Anti-hiv-1 polypeptides, derivatives and uses thereof

Abstract

The application discloses an anti-HIV-1 polypeptide and derivatives and application thereof. The amino acid sequence of the anti-HIV-1 polypeptide is shown as SEQ ID NO. 1 or SEQ ID NO. 2. The anti-HIV-1 polypeptide derivative is a polypeptide derivative obtained by modifying one or more of the amino terminal, the carboxyl terminal and the side chain of the anti-HIV-1 polypeptide with a chemical group, or a non-physiological salt of the anti-HIV-1 polypeptide. The anti-HIV-1 polypeptide and the derivative thereof disclosed by the application have significant and broad-spectrum anti-HIV activity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to an anti-HIV-1 polypeptide and derivatives and applications thereof. BACKGROUND

[0002] Currently, highly active anti-retroviral therapy (HAART) is mostly used in clinic, that is, two or more antiviral drugs are combined to inhibit HIV virus. This method needs to be taken for a lifetime, and due to the existence of latent viruses, this therapy cannot completely eradicate viral infection. Moreover, long-term use of HAART in clinic can cause drug resistance, adverse reactions, and high treatment costs. Therefore, developing effective drugs against AIDS is a major demand for global AIDS prevention and control.

[0003] The process of HIV-1 entering target cells depends on the envelope glycoprotein Env on the surface of the virus, which is composed of surface subunit gp120 and transmembrane subunit gp41 through non-covalent bonds. Gp120 has 5 variable regions (V1-V5) and 5 relatively conserved regions (C1-C5); gp41 is composed of three parts: extracellular region, transmembrane region and intracellular region. The extracellular region of gp41 is the functional region directly involved in the fusion of virus and target cells, which is divided into: N-terminal fusion peptide (FP), fusion peptide proximal region (FPPR), N-terminal heptad repeat region (NHR), C-terminal heptad repeat region (CHR) and membrane proximal external region (MPER), as shown in Figure 1 When the virus infects the target cell, first, gp120 binds to the CD4 receptor of the target cell membrane, the conformation of gp120 changes, and the binding site of the auxiliary receptor CCR5 or CXCR4 is exposed, so that gp120 binds to the auxiliary receptor. The binding of gp120 to the receptor and auxiliary receptor activates the fusion function of gp41, the fusion peptide FP is exposed and inserted into the target cell membrane, showing a so-called "intermediate state" (pre-hairpin intermediate, PHI); then, the CHR and NHR undergo reverse folding to form a "hairpin" like six-helix bundle core structure (6-HB), thereby shortening the virus membrane and cell membrane for fusion reaction, completing the infection process of virus entering host cells. During this process, gp41 is the key protein that mediates the fusion of HIV-1 and target cell membrane, and is an important target for drug development.

[0004] Currently, the most researched drugs based on gp41 are peptide HIV membrane fusion inhibitors. The first-generation HIV membrane fusion inhibitor T20 (Enfuvirtide) is a peptide drug derived from the natural CHR sequence of gp41, consisting of 36 amino acid residues. Its C-terminus also contains a tryptophan-enriched sequence of MPER, such as... Figure 1 As shown in B, T20 is the first and currently the only membrane fusion inhibitor approved by the US FDA for the treatment of viral infections. However, it suffers from relatively weak antiviral activity, a short half-life, a high risk of developing drug resistance, and high treatment costs, severely limiting its clinical application. Because T20 lacks a pocket binding site, C34 peptides derived from CHR containing pocket-binding regions (PBD) motifs have been widely used as sequence design templates for HIV membrane fusion inhibitors. Several membrane fusion inhibitors with significantly improved drug properties have been designed, such as T1249 and T2635, both containing PBD sequences. Domestically developed drugs such as sifviride (SFT) and avocado (ABT) are also designed based on C34 sequences. However, worryingly, inhibitors designed based on C34 are also prone to inducing viral resistance. Therefore, developing novel peptide drugs based on conserved sequences of gp41 has significant theoretical and practical value.

[0005] The discovery of T20 opens up a new field of using peptide drugs to inhibit HIV entry into target cells. It is worth noting that the sequence of the first generation HIV membrane fusion inhibitor T20 and the second generation HIV membrane fusion inhibitor T1249 both contain the N-terminal tryptophan-rich sequence of the MPER sequence of gp41 protein. MPER (665-KWASLWNWFNITNWLWYIK-683, HXB2 number) is a highly conserved and tryptophan-rich hydrophobic motif in the membrane-proximal external region of HIV-1 gp41, which plays an important role in the process of viral membrane fusion. MPER contains two conserved active elements: one is the N-terminal tryptophan-rich motif "WASLWNWF" (tryptophan-rich motif, TRM), which is also considered to be the lipid binding domain of T-20 and T1249 polypeptide; the other is the C-terminal cholesterol binding motif "LWYIK" (Cholesterol-recognition / interaction amino acid consensus, CRAC). At the same time, the MPER region also contains multiple broad-spectrum neutralizing antibody epitopes and is widely studied as a vaccine immunogen. Recently, a study reported that the broad-spectrum antibiotic drug dequalinium iodide and its derivative compound S2C3 can target MPER to exert anti-HIV effect, and NMR structure shows that this small molecule fusion inhibitor interacts with the hydrophobic pocket formed by the trimer MPER, providing important evidence for determining MPER as a target for small molecule compound design. Through research, it is found that in the previous research on MPER, most of the research is focused on the structure and function of MPER in the fusion process and its research as a vaccine target, and there is no research report that a polypeptide containing only the MPER sequence itself has anti-viral activity. gp41W polypeptide is a representative MPER polypeptide and is widely used as a model for MPER structure and function and vaccine immunogen research, but no research report has found that gp41W polypeptide itself has anti-HIV activity. Early research suggests that the polypeptide EK30 containing MPER and the N-terminal CHR partial sequence has weak anti-HIV activity, and its IC50 for inhibiting viruses is greater than 5000 nM. SUMMARY

[0006] In order to solve the problems of the prior art, the present application aims to provide an anti-HIV polypeptide and its derivatives and applications.

[0007] The specific technical scheme of the present application is as follows:

[0008] In one aspect, the present application provides an anti-HIV-1 polypeptide, wherein the amino acid sequence of the anti-HIV-1 polypeptide is as shown in SEQ ID NO. 1 (SLWNWFNITNWLWYIK) or SEQ ID NO. 2 (WASLWNWFNITNWLWYIK).

[0009] Wherein the abbreviations of the amino acids have the meanings commonly known in the art, for example: W is tryptophan, A is alanine, S is serine, L is leucine, N is asparagine, F is phenylalanine, I is isoleucine, T is threonine, Y is tyrosine, K is lysine, etc.

[0010] In the present application, the amino acid at any position of the polypeptide can be substituted by other amino acid to improve the bioavailability, stability and / or antiviral activity of the polypeptide.

[0011] In another aspect of the present application, there is also provided an anti-HIV-1 polypeptide derivative, which is a polypeptide derivative obtained by modifying one or more of the amino terminal, carboxyl terminal and side chain of the anti-HIV-1 polypeptide with a chemical group, or a non-physiological salt of the anti-HIV-1 polypeptide.

[0012] Further, the amino terminal is modified with acetyl group (Ac); and / or, the carboxyl terminal is modified with amino group (NH2).

[0013] Preferably, in the present application, the first amino acid residue of the anti-HIV-1 polypeptide is connected with an amino terminal protection group Ac, and the terminal is connected with a carboxyl terminal protection group NH2. Ac represents acetyl group, and NH2 represents amino group.

[0014] In another aspect of the present application, there is also provided a nucleic acid molecule encoding the anti-HIV-1 polypeptide.

[0015] Further, the nucleic acid molecule can be DNA (such as cDNA, genomic DNA or recombinant DNA, etc.) or RNA (such as mRNA or hnRNA, etc.).

[0016] In another aspect of the present application, there is also provided a fusion protein comprising the anti-HIV-1 polypeptide, a multimer formed by the anti-HIV-1 polypeptide, or a complex obtained by coupling or fusing the anti-HIV-1 polypeptide with other polypeptide, protein or carrier.

[0017] In another aspect of the present application, there is also provided a composition comprising: 1) one or more of the anti-HIV-1 polypeptide, the anti-HIV-1 polypeptide derivative, the fusion protein, the multimer, the complex; and 2) optionally, a pharmaceutically acceptable carrier or excipient.

[0018] Further, the composition further comprises one or more other anti-HIV drugs.

[0019] The polypeptide derivatives described herein include, but are not limited to, the polypeptides as shown in SEQ ID NO. 1 or SEQ ID NO. 2 and all of their pharmaceutically usable different forms. The pharmaceutically usable different forms of these polypeptides include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above-mentioned substances and any mixtures of the above-mentioned forms.

[0020] In practical applications, the drugs of the present application can be administered directly to patients or administered to patients after being mixed with suitable carriers or excipients to achieve the purpose of treating and / or preventing HIV infection. The carrier materials herein include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesteryl stearate, etc.), enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl cellulose, etc.). Among them, the water-soluble carrier materials are preferred. Using these materials, various dosage forms can be prepared, including but not limited to tablets, capsules, drop pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, suppositories, freeze-dried powder injections, etc. They can be ordinary preparations, sustained-release preparations, controlled-release preparations, and various microparticle drug delivery systems. In order to make unit administration dosage forms into tablets, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, white clay, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerol, polyethylene glycol, ethanol, propyl alcohol, starch paste, dextrin, sugar syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants such as dry starch, alginate, agar powder, alginic acid, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glycerol triestearate, cocoa butter, hydrogenated oil, etc.; absorption promoters such as quaternary ammonium salt, sodium dodecyl sulfate, etc.; lubricants such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can be further coated 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 unit administration dosage forms into pills, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth gum, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc. In order to make unit administration dosage forms into suppositories, various carriers known in the art can be widely used. Examples of carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc.For the purpose of preparing a unit dosage form into an injection preparation such as a solution, an emulsion, a lyophilized powder and a suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, etc. In addition, for the purpose of preparing an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injection preparation, and in addition, a conventional cosolvent, a buffer, a pH adjusting agent, etc. can be added. In addition, if necessary, a coloring agent, a preservative, a flavoring agent, a taste-correcting agent, a sweetening agent or other materials can be added to the pharmaceutical preparation. The above-mentioned dosage form can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; cavity administration such as rectal and vaginal administration. The above-mentioned administration route is preferably injection administration.

[0021] The administration dose of the pharmaceutical of the present application depends on many factors, for example, the nature and severity of the disease to be prevented or treated, the gender, age, body weight and individual response of the patient or animal, the specific active ingredient used, the administration route and the administration frequency, etc. The above-mentioned dose can be administered in a single dose form or divided into several, for example, two, three or four dose forms.

[0022] The pharmaceutical of the present application can be directly and solely used for the treatment and prevention of HIV infected persons, or can be used in combination with one or more other anti-HIV drugs for the purpose of improving the overall therapeutic effect. These anti-HIV drugs include but are not limited to reverse transcriptase inhibitors, protease inhibitors, invasion inhibitors, integration inhibitors and maturation inhibitors, etc. The above-mentioned reverse transcriptase inhibitors can be one or several of AZT, 3TC, ddl, d4T, ddT, TDF, Abacavir, Nevirapine, Efavirenz, Delavirdine, Azvudine, Elnoviren, etc.; the above-mentioned protease inhibitors can be one or several of Saquinavir mesylate, Idinavir, Ritonavir, Amprenavir, Kaletra and Nelfinavirmesylate, etc.; the above-mentioned invasion inhibitors can be one or several of Maraviroc, TAK-779, T20, T2635, Sifuvirtide, Albowertide, etc.; the above-mentioned integration inhibitors can be one or several of Raltegravir, Dolutegravir and Elvitegravi, etc.

[0023] For any given patient, the specific effective therapeutic dose level must be determined based on a number of factors, including the disorder being treated and its severity; the activity of the specific active ingredient used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific active ingredient; the duration of treatment; medications used in combination with or concurrently with the specific active ingredient; and similar factors known in the medical field. For example, it is practiced in the art to start the dose of the active ingredient below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0024] In another aspect, the present invention also provides the use of the aforementioned anti-HIV-1 polypeptide, the aforementioned anti-HIV-1 polypeptide derivative, the aforementioned fusion protein, the aforementioned polymer, the aforementioned complex, or the aforementioned composition in the preparation of a medicament for treating or preventing diseases caused by HIV infection.

[0025] Furthermore, the disease caused by the HIV infection is AIDS.

[0026] Furthermore, the HIV mentioned is HIV-1.

[0027] The beneficial effects of this invention are as follows:

[0028] This invention has surprisingly discovered that specific truncated MPER peptides possess significant and broad-spectrum anti-HIV activity. Therefore, this invention provides an anti-HIV-1 peptide and its derivatives. The MPER18 and MPER16 provided by this invention exhibit broad inhibitory activity against 25 HIV-1 pseudoviruses, with average IC50 values ​​of 1530.16 nM and 829.62 nM, respectively, among which the shorter sequence MPER16 shows significant superiority. Attached Figure Description

[0029] Figure 1 The structure of the HIV-1 fusion protein gp41 and its MPER-derived polypeptide;

[0030] Figure 2 MPER-derived peptide sequence characteristics and anti-HIV activity;

[0031] Figure 3 The in vitro cytotoxicity of MPER16 and MPER18 as described in this invention;

[0032] Figure 4 This invention describes the inhibitory activity of MPER16 and MPER18 against different HIV-1 subtypes. Detailed Implementation

[0033] The application will be further described in conjunction with the specific embodiments, the examples given are only for the purpose of illustrating the application, and are not intended to limit the scope of the application. The examples provided below can be used as a guide for further improvement by those skilled in the art, and in no way constitute a limitation on the application. Those skilled in the art can refer to the content herein, and appropriately improve the relevant parameters to achieve. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the scope of the application. The method of the application has been described by the preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the compounds and preparation methods described herein without departing from the content, spirit and scope of the application, to realize and apply the technical field of the application.

[0034] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0035] More details of the application are described below, or some can be embodied in the examples of the application.

[0036] Unless otherwise specified, the amounts of different components used herein to express the reaction conditions can be interpreted as "approximately" or "about" in any case. Accordingly, the numerical parameters cited in the following description and claims are approximations that can vary depending on the different standard errors in the respective experimental conditions, and different numerical parameters can be obtained.

[0037] In this text, when the chemical structure formula and the chemical name of a compound are inconsistent or doubtful, the chemical structure formula is used to define the compound. The compounds described in this text can have one or more chiral centers, and / or double bonds and the like structures, and stereoisomers can exist, including double bond isomers (such as geometric isomers), optical antipodes or diastereoisomers. Accordingly, any chemical structure described in this text, whether it contains the above-mentioned structures in part or as a whole, includes all possible enantiomers and diastereoisomers of the compound, including only one stereoisomer (such as only geometric isomer, only enantiomer or only diastereoisomer) and any mixture of these isomers. These racemic isomers and stereoisomer mixtures can be further separated into their constituent enantiomers or stereoisomers by the separation techniques or chiral molecule synthesis methods used by those skilled in the art.

[0038] The abbreviations of amino acids in the following examples have the meanings commonly known in the art, for example: W is tryptophan, A is alanine, S is serine, L is leucine, N is asparagine, F is phenylalanine, I is isoleucine, T is threonine, Y is tyrosine, K is lysine, etc.

[0039] Example 1: Design of polypeptides

[0040] GP41 MPER plays an important role in the process of HIV infection. Due to its special structure, most of the previous studies on MPER focused on the structure and function of MPER in the fusion process and its research as a vaccine target. There is no report on whether the polypeptide containing only MPER sequence itself has antiviral activity. In this example, a series of polypeptides with different degrees of extension and truncation of MPER were designed, which are TRM20, TRM19, TRM17, TRM15, TRM14, TRM12b, TRM11, MPER20, MPER18, MPER17, MPER16, MPER15, MPER14, MPER13, MPER12, MPER11, MPER10, MPER16R and MPER16ACR shown in B. Figure 1 The amino terminal of all polypeptides is connected with acetyl (Ac) as the amino terminal protecting group, and the carboxyl terminal is connected with amino (NH2) as the carboxyl terminal protecting group.

[0041] Example 2: Synthesis of polypeptides

[0042] 1. Chemical reagents required in the preparation process

[0043] All the chemical reagents such as various Fmoc amino acids, N, N'-diisopropyl carbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), N, N-dimethylformamide (DMF), piperidine (PIPE), indantrione, acetic anhydride (Ac2O), N, N-diisopropyl ethylamine (DIEA), hydrazine hydrate, trifluoroacetic acid (TFA), ethanedithiol (EDT), thioanisole (TA), triisopropylsilane (TIPS), phenol, etc. are purchased from major chemical reagent suppliers without further purification before use. The protected amino acid raw materials used in the polypeptide synthesis process include Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH. The abbreviations among them have well-known definitions, for example: Fmoc is 9-fluorenylmethyloxycarbonyl, OtBu is tert-butoxy, tBu is tert-butyl, Trt is trityl, Boc is tert-butoxycarbonyl.

[0044] 2. Synthesis of peptide resin

[0045] Using Rink Amide MBHA resin as the carrier resin, the peptide resin is prepared by sequentially coupling with the protected amino acids corresponding to the polypeptide amino acid sequence through Fmoc deprotection and coupling reaction.

[0046] (1) Accessing the first protected amino acid of the main chain

[0047] Take 0.3 mmol of the first protected amino acid and 0.3 mmol of HOBt, and dissolve them with an appropriate amount of DMF; take another 0.3 mmol of DIC, and slowly add it to the protected amino acid DMF solution under shaking, and shake at room temperature for 5 minutes to obtain an activated protected amino acid solution, which is ready for use.

[0048] Take 0.1 mmol of Rink Amide MBHA resin (0.35 mmol / g x 0.3 g), and deprotect it with 25% PIPE / DMF solution (volume ratio) for 20 minutes (twice), and then wash and filter to obtain the Fmoc-deprotected resin.

[0049] Add the activated first protected amino acid solution to the Fmoc-deprotected resin, and couple for 60 minutes, then filter and wash to obtain the resin containing the first protected amino acid.

[0050] (2) Incorporate other protected amino acids into the main chain

[0051] Using the same method as described above for adding the first protecting amino acid to the main chain, other protecting amino acids corresponding to the peptide were sequentially added to obtain a resin containing the main chain amino acids. Finally, the N-terminus was acetylated and capped with 0.3 mmol Ac₂O + 0.6 mmol DIEA to complete the synthesis of the main chain. Each reaction step was monitored using the Kaiser Test; if the condensation reaction of a certain amino acid was incomplete, the condensation was repeated once until the desired target peptide was obtained.

[0052] 3. Preparation of crude product

[0053] Take the above-mentioned peptide resin, add lysis reagent (15 mL / g resin), mix well, and react with shaking at 30°C for 3 hours to lyse the target peptide from the resin and remove the side chain protecting groups. Collect the filtrate of the reaction mixture, wash the resin three times with a small amount of TFA / DCM, combine the filtrates, add anhydrous diethyl ether to precipitate, and centrifuge. Wash the filter cake twice with cold anhydrous diethyl ether, and dry to obtain a white powder, which is the crude peptide. The composition of the lysis reagent is as follows: trifluoroacetic acid: 1,2-ethylenedithiol: benzyl sulfide: phenol: H2O: triisopropylsilane = 68.5:10:10:5:3.5:1 (volume ratio).

[0054] 4. Preparation of pure products

[0055] The crude polypeptide was dissolved in water / acetonitrile by stirring, and the insoluble matter was removed by centrifugation before use. Purification was performed using reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic column used was an Agela C18 (10 μm). The column (50 mm × 250 mm) was used. The mobile phase consisted of mobile phase A (0.05% TFA and 2% acetonitrile aqueous solution) and mobile phase B (90% acetonitrile / aqueous solution). The flow rate was 25 mL / min. The UV detection wavelength was 220 nm. The crude solution was loaded onto the column, and gradient elution was performed. The corresponding purified fractions were collected, and the solvent was removed by direct freeze-drying to obtain the loose, pure trifluoroacetate polypeptide.

[0056] The trifluoroacetate polypeptide was redissolved in water and acetonitrile, and a large amount of anion exchange resin (acetate form) was added and stirred for 3 hours. After filtration and rinsing the ion exchange resin with a water / acetonitrile mixture, the filtrates were combined and lyophilized to obtain a loose, pure polypeptide acetate.

[0057] The chemical structures of the synthesized peptides were characterized by MALDI-TOF mass spectrometry, and their purity was determined by analytical high-performance liquid chromatography (Agela C18-4.6×250mm, flow rate 1 mL / min). The results showed that the purity of all synthesized peptides was greater than 95%.

[0058] Example 3: Discovery and identification of highly active MPER antiviral polypeptides

[0059] 1. Experimental materials and methods

[0060] The test polypeptides were Figure 2 polypeptides shown in Table 1. The viruses used were pseudoviruses of HIV-1 strains NL4-3, JRFL and SF162, prepared by the laboratory; the target cells TZM-bl were products of the National Experimental Cell Resource Sharing Service Platform. The main experimental steps were as follows:

[0061] (1) Preparation of HIV pseudovirus: co-transfect HEK293T cells with plasmids expressing the envelope protein (Env) of NL4-3 or JRFL or SF162 and the HIV-1 backbone plasmid pSG3Δenv; place the transfected cells in a 37°C, 5% CO2 cell incubator for 48 hours, then collect the supernatant, filter and collect the filtrate, which is the NL4-3 pseudovirus or JRFL or SF162-containing pseudovirus liquid, which is titrated and stored at -80°C for future use.

[0062] (2) Dissolve the test polypeptides in deionized water or dimethyl sulfoxide (DMSO), then dilute the polypeptides to the starting concentration with DMEM medium, then perform 3-fold dilution of the polypeptides in a 96-well cell culture plate, obtaining polypeptide dilutions. Set 9 dilution degrees for each test polypeptide.

[0063] (3) In the 96-well cell culture plate, add polypeptide dilutions (50 μL / well) to the drug wells and DMEM medium (50 μL / well) to the control wells, setting 3 replicate wells for each. Then add 1000 TCID50 of HIV-1 pseudovirus liquid (adjusted to 50 μL / well), and incubate at room temperature for 30 minutes.

[0064] (4) Resuspend the pre-cultured target cells TZM-bl with DMEM medium, adjust the cell concentration to 10 x 10 4 cells / mL, then add DEAE-dextran to a final concentration of 15 μg / mL. Finally, add the cells (100 μL / well) to the 96-well plate containing the virus & polypeptide complex, and incubate at 37°C in a 5% CO2 cell incubator for 48 hours.

[0065] (5) Discard the cell culture supernatant, add 30 μL of cell lysis solution (Promega, Catalog No. E1531) to each well, and lyse at room temperature for 15 minutes, then add the luciferase detection substrate reagent (Promega, Catalog No. E1501), and measure the relative fluorescence units (RLU) using a microplate luminometer, make an inhibition rate curve and calculate the drug half-inhibitory concentration (IC50 ).

[0066] 2. Experimental results and analysis

[0067] The results are shown in Figure 2 Table 1. None of the TRM11-TRM20 polypeptides showed antiviral activity. The antiviral activity of the polypeptide MPER30 (EK30), which is a continuation of the C-terminus of TRM20, and the representative MPER polypeptide MPER19 (gp41W) were consistent with the literature reports, and neither showed effective inhibitory activity at a concentration of 5000 nM. However, surprisingly, the truncated polypeptide MPER18, which was obtained by removing one lysine (K) residue from the N-terminus of MPER19, showed significant antiviral activity, with IC50values of 242 nM, 596.47 nM, and 457.37 nM for the NL4-3, JRFL, and SF162 HIV-1 pseudoviruses, respectively. 50 More interestingly, the inhibitory activity of the MPER17 polypeptide, which was obtained by further truncating one tryptophan (W) residue, decreased significantly, while the inhibitory activity of the MPER16 polypeptide, which was obtained by further removing one alanine (A) residue, was further improved compared to MPER18. The IC50values of MPER16 for the three HIV-1 pseudoviruses were 197.37 nM, 324.93 nM, and 288.77 nM, respectively. 50 However, a series of polypeptides obtained by further truncation (MPER15-MPER10) and the MPER16R polypeptide, which was obtained by adding one arginine (R) residue to the C-terminus of MPER16, lost activity. Further studies confirmed that removing the CRAC motif "LWYIK" from the C-terminus of the functional polypeptide MPER16 completely eliminated the anti-HIV activity, indicating the determining role of CRAC in the antiviral effect of the polypeptide.

[0068] To determine the specificity and drugability of the MPER antiviral polypeptides MPER18 and MPER16 of the present application, their in vitro cytotoxicity was further detected.

[0069] 1. Experimental materials and methods

[0070] The test polypeptides were MPER18 and MPER16. The test cell TZM-bl was a product of the National Experimental Cell Resource Sharing Service Platform. The in vitro cytotoxicity of the test polypeptides was detected using a CCK-8 cell proliferation / toxicity detection kit (manufacturer: Abbkine, item number: KTC011001), and the specific experimental steps were as follows:

[0071] (1) 3-fold gradient dilution of the test polypeptide in a 96-well cell culture plate, 4 dilution levels, 3 replicates for each dilution level, 100 μL polypeptide solution in each well; DMEM medium (100 μL per well) control wells were set.

[0072] (2) Add about 10 x 10 4 cells / mL of the test cell TZM-bl suspension to the 96-well cell culture plate of step (1), 100 μL / well, incubate at 37°C, 5% CO2 for 48 hours.

[0073] (3) Add 20 μL CCK-8 solution to each well, continue to incubate the plate in the incubator for 2 hours, then measure the absorbance (OD450) at 450 nm with a microplate reader. Use GraphPad Prism software to make a cell survival rate column chart.

[0074] 2. Experimental results and analysis

[0075] The results are shown in Figure 3 , MPER18 and MPER16 did not show cytotoxicity in target cells TZM-bl at a high concentration of 25 μM. Through CC50 / IC50 analysis, it can be seen that MPER18 and MPER16 both have a high therapeutic selectivity index (SI).

[0076] Example 5: Broad-spectrum anti-HIV activity of MPER18 and MPER16

[0077] HIV has high variability, and in evolution, many subtypes and recombinant viruses have been generated, among which HIV-1 subtypes A, B and C are the main viruses causing the world's AIDS epidemic, and in China, A / E and B / C recombinant viruses are dominant. This example further evaluates the broad-spectrum anti-HIV activity of MPER18 and MPER16 inhibitors using a pseudovirus infection experiment of 25 representative HIV-1 strains. The anti-viral activity of the newly synthesized polypeptide was analyzed using the anti-viral experiment in Example 3 above.

[0078] The results of the inhibition of MPER18 and MPER16 on pseudovirus infection of 25 representative HIV-1 strains are shown in Figure 4 . MPER18 and MPER16 have broad inhibitory activity against 25 HIV-1 pseudoviruses, with average IC50 values of 1530.16 nM and 829.62 nM, respectively, and the shorter sequence MPER16 shows obvious superiority.

[0079] The present disclosure has been described in detail. Those skilled in the art who are not bound by the details of the present disclosure can implement the present disclosure within a wide range of equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present disclosure, and without unnecessary experiments. Although the present disclosure gives a specific example, it should be understood that further improvements can be made to the present disclosure. In summary, according to the principles of the present disclosure, the present application intends to include any changes, uses or improvements of the present disclosure, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. The use of an anti-HIV-1 polypeptide or a derivative thereof in the preparation of a medicament for treating diseases caused by HIV-1 infection, characterized in that, The amino acid sequence of the anti-HIV-1 polypeptide is shown in SEQ ID NO. 1 or SEQ ID NO. 2, the derivative of the anti-HIV-1 polypeptide is acetyl modification at the amino terminal of the anti-HIV-1 polypeptide; and / or, amino modification at the carboxyl terminal of the anti-HIV-1 polypeptide; or the derivative of the anti-HIV-1 polypeptide is a non-physiologically toxic salt of the anti-HIV-1 polypeptide.

2. Use of a nucleic acid molecule for the manufacture of a medicament for the treatment of a disease caused by HIV-1 infection, characterized in that, The nucleic acid molecule encodes the anti-HIV-1 polypeptide as claimed in claim 1.

3. Use of a composition for the manufacture of a medicament for the treatment of a disease caused by HIV-1 infection, characterized in that, It comprises: 1) the anti-HIV-1 polypeptide as claimed in claim 1; 2) optionally, a pharmaceutically acceptable carrier or excipient.

4. Use according to claim 3, characterized in that, The composition further comprises one or more other anti-HIV-1 drugs.

5. The use according to any one of claims 1 to 4, characterized in that, The disease caused by the HIV-1 infection is AIDS. The amino acid sequence of the anti-HIV-1 polypeptide is shown in SEQ ID NO. 1 or SEQ ID NO. 2, the derivative of the anti-HIV-1 polypeptide is acetyl modification at the amino terminal of the anti-HIV-1 polypeptide; and / or, amino modification at the carboxyl terminal of the anti-HIV-1 polypeptide; or the derivative of the anti-HIV-1 polypeptide is a non-physiologically toxic salt of the anti-HIV-1 polypeptide. The nucleic acid molecule encodes the anti-HIV-1 polypeptide as claimed in claim 1. It comprises: 1) the anti-HIV-1 polypeptide as claimed in claim 1; 2) optionally, a pharmaceutically acceptable carrier or excipient. The composition further comprises one or more other anti-HIV-1 drugs. The disease caused by the HIV-1 infection is AIDS.

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