Peptide substrates cleavable by matrix metalloproteinases

Abstract

To provide a peptide with an amino acid sequence capable of cleavage with various proteases (e.g., MMP-2, MMP-3 and MMP-9).SOLUTION: The peptide comprises an amino acid sequence represented by general formula: P2P1P1'P2', where P1 is a glycine residue (Gly), P2 and P1' are hydrophobic amino acid residues, and P2' is an alanine residue (Ala), a tryptophan residue (Trp), a valine residue (Val), an arginine residue (Arg) or a threonine residue (Thr).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to peptide substrates that are cleavable by matrix metalloproteases. [Background technology]

[0002] Matrix metalloproteinases (MMPs) are a group of enzymes that function in the remodeling of the extracellular matrix. They are classified into various families and subfamilies based on their structure and function. The concerted action of various MMPs can degrade almost all components of the extracellular matrix. MMPs are also deeply involved in physiological and pathological processes. The activation of gelatinases (MMP-2 and MMP-9) is known to trigger cancer invasion and metastasis, while MMP-3, expressed and secreted during synovial proliferation, triggers the autoimmune disease rheumatoid arthritis. Rapid detection of MMP activation may lead to early detection and treatment of these diseases.

[0003] The development of materials targeting MMPs is progressing in various fields, and includes a wide range of materials, such as microporous gel systems used for cell transplantation and wound dressings (Patent Document 1), cell carriers for three-dimensional culture (Patent Document 2), and biosensors (Patent Document 3).

[0004] Patent Document 1 reports a wound-healing material containing microgel particles with a poly(ethylene glycol) backbone. Crosslinking between these microgel particles with an MMP-degradable crosslinker is expected to provide the mechanical strength necessary for structural support and matrix degradability associated with tissue regeneration. Patent Document 2 reports a cell culture microenvironment array that can reproduce cell behavior in a natural environment by controlling various matrix parameters, such as mechanical and biochemical properties. Patent Document 3 reports a wound dressing and a biosensor capable of real-time detection of biomarker enzymes (e.g., MMP-2) in samples such as body fluids, secretions, or exudates. The biosensor uses two electrodes, each coated with a natural or synthetic substrate such as gelatin. The electrode surface is exposed by chemical decomposition by enzymes released from the wound site, allowing the wound healing process to be monitored by measuring electrical resistance. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2017-522113 [Patent Document 2] Special Publication No. 2016-517694 [Patent Document 3] Special Publication No. 2021-502149 Summary of the Invention [Problem to be solved by the invention]

[0006] However, the substrates described in Patent Documents 1 to 3 have only been specifically shown to be cleaved by one type of MMP, and there is room for improvement in order to achieve rapid cleavage and, ultimately, early detection of disease.

[0007] An object of the present invention is to provide peptides having amino acid sequences that are cleavable by various proteases (eg, MMP-2, MMP-3, and MMP-9). [Means for solving the problem]

[0008] The present invention relates to, for example, the following inventions [1] to

[14] . [1] A peptide comprising an amino acid sequence represented by the general formula: P2P1P1'P2', wherein P1 is a glycine residue (Gly), P2 and P1' are hydrophobic amino acid residues, and P2' is an alanine residue (Ala), a tryptophan residue (Trp), a valine residue (Val), an arginine residue (Arg), or a threonine residue (Thr). [2] The peptide according to [1], wherein, in the amino acid sequence, P2 is a leucine residue (Leu) or an alanine residue (Ala); when P2 is a leucine residue (Leu), P1' is a leucine residue (Leu) and P2' is an arginine residue (Arg); when P2 is an alanine residue (Ala), P1' is a leucine residue (Leu) or a methionine residue (Met), and P2' is an alanine residue (Ala), a tryptophan residue (Trp), a valine residue (Val), an arginine residue (Arg), or a threonine residue (Thr). [3] The peptide according to [1] or [2], wherein the amino acid sequence is the amino acid sequence represented by SEQ ID NO: 1, 4, 5, 9, 10 or 11. [4] A matrix metalloproteinase-responsive agent comprising an amino acid sequence having a matrix metalloproteinase cleavage site represented by the general formula: P2P1↓P1'P2', where ↓ is the matrix metalloproteinase cleavage site, P1 is a glycine residue (Gly), P2 and P1' are hydrophobic amino acid residues, and P2' is an alanine residue (Ala), a tryptophan residue (Trp), a valine residue (Val), an arginine residue (Arg), or a threonine residue (Thr). [5] The responder according to [4], wherein, in the above amino acid sequence, P2 is a leucine residue (Leu) or an alanine residue (Ala), and when P2 is a leucine residue (Leu), P1' is a leucine residue (Leu) and P2' is an arginine residue (Arg), and when P2 is an alanine residue (Ala), P1' is a leucine residue (Leu) or a methionine residue (Met), and P2' is an alanine residue (Ala), a tryptophan residue (Trp), a valine residue (Val), an arginine residue (Arg), or a threonine residue (Thr). [6] The responder according to [4] or [5], wherein the amino acid sequence is the amino acid sequence represented by SEQ ID NO: 1, 4, 5, 9, 10 or 11. [7] The response agent according to any one of [4] to [6], wherein the matrix metalloproteinase comprises at least one selected from the group consisting of MMP-2, MMP-3, and MMP-9. [8] The response agent according to any one of [4] to [7], wherein the matrix metalloproteinase is MMP-2, and when reacted with an MMP-2-containing solution in which the concentration of the MMP-2 is 0.45 nM or more, the response agent is cleaved by the matrix metalloproteinase. [9] The responding agent according to any one of [4] to [8], wherein the cleavage reaction with the matrix metalloproteinase is observed by the resonance energy transfer (RET) phenomenon.

[10] A cancer sensor or a cancer marker comprising the peptide according to any one of [1] to [3].

[11] A quantitative assay kit for matrix metalloproteinase, comprising the peptide according to any one of [1] to [3].

[12] A drug delivery system comprising the peptide according to any one of [1] to [3].

[13] A cell separation material comprising the peptide according to any one of [1] to [3].

[14] A substrate for three-dimensional cell culture, comprising the peptide according to any one of [1] to [3]. [Effects of the Invention]

[0009] According to the present invention, peptides can be provided that have an amino acid sequence that can be cleaved by various proteases (e.g., MMP-2, MMP-3, and MMP-9) and can be cleaved even by low concentrations of MMP-2. Use of the peptides of the present invention is expected to provide cancer sensors or cancer markers (for supplementing existing cancer markers or for primary testing), MMP quantification assay kits, DDS (Drug Delivery Systems), cell separation materials, cell fractionation materials, substrates for 3D cell culture, and the like. Furthermore, evaluation systems for cancer sensors and the like using the peptides of the present invention are less susceptible to lot-to-lot variation, unlike those using naturally occurring substrates such as gelatin, and are therefore considered to have stable evaluation quality. [Brief explanation of the drawings]

[0010] [Figure 1] Enzyme cleavage test results for MMP-2. [Figure 2] Enzyme cleavage test results for MMP-3. [Figure 3] Enzyme cleavage test results for MMP-9. [Figure 4] Catalytic efficiency for MMP-2; kcat / Km. [Figure 5] Results of enzyme cleavage test for MMP-2 at enzyme concentration [E] = 1.0 nM. [Figure 6] Results of enzyme cleavage test for MMP-2 at an enzyme concentration [E] = 0.5 nM. [Figure 7] Results of enzyme cleavage test for MMP-2 at enzyme concentration [E] = 0.45 nM. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, an embodiment for carrying out the present invention (hereinafter simply referred to as "the present embodiment") will be described in detail. The present embodiment is an example for explaining the present invention, and is not intended to limit the present invention to the following content. The present invention can be carried out by appropriately modifying it within the scope of its spirit.

[0012] As used herein, "amino acid" or "amino acid residue" refers to both naturally occurring and non-naturally occurring amino acids. The 20 naturally occurring amino acids are represented as follows: alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), and tyrosine (Tyr). The amino acids are broadly classified into D- and L-forms based on their configuration, and the amino acid residues constituting the amino acid sequence having a protease cleavage site herein are preferably L-forms.

[0013] The terms "peptide" and "protein" refer to a substance containing two or more amino acids dehydrated and condensed by a peptide bond, and in the amino acid sequences described herein, the left corresponds to the N-terminus and the right corresponds to the C-terminus.

[0014] The method for synthesizing peptides and proteins is not particularly limited, and may be an organic chemical synthesis method or a genetic engineering synthesis method using microorganisms or cultured cells. The organic chemical synthesis method is not particularly limited, and may include, for example, an Fmoc / Boc solid-phase or liquid-phase method using an N-terminal protected amino acid in which the N-terminal amino group is substituted with a 9-fluorenylmethyloxycarbonyl; Fmoc group or a tert-butoxycarbonyl; Boc group, or a native chemical ligation method using various peptide segments synthesized by the solid-phase or liquid-phase method.

[0015] As used herein, "protease" refers to a hydrolase that catalyzes the hydrolysis of peptide bonds in peptides and proteins, and "matrix metalloproteases (MMPs)" refer to enzymes that contain zinc ions (Zn 2+ ) indicates the coordinated protease, and the protease concentration is represented by [E].

[0016] As used herein, the terms "protease cleavage site" and "↓" refer to a site or domain that has an amino acid sequence specifically recognized by a protease and is specifically recognized and hydrolyzed in the presence of an active protease. The peptides and proteins of the present invention have at least one or more of these sites or domains, and may also have multiple protease cleavage sites. A protease cleavage site may consist of 2 to 20 amino acid residues, 3 to 20 amino acid residues, or 4 to 15 amino acid residues. When referring to an amino acid sequence that is enzymatically cleaved, the amino acid residues are referred to as P1 site, P2 site, etc. from the cleavage site toward the N-terminus, and P1' site, P2' site, etc. from the cleavage site toward the C-terminus. As is well known, the cleavage sites P1 and P1' and the residues P2 and P2' adjacent to the cleavage site can affect the substrate specificity and cleavage efficiency of various proteases.

[0017] The cleavage reaction may be monitored by any method, including, for example, changes in electrochemical potential, fluorescence, chemiluminescence, phosphorescence, absorbance, antibody binding, or mass. The method for monitoring the change may also include, for example, fluorescence resonance energy transfer (FRET), which occurs based on dipole-dipole interactions between a donor fluorescent molecule and an acceptor fluorescent molecule. Molecules that can cause the FRET phenomenon include, but are not limited to, 5-(2-aminoethylamino)-1-naphthalenesulfonic acid; EDANS / 4-((-4-(dimethylamino)-phenyl)-azo)-benzoic acid; DABCYL pair; 7-methoxycoumarin-4-acetic acid; Mca / 2,4-dinitrophenyl; Dnp pair; 7-amino-4-carbamoylmethylcoumarin; ACC / Dnp pair; tryptophan; Trp / 5-dimethylaminonaphthalene-1-sulfonic acid; Dancyl pair. The molecule that causes the FRET phenomenon and the unit containing the molecular structure thereof may be introduced into the peptide main chain or into the peptide side chain.

[0018] As used herein, the term "substrate" refers to a chemical substance whose reaction is catalyzed by an enzyme, and the peptide of the present invention corresponds to a "substrate" for MMP. The substrate concentration is indicated as [S]. "Substrate reactivity" and "cleavage ability" are terms used to indicate the reaction specificity of a substrate for various enzymes. The reaction specificity of a substrate can be quantified by standard reaction kinetic analysis, and according to known methods, it can be derived from, for example, an enzyme reaction curve showing the relationship between substrate concentration and reaction rate or a linear plot of the Michaelis-Menten equation, such as a Lineweaver-Burk plot. Generally, the reaction specificity of the substrate is indicated by the catalytic efficiency; kcat / Km value, where kcat indicates the number of enzyme reactions per unit time, called molecular activity, and Km indicates the substrate concentration at which the reaction rate reaches half of the maximum reaction rate Vmax, called the Michaelis constant. As used herein, catalytic efficiency is defined as 5.0 x 10 3 M -1 s -1 More than 8.0×10 is preferable. 3 M -1 s -1 More preferably, 1.0×10 4 M -1 s -1 More preferably, 1.2 × 10 4 M -1 s -1 The above is particularly preferred. Furthermore, when deriving catalytic efficiency according to the above procedure, the substrate concentration is preferably lower than the Km value, preferably 30 μM or less, and more preferably 20 μM or less. In addition, in order to observe the above-mentioned change before and after the enzymatic cleavage reaction, the substrate concentration is preferably 5 μM or more, and more preferably 10 μM or more. For the same reason, the enzyme concentration, for example, the concentration of MMP-2 in the MMP-2-containing solution, can be 0.1 nM or more, preferably 0.3 nM or more, more preferably 0.6 nM or more, and even more preferably 1.0 nM or more.

[0019] In the amino acid sequence represented by the general formula: P2P1P1'P2' or the amino acid sequence represented by the general formula: P2P1↓P1'P2', P1 is a glycine residue (Gly), P2 and P1' are hydrophobic amino acid residues, and P2' is an alanine residue (Ala), tryptophan residue (Trp), valine residue (Val), arginine residue (Arg), or threonine residue (Thr). In the amino acid sequence represented by the general formula: P2P1↓P1'P2', ↓ is a matrix metalloproteinase cleavage site.

[0020] Hydrophobic amino acid residues include Leu, Ala, Met, Ile, Val, Gly, Pro, Trp, and Phe.

[0021] In the amino acid sequence represented by the general formula: P2P1P1'P2' or the amino acid sequence represented by the general formula: P2P1↓P1'P2', P2 may be a leucine residue (Leu) or an alanine residue (Ala); when P2 is a leucine residue (Leu), P1' may be a leucine residue (Leu) and P2' may be an arginine residue (Arg); when P2 is an alanine residue (Ala), P1' may be a leucine residue (Leu) or a methionine residue (Met), and P2' may be an alanine residue (Ala), a tryptophan residue (Trp), a valine residue (Val), an arginine residue (Arg), or a threonine residue (Thr).

[0022] Specific examples of amino acid sequences represented by the general formula: P2P1P1'P2' or the general formula: P2P1↓P1'P2' include the amino acid sequences represented by SEQ ID NOs: 1 to 11. That is, the peptide of the present invention may comprise at least one selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 11. The amino acid sequences represented by SEQ ID NOs: 1 to 11 can be cleaved by at least MMP-2, MMP-3, and MMP-9. The amino acid sequences represented by SEQ ID NOs: 1 to 11 can also be cleaved by a solution containing a low concentration (0.45 nM) of MMP-2. In the amino acid sequences represented by SEQ ID NOs: 1 to 11, the cleavage site is located between P1 and P1'. There are other MMPs that can cleave the peptides of the present invention, and they are classified into the collagenase group (MMP-1, 8, 13), gelatinase group (MMP-2, 9), stromelysin group (MMP-3, 10, 11), and transmembrane MMPs (MMP-14; MT1-MMP, MMP-15; MT2-MMP, MMP-16; MT3-MMP, MMP-17; MT4-MMP) from the viewpoint of structure and function. The above MMPs may act alone, or two or more types of MMPs may act in concert. In this specification, two or more types of MMPs are collectively referred to as "MMPs."

[0023] In the present invention, the amino acid sequence represented by the general formula: P2P1P1'P2' or the general formula: P2P1↓P1'P2' is preferably the amino acid sequence represented by SEQ ID NO: 1, 4, 5, 9, 10, or 11. That is, the peptide of the present invention preferably comprises an amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 5, the amino acid sequence represented by SEQ ID NO: 9, the amino acid sequence represented by SEQ ID NO: 10, and the amino acid sequence represented by SEQ ID NO: 11.

[0024] The peptide of the present invention may contain an amino acid sequence represented by the general formula: P2P1P1'P2' or the general formula: P2P1↓P1'P2', and may have an additional amino acid sequence added to at least one or both of the N-terminus and C-terminus of the amino acid sequence represented by the general formula: P2P1P1'P2' or the general formula: P2P1↓P1'P2'. There are no particular limitations on the amino acid sequence to be added.

[0025] Examples of the peptides of the present invention include peptides containing the amino acids of SEQ ID NOs: 12 to 21 or 22. The peptides of the present invention preferably contain an amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 12, the amino acid sequence represented by SEQ ID NO: 15, the amino acid sequence represented by SEQ ID NO: 16, the amino acid sequence represented by SEQ ID NO: 20, the amino acid sequence represented by SEQ ID NO: 21, and the amino acid sequence represented by SEQ ID NO: 22.

[0026] The length of the peptide of the present invention can be 40 or less amino acid residues, 30 or less, 20 or less, 10 or less, or 8 or less amino acid residues. Peptides within this length range are easily applicable to three-dimensional cell culture substrates, MMP quantification kits, and the like.

[0027] The peptide preferably contains the amino acid Arg, Asn, Glu, Asp, Gln, Ser, Thr, or Lys. The inclusion of these amino acids increases hydrophilicity. The peptide preferably contains the amino acid Cys or Lys. The inclusion of these amino acids allows the peptide to be modified.

[0028] The peptides of the present invention can be cleaved by at least MMP-2, MMP-3, and MMP-9, and therefore can be used as responsive agents to proteases (e.g., MMP-2, MMP-3, and MMP-9). As used herein, the term "responsive agent" refers to a peptide that is site-specifically hydrolyzed and cleaved by a protease (e.g., MMP-2, MMP-3, and MMP-9) (responses to a protease). By utilizing this cleavage reaction, the responsive agents of the present invention can be used for a variety of purposes.

[0029] For example, as described in Jin C et al., Blood Droplet-Based Cancer Diagnosis via Proteolytic Activity Measurement in Cancer Progression, Theranostics 2017, Vol. 7, Issue 11, pp. 2878-2887; Akifumi Y et al., Rapid and highly efficient capture and release of cancer cells using polymeric microfibers immobilized with enzyme-cleavable peptides, Acta Biomaterialia, Volume 67, February 2018, Pages 32-41; Ricardo CA et al., Synthetic hydrogels for human intestinal organoid generation and colonic wound repair, Nature Cell Biology 19, 1326-1335 (2017), peptides containing an amino acid sequence cleaved by MMP-2, MMP-3, or MMP-9 can be used as cancer sensors or cancer markers (for supplementing existing cancer markers and for primary testing), MMP quantification assay kits, and DDS (Drug Delivery Systems). The peptides of the present invention can be used in cancer sensors or cancer markers (for supplementing existing cancer markers or for primary testing), MMP quantification assay kits, DDS (Drug Delivery Systems), cell separation materials, cell sorting materials, and substrates for 3D cell culture, as will be described in detail below. Furthermore, evaluation systems for cancer sensors and the like using the peptides of the present invention are less likely to experience lot-to-lot variation, unlike those using naturally occurring substrates such as gelatin, and are therefore considered to have stable evaluation quality.

[0030] It is known that cancers secrete large amounts of MMP-2 and MMP-9 to grow and metastasize, penetrate blood vessels, and spread via the bloodstream. Therefore, since the presence of cancer increases the concentrations of MMP-2 and MMP-9 in the blood, the peptides of the present invention can be used as a primary diagnostic agent for cancer by assaying and quantifying MMP-2 and MMP-9 in the blood. Taking advantage of this, the peptides of the present invention can be used to develop monitoring sensors or sensors combined with blood glucose and oxygen monitoring (multiplexed sensors, sensor cartridges, consumables). Furthermore, because the peptides of the present invention also exhibit the ability to cleave MMP-3, which is classified as a stromelysin, they can be used as a primary diagnostic agent not only for cancer but also for rheumatoid arthritis, for example.

[0031] The peptides of the present invention can also be applied to kits (for research use) for quantifying the expression levels of MMPs. The peptides of the present invention can also be applied to assays for evaluating related physiological phenomena and pathological conditions. Because the peptides of the present invention are highly sensitive, they are thought to have low LOD (Limit of Detection). Therefore, high accuracy and high sensitivity are expected in assays in the low concentration range.

[0032] Because the peptides of the present invention have the characteristic of reacting with and being cleaved by MMPs, they may be applicable to a field known as ADC (Antibody Drug Conjugate) (drug + peptide linker + antibody). For example, by using a molecular design such as the following structural formula, it is possible to develop a material for DDS applications that uses a peptide linker that is cleaved by an enzyme called cathepsin. By changing the peptide linker in the structural formula to the peptide of the present invention, it is thought that an ADC can be developed that, after reaching its target cancer cells, is quickly cleaved by MMPs released by the cancer cells, allowing the drug to be released appropriately. [ka]

[0033] The peptides of the present invention can be immobilized on surface modifiers, crosslinkers, coating materials, etc., and these materials can be packed into columns to be used as cell separation or fractionation materials. Furthermore, if there is a technology that can control the holes (pores) that are cleaved in response to MMPs, separation or fractionation by size would also be possible.

[0034] The peptides of the present invention can be used as crosslinkers for polymer matrices and can therefore be applied to the 3D culture of MMP-secreting cells. When MMP-secreting tumor cells are cultured in a polymer matrix gel, the gel matrix is ​​gradually degraded by the MMP produced by the cells, strengthening the interactions between the cells and promoting the formation of cell clusters (spheroids). In other words, the peptides of the present invention can be used for cell cluster (spheroid) formation, the creation of 3D cancer spheroid culture models, and other applications.

[0035] Since the peptide of the present invention can be degraded by enzymes, it is thought that it may be possible to develop enzymatically degradable plastics and apply it to post-operative sutures, etc. [Example]

[0036] The present invention will be described in detail below with reference to embodiments for carrying out the present invention. However, these are merely examples for explaining the present invention and are not intended to limit the present invention to the following content. Furthermore, the present invention can be practiced with appropriate modifications within the scope of the gist of the present invention. Unless otherwise specified, commercially available reagents were used.

[0037] <Composition of reaction solutions, buffer solutions, etc.> Deprotection solution: piperidine (Fujifilm Wako Pure Chemical Industries, Ltd., 25 vol%), dimethylformamide; DMF (Fujifilm Wako Pure Chemical Industries, Ltd., 75 vol%) Amino acid cocktail: N-terminal Fmoc-protected amino acids (Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Gln(OtBu)-OH, Fmoc-Val-OH, and Fmoc-Trp(Boc)-OH were manufactured by Merck, Fmoc-Met-OH was manufactured by Sigma-Aldrich, and Fmoc-Lys(Boc)-OH, Fmoc-Arg(pbf)-OH, and Fmoc-Thr(tBu)-OH were manufactured by Chemscene, 6 equivalents relative to the reaction site), ethyl cyanohydroxyiminoacetate; Oxyma pure™ (Merck, 6 equivalents relative to the reaction site), N,N'-diisopropylcarbodiimide (Fujifilm Wako Pure Chemical Industries, Ltd., 6 equivalents relative to the reaction site), 2,4,6-trimethylpyridine (Tokyo Chemical Industry Co., Ltd., 12 equivalents relative to the reaction site), DMF (solvent amount) Inactivation solution: acetic anhydride (Fujifilm Wako Pure Chemical Industries, Ltd., 25 vol%), DMF (75 vol%) Resin removal solution: Trifluoroacetic acid (TFA) (Tokyo Chemical Industry Co., Ltd., 95 mass%), triisopropylsilane (Fujifilm Wako Pure Chemical Industries, Ltd., 2.5 mass%), distilled water (2.5 mass%) Eluent A: 0.1% TFA / distilled water Eluent B: 0.1% TFA / acetonitrile (for high-performance liquid chromatography, Fujifilm Wako Pure Chemical Industries, Ltd.) 1.5mM peptide solution: FRET substrate, DMSO Tris-HCl buffer: 100 mM tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)aminomethane hydrochloride, 100 mM sodium chloride, 10 mM calcium chloride (pH 7.5) 1M 4-aminophenylmercuric acetate; APMA stock solution: APMA, dimethyl sulfoxide; DMSO (Fujifilm Wako Pure Chemical Industries, Ltd.) 1mM APMA reaction solution: 1M APMA stock solution, Tris-HCl buffer 3.6 μM MMP-2 solution: recombinant MMP-2 (manufactured by Biovision Inc.), Tris-HCl buffer

[0038] <Fmoc Peptide Solid Phase Synthesis> Peptide substrates having the amino acid sequences shown in SEQ ID NOs: 12, 15, 16, 20, 21, or 22, and peptide substrates having the amino acid sequence shown in SEQ ID NO: 24, an existing MMP response sequence (see Bremer C et al., In vivo molecular target assessment of matrix metalloproteinase inhibition, Nature Medicine, v7, 743-748 (2001)), were synthesized by Fmoc solid-phase synthesis. For the peptide substrate having the amino acid sequence shown in SEQ ID NO: 23, an existing MMP response sequence (Patent Document 1), a Lys residue bearing a Dnp group on its side chain was newly introduced at the C-terminus of the amino acid sequence shown in SEQ ID NO: 23, resulting in a peptide substrate consisting of a total of nine residues. All of the above peptide substrates were synthesized as FRET peptide substrates incorporating an Mca group and a Dnp group (hereinafter referred to as "FRET substrates"; see Table 1). An Econopack column (20 mL, BIO RAD) was installed in a multi-solid-phase synthesizer (KMS-3, Kokusan Chemical Co., Ltd.). Rink amide AM resin (100-200 mesh, 0.7 mmol / g, Merck) was weighed to a 0.05 mmol scale, and 2 mL of dichloromethane (DCM) (peptide synthesis grade, Fujifilm Wako Pure Chemical Industries, Ltd.) was added and allowed to swell for 12–24 hours under vortex agitation. After removing the swelling solution, 2 mL of deprotection solution was added to deprotect the Fmoc groups on the resin (2 mL x 2, 10 min each). After removing the deprotection solution, the resin was washed with DMF and DCM, and then an amino acid cocktail was added. The reaction was allowed to proceed under vortex agitation for 2–3 hours to couple the amino acids onto the resin support. After removing the reaction solution, 2 mL of deactivation solution was added and the resin was left to react under vortex agitation for 30 minutes to cap any unreacted sites. After removing the inactivation solution, the resin support was washed with DMF and DCM to obtain the Fmoc amino acid-incorporated resin support. The above-mentioned deprotection, coupling reaction, and inactivation of unreacted sites were then repeated to synthesize the peptide substrate having the target amino acid sequence.

[0039] <Rough purification, LC separation and purification> After synthesizing a peptide with a given amino acid sequence, the N-terminal Fmoc group was deprotected, washed with DMF, DCM, and methanol, and then left in a desiccator for 24 hours to obtain a dry resin support. 2 mL of chilled deprotection solution was added to the dry resin support and stirred under vortex flow for 3–4 hours to achieve deprotection of the resin and side chain protecting groups. After collecting the supernatant containing the peptide, another 2 mL of the deprotection solution was added to wash the resin support. Ten volumes of cold diethyl ether were added to the supernatant and washing solution (4 mL total), and the precipitate was collected by centrifugation (2,000 × g, 10 min). The precipitate was washed again with cold diethyl ether and then dried under reduced pressure to obtain the crude polypeptide. The obtained crude polypeptide was dissolved in eluent A, and then the target component was fractionated using eluents A and B on a medium pressure liquid chromatograph (model: EPCLC-AI-580S, Yamazen Co., Ltd.). The acetonitrile was removed under reduced pressure, and the resulting mixture was freeze-dried to obtain a purified peptide.

[0040] [Table 1]

[0041] Example 1: Verification of MMP-2 cleavage of peptides having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, or 22 To verify MMP-2 cleavage, an MMP-2 Inhibitor Screening kit (BioVision Inc.) was used, and cleavage tests were performed according to the manufacturer's recommended protocol. Purified peptides having the amino acid sequences shown in SEQ ID NOs: 12, 15, 16, 20, 21, and 22 were dissolved in DMSO to obtain six 1.5 mM peptide solutions. The resulting 1.5 mM peptide solutions were diluted 40-fold with the MMP-2 assay buffer provided with the kit to prepare measurement samples (substrate concentration: [S] = 37.5 μM). MMP-2 was dissolved in the above MMP-2 assay buffer to obtain an MMP-2 solution (enzyme concentration: [E] = 7 μM). The resulting MMP-2 solution was added at 50 μL / well to a 96-well black plate (Thermo Fisher Scientific) and incubated at 37°C for 30 minutes to activate MMP-2. 50 μL / well of the measurement sample was added to the plate, and fluorescence was measured at an excitation wavelength of 325 nm and a detection wavelength of 393 nm (100 μL / well, [S]=18.75 μM, [E]=3.5 μM, 37°C, 120 minutes).

[0042] The results for 60 minutes after the start of fluorescence measurement are shown in Figure 1. The horizontal axis of Figure 1 shows the time (minutes) after the start of fluorescence measurement, and the vertical axis shows the change in fluorescence intensity value relative to the fluorescence intensity value at the start of measurement (ΔRFU value). The greater the slope of the graph, the faster the reaction rate, i.e., the higher the cleavage ability. As is clear from Figure 1, in this evaluation system, peptide substrates having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, or 22 were shown to have the cleavage ability of MMP-2.

[0043] Comparative Example 1: Verification of MMP-2 cleavage of peptides having the amino acid sequences represented by SEQ ID NOs: 23 and 24 The MMP-2 cleavage ability was evaluated in the same manner as in Example 1, except that purified peptides having the amino acid sequences represented by SEQ ID NOs: 23 and 24 were used instead of purified peptides having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, or 22.

[0044] In this evaluation system, the peptide substrate having the amino acid sequence represented by SEQ ID NO: 23 or 24 exhibited the ability to cleave MMP-2, but the change in fluorescence intensity per unit time (ΔRFU value) due to the FRET phenomenon was lower than in Example 1. In other words, it was suggested that the peptide substrate having the amino acid sequence represented by SEQ ID NO: 23 or 24 had a slow reaction rate and low cleavage ability ( FIG. 1 ).

[0045] Example 2: Verification of MMP-3 cleavage of peptides having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, or 22 To verify MMP-3 cleavage, an MMP-3 Inhibitor Screening kit (manufactured by BioVision Inc.) was used, and a cleavage test was performed according to the manufacturer's recommended protocol. Measurement samples were obtained in the same manner as in Example 1. An MMP-3 solution was obtained in the same manner as in Example 1, except that MMP-3 was used instead of MMP-2 (enzyme concentration: [E] = 1.6 μM). The resulting MMP-3 solution was added to a 96-well black plate (manufactured by Thermo Fisher Scientific) at 50 μL / well and incubated at 37°C for 10 minutes to activate MMP-3. Measurement samples were added to the plate at 50 μL / well, and fluorescence measurements were performed at an excitation wavelength of 325 nm and a detection wavelength of 393 nm (100 μL / well, [S] = 18.75 μM, [E] = 0.8 μM, 37°C, 120 minutes).

[0046] The results for 60 minutes after the start of fluorescence measurement are shown in Figure 2. The horizontal axis of Figure 2 shows the time (minutes) after the start of fluorescence measurement, and the vertical axis shows the change in fluorescence intensity value relative to the fluorescence intensity value at the start of measurement (ΔRFU value). The greater the slope of the graph, the faster the reaction rate, i.e., the higher the cleavage ability. As is clear from Figure 2, in this evaluation system, peptide substrates having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, or 22 were shown to have the ability to cleave MMP-3.

[0047] Comparative Example 2: Verification of MMP-3 cleavage of peptides having the amino acid sequences represented by SEQ ID NOs: 23 and 24 The MMP-3 cleavage ability was evaluated in the same manner as in Example 2, except that purified peptides having the amino acid sequences represented by SEQ ID NOs: 23 and 24 were used instead of purified peptides having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, or 22.

[0048] In this evaluation system, neither of the peptide substrates having the amino acid sequences shown in SEQ ID NO: 23 or 24 exhibited any cleavage ability by MMP-3 (FIG. 2).

[0049] Example 3: Verification of MMP-9 cleavage of peptides having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, or 22 To verify MMP-9 cleavage, an MMP-9 Inhibitor Screening kit (manufactured by BioVision Inc.) was used, and a cleavage test was performed according to the manufacturer's recommended protocol. Measurement samples were obtained in the same manner as in Example 1. An MMP-9 solution was obtained in the same manner as in Example 1, except that MMP-9 was used instead of MMP-2 (enzyme concentration: [E] = 10.4 μM). The resulting MMP-9 solution was added to a 96-well black plate (manufactured by Thermo Fisher Scientific) at 50 μL / well and incubated at 37°C for 30 minutes to activate MMP-9. Measurement samples were added to the plate at 50 μL / well, and fluorescence measurements were performed at an excitation wavelength of 325 nm and a detection wavelength of 393 nm (100 μL / well, [S] = 18.75 μM, [E] = 5.2 μM, 37°C, 120 minutes).

[0050] The results for 60 minutes after the start of fluorescence measurement are shown in Figure 3. The horizontal axis of Figure 3 shows the time (minutes) after the start of fluorescence measurement, and the vertical axis shows the change in fluorescence intensity value relative to the fluorescence intensity value at the start of measurement (ΔRFU value). The greater the slope of the graph, the faster the reaction rate, i.e., the higher the cleavage ability. As is clear from Figure 3, in this evaluation system, substrates having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, or 22 were shown to have the cleavage ability of MMP-9.

[0051] Comparative Example 3: Verification of MMP-9 cleavage of peptides having the amino acid sequences represented by SEQ ID NOs: 23 and 24 The MMP-9 cleavage ability was evaluated in the same manner as in Example 3, except that purified peptides having the amino acid sequences represented by SEQ ID NOs: 23 and 24 were used instead of purified peptides having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, or 22.

[0052] In this evaluation system, it was confirmed that the substrate having the amino acid sequence shown in SEQ ID NO: 24 had the ability to be cleaved by MMP-9, whereas the substrate having the amino acid sequence shown in SEQ ID NO: 23 did not exhibit the ability to be cleaved by MMP-9 (Figure 3).

[0053] Example 4: Evaluation of MMP-2 enzyme activity for peptides having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, or 22 A 3.6 μM MMP-2 solution was diluted 500-fold with a 1 mM APMA reaction solution and then incubated at room temperature (25°C) for 1 hour to activate MMP-2. Next, a 1.5 mM peptide solution obtained in the same manner as in Example 1 was diluted with Tris-HCl buffer to prepare measurement samples with substrate concentrations of 50 μM, 25 μM, 12.5 μM, or 6.25 μM. The MMP-2 solution activated by the above procedure was added to a 96-well black plate at 50 μL / well and incubated at 37°C for 1 hour. Then, 50 μL / well of the measurement sample was added, and fluorescence measurement was performed at an excitation wavelength of 325 nm and a detection wavelength of 393 nm (100 μL / well, [E] = 3.6 μM, [S] = 25 μM, 12.5 μM, 6.25 μM, or 3.125 μM, 37°C, 120 minutes). The initial reaction velocity v0 (μM min) was calculated from the measured values. -1 ) was calculated, and then the catalytic efficiency kcat / Km was derived from the publicly known Lineweaver-Burk plot (Equation (1)) and Equation (2). In this specification, [E]0 in Equation (2) refers to the initial enzyme concentration and is used synonymously with [E]. 1 / v0=(Km / Vmax)·(1 / [S])+(1 / Vmax) ―(1) kcat = Vmax / [E]0―(2) (v: reaction rate (μM min -1 ), Km: Michaelis-Menten constant (μM), Vmax: Maximum reaction velocity (μM min -1 ), [S]: substrate concentration (μM), kcat: catalytic constant (s -1 ), [E]0: initial enzyme concentration (μM))

[0054] The catalytic efficiency of the peptide substrate having the amino acid sequence represented by SEQ ID NO: 12, 15, 16, 20, 21, or 22 is 1.0 × 10 4 (M -1 ·s -1 In particular, the peptide substrate having the amino acid sequence represented by SEQ ID NO: 15, 16 or 21 exhibited an activity of 1.5 × 10 4 (M -1 ·s -1 ), which was 2 to 4 times the value of Comparative Example 4 described later (FIG. 4).

[0055] Comparative Example 4: Evaluation of MMP-2 enzyme activity for SEQ ID NOs: 23 and 24 The enzymatic activity of MMP-2 was evaluated in the same manner as in Example 4, except that purified peptides having the amino acid sequences represented by SEQ ID NOs: 23 and 24 were used instead of purified peptides having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, or 22.

[0056] In this evaluation system, the catalytic efficiency of the peptide substrate having the amino acid sequence shown in SEQ ID NO: 23 or 24 for MMP-2 was lower than that in Example 4, confirming its low enzymatic activity (Figure 4).

[0057] Example 5: Evaluation of the cleavage ability of peptides having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, or 22 at different MMP-2 concentrations MMP-2 was activated using the same method as in Example 4 and then diluted with 1 mM APMA reaction solution to give enzyme concentrations of 2.0 μM, 1.0 μM, or 0.9 μM. Next, the 1.5 mM peptide solution obtained using the same method as in Example 1 was diluted 60-fold with Tris-HCl buffer to prepare a 25 μM measurement sample. The diluted MMP-2 solution was added to a 96-well black plate at 50 μL / well and incubated at 37°C for 1 hour. The 25 μM measurement sample was then added at 50 μL / well, and fluorescence measurements were performed at an excitation wavelength of 325 nm and a detection wavelength of 393 nm (100 μL / well, [E] = 1.0 nM, 0.5 nM, or 0.45 nM, [S] = 12.5 μM, 37°C, 60 minutes, or 120 minutes).

[0058] The results are shown in Figures 5 to 7. The horizontal axis of Figures 5 to 7 shows the time (minutes) from the start of fluorescence measurement, and the vertical axis shows the change in fluorescence intensity value relative to the fluorescence intensity value at the start of measurement (ΔRFU value). The steeper the slope of the graph, the faster the reaction rate, i.e., the higher the cleavage ability. As is clear from Figures 5 to 7, in this evaluation system, the progress of cleavage was confirmed for peptide substrates having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, or 22 under all tested enzyme concentration conditions ([E] = 1.0 nM, 0.5 nM, or 0.45 nM).

[0059] Comparative Example 5: Evaluation of the cleavage ability of peptides having the amino acid sequences represented by SEQ ID NOs: 23 and 24 at different MMP-2 concentrations The cleavage performance at different MMP-2 concentrations was evaluated in the same manner as in Example 5, except that purified peptides having the amino acid sequences represented by SEQ ID NOs: 23 and 24 were used instead of purified peptides having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, or 22.

[0060] In this evaluation system, the progress of cleavage of the peptide substrate having the amino acid sequence shown by SEQ ID NO: 23 or 24 was confirmed when the enzyme concentration was 1.0 nM and 0.5 nM, but the change in fluorescence intensity per unit time due to the FRET phenomenon was smaller than in Example 5 (Figures 5 and 6). Furthermore, the progress of cleavage could not be confirmed when the enzyme concentration was 0.45 nM (Figure 7).

Claims

1. The amino acid sequence has a matrix metalloproteinase cleavage site represented by the general formula: P2P1↓P1'P2', ↓ is the matrix metalloproteinase cleavage site, P1 is a glycine residue (Gly), P2 is a leucine residue (Leu) or an alanine residue (Ala), when P2 is a leucine residue (Leu), P1' is a leucine residue (Leu) and P2' is an arginine residue (Arg); A matrix metalloproteinase-responsive agent, wherein when P2 is an alanine residue (Ala), P1' is a leucine residue (Leu) or a methionine residue (Met), and P2' is an alanine residue (Ala), a tryptophan residue (Trp), a valine residue (Val), an arginine residue (Arg), or a threonine residue (Thr).

2. The response agent according to claim 1, wherein the amino acid sequence is the amino acid sequence represented by SEQ ID NO: 1, 4, 5, 9, 10 or 11.

3. The response agent according to claim 1 or 2, wherein the matrix metalloprotease comprises at least one selected from the group consisting of MMP-2, MMP-3 and MMP-9.

4. the matrix metalloproteinase is MMP-2; The response agent according to any one of claims 1 to 3, which is cleaved by the matrix metalloproteinase when reacted with an MMP-2-containing solution in which the MMP-2 concentration is 0.45 nM or more.

5. The responsive agent according to any one of claims 1 to 4, wherein the cleavage reaction with the matrix metalloprotease is observed by the resonance energy transfer (RET) phenomenon.

6. A cancer sensor or a cancer marker comprising the response agent according to any one of claims 1 to 5.

7. A quantitative assay kit for matrix metalloproteinase, comprising the response agent according to any one of claims 1 to 5.

8. A drug delivery system comprising the responsive agent according to any one of claims 1 to 5.

9. A cell separation material comprising the responsive agent according to any one of claims 1 to 5.

10. A substrate for three-dimensional cell culture comprising the responsive agent according to any one of claims 1 to 5.

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