鼠的热痛PWT稳定提高到正常水平。以上结果说明，在极低剂量下，Gex-2就具有非常好的镇痛作用，并且连续14天给药不会诱导大鼠产生依赖性。As can be seen from Figure 13, the pain thresholds of the rats in the control group and the sham-operated group were between 17-20s, while the pain thresholds of the rats in the normal saline group decreased to less than 10s, indicating that skin wounds and muscle injuries did not affect the rats. Thermal pain sensitivity has a significant effect, and the decrease in pain threshold in rats is mainly caused by chronic compression of the sciatic nerve after ligation. When CCI rats were injected with 0.3nM Gex-2 or 0.25μg/kg GeXIVA, the thermal pain thresholds of the rats were significantly increased, although the pain thresholds of the rats could not be restored to the normal level, and due to individual differences The data fluctuated greatly, but there were significant differences between the two groups and the saline group. And 0.25μg/kg Gex-2 produced stronger analgesic effect, which could stably increase the heat pain PWT of CCI rats to normal level. The above results show that Gex-2 has a very good analgesic effect at very low doses, and administration for 14 consecutive days does not induce dependence in rats.
综上，通过对CCI大鼠给药1h后PWT的测试，可以看出Gex-2在低剂量下就对该模型具有非常好的镇痛作用，无论对大鼠的机械痛增敏还是热痛增敏都具有明显的缓解效果。Gex-2在0.075μg/kg的用药剂量下产生的镇痛效果与 0.25μg/kg的GeXIVA类似，说明该化合物的镇痛活性比GeXIVA更为优越；在0.25μg/kg的用药剂量下几乎可以使CCI大鼠的机械痛PWT恢复到术前阈值，并显著改善大鼠跛行和足外翻的情况，长达14天的用药也并没有使大鼠产生药物依赖。In conclusion, through the test of PWT after 1 h of administration to CCI rats, it can be seen that Gex-2 has a very good analgesic effect on the model at low doses, no matter on the mechanical hyperalgesia or thermal pain in rats. Sensitization has obvious relief effect. The analgesic effect of Gex-2 at a dose of 0.075 μg/kg was similar to that of GeXIVA at 0.25 μg/kg, indicating that the analgesic activity of the compound was superior to that of GeXIVA; at a dose of 0.25 μg/kg, it was almost The mechanical pain PWT of CCI rats was restored to the preoperative threshold, and the claudication and foot valgus of the rats were significantly improved, and the drug did not cause drug dependence in the rats for up to 14 days.
实施例11：Gex-2多肽的长效镇痛实验Example 11: Long-acting analgesic experiment of Gex-2 polypeptide
为测试Gex-2的药效是否可以维持较长久的时间，从而对慢性疼痛起到更好的作用，我们对GeXIVA组、Gex-2组和生理盐水组进行连续14天的肌肉注射给药，在每次给药后24h测试大鼠的机械痛和热痛PWT，并进行了作图分析。In order to test whether the efficacy of Gex-2 can be maintained for a longer period of time and thus have a better effect on chronic pain, we administered intramuscular injections to the GeXIVA group, the Gex-2 group and the normal saline group for 14 consecutive days. Rats were tested for mechanical pain and thermal pain PWT 24 h after each administration, and a graph analysis was performed.
给药后24h后大鼠足底机械痛PWT结果如图14所示：如图14所示，横坐标为时间，单位为天；纵坐标是大鼠的机械痛PWT，单位为克(g)。蓝色为对照组大鼠足底机械痛PWT值，红色为假手术组，橙色为0.25μg/kg Gex-2组，紫色为0.075μg/kg Gex-2组，黑色为GeXIVA组，绿色为生理盐水组。折线上方的橙色、紫色和黑色标志分别代表对应组别与生理盐水组之间的差异。Figure 14 shows the PWT results of mechanical pain in the plantar of the rat 24 hours after administration: as shown in Figure 14, the abscissa is the time, the unit is day; the ordinate is the mechanical pain PWT of the rat, the unit is grams (g) . Blue is the PWT value of plantar mechanical pain in the control group, red is the sham-operated group, orange is the 0.25 μg/kg Gex-2 group, purple is the 0.075 μg/kg Gex-2 group, black is the GeXIVA group, and green is the physiological group saline group. The orange, purple and black marks above the broken line represent the difference between the corresponding group and the saline group, respectively.
由图14可知，在连续两周的给药过程中，0.25μg/kg的GeXIVA在每次给药24h后仍然发挥显著的镇痛作用，且第二周测得的大鼠PWT均值较第一周更高，说明GeXIVA展现出了一定的药物累积作用。对于我们的化合物Gex-2，当以0.075μg/kg的剂量连续给药时，发挥的镇痛效果与0.25μg/kg的GeXIVA相当，每次给药24h后仍可以发挥镇痛作用，大鼠的PWT仍然与生理盐水组有显著的差异；而当Gex-2以0.25μg/kg的剂量连续给药时，每次给药24h后的镇痛效果仍可以使CCI大鼠的疼痛阈值恢复到与正常大鼠一致的水平，相较于给药1h后的镇痛效果无明显减弱，说明0.25μg/kg的Gex-2具有强效持久的镇痛作用，且Gex-2给药组大鼠第二周的PWT均值较第一周更高，说明Gex-2发挥药效也具有累积效应。除此之外，在连续14天的给药过程中，大鼠的步态逐渐趋于正常大鼠，跛行和足外翻的现象得到了明显的改善，说明大鼠的痛感得到了显著的缓解。It can be seen from Figure 14 that during the two consecutive weeks of administration, 0.25 μg/kg of GeXIVA still exerted a significant analgesic effect 24 hours after each administration, and the mean PWT of the rats measured in the second week was higher than that of the first one. Weekly higher, indicating that GeXIVA showed a certain drug accumulation effect. For our compound Gex-2, when administered continuously at a dose of 0.075 μg/kg, the analgesic effect was comparable to that of GeXIVA at 0.25 μg/kg, and it could still exert analgesic effect 24 h after each administration. Rats The PWT was still significantly different from the normal saline group; and when Gex-2 was continuously administered at a dose of 0.25 μg/kg, the analgesic effect after 24 hours of each administration could still restore the pain threshold of CCI rats to Compared with the level of normal rats, the analgesic effect was not significantly weakened after administration for 1 h, indicating that 0.25 μg/kg of Gex-2 has a strong and durable analgesic effect, and the rats in the Gex-2 administration group The mean value of PWT in the second week was higher than that in the first week, indicating that Gex-2 also had a cumulative effect on its efficacy. In addition, during the 14 consecutive days of administration, the gait of the rats gradually tended to be normal, and the phenomenon of lameness and foot eversion were significantly improved, indicating that the pain of the rats was significantly relieved. .
给药后24h后大鼠足底热痛PWT结果如图15所示：如图15所示，横坐标为时间，单位为天；纵坐标是大鼠的热痛PWT，单位为秒(s)。蓝色为对照组大鼠足底热痛PWT值，红色为假手术组，橙色为0.25μg/kg Gex-2组，紫色为 0.075μg/kg Gex-2组，黑色为GeXIVA组，绿色为生理盐水组。折线上方的橙色、紫色和黑色标志分别代表对应组别与生理盐水组之间的差异显著性水平。Figure 15 shows the PWT results of plantar heat pain in rats 24 hours after administration: as shown in Figure 15, the abscissa is time, the unit is day; the ordinate is the heat pain PWT of the rat, the unit is second (s) . The blue is the PWT value of plantar heat pain in the control group, the red is the sham operation group, the orange is the 0.25μg/kg Gex-2 group, the purple is the 0.075μg/kg Gex-2 group, the black is the GeXIVA group, and the green is the physiological group saline group. The orange, purple and black marks above the broken line represent the significance level of the difference between the corresponding group and the normal saline group, respectively.
由图15可知，在连续给药14天的情况下，对于GeXIVA和不同剂量的Gex-2来说，每次给药24h后的热痛PWT与给药1h后的数据保持了很好的一致性，均与生理盐水组有显著性差异，这说明两者的镇痛作用在24h内均不会衰减，具有长效镇痛作用。As can be seen from Figure 15, in the case of continuous administration for 14 days, for GeXIVA and different doses of Gex-2, the thermal pain PWT after 24 hours of each administration is in good agreement with the data after 1 hour of administration. There were significant differences with the normal saline group, which indicated that the analgesic effects of the two groups would not be attenuated within 24 hours, and had long-acting analgesic effects.
综上，不同剂量的Gex-2在给药24h后仍可以对大鼠机械痛和热痛增敏有显著的缓解作用，并且活性较给药1h后并没有出现明显降低，说明该多肽在大鼠体内的活性可以维持至少24h。To sum up, different doses of Gex-2 can still significantly relieve mechanical pain and thermal pain sensitization in rats 24 hours after administration, and the activity did not decrease significantly compared with 1 hour after administration, indicating that the polypeptide is in the large The activity in mice can be maintained for at least 24h.
实施例12：Gex-2多肽对三叉神经痛活性评价实验Example 12: Activity evaluation experiment of Gex-2 polypeptide on trigeminal neuralgia
Gex-2在三叉神经疼痛大鼠模型上的镇痛活性研究发现Gex-2在0.25μg/kg剂量下通过腹腔注射用药，可显著降低三叉神经疼痛(图16)。Gex-2的使用既不影响动物的运动状态，也不影响动物的平衡能力。The analgesic activity of Gex-2 in a rat model of trigeminal neuralgia found that Gex-2 administered by intraperitoneal injection at a dose of 0.25 μg/kg could significantly reduce trigeminal neuralgia (Figure 16). The use of Gex-2 neither affected the animal's motor status nor the animal's balance ability.
实施例13：Gex-2多肽对骨癌疼痛评价实验Example 13: Evaluation experiment of Gex-2 polypeptide on bone cancer pain
按照下表9的方式进行骨癌疼痛评价实验，分别在给药1小时和24小时候测试结果，如图17-18所示，结果表明：(1)Gex-2起到了很好的镇痛作用，具有统计学意义，但给药24h后活性相对于给药1h后的活性有所降低；The bone cancer pain evaluation experiment was carried out according to the following table 9, and the results were tested at 1 hour and 24 hours after administration, respectively, as shown in Figures 17-18. The results showed that: (1) Gex-2 played a good analgesic effect. , with statistical significance, but the activity after 24 hours of administration was lower than that after 1 hour of administration;
(2)如文献所述，CIBP模型大鼠痛阈降低的持续时间在25天左右，25天后大鼠由于自身免疫因子的产生消灭部分癌细胞，从而导致痛阈逐渐恢复；(2) As described in the literature, the duration of pain threshold reduction in CIBP model rats was about 25 days, and after 25 days, the rats eliminated some cancer cells due to the production of autoimmune factors, resulting in gradual recovery of pain threshold;
(3)Gex-2并没有像在CCI模型中那样，在CIBP模型中表现出累积效应。(3) Gex-2 did not show a cumulative effect in the CIBP model as in the CCI model.
实施例14：Gex-2多肽的条件性位置偏爱与成瘾性评判实验Example 14: Conditioned Place Preference and Addiction Judgment Experiment of Gex-2 Polypeptides
1、条件性位置偏爱实验1. Conditioned place preference experiment
条件性位置偏爱实验采用的装置为三箱CPP系统，左箱内部全为黑色，右箱内侧为黑白等距间隔的条纹，两箱大小均为30cm×25cm×30cm，中间箱内侧为灰白色，大小为30cm×10cm×30cm，中间箱与左右两箱之间各有穿梭门一个，为10cm×10cm的圆拱形洞口，穿梭门可通过手动移动隔板来控制开关。进行 CPP实验时，要保证光线柔和，周围无噪声，整个实验分为预处理阶段、训练阶段和测试阶段三个阶段进行：The device used in the conditioned place preference experiment is a three-box CPP system. The inside of the left box is all black, and the inside of the right box is black and white with equally spaced stripes. It is 30cm x 10cm x 30cm. There is a shuttle door between the middle box and the left and right boxes, which is a 10cm x 10cm round arch hole. The shuttle door can be controlled by manually moving the partition. When conducting CPP experiments, it is necessary to ensure that the light is soft and there is no noise around. The whole experiment is divided into three stages: preprocessing stage, training stage and testing stage:
预处理阶段：待大鼠适应环境后的第1天，取走CPP箱的隔板使穿梭门打开，大鼠由中间箱进入，任大鼠在三箱内自由穿梭15min，此时不记录数据。第2-3天，记录下大鼠在15min内分别在每个箱体里的停留时间，取两天实验数据的平均值，选取带有不明显偏好性的大鼠进行后续实验，在后续实验中，以大鼠的非偏爱箱作为伴药箱。Pretreatment stage: On the first day after the rats adapt to the environment, the partition of the CPP box is removed to open the shuttle door, the rats enter from the middle box, and the rats are allowed to shuttle freely in the three boxes for 15 minutes, and no data is recorded at this time. . On days 2-3, record the stay time of the rats in each box within 15 minutes, take the average of the two-day experimental data, and select rats with no obvious preference for follow-up experiments. , the rat's non-preferred box was used as the companion box.
训练阶段：将大鼠分为四组，分别是以黑箱为伴药箱的吗啡给药组、以黑白箱为伴药箱的吗啡给药组、以黑箱为伴药箱的Gex-2给药组和以黑白箱为伴药箱的Gex-2给药组，每组6只。训练阶段的第1/3/4/7天每天上午9:30和第2/4/6/8天每天下午15:30，吗啡组和Gex-2组分别肌肉注射对应药物。训练过程中，吗啡组的给药量由5mg/kg逐渐增加至10mg/kg，Gex-2组剂量保持在1nM，对照组注射生理盐水，药物注射量为200μL/只，药物注射完成立即放入伴药箱让大鼠自由活动40min。而第1/3/4/7天每天下午15:30和第2/4/6/8天每天上午9:30，三组分别肌肉注射等量生理盐水，放入非伴药箱让大鼠自由活动40min。Training stage: The rats were divided into four groups, which were the morphine administration group with the black box as the medicine box, the morphine administration group with the black and white box as the medicine box, and the Gex-2 administration group with the black box as the medicine box group and Gex-2 administration group with black and white box as medicine box, 6 animals in each group. On the 1st/3rd/4th/7th day of the training phase, at 9:30 am every day and on the 2nd/4/6/8th day at 15:30 pm, the morphine group and the Gex-2 group were injected intramuscularly with the corresponding drugs. During the training process, the dosage of morphine group was gradually increased from 5 mg/kg to 10 mg/kg, the dosage of Gex-2 group was kept at 1 nM, and the control group was injected with normal saline, and the drug injection volume was 200 μL per animal. The rats were allowed to move freely for 40 min with the medicine box. On the 1st/3rd/4th/7th day at 15:30 pm and on the 2nd/4/6/8th day at 9:30am, the three groups were injected intramuscularly with the same amount of normal saline, and put the rats into the non-accompanied medicine box. 40 minutes of free time.
测试阶段：训练完成后的第一天，进行大鼠CPP score的测试。具体方法为：取出CPP箱的隔板使穿梭门打开，将大鼠由中间箱放入，使其在不同箱体之间自由穿梭，记录15min内大鼠在不同箱体中待的时间，观察大鼠偏好性的变化；训练结束后的第2天，每只大鼠注射0.01mg/kg纳洛酮，促使戒断反应的发生，再将大鼠由中间箱放入，使其在不同箱体之间自由穿梭，记录15min内大鼠在不同箱体中待的时间，观察大鼠偏好性的变化。测试过程中，实验人员执行双盲原则。Test phase: On the first day after the training is completed, the rat CPP score test is performed. The specific method is as follows: take out the partition of the CPP box to open the shuttle door, put the rat in the middle box to make it shuttle freely between different boxes, record the time the rat stays in the different boxes within 15 minutes, and observe the Changes in the preference of rats; on the second day after training, each rat was injected with 0.01 mg/kg naloxone to induce withdrawal reactions, and then the rats were placed in the middle box to make them in different boxes. Freely shuttle between the bodies, record the time that the rats stay in different boxes within 15 minutes, and observe the changes in the preference of the rats. During the test, the experimenter performed the double-blind principle.
2 Gex-2的成瘾性评判2 The addiction evaluation of Gex-2
成瘾性是临床常使用的阿片类镇痛药物的一个潜在副作用，药物成瘾会严重危害患者的身体健康和精神健康。前面的研究已表明，Gex-2在大鼠CCI模型上表现出强于吗啡的镇痛效果，因此我们非常关心Gex-2是否具备成瘾性。Addiction is a potential side effect of commonly used opioid analgesics, and drug addiction can seriously endanger the physical and mental health of patients. Previous studies have shown that Gex-2 has a stronger analgesic effect than morphine in the rat CCI model, so we are very concerned about whether Gex-2 is addictive.
我们选用了CPP模型来评判Gex-2的成瘾性。选取具有不同偏好性的大鼠分为6组，每组均以“注射药物-伴药箱颜色”进行命名，分别是生理盐水-黑组、 生理盐水-黑白组、吗啡-黑组、吗啡-黑白组、Gex2-黑组和Gex2-黑白组，6组均以大鼠的非天然偏爱箱作为伴药箱，进行了为期八天的训练。每一组都在第1/3/5/7天的上午9:30和第2/4/6/8天的下午15:30注射药物，置于伴药箱40min，在第1/3/5/7天的下午15:30和第2/4/6/8天的上午9:30注射等量生理盐水，置于非伴药箱40min。训练结束后第一天让大鼠在CPP箱内自由穿梭，记录15min的数据，第二天给大鼠注射0.01mg/kg纳洛酮，10min后将大鼠置于CPP箱中，记录15min内的数据。得到的结果如15图9所示：We chose the CPP model to judge the addictiveness of Gex-2. Rats with different preferences were selected and divided into 6 groups, and each group was named after "injection drug-accompanying medicine box color", namely normal saline-black group, normal saline-black and white group, morphine-black group, and morphine-black group. The black and white group, the Gex2-black group and the Gex2-black and white group, all 6 groups were trained for eight days with the rat's unnatural preference box as the companion medicine box. Each group was injected with drugs at 9:30 am on the 1/3/5/7 day and 15:30 pm on the 2/4/6/8 day, placed in the companion medicine box for 40 minutes, and on the 1/3/ The same amount of normal saline was injected at 15:30 pm on the 5th/7th day and at 9:30am on the 2nd/4th/6/8th day, and placed in the non-accompanied medicine box for 40min. On the first day after the training, the rats were allowed to shuttle freely in the CPP box, and the data was recorded for 15 minutes. On the second day, the rats were injected with 0.01 mg/kg naloxone. After 10 minutes, the rats were placed in the CPP box and recorded within 15 minutes. The data. The results obtained are shown in Figure 15:
如图19所示，纵坐标是大鼠在伴药箱的时间占总时间的比例，比例越高，说明大鼠在伴药箱待的时间越长，大鼠就产生了越强的偏好性。Natural Preference(白色)指的是实验前大鼠的自然偏好性，由于我们选择的伴药箱是大鼠的天然非偏爱箱，所以Natural Preference组数据均小于50％。随后是8天的持续给药训练，该过程中生理盐水组和Gex-2组大鼠行为正常，而吗啡组大鼠逐渐出现了毒品成瘾的症状，例如时而狂躁时而萎靡、给药后异常兴奋、动作幅度夸张等行为。经过八天连续给药训练后，停药第一天测得的CPP结果如CPP result(浅灰色)所示，由上图可知，生理盐水组和Gex-2组均没有改变大鼠的自然偏好性，而吗啡组则逆转了大鼠的自然偏好，使大鼠更喜欢在伴药箱探索，说明大鼠对吗啡已形成依赖性，而Gex-2同生理盐水一样不会导致大鼠成瘾。注射纳洛酮后测得的数据(深灰色)与停药第一天测得的CPP数据一致，且吗啡组大鼠在注射纳洛酮后，出现了明显的焦虑样行为，而Gex-2和生理盐水组大鼠正常，这进一步验证了吗啡的成瘾性，同时也说明Gex-2不具有导致动物药物成瘾的副作用。As shown in Figure 19, the ordinate is the ratio of the time the rat spends in the companion medicine box to the total time. The higher the ratio, the longer the time the rat spends in the medicine companion box, the stronger the preference for the rat. . Natural Preference (white) refers to the natural preference of the rats before the experiment. Since the medicine box we chose is the natural non-preference box of the rats, the data of the Natural Preference group are all less than 50%. This was followed by 8 days of continuous administration training. During this process, the rats in the saline group and Gex-2 group behaved normally, while the rats in the morphine group gradually developed symptoms of drug addiction, such as mania and malaise, and abnormal post-administration. Excited, exaggerated range of motion, etc. After eight days of continuous administration training, the CPP results measured on the first day of drug withdrawal are shown in the CPP result (light gray). As can be seen from the above figure, neither the normal saline group nor the Gex-2 group changed the natural preference of the rats The morphine group reversed the natural preference of the rats and made the rats more like to explore in the companion medicine box, indicating that the rats had become dependent on morphine, and Gex-2 did not cause the rats to become addicted like normal saline. . The data measured after naloxone injection (dark gray) were consistent with the CPP data measured on the first day of drug withdrawal, and the rats in the morphine group showed obvious anxiety-like behavior after naloxone injection, while Gex-2 The rats in the normal saline group and the rats in the normal saline group were normal, which further verified the addictiveness of morphine, and also indicated that Gex-2 did not have the side effects of drug addiction in animals.
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited to this. Substitutions should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims (31)

  1. 一种活性多肽，其特征在于与α-芋螺毒素活性单体肽相比具有一个或多个半胱氨酸残基的删除或缺失突变；An active polypeptide characterized by having a deletion or deletion mutation of one or more cysteine residues compared to the α-conotoxin active monomer peptide;
    其中，所述α-芋螺毒素活性单体肽具有结合nAChR的功能。Wherein, the α-conotoxin active monomer peptide has the function of binding nAChR.
  2. 如权利要求1所述活性多肽，其特征在于所述α-芋螺毒素活性单体肽包括至少三个连续的精氨酸残基，并具有1个、2个、3个、4个或5个半胱氨酸残基。The active polypeptide of claim 1, wherein the α-conotoxin active monomer peptide comprises at least three consecutive arginine residues, and has 1, 2, 3, 4 or 5 a cysteine residue.
  3. 如权利要求1所述活性多肽，其特征在于所述α-芋螺毒素活性单体肽包括α-芋螺毒素天然单体肽，以及在α-芋螺毒素天然单体肽氨基酸序列的基础上缺失C端和/或N端的一个或数个氨基酸残基。The active polypeptide of claim 1, wherein the α-conotoxin active monomer peptide comprises a natural α-conotoxin monomer peptide, and is based on the amino acid sequence of the α-conotoxin natural monomer peptide One or several amino acid residues at the C-terminus and/or the N-terminus are deleted.
  4. 如权利要求3所述活性多肽，其特征在于所述α-芋螺毒素天然单体肽来源于选自下组的α-芋螺毒素，GeXIVA、GeXXVIIA、Vc1.1、PeIA、RgIA、B-VxXXIVA、S-GVIIIB、D-GeXXA、O-GeXXVIIA、D--Lt28.1、Mr1.1、BuIA、ImI、或AuIB；所述α-芋螺毒素天然单体肽包括α-芋螺毒素的成熟肽单体、前体肽单体。The active polypeptide of claim 3, wherein the α-conotoxin natural monomer peptide is derived from α-conotoxin selected from the group consisting of GeXIVA, GeXXVIIA, Vc1.1, PeIA, RgIA, B- VxXXIVA, S-GVIIIB, D-GeXXA, O-GeXXVIIA, D--Lt28.1, Mr1.1, BuIA, ImI, or AuIB; the α-conotoxin natural monomeric peptides include the Mature peptide monomer, precursor peptide monomer.
  5. 如权利要求3所述活性多肽，其特征在于所述α-芋螺毒素天然单体肽包括SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列。The active polypeptide of claim 3, wherein the α-conotoxin natural monomer peptide comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  6. 如权利要求1所述活性多肽，其特征在于所述活性多肽不含有半胱氨酸残基、不形成分子内二硫键或分子间二硫键，且其氨基酸残基数>10。The active polypeptide according to claim 1, characterized in that the active polypeptide does not contain cysteine residues, does not form intramolecular disulfide bonds or intermolecular disulfide bonds, and has more than 10 amino acid residues.
  7. 如权利要求1所述活性多肽，其特征在于所述活性多肽包含SEQ ID NO:3所示的序列，且具有12-20个氨基酸残基。The active polypeptide of claim 1, wherein the active polypeptide comprises the sequence shown in SEQ ID NO: 3 and has 12-20 amino acid residues.
  8. 如权利要求1至7中任一项所述活性多肽，其包括具有选自SEQ ID NO:4-9中任一所示氨基酸序列结构的线性肽、环肽、或D型氨基酸替换衍生肽。The active polypeptide of any one of claims 1 to 7, which comprises a linear peptide, a cyclic peptide, or a D-form amino acid substitution derivative peptide having an amino acid sequence structure selected from any one of SEQ ID NOs: 4-9.
  9. 如权利要求1至7中任一项所述活性多肽，其包括SEQ ID NO:4-9中任一所示多肽氨基酸序列的基础上进行氨基酸扫描突变获得的突变体，所述氨基酸扫描突变包括正电荷氨基酸扫描突变、负电荷氨基酸扫描突变、中性氨基酸扫描突变、稀有氨基酸替换突变、以及D型氨基酸替换突变。The active polypeptide according to any one of claims 1 to 7, which comprises a mutant obtained by performing amino acid scanning mutation on the basis of the amino acid sequence of the polypeptide shown in any one of SEQ ID NOs: 4-9, wherein the amino acid scanning mutation comprises Positively charged amino acid scanning mutations, negatively charged amino acid scanning mutations, neutral amino acid scanning mutations, rare amino acid substitution mutations, and D-type amino acid substitution mutations.
  10. 如权利要求9所述活性多肽，其中，正电荷氨基酸扫描突变包括精氨酸扫描突变；负电荷氨基酸扫描突变包括天冬氨酸扫描突变；中性氨基酸扫描包括丙氨酸扫描突变；稀有氨基酸替换突变包括将所述多肽链上的精氨酸残基逐一用瓜氨酸残基替换获得突变体；D型氨基酸替换突变包括在SEQ ID NO:4-9所示序列的基础上逐一对每个氨基酸残基采用其相应的D型氨基酸残基替换获得突变体。The active polypeptide of claim 9, wherein positively charged amino acid scanning mutation comprises arginine scanning mutation; negatively charged amino acid scanning mutation comprises aspartic acid scanning mutation; neutral amino acid scanning comprises alanine scanning mutation; rare amino acid substitution Mutation includes replacing arginine residues on the polypeptide chain with citrulline residues one by one to obtain mutants; D-type amino acid substitution mutation includes one by one on the basis of the sequences shown in SEQ ID NOs: 4-9. The amino acid residues were replaced with their corresponding D-form amino acid residues to obtain mutants.
  11. 如权利要求10所述活性多肽，其具有SEQ ID NO:10-73任一所述氨基酸序列，优选具有SEQ ID NO:5、20、21、22、23、30、34、46、50、51、53、66、或69所示的氨基酸序列。The active polypeptide of claim 10, which has the amino acid sequence of any one of SEQ ID NO: 10-73, preferably has SEQ ID NO: 5, 20, 21, 22, 23, 30, 34, 46, 50, 51 , 53, 66, or 69 shown in the amino acid sequence.
  12. 一种制备活性多肽的方法，包括将α-芋螺毒素天然单体肽氨基酸序列中的一个或多个半胱氨酸残基删除，所述α-芋螺毒素天然单体肽具有结合nAChR的功能。A method of preparing an active polypeptide, comprising deleting one or more cysteine residues in the amino acid sequence of an alpha-conotoxin natural monomeric peptide, the alpha-conotoxin natural monomeric peptide having nAChR binding Function.
  13. 如权利要求12所述制备活性多肽的方法，其特征在于还包括将α-芋螺毒素天然单体肽在C端和或N端截短。The method for preparing an active polypeptide according to claim 12, further comprising truncating the α-conotoxin natural monomer peptide at the C-terminus and/or the N-terminus.
  14. 如权利要求12所述制备活性多肽的方法，其特征在于所述α-芋螺毒素天然单体肽来源于选自下组的α-芋螺毒素，GeXIVA、GeXXVIIA、Vc1.1、PeIA、RgIA、B-VxXXIVA、S-GVIIIB、D-GeXXA、O-GeXXVIIA、D--Lt28.1、Mr1.1、BuIA、ImI、或AuIB；所述α-芋螺毒素天然单体肽包括α-芋螺毒素的成熟肽单体、前体肽单体。The method for preparing an active polypeptide according to claim 12, wherein the α-conotoxin natural monomer peptide is derived from α-conotoxin selected from the group consisting of GeXIVA, GeXXVIIA, Vc1.1, PeIA, RgIA , B-VxXXIVA, S-GVIIIB, D-GeXXA, O-GeXXVIIA, D--Lt28.1, Mr1.1, BuIA, ImI, or AuIB; the α-conotoxin natural monomer peptide includes α-conotoxin Mature peptide monomer, precursor peptide monomer of spirotoxin.
  15. 一种制备权利要求1-11所述活性多肽的方法，包括重组表达法和化学合成法，其中所述化学合成法包括固相合成法、液相合成法、固相-液相联合合成法、自然化学连接法(NCL)。A method for preparing the active polypeptide of claims 1-11, comprising recombinant expression method and chemical synthesis method, wherein said chemical synthesis method comprises solid phase synthesis method, liquid phase synthesis method, solid phase-liquid phase combined synthesis method, Natural Chemical Ligation (NCL).
  16. 如权利要求15所述的制备活性多肽的方法，其特征在于线性多肽的固相合成包括Fmoc固相合成法、Boc固相合成法；固相合成的方式包括由C端至N端合成法、由N端至C端合成法。The method for preparing an active polypeptide according to claim 15, wherein the solid-phase synthesis of the linear polypeptide comprises Fmoc solid-phase synthesis, Boc solid-phase synthesis; the solid-phase synthesis comprises C-terminal to N-terminal synthesis, Synthesis from N-terminal to C-terminal.
  17. 如权利要求16所述的制备活性多肽的方法，其中C端至N端的Fomc固相合成法包括：The method for preparing active polypeptide according to claim 16, wherein the Fomc solid-phase synthesis method from C-terminal to N-terminal comprises:
    (1)固相树脂的预处理；(1) Pretreatment of solid phase resin;
    (2)脱除固相树脂上氨基的Fmoc保护基；(2) remove the Fmoc protecting group of amino group on solid-phase resin;
    (3)加入C端第一个氨基酸进行缩合反应，将C端第一个氨基酸连接到固相树脂上；(3) adding the first amino acid at the C-terminal to carry out a condensation reaction, and connecting the first amino acid at the C-terminal to the solid phase resin;
    (4)脱除C端第一个氨基酸氨基上的Fmoc保护基，加入C端第二个氨基酸进行缩合反应，将C端第二个氨基酸连接到第一个氨基酸的氨基上；循环往复，从C端至N端将氨基酸逐一连接形成多肽链；(4) removing the Fmoc protecting group on the amino group of the first amino acid at the C-terminal, adding the second amino acid at the C-terminal to carry out a condensation reaction, and connecting the second amino acid at the C-terminal to the amino group of the first amino acid; The amino acids are linked one by one from the C-terminus to the N-terminus to form a polypeptide chain;
    (5)将多肽链从固相树脂上切割回收、纯化。(5) The polypeptide chain is cut, recovered and purified from the solid phase resin.
  18. 如权利要求15所述的制备活性多肽的方法，其特征在于环形多肽采用NCL法合成，包括：The method for preparing an active polypeptide according to claim 15, wherein the cyclic polypeptide is synthesized by the NCL method, comprising:
    (1)固相合成直链肽，使N端为半胱氨酸；(1) Solid-phase synthesis of straight-chain peptides, making the N-terminal cysteine;
    (2)从固相载体上切下N端为半胱氨酸、C端含硫酯的线性肽；(2) excising a linear peptide whose N-terminal is cysteine and C-terminal contains thioester from the solid phase carrier;
    (3)通过酰基转移反应完成线性肽的环合。(3) The cyclization of the linear peptide is accomplished by an acyl transfer reaction.
  19. 一种融合蛋白或缀合物，其包含权利要求1至11中任一项所述的活性多肽序列，所述融合蛋白或缀合物具有结合nAChR的功能。A fusion protein or conjugate comprising the active polypeptide sequence of any one of claims 1 to 11, the fusion protein or conjugate having the function of binding nAChR.
  20. 一种多聚体，由两个或更多个多肽单体聚合而成，其中至少一个多肽单体为权利要求1至11中任一项所述的活性多肽；所述多聚体的各多肽单体之间通过共价连接。A multimer formed by the polymerization of two or more polypeptide monomers, wherein at least one polypeptide monomer is the active polypeptide according to any one of claims 1 to 11; each polypeptide of the multimer The monomers are linked covalently.
  21. 如权利要求20所述多聚体，其为同源二聚体或异源二聚体；The multimer of claim 20, which is a homodimer or a heterodimer;
  22. 如权利要求20所述多聚体，其中组成多聚体的各多肽单体之间通过柔性linker或PEG连接臂连接，优选的，各多肽单体在各自的氨基端通过柔性linker或PEG连接臂相互连接。The multimer according to claim 20, wherein each polypeptide monomer constituting the multimer is connected by a flexible linker or a PEG linker, preferably, each polypeptide monomer is connected by a flexible linker or a PEG linker at the respective amino terminus connected to each other.
  23. 如权利要求20所述多聚体，其靶向抑制人α9α10nAChR的活性高于单体形式的权利要求1至11中任一项所述活性多肽。The multimer of claim 20, which has a higher targeting activity for inhibiting human α9α10 nAChR than the active polypeptide of any one of claims 1 to 11 in monomeric form.
  24. 一种核酸分子，其编码权利要求1至11中任一项所述的活性多肽、或权利要求19所述融合蛋白或缀合物。A nucleic acid molecule encoding the active polypeptide of any one of claims 1 to 11, or the fusion protein or conjugate of claim 19.
  25. 一种构建体，其包含权利要求24所述的核酸分子。A construct comprising the nucleic acid molecule of claim 24.
  26. 一种宿主细胞，其包含权利要求24所述的核酸分子和/或权利要求25所述的构建体，或者所述宿主细胞被权利要求24所述的核酸分子和/或权利要求25所述的构建体转化或转染。A kind of host cell, it comprises the described nucleic acid molecule of claim 24 and/or the described construct of claim 25, or described host cell is by the described nucleic acid molecule of claim 24 and/or described in claim 25 Construct transformation or transfection.
  27. 一种药物组合物，所述药物组合物包含权利要求1至11中任一项所述的活性多肽、权利要求19所述融合蛋白或缀合物、权利要求20-23中任一所述多聚体、权利要求24所述核酸分子、权利要求25所述构建体、权利要求26所述宿主细胞，以及可选的药学上可接受的辅料。A pharmaceutical composition comprising the active polypeptide of any one of claims 1 to 11, the fusion protein or conjugate of claim 19, the polyprotein of any one of claims 20 to 23. The polymer, the nucleic acid molecule of claim 24, the construct of claim 25, the host cell of claim 26, and optional pharmaceutically acceptable excipients.
  28. 权利要求1至11中任一项所述的活性多肽、权利要求19所述融合蛋白或缀合物、权利要求20-23中任一所述多聚体、权利要求24所述核酸分子、权利要求25所述构建体、权利要求26所述宿主细胞、权利要求27所述药物组合物在 α9α10nAChR抑制剂中的用途，其中所述α9α10nAChR包括人α9α10nAChR、鼠α9α10nAChR。The active polypeptide of any one of claims 1 to 11, the fusion protein or conjugate of claim 19, the multimer of any one of claims 20-23, the nucleic acid molecule of claim 24, the Use of the construct of claim 25, the host cell of claim 26, and the pharmaceutical composition of claim 27 in an α9α10 nAChR inhibitor, wherein the α9α10 nAChR comprises human α9α10 nAChR, and murine α9α10 nAChR.
  29. 权利要求1至11中任一项所述的活性多肽、权利要求19所述融合蛋白或缀合物、权利要求20-23中任一所述多聚体、权利要求24所述核酸分子、权利要求25所述构建体、权利要求26所述宿主细胞、权利要求27所述药物组合物在镇痛、抗肿瘤、和/或治疗神经系统疾病中的应用。The active polypeptide of any one of claims 1 to 11, the fusion protein or conjugate of claim 19, the multimer of any one of claims 20-23, the nucleic acid molecule of claim 24, the Use of the construct described in claim 25, the host cell described in claim 26, and the pharmaceutical composition described in claim 27 in analgesia, antitumor, and/or treatment of nervous system diseases.
  30. 如权利要求29所述的应用，其中所述活性多肽、融合蛋白或缀合物、多聚体、核酸分子、构建体、宿主细胞、和/或药物组合物，可与任选的镇痛、抗肿瘤、和/或治疗神经系统疾病的活性成分联用或联合制药。The use of claim 29, wherein the active polypeptide, fusion protein or conjugate, multimer, nucleic acid molecule, construct, host cell, and/or pharmaceutical composition can be combined with optional analgesia, Combination or combination pharmaceuticals of active ingredients for antitumor and/or treatment of nervous system diseases.
  31. 如权利要求29或30所述的应用，其中所述疾病包括神经痛、成瘾、帕金森症、癫痫症、局部缺血、兴奋性神经元细胞死亡、痴呆、乳腺癌、肺癌、脑脊髓炎、癌症与癌症化疗、酒精中毒、坐骨神经痛、糖尿病、三叉神经痛、硬化症、带状疱疹、机械伤和手术伤、艾滋病、头部神经瘫痪、药物中毒、工业污染中毒、淋巴神经痛、骨髓瘤、多点运动神经痛、慢性先天性感觉神经病、急性剧烈自发性神经痛、挤压神经痛、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、复合神经痛、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。The use of claim 29 or 30, wherein the disease comprises neuralgia, addiction, Parkinson's disease, epilepsy, ischemia, excitatory neuronal cell death, dementia, breast cancer, lung cancer, encephalomyelitis , cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis, herpes zoster, mechanical and surgical injuries, AIDS, head nerve paralysis, drug poisoning, industrial pollution poisoning, lymphatic neuralgia, bone marrow Tumor, multipoint motor neuralgia, chronic congenital sensory neuropathy, acute severe idiopathic neuralgia, crush neuralgia, vasculitis, vasculitis, ischemia, uremia, childhood biliary liver disease, chronic respiratory disorder, complex Neuralgia, multiple organ failure, sepsis/sepsis, hepatitis, porphyria, vitamin deficiency, chronic liver disease, native bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, sensory perineuritis or allergies.
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