WO2022037681A1 - Antimicrobial peptides and application thereof - Google Patents

Abstract

A bioinformatic method is used to analyze, discover and predict, from human intestinal metagenomics, peptides having microorganism inhibiting activity, that is, antimicrobial peptides, and the activity thereof is confirmed by means of experiments. Disclosed are a variety of antimicrobial peptides and a use thereof.

Description

Antimicrobial Peptides and Their Applications technical field

The present invention relates to antimicrobial peptides and uses thereof.

Background technique

The antibiotic resistance of microorganisms has become a public health problem that needs to be solved urgently, and the abuse of antibiotics is a major factor causing the current problem. The World Health Organization (WHO) lists seven classes of multi-drug resistant (MDR) pathogens that most urgently require treatment: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., collectively referred to as ESKAPE. Of these, five are Gram-negative bacteria that have raised concerns about the rapid development of antibiotic resistance by pathogens, and these MDR bacteria include vancomycin-resistant Enterococcus (VRE) and carbapenicillin-resistant Enterobacteriaceae ( carbapenem-resistant Enterobacteriaceae, CRE).

Under the background that more and more antibiotics have weakened effects and the use of antibiotics should be avoided as much as possible in non-essential scenarios, a new type of antimicrobial substances is urgently needed as a supplement or replacement for existing antibiotics. Natural antimicrobial peptides are a class of short peptides with a length of 7 to 50 amino acid residues. Their sequence similarity is not strong, and they are widely distributed in various species. They not only act as direct antimicrobial agents, but also represent innate immunity. Important effectors and regulators of the system even profoundly modulate immune responses through a range of activities, including increased chemokine production. Presumably, bacteria have been exposed to antimicrobial peptides for millions of years, and with the exception of a few species (eg, Burkholderia spp.), widespread resistance has not been reported. Therefore, antimicrobial peptides have the advantages of strong activity and low drug resistance as antimicrobial agents.

Antibiotics are the most widely used antimicrobial drugs at present, but their long-term addition will bring human health risks, so they cannot be added to daily chemical products (such as skin care products, diapers, soaps, etc.). The addition of antibiotics to livestock feed poses potential food safety risks and therefore has its limitations.

SUMMARY OF THE INVENTION

The antimicrobial peptides (AMPs) (also known as "antibacterial peptides") involved in the present invention are obtained by analyzing and excavating from the human gut metagenome by using a bioinformatics method. Existing reports show that the human gut contains a large number of uncultivated and difficult-to-cultivate bacterial species. Mining antimicrobial peptides from metagenomic sequencing data can not only avoid culture problems, but also has a low cost compared with the traditional "strain isolation and screening, compound purification and identification". The flux is different. We use the machine learning method to construct multiple models for fusion prediction, and then use the information such as bacterial flora taxonomy to obtain reliable peptides to be tested (Figure 1). It was purified by liquid chromatography and its activity was determined. In the art, "antimicrobial peptide" and "antibacterial peptide" are sometimes used interchangeably, and the use of "antimicrobial peptide" in the present invention is intended to indicate that the peptide of the present invention has a broad-spectrum inhibitory effect on microorganisms, including bacteria , fungi, viruses, etc.

In one aspect, the present invention provides antimicrobial peptides. In some embodiments, the antimicrobial peptide of the present invention comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-178; An amino acid sequence of 90% sequence identity; or a fragment comprising the amino acid sequence shown in any one of SEQ ID NOs: 1-178; or a fragment containing the amino acid sequence shown in any one of SEQ ID NOs: 1-178 Than amino acid sequences with greater than 90% sequence identity. In some embodiments, the antimicrobial peptides of the invention comprise greater than 91%, 92%, 93%, 94%, 95%, 96%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence of 97%, 98% or 99% sequence identity; or a fragment comprising greater than 91%, 92%, 93%, 94% compared to the amino acid sequence shown in any one of SEQ ID NOs: 1-178 , 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences.

In some embodiments, the amino acid sequence of the antimicrobial peptide of the present invention is the amino acid sequence shown in any one of SEQ ID NOs: 1-178; or is the same as the amino acid sequence shown in any one of SEQ ID NOs: 1-178 An amino acid sequence with greater than 90% sequence identity; or a fragment of the amino acid sequence shown in any one of SEQ ID NOs: 1-178; or an amino acid sequence shown in any one of SEQ ID NOs: 1-178 fragments compared to amino acid sequences with greater than 90% sequence identity. In some embodiments, the amino acid sequence of the antimicrobial peptide of the present invention is greater than 91%, 92%, 93%, 94%, 95%, An amino acid sequence having a sequence identity of 96%, 97%, 98% or 99%; or a fragment having greater than 91%, 92%, 93% compared to a fragment of the amino acid sequence shown in any one of SEQ ID NOs: 1-178 , 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences.

In some embodiments, the antimicrobial peptides of the invention comprise chemical modifications on amino acids.

In some embodiments, the chemical modification on the amino acid is present on less than 10% of the amino acids.

In one aspect, the present invention provides antimicrobial compositions comprising the antimicrobial peptides of the present invention.

In some embodiments, the compositions of the present invention are pharmaceutical compositions and comprise a pharmaceutically acceptable carrier.

In one aspect, the present invention provides the use of a peptide or composition of the present invention for inhibiting microorganisms in vitro or in vitro.

In one aspect, the present invention provides the use of a peptide or composition of the present invention in the manufacture of an antimicrobial agent.

In one aspect, the present invention provides the use of a peptide or composition of the present invention in the manufacture of a medicament for treating microbial infections and/or modulating immune responses.

In one aspect, the present invention provides peptides or compositions of the present invention for use in inhibiting microorganisms.

In one aspect, the present invention provides peptides or compositions of the present invention for use in treating microbial infections and/or modulating immune responses.

In one aspect, the present invention provides a method for inhibiting microorganisms comprising administering to a subject an effective amount of a peptide or composition of the present invention.

In one aspect, the present invention provides a method for treating a microbial infection and/or modulating an immune response comprising administering to a subject an effective amount of a peptide or composition of the present invention.

In one aspect, the present invention provides a method of treating a microbial infection, the method comprising administering to a subject a therapeutically effective amount of a peptide or composition of the present invention. In some embodiments, the subject can be a human or a mammal. In some embodiments, the microorganisms inhibited by the present invention are fungi and/or bacteria.

In some embodiments, the microorganisms inhibited by the present invention are Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Acinetobacter baumannii, Klebsiella pneumoniae, Bacillus subtilis, Enterobacter cloacae and/or Enterococcus faecalis.

In some embodiments, the microbial infection treated by the present invention is a fungal and/or bacterial infection.

In some embodiments, the microbial infection treated by the present invention is Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Acinetobacter baumannii, Klebsiella pneumoniae, Bacillus subtilis , Enterobacter cloacae and/or Enterococcus faecalis infection.

Description of drawings

The present invention will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

Figure 1: Schematic diagram of the prediction method for antimicrobial peptides.

Figure 2: Sequences of antimicrobial peptides (SEQ ID NOs: 1-178).

Figure 3: Structure prediction results of some antimicrobial peptides.

Figure 4: 11 antimicrobial peptides against Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus faecium, Escherichia coli Distribution of MICs for Escherichia coli, Enterococcus cloacae and Staphylococcus aureus.

Figure 5: MIC of 11 antimicrobial peptides against different MDR strains.

Figure 6: Cytotoxicity assay results for 11 antimicrobial peptides. The Y-axis represents the percentage of cell survival.

Figure 7: Results of the hemolysis assay for 11 antimicrobial peptides. The Y-axis represents percent cell survival.

Figure 8: Three antimicrobial peptides are effective in treating Klebsiella pneumoniae-infected mice. The Y-axis represents percent weight loss and the X-axis represents days post-infection. * indicates p<0.05 in t-test, ** indicates p<0.01 in t-test.

detailed description

The terms "comprising" and "comprising" as used herein mean that the substance, composition, system or method, etc. contains at least, but is not limited to, the structure, ingredient, element, component, feature or step, etc.; "comprising" , "comprising" is intended to indicate the presence of the stated structure, ingredient, element, component, feature, step, etc., but does not exclude the presence of any other structure, ingredient, element, component, feature, step, etc. In this context, when it is mentioned that a compound contains some structure or structures, it should be understood that it also covers compounds composed of this structure or structures; when it is mentioned that a product or composition contains some structure or structures ingredient, it should be understood that it also covers a product or composition consisting of the ingredient(s); when a method is referred to as comprising one or some steps, it should be understood to also encompass the step(s) Methods.

As used herein, the term "and/or" is intended to include any possible combination of one or more of the listed items.

In this document, wherever appropriate, terms used in the singular also include the plural and vice versa.

Peptides:

The peptides of the present invention are antimicrobial peptides (AMPs), which include: peptides whose amino acid sequence is any one of SEQ ID NOs: 1-178, and variants of these peptides. The variants include: (a) peptides comprising the amino acid sequence shown in any one of SEQ ID NOs: 1-178; (b) fragments whose amino acid sequence is the amino acid sequence shown in any one of SEQ ID NOs: 1-178 (i.e., the amino acid sequence is a peptide whose amino acid sequence is truncated on the basis of the amino acid sequence shown in any one of SEQ ID NOs: 1-178); (c) comprises as shown in any one of SEQ ID NOs: 1-178 (i.e., a peptide comprising a truncated amino acid sequence based on the amino acid sequence shown in any of SEQ ID NOs: 1-178); (d) in any of SEQ ID NOs: 1-178 A peptide obtained by amino acid substitution on the basis of the indicated amino acid sequence, and a peptide obtained by amino acid substitution on the basis of any of the above-mentioned peptides (a), (b) and (c). For example: SEQ ID NOs: 1-178 sequences with high sequence similarity (sequence identity percentage greater than 90% and less than 100%) are summarized and classified (Table 2), it can be seen that the 12 listed In this category, the sequences all contain differences (including substitution, extension, truncation), but still show the same or even higher antibacterial activity.

In some embodiments, the length of the peptide in (a) above is 20-120 amino acids; preferably 20-115 amino acids, more preferably 20-110 amino acids; more preferably 20-105 amino acids more preferably 20-100 amino acids; more preferably 20-95 amino acids; more preferably 20-90 amino acids; more preferably 20-85 amino acids; more preferably 20-80 amino acids more preferably 20-75 amino acids; more preferably 20-70 amino acids; more preferably 20-65 amino acids; more preferably 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 amino acids. In some embodiments, the peptide in (a) above is 1, 2, 3, 4, 5, 6, 7, 8 longer than its corresponding peptide with a sequence of one of SEQ ID NOs: 1-178 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids; the "corresponding sequence is SEQ ID NO: 1 "Peptide of one of -178" refers to a peptide whose sequence is one of SEQ ID NOs: 1-178 as an extension of the peptide in (a). The sequences of the peptides in (a) above may be N-terminal and/or C-terminal to SEQ ID NOs: 1-178 relative to extensions of SEQ ID NOs: 1-178.

In some embodiments, the peptide in (a) above has the same antimicrobial and/or immune system modulating activity as its corresponding peptide having the sequence of one of SEQ ID NOs: 1-178, or maintains a certain amount of activity Said activity, or can improve said activity; said maintaining a certain amount of said activity is: maintaining 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more than 99.9% of the corresponding sequences are SEQ ID NOs: 1-178 The antimicrobial and/or immune system-modulating activity of the peptide of a; the "peptide corresponding to one of the sequences of SEQ ID NOs: 1-178" means that the sequence that is the extended basis of the peptide in (a) is The peptide of one of SEQ ID NOs: 1-178. In some embodiments, methods for determining antimicrobial and/or immune system modulating activity are well known to those skilled in the art, for example, peptides can be applied to different microorganisms and the bacteriostatic rate can be determined by measuring OD values.

In some embodiments, the sequence at both ends of the antimicrobial peptide is not the part that has a direct effect on the bacteriostatic activity, and thus may still have the same activity for the lengthening of the ends. For example, the difference between SEQ ID NO: 120 and SEQ ID NO: 124 is only that the latter has two more residues (FS, Phe-Ser, phenylalanine-serine) at the N-terminus than the former, both of which are uncharged amino acids, Therefore, the effect on the antibacterial activity is small. Another example is SEQ ID NO: 5, SEQ ID NO: 13 and SEQ ID NO: 20; SEQ ID NO: 15 and SEQ ID NO: 21; SEQ ID NO: 99 and SEQ ID NO: 145; SEQ ID NO: 167 and SEQ ID NO: 177 is the same.

In some embodiments, the length of the peptide in (b) above is preferably 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 amino acids. In some embodiments, the peptide in (b) above is truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids; the "peptide whose corresponding sequence is one of SEQ ID NOs: 1-178" refers to The sequence underlying the truncation of the peptide in (b) is the peptide of one of SEQ ID NOs: 1-178. The sequences of the peptides in (b) above may be truncated relative to SEQ ID NOs: 1-178 at the N-terminal and/or C-terminal ends of SEQ ID NOs: 1-178.

In some embodiments, the peptide in (b) above has the same antimicrobial and/or immune system modulating activity as its corresponding peptide having the sequence of one of SEQ ID NOs: 1-178, or maintains a certain amount of activity Said activity, or can improve said activity; said maintaining a certain amount of said activity is: maintaining 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more than 99.9% of the corresponding sequences are SEQ ID NOs: 1-178 Antimicrobial and/or immune system modulating activity of the peptide of a; the "peptide whose corresponding sequence is one of SEQ ID NOs: 1-178" refers to the sequence that is the basis for the truncation of the peptide in (b) is a peptide of one of SEQ ID NOs: 1-178. In some embodiments, methods for determining antimicrobial and/or immune system modulating activity are well known to those skilled in the art, for example, peptides can be applied to different microorganisms and the bacteriostatic rate can be determined by measuring OD values.

In some embodiments, the sequence at both ends of the antimicrobial peptide is not the part that has a direct effect on the bacteriostatic activity, and thus may still have the same activity for truncations at both ends. For example, the difference between SEQ ID NO: 120 and SEQ ID NO: 124 is only that the latter has two more residues (FS, Phe-Ser, phenylalanine-serine) at the N-terminus than the former, both of which are uncharged amino acids, Therefore, the effect on the antibacterial activity is small. Another example is SEQ ID NO: 5, SEQ ID NO: 13 and SEQ ID NO: 20; SEQ ID NO: 15 and SEQ ID NO: 21; SEQ ID NO: 99 and SEQ ID NO: 145; SEQ ID NO: 167 and SEQ ID NO: 177 is the same.

In some embodiments, the peptide in the above (c) comprises the amino acid sequence of the peptide in the above (b), and the length of the peptide in the above (c) is 8-120 amino acids; preferably 8-115 amino acids; More preferred is 8-110 amino acids; more preferred is 8-105 amino acids; more preferred is 8-100 amino acids; more preferred is 8-95 amino acids; more preferred is 8-90 amino acids; More preferred is 8-85 amino acids; more preferred is 8-80 amino acids; more preferred is 8-75 amino acids; more preferred is 8-70 amino acids; more preferred is 8-65 amino acids; More preferred are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 amino acids. The extension of the peptide in (c) above with respect to the peptide in (b) above may be at the N-terminal and/or C-terminal end of the peptide in (b) above.

In some embodiments, the peptide in (c) above has the same antimicrobial and/or immune system modulating activity as its corresponding peptide having the sequence of one of SEQ ID NOs: 1-178, or maintains a certain amount of activity Said activity, or can improve said activity; said maintaining a certain amount of said activity is: maintaining 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more than 99.9% of the corresponding sequences are SEQ ID NOs: 1-178 The antimicrobial and/or immune system modulating activity of a peptide of a The sequence of the truncated basis of the peptide in is the peptide of one of SEQ ID NOs: 1-178. In some embodiments, methods for determining antimicrobial and/or immune system modulating activity are well known to those skilled in the art, for example, peptides can be applied to different microorganisms and the bacteriostatic rate can be determined by measuring OD values.

In some embodiments, the peptide in (d) above comprises a substitution based on any of the amino acids in SEQ ID NOs: 1-178 (or truncated SEQ ID NOs: 1-178), the number of amino acid substitutions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The one or more amino acid substitutions are conservative or non-conservative.

In some embodiments, the peptide in (d) above has greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% and less than 100% sequence identity.

Amino acid sequence identity refers to the percentage of amino acids in a candidate sequence that are identical to amino acids in a reference sequence after aligning the sequences and introducing gaps where necessary (to achieve maximum percent sequence identity). Alignment for purposes of determining percent sequence identity can be accomplished in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as Needle, BLAST, BLAST-2, ALIGN, ALIGN-2, CD-HIT or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment can be determined by known methods, including any algorithms needed to achieve maximal alignment over the full length of the sequences to be aligned.

In some embodiments, amino acid sequence identity is determined using, for example, the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Heritage). Genet) 16:276-277) (preferably version 5.0.0 or newer) of the Needleman-Wunsch algorithm (Needleman and paper) implemented in the Needle program Wunsch, 1970, J. Mol. Biol. 48: 443-453). The parameters used were a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output (obtained with the -nobrief option) marked as "longest identity" was used as percent identity and calculated as follows: (identical amino acid residues x 100)/(alignment length - total number of gaps in the alignment) .

In some embodiments, the peptide in (d) above has the same antimicrobial and/or immune system modulating activity as its corresponding peptide having the sequence of one of SEQ ID NOs: 1-178, or maintains a certain amount of activity Said activity, or can improve said activity; said maintaining a certain amount of said activity is: maintaining 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more than 99.9% of the corresponding sequences are SEQ ID NOs: 1-178 The antimicrobial and/or immune system-modulating activity of the peptide of a and amino acid substitution basis or truncated and amino acid substitution basis sequence is a peptide of one of SEQ ID NOs: 1-178. In some embodiments, methods for determining antimicrobial and/or immune system modulating activity are well known to those skilled in the art, for example, peptides can be applied to different microorganisms and the bacteriostatic rate can be determined by measuring OD values.

In some embodiments, "substitution" refers to changing one or several amino acids to another amino acid. It is common to replace negatively charged amino acids with positively charged amino acids within a certain range to increase the positive charge of antimicrobial peptides to affect the binding ability of antimicrobial peptides to bacterial cell membranes. For example, in SEQ ID NO: 44 and SEQ ID NO: 46, only the 17th position differs between the two sequences, the former is K (Lys, lysine) and the latter is R (Arg, arginine), both of which are It is a positively charged amino acid; another example is SEQ ID NO: 54 and SEQ ID NO: 55, only the 3rd position has a difference, the former is R (Arg, arginine) and the latter is H (His, histidine acid) are also positively charged amino acids; for example, the two sequences of SEQ ID NO: 175 and SEQ ID NO: 176 are different at the 4th, 6th and 49th positions, and the former at the 4th position is I (Ile , isoleucine) the latter is T (Thr, threonine), the former is Q (Gln, glutamine) at the 6th position, the latter is E (Glu, glutamic acid), and the former at the 49th position is N ( Asn, asparagine) the latter is H (His, histidine), and SEQ ID NO: 92 and SEQ ID NO: 93 at position 29, SEQ ID NO: 146 and SEQ ID NO: 147 at position 10 bit, SEQ ID NO: 166 and SEQ ID NO: 167 are the same at bit 1.

In some embodiments, the substitution of the present invention is to use D-amino acid instead of L-amino acid: this method is mainly to prevent the degradation of endopeptidases from the middle of the amino acid and improve the stability. For example, Carmona et al. (Improved protease stability of the antimicrobial peptide Pin2 substituted with D-amino acids (https://doi.org/10.1007/s10930-013-9505-2)) used D-amino acid substitution in the study of the antimicrobial peptide Pin2 After L-amino acid, not only the antibacterial activity is not affected, but also can maintain a high antibacterial activity in the environment of serum and trypsin.

In some embodiments, the substitutions of the present invention are conservative substitutions.

In some embodiments, conservative substitutions refer to the replacement of amino acid residues with amino acid residues having similar side chains. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid) , uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg, alanine, valine) , leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched side chains (e.g. threonine, valine, isoleucine) and aromatic Side chains (eg tyrosine, phenylalanine, tryptophan, histidine).

Other amino acid substitution patterns not listed in the art are well known to those skilled in the art.

In some embodiments, the antimicrobial peptides of the invention (i.e., peptides having the amino acid sequence set forth in any of SEQ ID NOs: 1-178, and (a), (b), (c), and (d) above ) contain chemical modifications that include modification of one or more amino acids ( including terminal amino acids).

In some embodiments, the chemical modifications described above may be present at any one or more amino acids in the peptides of the invention, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.

In some embodiments, the chemical modifications described above may be present in less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 4%, on less than 3%, less than 2%, or less than 1% of amino acids.

In some embodiments, the chemically modified peptides of the invention have the same antimicrobial and/or immune system modulating activity as their corresponding non-chemically modified peptides, or can maintain a certain amount of said activity, or can increase the activity. In some embodiments, said maintaining an amount of said activity is: maintaining 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or more than 99.9% of their corresponding antimicrobial and/or immune system-modulating peptides that do not contain chemical modifications activity. In some embodiments, methods for determining antimicrobial and/or immune system modulating activity are well known to those skilled in the art, for example, peptides can be applied to different microorganisms and the bacteriostatic rate can be determined by measuring OD values.

In some embodiments, modifications that may be present in the peptides of the invention include acetylation, acylation, ADP ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme moieties Linking, covalent linking of polynucleotides or polynucleotide derivatives, covalent linking of lipids or lipid derivatives, covalent linking of phosphatidyl alcohols, cross-linking, cyclization, disulfide bond formation, demethylation Covalent crosslink formation, cystine formation, pyroglutamic acid formation, formulation, gamma-carboxylation, glycation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins ( Examples are arginylation), and ubiquitination.

In some embodiments, the peptides of the present invention comprise chemical modifications to N-terminal and C-terminal amino acids: mainly methods such as C-terminal amidation, N-terminal acetylation, and deamination, which are mainly directed against susceptible to carboxypeptidase and aminopeptidase-degraded antimicrobial peptides to achieve the purpose of end blocking and improving stability.

In some embodiments, the peptides of the present invention comprise polyethylene glycol (PEG) (ie, PEGylation): the method is to condense PEG with native antimicrobial peptides to generate amphiphilic block copolymers. In general, PEGylated protein products are not toxic, have good solubility in water and organic solvents, improve biocompatibility and reduce polypeptide aggregation, and also reduce the probability of protease degradation. Zhang et al. found that (Modification of Antimicrobial Peptide with Low Molar Mass Poly(ethylene glycol) (https://doi.org/10.1093/jb/mvn134)) PEGylated antimicrobial peptide MA (a derivative fragment of antimicrobial peptide Melittin) and unPEGylated Compared with the PEGylated antimicrobial peptide MA, the half-life of the PEGylated antimicrobial peptide increased by 3-4 times, indicating that the stability was greatly improved.

In some embodiments, the present invention includes the immobilization of the antimicrobial peptides of the present invention by chemical covalent bonding: the formation of peptide bonds between the free amino or carboxyl groups of the antimicrobial peptides and the surface groups of the modified material, or The conformation of the antimicrobial peptide is fixed by reacting the thiol group of the antimicrobial peptide with the thiol group, maleimide, and epoxy group on the surface of the modified material, which may improve the stability and antibacterial activity. It has been proved in the literature that maintaining the secondary structure of immobilized antimicrobial peptides such as α helix and β sheet is the key to the activity of immobilized antimicrobial peptides.

In some embodiments, the present invention includes peptides obtained by cyclizing linear peptides. The main way of cyclization is to use amide bonds, disulfide bonds, or natural chemical linkages to construct a head-to-tail circular backbone, which can effectively prevent free polypeptide ends from being bound and decomposed by proteases. For example, studies have shown that cyclization of the 18-amino acid peptide Gomesin significantly improves its stability (Cyclization of the antimicrobial peptide gomesin with native chemical ligation: influences on stability and bioactivity (https://doi. org/10.1002/cbic.201300034)).

In some embodiments, the peptides of the invention include monomeric peptides and multimeric peptides, which may be dimers, trimers, tetramers, pentamers, hexamers, Heptapolymer, etc. Multimers of the peptides of the present invention may introduce intermolecular disulfide bond formation, eg, by amino acid substitution. For example, Morrison et al. (Identification and characterization of a novel murine beta-defensin-related gene (https://doi.org/10.1007/s00335-002-3014-5)) in the mouse heart and testis existence of a novel β - Intermolecular disulfide bonds have been introduced into the defensins, making them more efficient at inhibiting both Gram-positive and negative bacteria.

In some embodiments, the peptides of the present invention include the same or different peptides of the present invention or peptides formed by splicing of fragments thereof: for example, Li Guodong et al. 03): 496-499 (https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbnane=CJFD2006&filename=WSXB200603032&v=MTkyMjBlWDFMdXhZUzdEaDFUM3FUcldNMUZyQ1VSN3FmYitadUZDdmdWN3pJTWo3VGJMRzRIdGZNckk5R1pvUjg=)) obtained CecropinA activity after splicing the two α helix and Melittin Stronger hybrid peptides. Methods of splicing two different or identical antimicrobial peptides are well known to those skilled in the art.

Antimicrobial peptides are broad-spectrum antimicrobial substances that can directly inhibit or kill bacteria, yeast, fungi, viruses, and even cancer cells. Antimicrobial peptides also have the ability to modulate immune responses, and in addition to direct antimicrobial activity, they can protect the host through a variety of mechanisms, such as: chemotactic activity; attracting leukocytes; modulating host cell responses to TLR ligands; stimulating blood vessels production; enhances leukocyte/monocyte activation and differentiation; and modulates the expression of pro-inflammatory cytokines/chemokines, among others. The antimicrobial peptides of the present invention have these activities and functions as described above.

Nucleic acid:

The present invention provides nucleic acids encoding the above-mentioned antimicrobial peptides. The nucleic acid includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), preferably messenger ribonucleic acid (mRNA). The nucleic acid of the present invention can be used to prepare antimicrobial peptides, and can also be used to express antimicrobial peptides in host cells in vivo or in vitro, for example, by introducing mRNA into host cells to directly express antimicrobial peptides, or by changing the genetic composition of host cells for expression Antimicrobial peptides for the purpose of inhibiting microbial growth, killing microorganisms, treating microbial infections, and/or modulating immune responses. The host cells may include microbial cells, plant cells, animal cells, human cells. Uses or methods associated with the nucleic acids of the present invention are well known to those skilled in the art.

combination:

The present invention provides pharmaceutical compositions comprising one or more of the peptides or nucleic acids of the present invention.

The peptides of the present invention can be fused or conjugated to another compound to improve pharmacokinetics or bioavailability without eliciting an immune response.

The pharmaceutical compositions of the present invention contain a therapeutically effective amount of one or more peptides or nucleic acids of the present invention. Once formed, the pharmaceutical composition of the present invention can be directly administered to a subject to inhibit and kill microorganisms (including treating microbial infections, maintaining microbial balance in the body, etc.).

The subjects to which the pharmaceutical composition of the present invention is administered are mammals and humans.

Methods of administration or administration of the pharmaceutical compositions of the present invention include topical, oral, parenteral, subcutaneous, sublingual, intralesional, intraperitoneal, intravenous, intramuscular, pulmonary or interstitial spaces, and the like.

The pharmaceutical compositions of the present invention may have capsules, tablets, lozenges, coated tablets, pills, drops, suppositories, powders, sprays, vaccines, ointments, pastes, creams, inhalants, patches, aerosols etc. form.

The pharmaceutical compositions of the present invention may contain one or more pharmaceutically acceptable carriers (additives) and/or diluents. The pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those suitable for: (1) oral administration, such as veterinary drenches (aqueous or non-aqueous or mixed; suspensions); tablets, such as those targeted for buccal, sublingual, and systemic absorption; boluses; powders; granules; Intramuscular, intravenous or epidural injection, such as sterile solutions or suspensions or sustained-release formulations; (3) topical application, such as creams, ointments or controlled-release patches or sprays applied to the skin; (4) intravaginal or rectal, such as vaginal suppositories, creams, or foams; (5) sublingual; (6) ocular; (7) transdermal; (8) nasal; (9) pulmonary; or ( 10) In the sheath.

The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation , allergic reactions or other problems or complications with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition or vehicle such as liquid or solid fillers, diluents, excipients, formulation aids such as lubricants, talc powder, magnesium stearate, calcium stearate or zinc stearate or stearic acid) or solvent encapsulating materials involved in carrying or transporting the peptides of the invention from one organ or part of the body to another organ or part of the body . Each carrier must be "acceptable" in the meaning of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) carbohydrates such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and derivatives thereof , such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (9) ethanol; (20) pH buffer solution; (21) polyester, polycarbonate Esters and/or polyanhydrides; and (22) other non-toxic compatible substances for pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents may also be included in the pharmaceutical compositions of the present invention agents, preservatives and antioxidants.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as Ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha tocopherol, etc.; and (3) metal chelators such as citric acid, EDTA (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

The pharmaceutical compositions of the present invention include those suitable for oral, nasal, topical (including topical, buccal and sublingual), rectal, vaginal and/or parenteral administration. The pharmaceutical compositions can be presented in unit dosage form and can be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending upon the host treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Typically, the amount will be from about 0.1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%, in percent.

Pharmaceutical compositions of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or in the form of In the form of solutions or suspensions in aqueous or non-aqueous liquids either as oil-in-water or water-in-oil liquid emulsions or as elixirs or syrups or pastilles (using inert bases such as gelatin and glycerol or sucrose and gum arabic) and/or in the form of a mouthwash, etc., each of which contains a predetermined amount of the peptide of the present invention as an active ingredient. The peptides of the invention can also be administered as a bolus, electuary or paste.

In solid dosage forms (capsules, tablets, pills, lozenges, powders, granules, trouches, etc.) of the present invention for oral administration, the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as Sodium citrate or dicalcium phosphate and/or any of the following: (1) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders such as Carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates, and sodium carbonate; (5) dissolution blockers, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds and surfactants, such as poloxamers and sodium lauryl sulfate; ( 7) Wetting agents such as cetyl alcohol, glycerol monostearate and nonionic surfactants; (8) Absorbents such as kaolin and bentonite clays; (9) Lubricants such as talc, calcium stearate, Magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof; (10) colorants; and (11) controlled release agents such as cross-polymerization Vidone or ethyl cellulose. In the case of capsules, tablets and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type can also be used as fillers in soft and hard shell capsules, using excipients such as lactose or milk sugar, high molecular weight polyethylene glycols, and the like.

A tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Binders (eg, gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (eg, sodium starch glycolate or croscarmellose sodium), surface-active agent or dispersing agent to prepare compressed tablets. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Tablets and other solid dosage forms such as dragees, capsules, pills and granules of the pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical arts. They can also be formulated to provide sustained or controlled release of the active ingredient therein, using, for example, hydroxypropyl methylcellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They can be formulated for rapid release, eg freeze drying. For example, they can be sterilized by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water or in Some other sterile injectable media for use immediately before use. These compositions may also optionally contain opacifying agents, and may be compositions that release the active ingredient only or preferably in some parts of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymers and waxes. The active ingredient may also be in microencapsulated form with one or more of the above-mentioned excipients, if appropriate.

Liquid dosage forms for oral administration of the peptides of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzoic acid Benzyl esters, propylene glycol, 1,3-butanediol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and Fatty acid esters of sorbitan and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

In addition to the active compounds, suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide , bentonite, agar and tragacanth and mixtures thereof.

Pharmaceutical compositions of the present invention for rectal or vaginal administration may be formulated as suppositories, which may be prepared by admixing one or more peptides of the present invention with one or more suitable non-irritating excipients or carriers, whereby Such suitable non-irritating excipients or carriers include, for example, cocoa butter, polyethylene glycols, suppository waxes or salicylates, and which are solid at room temperature but liquid at body temperature and thus in the rectum. or melt in the vaginal cavity and release the active compound.

Formulations of the invention suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or sprays containing, for example, such carriers as are known in the art to be suitable.

Dosage forms for topical or transdermal administration of the peptides of the invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers or propellants that may be required.

Ointments, pastes, creams and gels may contain, in addition to the peptides of the invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycol Alcohol, silicone, bentonite, silicic acid, talc and zinc oxide or mixtures thereof.

Powders and sprays can contain, in addition to the peptides of the invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can also contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of controlled delivery of the peptides of the invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flow of compounds across the skin. Such flow rates can be controlled by providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

Ophthalmic formulations, ophthalmic ointments, powders, solutions and the like are also within the scope of the present invention.

Pharmaceutical compositions of the invention suitable for parenteral administration comprise one or more peptides of the invention together with one or more pharmaceutically acceptable sterile isotonic or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powder combinations, which can be reconstituted into sterile injectable solutions or dispersions just before use, which may contain carbohydrates, alcohols, antioxidants, buffers, bacteriostatic agents, conferring agents and A solute or suspending or thickening agent that is intended to be isotonic in the blood of the recipient.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions/formulations of the present invention include water, ethanol, polyols (eg, glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the desired particle size in the case of dispersions and by the use of surfactants.

The pharmaceutical compositions of the present invention may contain other active compounds such as conventional antibiotics (eg vancomycin, streptomycin, tetracycline, penicillin) or other antimicrobially active compounds such as fungicides such as itraconazole or miconazole. Other compounds may also be added to relieve symptoms that accompany infection, such as fever (salicylic acid) or rash.

The present invention provides non-pharmaceutical compositions comprising one or more of the peptides or nucleic acids of the present invention.

The non-pharmaceutical compositions of the present invention include antibacterial agents, disinfectants, cleaning agents, and preservatives containing the peptides of the present invention.

The antibacterial agent is especially suitable for packaging materials (eg food packaging), daily chemical products (eg skin care products, diapers, soaps).

The non-pharmaceutical compositions of the present invention include compositions containing the nucleic acids of the present invention. This composition can be used to prepare the peptides of the present invention. The non-pharmaceutical compositions of the present invention include compositions of host cells containing the nucleic acids of the present invention. This composition can be used to prepare the peptides of the present invention.

treatment method:

The present invention includes a method of treating a subject/patient by administering the peptide or pharmaceutical composition of the present invention in an appropriate dose, mode of administration, and regimen. Such subjects include humans and mammals. The treatment method can achieve the purpose of inhibiting microorganisms (including treating microbial infections, maintaining the balance of microorganisms in the body, etc.) or regulating immune responses in vivo. The "inhibition" refers to a reduction in the number and/or activity of one or more microorganisms as compared to the situation without the use of the peptides of the invention. The "treatment" refers to the relief or complete disappearance of symptoms due to microbial infection as compared to the case where the peptides of the present invention are not used. A "therapeutically effective amount" refers to an amount that achieves a therapeutic effect.

use:

The peptide or nucleic acid of the present invention has applications in medicine or pharmacy, for example, as an active ingredient in compositions with the function of inhibiting and killing microorganisms, for preparing these compositions, for inhibiting microorganisms in vivo (including treatment of microbial infections, maintenance of microbial homeostasis, etc.) or modulation of immune responses. The peptides of the present invention can also be used in combination with other active compounds known to those skilled in the art to achieve better effects of treating microbial infections or modulating immune responses.

In addition to therapeutic uses, the peptides of the present invention can also be used in antibacterial, disinfectant, cleaning and/or preservative agents, which can be used to disinfect and/or clean surfaces and/or objects. Another area of application is packaging, where peptides can be bound to or incorporated into packaging materials, or as preservatives for other materials that are susceptible to microbial degradation. The peptides of the present invention are particularly suitable for addition to daily chemical products (such as skin care products, diapers, soaps, etc.) or food packaging, because they have no toxic effects when contacted or ingested.

The technical solutions of the present invention will be described in further detail below through the examples and in conjunction with the accompanying drawings, but the present invention is not limited to the following examples. Without departing from the spirit and scope of the present invention, those skilled in the art can Various changes and modifications have been made to the present invention in form and detail, and these are considered to fall within the scope of the present invention.

Example 1: Prediction of antimicrobial peptides

Figure 1 is a schematic diagram of the antimicrobial peptide prediction method of the present invention.

First, collect gut metagenomic data from a population cohort containing a large number of samples, each of which contains the genomic information of all microbes in the gut. To obtain the abundance information of each species in the sample, that is, the taxonomic profile. Predict the open reading frame (ORF) of the preprocessed data and translate it into amino acid sequence to obtain a large number of short peptides to be tested. In addition, metatranscriptomic data and metaproteomic data were collected, both representing genes and functional proteins that are truly expressed in the gut.

Then, the existing known AMP sequences were collected, and by training multiple deep learning models based on long short-term memory network (LSTM) and convolutional neural network (CNN), these models were used to predict a large number of short peptides to be tested, and the high probability was selected as Sequences of antimicrobial peptides, as the results of the initial screening. After that, the correlation between the macroproteome, macrotranscriptome, classification profile and the primary screening sequence was calculated, and the short peptides with significant correlation were obtained. ).

Example 2: Solid Phase Synthesis of Antimicrobial Peptides (pAMPs)

In Example 1, the amino acid sequence of the antimicrobial peptide (pAMP) to be tested was obtained by prediction, and the peptide was synthesized by the solid-phase synthesis method in this example for subsequent activity verification. The synthesis method is as follows:

Synthesis:

1. Resin swelling: put the resin into the reaction tube, add DMF (15ml/g), 30min;

2. Deprotection: remove DMF, add 20% piperidine DMF solution (15ml/g) for 5min, remove and add 20% piperidine DMF solution (15ml/g) for 15min;

3. Detection: remove the piperidine solution, take a dozen resins, wash three times with ethanol, add one drop each of ninhydrin, KCN and phenol solution, heat at 105℃-110℃ for 5min;

4. Washing: DMF (10ml/g) twice, methanol (10ml/g) twice, DMF (10ml/g) twice;

5. Condensation: three times excess of protected amino acid, three times excess of HBTU, dissolve with as little DMF as possible, add to reaction tube, immediately add ten times excess of NMM, and react for 30min;

6. Washing: DMF (10ml/g) once, methanol (10ml/g) twice, DMF (10ml/g) twice;

7. Repeat operations 2-6;

8. Washing: DMF (10ml/g) twice, methanol (10ml/g) twice, DMF (10ml/g) twice, DCM (10ml/g) twice;

9. Lysis 120min: prepare lysis solution (10/g) [TFA 94.5%; water 2.5%; EDT 2.5%; TIS 1%];

10. Drying and washing: dry the lysate with nitrogen, wash six times with ether, and then evaporate to dryness at room temperature.

Cutting and post-processing:

1. Preparation of cutting solution: the ratio of TFA, TIS, EDT, and H ₂ O is (95:2:1:2), and phenol, anisole, etc. are added as appropriate;

2. The cutting time is about 2 hours;

3. Precipitate with ether after cleavage to remove the cleaved side chain protecting groups, auxiliary reagents of cleavage solution, salts and other impurities.

Purification and identification:

1. Dissolve: dissolve in water first, add a small amount of acetonitrile depending on water solubility, and add ethylenediamine, acetic acid, etc. if necessary;

2. Purification: use a reversed-phase C8 column, the mobile phase is water containing 0.1% TFA and acetonitrile, and the gradient is 20%-80%;

3. Identification: liquid chromatography-mass spectrometry combined to determine molecular weight;

4. Lyophilization.

The liquid chromatography and mass spectrometry data of pAMP obtained by solid-phase synthesis were identical to the expected theoretical values, proving that the sequence of solid-phase synthesized pAMP was the same as expected. That is, the pAMP corresponding to that predicted in Example 1 was synthesized through Example 2, which can be further verified by experiments.

Example 3: Determination of activity of antimicrobial peptides (pAMPs)

In this example, the antibacterial activity of the antimicrobial peptide synthesized in Example 2 was measured to verify the prediction in Example 1. The specific measurement method is as follows:

Strain culture and pAMP stock solution preparation:

Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans were taken, streaked on LB plates, and cultured at 37°C overnight. Pick a single colony in liquid LB medium, and culture it on a shaker at 37°C overnight to obtain the original bacterial solution. After diluting to OD600=0.1, it was diluted 1000 times again for antibacterial assay.

The thawed pAMP lyophilized powder was dissolved in an appropriate amount of distilled water to prepare a 2.4 mM stock solution for later use.

Bacteriostatic assay:

For the single pAMP screening of a single strain, the antibacterial assay was divided into 4 groups, and the samples were loaded in 96-well plates, and each well was a 200 μL system:

(1) Medium: Medium 200 μL

(2) Control group: culture medium 100 μL + bacterial liquid 100 μL

(3) Low concentration of pAMP (60 μM): 100 μL of medium + 95 μL of bacterial solution + 5 μL of pAMP stock solution

(4) High concentration pAMP (180 μM): 100 μL of medium + 85 μL of bacterial solution + 15 μL of pAMP stock solution

The two concentrations were repeated in 3 groups, and the medium and the control group were repeated in 12 groups. After adding the samples, shake well, and after culturing at 37°C for 12 h, use a microplate reader to measure the OD600 of each well, and subtract the OD of the medium from each group of data as the final result of the well for subsequent data analysis. Thus, the experimental data of the predicted pAMP in Example 1 can be obtained, thereby further obtaining the bacteriostatic rate of each pAMP.

Bacteriostatic rate calculation:

Bacteriostatic rate (%)=(average OD of control group-average OD of experimental group)/average OD of control group×100%

For antimicrobial peptides SEQ ID NOs: 1-178, the results are shown in Table 1:

The percentage value in the table is the bacteriostatic rate of the corresponding concentration of antimicrobial peptide against the corresponding strain (the calculation formula is shown above), in which the shade of green (the filling color of the table) represents its bacteriostatic rate, and the darker the green (the filling color of the table), the The higher the bacteriostatic rate. The "activity" is the bacteriostatic activity of the antimicrobial peptide, which is measured by the bacteriostatic rate. "*" indicates that the antimicrobial peptide has bacteriostatic activity against this strain (bacteriostatic rate > 0), and "*" in the "full concentration" column indicates that the antimicrobial peptide has antibacterial activity in 5 strains (10 concentrations in total) ) is active at any place under ); "*" in the column "low concentration" indicates that the antimicrobial peptide is active at any place under the low concentration conditions (5 concentrations) of 5 strains.

Example 4: Determination of Minimum Inhibitory Concentrations of Antimicrobial Peptides (pAMP) against Various Bacteria

According to the measurement results of the above-mentioned antibacterial activity, 11 kinds of antimicrobial peptides with strong activity were selected to determine the minimum inhibitory concentration of different strains.

I. Experimental materials:

(1) Antimicrobial peptides:

peptide_2041 (SEQ ID NO: 11), peptide_660 (SEQ ID NO: 13), peptide_593 (SEQ ID NO: 28), peptide_575 (SEQ ID NO: 51), pAMP_1655 (SEQ ID NO: 71), pAMP_240 (SEQ ID NO: 71) : 104), pAMP_1043 (SEQ ID NO: 110), pAMP_67 (SEQ ID NO: 114), pAMP_69 (SEQ ID NO: 115), pAMP_250 (SEQ ID NO: 116), pAMP_518 (SEQ ID NO: 125).

(2) Strain:

Acinetobacter baumannii strain ATCC 19606, Pseudomonas aeruginosa strain ATCC 15692, Klebsiella pneumoniae strain NCTC 5056, Enterococcus faecium strain ATCC 19434, Escherichia coli strain DH5α, Enterococcus cloacae strain ATCC 13047, Bacillus subtilis strain ATCC 23857, Staphylococcus aureus ATCC 6538.

II. Experimental method:

Minimum inhibitory concentration (MIC) determination:

The method for determining the MIC of antimicrobial peptides is the broth microdilution method detailed in the CLSI Guidelines (Wayne, PA Performance Standards for Antimicrobial Disk Susceptibility Tests. CLSI (1991)). Briefly, the above strains were respectively inoculated in cation-adjusted Mueller-Hinton broth (Cation-adjusted Mueller-Hinton broth (CaMHB), QDRS BIOTEC, China, product number 11865) and placed at 37°C overnight. Cultures were diluted 1:100 with freshly prepared CaMHB, and the bacteria were grown to exponential growth phase ( _OD600 of 0.4-0.6). The bacterial concentration was then adjusted to approximately ⁵ x 105 colony forming units (CFU) per milliliter. For any concentration of any of the antimicrobial peptides used in this example, a 100 μL bacterial aliquot was transferred to a 96-well plate containing 100 μL of the antimicrobial peptide solution. The concentrations of antimicrobial peptides were serially diluted in two-fold concentrations ranging from 500 μM to 0.98 μM. After 16-18 hours of incubation at 37°C, bacterial growth was examined. The lowest concentration of antimicrobial peptide that results in no detectable bacterial growth is the MIC.

For Enterococcus faecium strain ATCC 19434, the assay was performed in the same way using brain heart infusion broth (BHI) as the medium and cultured under anaerobic conditions.

All assays were performed in triplicate. MIC determination experiments were performed in triplicate independently.

III. Experimental results:

The results of the MIC determination are shown in Table 3 and Figure 4. It can be seen that for Klebsiella pneumoniae, the MICs of all antimicrobial peptides in this embodiment are less than 25 μM; for Enterococcus faecalis, Acinetobacter baumannii, Enterococcus cloacae and Escherichia coli, at least one antimicrobial peptide of this embodiment has The MIC of the peptide reached 25 μM. In addition, all the antimicrobial peptides of this example can achieve MIC of <10 μM for at least one species. Further, the MIC of the antimicrobial peptides of this example against Escherichia coli is in the lowest range (less than about 99%) of all antimicrobial peptides compared to all antimicrobial peptides with E. coli MIC measurements in the CAMP database. MIC of known antimicrobial peptides).

Example 5: Determination of antibacterial activity of antimicrobial peptides (pAMPs) against multidrug-resistant bacteria

Multidrug-resistant (MDR) gram-negative bacteria, such as vancomycin-resistant Enterococcus (VRE) and carbapenicillin-resistant Enterobacter (CRE), as well as other bacteria in ESKAPE, have received extensive attention. This example determined the minimum inhibitory concentrations (MICs) of antimicrobial peptides against ten clinical isolates belonging to MDR Gram-negative bacteria.

I. Experimental materials:

(1) Antimicrobial peptides:

peptide_2041 (SEQ ID NO: 11), peptide_660 (SEQ ID NO: 13), peptide_593 (SEQ ID NO: 28), peptide_575 (SEQ ID NO: 51), pAMP_1655 (SEQ ID NO: 71), pAMP_240 (SEQ ID NO: 71) : 104), pAMP_1043 (SEQ ID NO: 110), pAMP_67 (SEQ ID NO: 114), pAMP_69 (SEQ ID NO: 115), pAMP_250 (SEQ ID NO: 116), pAMP_518 (SEQ ID NO: 125).

(2) MDR strains:

Acinetobacter baumannii strains Ab3, Ab8 and Ab11; Klebsiella pneumoniae strains NK04047, NK06129, NK08334 and NK01067; Escherichia coli strains E01-7-1, E02-7 -2 and E02-16-2.

II. Experimental method:

The minimum inhibitory concentration (MIC) was determined in the same way as in Example 4.

III. Experimental results:

All tested clinical isolates are known to be resistant to the third-generation cephalosporins ceftazidime (CAZ), ceftriaxone (CRO), cefepime (FEP), and sulbactam cefoperazone, SCF); all tested clinical isolates of Klebsiella pneumoniae and Escherichia coli, and clinical isolates of Acinetobacter baumannii Ab8 were resistant to at least one carbapenem (ertapenem, ETP ), impenem (IPM) or meropenem (meropenem, MEM)) are resistant.

Among the antimicrobial peptides used in this example, the MIC of pAMP_1043 (SEQ ID NO: 110) for all clinical isolates reached <10 μM, and the MIC of 7 antimicrobial peptides for at least 9 clinical isolates reached <20 μM ( Figure 5). The experimental results showed that various antimicrobial peptides (such as pAMP_1043, peptide_575, peptide_593, peptide_660 and peptide_2041) had strong inhibitory effects on all strains.

Therefore, the antimicrobial peptides of the present invention, although less similar to known antimicrobial peptides, have broad-spectrum, potent antibacterial activities, including activity against MDR Gram-negative bacteria.

Example 6: Determination of Cytotoxicity of Antimicrobial Peptides (pAMPs)

This example measures the toxicity of antimicrobial peptides to eukaryotic cells.

I. Experimental materials:

(1) Antimicrobial peptides:

peptide_2041 (SEQ ID NO: 11), peptide_660 (SEQ ID NO: 13), peptide_593 (SEQ ID NO: 28), peptide_575 (SEQ ID NO: 51), pAMP_1655 (SEQ ID NO: 71), pAMP_240 (SEQ ID NO: 71) : 104), pAMP_1043 (SEQ ID NO: 110), pAMP_67 (SEQ ID NO: 114), pAMP_69 (SEQ ID NO: 115), pAMP_250 (SEQ ID NO: 116), pAMP_518 (SEQ ID NO: 125).

(2) Cells: HCT116 cells (human colorectal cancer cell line) and human erythrocytes

II. Experimental method:

(1) Determination of cytotoxicity to mammalian cells:

The cytotoxicity of antimicrobial peptides was determined using the MTT cell proliferation and cytotoxicity detection kit (Solebo, China). HCT116 cells in exponential growth phase were seeded in appropriate cell culture medium in 96-well microtiter plates. After 24 hours of incubation at 37 °C and 5% _CO in an atmospheric condition, the medium was replaced with a freshly prepared medium and antimicrobial peptides (final concentration 40 μM) were added (for the control group, the same volume of distilled water was added), then Incubate for 48 hours. MTT solution was then added and after 4 hours the optical density at ₄₉₀ nm (OD490) was measured to detect cell viability.

All assays were performed in quadruplicate. For each antimicrobial peptide, three experiments were performed in independent replicates.

(2) Determination of hemolysis:

Freshly collected human erythrocytes were first washed with PBS until the upper phase was clear after centrifugation (2000 r/min). The washed human erythrocytes were added to the wells of a 96-well U-bottom plate. Each antimicrobial peptide was diluted and added to the wells at final concentrations of 8 μM and 40 μM, respectively. After 1 hour incubation at 37°C, cells were centrifuged at 3000 r/min for 10 minutes. The supernatant was diluted and the optical density at ₅₇₀ nm (OD570) was determined. For each antimicrobial peptide, the experiments were performed in triplicate independently.

III. Experimental results:

Among the antimicrobial peptides used in this example, 4 antimicrobial peptides were not toxic to HCT116 cells at a concentration of 40 μM (survival inhibition ≤ 20%, Figure 6), and 8 antimicrobial peptides showed no significant hemolytic activity at a concentration of 8 μM (<20% hemolysis, Figure 7), 7 antimicrobial peptides had no significant hemolytic activity at 40 [mu]M concentration (Figure 7).

Example 7: Determination of the minimum inhibitory concentration and in vivo inhibitory activity of antimicrobial peptide (pMAP) against Klebsiella pneumoniae

I. Experimental materials:

(1) Antimicrobial peptides:

peptide_2041 (SEQ ID NO: 11), peptide_660 (SEQ ID NO: 13), peptide_593 (SEQ ID NO: 28), peptide_575 (SEQ ID NO: 51), pAMP_1655 (SEQ ID NO: 71), pAMP_240 (SEQ ID NO: 71) : 104), pAMP_1043 (SEQ ID NO: 110), pAMP_67 (SEQ ID NO: 114), pAMP_69 (SEQ ID NO: 115), pAMP_250 (SEQ ID NO: 116), pAMP_518 (SEQ ID NO: 125).

(2) Strain: Klebsiella pneumoniae (Klebsiella pneumoniae) strain ATCC 700603

II. Experimental method:

(1) The method for measuring the minimum inhibitory concentration (MIC) was the same as that in Example 4.

(2) Treatment of Klebsiella pneumoniae pulmonary infection mouse model:

Klebsiella pneumoniae was first cultured to 10 ⁹ CFU/L, and 6-week-old C57BL/6J mice were infected with 50 μl nasal drops. On the second day post-infection, 50 μl of nasal drops containing pAMP_1043, peptide_593 or peptide_575 were administered to the infected mice at a concentration of 5x the MIC against Klebsiella pneumoniae (40 μM for pAMP_1043, peptide_593 and peptide_575 80 μM). The same volume of distilled water was administered to the infected control mice. Body weights of treated and untreated (control) mice were recorded daily. The sample size was seven mice per group, which was based on a preliminary study showing that seven mice were sufficient to observe significant differences between the treatment and control groups. Mice were randomly assigned to each group. All animal experiments were approved by the Ethics Committee of the Institute of Microbiology, Chinese Academy of Sciences (SQIMCAS2021005).

III. Experimental results:

The MICs of antimicrobial peptides against Klebsiella pneumoniae (Klebsiella pneumoniae) strain ATCC 700603 are shown in Table 4.

For Klebsiella pneumoniae-infected mice, body weight recovery was used as a phenotype of treatment effect, and the results are shown in Figure 8. It can be seen that compared with the control mice, the infected mice treated with pAMP_1043, peptide_593 or peptide_575 regained their body weight significantly faster, indicating that the degree of bacterial infection was significantly reduced. Half of the mice in the control group showed weight loss seven days after infection, but all mice in the treatment group had regained their original weight by this time.

Table 4: MIC of 11 different antimicrobial peptides against Klebsiella pneumoniae strain ATCC 700603

SEQ ID NO:	110	125	11	51	13	28	71	114	115	116	104
MIC(μM)	8	8	16	16	32	32	32	32	32	125	＞500

Claims (21)

1. An antimicrobial peptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 1-178; or comprising a sequence identity greater than 90% compared to the amino acid sequence shown in any one of SEQ ID NOs: 1-178 or a fragment comprising the amino acid sequence shown in any one of SEQ ID NOs: 1-178; or a fragment comprising greater than 90% of the amino acid sequence shown in any one of SEQ ID NOs: 1-178 The sequence identity of the amino acid sequence.
2. The antimicrobial peptide of claim 1, its amino acid sequence is the amino acid sequence shown in any one of SEQ ID NOs: 1-178; % amino acid sequence of sequence identity; or a fragment of the amino acid sequence shown in any one of SEQ ID NOs: 1-178; or compared with a fragment of the amino acid sequence shown in any one of SEQ ID NOs: 1-178 Amino acid sequences with greater than 90% sequence identity.
3. An antimicrobial peptide comprising the amino acid sequence shown in any of SEQ ID NOs: 11, 13, 28, 51, 71, 104, 110, 114, 115, 116 and 125; , 13, 28, 51, 71, 104, 110, 114, 115, 116 and 125 compared to the amino acid sequences shown in any one of the amino acid sequences with greater than 90% sequence identity; or comprising as SEQ ID NOs: 11, 13, 28, 51, 71, 104, 110, 114, 115, 116 and 125. Fragments of the amino acid sequences shown in any one; Fragments of the amino acid sequences shown in any of 114, 115, 116 and 125 are compared to amino acid sequences that have greater than 90% sequence identity.
4. The antimicrobial peptide of claim 3, its amino acid sequence is the amino acid sequence shown in any one of SEQ ID NOs: 11, 13, 28, 51, 71, 104, 110, 114, 115, 116 and 125; ID NOs: amino acid sequences with greater than 90% sequence identity compared to the amino acid sequences shown in any of 11, 13, 28, 51, 71, 104, 110, 114, 115, 116 and 125; or as SEQ ID NOs: fragments of the amino acid sequences shown in any of 11, 13, 28, 51, 71, 104, 110, 114, 115, 116 and 125; Fragments of the amino acid sequences shown in any of 104, 110, 114, 115, 116, and 125 have greater than 90% sequence identity compared to amino acid sequences.
5. 4. The antimicrobial peptide of any one of claims 1-4, wherein the fragment contains at least 7 amino acids.
6. 5. The antimicrobial peptide of any one of claims 1-5, comprising chemical modifications on amino acids that are present on less than 10% of the amino acids.
7. An antimicrobial composition comprising the antimicrobial peptide of any one of claims 1-6.
8. 8. The composition of claim 7, wherein the composition is a pharmaceutical composition and comprises a pharmaceutically acceptable carrier.
9. Use of the peptide of any one of claims 1-6 or the composition of claim 7 or 8 for inhibiting microorganisms in vitro or in vitro.
10. Use of a peptide of any one of claims 1-6 or a composition of claim 7 or 8 in the manufacture of an antimicrobial agent.
11. Use of a peptide of any one of claims 1-6 or a composition of claim 7 or 8 in the manufacture of a medicament for the treatment of microbial infections and/or modulation of immune responses.
12. The use of any one of claims 9-11, wherein the microorganism is a fungus and/or a bacterium.
13. The use of any one of claims 9-12, wherein the microorganism is Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Acinetobacter baumannii, Klebsiella pneumoniae, Bacillus subtilis Bacillus, Enterobacter cloacae and/or Enterococcus faecalis.
14. The peptide of any one of claims 1-6 or the composition of claim 7 or 8 for use in inhibiting microorganisms.
15. The peptide of any one of claims 1-6 or the composition of claim 7 or 8 for use in the treatment of microbial infections and/or the modulation of immune responses.
16. The peptide or composition of claim 14 or 15, wherein the microorganism is a fungus and/or a bacterium.
17. The peptide or composition of any one of claims 14-16, wherein the microorganism is Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Acinetobacter baumannii, Klebsiella pneumoniae bacteria, Bacillus subtilis, Enterobacter cloacae and/or Enterococcus faecalis.
18. A method for inhibiting a microorganism comprising administering to a subject an effective amount of the peptide of any one of claims 1-6 or the composition of claim 7 or 8.
19. A method for treating a microbial infection and/or modulating an immune response comprising administering to a subject an effective amount of the peptide of any one of claims 1-6 or the composition of claim 7 or 8.
20. The method of claim 18 or 19, wherein the microorganism is a fungus and/or a bacterium.
21. The method of any one of claims 18-20, wherein the microorganism is Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Acinetobacter baumannii, Klebsiella pneumoniae, Bacillus subtilis Bacillus, Enterobacter cloacae and/or Enterococcus faecalis.

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