Polypeptides and Uses Thereof

Abstract

The present invention relates to the technical field of biomedical peptides, and more particularly to polypeptides and their use in pharmaceuticals for preventing, treating, curing, or alleviating diseases associated with antibacterial and / or anti-inflammatory properties. The polypeptides of the present invention have short amino acid sequences, simple structures, are easy to synthesize, and have high antibacterial and anti-inflammatory activities, making them suitable for use in the prevention and treatment of diseases caused by microbial infections and effective in healing skin wounds.

Description

[Technical Field]

[0001] The present invention relates to the technical field of biomedical peptides, and in particular to polypeptides and their use in medicines for preventing, treating, curing or alleviating diseases associated with their antibacterial and / or anti-inflammatory properties. [Background technology]

[0002] Skin trauma is generally defined as an injury caused by direct external force or the presence of a soft tissue infection, which triggers a local inflammatory response and results in local symptoms such as redness, swelling, or painful suppuration. Typical skin injuries result in traditional signs of inflammation (redness, swelling, and heat) due to leakage of fluid and blood from blood vessels and obstruction of local lymphatic flow. Cellular damage results in the release of numerous vasoactive substances, including histamine, serotonin, and catecholamines, which cause transient vasoconstriction followed by vasodilation, allowing fluid and cells to enter the extravascular injury site. In clinical practice, antibiotics are the most widely used treatment for skin injuries. However, with the widespread use of antibiotics, bacterial resistance has become a major problem. Furthermore, bacterial infections trigger the body's own inflammatory response, which not only delays skin healing but also leads to scarring. Common skin injuries seen in daily life, such as burns, scalds, and ulcers, are also primarily treated with antibiotics, and there remains an unmet clinical need as appropriate treatments are not being provided.

[0003] Therefore, new antibacterial / anti-inflammatory drugs have become a hot research and development topic in fields such as human medicine, nutrition, food science, and immunology both at home and abroad. The development of biological antibacterial peptides will help solve the problem of bacterial drug resistance, and antibacterial peptides with anti-inflammatory properties are expected to effectively meet clinical needs in the field of skin trauma, with expanding prospects for their development and application.

[0004] Thrombin-derived C-terminal peptides (TCP) are approximately 2 kDa thrombin-derived C-terminal peptides selectively hydrolyzed by coagulation factors. They are 25 amino acids long and contain two targets. In addition to interacting with bacterial lipopolysaccharide (LPS), TCP-25 binds to a hydrophobic pocket on the surface of human leukocytes, CD14, a differentiation antigen. This peptide functions as an antiendotoxin in vitro and in vivo, preventing sepsis and other problems such as shock. Previous studies have shown that TCP-25 effectively inhibits Staphylococcus aureus, Gram-negative Pseudomonas aeruginosa, and subcutaneous Pseudomonas aeruginosa in vitro, and reduces macrophage responses to serum lipase (LPS) in vivo, potentially promoting wound healing and inhibiting scar formation. Therefore, TCP-25 is an antibacterial and anti-inflammatory peptide with a dual targeting mechanism of action, offering excellent specificity, clinical efficacy, and minimal toxic side effects. Based on the dual mechanism of action of TCP-25, its broad spectrum bacteriostasis, and low toxicity, the present invention provides modified antibacterial / anti-inflammatory polypeptides based on TCP-25, which are expected to be developed as wound-healing drugs. Summary of the Invention [Problem to be solved by the invention]

[0005] The following provides a general, non-limiting description of only some aspects of the present invention. These and other aspects will be described in more detail below. All references herein are incorporated by reference in their entirety. In the event of any inconsistency between the disclosure of this specification and the references, the disclosure of this specification shall prevail.

[0006] The object of the present invention is to provide new polypeptides with antibacterial and / or anti-inflammatory properties to address the unmet clinical need for skin wound treatment.

[0007] Alanine scanning technology takes advantage of the fact that alanine, due to its small size, has little effect on protein structure, but substituting other important amino acids with alanine weakens or reduces certain protein functions. By substituting 19 other non-alanine residues with alanine one by one to construct an alanine scanning library, we then used expression and screening processes to identify the roles played by specific amino acid residues in terms of protein function, active site, stability, and morphology. Alanine scanning of the TCP-25 sequence was performed to detect the antibacterial and anti-inflammatory activities of the molecule. By comparing the activity with the unmutated TCP-25 sequence, we identified amino acid sites important for activity. Based on the information obtained, we then performed further single-amino acid mutations and combinatorial mutations of multiple amino acids at multiple sites to obtain a series of polypeptides with antibacterial and / or anti-inflammatory properties. [Means for solving the problem]

[0008] Specifically, in a first aspect, the present invention provides a polypeptide characterized in that it consists of a sequence shown in formula (I) below: (X 0 ) n -X 1 -X 2 -X 3 -X 4 -LX 5 -KWIX 6 -KX 7 -X 8 -(X 9 ) m (I) or a pharmaceutically acceptable salt thereof, wherein X 0 are each independently selected from any amino acid; X 1 , X 4 and X 5 are each independently selected from K, dK, R, dR, a derivative of K or R; X 2 , X 3 and X 6 are each independently selected from any amino acid; X 7 and X 8 are each independently selected from the I, V, L, T or d configurations of said amino acids and derivatives thereof; each X 9 are each independently any amino acid, n is 0 or an integer from 1 to 7, m is 0 or an integer of 1 to 5.

[0009] In some embodiments, the X 0 is selected from G, K, Y, F, T, S, K, L, W, E or d structures of said amino acids and derivatives thereof.

[0010] In some embodiments, the X 2 is selected from the V, L, T, K, W, E or d structures of said amino acids and derivatives thereof.

[0011] In some embodiments, the X 3 is selected from F, Y, W or d structures of said amino acids and derivatives thereof.

[0012] In some embodiments, the X 6 is selected from the Q, N, T, K, W, L or d structures of said amino acids and derivatives thereof.

[0013] In some embodiments, the X 1 Lys, D Lys, Orn, D Orn, Dab, D Dab, hLys, D hLys, ACLys, D ACLys, NMeLys, NMe D Lys, NMeDab or NMe D Dab is selected.

[0014] In some embodiments, the X 9are each independently selected from C, Q, F, G, N, T, K, W, L, Y, S, D, E, the d-structures of the above amino acids or derivatives of the above amino acids, and preferred X 9 Phe, D Phe, Trp, D Trp, Tyr, D Tyr, Thi, D Thi, Bip, D Bip, Nva, D Nva, Aib, D Aib, Pip, D Pip, hPhe, D hPhe, hTrp, D hTrp, hTyr, D hTyr, hHis, D hHis, 1-Nal, 2-Nal, Dip, 1- D Nal, 2- D Nal, D Dip is selected.

[0015] In some embodiments, when m=5, (X 9 ) m The first and last amino acids are linked by a bond to form a ring, and preferably the bond is a disulfide bond, an amide bond, a carbon-hydrogen bond, a dithiocarbamate C 1-6 It is selected from an alkyl, thioether or olefinic thioether bond, more preferably said bond is one of the structures shown below.

[0016] [ka]

[0017] On the other hand, the present invention provides a polypeptide characterized by having the sequence shown in formula (II) below: GKX 10 -GX 11 -YX 12 -X 1 -X 2 -X 3 -X 4 -LX 5 -KWIX6 -KX 7 -X 8 -(X 9 ) m (II) or a pharmaceutically acceptable salt thereof, wherein X 1 , X 4 , X 5 are each independently selected from K, dK, R, dR, a derivative of K or R; X 2 , X 3 , X 6 , X 11 , X 12 are each independently selected from any amino acid; X 7 is selected from the V, L or d configurations of said amino acids and derivatives thereof; X 8 is selected from the I, V, L, T or d configurations of said amino acids and derivatives thereof; each X 9 are each independently any amino acid, X 10 is any aromatic amino acid, m is 0 or an integer of 1 to 5.

[0018] In some embodiments, the X 2 is selected from the V, L, T, K, W, E or d structures of said amino acids and derivatives thereof.

[0019] In some embodiments, the X 3 , X 11 are each independently selected from F, Y, W or d structures of said amino acids and derivatives thereof.

[0020] In some embodiments, the X 6 is selected from the Q, N, T, K, W, L or d structures of said amino acids and derivatives thereof.

[0021] In some embodiments, the X 12is T, S, K, L, W, E or d structure of said amino acids and derivatives thereof.

[0022] In some embodiments, the X 9 is selected from C, Q, F, G, N, T, K, W, L, Y, S, D, E, the d-structures of the above amino acids or derivatives of the above amino acids, and preferred X 9 Phe, D Phe, Trp, D Trp, Tyr, D Tyr, Thi, D Thi, Bip, D Bip, Nva, D Nva, Aib, D Aib, Pip, D Pip, hPhe, D hPhe, hTrp, D hTrp, hTyr, D hTyr, hHis, D hHis, 1-Nal, 2-Nal, Dip, 1- D Nal, 2- D Nal, D Dip is selected.

[0023] In some embodiments, the X 10 is Y, F, W, dY, dF, dW or a derivative of the above amino acid, preferably X 10 Phe, D Phe, Trp, D Trp, Tyr, D Tyr, Thi, D Thi, Bip, D Bip, hPhe, D hPhe, hTrp, D hTrp, hTyr, D hTyr, hHis, D hHis, 1-Nal, 2-Nal, Dip, 1- D Nal, 2- D Nal or D Dip is selected.

[0024] In some embodiments, when m=5, (X 9 )m The first and last amino acids are linked by a bond to form a ring, and the preferred bond is a disulfide bond, an amide bond, a carbon-hydrogen bond, a dithiocarbamate C 1-6 It is selected from an alkyl, thioether or olefinic thioether bond, more preferably said bond is one of the structures shown below.

[0025] [ka]

[0026] On the other hand, the present invention provides a polypeptide consisting of the sequence shown in formula (III) below: GKX 10 -GX 11 -YX 12 -X 1 -X 2 -X 3 -X 4 -LX 5 -KWIX 6 -KX 7 -X 8 -X 13 -X 14 -X 15 -GX 16 (III) or a pharmaceutically acceptable salt thereof, wherein X 1 , X 4 , X 5 are each independently selected from K, dK, R, dR, a derivative of K or R; X 2 , X 3 , X 6 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 or X 16 are each independently selected from any amino acid; X 7 is selected from the V, L or d configurations of said amino acids and derivatives thereof; X8 is selected from the I, V, L, T or d configurations of said amino acids and derivatives thereof.

[0027] In some embodiments, the X 2 is selected from the V, L, T, K, W, E or d structures of said amino acids and derivatives thereof.

[0028] In some embodiments, the X 3 , X 11 are each independently selected from F, Y, W or d structures of said amino acids and derivatives thereof.

[0029] In some embodiments, the X 6 is selected from the Q, N, T, K, W, L or d structures of said amino acids and derivatives thereof.

[0030] In some embodiments, the X 10 is Y, F, W or d structure of said amino acids and derivatives thereof.

[0031] In some embodiments, the X 12 is T, S, K, L, W, E or d structure of said amino acids and derivatives thereof.

[0032] In some embodiments, the X 13 and X 16 are each independently selected from C, K, W, L, S, D, E or d structures of the amino acids and derivatives thereof, and preferably, the X 13 and X 16 are each independently selected from C or dC.

[0033] In some embodiments, the X 13 and X 16 are linked by a bond to form a ring, and preferably, the bond is a disulfide bond, an amide bond, a carbon-hydrogen bond, a dithiocarbamate C 1-6It is selected from an alkyl, thioether or olefinic thioether bond, more preferably said bond is one of the structures shown below.

[0034] [ka]

[0035] In some embodiments, the X 14 is Q, N, T, K, W, L or d structure of said amino acids and derivatives thereof.

[0036] In some embodiments, the X 15 is Y, F, W or d structure of said amino acids and derivatives thereof.

[0037] Preferably, the present invention provides polypeptides having the following amino acid sequences: SEQ ID NO: 1 to SEQ ID NO: 99.

[0038] Another aspect of the present invention provides a polypeptide, the sequence of the polypeptide being one of SEQ ID NOs: 1 to 99, preferably the polypeptide sequence is selected from SEQ ID NO: 1 (corresponding to compound 1 below), 10 (corresponding to compound 17 below), sequence 23 (corresponding to compound 30 below), 25 (corresponding to compound 32 below), 30 (corresponding to compound 37 below), 74 (corresponding to compound 84 below), 76 (corresponding to compound 86 below), 78 (corresponding to compound 88 below), 80 (corresponding to compound 90 below), 95 (corresponding to compound 105 below) or 96 (corresponding to compound 106 below).

[0039] In another aspect of the present invention, there is provided a polypeptide, wherein the polypeptide has one of the following structures or a pharmaceutically acceptable salt thereof, wherein the numbers below the sequence indicate compound numbers, e.g., 1 indicates compound 1, 2 indicates compound 2, etc.

[0040] [ka]

[0041] [ka]

[0042] [ka]

[0043] [ka]

[0044] [ka]

[0045] [ka]

[0046] [ka]

[0047] [ka]

[0048] On the other hand, the present invention provides a pharmaceutical composition comprising any one of the polypeptides of the present invention. In some embodiments, the pharmaceutical composition of the present invention further comprises at least one of a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, and vehicle.

[0049] On the other hand, the present invention provides the use of a polypeptide or a pharmaceutical composition according to the present invention in the preparation of a medicament for preventing, treating, curing or alleviating a disease, wherein said medicament is used to prevent, treat, cure or alleviate a bacterial infection and / or an inflammatory infectious disease.

[0050] In some embodiments, the bacterial infection and / or inflammatory infection disease includes, but is not limited to, skin infection, skin wound infection, burn infection, pneumonia, tuberculosis, diabetic foot, tonsillitis, bacterial dysentery, meningitis, scarlet fever, pharyngitis, bronchitis, gastritis, appendicitis, glomerulonephritis, endocarditis, pericarditis, cystitis, pelvic inflammation, and cervicitis.

[0051] The present invention has the following beneficial effects: the polypeptides described in the present invention have significant antibacterial / anti-inflammatory effects, high affinity to the target, very low MIC values, large diameters of bacterial inhibition zones, and the cyclic structure of the polypeptides of the present invention contributes to improved polypeptide stability and does not cause obvious cytotoxicity to mammalian cells. [Brief explanation of the drawings]

[0052] [Figure 1] 1 is an MS diagram of compound 30. [Figure 2] FIG. 1 is an HPLC profile of compound 30. [Figure 3] FIG. 1 is an MS diagram of compound 105. [Figure 4] FIG. 1 is an HPLC profile of compound 105. [Figure 5] FIG. 1 is an MS diagram of compound 106. [Figure 6] FIG. 1 is an HPLC profile of compound 106. [Figure 7] FIG. 1 is an MS diagram of compound 108. [Figure 8] FIG. 1 is an HPLC profile of compound 108. [Figure 9] FIG. 1 is an MS diagram of compound 109. [Figure 10] FIG. 1 is an HPLC profile of compound 109. [Figure 11] FIG. 1 shows the inhibitory effect of polypeptide compounds on fluorescein reporter gene expression in LPS-activated TLR4 cells. DETAILED DESCRIPTION OF THE INVENTION

[0053] The meaning of the term "peptide" or "polypeptide" is well known to those skilled in the art. Typically, a peptide or polypeptide is two or more amino acids linked by an amide bond between the amino group of one amino acid and the carboxyl group of the adjacent amino acid. The polypeptides described herein may be composed of naturally occurring or non-naturally occurring amino acids. They may also be modified to form analogs, derivatives, functional mimetics, pseudopeptides, etc., consisting of at least two amino acids. Unless a specific modification of the N- or C-terminus is indicated, polypeptides consisting of a specific amino acid sequence may consist of unmodified and modified amino and / or carboxy termini, as known to those skilled in the art. Polypeptides consisting of a specific amino acid sequence may contain modified and / or additional amino acids, as long as the N- and / or C-terminus do not contain modifications that prevent the addition of additional amino acids. Such modifications include, for example, acetylation of the N-terminus and / or amidation of the C-terminus.

[0054] The polypeptides of the present invention may be modified to form polypeptide derivatives. As is well known to those skilled in the art, various transformation modifications can be made to polypeptides. Exemplary transformation modifications include, but are not limited to, N-terminal acetylation, C-terminal amidation, d-amino acid substitutions, unnatural amino acid substitutions, fatty acid modifications, or various combinations of the above modifications. The present invention encompasses well-known peptide modifications. For example, polypeptide derivatives can comprise chemical modifications of polypeptides, such as alkylation, acylation, carbamoylation, iodination, or any other transformation modification that produces a polypeptide derivative. Polypeptide modifications can be composed of modified amino acids, such as hydroxyproline or carboxyglutamic acid, or can be composed of amino acids linked by non-peptide bonds.

[0055] Other modifications to the polypeptides of the invention may involve the substitution of unnatural amino acids for natural amino acids in the polypeptide, including 2-amino fatty acids (Aad), 3-amino fatty acids (βAad), β-alanine, β-aminopropionic acid (βAla), 2-aminobutyric acid (Abu), 4-aminobutyric acid, piperidine carboxylic acid (4Abu), 6-aminocaproic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (βAib), 2-aminoheptanedioic acid (Apm), 2,4-diaminobutyric acid (Dbu), diclazurin (Des), 2,2'-diaminoheptanedioic acid (Dbu), 2,2'-diaminoheptanedioic acid (Dbu), 2,2'-diaminoheptanedioic acid (Dbu), 2,2'-diaminobut ... Modified α-amino acids include, but are not limited to, α-amino acids (Dpm), 2,3-diaminopropanoic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), isohydroxylysine (αHyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isoclaurine (Ide), isoisoleucine (αIle), N-methylglycine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), N-valine (Nva), N-leucine (Nle), and ornithine (Orn). Of course, all modified α-amino acids can be replaced with the corresponding β-, γ-, or ω-aminocarboxylic acids.

[0056] The term "amino acid" refers to a molecule containing both an amino group and a carboxyl group. Suitable amino acids include, but are not limited to, D- and L-isomers of naturally occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways. As used herein, the term amino acid includes, but is not limited to, α-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.

[0057] The term "naturally occurring amino acid" refers to any one of the 20 L-amino acids commonly found in peptides synthesized in nature, namely, the L-isomers of alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Glu or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0058] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains (e.g., A, V, L, I, P, F, M, W), β-branched side chains (e.g., T, V, I), and aromatic side chains (e.g., Y, F, W, H). Thus, for example, a predicted non-essential amino acid residue in a polypeptide is preferably replaced with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on considerations of electronic equivalence (e.g., methionine being replaced with n-leucine) or other properties (e.g., phenylalanine being replaced with 2-thienylalanine).

[0059] The polypeptides of the present invention can be prepared using methods well known to those skilled in the art, including well-known chemical synthesis methods. Thus, if a polypeptide or its derivative contains one or more non-standard amino acids, it is likely prepared by chemical synthesis. In addition to preparing a polypeptide or its derivative using chemical synthesis, it can also be prepared by encoding nucleic acid expression. This is particularly relevant to the preparation of polypeptides or their derivatives containing only natural amino acids, in which case well-known methods for preparing nucleic acid-encoded polypeptide sequences can be used (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). Polypeptides can be expressed in organisms and purified by well-known purification techniques.

[0060] The term "alkylidene" refers to a divalent alkyl group (ie, --R--).

[0061] The term "alkenyl" refers to a straight or branched hydrocarbon chain having one or more carbon-carbon double bonds. The alkenyl moiety contains a specific number of carbon atoms. For example, C2 to C 10 means that the group contains 2 to 10 carbon atoms (inclusive of the end values).

[0062] The term "alkynyl" means a straight or branched hydrocarbon chain having one or more carbon-carbon triple bonds.

[0063] Regarding sequence identity, according to methods known in the art, sequence identity is calculated by sequence comparison. To determine the percentage identity of two amino acid sequences, the sequences are compared for best comparison. For example, a null position may be introduced into the sequence of the first amino acid sequence to best compare it with the second amino acid sequence. Then, the amino acid residues at corresponding amino acid positions are compared. If a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, the molecules are identical at that position. The identity between two sequences is a function of the number of identical positions shared by the sequences. Therefore, identity % = number of identical positions / total number of overlapping positions multiplied by 100.

[0064] In this comparison, the sequences may be of the same length or of different lengths. The optimal sequence comparison for determining the comparison window can be performed using the Smith and Waterman local homology algorithm (J. Theor. Biol. 1981), the Needleman and Wunsch homology comparison algorithm (J. Mol. Biol., 1972), the Pearson and Lipman similarity algorithm (Proc. Natl. Acad. Sci. USA 1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, TFASTA, Wisconsin Genetics Software Package version 7.0, Genetic Computer Group, 575 Science Drive, Madison, Wisconsin), or publicly available computer software such as BLAST. When using such software, it is preferable to use default parameters such as null penalties and stretch penalties. The best comparison (i.e., the one with the highest percentage of identity across the entire comparison window) generated by various methods is selected.

[0065] In certain embodiments, the amino acid sequence of the stapled peptide has at least 90%, 93%, or 95% sequence identity to SEQ ID NO:1.

[0066] The term "pharmaceutical composition" refers to a pharmaceutical composition comprising a therapeutically effective amount of a peptide of the present invention and a pharmaceutically acceptable carrier or excipient. As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and physiologically compatible analogs. Examples of pharmaceutically acceptable carriers or excipients include one or more of water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, the like, and combinations thereof. In any case, it is preferable to include an isotonicity agent, for example, a sugar, a polyol such as mannitol or sorbitol, or sodium chloride, in the composition. Also included may be a wetting or minor amount of a pharmacologically acceptable substance, for example, a wetting or emulsifying agent, a preservative, or a buffer, which improves the shelf life and effectiveness of the antibody or antibody portion. Optionally, a disintegrating agent may be included, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, e.g., sodium alginate. In addition to excipients, pharmaceutical compositions may include one or more of carrier proteins such as serum albumin, buffers, binders, sweeteners and other flavoring agents, coloring agents, and polyethylene glycol.

[0067] The compositions can be in many forms, including, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the established route of administration and therapeutic use. In one embodiment, the composition is in the form of an injectable or infusible liquid, for example, a form similar to that used for passive immunization of humans with antibodies. In one embodiment, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular), and in one embodiment, the polypeptide is administered by intravenous injection or infusion. In another embodiment, the polypeptide is administered by intramuscular or subcutaneous injection.

[0068] Other suitable routes of administration for use of the pharmaceutical composition include, but are not limited to, rectal, transdermal, vaginal, transmucosal, or enteral administration.

[0069] The "pharmaceutically acceptable salts" of the present invention, i.e., pharmaceutical salts, can be synthesized from polypeptides, basic moieties, or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid forms of these polypeptides with a stoichiometric amount of an appropriate base (e.g., hydroxides, carbonates, bicarbonates, etc., of Na, Ca, Mg, or K), or by reacting the free base forms of these polypeptides with a stoichiometric amount of an appropriate acid. Such reactions are typically carried out in water or an organic solvent, or a mixture thereof. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, acetonitrile, etc., are required, as appropriate. Lists of other suitable salts can be found, for example, in "Remington's Pharmaceutical Sciences," 20th Edition, Mack Publishing Company, Easton, Pa., (1985), and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use," Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

[0070] Pharmaceutically acceptable salts may be pharmaceutically acceptable acid addition salts, which may be formed by the action of the polypeptide of the present invention with an inorganic acid and / or an organic acid, for example, salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, or sulfuric acid, or salts formed with organic acids such as acetic acid, trifluoroacetic acid, propionic acid, malonic acid, oxalic acid, maleic acid, fumaric acid, malic acid, citric acid, gluconic acid, mandelic acid, tartaric acid, stearic acid, succinic acid, sulfosalicylic acid, lactic acid, benzoic acid, benzenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, naphthalenedisulfonic acid, and the like.

[0071] Pharmaceutically acceptable salts include pharmaceutically acceptable base addition salts that can be formed by the reaction of a polypeptide of the present invention with an inorganic and / or organic base. Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from Groups I to XII of the periodic table. In some embodiments, salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper, with particularly suitable salts including ammonium, potassium, sodium, calcium, and magnesium salts. Organic bases from which salts can be derived include primary, secondary, and tertiary amines, and substituted amines include naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Specific organic amines include, for example, isopropylamine, benzylpenicillin, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, aminobutanetriol, and the like.

[0072] Alternatively, the polypeptides disclosed herein, including their salts, can exist in their hydrated forms or in forms containing their solvents (e.g., ethanol, DMSO, etc.) and can be used for crystallization. The compounds of the present disclosure can inherently or by design form solvates with pharmaceutically acceptable solvents, including water, and therefore the compounds of the present invention include solvated and unsolvated forms.

[0073] (Example) The polypeptide compounds and derivatives provided by the present disclosure were synthesized using solid-phase synthesis of their linear precursors, using 2,2'-dithiodipyridine-catalyzed intramolecular disulfide bond formation. Fmoc-Cys(Trt)-2-Chlotrityl Resin was used as the synthetic support. The synthesis process began with completely solubilizing the Fmoc-Cys(Trt)-2-Chlotrityl Resin in N,N-dimethylformamide (DMF). The solid support was then condensed with activated amino acid derivatives until the desired polypeptide chain length was reached, followed by washing, Fmoc deprotection, washing, and subsequent amino acid condensation. Finally, the polypeptide was cleaved from the solid support by reacting the resin with a mixture of trifluoroacetic acid, water, triisopropylsilane, and phenylmethylsulfide (90:2.5:2.5:5, v:v:v:v). The resulting solid crude linear precursor was then precipitated with frozen methyl tert-butyl ether. The crude cleaved linear precursor was subjected to disulfide bond oxidation in alkaline solution to obtain the crude peptide of interest, which was then purified and separated using a C-18 reversed-phase preparative column in an acetonitrile / water system with 0.1% trifluoroacetic acid to obtain the pure polypeptide and its derivatives.

[0074] (Experimental Reagents) Fmoc-Cys(Trt)-2-Chlotrityl Resin, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Gln(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-L ys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Rink Amide-AM Resin, Fmoc-Asn(Trt)-OH, and Fmoc-Ser(tBu)-OH are all Gill The reagents were purchased from Biotech, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), trifluoroacetic acid (TFA), 2,2'-dithiobispyridine, ammonium bicarbonate, dimethyl sulfoxide (DMSO), and guanidine hydrochloride were purchased from Aladdin, N,N-dimethylformamide (DMF) from Anegi, dichloromethane (DCM), 4-methylpiperidine from Maclean's, triisopropylsilane, methyl tert-butyl ether, 4-methylmorpholine (NMM), and phenylmethylsulfide from TCL, acetonitrile from Sigma-Aldrich, and 10x PBS from Solepol. All other reagents were commercially available unless otherwise specified.

[0075] Example 1. Preparation of Compound 30 (i.e., corresponding SEQ ID NO: 23)

[0076] [ka]

[0077] Step 1: Synthesis of linear precursor peptide chain (GKWGFYTKVFRLKKWIQKVIC-QFGC) 226 mg (0.1 mmol) of Fmoc-Cys(Trt)-2-Chlotrityl Resin was completely solubilized in DMF for 1 hour. The linear precursor sequence was then synthesized from the second G at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows: Fmoc deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), for 8 minutes each time. The resin was washed 6–8 times with DMF until a neutral pH was reached. 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 1 mmol 4-methylmorpholine (NMM) were dissolved in DMF, added to the resin, and the reaction was allowed to proceed at room temperature for 1 hour. The resin was washed 4–6 times with DMF before the next amino acid coupling to obtain the linear precursor peptide chain. After synthesis of the linear precursor polypeptide, the resin was washed five times with DMF and five times with DCM, and then the resin was suctioned under vacuum.

[0078] Step 2: Cleavage of the linear precursor peptide chain Fresh cutting cocktail (10 mL) trifluoroacetic acid:water:triisopropylsilane:phenylmethylsulfide (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 1 and the reaction was allowed to proceed with shaking at room temperature for 2 hours. After the reaction was complete, the reaction solution was filtered, the resin was washed with trifluoroacetic acid, and the resin was combined with the reaction solution. The crude product was then precipitated with four volumes of MTBE. The crude product was washed three times with MTBE and vacuum pumped.

[0079] Step 3: Intramolecular disulfide bond formation The crude product obtained in step 2 was dissolved in 50% aqueous ACN to a concentration of 4 mg / mL. 0.5 equivalents of 2,2'-dithiobispyridine was weighed and added to 50 mM ammonium bicarbonate buffer (pH = 8.0, containing 50% acetonitrile) to completely dissolve the 2,2'-dithiobispyridine. Finally, the polypeptide solution was slowly added dropwise to the 2,2'-dithiobispyridine solution to a final concentration of 2 mg / mL, and the mixture was shaken at room temperature for 16 hours. The reaction results were monitored using LC-MS, and the reaction was quenched by adding 0.5% (v:v) trifluoroacetic acid after completion.

[0080] Step 4: Purification and preparation of polypeptides After filtration through a 0.45 μm membrane, the product was separated using a reversed-phase high-performance liquid chromatography system. Buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile) were used. The chromatography column used was a BR C-18 (Syphen) reversed-phase chromatography column. The detection wavelength during the purification process was set to 230 nm, the flow rate was 15 mL / min, and the gradient was 15-30% acetonitrile over 40 min. The relevant distillation fractions of the product were collected, and after confirming the purity by HPLC, the distillation fractions with a purity above 95% were combined and lyophilized to obtain the pure polypeptide.

[0081] Step 5: Detection and feature evaluation methods The purified polypeptide product of step 4 is subjected to analytical high performance liquid chromatography / mass spectrometry to confirm its purity, and the compound forms an intramolecular disulfide bond. See Figure 2 and Figure 1.

[0082] The inventors performed a series of modifications (including the addition or substitution of one or more amino acids) on the compound 30 polypeptide and the modified polypeptide compounds 1-109 to obtain the specific sequences and structures of these polypeptides and polypeptide derivatives. The synthesis and purification were carried out by Hunan Zhongsheng Peptide Biochemical Co., Ltd. in the same manner as in Example 1, and the purity exceeded 95%.

[0083] Example 2 Preparation of Compound 105 (ie, corresponding SEQ ID NO: 95).

[0084] [ka]

[0085] Step 1: Synthesis of linear precursor peptide chain Synthesis of the linear precursor peptide chain of compound 105. GKWGFYTKVFRLKKWIKKVIC-TFGC 294 mg (0.2 mmol) of Rink Amide-AM Resin was completely solubilized in DMF for 1 hour. Then, the linear precursor sequence was synthesized from the carboxyl-terminal C1 position to the amino terminus. Each coupling cycle was performed as follows: Fmoc deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL) for 8 minutes each time. The resin was washed 6–8 times with DMF until a neutral pH was reached. The resin was dissolved in DMF, and 1.0 mmol Fmoc-AA, 1.0 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 2 mmol 4-methylmorpholine (NMM) were added and reacted at room temperature for 1 hour. Before the next amino acid coupling, the resin was washed 4–6 times with DMF. After the linear polypeptide was synthesized, the resin was washed 5 times with DMF and 5 times with DCM. The resin was then aspirated under vacuum.

[0086] Step 2: Cleavage of the linear precursor peptide chain Fresh cutting cocktail (10 mL) trifluoroacetic acid:water:triisopropylsilane:phenylmethylsulfide (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 1 and the reaction was allowed to proceed with shaking at room temperature for 2 hours. After the reaction was complete, the reaction solution was filtered, the resin was washed with trifluoroacetic acid, and the resin was combined with the reaction solution. The crude product was then precipitated with four volumes of cold MTBE. The crude product was washed three times with MTBE and vacuum pumped.

[0087] Step 3: Intramolecular disulfide bond formation The crude product obtained in step 2 was dissolved completely in DMSO (the DMSO volume was 20% of the total volume of the reaction system). A phosphate-guanidine hydrochloride buffer solution (pH 7.0, containing 50% ACN) was prepared. A 1x PBS:6M guanidine hydrochloride (80:20, v:v) buffer solution and acetonitrile (1:1) were mixed to obtain a phosphate-guanidine hydrochloride buffer solution (pH 7.0, 50% ACN). The polypeptide solution was then slowly added dropwise to the buffer solution to a final concentration of 1 mg / mL. The mixture was shaken at room temperature for 16 hours. The reaction results were monitored by LC-MS, and the product was directly purified and purified after the reaction was completed.

[0088] Step 4: Polypeptide purification and preparation After filtration through a 0.45 μm membrane, the product was separated using a reverse-phase high-performance liquid chromatography system. Buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile) were used. The chromatography column was a BR C-18 (Syphen) reverse-phase chromatography column. During the purification process, the chromatographic detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 25-45% acetonitrile over 40 min. The relevant distillation fractions of the product were collected, and after confirming the purity by HPLC, the distillation fractions with a purity above 95% were combined and lyophilized to obtain the pure polypeptide.

[0089] Step 5: Detection and feature evaluation methods The purified polypeptide from step 4 was subjected to analytical high performance liquid chromatography / mass spectrometry to confirm its purity, and the compounds formed intramolecular disulfide bonds, as shown in Figures 3 and 4.

[0090] Example 3. Preparation of compound 106 (i.e., corresponding SEQ ID NO: 96).

[0091] [ka]

[0092] Step 1: Synthesis of linear precursor peptide chain Synthesis of the linear precursor peptide chain of compound 106. GKWGFYTKVFRLKKWIRKVIC-TFGC 294 mg (0.2 mmol) of Rink Amide-AM Resin was completely solubilized in DMF for 1 hour. Then, the linear precursor sequence was synthesized from the carboxyl-terminal C1 position to the amino terminus. Each coupling cycle was performed as follows: Fmoc deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL) for 8 minutes each time. The resin was washed 6–8 times with DMF until a neutral pH was reached. 1.0 mmol Fmoc-AA, 1.0 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 2 mmol 4-methylmorpholine (NMM) were dissolved in DMF, added to the resin, and incubated at room temperature for 1 hour. Before the next amino acid coupling, the resin was washed 4–6 times with DMF. After the linear polypeptide was synthesized, the resin was washed 5 times with DMF and 5 times with DCM. The resin was then aspirated under vacuum.

[0093] Step 2: Cleavage of the linear precursor peptide chain Fresh cutting cocktail (10 mL) trifluoroacetic acid:water:triisopropylsilane:phenylmethylsulfide (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 1 and the reaction was allowed to proceed with shaking at room temperature for 2 hours. After the reaction was complete, the reaction solution was filtered, the resin was washed with trifluoroacetic acid, and the resin was combined with the reaction solution. The crude product was then precipitated with four volumes of cold MTBE. The crude product was washed three times with MTBE and vacuum pumped.

[0094] Step 3: Intramolecular disulfide bond formation The crude product obtained in step 2 was dissolved completely in DMSO (the DMSO volume was 20% of the total volume of the reaction system). A phosphate-guanidine hydrochloride buffer solution (pH 7.0, containing 50% ACN) was prepared. A 1x PBS:6M guanidine hydrochloride (80:20, v:v) buffer solution and acetonitrile (1:1) were mixed to obtain a phosphate-guanidine hydrochloride buffer solution (pH 7.0, 50% ACN). The polypeptide solution was then slowly added dropwise to the buffer solution to a final concentration of 1 mg / mL. The mixture was shaken at room temperature for 16 hours. The reaction results were monitored by LC-MS, and the product was directly purified and purified after the reaction was completed.

[0095] Step 4: Polypeptide purification and preparation After filtration through a 0.45 μm membrane, the product was separated using a reverse-phase high-performance liquid chromatography system. Buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile) were used. The chromatography column was a BR C-18 (Syphen) reverse-phase chromatography column. During the purification process, the chromatographic detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 25-40% acetonitrile over 40 min. The relevant distillation fractions of the product were collected, and after confirming the purity by HPLC, the distillation fractions with a purity above 95% were combined and lyophilized to obtain the pure polypeptide.

[0096] Step 5: Detection and feature evaluation methods The purified polypeptide from step 4 was subjected to analytical high performance liquid chromatography / mass spectrometry to confirm its purity, and the compounds formed intramolecular disulfide bonds, as shown in Figures 5 and 6.

[0097] Example 4. Preparation of compound 108 (i.e., corresponding SEQ ID NO: 98).

[0098] [ka]

[0099] Step 1: Synthesis of linear precursor peptide chain Synthesis of the linear precursor peptide chain of compound 108. GKWGFYTKVFRLKKWINKVIC-TFGC 294 mg (0.2 mmol) of Rink Amide-AM Resin was completely solubilized in DMF for 1 hour. Then, the linear precursor sequence was synthesized from the carboxyl-terminal C1 position to the amino terminus. Each coupling cycle was performed as follows: Fmoc deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL) for 8 minutes each time. The resin was washed 6–8 times with DMF until a neutral pH was reached. 1.0 mmol Fmoc-AA, 1.0 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 2 mmol 4-methylmorpholine (NMM) were dissolved in DMF, added to the resin, and incubated at room temperature for 1 hour. Before the next amino acid coupling, the resin was washed 4–6 times with DMF. After the linear polypeptide was synthesized, the resin was washed 5 times with DMF and 5 times with DCM. The resin was then aspirated under vacuum.

[0100] Step 2: Cleavage of the linear precursor peptide chain Fresh cutting cocktail (10 mL) trifluoroacetic acid:water:triisopropylsilane:phenylmethylsulfide (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 1 and the reaction was allowed to proceed with shaking at room temperature for 2 hours. After the reaction was complete, the reaction solution was filtered, the resin was washed with trifluoroacetic acid, and the resin was combined with the reaction solution. The crude product was then precipitated with four volumes of cold MTBE. The crude product was washed three times with MTBE and vacuum pumped.

[0101] Step 3: Intramolecular disulfide bond formation The crude product obtained in step 2 was dissolved completely in DMSO (the DMSO volume was 20% of the total volume of the reaction system). A phosphate-guanidine hydrochloride buffer solution (pH 7.0, containing 50% ACN) was prepared. A 1x PBS:6M guanidine hydrochloride (80:20, v:v) buffer solution and acetonitrile (1:1) were mixed to obtain a phosphate-guanidine hydrochloride buffer solution (pH 7.0, 50% ACN). The polypeptide solution was then slowly added dropwise to the buffer solution to a final concentration of 1 mg / mL. The mixture was shaken at room temperature for 16 hours. The reaction results were monitored by LC-MS, and the product was directly purified and purified after the reaction was completed.

[0102] Step 4: Polypeptide purification and preparation After filtration through a 0.45 μm membrane, the product was separated using a reverse-phase high-performance liquid chromatography system. Buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile) were used. The chromatography column was a BR C-18 (Syphen) reverse-phase chromatography column. During the purification process, the chromatographic detection wavelength was set to 230 nm, the flow rate was 15 mL / min, and the gradient was 30-50% acetonitrile over 40 min. The relevant distillation fractions of the product were collected, and after confirming the purity by HPLC, the distillation fractions exceeding 95% were combined and lyophilized to obtain the pure polypeptide.

[0103] Step 5: Detection and feature evaluation methods The purified polypeptide from step 4 was subjected to analytical high performance liquid chromatography / mass spectrometry to confirm its purity, and the compounds formed intramolecular disulfide bonds, as shown in Figures 7 and 8.

[0104] Example 5. Preparation of compound 109 (i.e., corresponding SEQ ID NO: 98).

[0105] [ka]

[0106] Step 1: Synthesis of linear precursor peptide chain Synthesis of the linear precursor peptide chain of compound 109. GKWGFYTKVFRLKKWISKVIC-TFGC

[0107] 294 mg (0.2 mmol) of Rink Amide-AM Resin was completely solubilized in DMF for 1 hour. Then, the linear precursor sequence was synthesized from the carboxyl-terminal C1 position to the amino terminus. Each coupling cycle was performed as follows: Fmoc deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL) for 8 minutes each time. The resin was washed 6–8 times with DMF until a neutral pH was reached. 1.0 mmol Fmoc-AA, 1.0 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 2 mmol 4-methylmorpholine (NMM) were dissolved in DMF, added to the resin, and incubated at room temperature for 1 hour. Before the next amino acid coupling, the resin was washed 4–6 times with DMF. After the linear polypeptide was synthesized, the resin was washed 5 times with DMF and 5 times with DCM. The resin was then aspirated under vacuum.

[0108] Step 2: Cleavage of the linear precursor peptide chain Fresh cutting cocktail (10 mL) trifluoroacetic acid:water:triisopropylsilane:phenylmethylsulfide (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 1 and the reaction was allowed to proceed with shaking at room temperature for 2 hours. After the reaction was complete, the reaction solution was filtered, the resin was washed with trifluoroacetic acid, and the resin was combined with the reaction solution. The crude product was then precipitated with four volumes of cold MTBE. The crude product was washed three times with MTBE and vacuum pumped.

[0109] Step 3: Intramolecular disulfide bond formation The crude product obtained in step 2 was dissolved completely in DMSO (the DMSO volume was 20% of the total volume of the reaction system). A phosphate-guanidine hydrochloride buffer solution (pH 7.0, containing 50% ACN) was prepared. A 1x PBS:6M guanidine hydrochloride (80:20, v:v) buffer solution and acetonitrile (1:1) were mixed to obtain a phosphate-guanidine hydrochloride buffer solution (pH 7.0, 50% ACN). The polypeptide solution was then slowly added dropwise to the buffer solution to a final concentration of 1 mg / mL. The mixture was shaken at room temperature for 16 hours. The reaction results were monitored by LC-MS, and the product was directly purified and purified after the reaction was completed.

[0110] Step 4: Polypeptide purification and preparation After filtration through a 0.45 μm membrane, the product was separated using a reverse-phase high-performance liquid chromatography system. Buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile) were used. The chromatography column was a BR C-18 (Syphen) reverse-phase chromatography column. During the purification process, the chromatographic detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 25-40% acetonitrile over 40 min. The relevant distillation fractions of the product were collected, and after confirming the purity by HPLC, the distillation fractions with a purity above 95% were combined and lyophilized to obtain the pure polypeptide.

[0111] Step 5: Detection and feature evaluation methods The purified polypeptide from step 4 was subjected to analytical high performance liquid chromatography / mass spectrometry to confirm its purity, and the compounds formed intramolecular disulfide bonds, as shown in Figures 9 and 10.

[0112] (Biological evaluation) Example 6 Molecular Minimum Bacterial Inhibitory Concentration (MIC) Functional Test The present invention determines the bacterial inhibitory activity of a compound by measuring the minimal inhibitory concentrations (MIC) of the molecule against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853.

[0113] Bacteria for the minimum bacterial inhibitory concentration test were frozen in a -80°C deep freezer and resuscitated two days before use. A small amount of the frozen bacterial suspension was scraped with a sterile inoculating loop and streaked onto a TSA solid medium plate. The plate was then placed in a conventional incubator at 35±2°C for approximately 20 hours. Five to ten colonies with similar morphology were picked from the plate with a sterile inoculating loop and streaked onto a corresponding solid medium plate. The plate was then placed in a conventional incubator at 35±2°C for approximately 20 hours. Five to ten single colonies of bacteria were removed from the plate and resuspended in 500 μL of sterile saline (0.9% NaCl). The OD600 was adjusted to approximately 0.15 using a spectrophotometer. A 1.02x CAMHB (containing 0.02% BSA) equilibrated to room temperature was used to inoculate the plate at an inoculum concentration of approximately 2 x 10. 5 Bacteria were diluted 300-fold to achieve CFU / mL.

[0114] The backup polypeptide was solubilized in DMSO to a 3.2 mg / mL backup solution and serially diluted in DMSO at 11 dilutions, using a two-fold gradient. 2 μL of the polypeptide compound was transferred to the corresponding well of the test plate, and 98 μL of the prepared bacterial inoculum was added to the test plate. The maximum detectable concentration of the compound was 256 μg / mL. The test plate was centrifuged at 800 rpm for 30 seconds, then placed on a shaking plate and shaken at 400 rpm for 1 minute to ensure uniform mixing. The plate was then placed in a conventional incubator and incubated at 35 ± 2°C for 20 hours. The test plate was placed in a plate reader, and the reflector was adjusted to observe and record the bacterial growth in each well. Photographs of each test plate were simultaneously taken using the QCount system, and the OD600 values ​​of the bacteria in each well were read using a SpectraMax Plus 384. The test results are shown in Table 1 below (the numbers in Table 1 indicate the compound numbers, as above).

[0115] [Table 1-1]

[0116] [Table 1-2]

[0117] Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa are common causative strains of skin wound infections. As can be seen from the data in Table 1, the polypeptide compounds provided by the present invention have good bacterial inhibitory effects against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853, with minimal inhibitory concentrations (MIC) of 2 to 64 μg / mL against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, and 32 to 256 μg / mL against Pseudomonas aeruginosa ATCC 27853.

[0118] Example 7 Radial Diffusion Assay (RDA) Functionality Test The present invention evaluates the antibacterial activity of different polypeptides by measuring the diameter of the molecule's bacterial inhibition zone against E. coli ATCC 25922, Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853.

[0119] Bacteria were frozen in a -80°C deep freezer and resuscitated two days before use. Using a sterile inoculating loop, the frozen bacteria were weighed and scraped onto TSA solid medium plates, streaked onto plates, and cultured in an incubator at 35±2°C for approximately 20 hours. Multiple single colonies of bacteria were picked from each plate and resuspended in 1 mL of TSB. The OD600 was adjusted to 1.0 using a spectrophotometer. The resuspended bacteria were reinoculated into 10 mL of TSB at a 1:100 ratio and cultured at 37°C at 200 rpm until mid-logarithmic phase. The cultures were centrifuged at 4000 rpm for 10 minutes, the supernatant discarded, and the pellet resuspended in an equal volume of 10 mM Tris buffer (pH 7.4). The OD600 was adjusted to ~0.15 using a spectrophotometer. The compounds to be measured were diluted to 0.8 mg / mL and used to detect Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, respectively. Bottom agar was prepared with a TSB concentration of 0.03% (W / V), hypotonic agarose concentration of 1% (W / V), and Tween-20 concentration of 0.02% (V / V). It was sterilized by autoclaving and cooled to 42–46°C. Bacteria with an OD600 of 0.15 were added to the bottom agar at a ratio of 1:300, mixed thoroughly, and poured into a culture dish. The dish was allowed to solidify at room temperature. Nine 4-mm diameter wells were punched into the culture dish using a gel punch. 6 μL of the compound to be measured (0.8 mg / mL) was added to each well according to Figure 1. The wells were then incubated at 37°C for 3 h to allow the sample to fully diffuse into the bottom agar. Top agar was prepared with a TSB concentration of 6% (W / V) and hypotonic agar concentration of 1% (W / V). It was sterilized by autoclaving and cooled to 42–46°C. 15 mL of the top agar was added to the culture dish. After solidification, the dish was incubated at 37°C for approximately 20 h. The agar containing Pseudomonas aeruginosa was placed upside down on a 15 cm culture dish, and the bottom agar containing the bacteria was exposed to air, allowing the bacteria to grow sufficiently. -1 From 10 -3 The bacteria were serially diluted to 100 μL. 100 μL of the above bacterial dilutions were evenly spread onto TSA dishes. After the medium was absorbed by the TSA for 10 minutes, the dishes were inverted and cultured in an incubator at 35±2°C for 20 hours. Photographs of each culture dish were taken using the QCount system, and the diameter of each bacterial inhibition zone was measured and recorded. The results are shown in Table 2, where the numbers in the table represent the compound numbers as above.

[0120] [Table 2-1]

[0121] [Table 2-2]

[0122] As can be seen from the data in Table 1, the compounds provided by the present invention have good antibacterial effects against E. coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853, and the bacterial inhibitory effect is maintained even after the molecules diffuse through the agar interlayer. The diameters of the antibacterial circles against E. coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853 are 5 to 11 mm.

[0123] Example 8 Inhibitory effect (IC50) of polypeptides on fluorescein reporter gene expression in LPS-activated TLR4 cells The experimental cells were HEK293 / TLR4 / NF-kB-Luc cells (Cat: CBP74128) manufactured by Nanjing Kebai Biotechnology Co., Ltd. The cells were resuscitated and cultured according to the cell culture requirements. The assay kit used was the One-Step Luciferase Assay System (Bioscience Cat: 60690), and the reagents were prepared according to the manufacturer's instructions.

[0124] HEK293 / TLR4 / NF-kB-Luc cells were cultured and expanded in medium (MEM, 10% FBS, 1% non-essential amino acids (NEAA), 1 mM Na-pyruvate, 100 μg / mL hygromycin B, 1% puromycin). When cell proliferation density reached 80–90% of the culture flask, the cells were first washed with DPBS, digested with 0.25% trypsin (containing 0.5 mM EDTA), collected in a centrifuge tube, centrifuged at 1000 rpm for 3 minutes, the supernatant medium was removed, and 6–8 mL of fresh growth medium was added to resuspend the cells. The cells were then passaged at a ratio of 1:3–1:8 and cultured at 37°C in a 5% CO2 incubator. After passage, the passaging medium was replaced or subcultured every 2–3 days.

[0125] HEK293 / TLR4 / NF-kB-Luc cells were passage-expanded until the required cell number was reached, digested, and resuspended. The cell suspension was then transferred to a 384-well white permeable cell culture plate at 6000 cells / well, 25 μL / well, and cultured at 37°C and 5% CO2 for 4 days before use in sample detection. Determine the concentration of LPS to use: Lipopolysaccharide (LPS) was dissolved in sterilized water at 5 mg / mL and diluted with 1x loading buffer to a concentration of 60 μg / mL (6x). This was then serially diluted in a 3-fold gradient with 1x loading buffer for a total of 12 concentrations. Five μL of the diluted LPS solution was transferred to a 384-well white permeable cell culture plate that had been incubated for 4 d and then incubated at 37°C for 6 h. One hour later, Luciferase Assay System reagent was added to the plate at a 100:1 volumetric ratio to Solution A and Solution B. 30 μL of Luciferase Assay System reagent was added to the plate and incubated at room temperature, protected from light, and shaken at 350 rpm for 15 min. Chemiluminescence activity was detected using the Cytation 5 enzyme marker. The EC80 range of LPS was 0.06–0.1 μg / mL, and the EC80 concentration was selected for the experiment.

[0126] Inhibitory effect of polypeptides on luciferase reporter gene expression in LPS-activated TLR4 cells: The polypeptide was dissolved in sterilized water to 500 μM and diluted with 1× loading buffer to a concentration of 120 μM (12×). This was then serially diluted three-fold with 1× loading buffer to a total of seven concentrations. LPS was diluted with 1× loading buffer to 1.2 μg / mL (12×).

[0127] Equal volumes of 25 μL of gradient-diluted polypeptide (12x) and 25 μL of LPS (12x) were premixed. 5 μL of the premix was added to a 384-well white permeable cell plate that had been incubated for 4 days and then incubated at 37°C for 6 hours. One hour prior, solutions A and B were mixed at a 100:1 volume ratio with the Luciferase Assay System reagent. 30 μL of Luciferase Assay System reagent was added to the cell plate. The plate was shaken at 350 rpm at room temperature, protected from light, for 15 minutes. Chemiluminescence activity was detected using the Cytation 5 enzyme marker. Specific values ​​are shown in Table 3 (the IC50 value of TCP-25 was 6.81 ± 1.4 μM; the numbers in the table indicate the compound numbers as above).

[0128] [Table 3]

[0129] As can be seen from Table 3, the compounds of the present invention have a significant anti-inflammatory effect compared to the positive control group (TCP-25), the IC50 value is significantly lower than that of the positive control group, and the activity fold is 1.04 to 27.24 times higher than that of the positive control group. Data processing: In order to more intuitively demonstrate the advantages of the compounds described in the present invention over the positive control (TCP-25), the data of the negative control group in which only 1× Loading buffer was added to the experimental group of compounds 1, 30, 78, 105, and 106, and the positive control group (TCP-25) in which only LPS was added, were calculated and analyzed according to the following formula, and Figure 11 was obtained.

[0130]

number

[0131] As can be seen from FIG. 3, the compounds of the present invention had a more significant anti-inflammatory effect than the positive group, and the IC50 values ​​were significantly lower than those of the positive control group.

[0132] Although the preferred embodiments of the present invention have been specifically described above, the present invention is not limited to the above embodiments. Those skilled in the art may make any kind of equivalent modifications or substitutions without departing from the spirit of the present invention, and all of these equivalent modifications or substitutions shall be included within the scope defined by the claims of this application.

[0133] (Addendum) (Appendix 1) It consists of a sequence represented by the following formula (III): GKX 10 -GX 11 -YX 12 -X 1 -X 2 -X 3 -X 4 -LX 5 -KWIX 6 -KX 7 -X 8 -X 13 -X 14 -X 15 -GX 16 (III) or a pharmaceutically acceptable salt thereof, wherein X 1 , X 4 , X 5 are each independently selected from K, dK, R, dR, a derivative of K or R; X 2 , X 3 , X 6 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 and X16 are each independently selected from any amino acid; X 7 is selected from the V, L or d configurations of said amino acids and derivatives thereof; X 8 is selected from I, V, L, T or d structures of said amino acids and derivatives thereof.

[0134] (Appendix 2) X 2 is selected from the V, L, T, K, W, E or d structures of said amino acids and derivatives thereof; X 3 , X 11 are each independently selected from F, Y, W or d structures of said amino acids and derivatives thereof; X 6 is selected from Q, N, T, K, W, L or d structures of said amino acids and derivatives thereof; X 10 is Y, F, W or the d structure of the above amino acids and derivatives thereof, X 12 is T, S, K, L, W, E or d structure of said amino acid and derivatives thereof, X 13 and X 16 are each independently selected from C, K, G, W, L, S, D, E or d configurations of said amino acids and derivatives thereof; X 14 is Q, N, T, K, W, L or d structure of said amino acids and derivatives thereof, X 15 is any amino acid, preferably Y, F, W or the d structure of said amino acid and derivatives thereof.

[0135] (Appendix 3) X 13 and X 16 and dC are each independently selected from C or dC.

[0136] (Appendix 4) X 13 and X 16 are linked by a bond to form a ring, and preferably, the bond is a disulfide bond, an amide bond, a carbon-hydrogen bond, a dithiocarbamate C 1-6 is selected from an alkyl, thioether bond or an olefinic thioether bond, more preferably said bond is one of the structures shown below: [ka] 2. The polypeptide according to claim 1,

[0137] (Appendix 5) The polypeptide has one of the following structures or a pharmaceutically acceptable salt thereof: [ka] [ka] [ka] [ka] [ka] [ka] [ka] 2. The polypeptide according to claim 1,

[0138] (Appendix 6) A polypeptide having one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 99.

[0139] (Appendix 7) A pharmaceutical composition comprising the polypeptide according to any one of appendices 1 to 6.

[0140] (Appendix 8) 8. The pharmaceutical composition of claim 7, further comprising at least one pharmaceutically acceptable carrier, excipient, diluent, adjuvant, or vehicle.

[0141] (Appendix 9) Use of a polypeptide according to any one of appendices 1 to 6, or a pharmaceutical composition according to appendices 7 or 8, in the preparation of a medicament for the prevention, treatment, cure or alleviation of a bacterial infectious disease and / or an inflammatory disease.

[0142] (Appendix 10) 10. The use of claim 9, wherein the bacterial infectious disease and / or inflammatory disease is any one of skin infection, skin wound infection, burn infection, pneumonia, tuberculosis, diabetic foot, tonsillitis, bacterial dysentery, meningitis, scarlet fever, pharyngopharyngitis, bronchitis, gastritis, appendicitis, glomerulonephritis, endocarditis, pericarditis, cystitis, pelvic inflammation, and cervicitis.

Claims

1. It consists of a sequence shown in formula (III): G-K-X １０ -G-X １１ -Y-X １２ -X １ -X ２ -X ３ -X ４ -L-X ５ -K-W-I-X ６ -K-X ７ -X ８ -X １３ -X １４ -X １５ -G-X １６ (III) or a pharmaceutically acceptable salt thereof, wherein X １ , X ４ , X ５ are each independently selected from a derivative of K, dK, R, dR, K, or R; X ２ , X ３ , X ６ , X １０ , X １１ , X １２ , X １３ , X １４ , X １５ and X １６ are each independently selected from any amino acid; X ７ is selected from the V, L or d configurations of said amino acids and derivatives thereof; X ８ is selected from the I, V, L, T or d structures of said amino acids and derivatives thereof.

2. The X ２ is selected from the V, L, T, K, W, E or d configurations of said amino acids and derivatives thereof; The X ３ , X １１ are each independently selected from F, Y, W or d structures of said amino acids and derivatives thereof; The X ６ is selected from Q, N, T, K, W, L or d structures of said amino acids and derivatives thereof; The X １０ is Y, F, W or the d structure of the amino acid and derivatives thereof; The X １２ is T, S, K, L, W, E or the d structure of the amino acid and derivatives thereof, The X １３ and X １６ are each independently selected from C, K, G, W, L, S, D, E or d configurations of said amino acids and derivatives thereof; The X １４ is Q, N, T, K, W, L or the d structure of the amino acid and derivatives thereof, The X １５ is any amino acid, preferably Y, F, W or the d structure of said amino acid and derivatives thereof.

3. The X １３ and X １６ and each independently selected from C or dC.

4. The X １３ and X １６ are linked by a bond to form a ring, and preferably, the bond is a disulfide bond, an amide bond, a carbon-hydrogen bond, a dithiocarbamate C １－６ It is selected from an alkyl, thioether bond or an olefinic thioether bond, more preferably said bond is one of the structures shown below: 【Chemistry 1】 The polypeptide of claim 1.

5. The polypeptide has one of the following structures or a pharmaceutically acceptable salt thereof: 【Chemistry 2-1】 【Chemistry 2-2】 [Chemistry 2-3] 【Chemistry 2-4】 【Chemistry 2-5】 【Chemistry 2-6】 【Chemistry 2-7】 The polypeptide of claim 1.

6. A polypeptide characterized by being one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

99.

7. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 6.

8. 8. The pharmaceutical composition of claim 7, further comprising at least one pharmaceutically acceptable carrier, excipient, diluent, adjuvant or vehicle.

9. Use of a polypeptide according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 7 or 8 in the preparation of a medicament for preventing, treating, curing or alleviating a bacterial infectious disease and / or an inflammatory disease.

10. 10. The use according to claim 9, wherein the bacterial infectious disease and / or inflammatory disease is any one of skin infection, skin wound infection, burn infection, pneumonia, tuberculosis, diabetic foot, tonsillitis, bacterial dysentery, meningitis, scarlet fever, pharyngopharyngitis, bronchitis, gastritis, appendicitis, glomerulonephritis, endocarditis, pericarditis, cystitis, pelvic inflammation, and cervicitis.

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