An anti-coronavirus polypeptide, pharmaceutical composition and its application

Abstract

The present invention relates to the field of pharmaceutical technology, and discloses an anti-coronavirus polypeptide, a pharmaceutical composition and their applications. The anti-coronavirus polypeptide is based on the template EK1: SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL-NH2 amino acid sequence. Through solid-phase synthesis, stapling and alanine modification are carried out on it, and the amino acids at specific positions are replaced with S5 and alanine. It is proved in the antiviral activity test that this kind of polypeptide molecule has anti-SARS-CoV-2 activity.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and specifically to an anti-coronavirus polypeptide, a pharmaceutical composition and their applications. It has the activity of inhibiting novel coronavirus and can be used to prepare related anti-coronavirus drugs. Background Art

[0002] The novel coronavirus (SARS-CoV-2) is a virus with extremely strong infectivity and pathogenicity, and the novel coronavirus infection caused by it has caused inestimable losses globally.

[0003] It is very important to find means to inhibit the novel coronavirus. At present, there are mainly three types of antiviral drugs: vaccines, small molecule drugs, and polypeptide drugs. Among them, polypeptides play an important role in drug development due to their high affinity, good selectivity, low toxicity and other advantages.

[0004] The S protein in coronaviruses plays a crucial role in cytotoxicity and pathogenesis. The S protein is composed of two subunits, S1 and S2. Among them, the heptapeptide repeat sequences HR1 and HR2 in S2 can form a six-helix bundle structure of 6-HB, and this structure is conducive to the fusion of the cell membrane and the virus membrane, thus facilitating the invasion of the virus. When a polypeptide fusion inhibitor binds to HR1 in S2, HR1 cannot bind to HR2 to generate 6-HB, thereby achieving the effect of inhibiting virus invasion.

[0005] Based on this theory, the Jiang Shibo research group intercepted OC43-HR2P from the HR2 of OC43 coronavirus, found that it has broad-spectrum antiviral effects, and modified it to obtain the broad-spectrum antiviral polypeptide EK1 (SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL-NH2).

[0006] Although polypeptides have advantages such as high affinity, good selectivity, and low toxicity, they have disadvantages such as short half-life, unstable conformation, easy to be hydrolyzed by enzymes, and poor membrane permeability. Therefore, multiple research groups have developed the strategy of stapled peptides to improve drug-likeness. Stapled peptides are a type of polypeptide mimetic with high stability and good membrane permeability. By introducing unnatural amino acids with different lengths of side chains carrying α-methyl at the i, i + 4 or i, i + 7 positions, and then through a metathesis reaction to synthesize stapled peptides, the stability, helicity and cell permeability of the stapled peptides synthesized in this way have been greatly improved.

[0007] Because alanine has a small volume and high structural energy in the secondary structure, which is more conducive to stabilizing the secondary structure. Therefore, we combined the advantages of stapled peptides and alanine substitution, and designed and synthesized a series of derivatives based on EK1, in order to obtain better antiviral activity. Summary of the Invention

[0008] (1) Technical problem to be solved

[0009] In view of the deficiencies of the prior art, the present invention provides EK1 and its derivatives, as well as their preparation methods and applications. By using the stapled peptide strategy and replacing corresponding residues, the derivatives have advantages such as good antiviral activity, and it is desired to improve the stability and membrane permeability of the derivatives.

[0010] (2) Technical solution

[0011] To achieve the above-mentioned objectives of enhancing its cell permeability, improving enzyme stability and antiviral properties, the present invention provides the following technical solution:

[0012] An anti-coronavirus polypeptide, which is one of the polypeptide active molecules having the structures shown in the following Formulas 1-4, and the structural formulas are as Figures 1-4 shown:

[0013] 1. Using SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL-NH2 as the peptide chain template, wherein the E at the 21st position is replaced by S5 and the 17K is replaced by S5 and cyclized, and the L at the 10th position is replaced by A.

[0014] 2. Using SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL-NH2 as the peptide chain template, wherein the E at the 21st position is replaced by S5 and the 17K is replaced by S5 and cyclized, and the L at the 12th position is replaced by A.

[0015] 3. Using SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL-NH2 as the peptide chain template, wherein the E at the 21st position is replaced by S5 and the 17K is replaced by S5 and cyclized. And the Y at the 14th position is replaced by A.

[0016] 4. Using SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL-NH2 as the peptide chain template, wherein the E at the 21st position is replaced by S5 and the 17K is replaced by S5 and cyclized. And the L at the 19th position is replaced by A.

[0017] Wherein, S5 represents (2R)-2-amino-2-methyl-6-heptenoic acid, and the paired (2R)-2-amino-2-methyl-6-heptenoic acids in the fragment are cyclized by olefin metathesis reaction.

[0018] In this article, the "polypeptide active molecule of the present invention" refers to the polypeptide having the structures shown in Formulas 1-4 in the present invention. In this article, such a polypeptide can be referred to as a "polypeptide fragment" or "the polypeptide of the present invention".

[0019] The amino group at the N-terminus, the carboxyl group at the C-terminus, and the amino acid side chain groups of the polypeptides in Formulas 1-4 may not be modified, or may be modified on the premise of basically not affecting the activity of the polypeptides of the present invention, such as forming "pharmaceutically acceptable esters". Modifications of the N-terminal amino group include, but are not limited to, deamination, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the C-terminal carboxyl group include, but are not limited to, amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications. The carboxyl group at the C-terminus of the polypeptides of the present invention is amidated, which is -NH2.

[0020] The representation methods of polypeptides, amino acids, and chemical groups used herein are all representation methods recognized in the art. The abbreviations of amino acids can refer to the definitions in Table 1. The structures of special amino acids can refer to the definitions in Table 2. In this article, unless otherwise specified, amino acids generally refer to L-type amino acids.

[0021] Table 1 Amino Acid Abbreviation Table

[0022] Amino acid Three-letter abbreviation One-letter abbreviation Amino acid Three-letter abbreviation One-letter abbreviation Alanine Ala A Leucine Leu L Arginine Arg R Lysine Lys K Asparagine Asn N Methionine Met M Aspartic acid Asp D Phenylalanine Phe F Cysteamine Cys C Proline Pro P Glutamine Gln Q Serine Ser S Glutamic acid Glu E Threonine Thr T Glycine Gly G Tryptophan Trp W Histidine His H Tyrosine Tyr Y Isoleucine Ile I Valine Val V

[0023] Table 2 Special Amino Acid Abbreviation Table

[0024] Amino acid Abbreviation (2R)-2-Amino-2-methyl-6-heptenoic acid <![CDATA[S5]]> (2R)-2-Amino-2-methyl-9-decenoic acid <![CDATA[R8]]>

[0025] "Pharmaceutically acceptable salts" refer to salts formed by some small molecule acidic or basic compounds and polypeptides, which generally can increase the solubility of the polypeptides, and the formed salts basically do not change the activity of the polypeptides.

[0026] For example, the acids that can usually form salts with the polypeptides of the present invention include hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid, succinic acid, maleic acid, and citric acid, etc.; the bases that can form salts with the polypeptides of the present invention include hydroxides of alkali metals or alkaline earth metals, ammonium, and carbonates, etc.

[0027] The antiviral effect of the polypeptides of the present invention can be verified by conventional experimental methods in the art, such as cytological experiments, etc. In the specific embodiments of the present invention, a pseudovirus neutralization test is preferably used to detect the preliminary antiviral effect of the polypeptides. Through this test, it is found that the polypeptide active molecules of Formulas 1-4 involved in the present invention all have an in vitro anti-pseudovirus effect.

[0028] In addition, another technical problem to be solved by the present invention is to provide a pharmaceutical composition containing the polypeptide active molecules with the structures of Formulas 1-4, which can be used for treating or preparing anti-SARS-CoV-2 drugs.

[0029] This composition may contain one or more of the cyclic peptide active molecules of the present invention, and preferably contains only one.

[0030] The composition may contain one or more pharmaceutically acceptable diluents, excipients or carriers. Preferably, the composition is in unit dosage form, such as tablets, films, pills, capsules (including sustained release or delayed release forms), powders, granules, syrups or emulsions, sterilized injectable solutions, suspensions or lyophilized powder injections, aerosols or liquid sprays, drops, auto-injection devices or suppositories.

[0031] The above active pharmaceutical ingredient can be combined with a non-toxic pharmaceutically acceptable inert carrier, such as ethanol, glycerol, water or a combination thereof. The polypeptide active molecule of formula (I) of the present invention preferably uses a sterilized aqueous injection solution.

[0032] The pharmaceutical composition of the present invention can be administered by administration methods well known to those skilled in the art, such as oral, rectal, sublingual, pulmonary, transdermal, iontophoresis, vaginal and intranasal administrations. The pharmaceutical composition of the present invention is preferably administered parenterally, such as subcutaneous, intramuscular or intravenous injection.

[0033] The names, structural formulas and mass spectrometry data of some preferred compounds synthesized in the present invention are shown in Table 3

[0034] Table 3 Names, structural formulas and mass spectrometry data of preferred polypeptide active molecules

[0035]

[0036] For ease of understanding, the present invention will be described below through specific examples and drawings. It should be particularly noted that these descriptions are merely exemplary descriptions and do not constitute a limitation on the scope of the present invention. Brief Description of the Drawings

[0037] Figure 1 Schematic diagram of the structural formula of EK1-1 in Table 3 of the present invention;

[0038] Figure 2 Schematic diagram of the structural formula of EK1-2 in Table 3 of the present invention;

[0039] Figure 3 Schematic diagram of the structural formula of EK1-3 in Table 3 of the present invention;

[0040] Figure 4 Schematic diagram of the structural formula of EK1-4 in Table 3 of the present invention;

[0041] Figure 5 Result diagram of the inhibitory activity of EK1 derivatives with different concentrations of the present invention against SARS-CoV-2 pseudovirus. Detailed Description of the Invention

[0042] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0043] Embodiment 1: Preparation method of polypeptide anti-coronavirus active compound. Solid-phase synthesis of polypeptide fragments, and the specific steps are as follows:

[0044] 1) Weigh a certain amount of amino resin and swell it in 4 mL of DCM solution for 20 min;

[0045] 2) Use 20% piperidine / DMF solution / 0.1 mol / L Oxyma to remove the Fmoc protecting group for 5 min, and repeat twice;

[0046] 3) Wash the resin 5 times with 5 mL of DMF, then wash the resin 5 times with 5 mL of DCM, and finally wash the resin 5 times with 5 mL of DMF;

[0047] 4) Use the reaction solution for natural amino acid condensation: 4 equivalents of Fmoc-AA-OH, 4 equivalents of Oxyma, 4 equivalents of DIC for condensation reaction, and react at 60 °C for 20 min. For Fmoc-S5-OH, use 2 equivalents of amino acid solution, 2 equivalents of Oxyma, 2 equivalents of DIC, and react at 60 °C for 2 h;

[0048] 5) Repeat the operations in (2)-(4) and couple them in sequence according to the amino acid sequence; among them, at some cyclization sites, S5 is used to replace the amino acids at the i and i+4 positions respectively;

[0049] 6) Acetylate the amino acid with the N-terminal deprotected using a solution of pyridine: acetic anhydride = 1:1;

[0050] 7) Treat the resin with a dichloroethane solution of Grubbs first-generation catalyst for cyclization reaction;

[0051] 8) Use a cleavage reagent (TFA / TIPS / H2O = 95 / 2.5 / 2.5, v / v / v) to cleave the peptide chain from the resin;

[0052] 9) Add ice ether (40 mL) to the crude peptide, centrifuge at 3500 r / min for 3 min, and repeat the operation 5 times;

[0053] 10) Purify the crude peptide that has been naturally evaporated to dryness after centrifugation using RP-HPLC;

[0054] 11) The purification method used is reverse high performance liquid chromatography, with the following conditions: Chromatographic column:

[0055] YMC-Pack ODS-AQ column; Mobile phase: Mobile phase A is 0.1% TFA / water, and mobile phase B is 0.1%

[0056] TFA / acetonitrile; Gradient elution program: Elute with 35% B for 0 - 5 min, 35% B - 65% B for 5 - 60 min; Flow rate is 20 mL / min, injection volume is 1 mL, detection wavelengths are 214 nm and 254 nm; The product is identified by LC-MASS.

[0057] Experimental Example 2: Detection of the antiviral effect of EK1 and its derivatives in vitro using pseudovirus transfection

[0058] Apply a high-content cell drug screening and analysis system to determine the effect of 5 peptides on the fusion of 293T cells expressing NCovS and Huh-7 cells expressing ACE2.

[0059] The sample to be tested is dissolved in DMSO to prepare a 30 mM stock solution, controlling DMSO below 1 / 1000. For Huh-7, a stable transfected strain expressing ACE2-GFP is used. For 293T, two types of cells transfected with pAAV-Hncovs-CAG-RFP and pAAV-CAG-RFP respectively are used. After transfection, culture for 72 h. Seed Huh-7 cells in a 96-well plate at 1×10 4 cells / well, culture for 4 h, then add 5×104 293T cells containing each concentration of the drug per well and culture for 5 h to obtain the fusion situation of the two types of 293T cells with Huh7 at concentrations of 30, 15, 7.5, 3.75, and 0 μM.

[0060] Statistics: A: The ratio of the number of double-fluorescent cells to the number of red-fluorescent cells in each well containing Huh-7 (ACE2-GFP) and 293T (pAAV-Hncovs-CAG-RFP) cells. The ratio at a drug concentration of 0 μM is denoted as A0. B: The ratio of the number of double-fluorescent cells to the number of red-fluorescent cells in each well containing Huh-7 (ACE2-GFP) and 293T (pAAV-CAG-RFP) cells. Calculate the cell fusion inhibition rate using Inhibition of cell-cell fusion%=1-(A - B) / (A0 - B)×100%

[0061] Experimental results: The pseudovirus activity is shown in Table 4. In the pseudovirus infection experiment, several derivatives of EK1 showed good inhibitory activity at a concentration of 7.5 μM. Through Figure 5It can be seen that EK1-1 shows better inhibitory effect on SARS-CoV-2 pseudovirus compared with EK1. And the IC50 of EK1-1 is 34.08 μM, while the IC50 of EK1 is 46.61 μM. All these results indicate that the polypeptide EK1-1 modified with stapled peptide and alanine has better inhibitory effect on SARS-CoV-2 pseudovirus.

[0062] Table 4 Inhibitory activities of EK1 derivatives against SARS-CoV-2 pseudovirus

[0063]

[0064]

[0065] The beneficial effects of the present invention are as follows: The polypeptide antiviral active compound, its preparation method and application, the stapled peptide strategy is a method to stabilize α-helical peptides. This is an α-helical peptide with a fully hydrocarbon scaffold invented by Verdine, which can enhance the α-helical structure, membrane permeability, anti-protease hydrolysis ability, anti-protein-protein interaction ability, etc. of the polypeptide. In addition, due to its small volume and high structural energy in the secondary structure, alanine is more conducive to stabilizing the secondary structure. We simultaneously use alanine modification to synthesize a series of EK1-based derivatives. The results show that the derivatives have certain anti-SARS-CoV-2 virus activity, providing a new method for the current research and development of anti-SARS-CoV-2 virus drugs.

[0066] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An anti-coronavirus polypeptide, characterized in that, Specifically refers to a polypeptide active molecule having the structure shown in formula (1). 1) Using SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL-NH2 as the peptide chain template, wherein E at position 21 is replaced by S5 and K at position 17 is replaced by S5 and cyclized, and L at position 10 is replaced by A; Among them, S5 represents (2R)-2-amino-2-methyl-6-heptenoic acid, and the paired (2R)-2-amino-2-methyl-6-heptenoic acid in the fragment is cyclized by olefin metathesis reaction.

2. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the anti-coronavirus polypeptide as described in claim 1.

3. The pharmaceutical composition according to claim 2, wherein, It also contains a pharmaceutically acceptable diluent, excipient or carrier.

4. The pharmaceutical composition according to claim 3, wherein Wherein the carrier is one or more of ethanol, glycerol or water.

5. Use of a pharmaceutical composition according to any one of claims 2-4 in the preparation of a drug against novel coronavirus.

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