CN113698460A - Escherichia coli lipid A binding motif PCK and preparation method and application thereof - Google Patents

Abstract

The invention discloses a polypeptide specifically combined with escherichia coli lipid A and application thereof in inhibiting gram-negative bacteria. The amino acid sequence of the polypeptide is SEQ ID No.1 in a sequence table. The polypeptide of the invention can specifically inhibit gram-negative bacteria by combining with lipid A, thereby achieving the effect of resisting gram-negative bacteria infection, has low or no hemolysis, and has the hemolysis rate of 0.4 percent at the concentration of 128 mu g/mL. Aiming at escherichia coli, the FIC index value of the combined drug of the polypeptide and the berberine is 0.34, the synergistic antibacterial effect is achieved, and hemolysis and low cytotoxicity are avoided when the combined drug is used. The polypeptide or the medicinal salt and the derivative thereof can be used as a novel antibacterial preparation, particularly can be used together with berberine, and can be applied to development of antibacterial drugs.

Description

Escherichia coli lipid A binding motif PCK and preparation method and application thereof

Technical Field

The invention relates to an Escherichia coli lipid A binding motif PCK and a preparation method and application thereof.

Background

The diarrhea disease of livestock and poultry caused by gram-negative pathogenic bacteria seriously affects the health of organisms and brings great harm to livestock and poultry breeding. Wherein, the pathogenic escherichia coli is one of main pathogenic bacteria in human and animal intestinal tracts and can cause diarrheal diseases. The long-term unreasonable use of antibiotics causes the problems of high tolerance, multiple tolerance, universal drug resistance and induction of double infection, so far, no satisfactory and effective therapeutic drug exists, and the development of a novel green alternative anti-product is urgently needed.

The E.coli cell membrane is composed of an inner membrane (i.e., cytoplasmic membrane) which is formed by a phospholipid bilayer, and an outer membrane which is asymmetric, with a monolayer of phospholipids forming the inner surface and Lipopolysaccharide (LPS) lining the outer surface. LPS can protect escherichia coli from antibiotics in the intestinal tract of a mammalian host. The ratio of phospholipids to LPS is critical for membrane function, with too much LPS being detrimental to the inner membrane of the cell and too little LPS damaging the outer membrane. The research shows that the Escherichia coli inner membrane protein PbgA is one of few essential proteins without definite functions in Escherichia coli, is also a signal transducer of LPS, can bind to the LPS and inhibit the growth of bacteria. The PbgA derived fragment LAB has activity to inhibit Escherichia coli. The search data shows that the research on the synthesis and cytotoxicity of the lipid A binding motif of the PbgA protein derived from the Escherichia coli is not reported.

Disclosure of Invention

The technical problem to be solved by the invention is how to develop a medicament for replacing antibiotics so as to resist infection.

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

In the polypeptide or the medicinal salt thereof provided by the invention, the name of the polypeptide is PCK, and the amino acid sequence of the polypeptide is SEQ ID No.1 in a sequence table.

Derivatives of the above polypeptides also fall within the scope of the present invention.

The derivative of the polypeptide can be d1, d2, d3, d4 or d 5;

the d1 is a connector obtained by connecting an amino terminal protecting group at the amino terminal of the polypeptide and/or connecting a carboxyl terminal protecting group at the carboxyl terminal of the polypeptide;

d2 is a compound obtained by adding an amino acid residue to the amino terminus and/or the carboxyl terminus of the polypeptide;

d3 is a compound obtained by connecting oligopeptide at the amino terminal and/or the carboxyl terminal of the polypeptide;

the d4 is a compound obtained by modifying the polypeptide by protein, polyethylene glycol or maleimide;

the d5 is a compound obtained by connecting a lipophilic compound to the amino terminal and/or the carboxyl terminal of the polypeptide.

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

PM1, multimers formed by said polypeptide or pharmaceutically acceptable salt thereof;

PM2, multimers formed by said derivatives.

In the above polypeptide, a pharmaceutically acceptable salt thereof, or a derivative thereof, each three letters in the sequence of the polypeptide are abbreviations for amino acids, the abbreviations for amino acids having art-known meanings, for example: ser is serine, Tyr is tyrosine, Pro is proline, Met is methionine, Ala is alanine, Arg is arginine, Phe is phenylalanine, Leu is leucine, Lys is lysine, Trp is tryptophan, Gly is glycine and the like. All amino acids in the polypeptide sequence can be L-type amino acids, and one or more amino acids (except Gly) can also be replaced by D-type amino acids, artificially modified amino acids, natural rare amino acids and the like, so as to improve the bioavailability, stability and/or biological activity of the polypeptide. Wherein the D-form amino acid is an amino acid corresponding to the L-form amino acid constituting the protein; the artificially modified amino acid refers to common L-type amino acid which is modified by methylation, phosphorylation and the like and forms protein; the rare amino acids existing in nature include unusual amino acids constituting proteins and amino acids not constituting proteins, such as 5-hydroxylysine, methylhistidine, gamma-aminobutyric acid, homoserine and the like.

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

In the polypeptide, the pharmaceutically acceptable salt thereof or the derivative thereof, the amino terminal of the polypeptide of the present invention may contain an amino terminal protecting group, wherein the amino terminal protecting group may be any one of acetyl, amino, maleoyl, succinyl, tert-butoxycarbonyl or benzyloxy or other hydrophobic group or macromolecular carrier group; the carboxyl terminus of the polypeptide of the invention may contain a carboxyl-terminal protecting group, which may be any of an amino, amide, carboxyl, or tert-butoxycarbonyl group or other hydrophobic group or a macromolecular carrier group.

Also within the scope of protection of the present invention is a composition Z, which is Z1), Z2) or Z3):

z1) a composition comprising C1) and C2);

z2) a composition comprising C1) and C3);

z3) compositions comprising C1), C2) and C3);

C1) is C11), C12) or/and C13); c11) is the polypeptide or the medicinal salt thereof; c12) is the derivative; the C13) is the polymer;

C2) is berberine;

C3) is a pharmaceutically acceptable carrier or auxiliary material;

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

F1) inhibiting gram negative bacteria;

F2) treating and/or preventing and/or adjunctively treating diseases caused by gram-negative bacteria infection;

F3) inhibiting gram-negative bacteria from invading cells.

In the composition Z, F1) -F3), the gram-negative bacteria can be any one or more of Escherichia coli and salmonella.

The application of the above C11), the above C12), the above C13) or/and the composition Z in the preparation of at least one of the products E1) to E3) also belongs to the protection scope of the invention:

e1) is a product inhibiting gram-negative bacteria, such as a medicament;

e2) is a product for treating and/or preventing and/or assisting in the treatment of diseases caused by gram-negative bacterial infection, such as medicaments;

e3) is a product for inhibiting the invasion of cells by gram-negative bacteria, such as a drug;

in the above application, in E1) -E3), the gram-negative bacteria may be any one or more of escherichia coli and salmonella.

The invention also provides the application of the polypeptide or the medicinal salt thereof, the derivative or the polymer in preparing products for enhancing the bactericidal activity and/or bacteriostatic activity of the antibacterial drugs.

In the above application, the antibacterial agent is a drug having any one of the following functions:

F1) inhibiting gram negative bacteria;

F2) treating and/or preventing and/or adjunctively treating diseases caused by gram-negative bacteria infection;

F3) inhibiting gram-negative bacteria from invading cells.

In the application, the antibacterial drug is a drug containing berberine.

The present invention provides pharmaceutical compounds.

The medicinal compound provided by the invention is the C11), C12) or C13).

The medicinal compound has at least one of the following U1) -U3):

u1) for inhibiting gram-negative bacteria;

u2) for the treatment and/or prophylaxis and/or adjunctive treatment of diseases caused by gram-negative bacterial infections;

u3) for inhibiting the invasion of cells by gram-negative bacteria.

U1) -U3) of the above pharmaceutical compound, the gram-negative bacteria may be any one or more of escherichia coli and salmonella.

As noted above, the inhibition of gram-negative bacterial cell entry may be the binding of lipid a (lipid a) or other inhibition means mediated gram-negative bacterial cell entry.

Pharmaceutically acceptable salts of polypeptides of the invention include acetate (acetate), lactobionate (lactobionate), benzenesulfonate (benzamate), laurate (laurate), benzoate (benzoate), malate (malate), bicarbonate (bicarbonate), maleate (maleate), bisulfate (bisulfate), mandelate (mandelate), bitartrate (bitartrate), mesylate (mesylate), borate (borate), methyl bromide (methybromate), bromide (bromide), methyl nitrate (methylnitrate), calcium edetate (calcium), methylsulfate (methylsulfate), dexcamphorsulfonate (camsylate), mucate (mucate), carbonate (carbonate), naphthalenesulfonate (napsylate), chloride (chloride), nitrate (nitrate), clavulanate (chloride), N-methyl-citrate (citrate), ammonium dihydrooleate (ammonium chloride), ammonium dihydrooleate (sulfate), ethylenediaminetetraacetate (acetate), oxalate (oxalate), ethanedisulfonate (edisylate), pamoate (pamoate), pamoate (embonate), propionate laurate (estolate), palmitate (palmoate), ethanesulfonate (esylate), pantothenate (panthenate), fumarate (fumarate), phosphate/diphosphate (phosphate/diphosphate), glucoheptonate (gluconate), polygalacturonate (polygalacturonate), gluconate (gluconate), salicylate (salicylate), glutamate (glutamate), stearate (stearate), glycollylarsanilate (glycolurilate), sulfate (sulfate), hydroxybenzoate (hexedronate), subacetate (subacetate), hydrabamine (hydrabamine), succinate (hydrobromide), hydrobromide (salicylate), salicylate (salicylate), salicylate (salicylate), salicylate (salicylate), salicylate (salicylate), salicylate (salicylate), salicylate (salicylate), sulfate (salicylate), salicylate (salicylate), salicylate (salicylate), sulfate (salicylate), salicylate (salicylate), hydrochloride (salicylate), salicylate (salicylate), sulfate (salicylate), hydrochloride (salicylate), hydrochloride (tartrate), hydrochloride (tartrate), hydrochloride (tartrate), hydrochloride (tartrate), hydrochloride (tartrate), hydrochloride (hydrochloride), hydrochloride (tartrate), hydrochloride (, iodide (iodide), tosylate (tosylate), triiodode (triiodode), lactic acid (lactate), valeric acid (valerate), and the like. Depending on the use, 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, or bases such as ammonia, ethylenediamine (ethylenediamine), N-methyl-glutamine (N-methyl-glutamine), lysine (lysine), arginine (arginine), ornithine (ornithine), choline (choline), N '-dibenzylethylenediamine (N, N' -dibenzylethylenediamine), chloroprocaine (chloroprocaine), diethanolamine (diethanolamine), procaine (procaine), diethylamine (diethylamine), piperazine (piperazine), tris (hydroxymethyl) aminomethane (trimethyl), tetramethylammonium hydroxide (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, acetate, pamoate and the like may be used as the dosage form; pharmaceutically acceptable esters such as acetate (acetate), maleate (maleate), chloromethyl (pivaloyloxymethyl) acetate, and the like, and 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 polypeptide, the derivative thereof or the medicinal salt thereof, the polymer, the composition or the medicinal compound provided by the invention can be used for treating gram-negative bacterial infection. The lipopeptide or polypeptide, the derivative thereof, or the medicinal salt thereof, the polymer, the composition or the medicinal compound provided by the invention can also be used for preventing gram-negative bacteria infection.

In practice, the polypeptide, its derivative, or its pharmaceutically acceptable salt, the polymer, the composition or the pharmaceutical compound of the present invention can be administered as a medicament directly to a patient or an animal patient, or can be administered to a patient or an animal patient after mixing with a suitable carrier or excipient, for the purpose of treating and/or preventing gram-negative bacterial infection. The carrier material herein includes, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose, etc.). Among these, water-soluble carrier materials are preferred. 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 injections and the like. Wherein the suppository can be vaginal suppository, vaginal ring, ointment, cream or gel suitable for vaginal application. Can be common preparation, sustained release preparation, controlled release preparation and various microparticle drug delivery systems. In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier 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 and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, 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 double-layer and multi-layer tablets. In order to prepare the dosage form for unit administration into a pill, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, glycerin ester, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc. In order to prepare the unit dosage form into suppositories, various carriers known in the art can be widely used. As examples of the carrier, there may be mentioned, for example, polyethylene glycol, lecithin, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like. In order to prepare the unit dosage form into preparations for injection, 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, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc., can be used. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added. In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired.

The preparation can be used for injection administration, including subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracisternal injection or infusion, and the like; for buccal administration, e.g., rectally, vaginally, and sublingually; administration to the respiratory tract, e.g., nasally; administration to the mucosa. The above route of administration is preferably by injection, and the preferred route of injection is subcutaneous injection.

The dose of the polypeptide of the present invention, its derivative, pharmaceutically acceptable salt, said multimer, said composition or said pharmaceutically acceptable compound to be administered depends on many factors, such as the nature and severity of the disease to be prevented or treated, sex, age, body 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 dosage may be administered in a single dosage form or divided into several, e.g. two, three or four dosage forms.

For any particular patient, the specific therapeutically effective dose level will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the particular active ingredient employed; the specific composition employed; the 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; the duration of treatment; drugs used in combination or concomitantly with the specific active ingredient employed; and similar factors known in the medical arts. For example, it is common in the art to start doses of the active ingredient at levels below those required to achieve the desired therapeutic effect and to gradually increase the dose until the desired effect is achieved.

The polypeptide, the derivative thereof, or the pharmaceutically acceptable salt thereof, the polymer, the composition or the pharmaceutically acceptable compound can be directly and independently used for treating and preventing gram-negative bacteria infected persons, can also be used together with one or more medicaments for resisting gram-negative bacteria infection, can be used simultaneously or at intervals, and achieves the aim of improving the overall treatment effect.

In the invention, the gram negative bacteria can be specifically gram negative bacteria such as escherichia coli and salmonella.

Experiments prove that the polypeptide can specifically inhibit gram-negative bacteria. The polypeptide of the invention can specifically inhibit gram-negative bacteria and achieve the effect of resisting gram-negative bacteria infection, has low or no hemolysis, and has the hemolysis rate of 0.4 percent at the concentration of 128 mu g/mL. Aiming at escherichia coli, the FIC index value of the combined drug of the polypeptide and the berberine is 0.34, the synergistic antibacterial effect is achieved, and hemolysis and low cytotoxicity are avoided when the combined drug is used. The polypeptide or the medicinal salt and the derivative thereof can be used as a novel antibacterial preparation, particularly can be used together with berberine, and can be applied to development of antibacterial drugs.

Drawings

FIG. 1 is a high performance liquid chromatogram of the Escherichia coli lipidA binding motif PCK.

FIG. 2 is a mass spectrum of the PCK binding motif of Escherichia coli lipdA.

FIG. 3 is a graph of raw data for the MIC assay of the Escherichia coli lipidA binding motif PCK against Escherichia coli CVCC1515 and Salmonella ATCC 14028. In the figure, the concentration of PCK in the 1-11 columns is sequentially from low to high (2. mu.g/mL, 4. mu.g/mL, 8. mu.g/mL, 16. mu.g/mL, 32. mu.g/mL, 64. mu.g/mL, 128. mu.g/mL, 256. mu.g/mL, 512. mu.g/mL, 1024. mu.g/mL, 2048. mu.g/mL); column 12 is blank control (PCK concentration 0. mu.g/mL); behavior A-C Escherichia coli CVCC 1515; D-F behavioral salmonella ATCC 14028; behavior G is escherichia coli negative CK; h behavior salmonella negative CK. ● for turbidity, germ; x represents clear, sterile.

FIG. 4 is a graph of raw data for the MIC assay of the Escherichia coli lipidA binding motif PCK against Staphylococcus aureus ATCC 43300. In the figure, the concentration of PCK in the 1-11 columns is sequentially from low to high (2. mu.g/mL, 4. mu.g/mL, 8. mu.g/mL, 16. mu.g/mL, 32. mu.g/mL, 64. mu.g/mL, 128. mu.g/mL, 256. mu.g/mL, 512. mu.g/mL, 1024. mu.g/mL, 2048. mu.g/mL); column 12 is blank control (PCK concentration 0. mu.g/mL); A-F is Staphylococcus aureus ATCC 43300; row G, row H are negative CK. ● for turbidity, germ; x represents clear, sterile.

FIG. 5 is a graph of the raw data of the results of 96-well plate with the combination of Escherichia coli lipdA binding motif PCK and berberine against Escherichia coli CVCC 1515. In the figure, the berberine concentrations of the 1 st to 7 th columns are sequentially from low to high (0 mug/mL, 2.4 mug/mL, 4.9 mug/mL, 9.8 mug/mL, 19.5 mug/mL, 39 mug/mL and 78 mug/mL), the 8 th to 12 th columns are blank, and no sample exists; the PCK concentrations in rows A-G were from low to high (0. mu.g/mL, 32. mu.g/mL, 64. mu.g/mL, 128. mu.g/mL, 256. mu.g/mL, 512. mu.g/mL, 1024. mu.g/mL), and row H was blank with no sample. ● for turbidity, germ; x represents clear, sterile.

FIG. 6 is a graph showing the measurement of the hemolysis rate of PCK as a binding motif of Escherichia coli lipdA.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the present invention. Although the gram-negative bacteria Escherichia coli CVCC1515 and Salmonella ATCC14028 are taken as examples to specifically illustrate the bacteriostatic effect of the technical scheme of the invention, the protection scope of the application of the polypeptide is not limited to Escherichia coli CVCC1515 and Salmonella ATCC 14028. Any suitable anti-E.coli, Salmonella, etc. are within the scope of the gram negative bacteria of the present invention, such as E.coli, other strains of Salmonella, and other gram negative bacteria.

In addition, it should be noted that, unless otherwise specified, various materials and reagents used in the following examples are those commonly used in the art and are commercially available in a usual manner; the methods used are conventional methods known to the person skilled in the art or according to the conditions recommended by the manufacturers.

Example 1

1. Preparation of Escherichia coli lipid A binding motif PCK

The preparation of the lipid A binding motif PCK was accomplished by Fmoc solid phase synthesis, trifluoroacetic acid (TFA) cleavage, lyophilization, HPLC purification and mass spectrometric identification.

The amino acid sequence of PCK is:

Ser Tyr Pro Met Tyr Ala Arg Arg Phe Leu Phe Lys Trp Gly Leu Leu Arg-NH₂(as shown in sequence 1 of the sequence table).

The specific steps of synthesizing the Escherichia coli lipidA binding motif PCK by the Fmoc solid phase synthesis method are as follows: the preparation of the lipdA binding motif PCK is carried out from the direction of C end → N end, firstly, the first amino acid at the C end, namely Fmoc-Arg-OH is activated and coupled to CTC resin, and then, the protecting group Fmoc is deprotected to obtain X-Arg + CTC resin; activating and coupling the second amino acid at the C terminal, namely Fmoc-Leu-OH; following this procedure amino acids were sequentially added from the C-terminus to the last, washed 3 times with N, N-Dimethylformamide (DMF), then with methanol, and dried overnight in a nitrogen desiccator. The dried resin was cleaved by adding aqueous solution containing Triisopropylsilane (TIPS) and TFA cleavage solution. After filtration, adding glacial ethyl ether into the filtrate to precipitate the PCK, and placing the precipitate in a rotary evaporator to carry out rotary evaporation to obtain the PCK. After dissolving PCK in water, the solution was separated and purified by a high performance liquid C18 column (mobile phase A: 0.1% TFA + water; mobile phase B: 0.075% TFA + acetonitrile), and then lyophilized for MS identification and analysis. The high performance liquid chromatogram is shown in figure 1, the mass spectrum is shown in figure 2, and the molecular weight of PCK is 2124.51Da, and the purity is 98.36%.

2. Determination of PCK bacteriostatic activity

The determination of the Minimum Inhibitory Concentration (MIC) values of PCK for bacteria is performed by the micro broth dilution method (CLSI,2007) with reference to the Clinical and Laboratory Standards Institute (CLSI).

The bacteria used were of the following 3 species:

coli CVCC 1515: gram negative bacteria, China veterinary culture Collection of microorganisms, Cat # 1511C 0006000001651.

Salmonella ATCC 14028: gram negative bacteria, american type culture collection library product, cat # CDC 6516-60.

Staphylococcus aureus ATCC 43300: gram positive bacteria, American type culture Collection stock product, cat # F-182.

The bacteriostatic experiments for each of the above bacteria were as follows:

the overnight cultured bacteria were transferred to fresh MHB medium (Mueller-Hinton Broth) (pH 7.0), shake-cultured at 37 ℃ to the half log phase, and the suspension was diluted to OD with fresh MHB medium_600nm0.01, then the bacterial liquid is further diluted to 5X 10⁵CFU/mL to obtain a bacterial liquid. The concentration of PCK in a 100. mu.L system after 10. mu.L of PCK solution was adjusted to 2. mu.g/mL, 4. mu.g/mL, 8. mu.g/mL, 16. mu.g/mL, 32. mu.g/mL, 64. mu.g/mL, 128. mu.g/mL, 256. mu.g/mL, 512. mu.g/mL, 1024. mu.g/mL, 2048. mu.g/mL, respectively, for each column of a 96-well microplate by dissolving PCK (solute) with ultrapure water by the double dilution method, followed by adding 90. mu.L of a bacterial suspension (5X 10. mu.g/mL) to each well of the 96-well microplate (plate)⁵CFU/mL), three-well replicates per concentration were set, and a negative control set was set (10. mu.L of ultrapure water and 90. mu.L of bacterial suspension (5X 10) was added to each well⁵CFU/mL), and culturing in an incubator at 37 ℃ for 16-18h until the negative control hole is visibly turbid with naked eyes. The MIC value was defined as the lowest compound concentration that completely inhibited bacterial growth.

After the MIC value was determined, 10. mu.L of each content was applied to an MHA plate (Mueller-Hinton Agar) in wells of the plate where no bacteria had grown, three times per well, and after complete absorption, incubation was carried out at 37 ℃ for 18 to 24 hours, and the Minimum Bactericidal Concentration (MBC) of the antibacterial substance was determined depending on whether or not there was any bacterial growth. The value of MBC is the lowest concentration of antimicrobial substance that prevents the formation of colonies of residual bacteria.

The results of the bacteriostatic effect are shown in fig. 3, fig. 4 and table 1:

TABLE 1 measurement results of antibacterial Activity of PCK

As is clear from table 1, fig. 3, and fig. 4, PCK has a certain bacteriostatic activity against gram-negative bacteria. MIC and MBC values of PCK to Escherichia coli CVCC1515 and Salmonella ATCC14028 are 512 mug/mL. However, PCK was not active against Staphylococcus aureus ATCC43300, both MIC and MBC values were greater than 2048. mu.g/mL.

3. Combined medicine of PCK and berberine

The effect of the combination is evaluated by using fractional inhibitory concentration FIC (fractional inhibition concentration) index, and MIC values of drug A (PCK) and drug B (berberine) are respectively MIC values when they are administered separately_AAnd MIC_BThe respective concentrations of the optimal aggregation points for drug A and drug B when administered in combination are represented by A and B, respectively, FIC index formula: FIC index is FIC_A+FIC_B＝A/MIC_A+B/MIC_B. Synergistic effect: FIC index is less than or equal to 0.5; adding: FIC index not less than 0.5 and not more than 1; unrelated effects: FIC index is more than or equal to 1 and less than or equal to 2; antagonism: FIC index is not less than 2.

Experiments were performed on E.coli CVCC1515 with MIC of drug A (PCK) for E.coli CVCC1515_A512 μ g/mL, MIC of drug B (i.e., berberine) for E.coli CVCC1515_B39 μ g/mL.

Respectively preparing PCK solution and berberine solution with different concentrations by using ultrapure water as a solvent:

PCK solution was set at 6 concentrations:

0μg/mL、1/16×MIC_A＝32μg/mL、1/8×MIC_A＝64μg/mL、1/4×MIC_A＝128μg/mL、1/2×MIC_A＝256μg/mL、1×MIC_A＝512μg/mL、2×MIC_A＝1024μg/mL。

the concentration of the berberine solution is set to be 6: 0 mugmL、1/16×MIC_B＝2.4μg/mL、1/8×MIC_B＝4.9μg/mL、1/4×MIC_B＝9.8μg/mL、1/2×MIC_B＝19.5μg/mL、1×MIC_B＝39μg/mL、2×MIC_B＝78μg/mL。

In a 96-well plate, berberine solution is added into 1-7 rows of the 96-well plate from low concentration to high concentration (0 mug/mL, 2.4 mug/mL, 4.9 mug/mL, 9.8 mug/mL, 19.5 mug/mL, 39 mug/mL and 78 mug/mL) according to the row by 5 mug/L respectively, the concentration of the berberine in the same row is equal, and each row is added with 7 wells. In lines A-G, 5. mu.L of PCK solution was added in lines from low concentration to high concentration (0. mu.g/mL, 32. mu.g/mL, 64. mu.g/mL, 128. mu.g/mL, 256. mu.g/mL, 512. mu.g/mL, 1024. mu.g/mL), with 7 wells per line, and the concentration of PCK was the same as in the same line. The bacterial liquid (10) of step 2 is added into each hole respectively⁵CFU/mL)90 μ L, 100 μ L per well. The 96-well plate was incubated at 37 ℃ in an incubator for 18-24h, 3 replicates. The FIC index was calculated and the results are shown in table 2.

TABLE 2 combination of PCK and berberine against E.coli CVCC1515

As can be seen from Table 2 and FIG. 5, the FIC index value of the combination of PCK and berberine is 0.34, which is less than 0.5, indicating that PCK and berberine have synergistic bacteriostatic effect on Escherichia coli CVCC 1515.

4. Cytotoxicity assay of E.coli lipid A binding motif PCK

Collecting fresh normal Beijing duck whole blood, and performing heparin anticoagulation; centrifuging at 4 deg.C and 3000r/m for 10min, collecting precipitate, washing with 0.9% physiological saline for 3 times, and resuspending with physiological saline at 8% (v/v) to obtain erythrocyte suspension for cytotoxicity determination.

4.1 cytotoxicity assays with PCK alone

mu.L of the erythrocyte suspension and 50. mu.L of PCK solution with different concentrations (control group 1 is replaced by 100. mu.L TritionX-100 with the concentration of 0.1%, and control group 2 is replaced by normal saline) are mixed evenly, and the 50. mu.L of the solution used in the treatment group and the control group is as follows:

treatment group 1: a solution of PCK at a concentration of 0.5. mu.g/mL, with the solute PCK and the solvent physiological saline (the solute sodium chloride, the sodium chloride at a concentration of 0.9%, the solvent distilled water).

Treatment group 2: a solution of PCK at a concentration of 1 μ g/mL, a solute of PCK, and a solvent of physiological saline (solute of sodium chloride, concentration of sodium chloride of 0.9%, solvent of distilled water).

Treatment group 3: a solution of PCK at a concentration of 2 μ g/mL, a solute PCK, and a solvent physiological saline (solute sodium chloride, concentration of sodium chloride is 0.9%, solvent distilled water).

Treatment group 4: a solution containing 4. mu.g/mL of PCK, a solute of PCK, and a solvent of physiological saline (a solute of sodium chloride, a concentration of sodium chloride of 0.9%, and a solvent of distilled water).

Treatment group 5: a solution containing PCK at a concentration of 8. mu.g/mL, a solute of PCK, and a solvent of physiological saline (a solute of sodium chloride, a concentration of sodium chloride of 0.9%, and a solvent of distilled water).

Treatment group 6: a solution containing PCK at a concentration of 16. mu.g/mL, a solute of PCK, and a solvent of physiological saline (a solute of sodium chloride, a concentration of sodium chloride of 0.9%, and a solvent of distilled water).

Treatment group 7: a solution of 32. mu.g/mL of PCK, a solute of PCK, and a solvent of physiological saline (a solute of sodium chloride, a concentration of sodium chloride of 0.9%, and a solvent of distilled water).

Treatment group 8: a solution of PCK at a concentration of 64. mu.g/mL, a solute of PCK, and a solvent of physiological saline (solute of sodium chloride, concentration of sodium chloride of 0.9%, solvent of distilled water).

Treatment group 9: a solution of 128. mu.g/mL of PCK, a solute of PCK, and a solvent of physiological saline (a solute of sodium chloride, a concentration of sodium chloride of 0.9%, and a solvent of distilled water).

Treatment group 10: a solution with a concentration of PCK of 256 μ g/mL, a solute of PCK, and a solvent of physiological saline (solute of sodium chloride, concentration of sodium chloride of 0.9%, solvent of distilled water).

Control group 1: 100 μ L Tritiox-100 at a concentration of 0.1% (hemolysis rate 100%).

Control group 2: physiological saline (hemolysis rate ═ 0%).

The final concentration of the red blood cells after mixing is 4% (v/v); placing the mixed system in a constant temperature box at 37 ℃ and incubating for 1 h; taking out the cells, and centrifuging the cells for 10min (4 ℃, 1500 r/m); the supernatant was collected and the absorbance at 540nm was measured using a microplate reader.

The percent hemolysis value is calculated by the following formula: hemolysis rate (OD)_{Drug group}-OD_{Control group 2})/(OD_{Control 1}-OD_{Control group 2})×100％。

The results showed that the hemolysis rates of PCK were 17% and 0.4% at 256. mu.g/mL and 128. mu.g/mL, respectively (FIG. 6), indicating that the concentration of PCK was 0.5-128. mu.g/mL, which was not toxic to red blood cells of Beijing duck. However, PCK has low hemolysis on beijing duck erythrocytes at high concentrations.

4.2 cytotoxicity assays with PCK in combination with Berberine

The 96-well plate is added with 100 mu L/well (10)⁴Individual cells/mL), placed in an incubator (37 ℃, 5% CO)₂) Culturing for 24h, and adding the substance to be tested (PCK solution + berberine solution). The specific test substance settings were as follows:

the PCK solution was set at 6 concentrations, diluted with DMEM cell culture medium:

0μg/mL、1/16×MIC_A＝32μg/mL、1/8×MIC_A＝64μg/mL、1/4×MIC_A＝128μg/mL、1/2×MIC_A＝256μg/mL、1×MIC_A＝512μg/mL、2×MIC_A＝1024μg/mL。

the berberine solution is set to have 6 concentrations and is respectively diluted by DMEM cell culture solution: 0. mu.g/mL, 1/16 XMIC_B＝2.4μg/mL、1/8×MIC_B＝4.9μg/mL、1/4×MIC_B＝9.8μg/mL、1/2×MIC_B＝19.5μg/mL、1×MIC_B＝39μg/mL、2×MIC_B＝78μg/mL。

In a 96-well plate, 5. mu.L of PCK solution was added in rows from low concentration to high concentration (0. mu.g/mL, 32. mu.g/mL, 64. mu.g/mL, 128. mu.g/mL, 256. mu.g/mL, 512. mu.g/mL and 1024. mu.g/mL), with the same row of PCK concentration being equal, 7 wells in each row, and row 1 was used as a berberine control without PCK. From low to high concentrations (0. mu.g/mL, 2.4. mu.g/mL, 4.9. mu.g/mL, 9.8. mu.g/mL, 19.5. mu.g/mL, 39. mu.g/mL and 78. mu.g/mL) berberine solutions were added in columns of 5. mu.L each with 7 wells, the berberine concentrations in the same column were the same, and column 1 was used as the PCK control without berberine.

Adding the substance to be tested and then continuing culturing for 24 h; to each well, 10. mu.L of CCK-8 solution was added, incubated at 37 ℃ for 1-4 hours, and the absorbance at a wavelength of 450nm was measured with a microplate reader.

Cell viability ═ 100% x [ (experimental-blank)/(control-blank).

1/16×MIC_AConcentration of PCK (32. mu.g/mL) with 1/8 × MIC_BThe hemolysis rate and the cell survival rate of the berberine (4.9 mug/mL) are respectively 0.07 percent and 91.7 percent when the berberine is used in combination with the concentration, and are lower than those of the berberine (the hemolysis rate is 1 percent and the cell survival rate is 53.99 percent), which indicates that the PCK and the berberine have no hemolysis and low cytotoxicity when the PCK and the berberine are used in combination.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> institute of feed of Chinese academy of agricultural sciences

<120> Escherichia coli lipid A binding motif PCK and preparation method and application thereof

<130> GNCSY212318

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ser Tyr Pro Met Tyr Ala Arg Arg Phe Leu Phe Lys Trp Gly Leu Leu

1 5 10 15

Arg

Claims (10)

1. A polypeptide or a pharmaceutically acceptable salt thereof, characterized by: the amino acid sequence of the polypeptide is SEQ ID No.1 in a sequence table.

2. A derivative of the polypeptide of claim 1 which is d1, d2, d3, d4 or d 5;

the d1 is a connector obtained by connecting an amino terminal protecting group at the amino terminal of the polypeptide and/or connecting a carboxyl terminal protecting group at the carboxyl terminal of the polypeptide;

d2 is a compound obtained by adding an amino acid residue to the amino terminus and/or the carboxyl terminus of the polypeptide;

d3 is a compound obtained by connecting oligopeptide at the amino terminal and/or the carboxyl terminal of the polypeptide;

the d4 is a compound obtained by modifying the polypeptide by protein, polyethylene glycol or maleimide;

the d5 is a compound obtained by connecting a lipophilic compound to the amino terminal and/or the carboxyl terminal of the polypeptide.

Multimers of PM1 or PM 2:

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

PM2, multimers formed by the derivatives of claim 2.

4. A composition, the composition Z being Z1), Z2), or Z3):

z1) a composition comprising C1) and C2);

z2) a composition comprising C1) and C3);

z3) compositions comprising C1), C2) and C3);

C1) is C11), C12) or/and C13); c11) is the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof; c12) is the derivative of claim 2; the C13) is the multimer of claim 3;

C2) is berberine;

C3) is a pharmaceutically acceptable carrier or auxiliary material;

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

F1) inhibiting gram negative bacteria;

F2) treating and/or preventing and/or adjunctively treating diseases caused by gram-negative bacteria infection;

F3) inhibiting gram-negative bacteria from invading cells.

5. The composition of claim 4, wherein: the F1) -F3), the gram-negative bacteria are any one or more of Escherichia coli and Salmonella.

6, C11), C12), C13) or/and Z in the preparation of at least one of the products E1) -E3):

c11) is the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof; c12) is the derivative of claim 2; the C13) is the multimer of claim 3;

said Z is the composition of claim 4 or 5;

e1) is a product for inhibiting gram-negative bacteria;

e2) is a product for the treatment and/or prevention and/or adjuvant treatment of diseases caused by gram-negative bacterial infection;

e3) is a product for inhibiting the invasion of cells by gram-negative bacteria.

7. Use according to claim 6, characterized in that: e1) -E3), wherein the gram-negative bacteria are any one or more of escherichia coli and salmonella.

8. Use of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, the derivative of claim 2, or the multimer of claim 3 for the preparation of a product that enhances the bactericidal and/or bacteriostatic activity of an antibacterial drug.

9. Use according to claim 8, characterized in that: the antibacterial drug is a drug with any one of the following functions:

F1) inhibiting gram negative bacteria;

F2) treating and/or preventing and/or adjunctively treating diseases caused by gram-negative bacteria infection;

F3) inhibiting gram-negative bacteria from invading cells.

10. Use according to claim 8 or 9, characterized in that: the antibacterial drug is a drug containing berberine.

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