CN113999284B - Cyclic polypeptides or pharmaceutically acceptable salts thereof for use against ebola virus - Google Patents

Abstract

The invention discloses a cyclic polypeptide or a pharmaceutically acceptable salt thereof for resisting Ebola virus. The amino acid sequence of the cyclic polypeptide is shown in the following (formula I): x ₁X₂X₃X₄X₅X₆X₇X₈ (formula i) wherein each of X ₁ to X ₈ is an amino acid residue, X ₁ is C, X ₂ is E, D, H or Y, X ₃ is Y, X ₄ is F, X ₅ is F, X ₆ is W or V, X ₇ is Y or H, and X ₈ is C; x ₁ in the formula I and X ₈ form disulfide bond connection to form a ring. The cyclic polypeptide can specifically inhibit ebola virus from entering cells by combining with a target protein EBOV-GPcl, so as to achieve the effect of resisting EBOV infection.

Description

Cyclic polypeptides or pharmaceutically acceptable salts thereof for use against ebola virus

The application is a divisional application with the application number 2020104602227, the application number 201810087839.1 and the application date 2018, 01 and 30 of the original application, and the application and the creation name are polypeptides specifically combined with the Ebola virus activated envelope glycoprotein and application thereof in antiviral.

Technical Field

The invention relates to a cyclic polypeptide for resisting Ebola virus or a medicinal salt thereof in the technical field of medicines.

Background

Ebola virus disease (formerly known as Ebola hemorrhagic fever) is an acute hemorrhagic infectious disease caused by Ebola virus (EBOV) of the family filoviridae, and has a mortality rate of up to 90% and is one of the most fatal viral infectious diseases in humans. EBOV can be classified into 5 species, zaire type (Zaire ebolavirus, zaire-EBOV, ZEBOV), sudan type (Sudan ebolavirus, SUDV), taisen type (Tai Forest ebolavirus, TAFV), bendibu type (Bundibugyo ebolavirus, BDBV) and rice type (Reston ebolavirus, RESTV). Among them, zaire type ebola virus is the strongest in pathogenicity. At present, symptomatic and supportive treatment is mainly adopted for EBOV infection, and no specific therapeutic drug and vaccine which are effective through systematic clinical verification are available. With the increasing global population fluidity and the existence of EBOV superdiffusers in combination with the strong pathogenicity of the virus itself and the susceptibility to genetic variation, EBOV has become a potential global health threat. Therefore, it is necessary to study anti-EBOV drugs with definite targets and brand new structural types, ensuring the ability to prevent EBOV infection and cope with new outbreaks of infectious diseases.

The EBOV is a single-strand negative strand RNA virus, the outermost layer is wrapped by a virus envelope, and the center is a spiral nucleocapsid. The genome is about 18.9kb in length, encodes seven structural proteins, and has the gene sequence of 3-NP-VP35-VP40-GP-VP30-VP24-L-5. Among these, the envelope glycoprotein (glycoprotein, GP) is the only protein responsible for viral entry into host cells. GP, whether it is covered on the surface of the virus or shed from infected cells during infection, can participate in a variety of immune response reactions, playing an important role in the viral life cycle and host-pathogen interactions, and is a potentially effective target for inhibiting EBOV infection. Since the viral entry phase is the first step in the viral replication cycle, blocking EBOV-GP mediated viral entry will effectively inhibit viral infection and cytotoxicity caused thereby, while also reducing the occurrence of drug resistance. Cleavage occurs after the EBOV-GP enters lysosomes, and activated glycoprotein (Primed GP, GPcl) after cleavage can directly interact with endocytosis receptor-human cholesterol transporter (Niemann-Pick C1, NPC 1), thereby initiating the process of membrane fusion of the virus with the inside of host cells.

Disclosure of Invention

The technical problem to be solved by the invention is how to specifically inhibit ebola virus from entering cells so as to resist EBOV infection.

In order to solve the technical problems, the invention provides a cyclic polypeptide or a pharmaceutically acceptable salt thereof.

In the cyclic polypeptide or the pharmaceutically acceptable salt thereof provided by the invention, the amino acid sequence of the cyclic polypeptide is shown in the following formula (I):

x ₁X₂X₃X₄X₅X₆X₇X₈ (formula I),

In the formula I, X ₁ to X ₈ are each an amino acid residue, wherein X ₁ is C, X ₂ is E, D, H or Y, X ₃ is Y, X ₄ is F, X ₅ is F, X ₆ is W or V, X ₇ is Y or H, and X ₈ is C; x ₁ in the formula I and X ₈ form disulfide bond connection to form a ring.

In the above cyclic polypeptide or pharmaceutically acceptable salt thereof, the cyclic polypeptide may be Pep-3.3, pep-3.2, pep-3.1 or Pep-3.10; the amino acid sequence of the Pep-3.3 is SEQ ID No.1 in a sequence table, the amino acid sequence of the Pep-3.2 is SEQ ID No.2 in the sequence table, the amino acid sequence of the Pep-3.1 is SEQ ID No.3 in the sequence table, and the amino acid sequence of the Pep-3.10 is SEQ ID No.4 in the sequence table.

Derivatives of the above cyclic polypeptides are also within the scope of the present invention.

The derivative of the cyclic polypeptide may be d1, d2, d3, d4 or d5; d1 is a linker obtained by connecting an amino-terminal protecting group to the amino terminus of the cyclic polypeptide and/or connecting a carboxyl-terminal protecting group to the carboxyl terminus of the cyclic polypeptide;

D2 is a polypeptide which is obtained by adding an amino acid residue at the amino terminal end and/or the carboxyl terminal end of the cyclic polypeptide and can be specifically combined with GPcl (activated envelope glycoprotein of virus);

D3 is a polypeptide which is obtained by connecting oligopeptide at the amino terminal end and/or the carboxyl terminal end of the cyclic polypeptide and can be specifically combined with GPcl;

D4 is a modifier obtained by modifying the cyclic polypeptide by protein, polyethylene glycol or maleimide;

The d5 is a polypeptide which is obtained by connecting a lipophilic compound to the amino terminal end and/or the carboxyl terminal end of the cyclic polypeptide and can be specifically combined with GPcl.

The following multimers of PM1 or PM2 are also within the scope of the invention:

PM1, a multimer formed from the cyclic polypeptide or a pharmaceutically acceptable salt thereof;

PM2, multimers formed from said derivatives.

In the above cyclic polypeptides, pharmaceutically acceptable salts thereof, or derivatives thereof, each capital letter in the sequence of the cyclic polypeptide is an abbreviation for an amino acid, which has the meaning well known in the art, for example: c is cysteine, D is aspartic acid, E is glutamic acid, Y is tyrosine, F is phenylalanine, W is tryptophan, V is valine, H is histidine, and the like. All amino acids in the cyclic polypeptide sequence can be L-type amino acids, and one or more (such as 2-5, 2-4 or 2-3) amino acids can be replaced by amino acids with D-type conformations, artificially modified amino acids, rare amino acids existing in nature and the like so as to improve the bioavailability, stability and/or antiviral activity of the cyclic polypeptide. Wherein the D-form amino acid refers to an amino acid corresponding to the L-form amino acid constituting the protein; artificially modified amino acids refer to common L-type amino acids which are modified by methylation, phosphorylation and the like to form proteins; rare amino acids that exist in nature include unusual amino acids that constitute proteins and amino acids that do not constitute proteins, such as 5-hydroxylysine, methylhistidine, gamma-aminobutyric acid, homoserine, and the like.

In the above cyclic polypeptide, a pharmaceutically acceptable salt thereof, or a derivative thereof, the lipophilic compound may be attached to the side chain of the terminal amino acid or may be directly attached to the peptide chain.

In the above cyclic polypeptide, a pharmaceutically acceptable salt thereof, or a derivative thereof, the amino-terminal end of the cyclic polypeptide of the present invention may contain an amino-terminal protecting group, which may be any of acetyl, amino, maleyl, succinyl, t-butoxycarbonyl or benzyloxy or other hydrophobic groups or macromolecular carrier groups; the carboxyl terminal of the cyclic polypeptides of the present invention may contain a carboxyl terminal protecting group, which may be any of amino, amido, carboxyl, or t-butoxycarbonyl groups or other hydrophobic groups or macromolecular carrier groups.

Compositions comprising the following C1) and C2) are also within the scope of the invention: c1 C11), C12) or/and C13); the C11) is the above cyclic polypeptide or a pharmaceutically acceptable salt thereof; the C12) is the derivative; the C13) is the above polymer;

C2 Pharmaceutically acceptable carriers or excipients;

The composition has at least one of the following functions F1) -F3):

F1 Antiviral;

F2 Treatment and/or prevention and/or adjuvant therapy of diseases caused by viral infections;

F3 Inhibiting viral entry into the cell.

In the above composition, the viruses F1) -F3) may be Ebola virus (Zaler type, sudan type, taisenia type, bendibuch type and/or Leston type), or Marburg virus (MARV).

The use of the abovementioned C11), the abovementioned C12), the abovementioned C13) or/and C14) for preparing at least one of the products E1) to E3) also falls within the scope of the invention:

the C14) is the composition;

the E1) is an antiviral product, such as a drug or vaccine;

The E2) is a product for treating and/or preventing and/or assisting in treating diseases caused by virus infection, such as medicines or vaccines;

The E3) is a product for inhibiting the invasion of virus into cells, such as a drug or vaccine;

In the above application, the viruses E1) -E3) may be Ebola virus (Zaler type, sudan type, taisen type, bendibuch type and/or Leston type), or Marburg virus.

The present invention provides pharmaceutical compounds.

The pharmaceutical compounds provided by the invention are C11), C12) or C13) as described above.

Of the above pharmaceutical compounds, the pharmaceutical compounds have at least one of the uses of U1) -U3) below:

u1) is used for resisting virus;

U2) for the treatment and/or prophylaxis and/or adjuvant treatment of diseases caused by viral infections;

U3) is used to inhibit viral invasion into cells.

In U1) -U3) of the above pharmaceutical compounds, the virus may be an ebola virus (zaire type, sudan type, taisen type, bunyak type and/or leston type), or a marburg virus.

Above, the inhibiting viral entry into the cell may be inhibiting GPcl-mediated viral entry into the cell.

The pharmaceutical salts of the cyclic polypeptides of the invention include acetate (acetate), lactobionic acid (lactobionate), benzenesulfonate (benzenesulfonate), laurate (laurate), benzoate (benzoate), malate (malate), bicarbonate (bicarbonate), maleate (maleate), bisulfate (bisulfate), mandelate (mandelate), bitartrate (bitartrate), methanesulfonate (mesylate), borate (borate), bromomethane (methylbromide), bromide (bromomide), methyl nitrate (METHYLNITRATE), calcium edetate (calcium acetate), methylsulfate (methylsulfate), camphorsulfonic acid (camsylate), mucic acid (mucate), carbonate (carbonate), naphthalene sulfonate (napsylate), chloride (chloride), nitrate (nitrate), clavulanate (clavulanate), N-methylglucamine (N-methylglucamine), citrate (citrate), ammonium salt (ammonium salt), dihydrochloride (dihydrochloride), oleate (oleate), ethylenediamine tetraacetate (edetate), oxalate (oxalate), ethanedisulfonate (edisylate), pamoate (pamoate) (pamoate embonate), propionate laurate (estolate), palmitate (palmate), ethanesulfonate (esylate), pantothenate (pantothhenate), fumarate (fumarate), phosphate/diphosphate (phosphate/diphosphate), glucoheptonate (polygalacturonate), glucoheptonate (gluconate), salicylate (salicylate), glutamate (glutamate), stearate (stearate), paracetamol (glycollylarsanilate), sulfate (sulfate), hydroxybenzoate (hexylresorcinate), basic acetate (subacetate), seabar (hydramine), succinate (succinate), hydrobromide (hydrobromide), tanniate (tannate), hydrochloride (hydrochloride), tartrate (tartrate), hydroxynaphthoate (hydroxynaphthoate), 8-chlorotheophyllinate (teoclate), iodide (iodate), tosylate (tosylate), triethyliodide (triemide), lactate (lactate), valerate (valinate) and the like. Depending on the application, pharmaceutically acceptable salts may be formed from cations such as sodium (sodium), potassium (potassium), aluminum (aluminum), calcium (calcium), lithium (lithium), manganese (magnesium) and zinc (zinc), bismuth (bismuth), and the like, and bases such as ammonia, ethylenediamine (ethylenediamine), N-methyl-glutamine (N-methyl-glutamine), lysine (lysine), arginine (arginine), ornithine (ornithine), choline (choline), N '-dibenzylethylenediamine (N, N' -dibenzylethylene-diamine), chloroprocaine (chloroprocaine), diethanol ammonia (diethanol amine), procaine (procaine), diethylamine (DIETHYLAMINE), piperazine (piperazine), trimethylol-aminomethane (tris (hydroxymethyl) aminomethane), and tetramethylammonium hydroxide (tetramethylammonium hydroxide), and the like. These salts can be prepared by standard methods, for example by reaction of the free acid with an organic or inorganic base. In the presence of a basic group such as an amino group, an acidic salt such as hydrochloride (hydrobromide), hydrobromide (hydrobromide), acetate (acetate), pamoate (pamoate) and the like can be used as the dosage form; pharmaceutically acceptable esters such as acetate, maleate, chloromethyl trimethylacetate (pivaloyloxymethyl) and the like, as well as esters known in the literature for improving solubility and hydrolyzability, in the presence of an acidic group such as-COOH or an alcohol group, can be used as sustained release and prodrug formulations.

The cyclic polypeptide, the derivative or the pharmaceutically acceptable salt thereof, the polymer, the composition or the pharmaceutically acceptable compound provided by the invention can be used for treating EBOV and/or MARV infection. The lipopeptides or polypeptides, derivatives thereof, or pharmaceutically acceptable salts thereof, the multimers, the compositions or the pharmaceutically acceptable compounds provided herein may also be used for the prevention of EBOV and/or MARV infections, including pre-exposure or post-suspected exposure, such as blood transfusion, organ transplantation, fluid exchange, bite, accidental needle stick or intra-operative exposure to a patient's blood, and the like.

In practical applications, the cyclic polypeptide, derivative or pharmaceutically acceptable salt thereof, the multimer, the composition or the pharmaceutical compound of the invention may be administered directly to a patient as a medicament or after being mixed with a suitable carrier or excipient to treat and/or prevent EBOV and/or MARV infection. The carrier materials herein include, but are not limited to, water soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethylcellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl ethyl cellulose, etc.). Among them, preferred is a water-soluble carrier material. The materials can be prepared into various dosage forms, 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 injection and the like. The suppository can be pessary, vaginal ring, ointment, cream or gel suitable for vaginal application. Can be common preparation, slow release preparation, controlled release preparation and various microparticle administration systems. For the purpose of shaping the unit dosage form into a tablet, 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, kaolin, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, dextrose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, and the like; disintegrants such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oils and the like; absorption promoters such as quaternary ammonium salts, sodium lauryl sulfate, and the like; lubricants such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer and multilayer tablets. For the purpose of formulating the unit dosage form into a pill, various carriers well 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 oils, polyvinylpyrrolidone, gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, and the like; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc. For preparing a unit dosage form into a suppository, various carriers well known in the art can be widely used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides, and the like. For preparing unit dosage forms into injectable preparations such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in the art, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. may be used. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and further, a conventional cosolvent, a buffer, a pH adjuster, and the like may be added. In addition, colorants, preservatives, flavors, flavoring agents, sweeteners, or other materials may also be added to the pharmaceutical formulation, if desired.

The preparation can be administrated by injection, including subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracisternal injection or infusion, etc.; administration via the luminal tract, such as rectally, vaginally, and sublingually; respiratory tract administration, such as via the nasal cavity; mucosal administration. The above route of administration is preferably injection, and the preferred route of injection is subcutaneous injection.

The dosage of the cyclic polypeptide of the present invention, its derivative, pharmaceutically acceptable salt, the multimer, the composition or the pharmaceutical compound to be administered 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-mentioned doses may be administered in a single dosage form or divided into several, for example two, three or four dosage forms.

For any particular patient, the particular therapeutically effective dose level will depend on a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; age, weight, general health, sex and diet of the patient; the time of administration, route of administration and rate of excretion of the particular active ingredient employed; duration of treatment; a medicament for use in combination or simultaneously with the particular active ingredient employed; and similar factors well known in the medical arts. For example, it is common in the art to start doses of the active ingredient below the level required to obtain the desired therapeutic effect and to gradually increase the dose until the desired effect is obtained.

The cyclic polypeptide, the derivative thereof or the pharmaceutically acceptable salt thereof, the polymer, the composition or the pharmaceutical compound can be directly and independently used for treating and preventing EBOV and/or MARV infected persons, can also be used in combination with one or more anti-EBOV and/or MARV infected medicaments, can be used simultaneously or at intervals, and can achieve the aim of improving the overall treatment effect.

In the present invention, the antiviral activity may also be referred to as a viral inhibitory activity, and specifically may be a viral entry inhibition into cells.

Experiments prove that the cyclic polypeptide can specifically inhibit the ebola virus from entering cells. The cyclic polypeptide can specifically inhibit ebola virus from entering cells by combining with a target protein EBOV-GPcl, so as to achieve the effect of resisting EBOV infection. The cyclic polypeptide or the medicinal salt and the derivative thereof can be used as a novel EBOV entry inhibitor and can be applied to the development of vaccines or medicaments for resisting the EBOV.

Drawings

FIG. 1 shows that Pep-3.1, pep-3.2, pep-3.3 and Pep-3.10 are capable of specifically inhibiting EBOV-Zaire GP/HIV-luc recombinant viral activity. In FIG. 1, VSVG represents VSV-G/HIV-luc, ebola-GP represents EBOV-GP/HIV-luc, viral infection rate=1-inhibition rate.

FIG. 2 is a graph showing the effect of a polypeptide on 293T cell growth as demonstrated by cell growth experiments.

FIG. 3 shows that Pep-3.1, pep-3.2, pep-3.3 and Pep-3.10 have good dose dependence on the inhibition of EBOV-Zaire GP/HIV-luc recombinant virus.

FIG. 4 shows the effect of polypeptide Pep-3.3 on the entry phase of virus.

FIG. 5 is a graph showing the kinetic binding curves of different concentrations of the polypeptide Pep-3.1, pep-3.2, pep-3.3 and Pep-3.10 to the target protein GPcl measured in vitro by biological membrane layer optical interferometry. In the figure, arrows show the concentrations of the respective samples.

Detailed Description

The invention is realized by the following technical scheme:

First, a structure-based polypeptide drug design was performed using the C-terminal loop structure (amino acid sequence DDFFVY) of the NPC1 domain as a template. To improve the stability of the polypeptide, it is protected from phosphatase, and a cysteine is introduced at each end of the amino acid sequence, thereby introducing a disulfide bond. Then, the stability of the polypeptide is further improved by amidation modification of the carbon end and acetylation modification of the nitrogen end. The polypeptide structure is Ac-cyclo (CDDFFVYC) -NH ₂. In order to improve the binding capacity of the polypeptide and the target protein EBOV-GPcl, the structure of the polypeptide is further optimized by constructing single-point, double-point and three-point amino acid mutations, so as to obtain the optimal combination sequence of the polypeptide. Then, the designed polypeptide was inoculated into the target protein GPcl, and a polypeptide having a superior free energy (Table 1) was selected for solid phase synthesis.

EBOV is classified as a dangerous quaternary virus, so that the polypeptides are evaluated for biological activity at the in vitro level using a safe and effective research approach, the pseudovirus technique. The replication-defective pseudovirus EBOV-GP/HIV-luc is prepared by wrapping HIV cores with GP proteins of the Tie-Emi type EBOV with the highest toxicity, and the antiviral activity of the sample is judged by a fluorescent reporter gene detection technology. Meanwhile, the specificity of the polypeptides was analyzed using a VSVG/HIV-luc recombinant virus model. After removal of cytotoxicity, the mechanism of action of the polypeptides was further verified using a drug action time point experiment. Finally, the binding capacity of the polypeptide and the target protein GPcl is measured in vitro by utilizing a biological film layer optical interference technology based on an optical fiber biosensor, and the targeting of the polypeptide is verified.

All polypeptides in Table 1 were modified by amino-terminal homoacetylation (Ac-), carboxy-terminal homoamidation (-NH ₂). "Cyclo" means that the amino acid sequence in brackets forms a cyclic polypeptide with the amino acid residue at position 1 and the amino acid residue at position 1 last. In Pep-1, the amino acid sequence in brackets is connected with the amino acid residue at the last 1 position through an amide bond to form a cyclic polypeptide; in Pep-3, pep-3.1, pep-3.2, pep-3.3, pep-3.6, pep-3.7 and Pep-3.10, the amino acid sequence in brackets is linked with the amino acid residue at the last 1 position through disulfide bond formation to form a cyclic polypeptide.

TABLE 1 sequence structure of polypeptides

Name of the name	Sequence structure
		Pep-1	Ac-cyclo(DDFFVY)-NH2
Pep-2	Ac-DDFFVY-NH2
		Pep-3	Ac-cyclo(CDDFFVYC)-NH2
Pep-3.1	Ac-cyclo(CHYFFVYC)-NH2
		Pep-3.2	Ac-cyclo(CDYFFWYC)-NH2
Pep-3.3	Ac-cyclo(CEYFFWYC)-NH2
		Pep-3.6	Ac-cyclo(CDRFFVYC)-NH2
Pep-3.7	Ac-cyclo(CDYFFRYC)-NH2
		Pep-3.10	Ac-cyclo(CYYFFVHC)-NH2

The polypeptides of table 1 were all synthesized by beijing Saint gene technologies, inc, with purity >98%. The specific acquisition means is the prior art, and the present invention is not particularly limited thereto.

The following detailed description of the invention is provided in connection with the accompanying drawings that illustrate the invention and are not intended to limit the scope of the invention. Although the following describes the antiviral mechanism of the present invention by taking zaire EBVO as an example, the scope of protection of the use of the polypeptide is not limited to EBOV. Any virus suitable for the above antiviral mechanism is within the scope of the present invention, and may be, for example, other four subtypes of EBOV, MARV, etc.

In addition, it should be noted that the various materials and reagents used in the following examples are those commonly used in the art and are commercially available in general terms unless otherwise indicated; the methods used are conventional methods known to those skilled in the art or according to the conditions recommended by the manufacturer.

Example 1, EBOV entry inhibitor screening model verifies that Pep-3.1, pep-3.2, pep-3.3 and Pep-3.10 are capable of specifically inhibiting EBOV activity.

The recombinant virus (EBOV-GP/HIV-luc) was prepared by coexpression of the GP of Zaire-EBOV with the HIV core plasmid (pNL 4-3. Luc) using a cell-level recombinant virus technique, and the antiviral activity of the polypeptides was evaluated using a high throughput screening model of EBOV entry inhibitors targeting the GP protein. The method comprises the following specific steps:

293T cells were cultured, and after the cells were grown in the flask, the old medium was discarded and digested with a digest containing 0.25% pancreatin and 0.02% EDTA. After the cells were rounded, the digests were discarded, and immediately high-sugar DMEM medium (GIBICO) containing 10% fbs (purchased from GIBCO) was added, the bottom was gently blown with a pipette, the cells were completely detached from the bottom and dispersed as a single cell suspension. After counting, the cell concentration was adjusted to 2.2X10 ⁵ cells/mL with medium and plated in 6-well plates at 2 mL/well. After 24h (cell abundance approximately 70%) transfection, plasmid dose: 2 mu g pZEBOV-GP and 3. Mu.g of HIV-luc plasmid pNL4-3.Luc. R-E-carrying a luciferase reporter gene, the transfection reagent was Lipofectamine2000 (Invitrogen company), and transfection was performed according to the instructions to generate Ebola pseudotype virus, which was designated EBOV-GP/HIV-luc or EBOV-Zaire GP/HIV-luc. Supernatants containing pseudotyped virus were collected 48 hours post-transfection, pooled, clarified from floating cells and cell debris by low-speed centrifugation, and filtered through a 0.45 μm pore size filter. The pseudo-viral particles were quantified by measuring virus-related HIV p24 levels using an ELISA assay.

Wherein pZEBOV-GP is a recombinant expression plasmid of Glycoprotein (GP) expressing Zaire-EBOV obtained by inserting the 5900-8305 th position of the Zaire ebolavirus isolate H.sapiens-wt/GIN/2014/Makona-Gueckedou-C07 GP gene (GenBank Accession No. KJ660347 (Update Date Dec 18, 2014: 25 PM) into the vector pcDNA3.1 (+).

EBOV-GP/HIV-luc pseudovirions were incubated with 293T cells in 96-well plates. After 48 hours, cells were collected and lysed to measure firefly luciferase activity. The value of luciferase activity represents a viral infection.

The polypeptides of Table 1 were dissolved in DMSO and mixed with EBOV-GP/HIV-luc pseudoviruses, respectively, and added to 293T cells to give a polypeptide content of 10. Mu.M. After 48 hours, 293T cells were lysed and the inhibition of the virus by the polypeptide was assessed by measuring luciferase activity. Solvent DMSO was used as a blank (DMSO) and EBOV entry inhibitor tetrandrine (TET) and HIV-1 reverse transcriptase inhibitor Efavirenz (EFV) were introduced simultaneously as controls. The tetrandrine and the efavirenz are respectively dissolved by DMSO and then respectively mixed with EBOV-GP/HIV-luc pseudovirus to be added into 293T cells, and the content of the tetrandrine and the efavirenz is 1 mu M. After 48 hours, 293T cells were lysed and the inhibition of the virus by the polypeptide was assessed by measuring luciferase activity.

Most of the currently known EBOV inhibitors are broad-spectrum antiviral drugs, and in order to find a narrow-spectrum inhibitor against EBOV, a specific analysis of the screened active polypeptide is required. Since vesicular stomatitis virus coat glycoprotein (vesicular stomatitis virus glycoprotein, VSVG) acts similarly to EBOV-GPcl, and plays an important role in the recognition of viruses and receptors, the screened active polypeptides were specifically analyzed using pseudoviruses expressing VSV-GP. After removal of the cytotoxic factors, the inhibitory activity of the active polypeptide against VSV-G/HIV-luc pseudovirus was also detected using luciferase-associated preparation, as described above. If the polypeptide has an obvious inhibition effect on the virus entry mediated by the EBOV-GPcl, but has no inhibition or low inhibition rate on VSV, the polypeptide is proved to have specificity on the EBOV.

As shown in FIG. 1, the inhibition rate of Pep-3.1, pep-3.2, pep-3.3 and Pep-3.10 on EBOV-GP/HIV-luc pseudoviruses is higher than 80%, and the inhibition rate on VSV-G/HIV-luc pseudoviruses at the same concentration is lower than 50%. This suggests that Pep-3.1, pep-3.2, pep-3.3 and Pep-3.10 have specific inhibitory effects on the EBOV-GP/HIV-luc pseudoviruses. Among them, pep-3.3 has the most remarkable specific inhibition effect. The EBOV entry inhibitor tetrandrine (TET) as a positive control has a similar selective inhibition to the active polypeptide, whereas the HIV-1 reverse transcriptase inhibitor Efavirenz (EFV) has an inhibitory effect on both pseudoviruses.

Wherein, the preparation method of pseudovirus VSV-G/HIV-luc expressing VSV-GP is different from the preparation method of EBOV-GP/HIV-luc only in that pZEBOV-GP in the preparation method of EBOV-GP/HIV-luc is replaced by pVSV-GP, and other operations are identical. pVSV-GP is a recombinant expression plasmid expressing vesicular stomatitis virus coat glycoprotein, which is obtained by inserting the 14 th to 1567 th positions of the vesicular stomatitis virus coat glycoprotein GP gene (GenBank Accession No. V01114 (Update Date Feb 4,2011)) into the vector pcDNA3.1+.

The antiviral activity of examples 2, pep-3.1, pep-3.2, pep-3.3 and Pep-3.10 was independent of their cytotoxicity.

To exclude non-specific differences due to polypeptide toxicity, the effect of the polypeptide on 293T cell growth was assessed using a cell counting kit-8 (ell Counting Kit-8, CCK-8).

The CCK-8 kit is a kit for detecting cell proliferation, cell survival and cytotoxicity, is a widely applied rapid high-sensitivity detection kit based on WST-8 (water-soluble tetrazolium salt, chemical name: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazolium monosodium salt), is an alternative method of MTT method, adopts water-soluble tetrazolium salt-WST-8 in the kit, and can be reduced by some dehydrogenases in mitochondria to generate orange formazan in the presence of an electronic coupling reagent. The more and the faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the shade of color and the number of cells are in good linear relationship. The method comprises the following specific steps:

293T cells were cultured in 96-well plates and incubated with the polypeptides of Table 1 (dissolved in DMSO) in a medium of 25. Mu.M. After 48 hours, the cell supernatant was changed to a cell culture medium containing 10% CCK-8 reagent and the cells were continuously cultured in a 5% CO ₂ incubator at 37℃for 1 hour. The Optical Density (OD) values per well at 450nm were recorded on a microplate reader (Thermo, varioskan Flash). The solvent DMSO was used as a blank (DMSO), tetrandrine (TET) was used as a control, and the content of tetrandrine in the medium was 3.125 μm (tetrandrine was not cytotoxic at this concentration, which was used as a positive control).

As shown in FIG. 2, at a concentration of 25. Mu.M (well above the measured IC ₅₀ value), the polypeptide had no significant effect on cell activity. Thus, it is demonstrated that the antiviral activity of the polypeptide is independent of its cytotoxicity.

Example 3, pep-3.1, pep-3.2, pep-3.3 and Pep-3.10 have good dose dependence on the inhibition of EBOV.

With reference to the procedure in example 1, different concentrations of Pep-3.1, pep-3.2, pep-3.3 and Pep-3.10 of Table 1 were dissolved in DMSO and mixed with EBOV-GP/HIV-luc pseudoviruses, respectively, to 293T cells. After 48 hours, 293T cells were lysed and peptides were assessed for anti-EBOV activity by measuring luciferase activity. Solvent DMSO was used as a control (DMSO), and luciferase activity of the control was taken as 100% of cell viability. As can be seen from FIG. 3, pep-3.1, pep-3.2, pep-3.3 and Pep-3.10 significantly inhibited the EBOV-GP/HIV-luc pseudoviral activity in a dose-dependent manner.

Example 4 determination of the entry phase of Pep-3.3 action on virus by drug action time point experiments.

The specific inhibition of the EBOV-GP/HIV-luc pseudoviruses by the polypeptides Pep-3.1, pep-3.2, pep-3.3 and Pep-3.10 suggests that they may act as EBVO entry inhibitors. To verify this, the stage of action of the polypeptide in the viral infection cycle was studied by a Time of day (TOA) experiment. Since in example 1 Pep-3.3 has the strongest specific inhibitory effect on the EBOV-GP/HIV-luc pseudovirus, the mechanism of action of Pep-3.3 was mainly studied. The method comprises the following specific steps:

The day before infection, 293T cells were seeded into 96-well plates at a cell number of 6X 10 ⁴/well and 50. Mu.L of the infected cells of EBOV-GP/HIV-luc of example 1 were added, respectively. Pep-3.3 (dissolved in DMSO at 1×10 ^-5mol·L^-1 in medium) of table 1, as an EBOV entry inhibitor tetrandrine (TET) (dissolved in DMSO at 1×10 ^-7mol·L^-1 in medium), non-nucleoside reverse transcriptase inhibitor efavirenz (efavirenz, EFV) (dissolved in DMSO at 1×10 ^-9mol·L^-1 in medium) as a control, and as a solvent control, was added at 0, 2,4, 6, 8, 10, 12, 14 and 16h time points during infection (-1 hour), during infection (0 hour), and after infection; 48h after infection, the luciferase activity of the reporter gene is detected to reflect the replication level of the recombinant virus.

The action link of the medicine can be primarily judged by measuring the failure time of the medicine in single infection of the EBOV. As shown in FIG. 4, pep-3.3 showed very strong inhibition early in viral entry, and no inhibition of viral infection after 4h (viral completed adsorption process). This is consistent with the time of action of EBOV into the inhibitor tetrandrine. The non-nucleoside reverse transcriptase inhibitor efavirenz still has an inhibiting effect on viruses at 6 hours. These results indicate that Pep-3.3 functions after the virus binds to the host and before membrane fusion of the virus to the host occurs.

Example 5 in vitro determination of binding Capacity of polypeptide to target protein GPc l Using biological Membrane layer optical interference technique

The surface glycoprotein GP of the ebola virus envelope is subjected to enzyme digestion treatment of host protease Cathepsin in endocytosis to become activated glycoprotein GPcl, and a receptor binding site is exposed. To verify that the polypeptide specifically inhibited viral entry by binding to the target protein GPcl, the binding capacity of the polypeptide to the target protein GPcl was determined in vitro using a fiber optic biosensor-based biofilm layer optical interference (BioLayer Interferometry, BLI) technique. The BLI technique is capable of tracking interactions between biomolecules in real time and is an ideal choice for studying interactions of proteins and other biomolecules. The method comprises the following specific steps:

The experiment was performed using a Ni-NTA (NTA) biosensor, using an Octet RED96 (ForteBio, inc.) instrument. The experiment is mainly carried out by the following steps: 1) Detecting a base line, immersing the NAT sensor in a buffer solution, and standing for 120s to achieve balance; 2) Incubating the protein onto the sensor, moving the sensor probe into a solution (50 mug/ml) of the purified GPcl protein with His tag, standing for 600s, and fixing the protein on the NTA sensor; 3) The sensor is moved into the buffer solution for standing for 120s to reach balance after the second detection baseline; 4) Measuring the Kon value in combination with moving the sensor into the polypeptide solution and standing for 60 s; 5) Dissociation the sensor was moved into buffer solution and allowed to stand for 60s to obtain Koff values. Four different concentrations of polypeptide were used in this process to obtain the final kinetic profile. Experimental data were analyzed using ForteBio data analysis software DATA ANALYSIS 9.0.0. Dissociation rate constant kd=k _off/K_on.

The abscissa in fig. 5 represents the reaction time in seconds. The ordinate is GPcl the signal intensity of the interaction with the polypeptide in nm. The results showed that Pep-3.1, pep-3.2, pep-3.3, and Pep-3.10 of Table 1 were each capable of binding to GPcl proteins, and that the fitted dissociation rate constants KD were 119.8. Mu.M, 97.4. Mu.M, 69.7. Mu.M, and 31. Mu.M, respectively.

Sequence listing

<110> Institute of medical biotechnology of the national academy of medical science

<120> Cyclic Polypeptides or pharmaceutically acceptable salts thereof for use against Ebola Virus

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 1

Cys Glu Tyr Phe Phe Trp Tyr Cys

1 5

<210> 2

<211> 8

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 2

Cys Asp Tyr Phe Phe Trp Tyr Cys

1 5

<210> 3

<211> 8

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 3

Cys His Tyr Phe Phe Val Tyr Cys

1 5

<210> 4

<211> 8

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 4

Cys Tyr Tyr Phe Phe Val His Cys

1 5

Claims (5)

1. A cyclic polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: the cyclic polypeptide is Pep-3.1 or Pep-3.10; the amino acid sequence of the Pep-3.1 is SEQ ID No.3 in a sequence table, and the amino acid sequence of the Pep-3.10 is SEQ ID No.4 in the sequence table.

2. A derivative of the cyclic polypeptide of claim 1, which is a linker obtained by linking an amino-terminal protecting group at the amino terminus of the cyclic polypeptide and/or a carboxy-terminal protecting group at the carboxy terminus of the cyclic polypeptide.

Multimers of PM1 or PM 2:

PM1, a multimer formed from the cyclic polypeptide of claim 1 or a pharmaceutically acceptable salt thereof;

PM2, a multimer formed from the derivative of claim 2.

4. A composition comprising C1) and C2): c1 C11), C12) or/and C13); the C11) is the cyclic polypeptide of claim 1 or a pharmaceutically acceptable salt thereof; the C12) is a derivative according to claim 2; the C13) is the multimer of claim 3;

C2 Pharmaceutically acceptable carriers or excipients;

The composition has at least one of the following functions F1) -F3):

F1 Antiviral;

F2 Treatment and/or prevention and/or adjuvant therapy of diseases caused by viral infections;

F3 Inhibiting viral invasion into cells;

In F1) -F3), the virus is Ebola virus.

Use of C11), C12), C13) or/and C14) for the preparation of at least one of the products E1) to E3):

the C11) is the cyclic polypeptide of claim 1 or a pharmaceutically acceptable salt thereof; the C12) is a derivative according to claim 2; the C13) is the multimer of claim 3;

the C14) is the composition of claim 4;

the E1) is an antiviral product;

The E2) is a product for treating and/or preventing and/or assisting in treating diseases caused by virus infection;

Said E3) is a product that inhibits viral entry into cells;

The E1) -E3), the virus is Ebola virus.