c-Met protein-binding peptide complex

Abstract

[Problem] This invention provides a peptide complex that binds to c-Met protein. [Solution] A peptide complex comprising peptide A, wherein peptide A is a peptide composed of the amino acid sequence described in sequence identification number 1 (X1-X2-X3-V-S-X4-D-X5-D-X6-P-R-W-X7-MeC), or composed of amino acid sequences consisting of 1 to 3 amino acids substituted, deleted, added, or inserted from the amino acid sequence described in sequence identification number 1. X1 is A, MeA, F, or MeF. X2 is V, T, E, Q, or W. X3 is A, R, Y, or D. X4 is F, MeF, L, or MeF. X5 is D, E, S, P, or V. X 6 is (S)-2-aminoheptanoic acid (Ahp), R, L-leucine (Nle), (S)-2,7-diaminoheptanoic acid (Hty), or S. X 7 is S, A, L-α-aminobutyric acid (Abu), D, Q, or V.

Description

Technical Field

[0001] This invention relates to a complex comprising a peptide that binds to a c-Met protein, etc. Prior Technology

[0002] In recent years, regenerative medicine, which aims to regenerate body tissues damaged by illness, injury, etc., has gained attention. Because regenerative medicine uses the patient's own cells to regenerate tissues, it has the advantage of being less likely to cause immune problems compared to previous methods such as organ transplantation. This type of regenerative medicine requires culturing human cells, such as stem cells, and differentiating them into the target tissue. Furthermore, basic research is ongoing on the use of cultured mammalian cells, especially human cells, to create further improved technologies for regenerative medicine. In regenerative medicine, research, and the like, the efficient process of culturing and multiplying target cells becomes crucial. In the culture of human cells, especially stem cells, the components of the culture medium play a crucial role, with growth factors (sometimes called GF) being one of the most important elements. However, growth factors are generally very expensive, and, for example, in stem cell research, large quantities of growth factors are needed to maintain undifferentiated cells. Furthermore, hepatocyte growth factor receptor (HGF), one of the growth factors, and its receptor, c-Met (sometimes also called c-Met or Met), are also being studied as targets for pharmaceuticals. If c-Met is a single-transmembrane receptor type tyrosine kinase and its ligand, HGF, binds to c-MET, then c-MET dimers and becomes activated. It is known that c-Met activation is necessary for embryonic development, organogenesis, and wound repair. For example, it is known that the binding of HGF to the c-Met receptor activates related signaling pathways, promotes / maintains the proliferation and function of specific endothelial cells, promotes angiogenesis, and further leads to collateral circulation. Therefore, compounds with c-Met activating effects are sought and are currently under investigation. Against this backdrop, Patent Document 1 reports an antibody with c-Met activating activity. Furthermore, Non-Patent Document 1 reports an HGF alternative currently under development. Furthermore, in recent years, the application of peptides, especially cyclic peptides, in pharmaceutical compositions has attracted attention as alternatives to low-molecular-weight compounds such as antibodies and other high-molecular-weight compounds. For example, Patent Document 2 and Non-Patent Document 2 describe peptide complexes that can be used as c-Met protein activators. These peptide complexes, which can be used as c-Met protein activators, are known to promote cell proliferation and migration, and their cell proliferation effect is expected to be used in various applications such as pharmaceuticals and as components of culture media for cell culture. Examples of usefulness include: growth factor substitutes added when culturing cells and tissues for regenerative medicine; organ protectants during organ transplantation; regeneration promoters; and therapeutic drugs for treating diseases that cause decreased expression of hepatocyte growth factors. [Known Technical Documents] [Patent Literature]

[0003] [Patent Document 1] Japanese Patent Publication No. 2021-50793 [Patent Document 2] Japanese Patent No. 6426103 [Non-patent literature] [Non-Patent Literature 1] THE CHEMICAL TIMES (Kanto Chemical Co., Ltd.) 2020 No.2 pp.6-11 Special Issue: Development of Chemically Synthesized Growth Factor Substitute Compounds for Regenerative Medicine Related Technologies Ryosuke Ueki and Shinsuke Toshiro [Non-Patent Literature 2] Ito, K. et al., Nature Communications, 6, Article number: 6373 (2015) Summary of the Invention

[0004] [The problem that the invention aims to solve] As mentioned earlier, the culture of cells, primarily stem cells, requires the addition of expensive growth factors, which hinders the medical applications of stem cells. Furthermore, since growth factors are also likely to be used as pharmaceuticals, there is a desire to develop low-cost and stably available alternatives. In this context, as described in the aforementioned publication, a peptide complex that functions as a c-Met protein agonist has been discovered, comprising a peptide that binds to the c-Met protein. However, the peptide complex described in the aforementioned publication contains components exhibiting lower activity than human HGF; therefore, there is a desire to develop a c-Met protein-binding complex other than this peptide complex.

[0005] The invention described in this specification is intended to solve one or more of the aforementioned problems.

[0006] [Technical means to solve the problem] The present invention is basically based on the following insights obtained from the embodiments: the peptide indicated by sequence identification number 1 binds to the c-Met protein.

[0007] Furthermore, the present invention is basically based on the following insights obtained from the embodiments: peptide complexes designed by using linkers to bind the above-mentioned peptides to obtain a dimer structure can function as c-Met protein agonists.

[0008] The first invention relates to a peptide complex comprising peptide A that binds to c-Met protein. Peptide A is a peptide composed of the amino acid sequence described in X1-X2-X3-VSX4-DX5-DX6-PRWX7-MeC (Sequence Identification Number 1), or a peptide that binds to c-Met protein composed of 1 to 3 amino acids in the amino acid sequence described in Sequence Identification Number 1 through substitution, deletion, addition, or insertion. X 1 is an amino acid that can be N-alkylated. X2 is any amino acid. X3 is any amino acid. X4 is a hydrophobic amino acid that can be N-alkylated. X5 is any amino acid. X6 is an amino acid or S with a substituted alkyl chain in the side chain. X 7 is any amino acid.

[0009] [Invention Benefits] According to the present invention, a complex comprising a novel peptide that binds to a c-Met protein may be provided.

[0010] Furthermore, according to the present invention, a novel peptide complex having c-Met activating activity can be provided.

[0011] Furthermore, according to the present invention, a cell or tissue culture medium composition can be provided as a substitute for growth factors added when culturing cells, tissues, etc., for regenerative medicine, comprising a novel peptide complex having c-Met activating activity. This can also be used, for example, as an organ protectant or regeneration promoter in organ transplantation.

[0012] Furthermore, according to the present invention, a novel c-Met protein agonist and a pharmaceutical composition comprising such c-Met protein agonist can be provided. For example, this can be used as a therapeutic agent for diseases causing decreased expression of hepatocyte growth factor (HGF).

[0013] Furthermore, according to the present invention, a pharmaceutical composition comprising a novel c-Met protein agonist that promotes cell proliferation can be provided. Simple Explanation of the Diagram

[0014] Figure 1 shows the results of c-Met activating activity measurements using the peptide complex of the present invention and human HGF by phosphoric acid-c-Met AlphaLISA. In the figure, black squares represent c-Met activating peptide complexes (the complex of peptide (sequence identification number 34) and linker (sequence identification number 37) (GFs_c-Met-00014336-PEG13 dimer; complex No. 49 in Table 4), and X represents human HGF. The horizontal axis represents the concentration (nM) of the peptide complex or human HGF, and the vertical axis represents the relative value of the activation signal when the maximum value of the activation signal induced by human HGF is set to 100. Figure 2 shows the results of c-Met activating activity measurements performed on HUVEC cells using the peptide complex of the present invention and human HGF. In the figure, black squares represent the c-Met activating peptide complex (a complex of peptide (sequence identification number 34) and linker (sequence identification number 37) (GFs_c-Met-00014336-PEG13 dimer; complex No. 49 in Table 4), and X represents human HGF. The horizontal axis represents the concentration (nM) of the peptide complex or human HGF, and the vertical axis represents the relative value of the activation signal when the maximum value of the activation signal induced by human HGF is set to 100. Figure 3 shows the results after 3 days in an evaluation test of lumen formation in human renal proximal tubule epithelial cells using the peptide complex of the present invention, human HGF, and human epidermal growth factor (EGF). Figure 4 shows the results after 8 days in an evaluation assay of lumen formation in human renal proximal tubule epithelial cells using the peptide complex of the present invention, human HGF, and human epidermal growth factor (EGF). In the figure, black arrows indicate branched lumen formation. Figure 5 shows the results of the evaluation using the human phospho-RTK array (HGF-RTK array) assay of the peptide complex and human HGF of the present invention. Figure 5(1) shows the array diagram, and Figure 5(2) shows the evaluation results. In the figures, the black boxes indicate wells with antibodies for HGFR immobilized. Implementation

[0015] The following describes a method for carrying out the present invention. The present invention is not limited to the methods described below, but also includes appropriate modifications made by those skilled in the art within the scope of the invention.

[0016] The first invention relates to a peptide complex comprising peptide A that binds to c-Met protein.

[0017] c-Met protein c-Met protein is a hepatocyte growth factor (HGF) receptor and possesses tyrosine kinase activity. The c-Met protein is a transmembrane receptor composed of α and β subunits linked by disulfide bonds. In vivo, c-Met protein dimers upon HGF binding, followed by autophosphorylation, activating various signal transduction pathways. This results in the promotion of cell proliferation through activation of signal transduction pathways such as the MAPK and Akt pathways, while simultaneously inhibiting apoptosis. If this function can be enhanced to promote cell proliferation and migration, it may facilitate the manufacture of cell preparations for regenerative medicine and the treatment of refractory organ diseases such as cirrhosis. c-Met proteins are also known as c-Met, MET, or HGFR. The GenBank accession number for the human c-Met protein is NP_000236, and the GenBank accession number for the mouse c-Met protein is NP_032617.

[0018] Hepatocyte growth factor (HGF) Hepatocyte growth factor (HGF) is a multifunctional cytokine that functions as a growth factor for a wide range of tissues and cell types. It has a heterodimer structure formed by disulfide bonds between a heavy chain of approximately 60,000 molecules and a light chain of approximately 35,000 molecules. HGF is known to promote the proliferation of epithelial cells, endothelial cells, and mesenchymal cells, and also has functions such as morphogenesis induction, increased cell motility, anti-apoptosis, and angiogenesis. The GenBank accession number for human HGF is NP_000592, and the GenBank accession number for mouse HGF is NP_001276387. Human HGF is preferred; unless otherwise specified in this specification, HGF refers to human HGF.

[0019] Binding to c-Met protein The term "binding to c-Met protein" refers to the binding of a peptide or peptide complex to a c-Met protein. Whether or not binding to a c-Met protein occurs can be measured using conventional methods for measuring intermolecular binding. These methods can be determined by any suitable, known method, including competitive binding analyses such as surface plasma resonance (SPR) analysis, Scatchard analysis and / or radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assay, as well as other variations known in the art. Preferably, the evaluation is performed, for example, by surface plasma resonance (SPR) spectroscopy as described in Japanese Patent No. 6426103 (Patent Document 2). Furthermore, compounds exhibiting c-Met activating activity do so by binding to c-Met proteins. Therefore, by evaluating c-Met activating activity—that is, the presence or absence of c-Met activating activity—one can indirectly evaluate whether the peptide or complex binds to c-Met proteins. In addition, within a complex, if part or all of the complex can bind to a c-Met protein, then the complex can be considered to be bound to a c-Met protein. For example, in the case where the complex contains peptide A, the portion of peptide A in the complex can be the site of binding to the c-Met protein, and other portions of the complex can also bind to the c-Met protein.

[0020] c-Met activating activity The term "c-Met activating activity" refers to the activity exhibited by binding to c-Met proteins and displaying effects similar to those of HGF. Whether a peptide or peptide complex possesses c-Met activating activity can be measured using conventional methods. For example, as shown in the examples, c-Met activating activity can be evaluated using a phosphorylated c-Met alpha ELISA assay or a HUVEC cell proliferation assay. Furthermore, the phosphorylation capacity of c-Met can also be evaluated using the ELISA method described in Japanese Patent No. 6426103 (Patent Document 2).

[0021] Diseases that can be cured by peptide complexes with c-Met agonist activity Examples of diseases that can be cured by peptide complexes with c-Met activating activity include ischemic heart disease, acute hepatitis, fulminant hepatitis, cirrhosis, biliary atresia, fatty liver, acute renal failure, chronic renal failure, diabetic nephropathy, acute pneumonia, pulmonary fibrosis, vascular disease, myocardial infarction, dilated cardiomyopathy, skin ulcers, cerebral infarction, occlusive arteriosclerosis, gastric ulcers, and amyotrophic lateral sclerosis (ALS).

[0022] peptide complex The peptide complex comprises a peptide complex containing peptide A that binds to the c-Met protein. This peptide complex contains one or more peptides A. Preferably, the peptide complex contains two peptides A. However, examples of peptide complexes containing only one peptide A are not limited; for example, it may contain peptide A and a substance (load) to be delivered to the c-Met protein, such as a conventional pharmaceutical composition, or a peptide complex containing peptide A and a fluorescent protein as a label. In the conventional case of a pharmaceutical composition and peptide A complex, the binding ability of peptide A to the c-Met protein can be used to deliver the pharmaceutical composition to the c-Met protein. The substance intended to be delivered to the c-Met protein is not particularly limited and can be any substance desired by those skilled in the art to which this invention pertains. Examples of such substances are not limited, but the following are some examples: Compounds: This includes not only low-molecular-weight and medium-molecular-weight compounds, but also any compounds that can be introduced through the cellular cytosis mechanism. Examples include well-known low-molecular-weight drugs. Peptide: A peptide that can bind to a target in the body and exhibit a certain effect; for example, it can be a cyclic peptide. RI: Any low- or medium-molecular-weight compound, antibody, or other compound that can be labeled with a radioactive isotope is acceptable. Examples include compounds used in positron emission tomography (PET) scans. Proteins: Any protein that performs useful functions in the body, such as antibodies or enzymes, is acceptable. Examples include enzymes used in enzyme supplementation therapy. Nucleic acids: Any substance containing a nucleotide sequence, such as DNA or RNA, is acceptable. Examples include nucleic acid pharmaceuticals. DDS: Can be liposomes, microcells, or other DDS molecules. These DDS molecules may also contain pharmaceutical compounds. And, can be a complex of the substances listed above.

[0023] Examples of peptide complexes include (1) a first peptide, (2) a second peptide and (3) a linker connecting the first peptide and the second peptide. This peptide complex can also be a peptide complex consisting only of (1) a first peptide, (2) a second peptide and (3) a linker connecting the first peptide and the second peptide. In this peptide complex, at least one of the first peptide and the second peptide is peptide A. The first and second peptides can be the same or different, but peptide A is preferred. That is, peptide complexes can be either heterodimers or homodimers. In a heterodimer system, the first and second peptides are different peptides (e.g., peptide A with different amino acid sequences). In a homodimer system, the first and second peptides are both peptide A. Homodimers are preferred peptide complexes.

[0024] The first and second peptides are preferably the same peptide A, and are peptide complexes formed by the C-terminus of the first and second peptides and a linker. More preferably, the first and second peptides are cyclic peptides.

[0025] Peptide A This specification describes not only complexes containing peptide A, but also peptide A itself. Peptide A is A peptide that binds to c-Met protein, consisting of the amino acid sequence described in X1-X2-X3-VSX4-DX5-DX6-PRWX7-MeC (Sequence Identification Number 1); or A peptide that binds to c-Met protein, consisting of an amino acid sequence formed by substitution, deletion, addition, or insertion of 1 to 3 (1, 2, or 3) amino acids in the amino acid sequence recorded in Sequence Identification Number 1. In this peptide (2), it is also preferred to maintain the V at position 4, the S at position 5, the D at position 7, the D at position 9, the P at position 11, the R at position 12, the W at position 13, and the MeC at position 15 in Sequence Identification Number 1.

[0026] X1 is an N-alkylatable amino acid, preferably an N-methylatable A or an N-methylatable F. More preferably, X1 is MeF (N-methylatable F). X2 can be any amino acid, preferably a hydrophilic amino acid, an aliphatic / branched chain amino acid, or an aromatic amino acid, and even more preferably V, T, E, Q, or W. The best X2 is T. X3 is any amino acid, which can be a hydrophilic amino acid, an aliphatic amino acid, or an aromatic amino acid, preferably A, R, Y, or D. The best X3 is A. X4 is an N-alkylatable hydrophobic amino acid, preferably an N-methylatable hydrophobic amino acid, and even more preferably an N-methylatable F or an N-methylatable L. The most suitable X4 is MeF (N-methylatable F). X5 can be any amino acid, preferably a hydrophilic amino acid, an aliphatic / branched chain amino acid, or P, and even more preferably D, E, S, P, or V. The best X5 is E. X6 is an amino acid or S having a substituted alkyl chain in the side chain, preferably (S)-2-aminoheptanoic acid (Ahp), R, L-leuconic acid (Nle), (S)-2,7-diaminoheptanoic acid (Hty), or S. More preferably, X6 is Ahp. X7 can be any amino acid, preferably a hydrophilic or aliphatic amino acid, and even more preferably S, A, L-α-aminobutyric acid (Abu), D, Q, or V. The best X7 is S.

[0027] The term "N-alkylatable amino acid" refers to an amino acid having an alkyl group on the nitrogen atom forming the peptide bond, i.e., an N-alkyl amino acid, or an amino acid without an alkyl group. Examples of N-alkyl amino acids include N-butylamino acids, N-ethylamino acids, and N-methylamino acids. Furthermore, the term "N-methylatable" indicates an amino acid that can be N-methylated. For example, "N-methylatable A" refers to alanine (A) or N-methylalanine (MeA).

[0028] The term "amino acid having a substituted alkyl chain in its side chain" refers to an amino acid having an alkyl chain in its side chain, such as amino acids belonging to the aliphatic amino acid group, or amino acids in which the functional group at the end of the alkyl group in the side chain of such amino acid is substituted, preferably an amino acid in which the alkyl group contains 5 or more carbon atoms. Examples include Ahp, Nle, and Hty.

[0029] A preferred example of peptide A is A. A peptide composed of the amino acid sequence recorded in MeF-TAVS-MeF-DED-Ahp-PRWS-MeC (sequence identification number 34); or B. A peptide that binds to c-Met protein, consisting of amino acid sequences consisting of 1 to 3 amino acids in the amino acid sequence recorded in Sequence Identification Number 34 through substitution, deletion, addition, or insertion.

[0030] Preservative amino acid substitution Substitution, deletion, addition, or insertion of one, two, or three amino acid residues from a specific sequence is preferably a conservative amino acid substitution. "Conservative amino acid substitution" refers to the substitution of an amino acid that is functionally equivalent or similar. Conservative amino acid substitution in a peptide results in a static change to the peptide's amino acid sequence. For example, one or more amino acids with the same polarity function equivalently, resulting in a static change to the peptide's amino acid sequence. Generally, substitutions within a certain group are considered conservative in terms of both structure and function. However, as those skilled in the art to which this invention pertains know, the function of a particular amino acid residue can be determined by the significance of the molecule containing that amino acid in its three-dimensional structure. For example, cysteine ​​residues can take the oxidized (disulfide) form, which is less polar than the reduced (thiol) form. Long aliphatic portions of arginine side chains can constitute structurally and functionally important features. Furthermore, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) facilitate ion-aromatic interactions or cation-π interactions. In these cases, even if the amino acid with such side chains is substituted with an amino acid belonging to the acidic or nonpolar group, structural and functional preservation is maintained. Residues such as proline, glycine, and cysteine ​​(in disulfide form) may have a direct effect on the stereostructure of the main chain and cannot be repeatedly substituted without structural deformation. As described below, preserveable amino acid substitutions include specific substitutions based on the similarity of the side chains (Lehninger, Biochemistry, 2nd edition, revised, 1975, pp. 73-75; Worth Publisher, New York (1975)) and typical substitutions.

[0031] Furthermore, the preservation of amino acid substitution, for example, preferably substitution into an amino acid that belongs to the same group as a certain amino acid in a group that classifies natural amino acids according to their common side chain properties. Hydrophobic (also known as nonpolar) amino acids: These are amino acids that are hydrophobic (nonpolar), including alanine (also referred to as "Ala" or simply "A"), glycine (also referred to as "Gly" or simply "G"), valine (also referred to as "Val" or simply "V"), leucine (also referred to as "Leu" or simply "L"), isoleucine (also referred to as "Ile" or simply "I"), proline (also referred to as "Pro" or simply "P"), phenylalanine (also referred to as "Phe" or simply "F"), tryptophan (also referred to as "Trp" or simply "W"), tyrosine (also referred to as "Tyr" or simply "Y"), and methionine (also referred to as "Met" or simply "M"). In addition, hydrophobic amino acids can be further divided into the following groups. Aliphatic amino acids: amino acids with fatty acid or hydrogen in their side chain, including Ala, Gly, Val, Ile, and Leu. Aliphatic / branched chain amino acids: Amino acids with branched fatty acids in their side chains, including Val, Ile, and Leu. Aromatic amino acids: Amino acids with an aromatic ring in their side chain, including Trp, Tyr, and Phe. Hydrophilic (also known as polar) amino acids: These are amino acids that are hydrophilic (polar), including: serine (also referred to as "S" or simply "S"), threonine (also referred to as "Thr" or simply "T"), cysteine ​​(also referred to as "Cys" or simply "C"), aspartic acid (also referred to as "Asn" or simply "N"), glutamic acid (also referred to as "Gln" or simply "Q"), aspartic acid (also referred to as "Asp" or simply "D"), glutamic acid (also referred to as "Glu" or simply "E"), lysine (also referred to as "Lys" or simply "K"), arginine (also referred to as "Arg" or simply "R"), and histidine (also referred to as "His" or simply "H"). In addition, hydrophilic amino acids can be further divided into the following groups. Acidic amino acids: Amino acids with acidic side chains, including Asp and Glu. Basic amino acids: Amino acids with basic side chains, including Lys, Arg, and His. Neutral amino acids: Amino acids with neutral side chains, including Ser, Thr, Asn, Gln, and Cys. Furthermore, Gly and Pro can also be classified as "amino acids that affect the orientation of the main chain". Amino acids with sulfur molecules in the side chain, Cys and Met can also be classified as "sulfur-containing amino acids".

[0032] In this specification, "amino acid" includes not only natural amino acids but also non-natural amino acids. Non-natural amino acids include, for example, N-alkyl amino acids formed by N-alkylation of the natural amino acids described above, and amino acids formed by modifying the nitrogen atoms that form peptide bonds with branched or unbranched lower-order alkyl groups (e.g., C1-C5, preferably C1-C3, more preferably C1). Among N-alkyl amino acids, N-ethylamino acids, N-butylamino acids, or N-methylamino acids are preferred, with N-methylamino acids being the most common. Furthermore, non-natural amino acids include chemically modified amino acids such as D-amino acids (also described as D-amino acids), β-amino acids, γ-amino acids, amino acid variants, and amino acid derivatives, as well as amino acids such as ornithine and ornithine that cannot serve as building blocks of proteins in vivo. Furthermore, it includes amino acids that have a further addition functional group to the side chain of a natural amino acid or have been substituted into another functional group (e.g., amino acids in which the aryl, alkyl, or other parts of the side chain have been substituted or added, amino acids in which the number of carbon atoms of the aryl, alkyl, or alkyl groups of the side chain has been increased, amino acids in which the aromatic ring of the side chain has been substituted, amino acids that have undergone heterocyclization or fused cyclization, etc.). Furthermore, by adding or substituting functional groups to the side chains of natural amino acids, different properties can be imparted to them. For example, A4p is alanine formed by adding a piperidinium group to its side chain, but by adding this piperidinium group, it differs from alanine, which belongs to the nonpolar amino acid group, and exhibits basic polarity. In other words, the aforementioned groups, which classify natural amino acids based on their common side-chain properties, may include non-natural amino acids with the same side-chain properties. For example, N-methylarginine (MeR), an N-methylamine that is a basic amino acid, is a non-natural amino acid, but it can be classified as a basic amino acid because it is basic. Thus, non-natural amino acids exhibiting the same side-chain properties as a certain amino acid can also be included as objects for preservation amino acid substitution. Without limitation, non-natural amino acids include N-methyl amino acids, Ahp, Nle, Hty, Abu, etc. For example, Ahp, Nle, Abu, and Hty can be classified as hydrophobic amino acids; furthermore, Ahp, Nle, and Abu can also be classified as aliphatic amino acids, and Hty can be classified as aromatic amino acids. In addition, N-methyl amino acids can also be classified as N-alkyl amino acids, or they can be classified according to the nature of the side chains of the un-N-methylated original amino acids.

[0033] Specifically, peptide A is preferably a peptide composed of the amino acid sequence described in any one of sequence identification numbers 2 to 34. Among these, it is more preferably a peptide composed of the amino acid sequence described in sequence identification number 34.

[0034] Peptide A is preferably a cyclic peptide. The term peptide refers to a structure composed of multiple amino acids in a continuous sequence, and its meaning also includes polypeptides and proteins. Furthermore, in this application, the term amino acid includes not only naturally occurring amino acids (natural amino acids) but also non-natural amino acids (non-natural amino acids). Furthermore, in this application, the peptide of the present invention also includes: a peptide that forms a cyclic portion by cyclization after synthesis, a peptide obtained by further chemical modification of the peptide, and a complex of the peptide and a substance bound to the peptide.

[0035] In this specification, the cyclization of a peptide involves altering a portion of its amino acid. Peptides also include those containing a portion of the altered amino acid. An example of an alteration used for cyclization is the addition of a chloroacetyl group to an N-terminal amino acid, which then binds to a cysteine ​​residue in the peptide, resulting in cyclization. Peptides containing various (natural / non-natural) amino acids formed by the addition of a chloroacetyl group are also included in the peptide of this application.

[0036] A cyclic peptide is defined as a peptide in which two amino acids are bound together, forming a ring structure, either entirely or partially. Furthermore, this application also includes peptides with cross-linked amino acids, peptides that form cyclic structures through cycloaddition or macrocyclization reactions, and peptides with a lasso peptide structure. That is, in this application, a cyclic peptide only needs to have a portion forming a cyclic structure; it may also have a linear portion.

[0037] Generally, peptides exhibit poor metabolic stability in vivo and, due to their large size, have difficulty penetrating cell membranes. To address these issues, methods have been employed to cyclize peptides. Cycling peptides enhances protease resistance and metabolic stability, while limiting conformational changes, thus suggesting increased rigidity and improved membrane permeability and affinity for target proteins.

[0038] Regarding peptide cyclization, conventional methods can be used. While not limited to these, for example, peptides can be designed to contain two or more cysteine ​​residues, forming a cyclic structure via disulfide bonds after translation. Furthermore, following the method of Goto et al. (Y. Goto, et al. ACS Chem. Biol. 3 120-129 (2008)), peptides with a chloroacetyl group at the N-terminus can be synthesized using genetic code reprogramming techniques, and cyclization can also be achieved by pre-positioning cysteine ​​residues within the peptide. In this way, after translation, the thiol group automatically performs a nucleophilic attack on the chloroacetyl group, and the peptide cyclizes via a thioether bond. Alternatively, genetic code reprogramming techniques can be used to incorporate combinations of other amino acids that would otherwise bind to form a cyclic structure into the peptide for cyclization. Alternatively, a peptide with a cycloacetylamine at the N-terminus can be synthesized, L-2-aminoadipic acid residues can be incorporated into the peptide, and cyclization can be achieved by binding between these residues. Thus, any known cyclization method can be used without particular restriction.

[0039] Peptide length of peptide A The peptide length (number of amide bonds) of peptide A is not particularly limited, but the total amino acid residues (in the case where the substance bound to the peptide or the linker that binds the substance to the peptide contains amino acids, excluding such amino acids) are preferably 20 residues or less. Preferably, the number of amino acids is 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more; more preferably, the number of amino acids is 19 or less, 18 or less, 17 or less, 16 or less, or 15 or less.

[0040] Nucleic acid encoding peptide A This specification also describes the nucleic acid encoding peptide A (c-Met protein-binding peptide). In this specification, "nucleic acid" can be natural or non-natural, including but not limited to DNA, RNA and chimeras of the like.

[0041] connector The type and length of linkers used for peptide complexes are not particularly limited as long as the peptide complex can bind to c-Met proteins. Examples of linkers include amino acid linkers (peptide linkers), chemical linkers, fatty acid linkers, nucleic acid linkers, glycan linkers, etc., and complexes of chemical linkers and peptide linkers are also possible. Examples of chemical linkers include PEG (Polyethylene glycol) linkers. Furthermore, linkers can be fatty acid linkers induced by fatty acids and containing a divalent chemical moiety. Amino acid (peptide) linkers are linkers containing at least one arbitrary amino acid, such as glycine-rich peptides with the sequence [Gly-Gly-Gly-Gly-Ser]n (where n is 1, 2, 3, 4, 5, or 6) as described in U.S. Patent No. 7,271,149, and serine-rich peptide linkers as described in U.S. Patent No. 5,525,491. These linkers can be bound to c-Met protein-binding peptides using conventional methods or methods thereof. For example, the linker can be bound to the c-Met protein-binding peptide by binding the linker to the terminal Cys residue. Alternatively, the linker can be bound to an amino acid other than the C-terminus of the c-Met protein-binding peptide.

[0042] A preferred example of a linker is a PEG linker. A PEG linker is a linker containing polyethylene glycol (PEG) or a PEG derivative. A PEG linker may contain amino acids. A PEG linker preferably contains eight or more PEG molecular units. Specific examples of linkers are (1) linkers having a sequence described in any one of sequence identification numbers 35 to 41, or (2) linkers consisting of a sequence formed by substitution, deletion, addition, or insertion of one to three amino acids (the amino acids are not particularly limited as long as they can be used as linkers, but may include, for example, S, G, and K, and any one or more of these amino acids) in a sequence described in any one of sequence identification numbers 35 to 41.

[0043] Peptide A, the linker, and the peptide complex can be prepared by conventional methods (e.g., Japanese Patent No. 6426103 (Patent Document 2)) or by appropriately modifying conventional methods.

[0044] c-Met protein activator The aforementioned peptide complex promotes autophosphorylation through the polymerization of c-Met proteins, thereby exhibiting HGF-like functions. Therefore, the aforementioned peptide complex can be used as a c-Met agonist, or in pharmaceutical compositions containing a c-Met protein agonist.

[0045] Pharmaceutical Composition This specification also provides a pharmaceutical composition comprising the aforementioned peptide complex and a pharmaceutically permissible carrier. The pharmaceutical composition comprising a c-Met protein agonist is intended for the treatment or prevention of diseases selected from the group consisting of ischemic heart disease, acute hepatitis, fulminant hepatitis, cirrhosis, biliary atresia, fatty liver, acute renal failure, chronic renal failure, diabetic nephropathy, acute pneumonia, pulmonary fibrosis, vascular disease, myocardial infarction, dilated cardiomyopathy, skin ulcers, cerebral infarction, occlusive arteriosclerosis, gastric ulcers, and amyotrophic lateral sclerosis (ALS).

[0046] The pharmaceutical composition of the present invention comprises the peptide complex of the present invention as an active ingredient. Due to its HGF-like function, it is highly useful for promoting cell proliferation, promoting cell migration, inhibiting apoptosis, inducing morphogenesis, angiogenesis, and regeneration or protection of tissues or organs, and can be used as a treatment or preventative agent for such related diseases. Examples of such diseases include, but are not limited to, acute hepatitis, fulminant hepatitis, cirrhosis, biliary atresia, fatty liver, acute renal failure, chronic renal failure, diabetic nephropathy, acute pneumonia, pulmonary fibrosis, vascular disease, myocardial infarction, dilated cardiomyopathy, skin ulcers, cerebral infarction, occlusive arteriosclerosis, gastric ulcers, and amyotrophic lateral sclerosis (ALS).

[0047] The administration method of the above-mentioned pharmaceutical composition is not particularly limited and can be administered orally or non-orally. Examples of non-oral administration include intramuscular injection, intravenous injection, subcutaneous injection, transdermal administration, and transmucosal administration (via nose, mouth, eye, lung, vagina, or rectum). Peptides in pharmaceutical compositions, due to their easy metabolism and excretion, can be modified in various ways. For example, the addition of polyethylene glycol (PEG) or sugar chains to peptides can prolong their retention time in the blood and reduce their antigenicity. Furthermore, biodegradable polymers such as polylactic-glycolic acid (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared from unsaturated fatty acids, nanoparticles, and nanospheres can be used as sustained-release bases to encapsulate peptides. In transdermal administration, a weak current can be passed through the skin surface and allowed to penetrate the stratum corneum (iontophoresis).

[0048] Pharmaceutical compositions can use the active ingredient directly, or they can be formulated by adding pharmaceutically permissible carriers, excipients, additives, etc. Examples of dosage forms include liquids (e.g., injections), dispersants, suspensions, lozenges, pills, powders, sachets, powders, fine granules, capsules, syrups, tablets, inhalers, ointments, eye drops, nasal drops, ear drops, and pastes. Formulation can be carried out by using appropriate excipients, binders, disintegrants, lubricants, solubilizers, dissolution aids, colorants, flavoring and odor-correcting agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, antioxidants, etc., and by conventional methods.

[0049] Examples of pharmaceutically permissible ingredients for formulation include distilled water, saline solution, phosphate buffer, glucose, glycerol, ethanol, and other pharmaceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, anhydrous silicate, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyethylene polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tragacanth gum, casein, agar, polyethylene glycol, diglycerides, glycerol, propylene glycol, petroleum ether, paraffin wax, octyl dodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, human serum albumin, etc., but are not limited to these.

[0050] As absorption enhancers to improve the absorption of poorly absorbed drugs, surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponins can be used; bile salts such as glycocholic acid, deoxycholic acid, and taurine; chelating agents such as EDTA and salicylic acids; fatty acids such as hexanoic acid, decanoic acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acetylated collagen peptides, N-acetylated amino acids, cyclodextrins, chitosans, and nitric oxide donors can be used.

[0051] Pills or tablets can also be coated with sugar, gastric-soluble, or enteric-soluble substances. Injectable preparations may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, dissolving aids, preservatives, etc., can be added.

[0052] The pharmaceutical composition of the present invention may also be administered in combination with other drugs or treatments useful for the above-mentioned diseases.

[0053] The dosage of the pharmaceutical composition of the present invention administered to mammals (e.g., humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, sheep, etc.), especially humans, varies depending on the symptoms, the patient's age, sex, weight, susceptibility differences, administration method, administration interval, type of active ingredient, and type of formulation, and is not particularly limited. However, for example, it may be administered in a single dose or in multiple doses of 30 μg to 1000 mg, 100 μg to 500 mg, or 100 μg to 100 mg. In the case of injection administration, depending on the patient's weight, it may be administered in a single dose or in multiple doses of 1 μg / kg to 3000 μg / kg or 3 μg / kg to 1000 μg / kg.

[0054] Treatment This specification also provides treatment methods for the various diseases mentioned above, which include the steps of administering an effective amount of the peptide complex or the pharmaceutical composition to a subject (e.g., a mammal, a patient, etc.). Furthermore, this specification also describes the use of peptide A or the aforementioned peptide complex in the manufacture of the aforementioned pharmaceutical compositions, and methods for manufacturing the aforementioned pharmaceutical compositions using peptide A or the aforementioned peptide complex.

[0055] Culture medium additives, culture medium This specification also provides culture medium additives containing the aforementioned peptide complexes, and culture media containing peptide complexes. These culture medium additives are used for culturing cells or tissues derived from mammals. Examples of cells for culture are not limited to these, but include somatic cells, germ cells, and cells capable of differentiating into all tissues or cells constituting a living organism, i.e., pluripotent stem cells. Examples of pluripotent stem cells include, for example, embryonic stem cells (ES cells), sperm stem cells, pluripotent reproductive stem cells, embryonic germ cells, induced pluripotent stem cells (iPS cells), and cells cultured from fibroblasts or bone marrow stem cells. Furthermore, these can be fused cells with somatic cells, pluripotent stem cells induced / selected by stress or cellular stimulation, or pluripotent stem cells established by culturing early embryos created through nuclear transfer of somatic cell nuclei. Examples of tissues for culture include tissues differentiated from cells and tissues taken from the body. For example, this culture medium additive can be used to protect regenerated tissues or for organs used in organ transplantation. Cells or tissues are preferably derived from primates such as humans, monkeys, and chimpanzees, and even more preferably from humans.

[0056] The culture medium is not particularly limited as long as it is used for culturing cells or tissues, but it is preferred to use a culture medium supplemented with human HGF. It can be a serum medium, but it is preferred to be a serum-free medium or a low-serum medium. Culture medium additives can be in solution form or in dried solid form (e.g., solid, powder, etc.). In the case of a solution, it can be used directly as a culture medium, or it can be diluted with a solvent and the aforementioned additives added as needed. Examples of solvents used for dilution include water, buffer solutions, physiological saline, and various culture media for cell or tissue culture; these can be used alone or in combination of two or more. In cases where the culture medium additives are in the form of dried solids, for example, a culture medium prepared by dissolving solvents such as water, buffer solutions, physiological saline, and culture media used for various cell or tissue cultures, and adding the above-mentioned additives as needed, may also be used as a culture medium. In the culture medium for culturing cells or tissues, or in the culture medium for cells obtained therefrom, the content of the peptide complex of the present invention can be arbitrarily set by those skilled in the art, depending on the cells or tissues being cultured. For example, the final concentration relative to the total amount of the composition or the total amount of the culture medium can be about 0.01 to about 10,000 nmol / L, more preferably about 0.1 to about 1,000 nmol / L, more preferably about 0.5 to about 1,000 nmol / L, and even more preferably about 1 to about 100 nmol / L.

[0057] DDS vector The aforementioned peptide A possesses the property of binding to c-Met proteins. Therefore, peptide A, or a peptide complex containing peptide A, functions as a DDS carrier (drug delivery carrier) or drug delivery complex by binding to a known substance delivered to c-Met proteins, such as a drug. This specification also discloses such a DDS carrier and drug delivery complex. Conventional methods can be used to bind peptide A or the peptide complex to a drug.

[0058] [Example] Abbreviation (general) Angstrom (unit: Å) Bovine serum albumin is BSA; Dimethyl sulfoxide is DMSO; Dimethylformamide is DMF; N,N-Diisopropylethylamine is either DIPEA or DIEA; 3,6-Dioxa-1,8-octane-dithiol is DODT; Dulbecco's Modified Eagle Medium is DMEM; 50% effective concentration is EC50; Epidermal growth factor (EGF) is used to identify epidermal growth factor. 9-fluorenylmethyloxycarbonyl is Fmoc; Fetal bovine serum is FBS; N2,N6-bis(((9H-fluorene-9-yl)methoxy)carbonyl)-L-lysine is Fmoc-Lys(Fmoc)-OH; Gram (unit) is g; O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate is HATU; The HGF receptor is HGFR; The liquid chromatography-mass spectrometer is either LC-MS or LC / MS; Milliliters (mL) are units of measurement; molar concentration (M) is a unit of measurement; microliters (μL) are units of measurement. Millimolar concentration (unit: mM); Milligram (unit) is mg; Acetonitrile is MeCN; Minutes (unit) are measured in min; Millimeters (unit: mm) Nanometers (unit: nm) are measured in nanometers. The basal medium used for renal epithelial cells was REBM; The number of rotations per minute (in rpm) is the unit. Trifluoroacetic acid is TFA; Triisopropylsilane is TIS.

[0059] Abbreviation (non-natural amino acids) MeF N-methyl-L-phenylalanine MeA N-methyl-L-alanine Ahp (S)-2-aminoheptanoic acid Nle L-leucine Hty (S)-2-amino-4-(4-hydroxyphenyl)butyric acid Abu (S)-2-aminobutyric acid MeC N-methyl-L-cysteine PEG4c 1-Amino-3,6,9,12-Tetraoxaspiro-15-pentadecanoic acid PEG8c 1-amino-3,6,9,12-tetraoxapentadecane-15-oic acid PEG12c 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanononatriacontane-39-oic acid cPEG1c 3,3'-Oxy dipropionic acid cPEG9c 4,7,10,13,16,19,22,25,28-nonaoxahentriacontanedioic acid cPEG17c 4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52-heptadecoxapentapentacontanedioic acid OCOPEG13OCO 3,6,9,12,15,18,21,24,27,30,33-undecaoxapentacontane-1,35-diyl bis(bicarbonate))

[0060] [Example 1] Chemical synthesis All raw materials, structural units, reagents, acids, bases, solid resins, and solvents used in the chemical synthesis in the following examples can be commercially available or synthesized by those skilled in the art using organic chemical methods. Furthermore, amino acids containing protecting groups can be commercially available unless otherwise specified. The elongation of peptide chains in the solid-phase resin was carried out using the resins described in the various examples as the initial raw materials, and under commonly used peptide coupling reaction conditions and Fmoc removal reaction conditions. The reaction was performed using an automated peptide synthesizer, namely Biotage's Siro I, in accordance with the manufacturer's instructions. A series of common amino acids used are listed below, with side-chain protecting groups shown in parentheses. Fmoc-Trp(Boc)-OH;Fmoc-Thr(tBu)-OH;Fmoc-N-Me-Gly-OH;Fmoc-Asp(OtBu)-OH;Fmoc-N-Me-Phe-OH;Fmoc-Ala-OH;Fmoc-N-Me-Ala-OH;Fmoc-His(Trt )-OH;Fmoc-Tyr(tBu)-OH;Fmoc-Val-OH;Fmoc-HydPro(tBu)-OH;Fmoc-Cys(Trt)-OH;Fmoc-Lys(Mtt)-OH;Fmoc-Ser(tBu)-OH;Fmoc-N-Me-Ser(tBu)-OH. The purification method for the obtained crude and refined peptides was performed using a Waters AutoPurification System-SQD2 single quadruple mass spectrometer with reversed-phase HPLC, monitoring the m / z ions from the target substance while performing dissolution. It was confirmed that the mass spectra obtained in ESI-positive scanning mode and the mass spectra containing the multivalent ions calculated from the molecular formula of the target substance were consistent within the error range of the mass analyzer used. Furthermore, the purification conditions including the column used are shown in the examples. The structure of chemically synthesized peptides was determined by confirming their molecular weight using ESI-MS(+) mass spectrometry. This molecular weight was calculated taking into account the amino acids used in the target sequence and, if necessary, the structural units used. Furthermore, "ESI-MS(+)" indicates electrolytic free mass spectrometry performed in positive ion mode. The detected mass is reported in m / z units. Additionally, compounds with molecular weights greater than approximately 1000 were detected at high frequency as divalent or trivalent ions.

[0061] [Example 2] Recognition of peptides with c-Met activating activity The c-Met agonist peptides were identified using Recombinant Human HGF R / c-MET Fc Chimera His-tag Protein (R&D Systems) as the target, and by excerpts of the methods described in International Publications WO2014 / 119600, WO2012 / 033154, or WO2007 / 066627. The peptides were chemically synthesized to confirm whether they actually possess c-Met agonist activity. Furthermore, they were synthesized as homodimers (peptide complexes) formed by linkers combining two peptides. The amino acid sequences of the peptide moieties in the synthesized peptide complexes are shown in Table 1, and the linker sequences are shown in Table 2. The synthesized peptide complexes (combinations of peptides and linkers) are shown in Tables 3 and 4.

[0062] Table 1: Amino acid sequences of peptides [Table 1-1]

[0063] [Table 1-2]

[0064] Table 2: Connector Subsequences [Table 2]

[0065] Table 3: Peptide Complexes [Table 3]

[0066] Table 4: Peptide Complexes [Table 4]

[0067] [Example 3] Chemical synthesis of peptides with c-Met activating activity [Example 3-1] Regarding the peptide complexes described in Table 3, they were synthesized in the same manner as the peptide complexes described below, provided there are no other synthetic examples. Furthermore, in Table 3, ESI-MS (m / z) represents ESI-MS (+) observations, and [M+XH]X+ represents the value of X when the proton addition number in this case is expressed as (M+XH)X+. Synthesis of the GFs_c-Met_0012_7 complex (GFs_c-Met_0012_7-PEG12c-K dimer: peptide complex No. 8 in Table 3)

[0068] [Chemical Formula 1]

[0069] The target peptide was synthesized using NovaPEG Rink Amide resin (Merck, 0.53 mmol / g, 0.005 g) following the general method described above, starting with the removal of Fmoc groups. A Biotage Siro I solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. The synthesis proceeded by introducing Fmoc-Lys(Fmoc)-OH into the solid-phase resin, removing both Fmoc groups, and simultaneously extending from the two amino groups on Lys. The introduction of each residue was carried out using Fmoc-AA / HATU / DIPEA (8.4 equivalents / 7.8 equivalents / 16.8 equivalents) relative to 1 equivalent of resin. Furthermore, the Fmoc removal system was reacted with a 20% piperidine DMF solution at 25°C for 5 minutes, the solution was removed, and then a 20% piperidine DMF solution was added again, and the reaction was carried out for another 15 minutes. The introduction of chloroacetyl groups was carried out as follows: For the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, after removing the Fmoc groups of the α-amino groups using the aforementioned method, a DMF solution of 0.3 M chloroacetic acid (8.4 equivalents), a DMF solution of 0.28 M HATU (7.8 equivalents), and a DMF solution of 1.05 M DIPEA (16.8 equivalents) were added to the solid-phase resin, and the mixture was shaken twice for 30 minutes at 25°C. Deprotection of the side chains and removal from the solid-phase resin were performed using the following method: First, the resin obtained after the chloroacetyl group introduction step was washed five times with DMF and three times with dichloromethane, and then dried under reduced pressure. Next, a reaction mixture-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin, and the mixture was shaken at 25°C for 60 minutes. The reaction solution was recovered by filtration from the frit. The remaining solid resin in the reaction vessel was shaken again with the ablation mixture to recover the solution components from the frit, and then mixed with the aforementioned filtrate. Adding this filtrate to excess diethyl ether / hexane (1 / 1) mixed solvent cooled to 0°C produced a turbid precipitate. This mixture was centrifuged (8500 rpm, 0°C, 30 seconds) to decant the solution. The resulting solid was washed again with a small amount of diethyl ether / hexane cooled to 0°C and dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. The peptide cyclization reaction was performed by dissolving the peptide in DMSO at a final concentration of 2.5 μm based on the molar number of the solid resin, adding 10 equivalents of triethylamine, and shaking at 25°C for 16 hours. The resulting reaction solution was concentrated under reduced pressure using EZ-2 Elite. The resulting mixture was subjected to solid-phase extraction using an ASPEC (registered trademark) C18 filter cartridge from Gilson. The extract was then concentrated under reduced pressure using an EZ-2 Elite filter. When analyzed by LC-MS, the mass spectrum of the target analyte will be observed in one of the main peaks. Analytical conditions: Hold time = 1.73 min; Column: Kinetex (registered trademark) EVO C18 1.7 μm 2.1 x 50 mm, 100 Å; Mobile phase: A = 0.025% TFA in H₂O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 5-95% over 2.10 min, followed by 95-95% over 0.75 min; Flow rate: 0.6 mL / min ESI-MS (+) observed value m / z = 1725.0(M+3H) 3 + theoretical value m / z = 5170.91

[0070] [Example 3-2] Regarding the peptide complexes described in Table 4, they were synthesized in the same manner as the peptide complexes described below, provided there are no other synthetic examples. Furthermore, in Table 4, ESI-MS (m / z) represents ESI-MS (+) observations, and [M+XH]X+ represents the value of X when the proton addition number in this case is expressed as (M+XH)X+. Synthesis of the GFs_c-Met-0012 complex (GFs_c-Met-0012-PEG12 dimer; peptide complex No. 17 in Table 4)

[0071] [Chemical Formula 2]

[0072] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g, 0.11 g) following the general method described above, starting with the removal of Fmoc groups. A Biotage Siro I solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. The Fmoc-Lys(Fmoc)-OH group was introduced into the solid-phase resin, removing both Fmoc groups and simultaneously extending from the two amino groups of Lys. The introduction of each residue was carried out using Fmoc-AA / HATU / DIPEA (8.4 equivalents / 8 equivalents / 16 equivalents) relative to 1 equivalent of the resin. Furthermore, the Fmoc removal system was reacted with a DMF solution containing 0.1 M HOBt and 5% piperidine at 25°C for 5 minutes, the solution was removed, and the system was reacted again with a DMF solution containing 0.1 M HOBt and 5% piperidine for 15 minutes. The introduction of chloroacetyl groups was carried out as follows: For the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, after removing the Fmoc groups of the α-amino groups using the aforementioned method, a 0.5M DMF solution (10 equivalents) of chloroacetic acid, a 0.49M DMF solution of HATU (9.8 equivalents), and a 0.5M DMF solution of DIPEA (10 equivalents) were added to the solid-phase resin, and the mixture was shaken at room temperature for 30 minutes. Deprotection of the side chains and removal from the solid-phase resin were performed using the following method: First, the resin obtained after the chloroacetyl group introduction step was washed 5 times with DMF and 3 times with dichloromethane, and then dried under reduced pressure. Next, a reaction mixture-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin, and the mixture was shaken at 25°C for 60 minutes. The reaction solution was recovered by filtration from the glass buffer. The remaining solid resin in the reaction vessel was shaken again with the ablation mixture to recover the solution components from the glass frit and mixed with the aforementioned filtrate. Adding this filtrate to excess diethyl ether / hexane (1 / 1) mixed solvent cooled to 0°C produced a turbid precipitate. This mixture was centrifuged (9000 rpm, 2 min) to decant the solution. The resulting solid was washed again with a small amount of diethyl ether / hexane cooled to 0°C and dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. The peptide cyclization reaction was performed by dissolving the peptide in DMSO / acetonitrile / water (18 / 1 / 1) at a final concentration of 1 Mm based on the mole number of the solid resin, followed by the addition of 20 equivalents of triethylamine and shaking at 25°C for 15 hours. The resulting reaction solution was concentrated under reduced pressure using EZ-2 Elite. The resulting mixture was purified under the following conditions (column: Waters XSelect (registered trademark) C18 19 x 150 mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (%B conc): 13-38% over 3 minutes, 38-43% over 8 minutes, and 43-60% over 1 minute; flow rate: 17 mL / min). The purity of the target analyte was calculated from the area ratio of LC / MS (UV wavelength 225 nm) chromatograms under the following analytical conditions, and was 59.97%.

[0073] Analytical conditions: Hold time = 5.75 min; Column: Kinetex (registered trademark) EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H₂O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 7.15 min 20-60%, then 0.30 min 60-95%, then 1.55 min 95-95%; Flow rate: 0.5 mL / min ESI-MS (+) observed value m / z = 1300.6(M+4H) / 4 + theoretical value m / z = 5196.56

[0074] [Example 4] Measurement of the c-Met activating activity of the peptide complex of the present invention by phosphoric acid-c-Met AlphaLISA assay The phosphorylation of c-Met was verified to evaluate the activation ability (c-Met activating activity) of the peptide complex of the present invention. A431 cells, which are human cells, were cultured in DMEM containing 10% FBS (Gibco), high glucose, GlutaMAX (trademark) supplement, and pyruvate (Thermo Fisher Scientific). After cell exfoliation using Accutase (Innovative Cell Technologies), the cells were seeded in 96-well plates at a density of 10,000 cells per well and cultured overnight. The following day, to induce starvation, the culture solution was replaced with DMEM containing 0.1% BSA (Sigma-Aldrich), high glucose, GlutaMAX (trademark) supplement, and pyruvate (Thermo Fisher Scientific), and cultured for another night. Subsequently, the peptide complex synthesized in Example 3 or the Recombinant Human HGF Protein (R&D Systems) as a control was added, and after stimulation for 15 minutes, cells were lysed with the lysis buffer (Lysis Buffer) included with the AlphaLISA SureFire Ultra TMMPhospho-c-Met (Tyr1234 / 1235) kit (PerkinElmer). Analysis was performed according to the kit's protocol, and signal detection was performed using a SpectraMax Paradigm multimode microplate reader (Molecular Devices, LLC.). Signals obtained from GraphPad Prism (MDF Co., Ltd.) analysis were analyzed, with the maximum value of the HGF-induced signal set as 100%. Activators exhibiting more than 50% activation were recorded as 2, and those exhibiting less than 50% activation were recorded as 1. The results are shown in Table 3. Furthermore, although activity measurements were performed using monomeric peptides, no c-Met activating activity was observed. Similarly, the C50 value was calculated using GraphPad Prism as the concentration of the peptide representing 50% activity of the HGF-induced signal. The results are shown in Table 4. Furthermore, in this analysis, the EC50 value of HGF as a control was 0.66 nM. Furthermore, regarding the complex of the peptide (sequence identification number 34) and the linker (sequence identification number 37) (GFs_c-Met-00014336-PEG13 dimer; complex No. 49 in Table 4), the graph of c-Met activating activity is shown in Figure 1. The EC50 of this peptide complex is 0.72 nM, exhibiting almost the same c-Met activating activity as HGF. These results demonstrate that the peptide complex of the present invention does indeed possess c-Met activating activity.

[0075] [Example 5] HUVEC cell proliferation evaluation To evaluate the bioactivity of the peptide complex of the present invention, cell proliferation-inducing activity was verified. Normal human umbilical vein endothelial cells (HUVECs) were cultured using HuMedia-EG2 (Kurashiki Bosho Co., Ltd.). Cells were dissected using Accutase (Innovative Cell Technologies) and then seeded in 96-well plates at a density of 10,000 cells per well using Medium without serum or growth factors (Cell Systems) containing 5% FBS (MBL). The cells were cultured overnight. The following day, a complex of the peptide (Sequence Identification No. 34) and linker (Sequence Identification No. 37) synthesized in Example 2 (GFs_c-Met-00014336-PEG13 dimer; peptide complex No. 49 in Table 4) or Recombinant Human HGF Protein (R&D Systems) as a control was added, and the cells were cultured for another 2 days. Afterward, the culture medium was removed, and cell counts were quantified using CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay reagent (Promega) according to the accompanying protocol. The testing system uses the SpectraMax (registered trademark) Paradigm (registered trademark) micro-disc analyzer (Molecular Devices). The signal values ​​were analyzed using GraphPad Prism, and the EC50 was calculated. The resulting activity graph is shown in Figure 2. The results showed that the EC50 of HGF was 0.37 nM, and the EC50 of the peptide complex was 0.55 nM. The peptide complex of this invention exhibited almost the same c-Met activating activity as HGF. The results show that the peptide complex of the present invention has the activity of inducing human cell proliferation.

[0076] [Example 6] Synthesize the following peptide complex. [Example 6-1] Synthesis of cyclic peptides having the amino acid sequence described by sequence identification number 34 in Table 1

[0077] [Chemical Formula 3]

[0078] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.65 mmol / g, 0.31 g) following the general method described above, starting with the removal of the Fmoc group. A Biotage Siro I solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. The introduction system for each residue was reacted with Fmoc-AA / HATU / DIPEA (3.2 equivalents / 3 equivalents / 6.3 equivalents) relative to 1 equivalent of the resin. Furthermore, the Fmoc removal system was reacted with a 20% piperidine DMF solution at 25°C for 5 minutes, the solution was removed, and the system was reacted again with a 20% piperidine DMF solution for 15 minutes. The introduction of chloroacetyl groups was carried out as follows: For the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, after removing the Fmoc groups of the α-amino groups using the aforementioned method, a 0.5M DMF solution of chloroacetic acid (5 equivalents), a 0.49M DMF solution of HATU (4.9 equivalents), and a 0.5M DIPEA DMF solution (5 equivalents) were added to the solid-phase resin, and the mixture was shaken at room temperature for 30 minutes. Deprotection of the side chains and removal from the solid-phase resin were performed using the following method: First, the resin obtained after the chloroacetyl group introduction step was washed 5 times with DMF and 3 times with dichloromethane, and then dried under reduced pressure. Next, a reaction mixture-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin, and the mixture was shaken at 25°C for 60 minutes. The reaction solution was recovered by filtration from the glass buffer. The remaining solid resin in the reaction vessel and the ablation mixture were shaken again to recover the solution components from the glass frit and mixed with the aforementioned filtrate. Adding this filtrate to excess diethyl ether / hexane (1 / 1) mixed solvent cooled to 0°C produced a turbid precipitate. This mixture was centrifuged (9000 rpm, 2 minutes) to decant the solution. The resulting solid was washed again with a small amount of diethyl ether / hexane cooled to 0°C and dried under reduced pressure. The resulting solid was used for subsequent cyclization reactions. The peptide cyclization reaction was performed by dissolving the peptide at a final concentration of 2.5 mM based on the molar number of the solid resin in DMSO / acetonitrile / water (18 / 1 / 1), adding 10 equivalents of triethylamine, and shaking at 25°C for 4 hours. The resulting reaction solution was concentrated under reduced pressure using EZ-2 Elite. The crude product was purified under the following conditions (column: Waters Xbridge (registered trademark) C18 5μm 50 x 150mm; mobile phase: A=0.1% TFA in H2O, B=0.1% TFA in MeCN; temperature: 40 ℃; gradient (%B conc): 3 min 5-30%, then 8 min 30-35%, then 1 min 35-60%; flow rate: 120 mL / min). The purity of the target analyte was calculated from the area ratio of LC / MS (UV wavelength 225 nm) chromatograms under the following analytical conditions, and was 96.90%. Analytical conditions: Hold time = 3.84 min; Column: Kinetex (registered trademark) EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H₂O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (%B conc): 7.15 min 20-60%, then 0.30 min 60-95%, then 1.55 min 95-95%; Flow rate: 0.5 mL / min ESI-MS (+) observation m / z = 1027.7(M+2H) 2 + The resulting cyclic peptides were used to synthesize peptide complexes.

[0079] [Example 6-2] Synthesis of GFs_c-Met-00014336 (GFs_c-Met-00014336-PEG13 dimer; peptide complex No. 49 in Table 4)

[0080] [Chemical Formula 4]

[0081] GFs_c-Met-00014234 (8.0 mg, 3.51 μmol) was dissolved in DMF (0.2 mL), and DMF solution of DIPEA (2.3 μL, 13 μmol) (23 μL) and DMF solution of bis(2,5-dioxopyrrolidine-1-yl)(3,6,9,12,15,18,21,24,27,30,33-undecoxapentadecano-1,35-diyl) dicarbonate (1.38 mg, 1.67 μmol) (14 μL) were added. The mixture was stirred at 25 °C for 16 hours. The resulting mixture was purified under the following conditions (column: Waters XSelect (registered trademark) C18 19 x 150 mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (%B conc): 13-38% over 3 minutes, 38-43% over 8 minutes, and 43-60% over 1 minute; flow rate: 17 mL / min). The purity of the target analyte was calculated from the area ratio of LC / MS (UV wavelength 225 nm) chromatograms under the following analytical conditions, and was 96.42%. Analytical conditions: Hold time = 5.07 min; Column: Kinetex (registered trademark) EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H₂O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (%B conc): 7.15 min 20-60%, then 0.30 min 60-95%, then 1.55 min 95-95%; Flow rate: 0.5 mL / min ESI-MS (+) observed value m / z = 1177.2(M+4H) 4 + theoretical value m / z = 4703.25

[0082] [Example 6-3] Synthesis of GFs_c-Met-00014305 (GFs_c-Met-00014305-PEG17 dimer; peptide complex No. 50 in Table 4)

[0083] [Chemical Formula 5]

[0084] The cyclic peptide (GFs_c-Met-00014234) synthesized in Example 6-1 (8.0 mg, 3.51 μmol) was dissolved in DMF (0.25 mL), and a DMF solution of triethylamine (2.0 μL, 14 mmol) (20 μL) and a DMF solution of bis(2,5-dioxopyrrolidone-1-yl)4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52-heptadecanoic acid methyl ester (1.75 mg, 1.65 μmol) (18 μL) were added. The mixture was stirred at 25 °C for 16 hours. The resulting mixture was purified under the following conditions (column: Waters XSelect (registered trademark) C18 5 μm 19 x 150 mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40 ℃; gradient (%B conc): 12-37% over 3 minutes, 37-42% over 8 minutes, and 42-60% over 1 minute; flow rate: 17 mL / min). The purity of the target analyte was calculated from the area ratio of LC / MS (UV wavelength 225 nm) chromatograms under the following analytical conditions, and was 94.19%. Analytical conditions: Hold time = 5.92 min; Column: Kinetex (registered trademark) EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H₂O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (%B conc): 7.15 min 20-60%, then 0.30 min 60-95%, then 1.55 min 95-95%; Flow rate: 0.5 mL / min ESI-MS (+) observed value m / z = 1235.3(M+4H) 4 + theoretical value m / z = 4935.41

[0085] [Example 6-4] Synthesis of GFs_c-Met-00014318 (GFs_c-Met-00014318-PEG9 dimer; peptide complex No. 51 in Table 4)

[0086] [Chemical Formula 6]

[0087] GFs_c-Met-00014234 (8.0 mg, 3.51 μmol) was dissolved in DMF (0.25 mL), and a DMF solution of triethylamine (2.0 μL, 14 μmol) (20 μL) and a DMF solution of bis(2,5-dioxopyrrolidone-1-yl)4,7,10,13,16,19,22,25,28-nonoxatricarboxylic acid methyl ester (1.17 mg, 1.65 μmol) (12 μL) were added. The mixture was stirred at 25 °C for 16 hours. The resulting mixture was purified under the following conditions (column: Waters XSelect (registered trademark) C18 19 x 150 mm; mobile phase: A = 0.1% TFA H₂O, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (%B conc): 11-36% over 3 minutes, 36-41% over 8 minutes, and 41-60% over 1 minute; flow rate: 17 mL / min). The purity of the target analyte was calculated from the area ratio of LC / MS (UV wavelength 225 nm) chromatograms under the following analytical conditions, and was 94.56%. Analytical conditions: Hold time = 5.03 min; Column: Kinetex (registered trademark) EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H₂O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (%B conc): 7.15 min 20-60%, then 0.30 min 60-95%, then 1.55 min 95-95%; Flow rate: 0.5 mL / min ESI-MS (+) observed value m / z = 1147.1(M+4H) 4 + theoretical value m / z = 4582.2

[0088] [Example 7] Tube formation evaluation Human renal proximal tubular epithelial cells (RPTEC) were cultured in proliferation medium (Renal Epithelial Cell Basal Medium (REBM) + 0.5% FBS + 2.4 mM L-Alanyl-L-Glutamine + 10 nM Triiodothyronine + 10 ng / mL rh EGF + 100 ng / mL Hydrocortisone Hemisuccinate + 5 μg / mL rh Insulin + 1 μm Epinephrine + 5 μg / mL Transferrin) (ATCC). Cells were dissected using Trypsin-EDTA and then resuspended in analytical medium (REBM + 0.5% FBS + 2.4 mM L-Alanyl-L-Glutamine + 10 nM Triiodothyronine + 100 ng / mL Hydrocortisone Hemisuccinate + 1 μm Epinephrine + 5 μg / mL Transferrin + 1.8 mM CaCl2). RPTEC was resuspended on ice in a solution prepared according to the instructions using Cellmatrix (registered trademark) Type IA (Nitta Gelatin Co., Ltd.), concentrated culture medium (Nitta Gelatin Co., Ltd.), and reconstruction buffer (Nitta Gelatin Co., Ltd.), to a density of 200,000 cells per well. 300 μL was injected into each well of a 48-well plate. The suspension was then incubated in a CO2 incubator (37°C, 5% CO2) for 60 minutes to allow gelation. 2 nM HGF, 1.6 nM EGF, or a complex of the peptide synthesized in Example 2 (Sequence Identification No. 34) and the linker (Sequence Identification No. 37) (GFs_c-Met-00014336-PEG13 dimer; peptide complex No. 49 in Table 4) was added to the analytical medium at concentrations of 0.08, 0.4, 2, and 10 nM, and 400 μL was added to each well of a 48-well plate containing the gelled suspension as described above. The plate was incubated in a CO2 incubator (37°C, 5% CO2), with the medium changed every 3 days. The presence of lumens was observed and evaluated using a phase-contrast microscope after 3 days and 8 days of incubation. The results are shown in Figures 3 and 4. Figure 3 shows the results after 3 days: a) MOCK (no additive), b) with 1.6 nM EGF, c) with 2 nM HGF, and d) observations of wells with 2 nM peptide complex (peptide complex No. 49 in Table 4). Figure 4 shows the results after 8 days: a) MOCK (no additive), b) with 2 nM HGF, c) with 0.08 nM, d) with 0.4 nM, e) with 2 nM, and f) observations of wells with a complex (GFs_c-Met-00014336-PEG13 dimer; peptide complex No. 49 in Table 4) of the peptide (sequence identification number 34) and linker (sequence identification number 37) synthesized in Example 2 at a concentration of 10 nM. These results show that the peptide complex of the present invention promotes lumen formation in the same way as HGF. Furthermore, Figure 4f shows that it promotes not only lumen formation but also branching. Since lumen formation requires multiple functions such as cell proliferation, migration, and collagen degradation, the peptide complex of the present invention demonstrates that it possesses the same physiological activity as HGF.

[0089] [Example 8] Human Phospho-RTK Array Analysis A431 cells were cultured in DMEM containing 10% FBS (Gibco), high glucose, GlutaMAX (trademark) supplement, and pyruvate (Thermo Fisher Scientific). Cells were seeded using Accutase (trademark) (Innovative Cell Technologies) at a density of 1,000,000 cells per well in 6-well plates and cultured overnight. The following day, to starve the cells, the medium was replaced with analytical medium (0.1% BSA (Sigma), DMEM, high glucose, GlutaMAX (trademark) supplement, pyruvate (Thermo Fisher Scientific)) and cultured for another night. Then, 7.8 nM of Recombinant Human HGF Protein (R&D Systems) or 2 nM of the peptide (Sequence Identification No. 34) and linker (Sequence Identification No. 36) synthesized in Example 2 (GFs_c-Met-00014336-PEG9 dimer; peptide complex No. 51 in Table 4) was added, and after stimulation for 10 minutes, the cells were lysed with the lysis buffer provided with the Proteome Profiler Human Phospho-RTK Array Kit (R&D Systems). The procedure was then followed according to the kit protocol, and the chemiluminescence signal of the WesternSure PREMIUM chemiluminescent matrix kit (LI-COR) was detected using C-DiGit (Scrum). The results are shown in Figure 5. Furthermore, in Figure 5(2), a represents the result without addition, b represents the result with HGF addition, and c represents the result with peptide complex addition. In both the addition of peptide complex and HGF, only HGF R was specifically phosphorylated. Therefore, it is shown that the peptide complex of the present invention has the same specific phosphorylation pattern as HGF.

[0090] [Industrial Utilization] This invention can be applied to the pharmaceutical industry, biotechnology industry, etc.

[0091] none [Sequence List] 21-068P_ST25.txt

[0092] Sequence List <![CDATA[ <110> PeptiDream INC. (Japan) <![CDATA[ <120> c-Met protein-binding peptide complex <![CDATA[ <130> 21-068P]]> <![CDATA[ <160> 40 <![CDATA[ <170> PatentIn version 3.5]]> <![CDATA[ <210> 1]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> misc_feature]]> <![CDATA[ <222> (1)..(3)]]> <![CDATA[ <223> Xaa can be any naturally occurring amino acid. <![CDATA[ <220> ]]> <![CDATA[ <221> misc_feature]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Xaa can be any naturally occurring amino acid. <![CDATA[ <220> ]]> <![CDATA[ <221> misc_feature]]> <![CDATA[ <222> (8)..(8)]]> <![CDATA[ <223> Xaa can be any naturally occurring amino acid. <![CDATA[ <220> ]]> <![CDATA[ <221> misc_feature]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa can be any naturally occurring amino acid. <![CDATA[ <220> ]]> <![CDATA[ <221> misc_feature]]> <![CDATA[ <222> (14)..(14)]]> <![CDATA[ <223> Xaa can be any naturally occurring amino acid. <![CDATA[ <400> 1]]> Xaa Xaa Xaa Val Ser Xaa Asp Xaa Asp Xaa Pro Arg Trp Xaa Cys 1 5 10 15 <![CDATA[ <210> 2]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 2]]> Phe Val Tyr Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 3]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 3]]> Ala Val Tyr Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 4]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 4]]> Phe Val Tyr Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 5]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 5]]> Ala Val Tyr Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 6]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 6]]> Phe Thr Tyr Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 7]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 7]]> Phe Trp Tyr Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 8]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 8]]> Phe Val Asp Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 9]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 9]]> Phe Val Ala Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 10]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 10]]> Phe Val Tyr Val Ser Phe Asp Val Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 11]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 11]]> Phe Val Tyr Val Ser Phe Asp Pro Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 12]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Nle]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 12]]> Phe Val Tyr Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 13]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Hty]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 13]]> Phe Val Tyr Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 14]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 14]]> Phe Val Tyr Val Ser Phe Asp Asp Asp Ser Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 15]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 15]]> Phe Val Tyr Val Ser Phe Asp Asp Asp Xaa Pro Ser Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 16]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (14)..(14)]]> <![CDATA[ <223> Xaa is Abu]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 16]]> Phe Val Tyr Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp Xaa Cys 1 5 10 15 <![CDATA[ <210> 17]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 17]]> Phe Val Tyr Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp Ala Cys 1 5 10 15 <![CDATA[ <210> 18]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 18]]> Phe Val Tyr Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp Asp Cys 1 5 10 15 <![CDATA[ <210> 19]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 19]]> Phe Val Tyr Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp His Cys 1 5 10 15 <![CDATA[ <210> 20]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 20]]> Phe Val Tyr Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp Gln Cys 1 5 10 15 <![CDATA[ <210> 21]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 21]]> Phe Val Tyr Val Ser Phe Asp Asp Asp Xaa Pro Arg Trp Val Cys 1 5 10 15 <![CDATA[ <210> 22]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 22]]> Phe Glu Ala Val Ser Phe Asp Ser Asp Xaa Pro Arg Trp Ala Cys 1 5 10 15 <![CDATA[ <210> 23]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 23]]> Phe Glu Ala Val Ser Phe Asp Pro Asp Xaa Pro Arg Trp Ala Cys 1 5 10 15 <![CDATA[ <210> 24]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 24]]> Phe Glu Ala Val Ser Phe Asp Pro Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 25]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 25]]> Phe Gln Ala Val Ser Phe Asp Ser Asp Xaa Pro Arg Trp Ala Cys 1 5 10 15 <![CDATA[ <210> 26]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 26]]> Phe Gln Ala Val Ser Phe Asp Pro Asp Xaa Pro Arg Trp Ala Cys 1 5 10 15 <![CDATA[ <210> 27]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 27]]> Phe Gln Ala Val Ser Phe Asp Pro Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 28]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 28]]> Phe Thr Arg Val Ser Phe Asp Ser Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 29]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 29]]> Phe Thr Ala Val Ser Leu Asp Ser Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 30]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 30]]> Phe Thr Ala Val Ser Phe Asp Ser Asp Arg Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 31]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 31]]> Phe Thr Ala Val Ser Phe Asp Ser Asp Xaa Pro Arg Trp Ala Cys 1 5 10 15 <![CDATA[ <210> 32]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 32]]> Phe Thr Ala Val Ser Phe Asp Pro Asp Xaa Pro Arg Trp Ala Cys 1 5 10 15 <![CDATA[ <210> 33]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 33]]> Phe Thr Ala Val Ser Phe Asp Pro Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 34]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (6)..(6)]]> <![CDATA[ <223> Methylation <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (10)..(10)]]> <![CDATA[ <223> Xaa is Ahp]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MOD_RES]]> <![CDATA[ <222> (15)..(15)]]> <![CDATA[ <223> Methylation <![CDATA[ <400> 34]]> Phe Thr Ala Val Ser Phe Asp Glu Asp Xaa Pro Arg Trp Ser Cys 1 5 10 15 <![CDATA[ <210> 35]]> <![CDATA[ <211> 3]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Xaa is PEG12c]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (3)..(3)]]> <![CDATA[ <223> Xaa is PEG12c]]> <![CDATA[ <400> 35]]> Xaa Lys Xaa 1 <![CDATA[ <210> 36]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (3)..(3)]]> <![CDATA[ <223> Xaa is cPEG9c]]> <![CDATA[ <400> 36]]> Gly Lys Xaa Lys Gly 1 5 <![CDATA[ <210> 37]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (3)..(3)]]> <![CDATA[ <223> Xaa is OCOPEG13OCO]]> <![CDATA[ <400> 37]]> Gly Lys Xaa Lys Gly 1 5 <![CDATA[ <210> 38]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (3)..(3)]]> <![CDATA[ <223> Xaa is cPEG17c]]> <![CDATA[ <400> 38]]> Gly Lys Xaa Lys Gly 1 5 <![CDATA[ <210> 39]]> <![CDATA[ <211> 3]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Xaa is PEG4c. <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (3)..(3)]]> <![CDATA[ <223> Xaa is PEG4c]]> <![CDATA[ <400> 39]]> Xaa Lys Xaa 1 <![CDATA[ <210> 40]]> <![CDATA[ <211> 3]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Xaa is PEG8c]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (3)..(3)]]> <![CDATA[ <223> Xaa is PEG8c]]> <![CDATA[ <400> 40]]> Xaa Lys Xaa 1 <![CDATA[ <210> 41]]> <![CDATA[ <211> 3]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polypeptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (3)..(3)]]> <![CDATA[ <223> Xaa is cPEG1c]]> <![CDATA[ <400> 41]]> Gly Lys Xaa Lys Gly 1

[0093]

Claims

1. A peptide complex comprising peptide A that binds to a c-Met protein, wherein, Peptide A is a peptide composed of the amino acid sequence described in X1-X2-X3-V-S-X4-D-X5-D-X6-P-R-W-X7-MeC (sequence identification number 1), where X1 is an N-methylated A or an N-methylated F, X2 is V, T, E, Q or W, X3 is A, R, Y or D, X4 is a methylated F or a methylated L, X5 is D, E, S, P or V, X6 is (S)-2-aminoheptanoic acid (Ahp), R, L-leucine (Nle), (S)-2,7-diaminoheptanoic acid (Hty) or S, and X7 is S, A, L-α-aminobutyric acid (Abu), D, Q or V.

2. The peptide complex of claim 1, wherein, Peptide A is a peptide composed of the amino acid sequence described in MeF-T-A-V-S-MeF-D-E-D-Ahp-P-R-W-S-MeC (Sequence Identification Number 34), or, peptide A is a variant of the peptide composed of the amino acid sequence described in Sequence Identification Number 34, wherein in the amino acid sequence described in Sequence Identification Number 34, the first amino acid is either N-methylated A or N-methylated F; the second amino acid is V, E, Q, or W; the third amino acid is R, Y, or D; the sixth amino acid is either methylated F or methylated L; the eighth amino acid is D, S, P, or V; and the tenth amino acid is R, L-leucine (Nle), (S)-2,7-diaminoheptanoic acid (Hty), or S. The fourteenth amino acid is A, L-α-aminobutyric acid (Abu), D, Q, or V.

3. The peptide complex of claim 1, wherein, The first and second peptides can be the same or different, and they are peptide A.

4. The peptide complex as claimed in claim 3, wherein, Peptide A is a cyclic peptide.

5. The peptide complex of claim 4, wherein, It has c-Met protein-promoting activity.

6. The peptide complex of claim 5, wherein, The first and second peptides are the same peptide A, and the C-terminus of the first and second peptides has been bound to the linker.

7. The peptide complex of claim 1, wherein, This linker is a PEG linker.

8. A pharmaceutical composition comprising a peptide complex as claimed in any one of claims 3 to 7 and a pharmaceutically permissible carrier.

9. The pharmaceutical composition of claim 8, used for the treatment or prevention of diseases selected from the group consisting of ischemic heart disease, acute hepatitis, fulminant hepatitis, cirrhosis, biliary atresia, fatty liver, acute renal failure, chronic renal failure, diabetic nephropathy, acute pneumonia, pulmonary fibrosis, vascular disease, myocardial infarction, dilated cardiomyopathy, skin ulcers, cerebral infarction, occlusive arteriosclerosis, gastric ulcers and amyotrophic lateral sclerosis.

10. A culture medium additive comprising a peptide complex as claimed in any one of claims 1 to 6.

11. The culture medium additive as requested in item 10, which is used to culture cells or tissues derived from the human body.

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