JP7739876B2 - Peptide substrates cleavable by matrix metalloproteinases - Google Patents

Description

The present invention relates to peptide substrates that can be cleaved by matrix metalloproteinases.

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, and the concerted action of various MMPs allows them to degrade almost all components that make up the extracellular matrix. MMPs are also deeply involved in physiological and pathological processes, with activation of gelatinases (MMP-2, MMP-9) triggering cancer invasion and metastasis, and MMP-3, which is expressed and secreted during synovial proliferation, triggering the autoimmune disease rheumatoid arthritis. Rapid detection of MMP activation is thought to lead to early detection and treatment of these diseases, for example.

Material development targeting MMPs is underway in a variety of fields, including microporous gel systems used in cell transplants and wound dressings (Patent Document 1), cell carriers for three-dimensional culture (Patent Document 2), and biosensors (Patent Document 3).

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 biosensor capable of real-time detection of enzymes (e.g., MMP-2) that serve as biomarkers 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, and the wound healing process can be monitored by detecting electrical resistance.

Special Publication No. 2017-522113 Special table 2016-517694 publication Special Publication No. 2021-502149

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.

The object of the present invention is to provide peptides having amino acid sequences that can be cleaved by various proteases (e.g., MMP-2, MMP-3, and MMP-9).

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); 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).
[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] In the 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). The responder according to [4].
[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 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].

The present invention provides peptides having amino acid sequences 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 lead to the provision of cancer sensors or cancer markers (for supplementing existing cancer markers and for primary testing), MMPs 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.

Enzyme cleavage test results for MMP-2. Enzyme cleavage test results for MMP-3. Enzyme cleavage test results for MMP-9. Catalytic efficiency for MMP-2; kcat/Km. Enzyme cleavage test results for MMP-2 at an enzyme concentration [E] = 1.0 nM. Results of enzyme cleavage test for MMP-2 at an enzyme concentration [E] = 0.5 nM. Enzyme cleavage test results for MMP-2 at an enzyme concentration [E] = 0.45 nM.

The following provides a detailed description of an embodiment of the present invention (hereinafter simply referred to as "the present embodiment"). The following 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 implemented with appropriate modifications within the scope of its intent.

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). Furthermore, the above amino acids are broadly classified as D- or L-isomers based on their configuration; however, the amino acid residues constituting the amino acid sequence having a protease cleavage site herein are preferably L-isomers.

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

There are no particular limitations on the method for synthesizing peptides and proteins, and they may be organic chemical synthesis methods or genetic engineering synthesis methods using microorganisms or cultured cells. The organic chemical synthesis method is not particularly limited, and examples include the 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, and native chemical ligation methods using various peptide segments synthesized by the solid-phase or liquid-phase method.

As used herein, "protease" refers to a hydrolytic enzyme that catalyzes the hydrolysis of peptide bonds in peptides and proteins, "matrix metalloproteinase (MMP)" refers to a protease with a zinc ion (Zn ²⁺ ) coordinated in the active center, and the protease concentration is expressed as [E].

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 such site or domain 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 the P1 site, P2 site, etc. from the cleavage site toward the N-terminus, and the 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 adjacent residues P2 and P2' can affect the substrate specificity and cleavage efficiency of various proteases.

There are no particular limitations on the method for observing the cleavage reaction, and it may be observed, for example, by changes in electrochemical potential, fluorescence, chemiluminescence, phosphorescence, absorbance, antibody binding, or mass. There are no particular limitations on the method for observing the change, and for example, fluorescence resonance energy transfer (FRET), which occurs based on dipole-dipole interactions between a donor fluorescent molecule and an acceptor fluorescent molecule, may be used. Molecules that 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, etc. The molecules that cause the FRET phenomenon and units containing their molecular structures may be introduced into the peptide backbone or into the peptide side chain.

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, for example, from 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. In this specification, the catalytic efficiency is preferably 5.0 × 10 ³ M ⁻¹ s ⁻¹ or more, more preferably 8.0 × 10 ³ M ⁻¹ s ⁻¹ or more, even more preferably 1.0 × 10 ⁴ M ⁻¹ s ⁻¹ or more, and particularly preferably 1.2 × 10 ⁴ M ⁻¹ s ⁻¹ or more. Furthermore, when deriving the 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 observing the above-mentioned change before and after the enzymatic cleavage reaction, the substrate concentration is preferably 5 μM or more, 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.

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.

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

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), tryptophan residue (Trp), valine residue (Val), arginine residue (Arg), or threonine residue (Thr).

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 peptides of the present invention may contain at least one amino acid sequence 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 from a structural and functional perspective, 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). 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."

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.

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 restrictions on the amino acid sequence to be added.

Examples of the peptides of the present invention include peptides containing the amino acids of SEQ ID NOs: 12 to 21 or 22. Preferably, the peptides of the present invention 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.

The length of the peptides of the present invention can be 40 amino acid residues or less, 30 amino acid residues or less, 20 amino acid residues or less, 10 amino acid residues or less, or 8 amino acid residues or less. Peptides within this length range are more suitable for use in three-dimensional cell culture substrates, MMP quantification kits, etc.

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.

The peptides of the present invention can be cleaved by at least MMP-2, MMP-3, and MMP-9, and can therefore be used as responsive agents to proteases (e.g., MMP-2, MMP-3, and MMP-9). As used herein, "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 in a variety of applications.

For example, Jin C et al. , Blood Droplet-Based Cancer Diagnosis via Proteolytic Activity Measurement in Cancer Progression, Theranostics 2017, Vol. 7, Issue 11, 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. As described in, for example, "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), MMPs quantification assay kits, DDS (Drug Delivery Systems), cell separation materials, cell separation materials, substrates for three-dimensional cell culture, and the like. Because the peptides of the present invention are cleaved by MMP-2, MMP-3, and MMP-9, they can be used as cancer sensors or cancer markers (for supplementing existing cancer markers and for primary testing), MMP quantification assay kits, DDS (Drug Delivery Systems), cell separation/sorting materials, and substrates for 3D cell culture, as 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.

It is known that in order to grow and metastasize, cancer secretes large amounts of MMP-2 and MMP-9, which penetrate into blood vessels and spread via the bloodstream. In other words, since the presence of cancer increases the concentrations of MMP-2 and MMP-9 in the blood, the peptides of the present invention could 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 could 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 are capable of cleaving MMP-3, which is classified as a stromelysin group, they could also be used as a primary diagnostic agent for rheumatoid arthritis, for example, in addition to cancer.

The peptides of the present invention can also be used in kits (for research use) to quantify the expression levels of MMPs. They can also be used in assays to evaluate related physiological phenomena and pathological conditions. Because the peptides of the present invention are highly sensitive, they are believed to have low LOD (Limit of Detection) values. Therefore, high accuracy and high sensitivity are expected in assays in the low concentration range.

Because the peptide of the present invention has the characteristic of reacting with and being cleaved by MMPs, it may also be applicable to a field known as ADC (Antibody Drug Conjugate) (drug + peptide linker + antibody). For example, by molecular design such as the following structural formula, it is possible to develop a DDS application material using a peptide linker that is cleaved by an enzyme called cathepsin. By changing the peptide linker in the following structural formula to the peptide of the present invention, it is thought that an ADC can be developed that reaches the target cancer cells, and then the peptide linker is quickly cleaved by MMPs released by the cancer cells, allowing the drug to be released appropriately.

By immobilizing the peptides of the present invention on surface modifiers, crosslinkers, coating materials, etc. and then filling these materials into columns, they can be used as cell separation or fractionation materials. Furthermore, if technology were available to control the holes (pores) that are cleaved in response to MMPs, separation or fractionation by size would also be possible.

The peptides of the present invention can be used as crosslinkers for polymer matrices, making them applicable to the three-dimensional 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 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, creating three-dimensional cancer spheroid culture models, and other applications.

Since the peptides of the present invention can be degraded by enzymes, they may also be applicable to the development of enzymatically degradable plastics and post-surgical sutures, etc.

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

<Composition of reaction solution, buffer solution, 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 Ltd., 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 cyano(hydroxyimino)acetate; 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.)
1.5 mM peptide solution: FRET substrate, DMSO
Tris-hydrochloride 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.)
1 mM APMA reaction solution: 1 M APMA stock solution, Tris-HCl buffer 3.6 μM MMP-2 solution: recombinant MMP-2 (manufactured by Biovision Inc.), Tris-HCl buffer

<Fmoc Peptide Solid Phase Synthesis>
Peptide substrates having the amino acid sequence represented by SEQ ID NO: 12, 15, 16, 20, 21, or 22, and peptide substrates having the amino acid sequence represented by 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)), respectively, were synthesized by Fmoc solid-phase synthesis. As a peptide substrate having the amino acid sequence represented by 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 represented by 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, manufactured by BIO RAD) was placed in a multi-solid-phase synthesizer (KMS-3, manufactured by Kokusan Chemical Co., Ltd.). Rink amide AM resin (100-200 mesh, 0.7 mmol/g, manufactured by Merck Ltd.) was weighed out to a 0.05 mmol scale, and 2 mL of dichloromethane (DCM) (peptide synthesis grade, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added and allowed to swell for 12 to 24 hours under vortex stirring. After removing the swelling solution, 2 mL of deprotection solution was added to deprotect the Fmoc group on the resin (2 mL x 2 times, 10 minutes 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 carried out under vortex stirring for 2-3 hours to couple the amino acid onto the resin support. After removing the reaction solution, 2 mL of inactivation solution was added, and the resin support was treated under vortex stirring for 30 minutes to cap any unreacted sites. After removing the inactivation solution, the resin support was washed with DMF and DCM to obtain an Fmoc amino acid-incorporated resin support. Subsequently, the above-mentioned deprotection, coupling reaction, and inactivation of unreacted sites were repeatedly carried out to synthesize a peptide substrate having the target amino acid sequence.

<Rough purification, LC separation and purification>
After synthesizing a peptide having a predetermined amino acid sequence, the N-terminal Fmoc group was deprotected, washed with DMF, DCM, and methanol, and then allowed to stand in a desiccator for 24 hours to obtain a dry resin support. Two mL of chilled deprotection solution was added to the dry resin support and treated with vortex stirring 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), followed by centrifugation (2,000 × g, 10 minutes) to collect the precipitate. 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 by a medium pressure liquid chromatograph (model: EPCLC-AI-580S, Yamazen Co., Ltd.). The acetonitrile was distilled off under reduced pressure, and the fraction was lyophilized to obtain a purified peptide.

Example 1: Verification of MMP-2 cleavage by 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 (manufactured by BioVision Inc.) was used, and cleavage tests were performed according to the manufacturer's recommended protocol. Purified peptides having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, and 22 were each dissolved in DMSO to obtain six 1.5 mM peptide solutions. The resulting 1.5 mM peptide solutions were each 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 MMP-2 assay buffer to obtain an MMP-2 solution (enzyme concentration: [E] = 7 μM). The resulting MMP-2 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-2. Measurement samples were added to the plate at 50 μL/well, 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).

Figure 1 shows the results for 60 minutes after the start of fluorescence measurement. 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.

Comparative Example 1: Verification of MMP-2 cleavage by peptides having 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.

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 due to the FRET phenomenon (ΔRFU value) was lower compared to Example 1. In other words, the peptide substrate having the amino acid sequence represented by SEQ ID NO: 23 or 24 had a slow reaction rate, suggesting low cleavage ability (Figure 1).

[Example 2: Verification of MMP-3 cleavage by 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 the plate was 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).

Figure 2 shows the results for 60 minutes after the start of fluorescence measurement. The horizontal axis of Figure 2 represents the time (minutes) after the start of fluorescence measurement, and the vertical axis represents 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.

Comparative Example 2: Verification of MMP-3 cleavage by 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.

In this evaluation system, neither peptide substrate having the amino acid sequence shown in SEQ ID NO: 23 nor 24 exhibited cleavage ability by MMP-3 (Figure 2).

[Example 3: Verification of MMP-9 cleavage by 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 obtained MMP-9 solution was added to a 96-well black plate (manufactured by Thermo Fisher Scientific) at 50 μL/well, and the MMP-9 was activated by incubating at 37°C for 30 minutes. Measurement samples were added to the plate at 50 μL/well, 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]=5.2 μM, 37° C., 120 minutes).

Figure 3 shows the results for 60 minutes after the start of fluorescence measurement. The horizontal axis of Figure 3 represents the time (minutes) after the start of fluorescence measurement, and the vertical axis represents 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.

Comparative Example 3: Verification of MMP-9 cleavage by 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.

In this evaluation system, it was confirmed that the substrate having the amino acid sequence represented by SEQ ID NO: 24 was capable of cleaving MMP-9. On the other hand, the substrate having the amino acid sequence represented by SEQ ID NO: 23 did not exhibit the ability to cleave MMP-9 (Figure 3).

Example 4: Evaluation of MMP-2 enzymatic activity of 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 activated MMP-2 solution was added to a 96-well black plate at 50 μL/well and incubated at 37°C for 1 hour. After incubation, 50 μL/well of the measurement sample was added. Fluorescence was measured 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). After calculating the initial reaction velocity v ₀ (μM min ⁻¹ ) from the measured values, 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/v ₀ = (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 rate (μM min ^-1 ), [S]: substrate concentration (μM), kcat: catalytic constant (s ^-1 ), [E] ₀ : initial enzyme concentration (μM))

The catalytic efficiency of the peptide substrate having the amino acid sequence represented by SEQ ID NO: 12, 15, 16, 20, 21, or 22 was 1.0 × ¹⁰ (M ^-1 ·s ^-1 ) or more. In particular, the peptide substrate having the amino acid sequence represented by SEQ ID NO: 15, 16, or 21 had a catalytic efficiency of 1.5 × ¹⁰ (M ^-1 ·s ^-1 ), which was 2 to 4 times higher than that of Comparative Example 4 described below (Figure 4).

[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.

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

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 in the same manner 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 in the same manner 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. Subsequently, 50 μL/well of the 25 μM 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] = 1.0 nM, 0.5 nM, or 0.45 nM, [S] = 12.5 μM, 37°C, 60 minutes, or 120 minutes).

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, cleavage progress was confirmed for peptide substrates having the amino acid sequences represented by SEQ ID NOs: 12, 15, 16, 20, 21, or 22 under all enzyme concentration conditions tested ([E] = 1.0 nM, 0.5 nM, or 0.45 nM).

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.

In this evaluation system, cleavage of the peptide substrate having the amino acid sequence represented by SEQ ID NO: 23 or 24 was confirmed at enzyme concentrations of 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, cleavage could not be confirmed at an enzyme concentration of 0.45 nM (Figure 7).

Claims (10)

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) . 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. The response agent according to claim 1 or 2 , wherein the matrix metalloproteinase comprises at least one selected from the group consisting of MMP-2, MMP-3 and MMP-9. 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. 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. A cancer sensor or a cancer marker comprising the response agent according to any one of claims 1 to 5 . A quantitative assay kit for matrix metalloproteinase, comprising the response agent according to any one of claims 1 to 5 . A drug delivery system comprising the responsive agent according to any one of claims 1 to 5 . A cell separation material comprising the responsive agent according to any one of claims 1 to 5 . A substrate for three-dimensional cell culture, comprising the responsive agent according to any one of claims 1 to 5 .