JP2020029403A - Peptides that strongly bind and activate cathepsin e - Google Patents

Abstract

To provide peptides that promote the biological activity of cathepsin E (CE), the peptide having an activation function higher than conventional peptides, and to provide methods for measuring CE activity and CE amount in a sample using this peptide.SOLUTION: Provided is a peptide having an amino acid sequence of formula 1: X1-C-X3-X4-X5-D-X7-X8-V-E-V-Q-X13-E-V-A-E-A-X19-X20-X21-X22-L-X24-L-X26-P-G-X29 (SEQ ID NO: 1) (in the formula 1, X1 is G, L, N, A or V; X3 is P or T; X4 is C, H or Y; X5 is I, A, E or V; X7 is F or L; X8 is K or M; X13 is V or M; X19 is L, T, Q or P; X20 is L, S, Q, E or P; X21 is S, T or A; X22 is A or R; X24 is S or H; X26 is S or H; X29 is W or S), but except for the amino acid sequence in which X8 is M and X29 is S.SELECTED DRAWING: Figure 5

Description

  The present invention relates to novel peptides and their uses. More specifically, the present invention relates to a peptide that affects enzyme activity, a medicine containing the peptide, and a method for detecting an enzyme using the peptide.

  Cathepsin E (hereinafter sometimes abbreviated as CE) is an intracellular aspartic protease, and is mainly expressed in specific cells such as immune cells, digestive epithelial cells, and skin tissues (Non-Patent Document 1). This enzyme is present in keratinocytes (keratinocytes) and the inner root sheath of hair follicles in normal skin, and is present mainly in organelles such as endosomes in antigen presenting cells such as macrophages of immune system cells (Patent Reference 1).

  It has been reported that a reduction in cathepsin E activity causes malignant tumors and immunoallergic diseases. Kawakubo et al. Found that cathepsin E regulates the expression of proteins related to signal transduction, development, differentiation and proliferation in mouse mammary gland, and that cathepsin E deficiency induces precancerous state in mouse mammary gland tissue (Non-patent Document 2) Furthermore, it has been reported that serum cathepsin E activity is significantly correlated with favorable prognosis of patients (Non-Patent Document 3). This study indicates that suppression of cathepsin E expression increases the risk of breast carcinogenesis and is associated with a poor prognosis for breast cancer. In addition, cathepsin E-deficient mice do not show any abnormalities when bred in a sterile environment, but are reported to exhibit atopic dermatitis-like symptoms (AD) when transferred to a normal environment. Decreased expression has also been observed in erythrocytes of both human and AD mouse models NC / Nga with AD (Non-Patent Document 4). On the other hand, although the clinical significance is controversial, there are reports suggesting the usefulness of cathepsin E as a biomarker for specific human cancers (Non-Patent Documents 5 to 9). However, this conflicting report is thought to arise from the difference in the expression level of non-functional cathepsin E in different types of cancer cells (Non-Patent Document 3).

  Cathepsin E is a protease having a strong anticancer effect. In mouse experiments, it has been found that the expression level of cathepsin E is positively correlated with the degree of tumor suppression, and it is thought that cathepsin E suppresses potent tumor growth and metastasis via multiple pathways (Non-Patent Document) 10). First, in vitro, cathepsin E specifically cleaves and releases the apoptosis-inducing molecule TRAIL from the surface of cancer cells and induces cancer cell-specific apoptosis by binding to the death receptor on the surface of cancer cells. It is clear (Non-Patent Document 10). Furthermore, it has been reported that cathepsin E is involved in the suppression of tumor-related angiogenesis and induces increased infiltration and activation of macrophages around tumors (Non-Patent Document 10). Cathepsin E has also been reported to induce growth arrest and apoptosis of human prostate cancer cells (Non-Patent Document 11). In a recent study, correlation analysis between serum levels of cathepsin E expression and clinicopathological parameters reported that activity levels but not protein levels were negatively associated with breast cancer stage and progression (non- Patent Document 3).

  Therefore, a drug that increases the expression / activity of cathepsin E is expected to be useful for treating cancer. In particular, cathepsin E is of great interest as a target for peptide drug discovery. Problems with antibody drugs that are currently the mainstream in drug discovery include the high molecular weight of antibodies and the high cost of synthesis. On the other hand, peptides are about one hundredth the size of antibodies and can be synthesized at low cost, and are easy to modify or chemically modify, so they can be applied to drug delivery systems or regulate the biological activity of target molecules by mimicking molecules. Is possible.

A peptide that specifically binds to cathepsin E and has an inhibitory effect on its activity has been reported (Patent Document 2). This peptide was produced by a molecular selection operation using an in-vitro-virus (IVV) method.
On the other hand, in the development of a peptide (peptide aptamer) that specifically binds to an enzyme, it is more difficult to develop a peptide having an enzyme activating effect than that having an enzyme activity inhibiting effect. Cathepsin E-activating peptide evolves the library using a development library method, which is a accelerated in vitro molecular evolution method, and screens out the enzyme activity function using the SF link method (Non-Patent Document 12). (Non-Patent Documents 13 and 14). Reported the development of a peptide having a length of 8 to 16 amino acid residues that activates cathepsin E under a neutral environment as a primary library and a secondary library (Non-Patent Document 13). Komatsu et al. Have reported the creation of a tertiary library in which two peptides each having a length of 8 amino acid residues are linked by a linker (Non-patent Document 14).

  Furthermore, the use of cathepsin E as a tumor marker has been proposed. A method of detecting breast cancer by measuring the amount of cathepsin E in a test sample and associating the measurement result with the possibility of breast cancer or the prognosis of breast cancer has been reported (Patent Document 3). It has been reported that the measurement of cathepsin E may be performed using a cathepsin E substrate, an antibody against cathepsin E, or a peptidic compound (aptamer) having specific affinity for cathepsin E (patent) Reference 3). A method for measuring the amount of cathepsin E using a peptide that specifically binds to cathepsin E has been reported (Patent Document 4 and Non-Patent Document 15). Furthermore, a pharmaceutical composition for improving, preventing or delaying the aging phenomenon, including a cathepsin E protein or a substance having a cathepsin E protein expression promoting action (interferon γ or lipopolysaccharide) has been reported (Patent Document 1). ).

JP 2011-105686A JP 2007-332085 JP 2011-137686 A JP 2011-115146A

Yamamoto K, Kawakubo T, Yasukochi A, Tsukuba T, Emerging roles of cathepsin E in host defense mechanisms, Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, Volume 1824, Issue 1, 2012, Pages 105-112. Kawakubo T. et al. (2008) Gene expression profiling of mammary glands of cathepsin E-deficient mice compared with wild-type littermates. Biochimie, 90, 396-404. Kawakubo T, Yasukochi A, Toyama T, et al.Repression of cathepsin E expression increases the risk of mammary carcinogenesis and links to poor prognosis in breast cancer.Carcinogenesis.2014 Mar; 35 (3): 714-26. Tsukuba T, Okamoto K, Okamoto Y et al. Association of cathepsin E deficiency with development of atopic dermatitis.J Biochem. 2003 Dec; 134 (6): 893-902. Ullmann, R. et al. (2004) Protein expression profiles in adenocarcinomas and squamous cell carcinomas of the lung generated using tissue microarrays.J. Pathol., 203, 798-807. Blaveri, E. et al. (2005) Bladder cancer outcome and subtype classification by gene expression.Clin. Cancer Res., 11, 4044-4055. Mota, F. et al. (1997) Cathepsin E expression by normal and premalignant cervical epithelium. Am. J. Pathol., 150, 1223-1229. Caruso, M. et al. (2009) Over-expression of cathepsin E and trefoil factor 1 in sessile serrated adenomas of the colorectum identified by gene expression analysis.Virchows Arch., 454, 291-302. Uno, K. et al. (2000) Clinical significance of cathepsin E in pancreatic juice in the diagnosis of pancreatic ductal adenocarcinoma.J. Gastroenterol. Hepatol., 15, 1333-1338. Kawakubo, T, Yasukochi, A, Nakamura S, and Yamamoto, K. Cathepsin E as a potent anticancer protease. J. Oral Biosci. 53 (2): 128-136, 2011. Kawakubo T, Okamoto K, Iwata JI, et al. Cathepsin E prevent tumor growth and metastasis by catalyzing the proteolytic release of soluble TRAIL from tumor cell surface.Cancer Research.2007; 67 (22): 10869-10878. Naimuddin M, Kitamura K, Kinoshita Y, et al. Selection-by-function: efficient enrichment of cathepsin E inhibitors from a DNA library. Journal of Molecular Recognition. 2007; 20 (1): 58-68. Biyani M, Futakami M, Kitamura K, et al. In vitro selection of cathepsin E-activity-enhancing peptide aptamers at neutral pH.International Journal of Peptides.International Journal of Peptides Volume 2011 (2011), Article ID 834525, 10 pages Komatsu M, et al., "Peptide-Modulated Activity Enhancement of Acidic Protease Cathepsin E at Neutral pH," International Journal of Peptides, vol. 2012, Article ID 316432, 7 pages, 2012. Tung et al., "1 Peptide aptamer-modified single walled carbon nanotube-based transistors for high-performance biosensors Scientific Reports 7: 17881 DOI: 10.1038 (2017).

  An object of the present invention is to provide a peptide that promotes the activity of cathepsin E, which has a higher activation function than conventional peptides, and / or has a higher binding affinity for cathepsin E than conventional peptides. And In addition, another object is to provide a method for measuring the enzyme activity of cathepsin E and a method for measuring the amount of cathepsin E using the above peptide. Furthermore, it is an object of the present invention to provide a drug for cancer treatment containing a peptide that promotes the activity of cathepsin E.

  We have based on the tertiary library and further evolved it by our unique approach to have a much higher activation function than the cathepsin E activating peptide with the highest activating power belonging to the tertiary library The peptide was found. In addition, a drug for cancer treatment containing this peptide was found.

  The peptide aptamers belonging to the tertiary library have a detection sensitivity of several tens of nM as a dissociation constant. Despite having a certain strength, it was not accurate enough to measure the concentration of cathepsin E in a biological sample (eg, in blood) (several nM or less). The peptide aptamer obtained this time is expected to detect low-concentration cathepsin E in blood with sufficient accuracy.

The present invention is as follows.
[1] Formula 1: X1-C-X3-X4-X5-D-X7-X8-VEEVQ-X13-EVAVEAEA-X19-X20-X21-X22- L-X24-L-X26-PG-X29
(In the formula 1, X1 is G, L, N, A or V;
X3 is P or T;
X4 is C, H or Y;
X5 is I, A, E or V;
X7 is F or L;
X8 is K or M;
X13 is V or M;
X19 is L, T, Q or P;
X20 is L, S, Q, E or P;
X21 is S, T or A;
X22 is A or R;
X24 is S or H;
X26 is S or H;
X29 is W or S) (SEQ ID NO: 1), except for the amino acid sequence where X8 is M and X29 is S.
[2] The peptide according to [1], wherein X8 in the above formula 1 is K (SEQ ID NO: 2).
[3] The peptide according to [1], wherein X8 in the above formula 1 is M and X29 is W (SEQ ID NO: 3).
[4] In the above formula 1,
X1 is G or L;
X3 is P,
X4 is C or H;
X5 is I or A;
X7 is F,
X19 is L,
X20 is L, S, Q or E;
X21 is S or T;
X22 is A (SEQ ID NOs: 4 to 6),
The peptide according to any one of [1] to [3].
[5] Assuming that the original enzyme activity of cathepsin E is 100%, the enzyme activity of cathepsin E is further increased by about 20% or more than that of the peptide (CEAP3) obtained from the tertiary library. 5] The peptide according to any one of the above.
[6] The peptide according to any one of [1] to [5], wherein the peptide has a dissociation constant (KD) for binding to cathepsin E of 1 nM or less.
[7] A medicament for treating cancer, comprising the peptide according to any one of [1] to [6].
[8] A method for measuring the enzymatic activity of cathepsin E in a sample,
(A) mixing the sample with the solid-phased peptide aptamer to obtain cathepsin E bound to the solid-phased peptide aptamer;
(B) contacting cathepsin E bound to the immobilized peptide aptamer with a labeled cathepsin E substrate; and (C) detecting cathepsin E substrate activity by cathepsin E cleavage to determine cathepsin E enzyme activity. A method, wherein the peptide aptamer is the peptide according to any one of [1] to [6].
[9] A method for measuring the amount of cathepsin E contained in a sample,
(A) a step of mixing the sample with the immobilized peptide aptamer to obtain cathepsin E bound to the immobilized peptide aptamer; and (b) detecting the peptide aptamer to which cathepsin E is bound and detecting the cathepsin E in the sample. A method for determining the amount, wherein the peptide aptamer is the peptide according to any one of [1] to [6].
[10] The method according to [9], wherein the method is performed using a carbon nanotube field effect transistor method.
In the above step (a), the immobilized peptide aptamer is a peptide aptamer immobilized on a carbon nanotube provided on an electrode, and in the above step (b), the binding of the peptide aptamer and cathepsin E is carried out. A method comprising measuring before and after a potential difference between the electrode and a standard electrode.

  The peptide of the present invention has a higher binding affinity for cathepsin E than conventional peptides. Therefore, the peptide of the present invention can be used as a peptide aptamer to detect low-concentration cathepsin E in a sample. The peptide of the present invention has a higher binding affinity for cathepsin E and a higher action of promoting cathepsin E activity than conventional peptides. Therefore, the peptide of the present invention can be used as a peptide aptamer to measure the enzymatic activity of cathepsin E in a sample with high sensitivity. The peptide of the present invention has a higher action of promoting cathepsin E activity than the conventional peptide. Therefore, the peptide of the present invention can further enhance the function of inducing apoptosis in cancer cells, and is expected to be applied to cancer treatment.

Construct for point replacement quaternary library (P109) (SEQ ID NO: 45). For each codon in the Variable region (bases 382 to 468), it is designed so that mutations are added to 19 amino acids excluding the original amino acid. Rally. Schematic diagram of in vitro selection using Selection-by-Function introduction type cDNA display. Gel electrophoresis diagram of the selection product of the point-substituted quaternary library. Lane 1: 100 bp ladder, Lane 2: No binding to cathepsin E, Lane 3: Washing with selection buffer 1, Lane 4: Washing with selection buffer 2, Lane 5: Washing with selection buffer 3, Lane 6: Cathepsin E enzyme reaction product, lane 7: washing with wash buffer, lane 8: eluate (each 524 bp band). Amino acid sequence of selection product of quaternary library of point substitution. The linker region is shaded. Amino acid point substitutions are indicated by □ (boxed letters). The number next to the amino acid sequence is the copy number obtained. Cathepsin E activity promotion degree of point-substituted peptide. The notation on the vertical axis indicates the degree of activity promotion. The activity of CE is obtained by adding 100% to this numerical value. Error bars indicate standard deviation (3 experiments). Gel electrophoresis results of the additive point-substituted quaternary library. Lane 1: 100 bp ladder, lane 2: reference band (545 bp), lane 3: additive point substitution library (524 bp). Lane 3 represents a 524 bp band including sequences necessary for subsequent transcription, ligation, translation, and selection. Nucleotide sequence waveform of the quaternary library of additive point substitution by direct sequencing. Base sequence waveforms of the additive point-substituted quaternary library before and after selection. The base sequence waveforms by the direct sequence method before and after selection are shown vertically. Amino acid sequence of the selection product of the quaternary library of the additive point substitution. Residues shown in bold and underlined indicate sites where mutations were confirmed as a result of introduction using the oligomers in Table 3 and selection using the prepared mutant library. Residues underlined only indicate sites containing mutations considered to be due to errors in the extension reaction from hybridization for each fragment during library construction. The number next to the amino acid sequence is the copy number obtained. Degree of enhancement of cathepsin E activity of additive point-substituted peptide. The activity evaluation includes 13 types of additive point-substituted quaternary library selection products and M8K mutant peptides whose activity on cathepsin E was not examined during the point-substitution library selection. The notation on the vertical axis indicates the degree of activity promotion. The activity of CE is obtained by adding 100% to this numerical value. Error bars indicate standard deviation (3 experiments).

  The description of the present invention described below may be made based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range represented by using “to” means a range including numerical values described before and after “to” as a lower limit and an upper limit.

(Definition)
“Cathepsin E” in this specification means a cathepsin E protein, and may be described as CatE or CE in this specification.
As used herein, "peptide aptamer" refers to a peptide that specifically binds to a particular molecule. As used herein, the term "peptide aptamer" is used to refer to whether it inhibits or promotes the biological activity of a particular molecule, or affects the biological activity of a particular molecule. A peptide that specifically binds to a particular molecule, whether or not it has, is meant. In this specification, the “peptide aptamer” may be simply referred to as an aptamer.

  As used herein, “cathepsin E activating peptide (CEAP)” directly, indirectly, substantially completely or partially induces, promotes, or enhances the biological activity of cathepsin E. And any peptide capable of doing so. Normally, cathepsin E functions at acidic pH, but in the present specification, the second activity of cathepsin E, which functions at neutral pH, is selected (screened) using a substrate effective for the second activity. Activate cathepsin E in a neutral pH environment.

  “CEAP3” herein is a tertiary library selection product and has the amino acid sequence “GCPCIDFMVEVQVEVAEALLTALSLSPGS” (SEQ ID NO: 7). Assuming that the activity of cathepsin E in the absence of the peptide is 100%, the activity of cathepsin E in the presence of CEAP3 is 150%. In Non-Patent Document 14, a peptide capable of increasing the activity of cathepsin E to 175% or 186% in the presence of a peptide as compared with the absence of the peptide is obtained.

  “X1” “X3” “X4” “X5” “X7” “X8” “X13” “X19” “X20” “X21” “X22” “X24” “X26” of Formula 1, 2 or 3 in this specification "" X29 "indicates an arbitrary amino acid residue, and the number attached to X indicates the position from the N-terminal side of the amino acid sequence described in Formula 1 for convenience. In addition, for example, the second amino acid residue from the N-terminus is not described as X2 in Formula 1, but is described as C (cys). Since an arbitrary amino acid residue may be added to the N-terminal side of the peptide of the present invention, in that case, X1 is not the first amino acid residue.

  The term "peptide" generally refers to a continuous, relatively short sequence of amino acids linked by peptide (amide) bonds. Typically, but not necessarily, the peptide has a length of about 2 to about 50 amino acids, about 4 to 40 amino acids, or about 10 to 30 amino acids.

The term "polypeptide" generally refers to long forms of the peptide, but "polypeptide" and "peptide" may be used interchangeably in some contexts herein.
The terms "amino acid" and "residue" are used interchangeably herein.

  A “region” of a peptide or polypeptide is typically a contiguous sequence of two or more amino acids. Consisting of at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 contiguous amino acids.

  As used herein, "library" refers to a group of molecules having different structures. The library of the present invention includes an mRNA display library, a cDNA display library, and the like. In the present specification, the step of selecting a molecule having a specific property from the library is referred to as “selection” because the step is selected particularly based on the functional activity of the molecule. In the present specification, “selection” and “selection” are used interchangeably.

(peptide)
As described above, the peptide of the present invention has a higher activation function than the tertiary library selection product (including CEAP3) by further evolving it based on the tertiary library selection product by a unique approach. Based on the finding of a peptide group having A further evolutionary approach is to create a quaternary library (stage 1) by introducing point substitutions in the sequence, the second stage being obtained by culling the library. Molecular library of mutants (multipoint substitution mutants) containing point substitutions that are thought to improve activity based on the sequence information of the dominant mutants (mutants with higher activity than the original ones) and including a plurality of them in any combination (Additional point substitution library), that is, a quaternary library (second stage) is prepared, and the selection of the quaternary library is to select a group of peptides having a high activation function for cathepsin E. In these two steps, the knowledge of the amino acid sequence suitable for the peptide aptamer to have a high activation function was obtained, and the peptide of the present invention represented by the following formula 1 was reached.

  In the preparation of the quaternary library using the point-substituted product in the first step, the amino acid sequence shown in FIG. 4 and the degree of cathepsin E activity promotion shown in FIG. 5 indicate that the point-substituted L20E is a peptide having an activation function higher than CEAP3. And a peptide having T21S. Furthermore, these results clarify the sites and amino acid types in the two aptamer regions where the activity is not significantly reduced even if amino acid substitutions are made.

  Furthermore, in the selection of the quaternary library of the additive point substitution product in the second step, a peptide group having a high activation function for cathepsin E is determined from the amino acid sequence shown in FIG. 9 and the degree of cathepsin E activity promotion shown in FIG. A group of peptides with point substitution M8K and / or S29W was found. Furthermore, these results show that in the two aptamer regions, the position where the biological activity possessed by the peptide does not significantly decrease even when amino acid substitution occurs and the type of the amino acid, and the position where the activity tends to decrease and the type of the amino acid Is revealed.

  From the results of these two-step experiments, it was found that one or both of M8K and S29W significantly improved the promotion of cathepsin E activity, and that L20E and T21S found in the first step significantly promoted cathepsin E activity. It can be seen that the peptide does not contribute to the improvement, but a peptide containing one or both of M8K and S29W can significantly improve the promotion of cathepsin E activity regardless of the amino acids before and after the substitution. I understand.

Similarly, based on the results of these two experiments, the present invention defined the following equation 1.
Formula 1: X1-C-X3-X4-X5-D-X7-X8-VE-V-Q-X13-E-V-A-E-A-A-X19-X20-X21-X22-L-X24 -L-X26-PG-X29
(X1 is G, L, N, A or V;
X3 is P or T;
X4 is C, H or Y;
X5 is I, A, E or V;
X7 is F or L;
X8 is K or M;
X13 is V or M;
X19 is L, T, Q or P;
X20 is L, S, Q, E or P;
X21 is S, T or A;
X22 is A or R;
X24 is S or H;
X26 is S or H;
X29 is W or S) (SEQ ID NO: 1), except for the amino acid sequence where X8 is M and X29 is S.

In a preferred first embodiment of the invention, X8 in Formula 1 is K.
The peptides in which X8 is K shown in the results of the two-step experiment all show a higher cathepsin E activity promoting effect than CEAP3. In particular, MP04, 02, 13, 01, 03, 10, 09, and 11 show significant differences. It has a higher cathepsin E activity promoting effect than CEAP3 (see FIG. 10). MP07 and MP06 also have a higher cathepsin E activity promoting effect than CEAP3, but have no significant difference. From the former results, the substitution of amino acids other than X8 in MP04, 02, 13, 01, 03, 10, 09, 11 does not prevent the effect of promoting cathepsin E activity obtained when X8 is K. Has the same cathepsin E activity promoting effect as MP04 and MP03. The amino acid substitution of MP07 and MP06 slightly reduces the cathepsin E activity promoting effect as compared to MP03, but shows a higher cathepsin E activity promoting effect than CEAP3. In MP07 and MP06, X29 is S (instead of W), and in comparison with the results of MP02, the peptide in which X29 is W in MP07 and MP06 has a higher cathepsin E activity than MP07 and MP06. It is presumed that the effect will be higher than that of CEAP3, with a significant difference in cathepsin E activity promoting effect.

In a second preferred embodiment of the invention, X29 in Formula 1 is W.
From the results of MP12 and MP05, for the peptide in which X8 is M instead of K, even if X29 is W instead of S, a significant effect of promoting cathepsin E activity cannot be obtained, but the activity of promoting cathepsin E activity is higher than that of CEAP3. It has an effect (see FIG. 10). However, there is no significant difference. A peptide in which X8 is K and X29 is W is preferable from the viewpoint of having a higher cathepsin E activity promoting effect.

Amino acids that have been experimentally confirmed to be substitutable with amino acids other than X8 and X29 are X1, X3, X4, X5, X7, X13, X19, X20, X21, X22, X24, and X26.
X1 is G, L, N, A or V. From the results of the second step, it is clear that the peptide in which X1 is G or L has a higher cathepsin E activity promoting effect than CEAP3. It is clear from the results of the first step that the peptide in which X1 is N, A or V does not substantially reduce the cathepsin E activity promoting effect of the peptide, X8 is K, and X29 is S or W. It is expected that a certain peptide has a high probability of exhibiting a cathepsin E activity promoting effect substantially similar to that of MP02 and MP03. Here, MP02 is a peptide having point substitutions M8K, V13M and S29W but not containing other substitutions, and MP03 is a peptide having point substitutions M8K and V13M but not containing other substitutions. MP02 and MP03 show a high cathepsin E activity promoting effect (FIG. 10).

  X3 is P or T. It is clear from the results of the second step that the peptide in which X3 is P has a higher cathepsin E activity promoting effect than CEAP3. As a peptide in which X3 is T, only MP08 (X8 = M (not K), X29 = S (not W)) results, but no significant decrease in cathepsin E activity promoting effect is observed. Therefore, it is expected that a peptide in which X8 is K and X29 is S or W will exhibit a cathepsin E activity promoting effect close to MP02 and MP03.

  X4 is C, H or Y. From the results of the second step, it is clear that the peptide in which X4 is C or H has a higher cathepsin E activity promoting effect than CEAP3. From the results of the first step, it is clear that the peptide in which X4 is Y does not substantially reduce the cathepsin E activity promoting effect of the peptide. In the peptide in which X8 is K and X29 is S or W, It is expected that MP02 and MP03 are likely to exhibit almost the same cathepsin E activity promoting effect as MP02 and MP03.

  X5 is I, A, E or V. It is clear from the results of the second step that the peptide in which X5 is I, A, E or V has a higher cathepsin E activity promoting effect than CEAP3. It has also been shown from the results of the first step that the peptide in which X5 is A does not substantially reduce the cathepsin E activity promoting effect of the peptide. In the peptide in which X8 is K and X29 is S or W, , MP02 and MP03 are expected to have an effect of promoting cathepsin E activity.

  X7 is F or L. It is clear from the results of the second step that the peptide in which X7 is F has a higher cathepsin E activity promoting effect than CEAP3. As a peptide in which X7 is L, only the results of MP08 (X8 = M (not K), X29 = S (not W)) are shown, but no significant decrease in cathepsin E activity promoting effect is observed. Therefore, it is expected that a peptide in which X8 is K and X29 is S or W will exhibit a cathepsin E activity promoting effect close to MP02 and MP03.

  X13 is V or M. It is clear from the results of the second step that the peptide in which X13 is V or M has a higher cathepsin E activity promoting effect than CEAP3. It has also been shown from the results of the first step that the peptide in which X13 is M does not substantially reduce the cathepsin E activity promoting effect of the peptide. In the peptide in which X8 is K and X29 is S or W, , MP02 and MP03 have an effect of promoting cathepsin E activity.

  X19 is L, T, Q or P. From the results of the second step, it is clear that the peptide in which X19 is L, T or Q has a higher cathepsin E activity promoting effect than CEAP3. The results of the first step show that the peptide in which X19 is P does not substantially reduce the cathepsin E activity promoting effect of the peptide. In the peptide in which X8 is K and X29 is S or W, It is expected that a cathepsin E activity promoting effect close to MP02 and MP03 will be exhibited.

  X20 is L, S, Q, E or P. It is clear from the results of the second step that the peptide in which X20 is L, S or Q has a higher cathepsin E activity promoting effect than CEAP3. The peptide in which X20 is E or P has been shown from the results of the first step to not substantially reduce the cathepsin E activity promoting effect of the peptide, and the peptide in which X8 is K and X29 is S or W Is expected to exhibit a cathepsin E activity promoting effect close to MP02 and MP03.

  X21 is S, T, or A. From the results of the second step, it is clear that the peptide in which X21 is S or T has a higher cathepsin E activity promoting effect than CEAP3. It is also shown from the results of the first step that the peptide in which X21 is S does not substantially reduce the cathepsin E activity promoting effect of the peptide. In the peptide in which X8 is K and X29 is S or W, , MP02 and MP03 are expected to have an effect of promoting cathepsin E activity. As a peptide in which X21 is A, only the results of MP08 (X8 = M (not K) and X29 = S (not W) are shown, but no significant decrease in cathepsin E activity promoting effect is observed. , X8 is K and X29 is S or W, and it is expected that the peptide will exhibit a cathepsin E activity promoting effect close to MP02 and MP03.

  X22 is A or R. From the results of the second step, it is clear that the peptide in which X22 is A has a higher cathepsin E activity promoting effect than CEAP3. It is also shown from the results of the first step that the peptide in which X22 is R does not substantially reduce the cathepsin E activity promoting effect of the peptide. In the peptide in which X8 is K and X29 is S or W, , MP02 and MP03 are expected to have an effect of promoting cathepsin E activity.

  X24 is S or H. From the results of the second step, it is clear that the peptide in which X24 is S or H has a higher cathepsin E activity promoting effect than CEAP3.

  X26 is S or H. From the results of the second step, it is clear that the peptide in which X26 is S has a higher cathepsin E activity promoting effect than CEAP3. The results of the first step show that the peptide in which X26 is H does not substantially reduce the cathepsin E activity promoting effect of the peptide. X8 is K, and X29 is S or W. It is expected that a certain peptide will exhibit a cathepsin E activity promoting effect close to MP02 and MP03.

  A preferred peptide of the present invention has an amino acid sequence wherein X29 in the above formula 1 is W or S when X8 is K and X29 is W when X8 is M. Another preferred peptide of the invention is an amino acid sequence wherein X8 is K or M when X29 in the above formula 1 is W and X8 is K when X29 in the above formula 1 is S. Having.

  In a preferred embodiment, the peptide of the present invention comprises, in Formula 1, X1 is G or L, X3 is P, X4 is C or H, X5 is I or A, and X7 is F , X8 is M or K, X13 is V or M, X19 is L, X20 is L, S, Q or E, X21 is S or T, X22 is A, X24 Is S or H, X26 is S or H, X29 is W or S, provided that X8 is M and X29 is S.

  In a preferred embodiment, the peptide of the present invention comprises, in Formula 1, X1 is G or L, X3 is P, X4 is C or H, X5 is I or A, and X7 is F , X8 is K, X13 is V or M, X19 is L, X20 is L, S, Q or E, X21 is S or T, X22 is A, and X24 is S Or H, X26 is S or H, and X29 is a peptide having an amino acid sequence of W or S.

  In a preferred embodiment, the peptide of the present invention comprises, in Formula 1, X1 is G or L, X3 is P, X4 is C or H, X5 is I or A, and X7 is F , X8 is K, X13 is V or M, X19 is L, X20 is L, S, or Q, X21 is S or T, X22 is A, X24 is S or H, X26 is S, and X29 is a peptide having an amino acid sequence of W or S.

  In a preferred embodiment, the peptide of the present invention comprises, in Formula 1, X1 is G, X3 is P, X4 is C, X5 is I or A, X7 is F, and X8 is K X13 is V or M, X19 is L, X20 is L, X21 is S or T, X22 is A, X24 is S or H, X26 is S , X29 is a peptide having an amino acid sequence of W.

  The peptide of the present invention has the amino acid sequence of the above formula 1, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, Consists of 46, 47, 48, 49 or 50 amino acid residues.

  The peptide of the present invention further comprises N amino acids (where N is an integer of 1 or more and 21 or less) on the N-terminal side of the amino acid sequence of the above formula 1, and / or M (C-terminal side) Wherein M is an integer of 1 to 21).

  The peptide of the present invention has, as a first aptamer region, a compound represented by the formula 2: X1-C-X3-X4-X5-D-X7-X8 (wherein X1 is G, L, N, A or V; X3 is P or T, X4 is C, H or Y, X5 is I, A, E or V, X7 is F or L, or X8 is K or M. 8). That is, the first aptamer region is at least 8 amino acid residues in length, and includes all of the amino acid sequences composed of the combinations of the amino acid types described at the positions (X1 to X8) in the following table.

  Examples of the first aptamer region of the peptide of the present invention include GCPCIDFK (SEQ ID NO: 9), GCPCADDFK (SEQ ID NO: 10), GCPHIDFK (SEQ ID NO: 11), LCPCIDFK (SEQ ID NO: 12), GCPCADFM (SEQ ID NO: 13), GCPCEDFM (SEQ ID NO: 14), GCPCVD FK (SEQ ID NO: 15), GCTCIDLM (SEQ ID NO: 16), GCCPIDFM (SEQ ID NO: 17), NCCPIDFM (SEQ ID NO: 18), ACPCIDFM (SEQ ID NO: 19), GCCPCADFM (SEQ ID NO: 20), GCPYIDFM (SEQ ID NO: 21) and VCPCIDFM (SEQ ID NO: 22).

  The peptide of the present invention has a formula: X19-X20-X21-X22-L-X24-L-X26-PG-X29 (where X19 is L, T, Q or P, X20 is L, S, Q, E or P, X21 is S, T or A, X22 is A or R, X24 is S or H, X26 is S or H X29 is W or S) (SEQ ID NO: 23). That is, the second aptamer region is at least 11 amino acid residues in length, and includes all of the amino acid sequences composed of combinations of the amino acid types described at the positions (X19 to X29) in the following table.

  Examples of the second aptamer region of the peptide of the present invention include LLSALLSSPGW (SEQ ID NO: 24), LLTALLSLSPGW (SEQ ID NO: 25), LLTALHLSPGW (SEQ ID NO: 26), LLTALSLSPGW (SEQ ID NO: 27), LLTALSLSPGS (SEQ ID NO: 28), LSTALSLSPGS (SEQ ID NO: 29), LLTALSLSPGW (SEQ ID NO: 30), LQTALLSLSPGW (SEQ ID NO: 31), TTLALLSLSPGW (SEQ ID NO: 32), QLTALSLSPGS (SEQ ID NO: 33), LLAALSLSPGS (SEQ ID NO: 34), LLTALLSLSPGLS (SEQ ID NO: 35) (SEQ ID NO: 36), LETALSLSPGS (SEQ ID NO: 37), PLTALSLSPGS (SEQ ID NO: 38), LPTALSL PGS (SEQ ID NO: 39), and the like LLTRLSLSPGS (SEQ ID NO: 40) and LLTALSLHPGS (SEQ ID NO: 41).

  VEEVQX13-EVA-EAA (X13 is V or M) (SEQ ID NO: 42) in the amino acid sequence of the above formula 1 is a linker region. The linker region is a certain spacer necessary to link two peptides (aptamer regions) that independently bind to the target molecule (protein). The amino acid constituting the linker used in the tertiary library was glycine (Gly-Gly-Gly-Ser) × 2 to 3 (Gly-Gly-Gly-Ser) (Non-patent Document 14). In the selection of the tertiary library, peptides using VEEVQVEVEVA-EA as a linker obtained by frame-shifting a linker mainly composed of glycine are selected. The construct for the quaternary library of the present invention was designed using the linker sequence: VEV-Q-V-E-V-A-E-A-A (SEQ ID NO: 43) since the peptide remained as a modified peptide. did. The linker region of the peptide of the present invention is VEEVQVEVEAAEA or VEEVQMEVAEVAE. -A (SEQ ID NO: 44). In the peptide of the present invention, the first aptamer region and the second aptamer region are linked by the linker. The combination of the first aptamer region, the linker region, and the second aptamer region is not limited to the 13 combinations shown in FIG. Naturally, the peptides of the invention have the formula 1: X1-C-X3-X4-X5-D-X7-X8-VEEV-Q-X13-E-V-A-E-A- X19-X20-X21-X22-L-X24-L-X26-P-G-X29 (wherein X1 is G, L, N, A or V, X3 is P or T, and X4 is C, H or Y, X5 is I, A, E or V, X7 is F or L, X8 is K or M, X13 is V or M, X19 is L, T, Q or P, X20 is L, S, Q, E or P, X21 is S, T or A, X22 is A or R, X24 is S or H, X26 is S or H, X29 is W or S) (first aptamer region, linker region and second aptamer region included in the amino acid sequence of (SEQ ID NO: 1)) Including any combination of In one embodiment, the peptide of the present invention comprises a first aptamer region, a linker region and a second aptamer region in the order from N-terminal to C-terminal. In another embodiment, the peptide of the present invention comprises a second aptamer region, a linker region and a first aptamer region in order from N-terminal to C-terminal.

  The oligonucleotides, polynucleotides, peptides, polypeptides, and small molecules used in the present invention and described herein can be produced using standard techniques known in the art.

  The peptides of the present invention may be conjugated to a carrier. The carrier is a biodegradable polymer (eg, PEG, polylactide, polyglycolide, polycaprolactone, and copolymers thereof, carbohydrate, cellulose, chitin, and lignin, or a polypeptide carrier such as Fc and serum albumin). . Peptides of the invention conjugated to a carrier have improved half-life and / or bioavailability as compared to those not conjugated to a carrier.

(Fourth library selection product)
The peptide of the present invention is a cathepsin E-activating molecule (cathepsin E-activating peptide (CEAP)) having a function of promoting cathepsin E enzymatic activity.
The peptide of the present invention can increase the activity of cathepsin E in the presence of the peptide to at least about 140 to 220%, about 170 to 220%, or about 190 to 220% as compared to the absence of the peptide. In particular, the value of "200% or more" (that is, the ability to double the activity of cathepsin E) gives great promise for the pharmaceutical use of the peptide of the present invention for treating cancer. It also leads to a method for specifically and highly sensitively detecting the activity of cathepsin E. Studies of serum cathepsin E activity and prognosis of breast cancer patients have shown that patients with favorable prognostic results have approximately twice as high cathepsin E activity as compared to patients with unfavorable prognostic results (Non-Patent Literature). 3, Fig.1 (E) etc.). Therefore, molecules (eg, peptides) that can activate cathepsin E activity to at least 200% in breast cancer patients are expected to be useful for treating patients with poor prognosis. The peptide of the present invention has a cathepsin E activity of 140% or more, 150% or more, 170% or more, 172% or more, 173% or more, 175% or more, 180% or more, compared to the activity in the absence of the peptide. It can be 185% or more, 188% or more, 190% or more, 195% or more, 200% or more, 205% or more, 208% or more, 210% or more, 215% or more, 218% or more, or 220% or more. The peptide of the present invention has an activity of cathepsin E of about 140 to 220%, about 150 to 220%, about 170 to 220%, about 172 to 220%, about 173 to 220% as compared to the activity in the absence of the peptide. %, About 175 to 220%, about 180 to 220%, about 185 to 220%, about 188 to 220%, about 190 to 220%, about 195 to 220%, about 200 to 220%, about 205 to 220%, It can be about 208 to 220%, about 210 to 220%, or about 215 to 220%.

  The peptide of the present invention further increases the activity of cathepsin E by at least about 20%, at least about 22%, and at least, as compared to the peptide (CEAP3) obtained from the tertiary library, assuming that the original enzyme activity of cathepsin E is 100%. About 23%, at least about 25%, at least about 30%, at least about 35%, at least about 38%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 58%, at least It can be increased by about 60%, at least about 65%, at least about 68, or at least about 70%.

  The peptide of the present invention can bind to cathepsin E with a dissociation constant (KD) of 1 nM or less. Since the peptide of the present invention has a dissociation constant of 1 nM or less, that is, on the order of pM, it is expected that low concentration cathepsin E in a sample is detected. The peptide of the present invention binds to different portions of cathepsin E, respectively. Since it has one aptamer region and the second aptamer region, it has high specificity for cathepsin E.

(Medicine)
The peptide of the present invention is provided as a medicine containing the peptide. In one embodiment, the medicament of the invention is for treating a disease or disorder in an individual. In some embodiments, the condition of the disease or disorder is an autoimmune disease. In another embodiment, the medicament of the invention is for treating cancer.

The cancer to be treated by the present invention is any type of cancer. In a preferred embodiment, the peptide of the present invention is particularly effective for cancer in which cathepsin E is considered to be involved in the release of TRAIL ligand and the accompanying binding to TRAIL receptor and subsequent cancer cell death due to apoptotic reaction. it is conceivable that. The peptide of the present invention is considered to be particularly effective for free cancer cells (blood system or planktonic cancer cells) to which cathepsin E is easily accessible. The peptide of the present invention is expected to reduce and eliminate the surface cells of the solid cancer cells in a form of successive cell death. The peptides of the present invention can be used in therapeutic methods. Further, the peptide of the present invention can be used in a method for producing or preparing a medicament.
The individual or patient to be treated according to the invention is a mammal, preferably a human.

  The present invention provides a medicament for treating cancer, comprising a cathepsin-activating peptide as an active ingredient. To promote cathepsin E enzyme activity, an effective amount of a medicament of the present invention is administered to a patient. The medicament of the present invention may contain a pharmaceutically acceptable carrier in addition to the peptide as an active ingredient.

  In another aspect, the medicament of the present invention comprises two or more cathepsin E activating peptides (CEAP). The first CEAP and the second CEAP may bind to the same site of cathepsin E or may bind to different sites.

  The medicament of the present invention may be administered orally, pulmonary, intranasally, intralesional, topically, or intraocularly by any suitable means. The medicament of the present invention may be administered by parenteral injection, which includes intramuscular, intravenous, intraarterial, or intraperitoneal injection, or subcutaneous injection. Dosage regimens are appropriately planned depending on whether the administration is short-term or long-term. The medicament of the present invention is formulated in a dosage form optimal for the purpose of treatment, and an optimal and clinically appropriate administration route, administration method, dosage and administration schedule for the treatment are determined based on the knowledge of the medical staff.

(Method for measuring enzyme activity of cathepsin E)
The peptide of the present invention can be used as a peptide aptamer in a method for measuring the enzymatic activity of cathepsin E in a sample, including the following (a) to (c).
(A) mixing the sample with the solid-phased peptide aptamer to obtain cathepsin E bound to the solid-phased peptide aptamer;
(B) contacting cathepsin E bound to the immobilized peptide aptamer with a labeled cathepsin E substrate; and (C) detecting cathepsin E substrate activity by cathepsin E to determine cathepsin E enzyme activity.
Even if the peptide of the present invention captures cathepsin E, the bond therewith is a so-called allosteric bond and does not hinder the activity of the enzyme, but rather has the function of improving the activity. For this reason, the measurement sensitivity of the cathepsin E enzyme activity increases.

  What correlates with the prognosis of breast cancer patients is not the total amount of cathepsin E but the activity of cathepsin E (Non-Patent Document 3). Possibly, but not limited to, cathepsin E in breast cancer patients may be partially inactivated or its expression level may be reduced in the first place, and the former may reduce both the substance amount and the activity amount of cathepsin E. It can be determined by measuring. In the prior art, it is difficult to measure the activity with high sensitivity, and there is a situation that only skilled experts can do so. The peptide of the present invention binds to cathepsin E with a dissociation constant of 1 nM or less, that is, a pM order, and improves the enzymatic activity of cathepsin E. Therefore, when used for the measurement of cathepsin E activity, the measurement sensitivity is lower than that of the conventional peptide. It rises remarkably as compared with the case where is used.

Method (a) for measuring enzyme activity of cathepsin E
In the method for measuring the enzymatic activity of cathepsin E of the present invention, the peptide aptamer is immobilized on a solid phase such as a plate, a plate well, an array, a bead, a tube, or a chip. The peptide aptamer may be immobilized on a solid phase via a linker such as a peptide or PEG, or may be immobilized directly.

  The sample used in the measurement method of the present invention is not particularly limited as long as it is a sample containing CE, and is usually a biological sample, such as mammalian blood, serum, plasma, urine, stool, cerebrospinal fluid, Body fluids such as saliva, swabs, various tissue fluids, cells (cancer cells, benign tumor cells, malignant tumor cells) and their cell lysates, or tissues (cancer tissues, benign tumor tissues, malignant tumor tissues) and their It can be a tissue lysate.

  The mixing of the immobilized peptide aptamer and the sample may be performed, for example, by injecting a buffer containing the sample into a container containing the solid phase on which the peptide aptamer is immobilized, or And a buffer containing a peptide aptamer and solid phase-immobilized beads. By this mixing, CE in the sample binds to the immobilized peptide aptamer. Thus, cathepsin E bound to the immobilized peptide aptamer is obtained. Mixing of the peptide aptamer and the sample is performed by appropriately selecting conditions under which the peptide aptamer and the cathepsin E can bind when the sample contains cathepsin E. That is, if the concentration of cathepsin E is excessively present in the measurement sample and exceeds the concentration of bindable peptide, the sample can be diluted with a buffer and used. In order to remove contaminants that are nonspecifically and weakly bound to the immobilized peptide aptamer, the solid phase or a container containing the solid phase (eg, a well or tube of a plate) is removed after removing the buffer containing the sample. Washing with distilled water or the like is preferred.

Step (b) of the method for measuring the enzyme activity of cathepsin E
Cathepsin E bound to the immobilized peptide aptamer is contacted with a labeled cathepsin E substrate. The label may use, for example, a coloring substance, a fluorescent substance, a chemiluminescent substance, an enzyme, or the like. The contact between the cathepsin E bound to the immobilized peptide aptamer and the labeled cathepsin E substrate is performed in a solution whose pH is adjusted to neutral. This is because the peptide of the present invention activates cathepsin E under a neutral pH environment.

Step (c) of the method for measuring the enzyme activity of cathepsin E
Cathepsin E enzyme activity is determined by detecting cathepsin E substrate cleavage by cathepsin E. Cathepsin E substrate cleavage by cathepsin E is detected by monitoring the chemical change of the labeling substance. The detection can be performed by a method known to those skilled in the art. For example, in the case of a fluorescent substance, it can be detected by a fluorometer, and in the case of a coloring substance, it can be detected by a spectrophotometer.
Specific examples of the method for measuring the enzymatic activity of cathepsin E in a sample are described below. However, it is not intended to be limited to this example.

<Immobilization of peptide aptamer>
The peptide (peptide aptamer) of the present invention is diluted to 1 mg / ml with a coupling buffer (0.2 M NaHCO ₃ , 0.5 M NaCl, pH 8.3), and 50 μl of the peptide solution is arrayed on a peptide-coated plate (TaKaRa). And immediately add 10 μl Reaction solution (TaKaRa) to each well and incubate at room temperature for 2 hours. After incubation, the solution is removed from the wells of the plate and the peptide-coated wells are washed three times with 200 μl of distilled water. Next, 200 μl of a blocking solution (0.5 M ethanolamine, 0.5 M NaCl (pH 8.3)) is added to each well, and at room temperature for 1 hour to inactivate non-specific binding substances present in the container. Incubate. After removing the blocking solution, the wells are washed three times with 200 μl of distilled water.

<Capture of cathepsin E by peptide aptamer>
The cathepsin E protease-containing tissue extract or serum is adjusted to pH 7.4 with buffer (500 mM sodium acetate, 1 M NaCl, pH 4.0). The precipitate formed is removed by centrifugation at 15,000 rpm for 5 minutes, and the supernatant is recovered. 30 μl of the supernatant is added to each recognition peptide-immobilized well, incubated at room temperature for 20 minutes, and the solution is removed. The solution remaining on the plate is washed twice with 200 μl of distilled water.

<Measurement with Enzyme on peptide>
The amount of cathepsin E captured on the peptide is measured by an assay using a fluorogenic substrate. Fluorogenic substrate labeled with cathepsin E substrate (Nma-Gly-Gly-Arg -Arg-Ser-Gly-Thr-Cys-Gly (Dnp) -D-Arg-NH 2: SEQ ID NO: 101), sodium acetate 50mM And a solution (pH 4.5) containing 100 mM NaCl is added to each well and then incubated at 40 ° C. for 10 minutes.
Using a fluorescence plate reader FluPOLO (TaKaRa), the increase in fluorescence intensity generated by substrate cleavage during incubation is measured at an excitation wavelength of 360 nm / fluorescence wavelength of 430 nm.

(Method of measuring cathepsin E amount)
The peptide of the present invention is used as a peptide aptamer in a method for measuring the amount of cathepsin E contained in a sample, including the following (a) and (b).
(A) a step of mixing the sample with the immobilized peptide aptamer to obtain cathepsin E bound to the immobilized peptide aptamer; and (b) detecting the peptide aptamer to which cathepsin E is bound and detecting the cathepsin E in the sample. The process of determining the quantity.

Step (a) of the method for measuring the amount of cathepsin E
In the method for measuring the amount of cathepsin E of the present invention, the peptide aptamer is immobilized on a solid phase such as a plate, a well of a plate, an array, a bead, a tube, a chip, a biosensor chip, or the like. Examples of the solid phase material include glass, plastic, metal, and carbon.

  The sample used in the measurement method of the present invention is not particularly limited as long as it is a sample containing CE, and is usually a biological sample, such as mammalian blood, serum, plasma, urine, stool, cerebrospinal fluid, Body fluids such as saliva, swabs, various tissue fluids, cells (cancer cells, benign tumor cells, malignant tumor cells) and their cell lysates, or tissues (cancer tissues, benign tumor tissues, malignant tumor tissues) and their It can be a tissue lysate.

  The mixing of the immobilized peptide aptamer and the sample may be performed by mixing the sample and the immobilized peptide aptamer in a buffer solution.For example, the sample may be placed in a container containing the solid phase. And a buffer solution. By this mixing, CE in the sample binds to the immobilized peptide aptamer. Thus, cathepsin E bound to the peptide aptamer is obtained. Mixing of the peptide aptamer and the sample is performed by appropriately selecting conditions under which the peptide aptamer and the cathepsin E can bind when the sample contains cathepsin E.

Step (b) of the method for measuring the amount of cathepsin E
For detection of the peptide aptamer to which cathepsin E is bound, for example, an appropriate label is previously bound to the peptide aptamer, and a signal obtained from the label can be measured. As the label that can be used here, for example, a chemiluminescent substance as a substrate, other than HRP (Horse Radish Peroxidase), ALP (Alkaline Phosphatase), and other known substances such as a fluorescent label can be used. .

In the peptide aptamer of the present invention, the first aptamer region and the second aptamer region bind to the same site of the cathepsin E protein or to different sites. In a preferred embodiment of the present invention, the first aptamer region and the second aptamer region of the peptide aptamer of the present invention bind to different sites of the cathepsin E protein, and the binding ability of the peptide aptamer to cathepsin E is additive. Or increase synergistically. Since the peptide aptamer of the present invention has a first aptamer region and a second aptamer region that respectively bind to different portions of cathepsin E, it has high specificity for cathepsin E, and can accurately determine the amount of cathepsin E contained in a sample. Can be measured.
Since the peptide aptamer of the present invention binds to cathepsin E with a dissociation constant (KD) of 1 nM or less and on the order of pM, the measurement accuracy of the amount of cathepsin E is increased as compared with conventional peptides.

  In a preferred embodiment, the method for measuring the amount of cathepsin E of the present invention can be carried out using a single-walled carbon nanotube field effect transistor method (SWCNT FET). Here, the peptide aptamer immobilized in step (a) of the method for measuring the amount of cathepsin E may be a peptide aptamer immobilized on a carbon nanotube provided on an electrode, and the step (b) May be a step including measuring a potential difference between the standard electrode and the electrode before and after the binding of the peptide aptamer and cathepsin E.

  In the method for measuring the amount of cathepsin E using the SWCNT FET method, a SWCNT FET is manufactured, and the peptide of the present invention is immobilized as a peptide aptamer to a carbon nanotube of a SWCNT channel via a linker, and the SWCNT FET sensor for detecting cathepsin E is used. Prepare The concentration of the peptide aptamer and the linker and the incubation conditions for immobilization can be systematically adjusted and determined based on known techniques in the art. The peptide aptamer-modified sensor can be placed in 0.0005 × PBS (pH 7.4) and measured at various concentrations (eg, 0.1 to 10 ng / mL) of cathepsin E. The method of measuring the amount of cathepsin E using the SWCNT FET method can be implemented with reference to Non-Patent Document 15.

(About the library)
The peptides of the present invention were developed from a quaternary library positioned at the final stage of cathepsin-activating peptide development using a developmental library method, which is a accelerated in vitro molecular evolution method. The in vitro evolution method is based on live selection through a process of selecting a population that has a desired function from a library that is a population of molecules, including populations with random sequences, and a clone selection process that selects individual clones. The rally will be narrowed down to a group with high activity. As selection progresses, weakly active, low-affinity molecules are removed and library diversity is reduced. In other words, although the types of constituent molecular groups decrease, molecules having high activity remain. In order to select a group of molecules having a higher activity from this, a library of the next stage is constructed by artificially adding a mutation. Among the developmental library methods, there is a step of using a Y-linked block shuffling (YLBS) method for library evolution. In this method, molecular diversity of N ¹⁶ (for example, 10 ¹⁶ when starting from 10 elements at the time of departure) is realized by linking only four times using N (for example, 10) blocks as raw materials. Four amino acid blocks are selected and ligated from a peptide molecule obtained after performing selection in a population and selection in individual clones to obtain a secondary library, followed by selection. The secondary library selection product obtained as a result can be considered as a functional unit (module) (Kitamura K et al. Journal of Molecular Biology. 2009; 387 (5): 1186-1198, Non-patent Document 13). . A tertiary library (paired peptide library) was prepared by combining this functional unit and a spacer sequence (Non-patent Document 14). In the present specification, a quaternary library of point substitutions obtained by further adding a point substitution to the amino acid sequence of the selection product of the tertiary library is prepared (FIG. 1). After the quaternary point-substituted library has been selected, DNA shuffling (Stemmer WP. Nature. 1994, Aug4; 370 (6488): 389-91) is further introduced into those point-substituted quaternary library selection products. I do.

(CDNA display method)
In order to create a peptide, it is necessary to first associate a phenotype peptide portion with a genotype DNA portion. FIG. 2 shows a procedure for obtaining a target peptide / protein from a quaternary library using the cDNA display method developed in this laboratory.
The DNA of the quaternary library encoding the peptide is amplified by PCR and transcribed to produce mRNA, which is linked to a small molecule puromycin linker that associates phenotype with genotype. Then, the peptide is linked to puromycin by performing translation. Thereafter, reverse transcription is performed to stabilize the mRNA. Subsequently, cDNA display is produced by purifying the peptide chain linked to puromycin. Finally, by performing selection on the target, a peptide having a higher function (binding ability) can be obtained (Non-Patent Document 12, Tsuji-Ueno S, et al. Protein and Peptide Letters. 2011; 18 (6): 642-650). In this conjugate, an SF link (Naimuddin et al. J. MOL, Recog. 2007) has been introduced and can be selected by function (eg, enzyme activity control).

The experimental method will be described below.
Experimental method 1: Preparation of cDNA display The PCR product obtained by amplifying the DNA of the construct of the quaternary library of the point-substituted product shown in FIG. 1 and a transcription reaction reagent are mixed as follows.
PCR product 1 pmol
25 mM r (A, T, U, G) TP mix (Promega) 3μl
5 × T7 Trans Buffer (Promega) 2μl
RNase-free water (Promega) up to 9μl
T7 Enz mix (Promega) 1μl

The solution containing the PCR product is incubated at 37 ° C. for 3 hours, and 1 μl of RQ1 RNase-Free DNase (1 U / μl, Promega) is added, followed by incubation at 37 ° C. for 15 minutes. RNA purification was performed using RNeasy (Qiagen) to a final solution volume of 14 μl. The mRNA product is quantified using a spectrophotometer Nano Drop ND-1000.
The mRNA product obtained above and the ligation reagent are mixed as follows.
mRNA product 20 pmol
SBP (streptavidin puromycin) linker (20 pmol / μl) 22 pmol
10 × T4 ligase buffer (TaKaRa) 2.5μl
RNase-free water up to 20μl

Next, the ligation reaction is performed on the solution containing mRNA in the order of 90 ° C .: 2 min → 70 ° C .: 2 min → 25 ° C .: 3 sec → 4 ° C .: Hold. After adding 1 μl of T4 RNK (10 U / μl; TaKaRa) and 1 μl of T4 RNA ligase (30 U / μl; TaKaRa), the mixture is incubated at 25 ° C. for 1 hour and 20 minutes. Perform RNA purification and bring the final solution volume to 27 μl. The ligation product is confirmed by electrophoresis (Gel composition: Polyacrylamide gel (6% T, 5% C) containing 8 M Urea, 1 × TBE; electrophoresis conditions 100 V, 55 ° C., 5 × TBE, 8 min).
The ligation product and the translation reagent are mixed as follows. Make two tubes in a 0.2 ml tube.
Ligation product (2.2pmol / μl) 4μl
20 × Master Mix-Me 1.25μl
20 × Master Mix-Leu 1.25μl
Reticulocyte lysate 34μl
RNase-free water up to 50μl

After incubating the solution containing the ligation product for translation at 30 ° C. for 20 minutes, 20 μl of 3M KCl and 6 μl of 1M MgCl ₂ are added, and the mixture is incubated at 37 ° C. for 1 hour and 20 minutes. Confirm the mRNA display product by SDS PAGE (Gel composition: Polyacrylamide gel (4 → 6% T 5% C) containing 8 M Urea, 0.1 SDS; electrophoresis conditions: current 10 mA, 2 hours, electrophoresis buffer 25 mM Tris, 192 mM Glycine, 0.1% SDS). The mRNA display product is purified using SA-beads (Dynabeads MyOne Streptavidin C1).
The SA-beads to which the mRNA display product is bound and the reagent for reverse transcription are mixed as follows.
Total amount of beads to which mRNA display product is bound
5 × ReverTraAce buffer (TOYOBO) 8μl
2.5 mM dNTP mix 16 μl
RNase-free water up to 15μl
ReverTraAce Enzyme (TOYOBO) 1μl

  After incubating the solution containing the beads at 42 ° C. with mixing for 20 minutes, the supernatant is removed. Wash twice with 100 μl of 5 mM imidazole in PBS500. Add 0.5 μl RNase T1 (1000 U / μl) to 5 mM imidazole in PBS500 containing 39.5 μl beads. Incubate at 37 ° C for 20 minutes and collect the supernatant. Further, the peptide chain linked to puromycin is purified (His Mag sepharose Ni-beads, biospin column, Xa) to obtain a cDNA display.

Experimental method 2: In vitro selection method For the obtained cDNA display, a cathepsin E-activating peptide is selected using a selection-by-function method. Prepare NHS-beads (NHS Mag Sepharose, GE Healthcare) on which cathepsin E is immobilized. The cDNA display is incubated at 25 ° C. for 10 minutes in Selection buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl 2, pH 7.4) containing cathepsin E-immobilized NHS-beads. Unbound DNA molecules are washed away three times with Selection buffer. The DNA molecules bound to the cathepsin E immobilized NHS-beads are incubated at a temperature (37 ° C.) suitable for cathepsin E proteolysis. Wash with Wash buffer (50 mM Tris-HCl, 1.0 mM NaCl pH 7.4) and elute with elution buffer (25% NH ₃ ) at 65 ° C. for 10 minutes. The DNA molecules eluted from the beads can be assumed to be largely produced by the enhanced proteolytic reaction of cathepsin E and are collected for the next evaluation step. This procedure is repeated three times (1 min, 20 sec, 10 sec) while shortening the reaction time for cathepsin E proteolysis. The final product was subjected to cloning and sequencing to identify the cathepsin E activating peptide, a product of the point-substituted quaternary library.

Experimental Method 3: Evaluation of Cathepsin E Activating Peptide To determine the enhancing activity of selected peptides on cathepsin E activity, peptides were synthesized by in vitro translation or chemical synthesis. 20 nM cathepsin E and the selected peptide are pre-incubated for 10 minutes at 25 ° C. in Selection buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.4). Next, 5 μM of a fluorescent substrate (MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys (Dnp) -D-Arg-NH ₂ : SEQ ID NO: 102) was added, and the mixture was added at 37 ° C. for 1 hour. Let react. The fluorescence intensity is observed at 440 nm (excitation 340 nm) using a fluorimeter. Cathepsin E normally functions at acidic pH, but a fluorogenic substrate sensitive at neutral pH (7.4) is described in SBP Athauda and K. Takahashi (Protein and Peptide Letters, vol.9, no.1, pp. 15-22, 2002).

The activity (%) of cathepsin E was derived from the following formula.
Sf is the fluorescence intensity of the cathepsin E reaction in the presence of the selected peptide, Cf is the fluorescence intensity of the cathepsin E reaction in the absence of the selected peptide, and Bf is the background fluorescence of a solution containing only the fluorescent substrate. . Cathepsin E activity becomes 100% in the absence of peptide. In the graphs of FIGS. 5 and 10, the notation on the vertical axis indicates the degree of activity promotion, and the activity of cathepsin E is obtained by adding 100% to this numerical value.

Experimental method 4: Design and construction of quaternary library of additive point-substituted substance A new library is prepared by performing DNA hybridization and extension reaction using oligomers. Mix the hybridization reagents as follows.
10 × Ex taq Buffer 2.5μl
2.5 mM dNTP mixture 2 μl
Hybridization oligomer 100 pmol each
Template DNA 500 ng
DDW up to 25μl
Ex Taq (5 U / μl) (TaKaRa) 0.125μl
In each hybridization step, a multi-step reaction is performed in the same tube.
Perform the reaction in the following cycle: 95 ° C = 2min → 25 ° C (35 ° C) = 5min → 40 ° C = 10min → 72 ° C = 5min. However, the hybridization temperature is changed with respect to the Tm of each oligomer.

Perform the above cycle 6 times and join the fragments. The final product is amplified by PCR using ordinary primers to obtain a quaternary library of additive point substitutions.
Hereinafter, examples of the present invention will be described, but the scope of the present invention is not limited to the examples.

Example 1: Functional selection type cDNA display A point-substituted quaternary library obtained by further adding a point substitution to an amino acid sequence to a selection product of a tertiary library obtained previously (Non-Patent Document 14) is shown in FIG. Made from construct.
In the in vitro selection (see the left inset of FIG. 2) in which the Selection-by-Function method is introduced in contrast to the normal cDNA display method, the so-called cDNA display portion is composed of a cDNA portion (information) + linker + peptide. (Function), and a cathepsin E substrate sequence is arranged at the head of this peptide portion. Therefore, when a peptide binds to cathepsin E, cathepsin E may be activated depending on the binding mode (inhibition / neutral / activation). At that time, “a state in which a carrot is hung on the nose of a horse” And the substrate portion is cleaved. In that case, the functional region of the peptide remains bound to cathepsin E, but the DNA portion becomes free by cleavage of the peptide in the substrate portion and comes out into solution. Conversely, when the peptide inhibits cathepsin E, no cleavage occurs and the entire cDNA display binds to cathepsin E and no DNA comes out in solution. FIG. 3 shows an experiment in which a cleaved DNA fragment was confirmed by acrylamide gel electrophoresis using this principle. The cDNA display (lane 6) selected by this series of in vitro selection methods showed a darker band (524 bp) as compared to the cDNA display (lane 5) which had not undergone the selection step after washing.

Example 2: Sequencing of selection product of quaternary library of point-substituted products Next, the sequence of selection products of quaternary library of point-substituted products was determined. A total of 62 clones and 42 types of sequences were determined. FIG. 4 shows the results. In particular, 10 copies of the sequence in which the 21st amino acid Thr was mutated to Ser and 3 copies of the sequence in which the 20th Leu was mutated to Glu were obtained. Although the linker sequence also had an amino acid substitution but was not originally a point-added site, it is considered to be an unintended advantageous mutation derived from PCR.

Example 3 Function Evaluation of Point-Substituted Peptide for Cathepsin E Of the 42 clone sequences selected as the selection product, 7 clones having the same sequence, 6 randomly selected clones, and a tertiary live without mutation Cell-free translation was performed on a total of 14 clones of the rally selection product (CEAP3), and the ability of the peptide to promote cathepsin E function was verified according to Experimental Method 3. The results are shown in FIG.
In particular, the peptide having the mutation T21S or L20E, which had a large copy number among the obtained clones, showed an increase of about 35% and 38%, respectively, as compared with the tertiary library selection product (CEAP3). On the other hand, the peptide having the mutation C4Y, G1V, or A22R exhibited a slightly lower activity promoting ability than CEAP3. In the peptide having the mutation V13M, a decrease of about 10% was confirmed. The 21st serine, which has a high appearance frequency, is the same hydrophilicity as the original threonine (T), and the 20th glutamic acid is an acidic polar side chain amino acid that exhibits acidity. The hydrophilic amino acid is involved in promoting the activity of cathepsin E. It is suggested that you do.

Example 4: Preparation of a quaternary library of additive point replacements Next, based on the results of Example 3, a quaternary library of additive point replacements combining a plurality of point replacements was prepared.
Using the oligomers shown in Table 3 (SEQ ID NOs: 46 to 74), a new library was prepared by performing the DNA hybridization and extension reaction described in Experimental Method 4. If all point substitution candidates have been introduced, the amino acid sequence: NCPYADFKVEVQMEVAEAPESALHLSPGW (SEQ ID NO: 103) is created.

  The quaternary library of additive point substitutions was confirmed by electrophoresis (FIG. 6, lane 3). In addition, it was confirmed by direct sequencing that the entire library by the extension reaction was correctly constructed (FIG. 7). It was confirmed that the sequence required for the next selection step could be constructed. For other point substitutions, sequences with mutations added at a minimum of 50% were obtained. In the point substitution of G1N, C4Y, and I5A, the sequence without mutation was predominant.

Example 5: Selection and evaluation of the quaternary library of the additive point substitution body For the quaternary library of the additive point substitution body prepared in Example 4, a cDNA display was prepared according to Experimental Methods 1 to 3, and cathepsin E and The selected products of the quaternary library of the additive point substitution product were obtained by the reaction. The selection product was analyzed by the direct sequencing method. FIG. 8 shows the obtained base sequence waveform.
From the waveforms of the DNA base sequence before and after selection, in the mutation of M8K, the second base showed almost the same intensity before selection, but the codon of AAG indicating Lys to the codon of AAG indicating Lys was stronger than that of ATG indicating Met. Showed strength. Similarly, in S29W, the codons of TCG indicating Ser and TGG indicating Trp had almost the same intensity before selection, but the codon corresponding to Trp showed higher intensity after selection. In addition, L20E and T21S showing the highest activity among the selection products of the quaternary library of the point-substituted product also converged to a sequence close to the wild type by the selection. These results suggest that the peptide having the mutation of M8K and S29W has an action of further promoting the activity of cathepsin E.

Example 6: Sequencing of the quaternary library of the additive point-substituted quaternary library The amino acid sequence of the culled product obtained by performing two rounds of selection on the quaternary library of the additive point-substituted substance is shown in FIG. . Interestingly, the T21S and L20E additive point substitutes with the highest activity promoting ability were not selected in the original point-substituted quaternary library selection product. This suggests that the complexity of the sequence space that cannot be inferred by simple inferences, that is, not only the contribution of individual amino acids but also their interrelationship, is important for the shape and function of the three-dimensional structure of proteins. ing.
As mutants, many mutations of M8K, V13M, and S29W were observed, and two-point and three-point additive point-substituted sequences were obtained. In addition, an additive point-substituted sequence to which the T21S mutation was added in the two clones having the highest activity among the selection products of the point-substituted quaternary library was also obtained.

Example 7: Evaluation activity of selection product of quaternary library of additive point-substituted product Activity for cathepsin E was evaluated according to Experimental Method 3 obtained by performing two rounds of selection. FIG. 10 shows the results of the 13 types of sequences obtained by the selection and the activity evaluation of a total of 14 types of M8K mutant peptides for which the activity against cathepsin E was not examined at the time of the point substitution library selection.
In particular, an additive point-substituted peptide containing a sequence having a mutation of M8K and a mutation of S29W activated cathepsin E at a high level. The addition activity was nonlinearly and positively correlated with the activity promotion obtained in the activity evaluation of the point-substituted peptide, which was a rational result in protein science (generally not simple addition). This demonstrated the effectiveness of the additive point replacement library.
The product of the quaternary library of the additive point substitution, which had the highest effect of promoting cathepsin E activity, increased the activity of cathepsin E to 218%. In comparison with CEAP3, the result of the third stage of the developmental library method, an increase of 68% was observed (MP04).

Example 8: Characterization by dissociation constant The binding affinity between cathepsin E and peptide MP04, which is a selection product of the quaternary library of the additive point-substituted quaternary library obtained in Example 7, was measured by using Biacore X100 (GE Healthcare). It was determined by the plasmon resonance method (SPR method). Cathepsin E was immobilized on a Biacore sensor chip NTA (GE Healthcare, UK) by an amine coupling method using an amine coupling kit (GE Healthcare, UK). Five different concentrations (200 μM, 40 μM, 8 μM, 1.6 μM and 0.32 μM) of peptide MP04 were used for the interaction to determine the dissociation constant. Since peptide MP04 is not very soluble, experiments were performed at high peptide concentrations using 10% DMSO in the experiments. The dissociation constant was calculated in Evaluation Software version 2.0.1, using a 1: 1 binding model. The results are shown in the table below.
The dissociation constant (KD) of peptide MP04 (SEQ ID NO: 91) was calculated to be 3.13 pM. In a conventional tertiary library, a peptide that binds to cathepsin E with a dissociation constant (KD) of 1 nM or less has not been developed. Since the peptide of the present invention binds to cathepsin E with a dissociation constant of 1 nM or less, cathepsin E (several nM or less) contained in a biological sample (for example, in blood) at a low concentration can be measured with sufficient accuracy. .

SEQ ID NOs: 1-41: Cathepsin E activating peptide SEQ ID NOs: 42-44: Linker SEQ ID NO: 45: Construct SEQ ID NOs: 46-74: Oligomeric SEQ ID NOS: 75-87 described in Table 3: Peptide SEQ ID NO: shown in FIG. 88 to 100: peptide SEQ ID NOs: 101 and 102 shown in FIG. 9: cathepsin E substrate SEQ ID NOs: 103 and 104: mutant peptide

Claims (10)

Formula 1: X1-C-X3-X4-X5-D-X7-X8-VE-V-Q-X13-E-V-A-E-A-A-X19-X20-X21-X22-L-X24 -L-X26-PG-X29
(In the formula 1, X1 is G, L, N, A or V;
X3 is P or T;
X4 is C, H or Y;
X5 is I, A, E or V;
X7 is F or L;
X8 is K or M;
X13 is V or M;
X19 is L, T, Q or P;
X20 is L, S, Q, E or P;
X21 is S, T or A;
X22 is A or R;
X24 is S or H;
X26 is S or H;
X29 is W or S) (SEQ ID NO: 1), except for the amino acid sequence where X8 is M and X29 is S.   The peptide according to claim 1, wherein X8 of the formula 1 is K (SEQ ID NO: 2).   The peptide according to claim 1, wherein X8 in the formula 1 is M and X29 is W (SEQ ID NO: 3). In the above equation 1,
X1 is G or L;
X3 is P,
X4 is C or H;
X5 is I or A;
X7 is F,
X19 is L,
X20 is L, S, Q or E;
X21 is S or T;
X22 is A (SEQ ID NOs: 4 to 6),
The peptide according to any one of claims 1 to 3.   Assuming that the original enzyme activity of cathepsin E is 100%, the enzyme activity of cathepsin E is further increased by about 20% or more as compared with the peptide (CEAP3) obtained from the tertiary library. The peptide according to claim 1.   The peptide according to any one of claims 1 to 5, wherein a dissociation constant (KD) for binding to cathepsin E is 1 nM or less.   A medicament for treating cancer, comprising the peptide according to any one of claims 1 to 6. A method for measuring the enzyme activity of cathepsin E in a sample,
(A) mixing the sample with the solid-phased peptide aptamer to obtain cathepsin E bound to the solid-phased peptide aptamer;
(B) contacting cathepsin E bound to the immobilized peptide aptamer with a labeled cathepsin E substrate; and (C) detecting cathepsin E substrate activity by cathepsin E cleavage to determine cathepsin E enzyme activity. A method, wherein the peptide aptamer is a peptide according to any one of claims 1 to 6. A method for measuring the amount of cathepsin E contained in a sample,
(A) a step of mixing the sample with the immobilized peptide aptamer to obtain cathepsin E bound to the immobilized peptide aptamer; and (b) detecting the peptide aptamer to which cathepsin E is bound and detecting the cathepsin E in the sample. A method comprising determining the amount, wherein the peptide aptamer is a peptide according to any one of claims 1 to 6. The method according to claim 9, wherein the method is performed using a carbon nanotube field effect transistor method,
In the step (a), the immobilized peptide aptamer is a peptide aptamer immobilized on a carbon nanotube provided on an electrode, and in the step (b), the binding of the peptide aptamer and cathepsin E is performed. Before or after the method comprising measuring a potential difference of the electrode relative to a standard electrode.