JP5971644B2 - Method for regulating function of TPR motif protein, method for screening for substance regulating function of TPR motif protein, and substance for regulating function - Google Patents

Description

  The present invention relates to a method for regulating the function of a TPR motif protein, a method for screening a functional regulator of a TPR motif protein, and a functional regulator.

  In general, the function of a cell is switched on by protein phosphorylation by protein phosphorylase, and turned off by protein dephosphorylation by protein dephosphorase. Protein phosphatase 5 (hereinafter referred to as PP5 or PP5 protein), which is a kind of protein having a TPR (tetratricopeptide repeat) motif (hereinafter referred to as “TPR motif protein”), is an enzyme involved in the dephosphorylation of the latter. It is known to exist in almost all cells (Non-patent Documents 1 to 6). However, PP5 is usually low in activity, but it is unclear what kind of activation mechanism the activity is enhanced.

  PP5 has been reported to be involved in cancer, neuronal cell death in Alzheimer's disease, and cellular metabolic regulation. Therefore, it is important to elucidate the activation mechanism of PP5 and to screen for PP5 activity modulators in both research fields and clinical fields such as biochemistry. The same applies to other TPR motif proteins as well as PP5.

Ramsey, A.J., and Chinkers, M. (2002) .Identification of Potential Physiological Activators of Protein Phosphatase 5. Biochem. 41, 5625-5632. Kang H, Sayner SL, Gross KL, Russell LC, Chinkers M. (2001) .Identification of amino acids in the tetratricopeptide repeat and C-terminal domains of protein phosphatase 5 involved in autoinhibition and lipid activation. Biochemistry. 2001 Sep 4; 40 (35): 10485-90. Chinkers, M. (2001). Protein phosphatase 5 in signal transduction. Trends Endocrinol. Metab. 12, 28-32. Morita, K., Saitoh, M., Tobiume, K, Matsuura, H., Enomoto, S., Nishitoh, and H., Ichijo, H. (2001). Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress.EMBO J. 20 (21), 6028-6036. Liu F, Grundke-Iqbal I, Iqbal K, Gong CX. (2005). Contributions of protein phosphatases PP1, PP2A, PP2B and PP5 to the regulation of tau phosphorylation. Eur J Neurosci. 22 (8), 1942-50. Sanchez-Ortiz, E., Hahm, B.K., Armstrong, D.L., and Rossie, S. (2009). Protein phosphatase 5 protects neurons against amyloid-βtoxicity. J. Neurochem. 111, 391-402.

  Accordingly, an object of the present invention is to provide a method for regulating the function of TPR motif proteins such as PP5, a method for screening a functional regulator of TPR motif protein, and a functional regulator of TPR motif protein.

  In order to achieve the above object, the regulation method of the present invention is a method for regulating the function of a TPR motif protein, characterized in that an S100 protein (hereinafter also referred to as “S100”) is bound to the TPR motif protein. To do.

  The screening method of the present invention is a screening method for a TPR motif protein function-regulating substance, and includes an analysis step of analyzing the function of the TPR motif protein in the presence of a candidate substance.

  The function regulator of the present invention is a function regulator of a TPR motif protein and is characterized by containing at least one compound selected from the group consisting of formulas (1) to (50).

  The screening method of the present invention is a screening method for a substance that modulates the function of S100, and includes an analysis step of analyzing the function of the TPR motif protein in the presence of S100 and a candidate substance.

  As a result of intensive studies, the present inventors have found that PP5 activity is enhanced by binding of S100, which is an intracellular calcium receptor protein, to PP5, which is a kind of TPR motif protein. Based on this finding, a method for regulating the function of the TPR motif protein by S100 has been achieved. Furthermore, based on this finding, for example, a screening method for various functional regulators of TPR motif proteins such as S100 antagonist compounds was constructed, and this method has led to the provision of various functional regulators. Since TPR motif proteins such as PP5 are related to diseases such as cancer as described above, the present invention can be said to be an extremely important technique in both research fields such as biochemistry and clinical fields.

FIG. 1 shows the results of the pull-down assay in Example 1. FIG. 2 shows the result of SPR in Example 1. FIG. 3 (A) is an outline of the GST fusion protein in Example 1, and FIG. 3 (B) is the result of the pull-down assay. FIG. 4 (A) shows the results of the pull-down assay in Example 1, and FIG. 4 (B) shows the results of Western blotting. 5A and 5B are the results of the pull-down assay in Example 1. FIG. 6A to 6C are graphs showing PP5 dephosphorylation activity in Example 1. FIG. 7 (A) and (B) are the results of Western blotting in Example 1, and FIG. 7 (C) is a graph showing the relative value of dephosphorylation relative to the control. 8A to 8C are the results of Western blotting in Example 1. FIG. FIG. 9 is a graph showing the dephosphorylation activity of PP5 in the presence of a regulator in Example 2. 10 (A) and (B) are graphs showing the dephosphorylation activity of PP5 in Example 2 in the presence of a regulator. 11A to 11C are graphs showing the dephosphorylation activity of PP5 in Example 3 in the presence of a regulator. 12 (A) to 12 (D) are graphs showing the dephosphorylation activity of PP5 in Example 3 in the presence of a regulator. FIG. 13 is a graph showing the dephosphorylation activity of PP5 in the presence of a modulator in Example 3. 14 (A) to 14 (D) are graphs showing the dephosphorylation activity of PP5 in Example 3 in the presence of a regulator. FIG. 15 is a graph showing PP5 dephosphorylation activity in the presence of a modulator in Example 4. FIGS. 16A and 16B are graphs showing the dephosphorylation activity of PP5 in Example 5 in the presence of a regulator. FIGS. 17A and 17B are graphs showing the dephosphorylation activity of PP5 in Example 5 in the presence of a modulator. 18 (A) to 18 (C) are graphs showing the dephosphorylation activity of PP5 in Example 6 in the presence of a regulator. 19A and 19B are graphs showing the dephosphorylation activity of PP5 in Example 7 in the presence of a regulator. FIG. 20 is a graph showing the relative value of PP5 dephosphorylation activity in the presence of a modulator in Example 8. FIG. 21 is a graph showing the relative value of PP5 dephosphorylation activity in the presence of a modulator in Example 8. 22 is a graph showing the relative value of the dephosphorylation activity of PP5 in the presence of a modulator in Example 8. FIG. FIG. 23 is a graph showing the relative dephosphorylation activity of PP5 in the presence of a modulator in Example 8. FIG. 24 is a graph showing the relative value of PP5 dephosphorylation activity in the presence of a modulator in Example 8. FIG. 25 is a graph showing the relative value of PP5 dephosphorylation activity in the presence of a modulator in Example 8. FIG. 26 is a graph showing the relative value of PP5 dephosphorylation activity in the presence of a modulator in Example 8. FIG. 27 is a graph showing the relative dephosphorylation activity of PP5 in the presence of a modulator in Example 8.

(Method for regulating the function of TPR motif protein)
As described above, the method for regulating the function of the TPR motif protein of the present invention is characterized in that S100 is bound to the TPR motif protein.

  The binding between the TPR motif protein and S100 can be performed by bringing the TPR motif protein into contact with S100. The contact between the TPR motif protein and S100 may be, for example, in vivo or in vitro. In the case of in vivo, for example, it can be performed by administering a TPR motif protein and / or S100 to a living body. In this case, one of the TPR motif protein and S100 (for example, TPR motif protein) may be derived from the living body, and the other (for example, S100) may be administered from the outside of the living body, or both may be administered from the outside of the living body. Also good. In the case of in vitro, for example, the contact between the TPR motif protein and S100 may be performed in an artificial environment such as a test tube or an incubator, or in cells such as cultured cells (including the meaning of tissue). it can. In the latter case, for example, both the TPR motif protein and S100 may be derived from a cell, one may be derived from a cell, the other may be administered from outside the cell, or both may be derived from the cell. It may be administered. The administration may be, for example, administration as a protein or administration of a coding gene of the protein. In the latter case, for example, the protein may be expressed from the coding gene. The coding gene may be administered by, for example, a vector.

  The TPR motif protein is a protein having a TPR motif as described above. In the present invention, the type of the TPR motif protein is not particularly limited, and examples thereof include PP5, FKBP52, Cyclophilin 40, and kinesin light chain. In the present invention, it is understood that S100 regulates its function by binding to the TPR domain of the TPR motif protein.

  PP5 is a protein phosphatase. PP5 has a TPR domain and a catalytic domain (CD). In the case of PP5, it is understood that S100 enhances the dephosphorylation activity of PP5 by binding to its TPR domain.

  The origin of the TPR motif protein is not particularly limited, and examples thereof include human origin and non-human animal origin other than human. Examples of the non-human animal include non-human mammals such as mice, rats, rabbits, dogs, and sheep. As a specific example, human-derived PP5 is registered with NCBI accession number BC001970, for example, and can be represented by SEQ ID NO: 1. In SEQ ID NO: 1, the first to 181st regions (underlined portions) are TPR domains, and the 182nd to 499th regions are catalytic domains.

SEQ ID NO: 1 (PP5): BC001970
MAMAEGERTECAEPPRDEPPADGALKRAEELKTQANDYFKAKDYENAIKF
YSQAIELNPSNAIYYGNRSLAYLRTECYGYALGDATRAIELDKKYIKGYY
RRAASNMALGKFRAALRDYETVVKVKPHDKDAKMKYQECNKIVKQKAFER
AIAGDEHKRSVVDSLDIESMTIEDEYSGPKL EDGKVTISFMKELMQWYKD
QKKLHRKCAYQILVQVKEVLSKLSTLVETTLKETEKITVCGDTHGQFYDL
LNIFELNGLPSETNPYIFNGDFVDRGSFSVEVILTLFGFKLLYPDHFHLL
RGNHETDNMNQIYGFEGEVKAKYTAQMYELFSEVFEWLPLAQCINGKVLI
MHGGLFSEDGVTLDDIRKIERNRQPPDSGPMCDLLWSDPQPQNGRSISKR
GVSCQFGPDVTKAFLEENNLDYIIRSHEVKAEGYEVAHGGRCVTVFSAPN
YCDQMGNKASYIHLQGSDLRPQFHQFTAVPHPNVKPMAYANTLLQLGMM

Although PP5 is illustrated below, this invention is not restrict | limited to this. Examples of PP5 include at least one protein selected from the group consisting of the following (A1) to (A3).
(A1) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (A2) comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of (A1) And a protein having the same function as the protein of (A1) (A3) comprising an amino acid sequence having 80% or more identity with the amino acid sequence of (A1), and identical to the protein of (A1) Protein with the function of

  In the above (A2) and (A3), “same function” means dephosphorylation activity.

  In the above (A2), “one or several” is not particularly limited, and is, for example, 1 to 30, preferably 1 to 20, more preferably 1, 2, 3, 4, 5 , 6, 7, 8, 9, or 10.

  In the above (A3), “identity” is, for example, the degree of identity when the sequences to be compared are appropriately aligned, and means the appearance rate (%) of an exact match of amino acids between the sequences. . The identity can be performed, for example, by using an arbitrary algorithm, and specifically, BLAST or the like can be used. For example, the default parameters can be used for calculating the identity. The same applies hereinafter. The identity is, for example, 80% or more, more preferably 85% or more, further preferably 90% or more, still more preferably 95% or more, particularly preferably 96% or more, 97% or more, 98% or more, or 99%. That's it.

  The origin of S100 is not particularly limited, and examples thereof include human origin and non-human animal origin other than human. Examples of the non-human animal include non-human mammals as described above. Examples of human-derived PP5 include S100A1, S100A2, S100A6, S100B, and S100P. For example, S100A1 is registered with NCBI accession number BC014439 and can be represented by SEQ ID NO: 2, S100A2 is registered with NCBI accession number BC105787 and can be represented by SEQ ID NO: 3, and S100A6 is , NCBI accession number BC001431, and can be represented by SEQ ID NO: 4, S100B can be registered by NCBI accession number BC001766, and can be represented by SEQ ID NO: 5, and S100P is NCBI accession It is registered under the number BC006819 and can be represented by SEQ ID NO: 6.

SEQ ID NO: 2 (S100A1) BC014439
MGSELETAMETLINVFHAHSGKEGDKYKLSKKELKELLQTELSGFLDAQKDVDAVDKVMKELDENGDGEVDFQEYVVLVAALTVACNNFFWENS
Sequence number 3 (S100A2) BC105787
MMCSSLEQALAVLVTTFHKYSCQEGDKFKLSKGEMKELLHKELPSFVGEKVDEEGLKKLMGSLDENSDQQVDFQEYAVFLALITVMCNDFFQGCPDRP
Sequence number 4 (S100A6) BC001431
MACPLDQAIGLLVAIFHKYSGREGDKHTLSKKELKELIQKELTIGSKLQDAEIARLMEDLDRNKDQEVNFQEYVTFLGALALIYNEALKG
Sequence number 5 (S100B) BC001766
MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMVTTACHEFFEHE
SEQ ID NO: 6 (S100P) BC006819
MTELETAMGMIIDVFSRYSGSEGSTQTLTKGELKVLMEKELPGFLQSGKDKDAVDKLLKDLDANGDAQVDFSEFIVFVAAITSACHKYFEKAGLK

Although S100 is illustrated below, this invention is not limited to this. Examples of S100 include at least one protein selected from the group consisting of the following (B1) to (B3).
(B1) A protein comprising the amino acid sequence represented by any of SEQ ID NOs: 2 to 6 (B2) In the amino acid sequence of (B1), one or several amino acids are deleted, substituted, inserted and / or added. A protein having the same function as the protein of (B1) (B3), an amino acid sequence having 80% identity or more with the amino acid sequence of (B1), and (B1 Protein with the same function as

  In the above (B2) and (B3), the “same function” means that the function of the TPR motif protein is regulated by binding to the TPR motif protein. When the TPR motif protein is, for example, PP5, “same function” means that PP5 dephosphorylation activity is enhanced by binding to PP5.

  In the above (B2), “one or several” is not particularly limited, and is, for example, 1 to 30, preferably 1 to 20, more preferably 1, 2, 3, 4, 5 , 6, 7, 8, 9, or 10.

  In (B3), the identity is, for example, 80% or more, more preferably 85% or more, still more preferably 90% or more, still more preferably 95% or more, particularly preferably 96% or more, 97% or more, 98% or more or 99% or more.

  In the present invention, the combination of the TPR motif protein and S100 is not particularly limited. For example, it is preferable that the combination is derived from the same TPR motif protein and S100.

S100 is a calcium receptor protein involved in the intracellular calcium signal system. S100 becomes an apo-type in the absence of calcium ions (Ca ²⁺ ) and a holo-type in the presence of calcium ions. For example, since S100 binds to the TPR motif protein in a holo form, the binding of S100 to the TPR motif protein is preferably performed in the presence of calcium ions. Alternatively, S100 is preferably a holo type S100.

  In the present invention, S100 can regulate the function of the TPR motif protein as described above. Therefore, the function regulator of the present invention is a TPR motif protein function regulator and is characterized by containing S100. The function regulator of the present invention is characterized by containing S100, and other configurations are not limited at all. When the TPR motif protein is, for example, PP5, the function regulator of the present invention can be said to be a PP5 activity enhancer.

  S100 can be used, for example, for the treatment of diseases caused directly or indirectly by the function of the TPR motif protein. When the TPR motif protein is, for example, PP5, examples of the disease include cancer and Alzheimer's disease. Therefore, S100 can be applied to the following therapeutic agents and treatment methods.

  The therapeutic agent for a disease of the present invention comprises S100. The therapeutic agent of the present invention is characterized by containing S100, and other configurations are not limited at all.

  The therapeutic agent of the present invention may further contain, for example, a pharmaceutically acceptable additive as necessary. The additive is not particularly limited, and examples thereof include base materials, excipients, colorants, stabilizers, preservatives, and fragrances. In the present invention, the blending amount of S100 and the blending amount of the additive are not particularly limited as long as they do not hinder the function of S100.

  The administration conditions of the therapeutic agent of the present invention are not particularly limited, and the dosage form, administration timing, dosage, etc. can be appropriately set according to the type and degree of the target disease.

  The administration form of the therapeutic agent of the present invention is not particularly limited, and may be, for example, oral administration or parenteral administration. Examples of the parenteral administration include intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, intraperitoneal administration, and local administration. In addition, the administration target of the therapeutic agent of the present invention is not particularly limited, and examples thereof include humans and non-human animals. Examples of non-human animals include the non-human mammals described above.

  The dosage form of the therapeutic agent of the present invention is not particularly limited, and can be appropriately determined according to, for example, the administration form. The dosage form is not particularly limited, and examples thereof include a liquid form and a solid form. In the case of oral administration, examples of the dosage form include tablets, coated tablets, pills, fine granules, granules, powders, capsules, syrups, emulsions, suspensions and the like. In the case of parenteral administration, examples of the dosage form include an injectable preparation and an infusion preparation.

  The disease treatment method of the present invention includes a step of administering S100 to a patient. The present invention is characterized by administering S100, and other conditions and steps are not particularly limited. Administration conditions and the like are not particularly limited, and for example, the description of the therapeutic agent of the present invention can be used. Examples of the patient include humans and non-human animals as described above.

(Screening method for functional regulator of TPR motif protein)
As described above, the screening method of the present invention is a screening method for a TPR motif protein function-regulating substance, and includes an analysis step of analyzing the function of the TPR motif protein in the presence of a candidate substance.

  In the analysis step, the function of the analysis target is not particularly limited, and can be set according to the type of the target TPR motif protein.

According to the screening method of the present invention, for example, a function-regulating substance having the following action can be screened.
(X1) An enhancer that enhances the function of S100 for the TPR motif protein.
(X2) A substance that binds to S100 and loses the function of S100 with respect to the TPR motif protein.
(X3) A substance that competitively binds to S100 with respect to the TPR motif protein and inhibits the binding of S100 to the TPR motif protein.
(X4) A substance that enhances the function of the TPR motif protein.
(X5) A substance that acts on the functional regulatory site of the TPR motif protein to reduce the function of the TPR motif protein.
(X6) A substance that acts on the functional site of the TPR motif protein to inhibit the function of the TPR motif protein.

  The candidate substance is not particularly limited, and examples thereof include compounds such as low molecular compounds and high molecular compounds. The candidate substance may be, for example, a natural product or a synthetic product. The compound may be, for example, a salt or a hydrate.

  The screening method of the present invention preferably includes, for example, a step of comparing the function of the TPR motif protein in the presence of the candidate substance and the function of the TPR motif protein in the absence of the candidate substance in the analysis step. Moreover, it is preferable that the analysis step further includes a selection step of selecting, as the function-regulating substance, a substance that exhibits a different function with respect to the absence of the candidate substance. Thus, the ability to regulate the candidate substance can be determined by comparing functions between the presence and absence of the candidate substance.

  In the screening method of the present invention, in the analysis step, for example, the TPR motif protein and the candidate substance may be contacted in the presence of S100, or the TPR motif protein and the candidate substance may be contacted in the absence of S100. You may make it contact. When the contact step is performed in the presence of S100, for example, it is preferably performed in the coexistence of S100 and calcium ions, or it is preferable to use holo-type S100 combined with calcium ions as S100.

  When the contact step is performed in the presence of S100, for example, an activator that activates the function of the TPR motif protein or an inactivator that inactivates PP5 can be screened. The former is, for example, the substance (x1), and the latter is, for example, the substance (x2) or (x3). In the comparison step, when the function of the TPR motif protein in the presence of the candidate substance is higher than the function of the TPR motif protein in the absence of the candidate substance, the candidate substance can be determined as the activating substance. Further, in the comparison step, when the activity of the TPR motif protein in the presence of the candidate substance is lower than the activity of the TPR motif protein in the absence of the candidate substance, the candidate substance can be determined as the inactivated substance. .

  When the contact step is performed in the absence of S100, for example, an activator that activates the function of the TPR motif protein and an inactivator that inactivates the function of the TPR motif protein can be screened. Examples of the former include the substance (x4). Examples of the latter include the substance (x5) or the substance (x6). In the comparison step, when the function of the TPR motif protein in the presence of the candidate substance is higher than the function of the TPR motif protein in the absence of the candidate substance, the candidate substance can be determined as the activating substance. Further, in the comparison step, when the activity of the TPR motif protein in the presence of the candidate substance is lower than the activity of the TPR motif protein in the absence of the candidate substance, the candidate substance can be determined as the inactivated substance. .

  The contact step may be performed in vivo or in vitro, for example. In the case of in vivo, for example, it can be performed by administering the candidate substance to a living body. In this case, both the TPR motif protein and S100 may be derived from the living body, or both may be administered from the living body, or one of the TPR motif protein and S100 is derived from the living body, and the other is derived from the living body. It may be administered. In the case of in vitro, for example, the contact between the TPR motif protein and S100 may be performed in an artificial environment such as a test tube or a culture vessel, or may be performed in a cell such as a cultured cell or a tissue such as a cultured tissue. . In the latter case, the TPR motif protein and S100 may be derived from the cell or the tissue, respectively, or may be administered from outside the cell or the tissue.

  In the screening method of the present invention, the TPR motif protein may be, for example, a protein consisting of a full-length amino acid sequence or a deletion protein in which a part of the region is deleted. Examples of the deletion protein include a protein lacking a TPR domain, a protein lacking a functional domain, and the like.

  In the screening method of the present invention, the type of TPR motif protein is not particularly limited, and examples thereof include the proteins described above. Hereinafter, as a specific example, an example in which the target TPR motif protein is PP5 is shown.

  In the screening method of the present invention, when the TPR motif protein is PP5, it is preferable that the function-regulating substance is the activity-regulating substance of PP5, and the dephosphorylation activity of PP5 is analyzed in the analysis step.

According to the screening method of the present invention, for example, an activity-regulating substance that exhibits the following action on PP5 can be screened.
(X1 ′) Enhancer that enhances PP5 activation by S100. Hereinafter, it is also referred to as an S100 potentiator.
(X2 ′) A substance that binds to S100 and loses the activation function of S100. Hereinafter also referred to as S100 antagonist.
(X3 ′) A substance that competitively binds to S100 with respect to PP5 and inhibits S100 from binding to PP5. Hereinafter, it is also referred to as an S100 binding antagonist.
(X4 ′) A substance that enhances the activity of PP5. Hereinafter, it is also called PP5 activator, S100 mimic or S100 agonist.
(X5 ′) A substance that acts on the activity-regulating site of PP5 and decreases the activity of PP5. Hereinafter, it is also referred to as an allosteric PP5 inhibitor.
(X6 ′) A substance that acts on the active site of PP5 to inhibit the activity of PP5. Hereinafter, it is also referred to as a PP5 catalytic inhibitor.

  The screening method of the present invention further includes, for example, a step of comparing the activity of PP5 in the absence of the candidate substance with the activity of PP5 in the presence of the candidate substance. In the comparison step, when the activity of PP5 in the presence of the candidate substance is higher than the activity of PP5 in the absence of the candidate substance, the candidate substance can be determined as an activator of PP5, and the candidate substance When the activity of PP5 in the presence is lower than the activity of PP5 in the absence of the candidate substance, the candidate substance can be determined as a PP5 inactivating substance.

  In the screening method of the present invention, in the contacting step, for example, PP5 and the candidate substance may be contacted in the presence of S100, or PP5 and the candidate substance may be contacted in the absence of S100. Good. When the contact step is performed in the presence of S100, for example, it is preferably performed in the coexistence of S100 and calcium ions, or it is preferable to use holo-type S100 combined with calcium ions as S100.

  When the contact step is performed in the presence of S100, for example, an activating substance that activates PP5 and an inactivating substance that inactivates PP5 can be screened. The former includes, for example, an enhancer (S100 potentiator) that enhances the activation of PP5 by (x1 ′) S100. The latter is, for example, a substance (S100 antagonist) that binds to (x2 ′) S100 and loses the activation function of S100, and (x3 ′) PP5 that competitively binds to S100 and PP5 of S100. And a substance that inhibits binding (S100 binding antagonist). In the comparison step, when the activity of the PP5 protein in the presence of the candidate substance is higher than the activity of the PP5 protein in the absence of the candidate substance, the candidate substance can be determined as the activation substance. In the comparison step, when the activity of the PP5 protein in the presence of the candidate substance is lower than the activity of the PP5 protein in the absence of the candidate substance, the candidate substance can be determined as the inactivated substance.

  When the contact step is performed in the absence of S100, for example, an activating substance that activates PP5 and an inactivating substance that inactivates PP5 can be screened. The former includes, for example, a substance (PP5 activator, S100 mimic or S100 agonist) that enhances the activity of (x4 ′) PP5. The latter, for example, acts on the (x5 ′) PP5 activity-regulating site to decrease the PP5 activity (allosteric PP5 inhibitor) or the (x6 ′) PP5 active site, and acts on the PP5 activity. (PP5 Catalytic Inhibitor) can be mentioned. In the comparison step, when the activity of PP5 in the presence of the candidate substance is higher than the activity of PP5 in the absence of the candidate substance, the candidate substance can be determined as the activated substance. In the comparison step, when the activity of PP5 in the presence of the candidate substance is lower than the activity of PP5 in the absence of the candidate substance, the candidate substance can be determined as the inactivated substance.

  In the analysis step, a method for analyzing the phosphatase activity of PP5 is not particularly limited, and a known method can be adopted. The analysis of the activity can be performed, for example, using a commercially available kit according to the protocol. Examples of the kit include Ser / Thr Phosphatase Assay kit (Upstate).

The analysis of phosphatase activity can be performed, for example, by the following method. A phosphorylated phosphopeptide (SEQ ID NO: 7: KRpTIRR) is used as a substrate. Then, the phosphopeptide, the candidate substance and / or PP5 are added to a buffer solution, and this mixed solution is incubated. The composition of the buffer solution is, for example, 20 mmol / L Tris-HCl (pH 7.5), 20 mmol / L MgCl ₂ , 0.01% Tween 20, and 1 mmol / L CaCl ₂ . After incubation, a predetermined concentration of S100 is added to the mixture and incubated. Then, after adding malachite green solution to the reaction solution after incubation, the absorbance of the sample at 630 nm is measured. The composition of the malachite green solution is, for example, 0.034% malachite green, 10 mmol / L ammonium molybdenum, 1 mol / L HCl, 3.4% ethanol, and 0.01% Tween20. Then, the amount of released phosphate is calculated using a phosphate standard curve prepared from a known amount of phosphate. This can be evaluated as phosphatase activity.

  In the screening method of the present invention, PP5 may be, for example, the protein described in the regulatory method of the present invention or, for example, the deletion PP5 shown below. Examples of the deletion PP5 include PP5-ΔTPR lacking the TPR domain, PP5-ΔCD lacking the catalytic domain (CD), and the like.

  In the screening method of the present invention, examples of S100 include the proteins described in the regulatory method of the present invention.

(Functional regulator of TPR motif protein)
The function regulator of the TPR motif protein of the present invention is a regulator that regulates the function of the TPR motif protein, and activates the function of the TPR motif protein or inactivates the function of the TPR motif protein. Inactivating agents.

  When the TPR motif protein is PP5, the function regulator of the present invention is a regulator that regulates the activity of PP5, an activator that activates PP5 activity, or an inactivation that inactivates PP5 activity. Agent.

  Examples of the function regulator of the present invention include compounds represented by the following formulas (1) to (50). When the TPR motif protein is PP5, the following compounds lose the activation function of S100 by binding to the S100 potentiator that enhances the activation of PP5 by (x1 ′) S100 and (x2 ′) S100, respectively. An S100 antagonist that binds to S100 competitively with S100 and inhibits S100 binding to PP5, a PP5 activator that enhances the activity of (x4 ′) PP5, (X5 ′) Allosteric PP5 inhibitor that acts on the activity-regulating site of PP5 to decrease the activity of PP5, and (x6 ′) PP5 catalytic inhibitor that acts on the active site of PP5 and inhibits the activity of PP5 it can. The present invention is not limited to these.

Among these compounds, those shown below can be represented by general formulas.

  The activator preferably includes at least one compound selected from the group consisting of formulas (1) to (5) and (12) to (36), for example. The activator may contain, for example, one type of compound, or two or more types may be used in combination.

  The deactivator preferably contains at least one compound selected from the group consisting of formulas (6) to (11) and (37) to (50). The deactivator may contain, for example, one type of compound, or two or more types may be used in combination.

  The function regulator of the present invention is not limited to these examples. The function regulator may be, for example, a derivative of the exemplified compound. As a specific example, in the compound, for example, a sulfone group and an azo group may be arbitrary, for example.

  If these function regulators are used in combination, for example, it is possible to freely set ON / OFF of the function of the TPR motif protein.

  The function regulator of this invention should just contain the above compounds, for example, and another structure is not restrict | limited at all.

  The function-regulating agent of the present invention can be used, for example, for the treatment of diseases caused directly or indirectly by the function of TPR motif protein. When the TPR motif protein is PP5, examples of the disease include cancer and Alzheimer's disease. Therefore, the function regulator can be applied to the following therapeutic agents and treatment methods.

  The therapeutic agent for a disease of the present invention is characterized by containing the function regulator. The therapeutic agent of the present invention is characterized by containing the function-regulating agent, and other configurations are not limited at all.

  The therapeutic agent of the present invention may further contain, for example, a pharmaceutically acceptable additive as necessary. The additive is not particularly limited, and examples thereof include those described above. In the present invention, the blending amount of the function regulator and the blending amount of the additive are not particularly limited as long as they do not interfere with the function of the function regulator. The administration conditions, dosage form, and dosage form of the therapeutic agent of the present invention are not particularly limited, and for example, the above description can be used.

  The disease treatment method of the present invention includes a step of administering the function-regulating agent to a patient. The present invention is characterized by administering the function regulator, and other conditions and steps are not particularly limited. Administration conditions, administration subjects, and the like are not particularly limited, and for example, the above description can be used.

(Screening method of S100 function regulator)
As described above, the screening method of the present invention is a screening method for a S100 function-regulating substance, and includes an analysis step of analyzing the function of a TPR motif protein in the presence of S100 and a candidate substance. .

According to the screening method of the present invention, for example, a function-regulating substance that regulates the function of S100 for a TPR motif protein can be screened. Examples of the function regulating substance include the substances (x1) to (x3) as described above.
(X1) An enhancer that enhances the function of S100 for the TPR motif protein.
(X2) A substance that binds to S100 and loses the function of S100 with respect to the TPR motif protein.
(X3) A substance that competitively binds to S100 with respect to the TPR motif protein and inhibits the binding of S100 to the TPR motif protein.

As a specific example, when the TPR motif protein is PP5, according to the screening method of the present invention, for example, a regulator for the function of S100 to enhance the activity of PP5 can be screened. Examples of the regulating substance include the substances (x1 ′) to (x3 ′) as described above.
(X1 ′) S100 potentiator that enhances the activation of PP5 by S100.
(X2 ′) An S100 antagonist that binds to S100 and loses the activation function of S100.
(X3 ′) An S100 binding antagonist that binds to PP5 competitively with S100 and inhibits the binding of S100 to PP5.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples. Commercially available reagents were used based on their protocol unless otherwise indicated.

1. The materials glutathione sepharose and Nickel-nitrilotriacetic acid-agarose were purchased from GE Healthcare and Qiagen, respectively. Anti-S100A1 antibody (R & D systems), anti-S100A2 antibody (Sigma), anti-S100A6 antibody (Sigma), anti-S100B antibody (QED), anti-Hsp90 antibody (Stressgen), anti-PP5 antibody (BD transduction laboratories), anti-pSer396 Tau antibody (Abcam), anti-pSer409 antibody (Invitrogen), anti-tau antibody (Invitrogen), anti-pThr845 ASK1 antibody (Cell Signaling), HRP-labeled anti-mouse IgG (Cell Signaling), HRP-labeled anti-rabbit IgG (Cell Signaling) Anti-HA-HRP labeled antibody (Sigma) was used. Geneticin was purchased from Invitrogen and ionomycin, anti-HA antibody agarose and other chemicals were purchased from Sigma.

2. Cell culture and transfection COS-7 cells were purchased from JCRB. COS-7 cell culture used a humidified chamber of 95% O ₂ and 5% CO ₂ and DMEM (Sigma) supplemented with 10% fetal calf serum, 1% penicillin and 1% streptomycin. The transfection was performed according to the protocol using Fugene 6 (Invitrogen) or HD transfection reagent (Invitrogen) as a transfection reagent.
In addition, in order to prepare COS-7 cells that stably express the Tau protein, COS-7 cells were cultured in the presence of 500 ng / ml geneticin for several weeks after transfection. The expression of tau was confirmed by Western blot.

3. As the recombinant protein S100, S100A1, S100A2, S100A4, S100A6, S100A10, S100A11, S100A13, S100B and S100P were expressed and purified based on the following literature.
Okada, M. et al., (2004) J. Biol. Chem. 279, 4221-4233
Yamashita, K. et al., (1999) Protein Expr. Purif. 16, 47-52
GST-PP5 conjugated with GST and His-PP5 conjugated with His tag (6 × His) were expressed and purified according to the manufacturer's protocol (GE Healthcare and Qiagen). Recombinant GSK-3β and PKA were purchased from SignalChem and Promega, respectively. Human HSP90 was prepared based on the following literature.
Shimamoto, S. et. Al., (2008) J. Biol. Chem. 283, 28246-28258
Recombinant tau protein was purchased from ENZO life sciences.

4). The plasmid human PP5 gene was amplified by PCR from a human cDNA library and cloned into pET16a, PGEX-6P1, pME18S-Flag and pME18S-HA vectors. A mutant of GST-PP5 gene was prepared by PCR. The mutant is a deletion mutant (ΔTPR) in which 28-160 is deleted among 1-181 residues of TPR in PP5, and the amino acid residue at positions 32, 74, 97, or 101 is alanine. Four mutated alanine mutants (K32A, R74A, K97A, and R101A) were prepared. Moreover, the 171-486th variant in human PP5 was produced as a variation | mutation (His-PP5-CD) of the catalytic domain of His-PP5. The human Hsp90 gene was cloned based on the literature (Shimamoto, S. et. Al., (2008) J. Biol. Chem. 283, 28246-28258). The mouse ASK1 gene was amplified from mouse brain cDNA by PCR and cloned into the pME18S-cMyc vector. Construction of the pET11a-S100 plasmid and the pME18S-S100 plasmid was performed based on the literature (Okada, M. et al., (2004) J. Biol. Chem. 279, 4221-4233). The gene for permanently active S100P (S100P-PA) was prepared based on the literature (Austermann, J. et. Al., (2009) Biochim. Biophys. Acta 1793, 1078-1085). The pcDNA3-tau plasmid was prepared by cloning human tau441 cDNA (Addgene) into the pcDNA3 vector.

5. The GST pull-down S100 or HSP90 was analyzed for binding to PP5 as follows. GST-PP5 (12.5 μg), S100 protein (25 μg) or Hsp90 protein (50 μg) and glutathione sepharose beads (50 μl) were mixed in a buffer solution at room temperature for 1 hour. The GST-PP5 used was wild type (WT) or the mutant. The composition of the buffer was 100 mmol / L KCl, 20 mmol / L Tris-HCl (pH 7.5), 0.01% Tween 20, and 1 mmol / L CaCl ₂ or EGTA.

  The S100B binding assay used 50 μg GST-PP5 and 50 μg S100B. The beads were washed several times, then boiled with SDS sample buffer, separated by 10% Tricine SDS-PAGE gel, and stained with Coomassie Brilliant Blue (CBB). In the competitive binding assay, GST-PP5 (20 μg), S100 (50 μg) and a predetermined amount (0-150 μg) of HSP90 were incubated with glutathione sepharose (25 μl) as described above. Similar experiments were performed with GST-PP5 (20 μg), HSP90 (50 μg) and a predetermined amount (0-150 μg) of S100.

6). Surface plasmon resonance (SPR)
Protein binding interactions were performed using the SPR Biacore 2000 system (Biacore). N-ethyl-N ′-(3-diethylaminopropyl) carbodiimide, N-hydroxysuccinimide and ethanolamide-HCl (Biacore) were used for amine coupling of PP5 to the dextran surface of CM5 chip (Biacore). His-PP5 (100 μg / ml, 140 μl) was immobilized in 20 mmol / L ammonium acetate (pH 4.2) and bound to response unit 2600 (0.3 pmol) to obtain a stable baseline. In all steps, 20 mmol / L HEPES (pH 7.4), 150 mmol / L NaCl, 0.005% Tween 20, 1 mmol / L CaCl ₂ was used at a flow rate of 20 μl / min. Recombinant proteins S100A1, S100A2, S100A6 and S100B were injected at predetermined concentrations (1.25 μmol / L, 625 nmol / L, 313 nmol / L, 156 nmol / L). The sensor chip coupled with His-PP5 was regenerated during protein injection by a brief (60 sec) wash with 50 mmol / L NaOH until the response unit returned to baseline prior to unit injection. Response curves were prepared by subtracting signals generated simultaneously in the control flow cell. Biacore sensorgram curves were evaluated in BIA evaluation 3.0 using a 1: 1 Langmuir model.

7). In vivo competitive binding assay For PP5 binding assay, pME18S-S100 and pME18S-HA-PP5 plasmids were co-transfected into COS-7 cells. 48 hours after transfection, the cells were washed once with PBS and lysed with buffer. The composition of the buffer solution was 50 mmol / L Tris-HCl (pH 7.5), 150 mmol / L NaCl, 0.5% Triton-X100, 0.5% NP40, and protease inhibitor cocktail (Roche). The sample after lysis was sonicated and centrifuged at 14,000 rpm for 10 minutes. The obtained supernatant was incubated with 30 μl of anti-HA antibody agarose in the presence of 1 mmol / L CaCl ₂ or EGTA for 1 hour at room temperature. Then, after washing the anti-HA antibody agarose, the obtained sample was analyzed by Western blotting with an anti-HSP90 antibody, anti-HA-HRP antibody or anti-S100 protein antibody. Also, for ASK1 binding assay, pME18S-Flag-PP5, pME18S-cMyc-ASK1 and pME18S-S100A1 or pME18S-S100P or pME18S-S100P-PA plasmids were co-transfected. The protein was purified and incubated with 30 μl of anti-Flag antibody agarose. The obtained samples were analyzed by Western blot with anti-PP5 antibody or anti-cMyc-HRP antibody.

8). In Vitro Phosphatase Assay The phosphatase activity of PP5 protein (WT) or PP5 variant protein (PP5-CD) was measured using Ser / Thr Phosphatase Assay kit (Upstate) according to its protocol. Phosphopeptide (SEQ ID NO: 7: KRpTIRR) 100 μmol / L was incubated with 250 ng His-PP5 (WT) or 250 ng His-PP5-CD in 50 μl buffer. The composition of the buffer was 20 mmol / L Tris-HCl (pH 7.5), 20 mmol / L MgCl ₂ , 0.01% Tween 20, and 1 mmol / L CaCl ₂ or EGTA. A predetermined concentration (0-50 μmol / L) of S100 protein or HSP90 was added and incubated at 37 ° C. for 10 minutes. HSP90 used HSP90-WT and HSP C90, respectively. Then, after adding 100 μl of malachite green solution, the absorbance of the sample at 630 nm was measured with a microplate reader. The composition of the malachite green solution was 0.034% malachite green, 10 mmol / L ammonium molybdenum, 1 mol / L HCl, 3.4% ethanol and 0.01% Tween20. The amount of phosphate released was calculated using a phosphate standard curve prepared from known amounts of phosphate. This was evaluated as phosphatase activity.

9. Tau dephosphorylated recombinant tau441 in vitro was phosphorylated with GSK-3β or PKA. For tau Ser396 phosphorylation, 10 μg of tau was incubated in buffer with 1 μg of GSK-3β for 90 minutes at 30 ° C. The composition of the buffer was 40 mmol / L HEPES (pH 7.5), 10 mmol / L MgCl ₂ , 0.5 mmol / L DTT, and 0.2 mmol / L ATP. For phosphorylation of tau Ser409, the protein was incubated with 250 units of PKA in buffer for 90 minutes at 30 ° C. The composition of the buffer was 40 mmol / L HEPES (pH 6.8), 10 mmol / L β-mercaproethanol, 10 mmol / L MgCl ₂ and 0.2 mmol / L ATP. After the phosphorylation reaction, the sample was washed with an Amicon ultra spin column (Millipore), and washed with 500 μl of 20 mmol / L Tris-HCl (pH 7.5) and 0.01% Tween20. Phosphorylated tau was incubated with 100 ng His-PP5 in buffer. The composition of the buffer was 20 mmol / L Tris-HCl (pH 7.5), 20 mmol / L MgCl ₂ , 0.01% Tween 20, and 1 mmol / L CaCl ₂ or EGTA. S100A1, S100A2 or S100A6 was added to the reaction mixture and incubated for 10 minutes at 37 ° C. The obtained samples were separated by SDS-PAGE and analyzed by Western blot using anti-pSer396 antibody, anti-pSer409 antibody or anti-tau antibody.

10. For dephosphorylation of dephosphorylated tau in vivo, COS-7 cells stably expressing tau were transfected with pME18S or pME18S-S100A1 expression plasmids. After 48 hours, 5 μmol / L ionomycin was applied, and further incubated for 15 or 60 minutes. The expressed protein was purified using a lysis buffer containing protease inhibitor cocktail and phosphate inhibitor cocktail (Pierce). After sonication and centrifugation, the resulting samples were subjected to Western blot analysis using anti-pSer396 tau antibody, anti-tau antibody, anti-S100A1 antibody or anti-PP5 antibody.

  For dephosphorylation of ASK1, pME18S-Flag-PP5, pME18S-cMyc-ASK1, and pME18S-S100P or pME18S-S100P-PA plasmids were co-transfected into COS-7 cells. After 48 hours, the expressed protein was purified, and the obtained sample was subjected to Western blot analysis using anti-pThr845-ASK1 antibody.

11. For statistical statistics, an unpaired t test and a paired t test were performed. The confidence level p <0.05 was considered statistically significant.

[Example 1]
S100 binding to the TPR domain of PP5 was confirmed.

(1) Pull-down assay In the presence of 1 mmol / L CaCl ₂ or EGTA, whether or not S100 directly binds to PP5 was confirmed by a pull-down assay. These results are shown in FIG. FIG. 1 shows the results of a pull-down assay showing the binding between various types of S100 and GST-PP5. As shown in FIG. 1 (A), S100A2 and S100A6 bound to PP5 in the presence of Ca ²⁺ . S100A1 was confirmed to bind to PP5 not only in the presence of Ca ²⁺ but also in the absence of Ca ²⁺ . When a large amount of GST-PP5 (50 μg) was used, S100B bound to PP5 in the presence of Ca ²⁺ as shown in FIG. 1 (B).

(2) SPR
For S100A1, S100A2, S100A6 and S100B binding to PP5, real-time binding kinetics were analyzed by SPR. The results are shown in FIG. 2 and Table 1 below. As shown in Table 1, the association rate constant (Ka) of each S100 to PP5 was similar. Moreover, it can be seen that the dissociation of S100A6 and S100A2 occurs more slowly than the dissociation of S100B and S100A1, and the overall binding affinity is relatively high. From this result, it can be considered that different interaction mechanisms exist between S100A1 and S100B and between S100A2 and S100A6.

(3) Determination of S100 binding domain in PP5 PP5 is composed of an N-terminal TPR and a C-terminal catalytic domain (CD). Therefore, the binding domain of S100 in PP5 was determined by pull-down assay.

  For the pull-down assay, four types of proteins fused with GST were used. An outline of these proteins is shown in FIG. The wild type (WT) is a fusion protein of PP5 and GST, the deletion (TPR) is a fusion protein of TPR and GST that lacks the catalytic domain, and the deletion mutant (ΔTPR) is a deletion of TPR. GST, a fusion protein of catalytic domain and GST, is a protein composed only of GST.

  These results are shown in FIG. FIG. 3B shows the results of the pull-down assay. As shown in FIG. 3 (B), none of S100 could confirm binding to the mutant lacking TPR. From this result, it was found that S100 interacts with the TPR domain of PP5.

(4) Inhibition of interaction between PP5 and HSP90 by S100 in vivo or in vitro HSP90 is known to bind to the TPR domain of PP5 via its C-terminal EEVD residue. S100 and HSP90 share the TPR domain. Therefore, it was confirmed by pull-down assay whether the association with PP5 is inhibited in vitro with an increase in the amount of HSP90 or S100.

  These results are shown in FIG. FIG. 4 (A) shows the results of the pull-down assay. As shown in FIG. 4A, as the amount of HSP90 was increased, substitution of S100A1 and S100A2 from PP5 occurred in an amount-dependent manner. And S100A6 showed weak binding inhibition. On the other hand, with the increase of S100A1 and S100A2, the association of HSP90 with PP5 was inhibited.

  In addition, co-immunoprecipitation was performed to confirm competitive binding of PP5 in mammalian cells. Specifically, lysate of COS-7 cells co-expressed with HA-PP5 and S100A1 or S100A2 was performed. Endogenous HSP90 co-precipitated with HA-PP5 was detected by Western blot analysis using anti-HSP90 antibody.

  These results are shown in FIG. As shown in FIG. 4B, similar to the in vitro results described above, the overexpression of S100A1 or S100A2 inhibited the interaction between PP5 and HSP90.

(5) Binding of S100 and HSP90 to PP5 mutant HSP90 binds to a motif called carboxylate clamp in the TPR domain of PP5. Carboxylate clamp is composed of four basic amino acid residues, namely K32, R74, K97 and R101. When the binding between PP5 and HSP90 was inhibited by S100, it was confirmed whether S100 competitively binds to one of the four types of amino acid residues in the carboxylate clamp.

  Alanine mutants (K32A, R74A, K97A, and R101A) were used as the alanine substitution mutants of the carboxylate clamp, and a pull-down assay was performed. These results are shown in FIG. As shown in FIG. 5 (A), HSP90 did not bind to any alanine mutant. In contrast, as shown in FIG. 5 (B), S100 bound to any alanine mutant. From this result, it was found that the carboxylate clamp point mutation in PP5 does not affect the binding of S100.

(6) Activation of PP5 by Ca2 ⁺ -dependent S100 The effect on the dephosphorylation activity of PP5 by S100 was confirmed. Specifically, a predetermined concentration of His-PP5 and a synthetic phosphopeptide (SEQ ID NO: 7: KRpTIRR) serving as a substrate were incubated, and phosphatase activity was measured. These results are shown in FIG.

In FIG. 6 (A), the black circles indicate the result of ^{Ca 2+} presence, white circle indicates a result of ^{Ca 2+} absence. As shown in FIG. 6 (A), the basal phosphatase activity of PP5 was 10 nmol / min / mg protein. S100A1, S100A2, S100A6 and S100B each significantly activated PP5 activity in a Ca ²⁺ dependent manner.

  The TPR domain protects the catalytic domain (CD) of PP5 from activation and removal of the TPR domain. Therefore, a mutant His-PP5 was prepared by removing the N-terminal TPR domain and leaving the C-terminal catalytic domain (CD). And activation of each S100 with respect to this mutant His-PP5 was confirmed. These results are shown in FIG.

  In FIG. 6 (B), the control is the result of mutant His-PP5 in the absence of S100. From this result, PP5 became structurally active by removal of the TPR domain, and showed about 650 nmol / min / mg protein. The activity of this mutant was 65 times the basal activity of the wild type enzyme.

  Next, it was confirmed whether full-length HSP90 (12 kDa) and HSP C90 (C-terminal domain of HSP90) can activate the activity of PP5. The result is shown in FIG. In each graph of FIG. 6 (C), the small graph in the upper right indicates the result when the amount of protein added is 0 to 20 μg / ml. As shown in FIG. 6 (C), dose-dependent activation of His-PP5 by HSP90 and HSP C90 was shown. However, as a result of comparison with S100, activation of PP5 by HSP90 was weak and was only about 6.5 times. On the other hand, HSP C90 showed a 17.3-fold induction of activity.

(7) PP5 activation and tau dephosphorylation by S100 in vivo and in vitro PP5 plays a key role in tau dephosphorylation. Excessive phosphorylation causes Alzheimer's disease. Therefore, by using the tau phosphorylated as a substrate, according to ^S100 (Ca 2+ / S100) in the ^{Ca 2+} presence was confirmed activation of PP5.

  Multiple sites of tau are phosphorylated by tau kinases such as PKA, GSK-3β, cdk5 and CaMKII. Therefore, for in vitro PP5 phosphatase assay, GSK-3β and PKA were used for phosphorylation of tau Ser396 and Ser409, respectively. The phosphorylated tau was then incubated with His-PP5 and S100A1, S100A2 or S100B. The phosphorylation state of tau was detected by Western blot using anti-pSer396 tau antibody or anti-pSer409 tau antibody. These results are shown in FIG.

  As shown in FIG. 7A, each S100 activated PP5 and increased tau dephosphorylation.

  In vivo confirmation of activation of PP5 by S100A1 and dephosphorylation of tau was performed. The cells used were transfected COS-7 cell lines that stably express high levels of tau441. These results are shown in FIGS. 7B and 7C.

As shown in FIGS. 7B and 7C, the control in the absence of S100 showed no change in the tau phosphorylation state in pSer396 even after treatment with ionomycin (95.3 ± 25.9%). N = 5). When S100A1 was overexpressed, no change was observed in the phosphorylation state in the absence of ionomycin (106.0 ± 15.7%, N = 5). However, 15 minutes of ionomycin treatment reduced the level of pSer396 tau (69.0 ± 12.6%, P <0.05, N = 4), and 60 minutes of treatment showed a significant reduction. (46.4 ± 4.6%, P <0.01, N = 5). From these results, it was found that S100A1 activates PP5 and increases tau dephosphorylation in a Ca ²⁺ dependent manner.

(8) Inhibition of the interaction between PP5 and ASK1 by S100 in vivo ASK1 is MAP3K that activates the JNK kinase cascade and p38 MAP kinase cascade, and is activated in response to oxidative stress such as H ₂ O ₂ . Is done. Activation of ASK1 is associated with autophosphorylation of Thr845 located in the kinase domain. Then, following activation of ASK1 by oxidative stress, PP5 binds to ASK1, whereby ASK1 is dephosphorylated and returns to an inactivated state.

  Therefore, S100A1, PP5 and ASK1 were co-expressed in COS-7 cells. It was confirmed by Western blot that the binding of PP5 and ASK1 was inhibited by ionomycin treatment of the cells. These results are shown in FIG.

As shown in FIG. 8 (A), inhibition was not confirmed when not treated with ionomycin. This is presumably because the increase of intracellular Ca ²⁺ concentration by ionomycin stimulates the binding of S100A1 to PP5, thereby inhibiting the interaction of PP5-ASK1.

  Moreover, wild type S100P was used as S100. The result is shown in FIG. As shown in FIG. 8 (B), wild-type S100P bound to GST-PP5 in vitro, similar to S100A1, S100A2, S100A6, and S100B.

  For COS-7 cells, wild-type S100P or S100P-PA was co-expressed with PP5 and ASK1. The result is shown in FIG. As shown in FIG. 8 (C), S100P-PA inhibited the binding of PP5 and ASK1. On the other hand, the wild type S100P was not inhibited. Due to the increase in pThr845 of phosphorylated ASK1, it is understood that S100P-PA inhibits dephosphorylation by dissociation of PP5 from ASK1.

[Example 2]
In the presence of S100 and a regulator, the phosphatase activity of PP5 was evaluated to confirm the function of the regulator.

  The phosphatase activity of PP5 (PP5-WT) was measured. The phosphatase activity was measured according to the above-mentioned “8. In vitro phosphatase assay” unless otherwise specified. Specifically, the phosphopeptide (100 μmol / L), the modulator at a predetermined concentration (0-200 μmol / L), 0.25 μmol / L S100A1, 0.02 μmol / L PP5 in 37 μl of buffer solution are added. Incubated for 10 minutes at ° C. Then, after adding 100 μl of malachite green solution, the absorbance of the sample at 630 nm was measured with a microplate reader. The buffer solution and the malachite green solution were as described above. The amount of released phosphate was then calculated using a phosphate standard curve prepared from known amounts of phosphate. This was evaluated as phosphatase activity.

(1) S100 binding antagonist Ponceau-3R was used as a modulator. The result is shown in FIG. As shown in FIG. 9, as a result of addition of Ponceau-3R, the binding of S100 to PP5 was blocked, and the activity of PP5 was suppressed to a steady activity. From this result, it can be said that Ponceau-3R is an S100 binding antagonist.

(2) S100 potentiator Erythrosine B and Phlosine B were used as regulators. These results are shown in FIG. As shown in FIG. 10, as a result of the coexistence of S100A1 and the modulator in PP5-WT, as compared with the activity of the regulator not added (0 μmol / L), as shown by the arrow in the graph, Enhanced activity was confirmed. From these results, it is understood that the modulator enhances the activation of PP5 by S100. Therefore, it can be said that the regulating substance is an S100 potentiator.

[Example 3]
In the presence of the regulator, PP5 phosphatase activity was evaluated to confirm the function of the regulator.

  Phosphatase activity was measured for PP5 (PP5-WT) and PP5 lacking the TPR domain (PP5-ΔTPR). The phosphatase activity was measured according to the above-mentioned “8. In vitro phosphatase assay” unless otherwise specified. Specifically, a modulator at a predetermined concentration (0-200 μmol / L), 0.25 μmol / L S100A1, 0.02 μmol / L PP5-WT or PP5-ΔTPR in 10 μl of a buffer solution at 37 ° C. The phosphatase activity was evaluated in the same manner as in Example 2 except that the incubation was performed for a minute.

(1) PP5 activator Thioflavin-S, α-naphthol orange, and Direct red 80 were used as regulators.

  These results are shown in FIG. As shown in FIG. 11 (A), thioflavin-S, like S100, bound to PP5 and enhanced the activity of PP5. In addition, since the activity of PP5-ΔTPR was also enhanced, it is considered that thioflavine-S can also enhance the activity by binding to the catalytic domain (CD) in PP5. As shown in FIGS. 11 (B) and (C), α-naphthol orange and Direct red 80 enhanced PP5 activity by binding to TPR of PP5, similar to S100. From these results, it can be said that each modulator is a PP5 activator, S100 mimic or S100 agonist.

(2) Regulatory substance serving as both PP5 activator and PP5 catalytic inhibitor As regulators, Congo-Red, Chrysamine-G, Evans-blue, and 4,4-bis (2-sulfonatostyryl) biphenyl were used. These results are shown in FIG. As shown in FIGS. 12 (A) to (D), any of the modulators enhanced PP5 activity when added at a low concentration, and inhibited PP5 activity when added in a certain amount or more. From these results, it is understood that each modulator has an ability to enhance PP5 activity by binding to TPR and an ability to inhibit PP5 activity by binding to catalytic domain (CD).

(3) Allosteric PP5 inhibitor Proflavine was used as a modulator. These results are shown in FIG. As shown in FIG. 13, the modulator inhibits PP5 activity by binding to TPR. From this result, it can be said that the modulator is an allosteric PP5 inhibitor that acts on the activity-regulating site of PP5.

(4) PP5 Catalytic Inhibitor Ethyl violet, Methyl green, Acridine Orange and Acridine Yellow were used as modulators. These results are shown in FIG. As shown in FIGS. 14 (A) to (D), any of the modulators inhibited PP5 activity. From these results, it can be said that each modulator is a PP5 catalytic inhibitor.

[Example 4]
Using TBPE, which is a triphenylmethane compound, as a regulator, the activity of PP5 was confirmed in the presence of S100 in the same manner as in Example 2. These results are shown in FIG. As shown in FIG. 15, in PP5-WT, as a result of coexistence of S100A1 and the regulator, an enhancement of activity was confirmed as compared with the activity of the regulator not added (0 μmol / L).

[Example 5]
As the regulator, Fast green FCF, Acid violet 6B, Guinea green B, and Brilliant milling green, which are triphenylmethane compounds, were used. These results are shown in FIG. 16 and FIG. As shown in FIGS. 16 (A) and (B), Fast green FCF and Acid violet 6B were confirmed to have decreased PP5 activity. Further, as shown in FIGS. 17A and 17B, Guinea green B and Brilliant milling green were confirmed to have enhanced PP5 activity.

[Example 6]
As regulators, acridine derivatives Acridine Orange, Acridine Yellow and Proflavin were used, and the activity of PP5 was confirmed in the absence of S100 in the same manner as in Example 3. These results are shown in FIG. As shown in FIG. 18, each of the modulators similarly inhibited PP5 activity for PP5-WT, but for PP5-ΔTPR, inhibition of PP5 activity was alleviated in the order of Acridine Orange, Acridine Yellow, and Proflavin. It was.

[Example 7]
Using xanthene compounds Eosin and Rose Bengal as regulators, the activity of PP5 was confirmed in the presence of S100 in the same manner as in Example 2. These results are shown in FIG. As shown in FIG. 19 (A), Eosin did not change PP5 activity in the presence of S100, and inhibited PP5 in the absence of S100 . On the other hand, as shown in FIG. 19 (B), Rose Bengal enhanced PP5 activity in the presence of S100, and inhibited PP5 activity when exceeding a predetermined amount.

[Example 8]
Further, the functions of various regulatory substances were confirmed by the same method as in Example 2 and Example 3.

Specifically, phosphatase activity was measured for each condition of PP5 (WT), PP5-ΔTPR, PP5 (WT) + S100A1. For PP5 (WT) and PP5-ΔTPR, the relative values were determined with the activity of no addition of the regulator as 100%. For PP5 (WT) + S100A1, a relative value was obtained based on the following formula.
Relative value (%) = [XY] / [ZY] × 100
X: Activity in the presence of PP5 (WT), S100A1 and regulator Y: Activity in PP5 (WT) Z: Activity in the presence of PP5 (WT) and S100A1

  These results are shown in FIGS. As shown in each figure, all showed a regulatory function for PP5 activity.

  As described above, according to the present invention, the function of the TPR motif protein can be regulated by S100. Further, according to the screening method of the present invention, for example, various TPR motif protein function regulators such as S100 antagonist compounds can be screened, and various function regulators can be provided. Since PP5, which is a kind of TPR motif protein, is related to diseases such as cancer, the present invention can be said to be a very important technique in both research fields and clinical fields such as biochemistry.

Claims (13)

A method for regulating the function of protein phosphatase 5, comprising:
A regulation method comprising enhancing the activity of protein phosphatase 5 by binding S100 protein to protein phosphatase 5 in the presence of calcium ions. However, human treatment is excluded. A method for screening a protein phosphatase 5 function regulator,
Candidate substance, the presence of calcium ions and S100 proteins, see contains an analysis step of analyzing the dephosphorylation activity of protein phosphatase 5,
In the analysis step, the dephosphorylation activity of protein phosphatase 5 in the presence of the candidate substance is compared with the dephosphorylation activity of protein phosphatase 5 in the absence of the candidate substance. Selecting those having enhanced activity as activators of protein phosphatase 5, and selecting those having decreased activity relative to the absence of the candidate substance as inhibitors of protein phosphatase 5. A screening method characterized. Including at least one compound selected from the group consisting of formulas (3), (4), (13) to (15), (17) to (21), (23), and (32), An activator for protein phosphatase 5, which enhances the activity of protein phosphatase 5 in the presence of S100 protein. Including at least one compound selected from the group consisting of formulas (6), (11), (42) to (43), and (49), and having the activity of protein phosphatase 5 in the presence of calcium ions and S100 protein. An inhibitor of protein phosphatase 5, which is characterized by reducing. Comprising at least one compound selected from the group consisting of formulas (10), (16), (22), and (24), enhancing the activity of protein phosphatase 5 in the absence of S100 protein, A function regulator of protein phosphatase 5, which reduces the activity of protein phosphatase 5 in the presence of S100 protein. Containing at least one compound of formulas (1) and (5), reducing the activity of protein phosphatase 5 in the absence of S100 protein and enhancing the activity of protein phosphatase 5 in the presence of calcium ions and S100 protein A protein phosphatase 5 function regulator. Containing at least one compound selected from the group consisting of formulas (12), (25) to (31), and (33) to (36), and enhancing the activity of protein phosphatase 5 in the absence of S100 protein An activator of protein phosphatase 5, characterized in that Comprising at least one compound selected from the group consisting of formulas (37), (38), (40), (41), and (45) to (48), and in the absence of S100 protein, protein phosphatase 5 An inhibitor of protein phosphatase 5, which reduces activity. A protein phosphatase 5 activator comprising a compound of the formula (2), which enhances the activity of protein phosphatase 5 in the presence of calcium ions and S100 protein. Including at least one compound selected from the group consisting of formulas (7) to (9), (39), and (50), and reducing the activity of protein phosphatase 5 in the presence of calcium ions and S100 protein. Inhibitor of protein phosphatase 5 characterized. An activator for an S100 protein comprising a compound of the formula (2) and enhancing the activity of the S100 protein in the presence of calcium ions. S100 comprising at least one compound selected from the group consisting of formulas (7) to (9), (39), and (50), wherein the activity of S100 protein is reduced in the presence of calcium ions. Protein inhibitor. A screening method for a substance that regulates the function of S100 protein, comprising:
Candidate substance, the presence of calcium ions and S100 proteins, see contains an analysis step of analyzing the dephosphorylation activity of protein phosphatase 5,
In the analysis step, the activity of protein phosphatase 5 in the absence of the S100 protein and in the presence of the candidate substance, and the protein phosphatase 5 in the absence of the S100 protein and in the absence of the candidate substance The activity of protein phosphatase 5 in the coexistence of the S100 protein and the candidate substance, and the presence of the S100 protein. A substance that enhances or decreases the activity of the protein phosphatase 5 in the absence of the candidate substance and increases or decreases the activity relative to the absence of the candidate substance, Select a substance that reduces the activity as an S100 inhibitor Screening wherein the that.