Method for designing polypeptide drug and application

Abstract

The invention belongs to the field of biological medicine, and relates to a method for designing a polypeptide drug and application. On one hand, the invention provides a novel method for designing the polypeptide drug, and the polypeptide drug prepared by adopting the method or strategy has a remarkably increased alpha-helical structure, thermal stability and biological activity. On the other hand, the invention provides a polypeptide compound for preventing and treating diseases, and the polypeptide compound comprises a polypeptide and an 'EAAAKK' sequence which is connected to the C terminal and / or N terminal of the polypeptide and has an alpha-helical structure.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a method for designing polypeptide drugs and applications. Background Art

[0002] Peptide drugs refer to a class of compounds with specific therapeutic effects that are extracted through chemical synthesis, genetic recombination or from plants and animals and are composed of amino acids connected by peptide bonds. Peptides have a wide range of biological activities and can participate in and regulate many physiological and pathological processes. In recent years, with the rapid development of biotechnology, peptide drugs have become one of the hot spots in the field of drug research and development. More and more peptide drugs have been developed and have been widely used in the prevention, diagnosis and treatment of diseases such as tumors, hepatitis, diabetes, and AIDS. The research and application of peptide drugs are of great significance for innovative drug development, improving disease treatment effects, and reducing drug side effects.

[0003] The molecular size of peptide drugs is between that of small molecule drugs and large molecule protein drugs. Compared with small molecule drugs, peptide drugs have higher activity and stronger selectivity; compared with protein drugs, peptide drugs have advantages such as low immunogenicity, high purity and low production cost. However, the number of amino acids in peptides is less than that of proteins, and they are easily degraded by proteases in the body, especially on the main chain of peptides, which is prone to deamidation reactions, causing changes in the conformation of amino acid residues on the side chains, such as cleavage of α-helix and β-sheet structures, and breaking of disulfide bonds and hydrogen bonds. These factors make most peptide drugs poorly stable in the body and have low bioavailability. Therefore, one of the focuses of peptide drug research and development is to transform or modify the structure of peptide drugs, improve their physical and chemical properties, and thus improve drug stability and biological activity.

[0004] Therefore, this field still needs to continue to develop stable and efficient peptide drugs to meet clinical needs. Summary of the invention

[0005] In order to solve the problem of poor stability and low activity of polypeptide drugs caused by easy deamidation reaction on the polypeptide main chain in the prior art, the present invention provides a method for designing polypeptide drugs and application thereof.

[0006] The present invention provides a polypeptide compound, comprising a polypeptide and an "EAAAKK" sequence having an alpha-helical structure connected to the C-terminus and / or the N-terminus of the polypeptide.

[0007] The present invention creatively connects the "EAAAKK" sequence with an α-helical structure to the C-terminus and / or N-terminus of the polypeptide, thereby giving the polypeptide significantly increased α-helical structure, thermal stability and biological activity.

[0008] One or more amino acids of the polypeptide compound of the present invention can be replaced with amino acids with D-type conformation, artificially modified amino acids, rare amino acids existing in nature, etc., to improve the bioavailability, stability and / or biological activity of the polypeptide. Wherein D-type amino acids refer to amino acids corresponding to L-type amino acids constituting proteins; artificially modified amino acids refer to common L-type amino acids constituting proteins that have been modified by methylation, phosphorylation, etc.; rare amino acids existing in nature include uncommon amino acids constituting proteins and amino acids that do not constitute proteins, such as 5-hydroxylysine, methylhistidine, x-aminobutyric acid, homoserine, etc.

[0009] The polypeptide compound includes different optical isomers, racemates and / or mixtures thereof. In the above case, a single enantiomer or diastereomer, such as an optically active isomer, can be obtained by asymmetric synthesis or by racemate resolution. The resolution of the racemate can be achieved by different methods, such as conventional recrystallization with a reagent that assists resolution, or by chromatography.

[0010] The polypeptide compound includes different cis and / or trans isomers with double bonds.

[0011] The derivative of a polypeptide compound may be a solvate of the compound, a complex of the compound, a chelate of the compound or a non-covalent complex of the compound. The derivative of the compound may also be a prodrug based on the compound.

[0012] When preparing drugs, any mixture of the above polypeptide compounds can be used.

[0013] The prepared medicine may contain any mixture of the above polypeptide compounds.

[0014] Further, the polypeptide compound is a compound having formula I, wherein formula I is: X1-X2-Xn-X3-X4; in formula I, X1 is an amino terminal protecting group; Xn is any polypeptide sequence; X4 is a carboxyl terminal protecting group; X2 is an "EAAAKK" sequence or is absent, X3 is an "EAAAKK" sequence or is absent, and at least one of X2 and X3 is an "EAAAKK" sequence.

[0015] Exemplarily, X1 is any one of acetyl (Ac), amino (NH2), maleyl, succinyl, tert-butyloxycarbonyl or benzyloxy and other hydrophobic groups or macromolecular carrier groups.

[0016] Exemplarily, the Xn is a "SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK" polypeptide sequence or a "VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK" polypeptide sequence or any other polypeptide sequence;

[0017] Exemplarily, X4 is any one of an amino group (NH2), a carboxyl group, a hydroxyl group, an amide group or a tert-butyloxycarbonyl group or other hydrophobic groups or macromolecular carrier groups.

[0018] Exemplarily, the polypeptide compound is polypeptide P43; polypeptide P43 is the compound represented by formula I, X1 is Ac, X2 is not present, Xn is the "SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK" polypeptide sequence, X3 is the "EAAAKK" sequence, and X4 is NH2.

[0019] Exemplarily, the polypeptide compound is polypeptide P54; polypeptide P54 is the compound represented by formula I, X1 is Ac, X2 is not present, Xn is the polypeptide sequence of "VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK"; X3 is the sequence of "EAAAKK", and X4 is NH2.

[0020] The peptides P43 and P54 obtained by adding the "EAAAKK" sequence to the C-terminus of the anti-new coronavirus peptides P37 and P48 can significantly improve the antiviral activity of the peptides, the α-helix content of the peptides and their thermal stability, while the ability of the peptides to bind to the target sequence is also significantly improved.

[0021] The present invention also protects the derivatives obtained by modifying the amino end and / or carboxyl end of the polypeptide compound with a lipophilic compound; or different pharmaceutically usable forms of the polypeptide compound.

[0022] Among them, the lipophilic compounds include cholesterol succinate, 2-cholesterol acetic acid, 2-cholesterol propionic acid, 3-cholesterol propionic acid, 2-cholesterol butyric acid, 2-cholesterol isobutyric acid, 3-cholesterol butyric acid, 3-cholesterol isobutyric acid, 4-cholesterol butyric acid, 2-cholesterol valeric acid, 2-cholesterol isovaleric acid, 3-cholesterol valeric acid, 5-cholesterol valeric acid, 2-cholesterol hexanoic acid, 6-cholesterol hexanoic acid, 2-cholesterol heptanoic acid, 7-cholesterol heptanoic acid, 2-cholesterol octanoic acid, 8-cholesterol octanoic acid, cholesterol bromoacetate, fatty acids containing 8 to 20 carbon atoms (such as octadecanoic acid), dihydrosphingosine, vitamin E and other lipid compounds.

[0023] The different pharmaceutically usable forms of the polypeptide include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, drug proformas based on the above substances, and any mixtures of the above forms.

[0024] The present invention also protects a polymer formed by any of the above polypeptide compounds and / or their pharmaceutically acceptable salts and / or their derivatives; or a fusion protein of any of the above polypeptide compounds and / or their pharmaceutically acceptable salts and / or their derivatives.

[0025] The present invention also protects a pharmaceutical compound with biological activity, which is any of the above-mentioned polypeptide compounds or pharmaceutically acceptable salts or derivatives thereof or any of the above-mentioned polymers or fusion proteins.

[0026] The pharmaceutical compound is used to treat and / or prevent corresponding diseases.

[0027] The diseases described in the present invention include but are not limited to tumors, hepatitis, diabetes, AIDS, diseases caused by coronavirus infection, etc.

[0028] The modified polypeptide drug prepared by the method of the present invention has significantly increased -helix structure, thermal stability and / or biological activity.

[0029] The present invention also protects the use of any of the above polypeptides or pharmaceutically acceptable salts or derivatives thereof in the preparation of drugs for treating and / or preventing corresponding diseases.

[0030] The present invention also protects the use of the "EAAAKK" sequence in the preparation of compounds that enhance the α-helical structure, thermal stability and biological activity of polypeptide drugs.

[0031] The present invention also provides a nucleic acid molecule encoding the polypeptide compound.

[0032] Furthermore, the nucleic acid molecule may be DNA (such as cDNA, genomic DNA or recombinant DNA, etc.) or RNA (such as mRNA or hnRNA, etc.).

[0033] The present invention also provides a method for protecting and enhancing the α-helical structure and / or thermal stability and / or biological activity of a polypeptide drug, comprising the following steps: connecting the "EAAAKK" sequence to the N-terminus and / or C-terminus of the polypeptide.

[0034] The present invention provides a method for designing a polypeptide drug, comprising the following steps:

[0035] The "EAAAKK" sequence having an α-helical structure is linked to the C-terminus and / or N-terminus of the polypeptide.

[0036] The polypeptide drug prepared by the method or strategy has significantly increased α-helical structure, thermal stability and / or biological activity.

[0037] More details of the present invention are described in detail below, or some of them can also be reflected in the embodiments of the present invention. Unless otherwise specified, the quantities of different components and reaction conditions used herein can be interpreted as "roughly" or "approximately" in any case. Accordingly, unless otherwise specified, the numerical parameters cited below and in the claims are approximate parameters, and different numerical parameters may be obtained under respective experimental conditions due to different standard errors.

[0038] Herein, when there is a disagreement or ambiguity between the chemical formula and chemical name of a compound (including a polypeptide compound), the chemical formula is used to accurately define the compound. The compounds described herein may contain one or more chiral centers, and / or double bonds and structures such as these, and may also exist as stereoisomers, including double bond isomers (such as geometric isomers), optical enantiomers or diastereomers. Accordingly, any chemical structure within the scope of the description herein, whether it contains the above-mentioned similar structures in part or in its entirety, includes all possible enantiomers and diastereomers of the compound, including any single stereoisomer (such as a single geometric isomer, a single enantiomer or a single diastereomer) and any mixture of these isomers. These racemic isomers and mixtures of stereoisomers can also be further separated into their constituent enantiomers or stereoisomers by those skilled in the art using chiral separation techniques or chiral molecular synthesis methods.

[0039] In practical applications, the medicine of the present invention can be directly administered to a patient, or can be administered to a patient after being mixed with a suitable carrier or excipient to achieve the purpose of treating and / or preventing a disease. The carrier material here includes, but is not limited to, a water-soluble carrier material (such as polyethylene glycol, polyvinyl pyrrolidone, organic acid, etc.), a poorly soluble carrier material (such as ethyl cellulose, cholesterol stearate, etc.), and an enteric carrier material (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, a water-soluble carrier material is preferred. Using these materials, a variety of dosage forms can be made, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a common preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle delivery systems. In order to make a unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make the unit dosage form into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin, talc, etc.; binders, such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare a unit dosage form as a suppository, various carriers known in the art can be widely used. Examples of carriers include, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc.In order to prepare the unit dosage form into an injection preparation, such as a solution, emulsion, lyophilized powder injection and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injection preparation. In addition, conventional cosolvents, buffers, pH regulators, etc. can also be added. In addition, if necessary, colorants, preservatives, spices, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparation. The above dosage forms can be used for administration by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; cavity administration, such as rectal and vaginal; respiratory tract administration, such as nasal; mucosal administration, etc.

[0040] The dosage of the drug of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the specific active ingredient used, the route of administration and the number of administrations, etc. The above dosage can be administered in a single dosage form or divided into several, such as two, three or four dosage forms.

[0041] The drug of the present invention can be used directly alone for the treatment and prevention of diseases, or can be used in combination with one or more other antiviral drugs to achieve the purpose of improving the overall therapeutic effect.

[0042] For any particular patient, the specific therapeutically effective dosage level must be determined based on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific active ingredient being employed; the specific composition being employed; the patient's age, weight, general health, sex and diet; the time of administration, route of administration and excretion rate of the specific active ingredient being employed; the duration of treatment; drugs used in combination or concomitantly with the specific active ingredient being employed; and similar factors well known in the medical arts. For example, it is practice in the art to start the dose of the active ingredient at a level lower than that required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0043] Beneficial effects:

[0044] The present invention creatively connects the "EAAAKK" sequence with an α-helical structure to the C-terminus and / or N-terminus of the anti-new coronavirus polypeptide, giving the polypeptide significantly increased α-helical structure, thermal stability and biological activity.

[0045] The antiviral activity of peptides P43 and P54 was increased by about 20 times compared with the peptides without the "EAAAKK" sequence connected to the C-terminus. At the same time, the introduction of the "EAAAKK" sequence changed the secondary structure of the peptide, significantly increased the α-helix content and thermal stability of the peptide, and significantly improved the ability to bind to the target sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The activity of peptides P37 and P43 in inhibiting SARS-CoV-2 infection of 293T / ACE2 cells (left) or Huh-7 cells (right);

[0047] Figure 2 The activity of peptides P48 and P54 in inhibiting SARS-CoV-2 infection of 293T / ACE2 cells (left) or Huh-7 cells (right);

[0048] Figure 3 α-helical content (left) and thermal stability (right) of P37, P43, P48, and P54 polypeptides;

[0049] Figure 4 The α-helix content (left) and thermal stability (right) of the complexes of P37, P43, P48 and P54 polypeptides with target sequence polypeptides. DETAILED DESCRIPTION

[0050] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are described in detail below with reference to the accompanying drawings, but they should not be construed as limiting the applicable scope of the present invention.

[0051] The experimental methods in the following examples, unless otherwise specified, are conventional methods and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0052] Unless otherwise specified, reagents and the like can be obtained from commercial sources.

[0053] Example 1. Synthesis of polypeptide

[0054] The present invention adopts the novel method for designing peptide drugs to prepare anti-new coronavirus (SARS-CoV-2) peptides P43 and P54, and also synthesizes template peptides P37 and P48 for comparative studies, as well as target mimic peptide N57. The amino termini of all peptides are connected to acetyl (Ac) as an amino terminal protecting group, and the carboxyl termini are connected to amino (NH2) as a carboxyl terminal protecting group. The specific sequence structure is as follows:

[0055]

[0056] The preparation method includes: preparing peptide resin by solid phase peptide synthesis method, acid hydrolyzing the peptide resin to obtain a crude product, and finally purifying the crude product to obtain a pure product. The specific steps are as follows:

[0057] 1. Synthesis of peptide resin

[0058] Rink Amide BHHA resin was used as a carrier resin, and the peptide resin was prepared by coupling with the corresponding protected amino acids in the polypeptide amino acid sequence in sequence through Fmoc removal and coupling reaction.

[0059] (1) Insertion of the first protected amino acid in the main chain

[0060] Take 0.3 mmol of the first protected amino acid and 0.3 mmol of HOBt, dissolve them in an appropriate amount of DMF; take another 0.3 mmol of DIC, slowly add it to the DMF solution of the protected amino acid under shaking, and shake the reaction at room temperature for 5 minutes to obtain an activated protected amino acid solution for use.

[0061] 0.1 mmol of Rink Amide MBHA resin (0.35 mmol / g×0.3 g) was taken and deprotected with 25% PIPE / DMF solution (volume ratio) for 20 minutes (twice), and then washed and filtered to obtain the Fmoc-free resin.

[0062] The activated first protected amino acid solution was added to the Fmoc-removed resin, and the coupling reaction was carried out for 60 minutes. The resin containing the first protected amino acid was filtered and washed to obtain the resin.

[0063] (2) Adding other protected amino acids to the main chain

[0064] The same method as the first protected amino acid in the main chain was used to sequentially connect other protected amino acids corresponding to the polypeptide to obtain a resin containing main chain amino acids. Finally, 0.3mmol Ac2O+0.6mmol DIEA was used to acetylate the N-terminus to complete the synthesis of the main chain. After each step of the above reaction, the Kaiser Test was used to control the reaction. If the condensation reaction of a certain amino acid was incomplete, the condensation was repeated once until the desired target peptide was obtained.

[0065] 2. Preparation of crude product

[0066] Take the above peptide resin, add the cleavage reagent (cleavage reagent 15mL / gram resin), mix well and shake at 30°C for 3 hours to cleave the target polypeptide from the resin and remove the side chain protecting group. Collect the filtrate of the reaction mixture, wash the resin with a small amount of TFA / DCM three times, combine the filtrate, add anhydrous ether to precipitate, and centrifuge. Wash the filter cake with cold anhydrous ether and precipitate twice, and drain to obtain an off-white powder as the crude polypeptide. The composition of the cleavage reagent is as follows: trifluoroacetic acid: 1,2-ethanedithiol: anisyl thioether: phenol: H2O: triisopropylsilane = 68.5:10:10:5:3.5:1 (volume ratio).

[0067] 3. Preparation of pure product

[0068] Take the above crude polypeptide, add water / acetonitrile, stir and dissolve, centrifuge to remove insoluble matter and set aside. Reverse phase high performance liquid chromatography was used for purification. The chromatographic column model used was Agela C18 (10 μm, 50mm×250mm), the mobile phase consists of mobile phase A (0.05% TFA and 2% acetonitrile in water) and mobile phase B (90% acetonitrile / water solution). The mobile phase flow rate is 25mL per minute. The UV detection wavelength is 220 nanometers. The crude solution is loaded on the chromatographic column, gradient elution is performed, the corresponding purified components are collected, and the solvent is directly freeze-dried to obtain a fluffy trifluoroacetate peptide pure product.

[0069] The trifluoroacetate peptide pure product was redissolved with water and acetonitrile, and a large amount of anion exchange resin (acetate form) was added and stirred for 3 hours. After filtering and washing the ion exchange resin with a water / acetonitrile mixed solvent, the filtrate was combined and freeze-dried to obtain a fluffy peptide acetate pure product.

[0070] The chemical structure of the synthesized peptides was characterized by MALDI-TOF mass spectrometry, and the purity was determined by analytical high performance liquid chromatography (Agela C18-4.6×250 mm, flow rate 1 mL per minute). The results showed that the purity of the synthesized peptides was greater than 95%.

[0071] Example 2: Adding the "EAAAKK" sequence to the C-terminus of a polypeptide significantly improves its antiviral activity

[0072] The P37 polypeptide is a SARS-CoV-2 membrane fusion inhibitor containing an MPER sequence, and its antiviral activity is relatively low. The present invention creatively adds an "EAAAKK" sequence capable of forming an α-helix to the C-terminus of P37 to obtain the polypeptide P43. This example compares the antiviral activities of P37 and P43 using a SARS-CoV-2 pseudovirus inhibition experiment.

[0073] 1. Experimental Materials and Methods

[0074] The test polypeptides were P37 and P43 synthesized in Example 1. The plasmid expressing SARS-CoV-2 S protein was constructed by this laboratory, the HIV backbone plasmid pNL4-3.luc.RE was provided by the AIDS Reagent and Reference Project of the National Institutes of Health; 293T cells were purchased from the American Model Culture Collection; 293T / ACE2 was prepared and preserved by this laboratory; Huh-7 was a product of the National Laboratory Cell Resource Sharing Service Platform.

[0075] The basic steps of the experiment are as follows:

[0076] ① Preparation of pseudovirus: The plasmid encoding SARS-CoV-2 S protein and HIV-1 backbone plasmid pNL4-3.luc.R were co-transfected into 293T cells at a ratio of 1:1. After culturing in a cell culture incubator at 37°C and 5% CO2 for 48 hours, the supernatant containing the pseudovirus was collected, filtered, and stored at -80°C for later use.

[0077] ②Use deionized water to dissolve the peptide to be tested and determine the concentration, then dilute the peptide to the starting concentration with DMEM medium, and perform 3-fold dilution in the wells of a 96-well cell culture plate. The final volume of the peptide solution is 50 μL / well, with 3 replicate wells and 9 dilution gradients. DMEM medium without peptide was used as a control.

[0078] ③ Add 50 μL of pseudovirus into the drug dilution plate wells and incubate at room temperature for 30 minutes.

[0079] ④ Adjust the concentration of pre-cultured 293T / ACE2 or Huh-7 cells to 10×10 4 / mL suspension, and add DEAE-dextran with a final concentration of 15μg / mL, then add the cells to the 96-well plate containing the virus, 100μL / well, and culture in a cell culture incubator at 37°C and 5% CO2 for 48 hours.

[0080] ⑤ After discarding the supernatant, add 30 μL / well of cell lysis buffer, lyse at room temperature for 15 minutes, add luciferase substrate (Promega), measure the relative fluorescence unit (RLU) with a microplate photometer, and calculate the inhibition rate curve and drug half-inhibitory concentration (IC 50 ).

[0081] 2. Experimental results and analysis

[0082] The experimental results are shown in Figure 1 , P37 and P43 inhibited the IC of SARS-CoV-2 infected 293T / ACE2 cells 50 The values ​​were 49711.00nM and 2853.00nM respectively; P37 and P43 inhibited SARS-CoV-2 infection of Huh-7 cells with IC50 The values ​​were 47537.67nM and 1166.10nM respectively; in comparison, the antiviral activity of P43 in 293T / ACE2 cells was 17 times higher than that of P37, and the antiviral activity of P43 in Huh-7 cells was 41 times higher than that of P37. The results showed that adding the "EAAAKK" sequence to the C-terminus of the peptide can significantly improve the antiviral activity of the peptide.

[0083] Example 3. Further optimization of anti-SARS-CoV-2 polypeptides

[0084] The research team of the present invention has previously discovered that the membrane proximal extracellular region (MPER) sequence of the SARS-CoV-2 S protein and the N-terminal extension motif of HR2 play a key role in the viral infection and cell-cell fusion process mediated by the S protein. In order to further optimize the antiviral peptide, the present invention designed a peptide P48 containing both the MPER sequence and the HR2 N-terminal extension sequence, and further added the "EAAAKK" sequence to the C-terminus of P48 to obtain the peptide P54. The antiviral activity of the newly prepared peptides P48 and P54 was analyzed using the SARS-CoV-2 pseudovirus inhibition experiment in Example 2 above.

[0085] The results are as follows Figure 2 As shown, in 293T / ACE2 cells, P48 inhibited the IC of SARS-CoV-2 50 The value was 1864.67nM, while the IC of P54 inhibiting SARS-CoV-2 50 The value was 97.10nM, and the antiviral activity of P54 was 19 times higher than that of P48; in Huh-7 cells, P48 inhibited SARS-CoV-2 with an IC 50 The value was 1656.67nM, while the IC of P54 inhibiting SARS-CoV-2 50 The value is 82.31nM, and the antiviral activity of P54 is 20 times higher than that of P48. This experimental result further shows that the addition of the "EAAAKK" sequence significantly improves the inhibitory activity of the peptide as a SARS-CoV-2 membrane fusion inhibitor.

[0086] Example 4. Helical structural characteristics and binding stability analysis of anti-SARS-CoV-2 polypeptides

[0087] 1. Experimental Materials and Methods

[0088] Circular dichroism (CD) spectroscopy was used to determine the secondary structure (α-helix) and thermal stability of the peptide inhibitor as well as the interaction between the peptide and the target sequence mimicking peptide. The target sequence mimicking peptide used was N57

[0089] (Ac-NQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDIL-NH2) is derived from the HR1 sequence of the S2 subunit of the SARS-CoV-2 spike protein. The test polypeptides are P37, P43, P48 and P54 synthesized in Example 1, and the test complex is a mixture of the test polypeptide and N57.

[0090] The specific experimental steps are:

[0091] (1) Prepare a 10 μM solution of the test peptide (or test complex) using phosphate buffered saline (PBS, pH 7.2) (for the test complex, 10 μM means that the concentrations of the test peptide and N57 are both 10 μM) and place in a 37°C water bath for 30 minutes.

[0092] (2) The solution obtained in step 1 was transferred to a corresponding cuvette, and the change of the molar ellipticity [θ]λ of the solution in the wavelength range of 195-270 nm was scanned using a Jasco spectropolarimeter (model J-815). The typical α-helical structure can show maximum negative peaks at 208 nm and 222 nm. The PBS blank control was subtracted to correct the spectrum value. During the calculation process, the peak value of -33000 degree.cm2.dmol-1 was used as the standard of 100% α-helical content, and the percentage of α-helical content was calculated based on the molar ellipticity of the solution at 222 nm.

[0093] (3) Add the solution obtained in step 1 to the thermal stability detection cuvette, and adjust the CD temperature control module to scan the change of [θ]222 of the solution at 2-98°C at a speed of 2°C per minute. Smooth the melting curve, and calculate the midpoint temperature (Tm) of the thermal dissociation transition using Origin software to reflect the thermal stability of the helix.

[0094] 2. Experimental results and analysis

[0095] The CD results of the tested peptides alone are as follows Figure 3 As shown, the α-helix content of P37 alone is 31%, while the α-helix content of P43 with the addition of the "EAAAKK" sequence is increased to 51%; the α-helix content of P48 alone is 37%, and the Tm value is 34°C, while the α-helix content of P54 with the addition of the "EAAAKK" sequence is increased to 56%, and the Tm value is increased to 43°C; This shows that the introduction of the "EAAAKK" sequence significantly increases the α-helix structure and thermal stability of the polypeptide. The relatively high self-α-helix content can effectively reduce the binding entropy of the polypeptide with the target, increase the binding constant, and improve the activity; the relatively stable secondary structure can also improve the stability of the polypeptide in vivo, which is beneficial to the antiviral activity.

[0096] The CD results of the complex of the test peptide and N57 are as follows Figure 4 As shown, all four peptides can interact with the target sequence mimic peptide N57 to form a stable α-helical structure. The α-helical content of the P37-N57 and P43-N57 complexes is 39% and 46%, respectively, and the Tm values ​​are 67°C and 67°C, respectively; the α-helical content of the P48-N57 and P54-N57 complexes is 39% and 49%, respectively; the Tm values ​​are 82°C and 83°C, respectively; this indicates that the addition of the "EAAAKK" sequence can significantly enhance the ability of the antiviral peptides P43 and P54 to bind to the viral target sequence, while maintaining stable binding to the target.

[0097] Example 5. Broad-spectrum anti-SARS-CoV-2 activity of P43 and P54 polypeptides

[0098] SARS-CoV-2 continues to produce mutant strains of particular concern during the epidemic, especially the current Omicron strain has multiple sub-lineages and has become the dominant strain in the world. This example uses SARS-CoV-2 WT strain and 6 representative Omicron mutant strains, and uses the fusion inhibition experiment based on the DSP system and the SARS-CoV-2 pseudovirus inhibition experiment in Example 2 to further evaluate the broad-spectrum anti-SARS-CoV-2 activity of P43 and P54 polypeptides.

[0099] 1. Experimental Materials and Methods

[0100] (1) Fusion suppression experiment based on DSP system

[0101] The tested polypeptides were P37 and P48 synthesized in Example 1. The plasmids expressing SARS-CoV-2 WT and its mutant virus S protein were constructed by our laboratory; pDSP 1-7 and pDSP 8-11 The fluorescent reporter plasmid was provided by Professor Zene Matsuda of the University of Tokyo, Japan, and is routinely used and stored in our laboratory. 293T cells were purchased from the American Model Culture Collection. The target cell 293T / ACE2 was prepared and stored by our laboratory.

[0102] The basic steps of the experiment are as follows:

[0103] ① 293T effector cell suspension (1.5×10 4 / 100 μL / well) were plated in a 96-well plate, and 293T / ACE2 target cell suspension (1.5×10 5 The cells were plated in a 10-cm cell culture dish and cultured at 37°C with 5% CO2.

[0104] ② After 16 hours of culture, the plasmid expressing SARS-CoV-2WT and its mutant virus S protein and pDSP 1-7 The plasmid was co-transfected into 293T effector cells, and pDSP 8-11 The plasmid was transfected into 293T / ACE2 target cells, and then the cells were cultured.

[0105] ③ After 24 hours, the peptide was diluted 3-fold in a 96-well plate, with 3 replicate wells and 9 dilution gradients. The diluted peptide was added to the effector cells and incubated in a cell culture incubator at 37°C and 5% CO2 for 1 hour.

[0106] ④ Preheat DMEM complete medium and add EnduRen live cell substrate (Promega) at a ratio of 1:4000, and then use it to resuspend the 293T / ACE2 target cells collected by centrifugation to adjust the cell concentration to 3×10 5 / mL and incubate at 37°C, 5% CO2 for 30 minutes.

[0107] ⑤ Add 293T / ACE2 target cells to 293T effector cells at 100 μL / well, then centrifuge at 300 g for 1 minute to allow the effector cells and target cells to fully contact, and then culture the mixed cells for 2 hours.

[0108] ⑥ Read the luciferase activity (RLU) in a microplate luminometer and calculate the inhibition rate and IC50 value.

[0109] (2) SARS-CoV-2 pseudovirus inhibition experiment

[0110] The test polypeptide is P54 synthesized in Example 1, the target cells are Huh-7 or Caco-2, and the experimental method is the same as in Example 2.

[0111] 2. Experimental results and analysis

[0112] The results of P43 and P54 inhibition of fusion mediated by the S protein of SARS-CoV-2WT and six Omicron strains are shown in Table 1. As viral membrane fusion inhibitors, P43 and P54 have broad-spectrum fusion inhibition activity against SARS-CoV-2 mutants, especially P54, which shows strong and broad-spectrum advantages, inhibits the fusion IC of WT, BA.4 / 5, BF.7, BQ.1.1, XBB, XBB.1.5 and CH.1.1 S proteins. 50 They were 2.2nM, 0.29nM, 0.28nM, 0.41nM, 0.3nM, 0.29nM and 0.32nM respectively; it is worth noting that the fusion inhibition activity of P54 against Omicron subtype strains was significantly higher than that against WT strains.

[0113] Table 1

[0114]

[0115]

[0116] Furthermore, the present invention evaluated the inhibitory activity of P54 on pseudovirus infection of SARS-CoV-2WT and 6 Omicron strains in two target cells, Huh-7 and Caco-2. The results are shown in Table 2. In Huh-7 cells, the IC values ​​of P54 for inhibiting pseudovirus infection of WT, BA.4 / 5, BF.7, BQ.1.1, XBB, XBB.1.5 and CH.1.1 were 50 The values ​​were 85.67nM, 36.78nM, 28.40nM, 38.33nM, 27.59nM, 39.01nM and 30.57nM, respectively, with an average IC 50 The value is 40.91nM; in Caco-2 cells, P54 inhibits the IC of the above six SARS-CoV-2 pseudovirus infections 50 The values ​​were 25.32nM, 36.71nM, 24.15nM, 32.53nM, 26.54nM, 27.4nM and 28.23nM, respectively, with an average IC 50 The value is 28.70nM, indicating that P54 has good inhibitory activity against various SARS-CoV-2, especially the Omicron mutant strain, and can be further developed into a clinically applicable broad-spectrum antiviral drug.

[0117] Table 2

[0118]

[0119] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A polypeptide compound, characterized in that: It comprises a polypeptide and an "EAAAKK" sequence having an alpha-helical structure connected to the C-terminus and / or N-terminus of the polypeptide.

2. The polypeptide compound according to claim 1, characterized in that The polypeptide compound is a compound having a structure shown in Formula I, wherein Formula I is: X1-X2-Xn-X3-X4; in Formula I, X1 is an amino terminal protecting group; Xn is any polypeptide sequence; X4 is a carboxyl terminal protecting group; X2 is an "EAAAKK" sequence or is absent, X3 is an "EAAAKK" sequence or is absent, and at least one of X2 and X3 is an "EAAAKK" sequence; Optionally, X1 is any one of acetyl, amino, maleyl, succinyl, tert-butyloxycarbonyl or benzyloxy and other hydrophobic groups or macromolecular carrier groups; The X4 is any one of an amino group, a carboxyl group, a hydroxyl group, an amide group or a tert-butyloxycarbonyl group and other hydrophobic groups or macromolecular carrier groups.

3. The polypeptide compound according to claim 2, characterized in that The polypeptide compound is polypeptide P43 and / or polypeptide P54, In the polypeptide P43, X1 is Ac, X2 is absent, Xn is a polypeptide sequence of "SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK", X3 is a sequence of "EAAAKK", and X4 is NH2; In the polypeptide P54, X1 is Ac, X2 is absent, Xn is the polypeptide sequence of "VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK"; X3 is the sequence of "EAAAKK", and X4 is NH2.

4. A polypeptide compound derivative or a different pharmaceutically usable form of a polypeptide compound, characterized in that: The polypeptide compound is the polypeptide compound as claimed in claim 1.

5. The polypeptide compound derivative or different pharmaceutically usable forms of the polypeptide compound according to claim 4, characterized in that: The polypeptide compound derivative is a derivative obtained by modifying the amino end and / or carboxyl end of the polypeptide compound with a lipophilic compound. Optionally, the lipophilic compound is selected from one or more lipid compounds such as cholesterol succinate, 2-cholesterol acetic acid, 2-cholesterol propionic acid, 3-cholesterol propionic acid, 2-cholesterol butyric acid, 2-cholesterol isobutyric acid, 3-cholesterol butyric acid, 3-cholesterol isobutyric acid, 4-cholesterol butyric acid, 2-cholesterol valeric acid, 2-cholesterol isovaleric acid, 3-cholesterol valeric acid, 5-cholesterol valeric acid, 2-cholesterol hexanoic acid, 6-cholesterol hexanoic acid, 2-cholesterol heptanoic acid, 7-cholesterol heptanoic acid, 2-cholesterol octanoic acid, 8-cholesterol octanoic acid, cholesteryl bromoacetate, fatty acids containing 8 to 20 carbon atoms, dihydrosphingosine, and vitamin E; Optionally, the different pharmaceutically acceptable forms of the polypeptide compound include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above substances, and any mixtures of the above forms.

6. A polymer or fusion protein, characterized in that The multimer or fusion protein comprises the polypeptide compound according to claim 1 and / or the polypeptide compound derivative according to claim 4 or one or more of different pharmaceutically usable forms of the polypeptide compound.

7. A pharmaceutical compound or pharmaceutical composition, characterized in that It is one or more of the polypeptide compound according to claim 1, the polypeptide compound derivative or different pharmaceutically usable forms of the polypeptide compound according to claim 4, and / or the multimer or fusion protein according to claim 6.

8. Use of the polypeptide compound according to claim 1, the polypeptide compound derivative according to claim 4, or different pharmaceutically acceptable forms of the polypeptide compound in the preparation of a drug for treating and / or preventing a corresponding disease, characterized in that: The diseases include, but are not limited to, tumors, hepatitis, diabetes, AIDS and / or diseases caused by coronavirus infection.

9. A nucleic acid molecule, characterized in that Encoding the polypeptide compound according to any one of claims 1 to 3, wherein the nucleic acid molecule is DNA or RNA.

10. A method for enhancing the α-helical structure and / or thermal stability and / or biological activity of a polypeptide, characterized in that: The method comprises the following steps: connecting the "EAAAKK" sequence to the N-terminus and / or C-terminus of the polypeptide.

11. A method for designing a polypeptide drug, comprising the following steps: The "EAAAKK" sequence having an α-helical structure is connected to the C-terminus and / or N-terminus of the polypeptide.

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