Conus polypeptide, pharmaceutical composition and application thereof

Abstract

The present invention belongs to the field of biomedicine technology, and relates to a conus polypeptide, a pharmaceutical composition and its application. Its amino acid sequence is a parent sequence or a parent sequence mutant; the parent sequence is shown in SEQ ID NO.1, and the two cysteines in the parent sequence are connected by forming a disulfide bond; the mutation mode of the parent sequence mutant is a combination of one or more of the following: (1) deleting one or two amino acids other than cysteine ​​in the parent sequence, and the number of amino acids between the two cysteines remains unchanged, (2) replacing up to three amino acids in the parent sequence with other amino acids, (3) adding one or two amino acids in the parent sequence, and the number of amino acids between the two cysteines remains unchanged. The conus polypeptide provided by the present invention contains a pair of disulfide bonds or does not contain disulfide bonds, and the number of amino acids is 9 to 13, has strong α7 nAChR inhibitory activity, and has the potential to be developed into an anti-tumor or targeted drug.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a conus polypeptide, a pharmaceutical composition and applications thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Nicotinic acetylcholine receptors (nAChRs) belong to the ligand-gated pentameric ion channel family, which are widely distributed in the central and peripheral nervous systems and non-neuronal tissues, mediating rapid synaptic transmission. Among the 17 subunits of nAChRs (α1-α10, β1-β4, γ, δ, and ε), only α7 and α9 can form homopentamers alone or heteropentamers with other subunits. Homopentamer α7 nAChR is mainly distributed in the central nervous system and is essential for learning and memory. Its dysfunction is closely related to neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. In addition, α7 nAChR is overexpressed in macrophages and tumor cells, which can significantly affect cholinergic anti-inflammatory responses and the occurrence of cancer. Therefore, the development of new ligands targeting α7 nAChR is crucial to advance the treatment of related diseases.

[0004] Conus peptides are derived from marine cone snails, including disulfide-rich conus toxins and non-disulfide-rich conus peptides, which provide rich resources for drug development and neurochemical tool design. According to the inventors' research, a series of conus peptides targeting α7 nAChR have been developed. However, the existing conus peptides that can effectively inhibit α7 nAChR have the problem of being difficult to synthesize due to their long sequences. For example, the half-maximal inhibitory concentration (IC50) of the homodimeric protein VxXXB on α7 nAChR is 50 ) is 0.4 nM, while its molecular weight is as high as 11 kDa; alternatively, cono peptides with shorter sequences have weak inhibitory effects on α7 nAChR, for example, Czon1107, a 10-amino acid cono peptide with a single disulfide bond, has an IC 50is 77.1 μM. In addition, most of the cono peptides that antagonize α7 nAChR discovered to date contain four cysteine ​​residues and have highly similar sequences and disulfide bond frameworks. For example, ImI, ImII, and RgIA share the same 4 / 3 disulfide bond framework and globular connection pattern, while Mr1.1, RegIIA, and CIB share a 4 / 7 disulfide bond framework. This structural similarity limits the diversity of peptide ligands for α7 nAChR, thereby limiting their potential as drug leads or neurochemical tools. Therefore, there is an urgent need to discover potent α7 nAChR inhibitor ligands with simpler disulfide bond frameworks and shorter sequences. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a conus polypeptide, a pharmaceutical composition and its application. The conus polypeptide provided by the present invention contains a pair of disulfide bonds or does not contain a disulfide bond, the number of its amino acids is 9 to 13, has a strong α7 nAChR inhibitory activity, and has the potential to be developed into an anti-tumor or targeted drug.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] In the first aspect, a conus polypeptide, whose amino acid sequence is a parent sequence or a mutant of the parent sequence; the parent sequence is as shown in SEQ ID NO.1, and two cysteines in the parent sequence are connected by forming a disulfide bond; the mutation mode of the parent sequence mutant is one or more combinations of the following: (1) deleting one or two amino acids other than cysteine ​​in the parent sequence, and the number of amino acids between the two cysteines remains unchanged, (2) replacing up to three amino acids in the parent sequence with other amino acids, (3) adding one or two amino acids in the parent sequence, and the number of amino acids between the two cysteines remains unchanged.

[0008] In the present invention, when one cysteine ​​or two cysteines in the parent sequence mutant are replaced, it is a linear polypeptide.

[0009] The end of the parent sequence that ends with cysteine ​​is the C-terminus. In some embodiments, the C-terminus of the parent sequence is amidated. The amidation refers to the modification of -COOH to -CONH2.

[0010] In some embodiments, one or two amino acids at the N-terminus of the parent sequence are deleted.

[0011] In some embodiments, the mutations replacing other amino acids are: Q1A, Q1R, Q1D, Q1Pyr, P2A, P2R, P2D, G3A, G3R, G3D, C4A, C4S, R5A, R5Dap, R5Dab, R5D, W6A, W6R, W6D, W7A, W7R, W7D, S8A, S8R, S8Dap, S8D, R9A, R9D, W10A, W10R, W10D, C11A, C11S.

[0012] Among them, Pyr is the unnatural amino acid (S)-5-oxopyrrolidine-2-carboxylic acid, Dap is the unnatural amino acid (S)-2,3-diaminopropionic acid, and Dab is the unnatural amino acid (S)-2,4-diaminobutyric acid.

[0013] In some embodiments, 1 to 2 Args are introduced at the C-terminus of the parent sequence.

[0014] In some embodiments, the amino acid sequence of the parent sequence mutant is shown in any one of SEQ ID NOs. 2 to 17 and SEQ ID NOs. 19 to 40.

[0015] In a second aspect, a method for synthesizing the conus polypeptide described in the first aspect of the present invention comprises the following process for preparing a linear peptide: using the Fmoc solid phase peptide synthesis method, subjecting the Fmoc amino acid to repeated deprotection-coupling cycles to form a resin peptide on a resin, and then cleaving the resin peptide to obtain a linear peptide.

[0016] In some embodiments, the resin used is Rink-Amide resin or 2-Cl-CTC resin. Rink-Amide resin is used to prepare C-terminal amidated polypeptides. 2-Cl-CTC resin is used to prepare C-terminal free polypeptides. C-terminal free means that the C-terminal carboxyl group is not modified or chemically modified.

[0017] In some embodiments, the thiol group of cysteine ​​is protected by a protecting group, specifically, the protecting group is trityl (Trt) or acetylaminomethyl (Acm).

[0018] In some embodiments, a disulfide bond formation process is included, and the disulfide bond formation process adopts air oxidation method or iodine oxidation method.

[0019] In a third aspect, a pharmaceutical composition comprises at least one cono polypeptide according to the first aspect of the present invention.

[0020] The pharmaceutical composition of the present invention can be selected from the following administration methods: oral administration, intravenous injection, intramuscular injection, peritoneal injection, subcutaneous injection, etc., to inhibit α7 nAChR.

[0021] In some embodiments, it further comprises a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient of the present invention is a pharmaceutical carrier and / or excipient. The pharmaceutical carrier may be serum protein, physiological saline, buffer solution, etc. The excipient may be a binder, a filler, a disintegrant, a lubricant, a preservative, etc.

[0022] In a fourth aspect, a use of the cono polypeptide described in the first aspect of the present invention or the pharmaceutical composition described in the third aspect of the present invention in the preparation of a drug for blocking or inhibiting α7 nAChR.

[0023] Since the dysfunction of α7 nAChR is closely related to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and can also significantly affect the cholinergic anti-inflammatory response and tumorigenesis, therefore, in the fifth aspect, a use of the cono polypeptide described in the first aspect of the invention or the pharmaceutical composition described in the third aspect of the invention in the preparation of drugs for the treatment of neurodegenerative diseases, anti-inflammatory or anti-tumor.

[0024] In some embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, etc.

[0025] The beneficial effects of the present invention are:

[0026] The present invention provides a novel conus polypeptide that can effectively inhibit α7 nAChR. The conus polypeptide contains 9 to 13 amino acids and contains a pair of disulfide bonds or does not contain disulfide bonds. Compared with other known α7 nAChR potent inhibitory polypeptides, the synthesis process is simpler and the cost is lower. Among them, the conus toxin sequence SS1[ΔQP,S8R] containing 9 amino acids and a pair of disulfide bonds is the shortest conus polypeptide mutant that can potently act on α7 nAChR found so far; and compared with the wild type, the mutant has a 29-fold increase in the inhibitory effect on α7 nAChR, and the IC 50 The kinase activity of 49.1 nM is favorable for its development into anti-tumor drugs and targeted therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0028] Figure 1The AF2 predicted structure of the conus peptide SS1 in the embodiment of the present invention and the changes of SS1 during the MD process. a: The structure of SS1 predicted by AF2 and the corresponding amino acid sequence; b: The RMSD of SS1 in the molecular dynamics simulation at 500 ns; c: The overall RMSD of the molecular dynamics simulation of SS1 and α7 nAChR complex at 50 ns; d: The conformation extracted by SS1 during the molecular dynamics simulation at 500 ns; e: The conformation extracted by SS1 during the molecular dynamics simulation at 500 ns; f: The conformation extracted by SS1 during the molecular dynamics simulation at 500 ns.

[0029] Figure 2 The diagram is a diagram of the binding mode of the conus peptide SS1 and the human α7 nAChR complex in the embodiment of the present invention. a: The binding mode of the conus peptide SS1 on the human α7 nAChR; b: The interaction between A185, E184 and R5; c: The interaction between L78 and W6; d: The interaction between Y217 and S8; e: The interaction between W171, L141 and R9; f: The interaction between N133, Y217 and W10; g: The interaction between Y217 and C11.

[0030] Figure 3 It is the ESI-MS spectrum of SS1 in the embodiment of the present invention.

[0031] Figure 4 It is the HPLC spectrum of SS1 in the embodiment of the present invention.

[0032] Figure 5 It is the ESI-MS spectrum of SS1[S8R] in the embodiment of the present invention.

[0033] Figure 6 It is the HPLC spectrum of SS1[S8R] in the embodiment of the present invention.

[0034] Figure 7 This is the ESI-MS spectrum of SS1[ΔQP, S8R] in the embodiment of the present invention.

[0035] Figure 8 HPLC spectrum of SS1[ΔQP, S8R] in the embodiment of the present invention.

[0036] Fig. 9 The relative inhibition rates of SS1 and its analogs on human α7 nAChR at a concentration of 1 µM in the examples of the present invention (n≥4); a: relative inhibitory activity of alanine and arginine scanning analogs; b: activity of analogs modified based on the complex model; c: activity of double mutation and multiple mutation analogs; d: activity of mutant analogs of SS1[S8R].

[0037] Fig.10 The inhibitory concentration effect curves of SS1 (a), SS1[S8R] (b) and SS1[ΔQP, S8R] (c) on human α7nAChR in the examples of the present invention (n≥4). DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0039] Example 1 Design, synthesis and structure-activity relationship study of conus peptide SS1 and its analogs

[0040] This example provides a new conus peptide sequence, namely, conus peptide SS1 (SS1 for short), which can inhibit the activity of human α7 nAChR, and the sequence is QPGCRWWSRWC, * represents terminal amidation, and a disulfide bond is formed between the two cysteines, such as Figure 1 As shown in a, the stability of SS1 was evaluated by molecular dynamics simulation. After 500ns MD, the overall structure still could not reach a stable state, and it switched back and forth between partial α-helix and random coil, revealing the high dynamics of its structure. Figure 1 As shown in b, d, e and f. In order to study the structure-activity relationship between SS1 and human α7 nAChR, the AF2-Multimer was first used to construct a model of the SS1 and human α7 nAChR complex. Subsequently, Pymol was used to modify the C-terminus of the peptide, and a 50 ns molecular dynamics simulation was performed in the Amber20 software for full relaxation. The ff19SB force field was used throughout the dynamics simulation, with a 2 fs experimental step size, and the SHAKE algorithm was used to process the hydrogen bonds encountered. After the simulation, VMD software was used to analyze the conformational change trajectory after MD, and the RMSD value was calculated. The overall complex structure reached a steady state in 25 ns, indicating that the constructed model tends to be stable, as shown in Figure 1 As shown in c. The last conformation of the simulation was used to analyze the binding mode of conus peptide SS1 and hα7nAChR. The specific interaction is shown in Figure 2 As shown: Q1, P2, G3, and W7 are exposed to the solvent and do not participate in binding with the receptor, such as Figure 2 As shown in a; R5, W6, S8, R9, W10, and C11 all interact with the receptor and make different contributions to inhibiting the activity of α7 nAChR, such as Figure 2 As shown in b, c, d, e, f and g.

[0041] In this example, the electrophysiological data of alanine and arginine scanning (SEQ ID NO.1-17) performed on SS1 verified the rationality of the binding mode: the conus peptide SS1 binds to the orthosteric site of α7 nAChR, forming extensive hydrogen bonds, van der Waals forces and cation-π interactions with the C-Loop of the main subunit and the β-Sheet of the auxiliary subunit. SS1 is partially embedded in the binding pocket, and its N-terminal tail consists of three residues, which are exposed to the solvent and do not interact with the receptor. The key amino acid sites of the receptor are R5, W6, S8, R9 and W10, etc. Site-directed mutations of these amino acids have a greater impact on activity, and mutations of the remaining amino acid sites have less impact on activity. Combined with the above analysis. Finally, 23 single mutation and double mutation analogs (SEQ ID NO.18-40) were designed in this example.

[0042] Table 1 Number, name and amino acid sequence of SS1 and its series mutants acting on α7 nAChR

[0043]

[0044]

[0045]

[0046] * represents terminal amidation

[0047] Example 2 Synthesis of Conus polypeptide SS1 and its analogs

[0048] 1. Synthesis of conus peptide SS1 and its mutant linear peptides

[0049] The peptides involved in the present invention were synthesized using solid phase peptide synthesis (SPPS) and Fmoc chemical strategy. Ca-73 and SS1[S8R]-1 used Wang resin preloaded with Fmoc-Cys(Trt)-OH (Loading: 0.372 mmol / g), and the other peptides used Rink-Amid resin (Loading: 0.637 mmol / g). The resin (0.1 mmol) was swollen in N,N-dimethylformamide (DMF) for 30 minutes. The Fmoc protecting group on the resin was removed by treatment with 20% piperidine for 30 minutes. During the coupling process, 4 times of Fmoc-protected amino acids, 4 times of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 8 times of N,N-diisopropylethylamine (DIPEA) were added and reacted at room temperature for 60 min. The mixture was washed repeatedly with DMF and dichloromethane (DCM) three times. Repeat the deprotection-coupling cycle until the last amino acid protecting group is removed. The linear peptide is released from the resin by treating with TFA cleavage solution (TFA / H2O / TIPS, 90:5:5) at room temperature for 3 hours. Then, add excess cold ether, and a large amount of precipitate will be precipitated immediately. Centrifuge at 5000 rpm for 3 minutes, and discard the supernatant to obtain the crude peptide.

[0050] 2. Disulfide bond oxidation process of cono polypeptide SS1 and its mutants:

[0051] Conus polypeptide SS1 and its mutants contain at most one pair of disulfide bonds. During the synthesis process, the protecting group on cysteine ​​generally adopts Trt protecting group, and the disulfide bond can be constructed by air oxidation method / iodine oxidation.

[0052] Iodine oxidation method: Dissolve the crude product in 10% acetonitrile aqueous solution, sonicate to fully dissolve the polypeptide, slowly drop 5 mg / mL iodine solution until the solution turns light yellow, stop dropping, and shake at room temperature for 20 min.

[0053] The above oxidation process requires LC-MS to monitor the oxidation endpoint in real time, and L-ascorbic acid aqueous solution is added after the reaction is completed to reduce excess iodine.

[0054] 3. Purification and separation of conus peptide SS1 and its mutants:

[0055] Taking SS1, SS1[S8R], and SS1[ΔQP, S8R] as examples, the crude peptides were purified by reverse phase high performance liquid chromatography (RP-HPLC). The mobile phase composition of HPLC was: phase A was 10% acetonitrile aqueous solution, with 0.05% trifluoroacetic acid (TFA) added, and phase B was 10% water acetonitrile solution, with 0.05% TFA added. The mobile phase gradient was 100% mobile phase A, which changed uniformly to 50% mobile phase and 50% mobile phase B within 40 min. The semi-preparative column specifications were 20*250 mm, 10 μM, and the analytical column specifications were 4.6*250 mm, 5 μM.

[0056] The product peak was collected and freeze-dried. The identity and purity of the peptide were confirmed by ESI-MS and HPLC, requiring the molecular weight to be consistent with the expectation and the purity to be ≥95%. Among them, the theoretical mass of SS1 was 1461.69, and the detected mass-to-charge ratio (m / z) was 731.54 [M+2H] 2+ 、488.03[M+3H] 2+ ,like Figure 3 As shown; the purity is 95.05%, such as Figure 4 As shown; the theoretical mass of SS1[S8R] is 1531.80, and the detected mass-to-charge ratio (m / z) is 765.94[M+2H] 2+ 、510.94[M+3H] 3+ 、383.41[M+4H] 4+ ,like Figure 5 As shown; the purity is 98.07%, such as Figure 6 As shown; the theoretical mass of SS1[ΔQP, S8R] is 1305.51, and the detected mass-to-charge ratio (m / z) is 653.23[M+2H] 2+ 、435.75[M+3H] 3+ 、327.24[M+4H] 4+ ,like Figure 7 As shown; the purity is 98.57%, such as Figure 8 shown.

[0057] 4. In vitro activity test of conus peptide SS1 and its mutants

[0058] Taking SS1, SS1[S8R], and SS1[ΔQP, S8R] as examples, mature African clawed frogs in good survival condition were taken and buried deep in crushed ice for cryoanaesthesia. Mature oocytes were surgically obtained and placed in OR-2 solution. The cell mass was carefully cut into pieces with scissors and digested with 2 mg / mL Clostridium collagenase. After the oocytes were fully dispersed, single frog eggs were selected. Then, the cRNA of α7 nAChR was injected into the frog eggs in proportion using microinjection technology, and the corresponding receptors were expressed by culturing at 18°C ​​in a ND96 solution culture dish containing antibiotics for 2 days. After the two-electrode voltage clamp equipment was debugged, it was confirmed that the corresponding receptor was successfully expressed, and then the recording electrode was inserted into the oocyte to activate the channel opening with the corresponding concentration of Ach. When the excitation current reached the corresponding amplitude and existed stably, the cells were incubated with the test solution of the corresponding concentration for 10 min, and the current changes were observed. The experiment was repeated three times and the experimental results were recorded.

[0059] The electrophysiological activity of this series of conus peptide SS1 mutants was tested, and their results at 1 µM concentration are shown in Figure 3 As shown; among the mutants, SEQ ID NO.16, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.25, and SEQID NO.31 exhibited superior inhibitory activity, as shown Fig. 9 shown.

[0060] By testing the inhibitory ability of α7 nAChR-mediated acetylcholine-activated current at different peptide concentrations, the inhibitory concentration-effect curve was fitted, and the half-maximal inhibitory concentrations of SS1, SS1[S8R], and SS1[ΔQP, S8R] on human α7 nAChR were found to be 1.43 μM, 63.2 nM, and 49.1 nM, respectively. Fig.10 shown.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A conus polypeptide, characterized in that: Its amino acid sequence is a mutant of the parent sequence; the amino acid sequence of the mutant of the parent sequence is shown in any one of SEQ ID NO.16 and SEQ ID NO.

35.

2. A method for synthesizing the conus polypeptide according to claim 1, characterized in that: The method comprises the following steps of preparing the linear peptide: adopting the Fmoc solid phase peptide synthesis method, subjecting the Fmoc amino acid to repeated deprotection-coupling cycles to form a resin peptide on a resin, and then cleaving the resin peptide to obtain a linear peptide.

3. A pharmaceutical composition, characterized in that: It contains at least one cono polypeptide according to claim 1.

4. The pharmaceutical composition according to claim 3, characterized in that: It also includes pharmaceutically acceptable excipients.

5. Use of the cono polypeptide according to claim 1 or the pharmaceutical composition according to claim 3 or 4 in the preparation of a medicament for treating neurodegenerative diseases.

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