KR20090058630A - Screening method for substance inhibiting the interaction between rho g protein rac or cdc42 and pbd - Google Patents

Abstract

A method for screening an inhibitor of interaction between Rac or Cdc42 and PBD is provided to manufacture an anticancer drug for signaling of Rac/Cdc42 or inhibitor of Rac/Cdc42 signaling. A method for screening a material which inhibits the interaction between Rac or Cdc42 and PBD comprises: a step of contacting Rac-GTP or Cdc42-GTP to fixed PBD of plate under the presence of candidate material and measuring the amount of Rac-GTP or Cdc42-GTP which is binding with PBD; a step of contacting Rac-GTP or Cdc42-GTP to fixed PBD of plate without candidate material and measuring the amount of Rac-GTP or Cdc42-GTP which is binding with PBD; and a step of determining inhibitor between Rac or Cdc42 and PBD.

Description

Screening method for Substance Inhibiting the Interaction between Rho G protein Rac or Cdc42 and PBD}

The present invention relates to a method for screening a substance that inhibits binding between Rac or Cdc42 (Cell Division Cycle 42) protein and PBD (p21 binding domain).

Small molecule GTPases proteins, composed of a single polypeptide, are called low molecular G proteins because they bind to guanosine diphosphates (GDP) or guanosine triphosphates (GTP) at low molecular weights of 20 to 40 kDa. Small molecule GTPases can generally be classified into five groups: Ras, Rho, Rab, Arf, and Ran. Intracellular small molecule GTPases regulate various life phenomena such as gene transcription control, cell proliferation and migration, and cytoskeletal reconstitution. Small molecule GTPases are inactive when bound to GDP, active when bound to GTP, and exhibit biological activity in the active form. The activation of these small molecule GTPases regulates vital vital phenomena, which are tightly and precisely regulated by various signaling molecules in the cell. The protein that induces the activation of small molecule GTPases is a guanine nucleotide exchange factor (GEF) that is involved in the conversion of small molecule GTPases to GTP binding. On the other hand, GTPase activating protein (GAP) acts as a protein that induces the inactivation of low molecular weight GTPases and is involved in the conversion to inactive form of GDP binding state. Inactive low molecular weight GTPases also inhibit the binding and activation of GDP dissociation inhibitors (GDI).

About 20 Rho G proteins are known in the human body. Rho G protein has a variety of functions that regulate the assembly of actin cytoskeleton, smooth muscle contraction, cell-to-cell adhesion, cell movement and migration, gene transcription control, and enzyme activity. Among Rho G proteins, studies on Rac, RhoA, and Cdc42 have been actively conducted. Their function of regulating actin cytoskeleton shows that RhoA forms stress fibers and focal adhesions, Rac forms lamellipodia around the cells, and Cdc42 forms microspikes. Form microspike or filopodia. These factors are expressed by growth factors or stimulants. RhoA is activated by lysophatidic acid (LPA), Rac by PDGF, insulin (insulin), and Cdc42 by bradykinin (ILy). In humans, Rho G protein is regulated by 85 activators (GEFs) and 70 inactivators (GAPs). GDIs are currently known to Rho GDI α, β, and γ. These modulators are not yet precisely known for their function.

In addition to the Rho G protein, proteins involved in the Rho signaling pathway are important for the regulation of cytoskeleton. The most well known effect factor is PAK (p21-associated kinase). In particular, PBD (p21 binding domain) portion of the binding domain of PAK has a characteristic of specifically binding to Rac-GTP or Cdc42-GTP. Activated PAK leads to stabilization of actin filaments and actin polymer formation through inhibition of cofilin. Phosphorylation also inhibits MLC (Myosin light chain) and MLC kinase. The GST-PBD pulldown assay is being actively used to confirm the presence of these active forms of G protein in cells using the specific binding of active forms of Rac or Cdc42 to PBDs. However, this method requires an individual reaction for each candidate and includes a step of western blotting to identify HTS that can detect Rac or Cdc42 inhibitors in terms of time, cost and efficiency. There is a disadvantage that it is not suitable for development into a high throughput system.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors can search for a substance that inhibits the specific binding of the Rac protein or the Cdc42 protein, which is a kind of Rho G protein, with the p21 binding domain (PBD), which is a binding domain of p21-associated kinase (PAK), in a high-speed, high-capacity manner. Research efforts have been made to develop screening systems. As a result, the amount of Rac-GTP or Cdc42-GTP protein bound to PBD was measured by immobilizing the PBD on the plate and contacting the Rac-GTP or Cdc42-GTP protein in the presence or absence of a candidate. The present invention was completed by experimentally confirming that a substance that inhibits the interaction between PBD and Rac-GTP or Cdc42-GTP protein can be screened by a high-throughput high-throughput (HTS, highthroughput system).

Accordingly, an object of the present invention is to provide a method for screening a substance that inhibits binding between Rac or Cdc42 (Cell Division Cycle 42) protein and PBD (p21 binding domain).

The objects and advantages of the invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for screening a substance that inhibits binding between a Rac or Cdc42 protein and a PBD (p21 binding domain) comprising the following steps: (1) (a) plate ( contacting Rac-GTP or Cdc42-GTP with a candidate substance to a PBD immobilized on a plate) and measuring the amount of Rac-GTP or Cdc42-GTP bound to the PBD; (b) contacting the PBD immobilized on the plate with Rac-GTP or Cdc42-GTP in the absence of candidates and measuring the amount of Rac-GTP or Cdc42-GTP bound to the PBD; And (2) the amount of Rac or Cdc42 protein bound to PBD measured in step (1) (a) is greater than the amount of Rac or Cdc42 protein bound to PBD measured in step (1) Determining the candidate as a substance that inhibits binding between Rac or Cdc42 protein and PBD, if at all.

The present inventors can search for a substance that inhibits the specific binding of the Rac protein or the Cdc42 protein, which is a kind of Rho G protein, with the p21 binding domain (PBD), which is a binding domain of p21-associated kinase (PAK), in a high-speed, high-capacity manner. Research efforts have been made to develop screening systems. As a result, the amount of Rac-GTP or Cdc42-GTP protein bound to PBD was measured by immobilizing the PBD on the plate and contacting the Rac-GTP or Cdc42-GTP protein in the presence or absence of a candidate. The present invention was completed by experimentally confirming that a substance that inhibits the interaction between PBD and Rac-GTP or Cdc42-GTP protein can be screened by a high-throughput high-throughput (HTS, highthroughput system).

In the present specification, the term “Rac protein” or “Cdc (Cell Division Cycle) 42 protein” is a protein belonging to the Rho G protein group, which is a group of low molecular weight GTPases, and an active type of guanosine triphosphate (GTP) binding form and an inactive GDP ( Guanosine diphosphate binding cycle is repeated, and refers to a protein known to be involved in signal transduction pathways that regulate the remodeling (remodeling) and reorganization of the actin (skeletal cytoskeleton).

According to a preferred embodiment of the present invention, the Rac protein used in the method of the present invention comprises the amino acid sequence of SEQ ID NO: 2 sequence, the Cdc42 protein comprises the amino acid sequence of SEQ ID NO: 3 sequence .

As used herein, the term “pBD (p21 binding domain)” refers to a binding domain of a p21-associated kinase (PAK) protein. The term “PAK protein” is a kinase protein that acts as an effector by engaging in a lower signaling pathway that binds to the Rho G protein and regulates the cytoskeleton.

According to a preferred embodiment of the present invention, the "PBD (p21 binding domain)" used in the method of the present invention comprises the amino acid sequence of SEQ ID NO: 1 sequence.

According to a preferred embodiment of the invention, in order to facilitate the purification of the recombinant peptide or protein, in the present invention Rac protein, Cdc42 protein or PBD is used in the form of a fusion protein fused with an additional peptide or polypeptide.

Preferably, in the present invention, the additional peptide or polypeptide fused to the Rac protein, Cdc42 protein or PBD is glutathione S-transferase (GST, Glutathione S-transferase), 6x His (hexahistidine), thioredoxin (Trx, Thioredoxin), Maltose binding protein (MBP), FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Lys) or Nu utilization A (N utilization substance A). Because of the additional polypeptide for isolation and purification, proteins expressed in host cells can be purified quickly and easily through affinity chromatography. For example, a recombinant peptide or protein can be obtained quickly and easily using a resin column bound to glutathione when fused to GST, or a Ni-NTA His-bound resin column when fused to 6 × His.

As used herein, the term “Rac-GTP” or “Cdc42-GTP” refers to a GTP-bound form of Rac or Cdc42 protein, and “Rac-GDP” or “Cdc42-GDP” refers to GDP binding of Rac or Cdc42 protein. It means the form.

As used herein, the term "candidate" refers to a substance that is expected to inhibit the binding or interaction of Rac or Cdc42 protein with PBD. The candidates include, but are not limited to, low molecular weight compounds, high molecular weight compounds, nucleic acid molecules (eg, DNA, RNA, PNA and aptamers), proteins, sugars, and lipids.

The term "plate" in the context of the present invention refers to a solid support in the form of a flat to which the PBD can be immobilized. The material of the plate is not limited to a specific material, but is preferably a hydrocarbon polymer (such as polystyrene and polypropylene), glass, metal or gel, preferably a material of hydrocarbon polymer, most preferably polystyrene Use materials. The shape of the plate is not limited to a particular shape, but preferably, a flat plate having a plurality of wells serving as small test tubes is used, and more preferably a microtiter plate or a microplate. (microplate) is used.

PBD immobilization onto the plate can be done by simple adsorption to the surface of the plate (nonspecific immobilization) or by capturing antibodies to the PBD that have been previously immobilized on the plate surface (specifically). Ever immobilized).

The amount of Rac-GTP or Cdc42-GTP protein bound to PBD in the method of the present invention can be measured according to various immunoassays or immunostaining methods described below.

The immunoassay or method of immunostaining may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA (enzyme-linked immunosorbent assay), capture-ELISA, inhibition or competition analysis, sandwich analysis, flow cytometry, immunofluorescence Staining and immunoaffinity tablets include, but are not limited to.

The method of immunoassay or immunostainingEnzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W.,Enzyme - linked immunosorbent assay ( ELISA ), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane,Using Antibodies : A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

According to a preferred embodiment of the invention, the method of the invention binds to an immobilized PBD using a Rac-GTP or Cdc42-GTP protein, or a primary antibody against a peptide or polypeptide fused to these proteins. The amount of Rac-GTP or Cdc42-GTP protein can be measured.

According to another preferred embodiment of the present invention, Rac- bound to PBD immobilized using a primary antibody against Rac-GTP or Cdc42-GTP protein and a secondary antibody against the primary antibody in the method of the present invention. The amount of GTP or Cdc42-GTP protein can be measured.

When measuring the amount of Rac-GTP or Cdc42-GTP protein using only the primary antibody in the method of the present invention, the primary antibody may be used to which a label that generates a detectable signal is bound. Labels that bind to the antibody include chemicals (eg, biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P ₄₅₀ ), radioisotopes (E.g., C ¹⁴ , I ¹²⁵ , P ³² And S ³⁵ ), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent and fluorescence resonance energy transfer (FRET). Various signs and labeling methods are available in Ed Harlow and David Lane, Using Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999.

Enzymes labeled (bound) to the primary antibody include, but are not limited to, enzymes catalyzing color reaction, fluorescence, luminescence or infrared reaction, for example, alkaline phosphatase, β-galacto Sidase, horse radish peroxidase, luciferase and cytochrome P ₄₅₀ . When alkaline phosphatase is used as the enzyme that binds the primary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (substrate) When chromogenic reaction substrates such as naphthol-AS-B1-phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucifer Genine (bis-N-methylacridinium nitrate), resorphin benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), TMB (3,3,5,5 Substrates such as -tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]) and o -phenylenediamine (OPD) can be used.

When measuring the amount of Rac-GTP or Cdc42-GTP protein using the primary antibody against Rac-GTP or Cdc42-GTP protein and the secondary antibody against the primary antibody in the method of the present invention, the secondary antibody is a detectable signal It is possible to use a combination of a label to generate a.

Labels, enzymes and substrates bound to the secondary antibody are the same as those described in the description of labels, enzymes and substrates bound to the primary antibody, and are described in duplicate so as to avoid excessive complexity of the specification. I never do that.

In the measurement of the amount of Rac-GTP or Cdc42-GTP protein using the antibody, the measurement of the final enzyme activity or signal can be carried out according to various methods known in the art. For example, signals can be easily detected with streptavidin when biotin is used as a label, and luciferin when luciferase is used.

On the other hand, the antibody used in the screening method of the present invention, methods commonly practiced in the art, for example, a fusion method (Kohler and Milstein, European Journal of Immunology , 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) or phage antibody library methods (Clackson et al, Nature , 352: 624-628 (1991) and Marks et al, J. . Mol Biol, 222:.. 58, can be prepared by 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies : A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies : A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN It is described in detail in IMMUNOLOGY , Wiley / Greene, NY, 1991, which is incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing immortalized cell lines with antibody-producing lymphocytes, and the techniques required for this process are well known to those skilled in the art and can be readily implemented. Polyclonal antibodies can be obtained by injecting an antigen into a suitable animal, collecting antisera from the animal, and then isolating the antibody from the antisera using known affinity techniques.

According to the method of the present invention, the amount of Rac or Cdc42 protein bound to PBD measured in step (a) of step (1), Rac or Cdc42 bound to PBD measured in step (b) of step (1) If less than the amount of protein, the candidate is determined to inhibit the binding between Rac protein or Cdc42 protein and PBD. Such screened candidates are presumed to have an activity that inhibits the binding or interaction between Rac or Cdc42 protein and PBD, an inhibitor that inhibits Rac / Cdc42 signaling or an anticancer agent that acts on Rac / Cdc42 signaling. It can be developed as a new drug substance.

The present invention relates to a method for screening a substance that inhibits binding between Rac or Cdc42 (Cell Division Cycle 42) protein and PBD (p21 binding domain). According to the method of the present invention, substances that inhibit the binding between Rac or Cdc42 protein and PBD can be screened in a high-throughput manner. Candidates screened by the present invention can be developed as inhibitors that inhibit the signaling of Rac / Cdc42 or as new drugs of anticancer agents that act on the signaling of Rac / Cdc42.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Expression and Purification of PBD and GST - Rac / Cdc42 Fusion Proteins Rho G Protein Binding Domains

G protein binding domain PBD of PAK that binds GST-Rac or GST-Cdc42 protein was expressed in His-labeled Escherichia coli ( E. coli , DH5α) using pET-24a + (Novagen) vector (His- Amino acid sequence of the fused PBD protein: see SEQ ID NO: 4). Separation and purification was performed using Ni-NTA chromatography.

Rac and Cdc42 proteins were expressed by GST-labeling using pGEX4T-1 (Amersham Pharmacia) vector in Escherichia coli ( E. coli , DH5α) (amino acid sequence of GST-fused Rac or Cdc42 protein: SEQ ID NO: 5 and See section 6.) GST-fused Rac or Cdc42 protein was isolated and purified using glutathione affinity chromatography (see FIGS. 1A, 1B, and 1C).

Example 2 GST pull - down Confirmation of binding between PBD and Rac - GDP / GTP or Cdc42 - GDP / GTP by assay

GST-Rac and GST-Cdc42 expressed and purified in Example 1 were subjected to dialysis in 20 mM Tris, 20 mM NaCl, 5 mM EDTA, and 0.1 mM DTT solution. After exchange of buffer, GDPβs and GTPγs were respectively loaded at a final concentration of 100 μM, and PBD-His (prepared in Example 1) was added to perform a GST pull-down assay. As a result, it was confirmed that the PBD prepared in Example 1 and the GST-Rac / Cdc42 protein bind to each other (see FIGS. 2A and 2B).

Example 3 Confirmation of Binding Between Rac / Cdc42 and PBD Using the Method of the Present Invention

PBD was immobilized in a Maxisorp (Nunc, Netherland) 96 well plate for 12 hours using sodium bicarbonate (50 mM, pH 9.6) at a concentration of 10 μg / ml, followed by PBST (1% Tween- 20 μl was washed three times with 20 in phosphate buffered saline. PBD immobilized 98 well plates were then blocked for 2 hours at room temperature using PBST containing 1% skim milk powder.

The plate prepared as described above was treated with GST-Rac and GST-Cdc42 proteins as follows. GST-Rac and GST-Cdc42 proteins were diluted five-fold with PBS at a concentration of 50 μg / ml on the prepared plates and prepared in three sets of the same experimental groups. At this time, GTP-coupled GST-Rac and GST-Cdc42 protein, as a comparison group GDP-bound GST-Rac and GST-Cdc42 protein each 100μL loaded in each well (well), 1 at 37 ℃ The reaction was carried out for a time.

Subsequently, 200 μl of PBST (1% tween-20 in phosphate buffered saline) was washed three times, and anti-GST-HRP (horseradish peroxidase) IgG was prepared in PBST at a concentration of 1.25 μg / ml, and then 100 μl of each well ( well) and then reacted at 37 ° C. for 1 hour.

After washing 200 μl three times with PBST (1% tween-20 in phosphate buffered saline) three times, 2,2′-Azino-bis prepared at 100 μg / ml in 50 mM Phosphate-citrate buffer with sodium perborate (Sigma) (3-ethylbenzoline-6-sulfonate) iammonium salt (Sigma) was developed for 30 minutes at 37 ℃.

Absorbance was measured at 405 nm using a Microplate reader (Mutiskan plus, Germany). At this time, the data is plotted as a graph obtained by subtracting the blank value (see FIGS. 3A and 3B). According to the experimental results shown in FIGS. 3A and 3B, it was found that Rac and Cdc42 proteins had better binding capacity with PBD than GTP-bound state (active state) than GDP-bound state (inactive state).

According to the above experimental results, the system of the present invention enables screening of a substance that inhibits the binding (or interaction) between the GST-Rac-GTP or GST-Cdc42-GTP protein and the PBD polypeptide in a high-speed, high-capacity manner. You can see that.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

references

Shin EY, Woo KN, Lee CS, Koo SH, Kim YG, Kim WJ, Bae CD, Chang SI, Kim EG. Phosphorylation of p85 beta PIX, a Rac / Cdc42-specific guanine nucleotide exchangefactor, via the Ras / ERK / PAK2 pathway is required for basic fibroblast growthfactor-induced neurite outgrowth. J Biol Chem . 2004. 16; 279 (3): 1994-2004.

Shin EY, Shin KS, Lee CS, Woo KN, Quan SH, Soung NK, Kim YG, Cha CI, Kim SR, Park D, Bokoch GM, Kim EG. Basic fibroblast growth factor stimulates activation of Rac1 through a p85betaPIX phosphorylation-dependent pathway. J Biol Chem . 2002; 15; 277 (46): 44417-30.

Schmitz U, Thommes K, Beier I, Wagner W, Sachinidis A, Dusing R, Vetter H. Angiotensin II-induced stimulation of p21-activated kinase and c-Jun NH2-terminalkinase is mediated by Rac1 and Nck. J Biol Chem . 2001. 22; 276 (25): 22003-10.

4. Wen Y, Gu J, Knaus UG, Thomas L, Gonzales N, Nadler JL. Evidence that 12-lipoxygenase product 12-hydroxyeicosatetraenoic acid activates p21-activated kinase. Biochem J. 2000. 15; 349 (Pt 2): 481-7.

5. Benard V, Bohl BP, Bokoch GM. Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J Biol Chem . 1999; 7; 274 (19): 13198-204.

Figure 1a is an electrophoresis picture showing the results of the isolation and purification of the PBD protein that can specifically bind the active Rho G protein Rac and Cdc42.

Figure 1b is an electrophoresis picture showing the result of the separation and purification of GST-Rac protein.

Figure 1c is an electrophoresis picture showing the result of the separation and purification of GST-Cdc42 protein.

Figure 2a is a GST pull-down assay results showing that the PBD purified in the present invention specifically binds to GST-Rac-GTP, an active G protein.

Figure 2b is a GST pull-down assay results showing that the PBD purified in the present invention specifically binds to GST-Cdc42-GTP, an active Rho G protein.

Figure 3a is a control graph showing the concentration-dependent change in binding between Rac protein and PBD according to GDP / GTP binding.

Figure 3b is a control graph showing the concentration-dependent change in binding between Cdc42 protein and PBD according to GDP / GTP binding.

<110> Chung-buk National University <120> Screening Method for Substance Inhibiting the Interaction between          Rho G protein Rac or Cdc42 and PBD <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 95 <212> PRT <213> Macaca mulatta <400> 1 Met Lys Lys Glu Lys Glu Arg Pro Glu Ile Ser Leu Pro Ser Asp Phe   1 5 10 15 Glu His Thr Ile His Val Gly Phe Asp Ala Val Thr Gly Glu Phe Thr              20 25 30 Gly Met Pro Glu Gln Trp Ala Arg Leu Leu Gln Thr Ser Asn Ile Thr          35 40 45 Lys Ser Glu Gln Lys Lys Asn Pro Gln Ala Val Leu Asp Val Leu Glu      50 55 60 Phe Tyr Asn Ser Lys Lys Thr Ser Asn Ser Gln Lys Tyr Met Ser Phe  65 70 75 80 Thr Asp Lys Ser Ala Glu Asp Tyr Asn Ser Ser Asn Ala Leu Asn                  85 90 95 <210> 2 <211> 192 <212> PRT <213> Homo sapiens <400> 2 Met Gln Ala Ile Lys Cys Val Val Val Gly Asp Gly Ala Val Gly Lys   1 5 10 15 Thr Cys Leu Leu Ile Ser Tyr Thr Thr Asn Ala Phe Pro Gly Glu Tyr              20 25 30 Ile Pro Thr Val Phe Asp Asn Tyr Ser Ala Asn Val Met Val Asp Gly          35 40 45 Lys Pro Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln Glu Asp Tyr      50 55 60 Asp Arg Leu Arg Pro Leu Ser Tyr Pro Gln Thr Asp Val Phe Leu Ile  65 70 75 80 Cys Phe Ser Leu Val Ser Pro Ala Ser Phe Glu Asn Val Arg Ala Lys                  85 90 95 Trp Tyr Pro Glu Val Arg His His Cys Pro Asn Thr Pro Ile Ile Leu             100 105 110 Val Gly Thr Lys Leu Asp Leu Arg Asp Asp Lys Asp Thr Ile Glu Lys         115 120 125 Leu Lys Glu Lys Lys Leu Thr Pro Ile Thr Tyr Pro Gln Gly Leu Ala     130 135 140 Met Ala Lys Glu Ile Gly Ala Val Lys Tyr Leu Glu Cys Ser Ala Leu 145 150 155 160 Thr Gln Arg Gly Leu Lys Thr Val Phe Asp Glu Ala Ile Arg Ala Val                 165 170 175 Leu Cys Pro Pro Val Lys Lys Arg Lys Arg Lys Cys Leu Leu Leu             180 185 190 <210> 3 <211> 235 <212> PRT <213> Homo sapiens <400> 3 Met Gln Ala Ile Lys Cys Val Val Val Gly Asp Gly Ala Val Gly Lys   1 5 10 15 Thr Cys Leu Leu Ile Ser Tyr Thr Thr Asn Ala Phe Pro Gly Glu Tyr              20 25 30 Ile Pro Thr Val Phe Asp Asn Tyr Ser Ala Asn Val Met Gln Thr Ile          35 40 45 Lys Cys Val Val Val Gly Asp Gly Ala Val Gly Lys Thr Cys Leu Leu      50 55 60 Ile Ser Tyr Thr Thr Asn Lys Phe Pro Ser Glu Tyr Val Pro Thr Val  65 70 75 80 Phe Asp Asn Tyr Ala Val Thr Val Met Ile Gly Gly Glu Pro Tyr Thr                  85 90 95 Leu Gly Leu Phe Asp Thr Ala Gly Gln Glu Asp Tyr Asp Arg Leu Arg             100 105 110 Pro Leu Ser Tyr Pro Gln Thr Asp Val Phe Leu Val Cys Phe Ser Val         115 120 125 Val Ser Pro Ser Ser Phe Glu Asn Val Lys Glu Lys Trp Val Pro Glu     130 135 140 Ile Thr His His Cys Pro Lys Thr Pro Phe Leu Leu Val Gly Thr Gln 145 150 155 160 Ile Asp Leu Arg Asp Asp Pro Ser Thr Ile Glu Lys Leu Ala Lys Asn                 165 170 175 Lys Gln Lys Pro Ile Thr Pro Glu Thr Ala Glu Lys Leu Ala Arg Asp             180 185 190 Leu Lys Ala Val Lys Tyr Val Glu Cys Ser Ala Leu Thr Gln Lys Gly         195 200 205 Leu Lys Asn Val Phe Asp Glu Ala Ile Leu Ala Ala Leu Glu Pro Pro     210 215 220 Glu Pro Lys Lys Ser Arg Arg Cys Val Leu Leu 225 230 235 <210> 4 <211> 101 <212> PRT <213> Macaca mulatta <400> 4 Met Lys Lys Glu Lys Glu Arg Pro Glu Ile Ser Leu Pro Ser Asp Phe   1 5 10 15 Glu His Thr Ile His Val Gly Phe Asp Ala Val Thr Gly Glu Phe Thr              20 25 30 Gly Met Pro Glu Gln Trp Ala Arg Leu Leu Gln Thr Ser Asn Ile Thr          35 40 45 Lys Ser Glu Gln Lys Lys Asn Pro Gln Ala Val Leu Asp Val Leu Glu      50 55 60 Phe Tyr Asn Ser Lys Lys Thr Ser Asn Ser Gln Lys Tyr Met Ser Phe  65 70 75 80 Thr Asp Lys Ser Ala Glu Asp Tyr Asn Ser Ser Asn Ala Leu Asn His                  85 90 95 His His His His His             100 <210> 5 <211> 418 <212> PRT <213> Homo sapiens <400> 5 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro   1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu              20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu          35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys      50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn  65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu                  85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser             100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu         115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn     130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu                 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr             180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala         195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg     210 215 220 Gly Ser Met Gln Ala Ile Lys Cys Val Val Gly Asp Gly Ala Val 225 230 235 240 Gly Lys Thr Cys Leu Leu Ile Ser Tyr Thr Thr Asn Ala Phe Pro Gly                 245 250 255 Glu Tyr Ile Pro Thr Val Phe Asp Asn Tyr Ser Ala Asn Val Met Val             260 265 270 Asp Gly Lys Pro Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln Glu         275 280 285 Asp Tyr Asp Arg Leu Arg Pro Leu Ser Tyr Pro Gln Thr Asp Val Phe     290 295 300 Leu Ile Cys Phe Ser Leu Val Ser Pro Ala Ser Phe Glu Asn Val Arg 305 310 315 320 Ala Lys Trp Tyr Pro Glu Val Arg His His Cys Pro Asn Thr Pro Ile                 325 330 335 Ile Leu Val Gly Thr Lys Leu Asp Leu Arg Asp Asp Lys Asp Thr Ile             340 345 350 Glu Lys Leu Lys Glu Lys Lys Leu Thr Pro Ile Thr Tyr Pro Gln Gly         355 360 365 Leu Ala Met Ala Lys Glu Ile Gly Ala Val Lys Tyr Leu Glu Cys Ser     370 375 380 Ala Leu Thr Gln Arg Gly Leu Lys Thr Val Phe Asp Glu Ala Ile Arg 385 390 395 400 Ala Val Leu Cys Pro Pro Pro Val Lys Lys Arg Lys Arg Lys Cys Leu                 405 410 415 Leu Leu         <210> 6 <211> 461 <212> PRT <213> Homo sapiens <400> 6 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro   1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu              20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu          35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys      50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn  65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu                  85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser             100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu         115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn     130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu                 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr             180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala         195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg     210 215 220 Gly Ser Met Gln Ala Ile Lys Cys Val Val Gly Asp Gly Ala Val 225 230 235 240 Gly Lys Thr Cys Leu Leu Ile Ser Tyr Thr Thr Asn Ala Phe Pro Gly                 245 250 255 Glu Tyr Ile Pro Thr Val Phe Asp Asn Tyr Ser Ala Asn Val Met Gln             260 265 270 Thr Ile Lys Cys Val Val Val Gly Asp Gly Ala Val Gly Lys Thr Cys         275 280 285 Leu Leu Ile Ser Tyr Thr Thr Asn Lys Phe Pro Ser Glu Tyr Val Pro     290 295 300 Thr Val Phe Asp Asn Tyr Ala Val Thr Val Met Ile Gly Gly Glu Pro 305 310 315 320 Tyr Thr Leu Gly Leu Phe Asp Thr Ala Gly Gln Glu Asp Tyr Asp Arg                 325 330 335 Leu Arg Pro Leu Ser Tyr Pro Gln Thr Asp Val Phe Leu Val Cys Phe             340 345 350 Ser Val Val Ser Pro Ser Ser Phe Glu Asn Val Lys Glu Lys Trp Val         355 360 365 Pro Glu Ile Thr His His Cys Pro Lys Thr Pro Phe Leu Leu Val Gly     370 375 380 Thr Gln Ile Asp Leu Arg Asp Asp Pro Ser Thr Ile Glu Lys Leu Ala 385 390 395 400 Lys Asn Lys Gln Lys Pro Ile Thr Pro Glu Thr Ala Glu Lys Leu Ala                 405 410 415 Arg Asp Leu Lys Ala Val Lys Tyr Val Glu Cys Ser Ala Leu Thr Gln             420 425 430 Lys Gly Leu Lys Asn Val Phe Asp Glu Ala Ile Leu Ala Ala Leu Glu         435 440 445 Pro Pro Glu Pro Lys Lys Ser Arg Arg Cys Val Leu Leu     450 455 460  

Claims (8)

Screening methods for substances that inhibit binding between Rac or Cdc42 (Cell Division Cycle 42) protein and PBD (p21 binding domain) comprising the following steps: (1) (a) contacting Rac-GTP or Cdc42-GTP with a candidate substance to a PBD immobilized on a plate, followed by Rac-GTP or Cdc42-GTP bound to the PBD. Measuring the amount; (b) contacting Rac-GTP or Cdc42-GTP in the absence of a candidate to PBD immobilized on a plate, and then measuring the amount of Rac-GTP or Cdc42-GTP bound to the PBD; And (2) the amount of Rac or Cdc42 protein bound to the PBD measured in step (1) (a) is less than the amount of Rac or Cdc42 protein bound to PBD measured in step (b) If the candidate is determined to inhibit the binding between Rac or Cdc42 protein and PBD. The method of claim 1, wherein the PBD comprises the amino acid sequence of SEQ ID NO: 1, wherein the Rac protein comprises the amino acid sequence of SEQ ID NO: 2, the Cdc42 protein is the amino acid sequence of SEQ ID NO: 3 Method comprising a. The method of claim 1, wherein the Rac protein, Cdc42 protein or PBD is in the form of a fusion protein fused with an additional peptide or polypeptide. The method of claim 3, wherein the additional peptide or polypeptide fused to Rac protein, Cdc42 protein or PBD is glutathione S-transferase (GST), 6 × His (hexahistidine), thioredoxin (Thioredoxin). ), Maltose binding protein (Maltose binding protein), FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Lys), or NusA (N utilization substance A). The method according to any one of claims 1 to 4, wherein the determination of the amount of Rac-GTP or Cdc42-GTP protein bound to the PBD is based on the primary antibody against the peptide or polypeptide fused to Rac, Cdc42, Rac or Cdc42. primary antibody). The method according to any one of claims 1 to 4, wherein the determination of the amount of Rac-GTP or Cdc42-GTP bound to the PBD comprises a primary antibody against a peptide or polypeptide fused to Rac, Cdc42, Rac or Cdc42 and Characterized in that it is carried out using a secondary antibody against the primary antibody. 6. The method of claim 5, wherein said primary antibody is labeled with a radioisotope, enzyme, or fluorescent substance. The method of claim 6, wherein the secondary antibody is labeled with a radioisotope, an enzyme, or a fluorescent substance.

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