Screening methods, screening kits, in silico analysis methods, recombinant microorganisms or cells, and cancer therapeutics.

Abstract

To provide a screening method for cancer cell proliferation inhibitory compounds that is highly accurate and easy to use. [Solution] A screening method for cancer cell proliferation inhibitory compounds, comprising a reaction step of contacting a candidate compound with a composite structure and a detection step of detecting the destruction of the composite structure, wherein the composite structure used in the reaction step comprises a first peptide including a first binding region consisting of a part of the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, and a second peptide including a second binding region consisting of a part of the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, and the first binding region and the second binding region are bound by interaction, and in the detection step, the maintenance or cleavage of the bond between the first binding region and the second binding region of the composite structure is detected.

Description

Technical Field

[0001] The present invention relates to a method for screening a cancer cell growth inhibitory compound, a kit for screening, an in-silico analysis method, a recombinant microorganism or cell, and a cancer therapeutic agent.

Background Art

[0002] In recent years, in addition to the genetic carcinogenesis mechanism involving direct mutations of oncogenes and tumor suppressor genes, an epigenetic carcinogenesis mechanism due to factors other than changes in the base sequence has attracted attention.

[0003] And, as a chromatin remodeling complex that controls epigenetic gene expression, a complex of BRG- / BRM-associated factor (BAF) and Polybromo-associated BAF (PBAF) (hereinafter sometimes referred to as the "BAF / PBAF complex") is known. The BAF / PBAF complex has SMARCC1 (BAF155), SMARCC2 (BAF170), and SNF5 (INI1 / BAF47) as core subunits, and is a complex having an ATPase domain such as BRG1 or BRM. Although the BAF / PBAF complex itself is not a direct oncogene or tumor suppressor gene, its subunits frequently mutate in solid cancers, thereby causing epigenetic changes in oncogenes and tumor suppressor genes and being considered to be involved in the progression of solid cancers (Non-Patent Documents 1 to 3).

[0004] Furthermore, a method has been proposed to screen for compounds that have cancer cell proliferation inhibitory effects by focusing on the protein interactions of the BAF / PBAF complex (Patent Document 1). Specifically, Patent Document 1 describes screening for cancer cell proliferation inhibitory compounds by contacting a candidate compound with a modified BAF / PBAF complex and detecting or quantifying the labeled substance-fused BAF155 or the subunit assemblies containing it that are produced by the disruption of the modified BAF / PBAF complex. Patent Document 1 also describes that when the BAF / PBAF complex is disrupted, it is broken down into labeled substance-fused BAF155 or the subunit assemblies containing it and subunit assemblies containing protein tag-fused BRG1. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2023-82308 [Non-patent literature]

[0006] [Non-Patent Document 1] Halliday, GM, Bock, VL, Moloney, FJ, and Lyons, JG (2009). SWI / SNF: a chromatin-remodelling complex with a role in carcinogenesis. Int J Biochem Cell Biol 41, 725-728. [Non-Patent Document 2] Hodges, C., Kirkland, JG, and Crabtree, GR (2016). The Many Roles of BAF (mSWI / SNF) and PBAF Complexes in Cancer. Cold Spring Harbor perspectives in medicine 6. [Non-Patent Document 3] Reisman, D., Glaros, S., and Thompson, EA (2009). The SWI / SNF complex and cancer. Oncogene 28, 1653-1668. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] However, Patent Document 1 does not necessarily clarify the specific relationship between the interactions between the subunits constituting the BAF / PBAF complex and its inhibitory effect on cancer cell proliferation, leaving room for further investigation in this regard. Furthermore, in the case of the screening method described in Patent Document 1, since it detects and quantifies subunit assemblies including other subunits surrounding BRG1 and BAF155, it was considered that there were areas for improvement in terms of the accuracy and simplicity of the screening.

[0008] This invention has been made in view of the above circumstances, and aims to provide a screening method, a screening kit, and an in silico analysis method for cancer cell proliferation inhibitory compounds that are excellent in accuracy and ease of use. Furthermore, this invention aims to provide recombinant microorganisms or cells that produce peptides usable in the aforementioned screening method and screening kit. Finally, this invention aims to provide a cancer therapeutic agent containing a peptide or mRNA encoding a peptide discovered in the study of the interaction between BRG1 and BAF155. [Means for solving the problem]

[0009] To address the above challenges, the following screening methods, screening kits, in silico analysis methods, and recombinant microorganisms or cells are provided. [1] A method for screening compounds that inhibit the proliferation of cancer cells, A reaction step in which a candidate compound is brought into contact with a composite structure, A detection step for detecting the failure of the composite structure, Includes, The composite structure used in the reaction step is A first peptide comprising a first binding region consisting of a part of the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, A second peptide containing a second binding region consisting of a portion of the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, and It includes and the first binding region and the second binding region are bound by interaction, The detection step involves detecting whether the bond between the first bond region and the second bond region of the composite structure is maintained or broken. Screening method. [2] Furthermore, the process includes a determination step for determining the candidate compound, In the determination step, the candidate compound that cleaves the bond between the first binding region and the second binding region of the composite structure is determined to be a cancer cell proliferation inhibitory compound. The screening method described in [1] above. [3] A screening kit for cancer cell proliferation inhibitory compounds, A first peptide comprising a first binding region consisting of a part of the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, A second peptide comprising a second binding region consisting of a portion of the amino acid sequence represented by Sequence ID No. 2, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by Sequence ID No. 2, Equipped with reagents including, Screening kit. [4] An in silico analysis method for identifying cell proliferation inhibitory compounds, In silico, Information on a portion of the amino acid sequence represented by Sequence ID No. 1, or a first amino acid sequence that has 80% or more sequence identity with the amino acid sequence represented by Sequence ID No. 1, Part of the amino acid sequence represented by SEQ ID NO: 2, or information on a second amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, and selecting a compound capable of cleaving the bond due to the interaction between the first amino acid sequence and the second amino acid sequence based on at least any one of the information, Method. [5] A first peptide comprising a first binding region consisting of part of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, or A second peptide comprising a second binding region consisting of part of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 A recombinant microorganism or cell comprising a nucleotide sequence encoding the same. [6] A cancer therapeutic agent containing a peptide comprising part of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1. [7] A cancer therapeutic agent comprising mRNA encoding a peptide comprising part of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1. [Advantages of the Invention]

[0010] According to the screening method, screening kit, and in silico analysis method of the present invention, a compound having an inhibitory effect on cancer cell growth can be accurately and easily extracted from candidate compounds. The recombinant microorganism or cell of the present invention can efficiently produce a peptide that can be used in the above screening method and screening kit. Further, the cancer therapeutic agent of the present invention can suppress the growth of cancer cells. [Brief Description of the Drawings]

[0011] [Figure 1] ​This figure shows the location of the N-terminal domain (NTD) and its fragments in BRG1. [Figure 2] This figure shows the location of the BAF155 SANT domain and its fragments. [Figure 3] This figure shows the results of immunoprecipitation performed on a mixture of Flag-A4-FL and HA-C1-FL expressed in baculovirus and fragments expressed in bacteria, followed by detection of the precipitated proteins by SDS-PAGE and Western blotting. [Figure 4] This figure shows the results of immunoprecipitation performed on a mixture of Flag-A4-FL and HA-C1-FL expressed in baculovirus and fragments expressed in bacteria, followed by detection of the precipitated proteins by SDS-PAGE and Western blotting. [Figure 5] This figure shows the results of mixing fragments expressed in bacteria, performing immunoprecipitation, and detecting the precipitated proteins by SDS-PAGE and Western blotting. [Figure 6] This figure shows the results of mixing fragments expressed in bacteria, performing immunoprecipitation, and detecting the precipitated proteins by SDS-PAGE and Western blotting. [Figure 7] The top figure shows the structure of the BAF155(621-673)-BRG1(368-427) fusion protein. The bottom figure shows the crystal structure of the BAF155-BRG1 fusion protein. The 10-amino acid linker between the two segments is shown by a dashed line and is not visible in the crystal structure shown in the bottom figure. [Figure 8] This figure shows the intermolecular interactions within the BAF155-BRG1 complex. The left figure shows hydrogen bonds or salt bridges between BAF155 and BRG1, represented by dotted lines. The right figure is the same as the left figure rotated 180 degrees on the vertical axis, showing van der Waals contacts between Ile666 and Tyr670 of BAF155 and BRG1, with residues represented by spheres. [Figure 9] This figure shows the intermolecular interactions within the BAF155-BRG1 complex. [Figure 10]The following figures show the results of immunoprecipitation using HA-tagged BRG1 (HA-A4-FL), Flag-tagged BAF155 (Flag-C1-FL), and HA-tagged BAF60 expressed in baculovirus, which were prepared in vitro and immunoprecipitated using anti-Flag resin. BAF155 SANT peptide (SANT) was added and eluted, and the eluted products were detected along with a buffer control. The eluted products and precipitates were then detected by SDS-PAGE and Western blotting. [Figure 11] pCDNA3-dsRed (Red Cont) and pCDNA3-dsRed fusion Flag-tagged SANT peptide (Red-Flag-SANT) were introduced into NUGC3 cells. The results of fractionation into cytoplasm, nucleus, and chromatin, and detection of the fractionated proteins by SDS-PAGE and Western blotting are shown in the figure. [Figure 12] pCDNA3-dsRed (Red Cont) and pCDNA3-dsRed fusion Flag-tagged SANT peptide (Red-Flag-SANT) were introduced into NUGC3 cells. The localization of the introduced peptides was observed using a fluorescence microscope. [Figure 13] pCDNA3-dsRed (Red Cont) and pCDNA3-dsRed fusion Flag-tagged SANT peptide (Red-Flag-SANT) were introduced into NUGC3 cells. The figure shows the results of anti-Flag immunoprecipitation performed after peptide introduction. [Figure 14] This figure shows the results of counting the number of cells in NUGC3 cells after introducing pCDNA3-eGFP (EGFP-Cont) and pCDNA3-EGFP fusion Flag-tagged SANT peptide (EGFP-Flag-SANT) from the first day to the third day after introduction. [Figure 15] This figure shows the results of counting the number of cells in MEL28 cells after introducing pCDNA3-eGFP (EGFP-Cont) and pCDNA3-EGFP fusion Flag-tagged SANT peptide (EGFP-Flag-SANT) from the first day to the third day after introduction. [Figure 16]This figure shows cell migration observed within 24 hours after scratching the cell surface of NUGC3 cell lines for EGFP-Cont and EGFP-Flag-SANT. [Figure 17] This figure shows the results of observing cell migration within 24 hours after scratching the cell surface of NUGC3 cell lines for EGFP-Cont and EGFP-Flag-SANT, visualizing the migrated cells with a fluorescence microscope, and counting the number of migrated cells. [Figure 18] This figure shows the results of RT-qPCR analysis of POLA2, MTHFD1, RRM2, and CDK1 mRNA in NUGC3 cells 48 hours after knockdown of each gene. The y-axis shows the relative expression level of each gene (normalized by GAPDH). Error bars indicate the standard deviation of three independent experiments. [Figure 19] This figure shows the results of counting NUGC3 cells for 3 days after knockdown of POLA2, MTHFD1, RRM2, and CDK1. Error bars indicate standard deviations (SD) of three independent experiments. [Figure 20] NUGC3 cells were transfected twice with either capGFP-polyA mRNAs (0.25-3.0 μg / well) or capGFP-NLS-SANT-polyA mRNAs (0.25-3.0 μg / well) using the TransIT-mRNA transfection kit on Day 0 and Day 1. The image shows the cells 48 hours after the first transfection. [Figure 21] NUGC3 cells were transfected twice using the TransIT-mRNA transfection kit on Day 0 and Day 1 with capGFP-polyA mRNAs (0.25-3.0 μg / well) or capGFP-NLS-SANT-polyA mRNAs (0.25-3.0 μg / well). The figure shows the results of RT-qPCR analysis of POLA2, MTHFD1, RRM2, and CDK1 48 hours after transfection with capGFP-polyA mRNAs or capGFP-NLS-SANT-polyA mRNAs. [Figure 22]This figure shows the results of cut-and-run qPCR analysis of the TSS region of the POLA2, MTHFD1, RRM2, and CDK1 genes 48 hours after transfection with capGFP-polyA mRNAs or capGFP-NLS-SANT-polyA mRNAs. [Figure 23] This figure shows the results of subcutaneous injection of 3 × 10⁶ NUGC3 cells into the right dorsal skin of mice, followed by daily injection of pGFP-BspQ1 or pGFP-C1-SANT mRNA (2 μg) into the tumor, and then imaging the tumor 10 days later. [Figure 24] This figure shows the results of subcutaneous injection of 3 × 10⁶ NUGC3 cells into the right dorsal skin of mice, followed by daily injection of pGFP-BspQ1 or pGFP-C1-SANT mRNA (2 μg) into the tumors, and then measurement of tumor size and weight after 10 days. [Modes for carrying out the invention]

[0012] The following describes an embodiment of the screening method, screening kit, in silico analysis method, recombinant microorganisms or cells, and cancer therapeutic agent of the present invention.

[0013] <Screening Method> The present invention's screening method for cancer cell proliferation inhibitory compounds is: A reaction step in which a candidate compound is brought into contact with a composite structure, A detection process for detecting the failure of a composite structure Includes.

[0014] The following describes each step.

[0015] (Reaction process) In the reaction process, candidate compounds are brought into contact with the composite structure.

[0016] Candidate compounds are any compounds whose presence or absence and strength of cancer cell proliferation inhibitory effect should be determined, and may be a single compound or a mixture of two or more compounds. Furthermore, there are no particular limitations on the method or amount of contact between the candidate compounds and the composite structure, and these can be designed as appropriate.

[0017] The composite structure used in the reaction process includes a first peptide having a first binding region and a second peptide having a second binding region. The first binding region and the second binding region of the composite structure are bound together by interaction. More specifically, the first peptide and the second peptide are bound together in the composite structure via interactions (hydrostatic bonds, hydrogen bonds, ionic bonds) between the first and second binding regions.

[0018] The first binding region of the first peptide consists of a portion of the amino acid sequence W1 represented by Sequence ID No. 1 below, or an amino acid sequence X1 that has 80% or more sequence identity with amino acid sequence W1. Amino acid sequence W1 is a partial sequence derived from BAF155, one of the subunits that make up the BAF / PBAF complex (the sequence of "BAF-SANT" described later). (Sequence ID 1) REWTEQETLLLLEALEMYKDDWNKVSEHVGSRTQDECILHFLRLPIEDPYLEN

[0019] The amino acid sequence X1 constituting the first binding region may have one or more amino acid residue substitutions, deletions, insertions, additions, or combinations thereof compared to the amino acid sequence W1 represented by Sequence ID No. 1. In this case, the amino acid sequence X1 constituting the first binding region may have sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% with respect to the amino acid sequence W1.

[0020] Furthermore, for example, if the first binding region of the first peptide is composed of a portion of the amino acid sequence W1, the first binding region may consist of 20 or more, 30 or more, 40 or more, or 50 or more consecutive amino acid residues from the amino acid sequence W1.

[0021] The first peptide only needs to include a first binding region consisting of a portion of the amino acid sequence W1, or an amino acid sequence X1 having 80% or more sequence identity with amino acid sequence W1. This also includes forms in which an amino acid sequence of appropriate length is added to amino acid sequence X1, as long as it does not impair the interaction between the first and second binding regions. Specifically, for example, the first peptide may have an amino acid sequence related to a heterologous protein, such as a labeling molecule (e.g., luciferase or alkaline phosphatase), added to amino acid sequence X1.

[0022] More specifically, when amino acid sequences X1 constituting the first binding region of the first peptide are replaced, deleted, or inserted from amino acid sequence W1, the number of amino acid residues is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. Furthermore, when an amino acid sequence relating to a different protein is added to the end of amino acid sequence X1 of the first peptide, for example, the number of amino acid residues added is not particularly limited, but may be, for example, 5 to 100, 5 to 1000, or more.

[0023] The second binding region of the second peptide consists of a portion of the amino acid sequence W2 represented by Sequence ID No. 2 below, or an amino acid sequence X2 that has 80% or more sequence identity with amino acid sequence W2. Amino acid sequence W2 is a partial sequence (the "NTD" sequence described later) derived from BRG1, one of the subunits that make up the BAF / PBAF complex. (Sequence 2) QEREYRLQARIAHRIQELENLPGSLAGDLRTKATIELKALRLLNFQRQLRQEVVVCMRRD

[0024] The amino acid sequence X2 constituting the second binding region may have one or more amino acid residue substitutions, deletions, insertions, additions, or combinations thereof compared to the amino acid sequence W2 represented by Sequence ID No. 2. In this case, the amino acid sequence X1 constituting the first binding region may have sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% with respect to the amino acid sequence W1.

[0025] Furthermore, for example, if the second binding region of the second peptide is composed of a portion of the amino acid sequence W2, the second binding region may be composed of 20 or more, 30 or more, 40 or more, or 50 or more consecutive amino acid residues from the amino acid sequence W1.

[0026] The second peptide only needs to include a second binding region consisting of a portion of the amino acid sequence W2, or an amino acid sequence X2 having 80% or more sequence identity with amino acid sequence W2. This also includes forms in which an amino acid sequence of appropriate length is added to amino acid sequence X2, as long as it does not impair the interaction between the first and second binding regions. Specifically, for example, the second peptide may have an amino acid sequence related to a heterologous protein (e.g., luciferase or alkaline phosphatase) added to amino acid sequence X2.

[0027] More specifically, when amino acid sequences X2 constituting the second binding region of the second peptide are substituted, deleted, or inserted from amino acid sequence W2, the number of amino acid residues is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. Furthermore, when an amino acid sequence relating to a different protein is added to the end of amino acid sequence X2 of the second peptide, for example, the number of amino acid residues added is not particularly limited, but may be, for example, 5 to 100, 5 to 1000, or more.

[0028] The method for producing the first and second peptides is not particularly limited. The first and second peptides can be produced by conventionally known methods, such as methods utilizing cell engineering techniques or methods using peptide synthesis equipment.

[0029] The recombinant microorganism or cell of the present invention contains a nucleotide sequence encoding a first peptide or a second peptide. Here, "cell" refers to a cell line widely used in cell engineering techniques, such as Escherichia coli, yeast, or animal cells. The recombinant microorganism or cell of the present invention allows for the efficient acquisition of the first and second peptides of the present invention.

[0030] Furthermore, as mentioned above, the first and second peptides may be amino acid sequences X1 or X2 to which a labeling molecule is fused. Examples of labeling molecules include enzymes, fluorescent proteins, and protein tags. Specifically, examples of enzymes include luciferase, alkaline phosphatase, horseradish peroxidase, invertase, β-galactosidase, β-glucuronidase, and modified versions of these enzymes. Examples of fluorescent proteins include green fluorescent protein (GFP) and modified versions of GFP. Examples of protein tags include FLAG tags, HA tags, GST tags, Protein A tags, polyhistidine tags, V5 tags, Myc tags, SBP tags, Halo tags, and Strep tags.

[0031] Furthermore, whether or not the first peptide and the second peptide form a composite structure can be confirmed by known methods such as X-ray crystallography.

[0032] (Detection process) The detection process detects the failure of the composite structure. In other words, the detection process detects whether the bond between the first and second bonding regions of the composite structure is maintained or broken.

[0033] The method for detecting the maintenance or cleavage of the bond between the first and second binding regions is not particularly limited, but known methods that use the function of the labeled molecule bound to the first and / or second peptide as an indicator can be appropriately employed.

[0034] The "function of a labeled molecule" refers to directly or indirectly producing a detectable signal that is visually observable, electrically detected, or recorded by other means. Specifically, this includes, for example, luminescence, radiation, coloration, aggregation, and changes in magnetic and electrical properties. Examples of luminescence include fluorescence and phosphorescence, but fluorescence is typically used for detection. When a labeled molecule exhibits luminescence, examples include fluorescent dyes, luminescent proteins (e.g., luciferase and fluorescent proteins), and proteins that catalyze the luminescence reaction of the molecule. When a labeled molecule exhibits radiation, examples include radioactive isotopes. When a labeled molecule exhibits coloration, examples include color-developing dyes or enzymes that can amplify the signal by coloration (e.g., peroxidase, alkaline phosphatase, galactosidase, etc.). In addition, colored high molecular weight colloidal materials, such as particulate matter like latex beads, and magnetic and paramagnetic beads can also be used. Furthermore, these can be used in combination with methods that amplify signals through interaction with biosensors.

[0035] Alternatively, known carriers that bind to protein tags can be used. Examples of carriers include materials made of plastic, glass, gel, celluloid, paper, magnetic resin, polyvinylidene fluoride, nylon, nitrocellulose, agarose, latex, and polystyrene. Specifically, examples of carriers include ELISA plates, dipsticks, microtiter plates, radioimmunoassay plates, beads, agarose beads, plastic beads, latex beads, and magnetic beads.

[0036] Furthermore, in this invention, the term "detection" also encompasses the concept of "quantification." That is, the detection step includes a method for measuring the amount of the first peptide or the second peptide produced by the dissociation of the first and second peptides of the composite structure. Therefore, it is possible to determine whether a candidate compound has sufficient cancer cell proliferation inhibitory activity depending on the quantified amount of the first or second peptide. For example, a candidate compound in which a large amount of the first or second peptide is detected can be determined to have a high probability of having excellent cancer cell proliferation inhibitory activity. It is also possible to set a threshold related to the amount of the first or second peptide detected and extract candidate compounds that are above this threshold.

[0037] The BAF / PBAF complex (including those with mutations) can suppress the function of tumor suppressor genes in vivo. However, compounds that cleave the bond between the first and second binding regions of the complex structure disrupt the BAF / PBAF complex, and thus can release the suppression of tumor suppressor gene function in vivo. In other words, in the screening method of the present invention, candidate compounds that cleave the bond between the first and second binding regions of the complex structure can suppress the proliferation of cancer cells.

[0038] Therefore, the screening method of the present invention may include a determination step in which candidate compounds that cleave the bond between the first and second binding regions of a composite structure are determined to be cancer cell proliferation inhibitory compounds.

[0039] The screening method of the present invention can accurately and easily extract compounds having cancer cell proliferation inhibitory activity from candidate compounds without being affected by other subunits surrounding BRG1 and BAF155.

[0040] The screening method of the present invention may include various steps in addition to those described above.

[0041] <Screening Kit> The screening kit for cancer cell proliferation inhibitory compounds of the present invention uses the following reagents: A first peptide comprising a first binding region consisting of a portion of the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, A second peptide comprising a second binding region consisting of a portion of the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, Includes.

[0042] Since the first and second peptides are the same as those described in the screening method of the present invention, a detailed explanation will be omitted.

[0043] The first and second peptides may form a composite structure or be held separately. In the case of a composite structure, the first and second peptides are bound together via interactions (hydrogen bonds, ionic bonds) between the first and second binding regions. In the case of separately held peptides, a composite structure can be formed by coexisting the first and second peptides, for example, when used for screening.

[0044] Furthermore, the screening kit of the present invention may include known labeled molecules, carriers, etc., as described above.

[0045] By using the screening kit of the present invention, compounds having cancer cell proliferation inhibitory effects can be extracted from candidate compounds accurately and easily.

[0046] <In silico analysis method> The in silico analysis method of the present invention is a method for identifying cancer cell proliferation inhibitory compounds in in silico. Explanations of aspects common to the screening method of the present invention described above will be omitted.

[0047] The in silico analysis method of the present invention, in in silico, Information on a portion of the amino acid sequence represented by Sequence ID No. 1, or a first amino acid sequence that has 80% or more sequence identity with the amino acid sequence represented by Sequence ID No. 1, Information on a portion of the amino acid sequence represented by Sequence ID No. 2, or a second amino acid sequence that has 80% or more sequence identity with the amino acid sequence represented by Sequence ID No. 2, and The method includes selecting a compound capable of cleaving the bond between the first amino acid sequence and the second amino acid sequence due to interaction, based on at least one of the following pieces of information.

[0048] Regarding the interaction between the first and second amino acid sequences, hydrostatic bonds, hydrogen bonds, and ionic bonds can be observed at specific locations. For example, the in silico analysis method of the present invention can predict and select compounds capable of cleaving these bonds in silico. As described above, compounds that cleave the bonds between the first and second amino acid sequences disrupt the BAF / PBAF complex and are therefore considered highly likely to exert an inhibitory effect on cancer cell proliferation.

[0049] The in silico analysis method of the present invention can utilize, for example, known data on bioinformatics and / or the biological function of proteins when predicting or selecting compounds. Typically, the prediction or selection of compounds can be optimized and verified by docking simulations. Known software can be used for docking simulations. Furthermore, the in silico analysis method of the present invention can also utilize information such as the structure of surfactant and the structure of cyclic peptides that have cancer cell proliferation inhibitory effects, as described in Patent Document 1.

[0050] Furthermore, the in silico analysis method and the screening method of the present invention can be combined. That is, for example, a compound selected by the in silico analysis method of the present invention can be brought into contact with a composite structure as a candidate compound in the screening method of the present invention, and the failure of the composite structure can be detected.

[0051] The screening method, screening kit, in silico analysis method, and recombinant microorganisms or cells of the present invention are not limited to the embodiments described above.

[0052] <Cancer treatment drugs> One embodiment of the cancer therapeutic agent of the present invention contains a peptide comprising a portion of the amino acid sequence W1 represented by Sequence ID No. 1, or an amino acid sequence X1 having 80% or more sequence identity with amino acid sequence W1. The amino acid sequence W1 is the sequence of a peptide (described later as "BAF-SANT") derived from BAF155, one of the subunits constituting the BAF / PBAF complex. This peptide can disrupt the protein-protein interaction (PPI) between BRG1 and BAF155 and has an inhibitory effect on the proliferation of cancer cells, thus serving as an active ingredient in the cancer therapeutic agent. (Sequence ID 1) REWTEQETLLLLEALEMYKDDWNKVSEHVGSRTQDECILHFLRLPIEDPYLEN

[0053] The amino acid sequence X1 may have one or more amino acid residue substitutions, deletions, insertions, additions, or combinations thereof compared to the amino acid sequence W1 represented by Sequence ID No. 1. In this case, the amino acid sequence X1 constituting the first binding region may have a sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% with respect to the amino acid sequence W1.

[0054] Furthermore, for example, if amino acid sequence X1 is composed of a portion of amino acid sequence W1, amino acid sequence X1 may consist of 20 or more, 30 or more, 40 or more, or 50 or more consecutive amino acid residues from amino acid sequence W1.

[0055] The peptide contained in the cancer therapeutic agent of the present invention only needs to contain a part of the amino acid sequence W1, or an amino acid sequence X1 that has 80% or more sequence identity with the amino acid sequence W1, and also includes a form in which an amino acid sequence of an appropriate length is added to this amino acid sequence X1.

[0056] More specifically, when amino acid residues are substituted, deleted, or inserted into the amino acid sequence X1 constituting the peptide contained in the cancer therapeutic agent of the present invention, the number of amino acid residues is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. Furthermore, when an amino acid sequence relating to a different protein is added to the end of the amino acid sequence X1 of the peptide, for example, the number of amino acid residues added is not particularly limited, but may be, for example, 5 to 100, 5 to 1000, or more.

[0057] The administration method of cancer treatment drugs in this embodiment is not particularly limited and may be either oral or parenteral. Examples of parenteral administration include injection (such as intramuscular injection, intravenous injection, or subcutaneous injection), transdermal administration, and transmucosal administration (through the nose, oral cavity, or lung).

[0058] The cancer therapeutic agent of the present invention may use the peptide as an active ingredient as is, or it may be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Examples of dosage forms include liquids (e.g., injections), dispersants, suspensions, tablets, pills, powders, suppositories, powders, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, poultices, etc.

[0059] Formulation can be carried out by conventional methods, for example, by using excipients, binders, disintegrants, lubricants, solvents, solubilizers, colorants, flavoring and odor-correcting agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc., as appropriate.

[0060] Examples of ingredients used in formulation include purified water, saline solution, phosphate buffer, dextrose, glycerol, pharmaceutically acceptable organic solvents such as ethanol, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropylcellulose, starch, corn starch, anhydrous silicic acid, aluminum magnesium silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, acacia gum, tragacanth, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, and human serum albumin.

[0061] Another embodiment of the cancer therapeutic agent of the present invention includes mRNA encoding a peptide containing a portion of the amino acid sequence W1 represented by SEQ ID NO: 1, or an amino acid sequence X1 having 80% or more sequence identity with amino acid sequence W1. In other words, this embodiment is an mRNA drug for cancer treatment.

[0062] The typical administration method for cancer treatment drugs in this form is injection, such as intramuscular injection, intravenous injection, or subcutaneous injection. However, oral administration is also possible through combination with formulation technologies such as encapsulation. Similarly, transdermal administration and transmucosal administration (through the nose, mouth, or lung) are also possible.

[0063] In this embodiment of cancer therapeutics, the mRNA may include a 5' cap, a 3' untranslated region, a poly-A tail, etc., in addition to the coding region. Furthermore, in this embodiment of cancer therapeutics, from the viewpoint of maintaining the stability of the mRNA molecule itself, supporting efficient delivery to cells, and suppressing immune responses, the mRNA may contain, for example, lipid nanoparticles, modified nucleotides, pH buffers, sugars, preservatives, antioxidants, etc.

[0064] The cancer treatment drug of the present invention is not limited to the embodiments described above. [Examples]

[0065] The present invention will be described below with reference to examples, including the screening method and cancer treatment drug of the present invention, but the present invention is not limited in any way to the following examples.

[0066] 1. Experimental Method <Complex Disruption Assay> Human BRG1 was cloned with flag and HA tags and inserted into the vector pCAGIpuro (Iseki et al., Stem Cell 34(2):322-333, 2015). This yielded pCAGIPuro-Flag-HA-BRG1. Furthermore, 8His-tagged BAF155 fused with Luciferase-SV40 polyA was subcloned into pcDNA3 to create pcDNA3-8His-BAF155-Luc. 293T cells were sequentially transcribed with pCAGIPuro-Flag-HA-BRG1 and pcDNA3-8His-BAF155-Luc using Lipofectamine 3000. Transcribed cells were selected with 2 μg / ml puromycin and 0.5 mg / ml G418 to establish 293T-FlagBRG1-BAF155-Luc cells. 293T-FlagBRG1-BAF155-Luc cells were harvested from five 10 cm sheets of culture medium and collected in 1 ml of 0.2 M KCl HEG buffer (10 mM Hepes, pH 7.6, 0.5 mM EDTA, 10% glycerol). Homogenization was performed 5 times for 15 strokes using a Dounce homogenizer (WHEATOM 2 ml), followed by 3 sonications at 10% power using a BRANSON digital sonafire. The homogenates were centrifuged at 15,000 rpm for 10 minutes, and the resulting supernatant was frozen in liquid nitrogen and stored at -80°C. For the complex disruption assay, 11 μl of supernatant was incubated with 2 μl of anti-Flag resin that had been pre-washed three times with 500 μl of 0.2 M KCl HEG buffer. After 10 minutes of incubation, the anti-Flag resin was washed three times with 0.2 M KCl HEG buffer. The resin-bound complex was eluted twice with 12 μl of elution compound in 0.15 M KCl HEG buffer. The resulting elute (20 μl) was mixed with 20 μl of Britlite Plus (PerkinElmer) and measured using a FLUOstar OPTIMA microplate reader. For complex disruption assays using recombinant proteins, Flag- and His-tagged human BRG1, HA- and His-tagged human BAF60A, and HA- and His-tagged N-terminal Nluc-BAF155 were cloned into the pFastBac™1 (Invitrogen Life Technologies) vector to obtain pFastBac1-Flag-His-BRG1, pFastBac1-HA-His-BAF60A, and pFastBac1-HA-His-NLuc-BAF155, respectively. Baculoviruses were prepared using the Bac-to-BAC® baculovirus system according to the manufacturer's protocol. Baculoviruses expressing Flag-His-BRG1, HA-His-BAF60A, and HA-His-NLuc-BAF155 were coin-infected into two 10 cm dishes of Sf9 cells. Cells were harvested on day 3 after infection and collected in 2 ml of 0.5 M KCl HEG buffer. Homogenization was performed five times for 20 strokes using a Dounce homogenizer, and the homogenate was then centrifuged at 15,000 rpm for 10 minutes. The resulting supernatant was frozen in liquid nitrogen and stored at -80°C. For a complex disruption assay using the recombinant BAF complex, 10 μl of supernatant was incubated with 2 μl of anti-Flag resin that had been pre-washed three times with 500 μl of 0.5 M KCl HEG buffer. After 10 minutes of incubation, the anti-Flag resin was washed three times with 0.5 M KCl HEG buffer and once with 0.2 M KCl HEG buffer. The resin-bound complex was eluted twice with 12 μl of elution compound in 0.15 M KCl HEG buffer. The resulting elute (15 μl) was mixed with 25 μl of NanoGlo® (Promega) and measured using a FLUOstar OPTIMA microplate reader.

[0067] <rt-qpcr> cDNA was synthesized using 0.5 μg of total RNA with oligo(dT) primers (Life Technologies), random hexamers (Takara), and M-MuLV reverse transcriptase (NEB). Real-time RT-PCR was performed using reagents containing SYBR Green on an ABI PRISM 7900HT instrument (Applied Biosystems). The expression level of the target gene was normalized based on the expression of GAPDH, and the relative expression level was calculated compared to the DMSO control. The PCR primers used were as follows. forward 5’-AGGCGGCGATTTTTGTATGT-3’ (SEQ ID NO: 3), backward 5’-GGGCAATAAAGCGCATTCAA-3’ (SEQ ID NO: 4) for EGR1, forward 5’-TTATTTATTAAGATGGATTCTCAG-3’ (SEQ ID NO: 5), backward 5’-CTTGGAACAATAAGCAAACAATGC-3’ (SEQ ID NO: 6) for FOS, forward 5’-AGTGAGACTGAGGGATCGTAGA-3’ (SEQ ID NO: 7), backward 5’- GGGGTCGGGGATTCATTGAA-3’ (SEQ ID NO: 8) for FOSB, forward 5’-GGAGCGAGATCCCTCCAAAAT-3’ (SEQ ID NO: 9), backward 5’-GGCTGTTGTCATACTTCTCATG -3’ (SEQ ID NO: 10) for GAPDH.

[0068] <mRNA synthesis in vitro> As templates, the coding regions of eGFP and eGFP fused with NLS (nuclear localization signal) and C1-SANT peptide were subcloned into a template vector (BspQ I) for T7 mRNA synthesis to obtain pGFP-BspQ1 and pGFP-C1-SANT-BspQ1, respectively. Capped and modified mRNAs were synthesized by in vitro transcription (IVT) using BspQ1-linearized pGFP-BspQ1 and pGFP-C1-SANT-BspQ1 plasmid DNAs, the IVTpro T7 mRNA Synthesis Kit (Takara), CleanCap® Reagent M6 (TriLink Biotechnologies), and N1-methyluridine-5'-triphosphate (TriLink Biotechnologies). After IVT, the RNA was treated with DNase I (Invitrogen) and purified using the RNeasy® Mini Kit (Qiagen). The concentration of the mRNA was determined by measuring the optical density at 260 nm.

[0069] <Anti-tumor assay (in vivo) using mRNA> The athymic nude mice were obtained from Charles River Laboratories International, Inc. The mice were regularly housed in a temperature-controlled room under a 12-hour light / dark cycle and were given free access to food and water. In the nude mouse xenograft assay, 3 × 10^6 NUGC3 cells were xenografted into the athymic nude mice. Briefly, tumorigenicity was assayed by subcutaneous injection of 3 × 10^6 cells into the flank of the nude mice. The mice were observed for 5 weeks until the tumors reached 5 mm in size. After tumors of 5 mm size were formed, 2 μg of mRNA (pGFP-BspQ1 or pGFP-C1-SANT coding) was mixed with 100 μl of serum-free medium, mRNA boost reagent, and TransITR mRNA reagent and injected into the tumors the next day. Tumor size was examined 10 days after treatment. Images of the tumors were taken using a digital camera. Tumor samples were fixed in 10% neutral buffered formalin solution (Sigma-Aldrich) and stained with HE.

[0070] <Anti-tumor assay (in vitro) using mRNA> NUGC3 and MEL28 cells were cultured in RPMI medium supplemented with 10% FBS. One day prior to counting, the cells were seeded at a density of 0.4 × 10 4 cells / cm 2 in 24-well plates. The next day, 1 μg of mRNA (pGFP-BspQ1 or pGFP-C1-SANT coding) was mixed with 50 μl of serum-free medium, 0.5 μl of mRNA boost reagent, and 0.5 μl of TransITR mRNA reagent, and the cells were treated in duplicate and defined as "0" on that day. The mRNA was administered on days 0, 1, and 2. Four images were collected from each well on days 0, 1, 2, and 3, and the cell number was counted using a 1 mm 2 square. Three days later, the cells were trypsinized and counted by trypan blue staining.

[0071] <Anti-tumor assay (in vitro) using plasmid expression vector> NUGC3 and MEL28 cells were cultured in RPMI medium with 10% FBS. Cells were transfected with 1 μg of plasmid DNA (eGFP or eGFP-C1-SANT coding) using Lipofectamine 3000 according to the manufacturer's protocol. 24 hours after transfection, cells were sorted using a cell sorter and placed in 0.4 × 10⁶ well plates. 4 cells / cm 2 Seeds were sown at the following density. Four images were collected from each well at 0, 1, 2, and 3 days, at 1 mm. 2 The number of cells was counted using the square shape.

[0072] <Crystal structure analysis> For structural analysis, we designed a fusion protein by fusing human BAF155 (621-673) and human BRG1 (368-427) as C1-N2-4 and A4-N3-4 fragments, respectively, and linking them via a flexible link of two repeating Gly-Gly-Gly-Gly-Ser (2x(GGGGS)). This structure was inserted into a modified pET-15b vector (Novagen) expressing glutathione S-transferase (GST) and a His tag at the N-terminus, and equipped with an HRV 3C protease cleavage site for removing the tag after purification. X-ray diffraction experiments performed on selenomethionine (SeMet) derivative crystals yielded a different space group than that of the native crystal. Unexpectedly, this improved crystal packing with the search model, and we successfully determined the phase using molecular substitution. Expression vectors were introduced into methionine-dependent B834(DE3) strains and cultured in LeMaster medium 3 68 (supplemented with 150 μg / ml ampicillin) at 37°C. Expression of the SeMet-containing protein was induced with 1 mM IPTG when the OD 600 was 0.6, and the cultures were incubated overnight at 30°C. All purification steps were performed at 4°C. Crystallization was performed using the sitting drop vapor diffusion method. A 3 mg / ml BAF155-BRG1 fusion protein solution (dissolved in 100 mM NaCl, 10 mM Hepes pH 7.6, and 1 mM DTT) was divided into 100 nl drops and mixed with a 100 nl reservoir solution containing 20% ​​PEG6000 and 0.1 M MES pH 6.0, and incubated at 10°C. After several days, the grown rod-shaped crystals were cryoprotected in a solution containing 23% PEG6K, 0.1 M MES pH 6.0, and 20% glycerol, and then rapidly frozen in liquid nitrogen. X-ray diffraction data were collected at the SPring-8 BL32XU beamline using the ZOO 4 69 automated data acquisition system. The synchrotron radiation wavelength was set to 1A, and diffraction data were collected with a vibration angle of 0.1° per image, for a total angular range of 540°. The datasets obtained from five crystals were automated and integrated using the XDS 5 70 and KAMO 6 71 programs. The initial phase was determined by molecular substitution using the Phaser 7 72 program, with the model predicted by AlphaFold2 as the exploratory model. Model construction and refinement were performed using Coot 8 73 and Phenix 9 74. Structural diagrams were created using PyMOL (Schrodinger, Inc.).

[0073] 2.Results <1> Interaction between BRG1 and BAF155 To further investigate the direct interaction between BRG1 and BAF155, various protein fragments were designed and expressed in E. coli (Figures 1 and 2), and their interactions were examined in vitro using Flag-tagged IP (immunoprecipitation) or HA-tagged IP (immunoprecipitation). Full-length BRG1 (A4-FL) was confirmed to interact with full-length BAF155 (C1-FL), C1-N2, and C1-N3 (Figure 3: lanes 7, 9, and 10).

[0074] Background affinity was observed between BAF155 fragments C1-N2-1, 2, 3, and 4 and the resin, so a competitive assay was performed to further evaluate the affinity of the interaction (Figure 4). The BAF155 C1-N2-4 fragment inhibited the interaction between BRG1 and BAF155 (Figure 4: lane 17). The BAF155 C1-N2-4 fragment interacted with the BRG1 A4-N3 fragment (Figure 5: lane 13; Figure 6, lane 12), and specifically interacted with the A4-N3-4 fragment, a subregion of A4-N3 within SMARCA4(BRG1) (Figure 6, lane 16). This confirmed that the BRG1 A4-N3-4 fragment and the BAF155 C1-N2-4 fragment form a core interaction of the complex, and that the BAF155 C1-N2-4 fragment strongly and competitively inhibits this protein-protein interaction.

[0075] The A4-N3-4 fragment of BRG1 contains the region from amino acid 368 to 427, which is also called the NTD region or pre-HSA region. Hereafter, the A4-N3-4 fragment may be referred to as "NTD". Additionally, the C1-N2-4 fragment of BAF155 contains amino acids from 621 to 672 and matches the SANT domain, which has the same amino acid sequence as SMARCC2 (BAF170). Hereafter, the C1-N2-4 fragment of BAF155 may be referred to as "BAF-SANT".

[0076] The amino acid sequences of BAF-SANT and NTD are as follows: <baf-sant> (Sequence ID 1) REWTEQETLLLLEALEMYKDDWNKVSEHVGSRTQDECILHFLRLPIEDPYLEN <ntd> (Sequence 2) QEREYRLQARIAHRIQELENLPGSLAGDLRTKATIELKALRLLNFQRQLRQEVVVCMRRD

[0077] The addition of BAF-SANT disrupted the interaction between BRG1 and BAF155 in competitive experiments using proteins expressed in E. coli (Figure 6: lane 16). Furthermore, BAF-SANT also induced dissociation between BAF155 and BRG1 in complex disruption assays using proteins expressed in 293T cells and Sf9 cells. These results demonstrate that surfactin, as described in Patent Document 1, disrupts the binding of BRG1 and BAF155 by competing with this interaction.

[0078] <2> Structural analysis of the BAF / PBAF complex The structure of the human BAF / PBAF complex has been elucidated to date (for example, (1) Yuan, J., Chen, K., Zhang, W. & Chen, Z. Structure of human chromatin-remodeling PBAF complex bound to a nucleosome. Nature 605, 166-171, doi:10.1038 / s41586-022-04658-5 (2022). (2) He, S. et al. Structure of nucleosome-bound human BAF complex. Science, doi:10.1126 / science.aaz9761 (2020). (3) Mashtalir, N. et al. Chromatin landscape signals differentially dictate the activities of mSWI / SNF family complexes. Science 373, 306-315, doi:10.1126 / science.abf8705 (2021).). The NTD of SMARCA4(BRG1) is anchored to the basal module of the BAF complex, which consists of seven subunits, including SMARCD1(BAF60A). The portion of SMARCA4(BRG1) that is anchored to the basal module is composed of the SANT domain of SMARCC, the pre-HSA of SMARCA4(BRG1), and the C-terminal helix of SMARCD1.

[0079] To elucidate the details of the PPI of BAF155 and BRG1 disrupted by BAF-SANT or surfactin, a fusion protein was created using a 10-amino acid linker sequence of BAF-SANT (621-673 region of human BAF155) and NTD (368-427 region of human BRG1) (Figure 7, top). Next, the crystal structure of this fusion protein was analyzed at a resolution of 2.59 Å (Figure 7, bottom). The BAF155 segment of this fusion protein consists of an elongated loop with three α-helices (Ha-c) and a 310-helix at the C-terminus, followed by the BRG1 segment, which contains three α-helices (H1-3). Interactions between these components form a structurally compact BAF155-BRG1 complex (Figure 7, bottom and Figure 8).

[0080] Within the BAF155-BRG1 complex, several residues were identified that form ionic and hydrogen bonding interactions. Glu636 on the Ha helix of BAF155 was found to form a salt bridge with Lys405 and Arg408 on the H2 helix of BRG1. Furthermore, Glu667 on the elongation loop of BAF155 was found to form a salt bridge with Arg414 and Arg417 on the H3 helix of BRG1. Additionally, Gln413 on the H3 helix of BRG1 was found to perform helix capping through hydrogen bonding with the Hc helix of BAF155, interacting with the main chain carbonyl oxygens Leu664 and Phe661 of BAF155 (Figure 8 left, Figure 9).

[0081] Furthermore, for example, it was confirmed that Ile666 in the extension loop of BAF155 hydrophobically interacts with Ala406, Leu407, Leu410, and Gln413 of BRG1. In addition, it was confirmed that Tyr670 in the Hc helix of BAF155 is in contact with Leu374, Arg377, Ile378, Arg381, and Leu407 of BRG1 (Figure 8 right, Figure 9).

[0082] <3> X-ray crystallography analysis of the complex of BAF-SANT and NTD X-ray crystallography confirmed the structures of BAF-SANT and NTD, and their interaction (PPI) was observed both in vivo and in vitro. Full-length HA-tagged SMARCA4 (BRG1), HA-tagged full-length SMARCD1 (BAF60), and Flag-tagged full-length SMARCC1 (BAF155) were expressed in Sf9 cells, and core complexes were generated using these. The purified complexes showed dissociation upon the use of BAF-SANT. In the absence of BAF-SANT, the Flag resin precipitated all subunits via Flag-tagged full-length SMARCC1 (BAF155) (Figure 10: compare lanes 1 and 4). Adding BAF-SANT caused SMARCA4 (BRG1) to dissociate, while SMARCD1 (BAF60) did not (Figure 10: lanes 2 and 3). A decrease in SMARCA4 (BRG1) within the complex was also observed (Figure 10: lanes 5 and 6).

[0083] <4> The disruptive effect of BAF-SANT on protein-protein interactions (PPIs). NUGC3 cells were overexpressed with DsRed-fused NLS Flag-tagged BAF-SANT, while DsRed alone was used as a control. Fractionation of cell extracts revealed that SANT was localized in chromatin and the nucleus / cytoplasm, whereas this localization was not observed in the DsRed control (Figure 11). In NUGC3 cells overexpressing NLS-Flag-tagged BAF-SANT, it was concentrated in the nucleus rather than diffused within the cell as in DsRed alone (Figure 12). Analysis of whole cell extracts from NUGC cells using anti-Flag immunoprecipitation confirmed that BAF-SANT bound to BRG1 but not to BAF155 (Figure 13: lanes 3 and 6). These results indicate that BAF-SANT (Figures 10, 11, and 13) disrupts the protein-protein interaction (PPI) between SMARCA4 (BRG1) and SMARCC1 (BAF155) in vitro and in vivo.

[0084] <5> The effects of BAF-SANT on cancer cells pCDNA3-eGFP (EGFP-Cont) and pCDNA3-EGFP fusion flag-tagged SANT peptide (EGFP-Flag-SANT) were introduced into NUGC3 and MeL28 cells, and the number of cells was counted from the first day to the third day after introduction. BAF-SANT was shown to significantly suppress cell proliferation in NUGC3 and MeL28 cell lines with mutations in the BAF / PBAF complex subunit (Figures 14 and 15). This result indicates the importance of the BAF / PBAF complex in regulating cell growth. In addition, BAF-SANT significantly suppressed cell migration within 24 hours after scratching the cell surface in the NUGC3 cell line (Figures 16 and 17).

[0085] To elucidate the common mechanisms of surfactant and BAF-SANT, NUGC3 and MeL28 cell lines were treated with surfactant and BAF-SANT was overexpressed. The results showed genetic changes, and upregulated and downregulated genes were identified (Figure 18). There were very few genes that were overexpressed in common; only genes that were significantly suppressed in common were shown (Figure 19). POLA2, MTHFD1, and RRM2, among others, were confirmed to be downregulated by the overexpression of surfactant and BAF-SANT peptides. Furthermore, CDK1, a gene that plays a crucial role in mitotic progression, was also downregulated by surfactant and BAF-SANT peptides. POLA2, MTHFD1, RRM2, and CDK1 are all known to be involved in cancer progression. For example, POLA2 is a regulatory subunit of the DNA polymerase α complex and is associated with ovarian cancer and gastrointestinal stromal tumors, while RRM2 and CDK1 are enzymes that play important roles in DNA synthesis and repair, and their overexpression is associated with cancer progression. BAF-SANT has been shown to have an inhibitory effect on cancer cell proliferation.

[0086] <6> mRNA encoding BAF-SANT mRNA encoding BAF-SANT with a CAP structure, pseudo-uridine introduction, and a Poly A tail was generated. When this mRNA was supplied to NUGC3 cells, BAF-SANT fused with EGFP-NLS was expressed in the NUGC3 cells (Figure 20), and it was confirmed that this caused a decrease in cell proliferation and suppression of MTHFD1, RRM2, and CDK1 expression (Figures 21 and 22). Furthermore, direct injection of BAF-SANT mRNA into NUGC3 tumors significantly suppressed tumor growth (Figures 23 and 24). The mRNA encoding BAF-SANT has been confirmed to be usable as an mRNA drug for cancer treatment.< / ntd>

Claims

1. A screening method for cancer cell proliferation inhibitory compounds, A reaction step in which a candidate compound is brought into contact with a composite structure, A detection step for detecting the failure of the composite structure, Includes, The composite structure used in the reaction step is A first peptide comprising a first binding region consisting of a part of the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, A second peptide comprising a second binding region consisting of a part of the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, and It includes, and the first binding region and the second binding region are bound by interaction, The detection step involves detecting whether the bond between the first bonding region and the second bonding region of the composite structure is maintained or broken. Screening method.

2. Furthermore, the process includes a determination step for determining the candidate compound, In the determination step, the candidate compound that cleaves the bond between the first binding region and the second binding region of the composite structure is determined to be a cancer cell proliferation inhibitory compound. The screening method according to claim 1.

3. A screening kit for cancer cell proliferation inhibitory compounds, A first peptide comprising a first binding region consisting of a part of the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, A second peptide comprising a second binding region consisting of a part of the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, Equipped with reagents including, Screening kit.

4. An in silico analysis method for identifying compounds that inhibit cancer cell proliferation, In silico, Information on a portion of the amino acid sequence represented by SEQ ID NO: 1, or a first amino acid sequence that has 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, Information on a portion of the amino acid sequence represented by Sequence ID No. 2, or a second amino acid sequence that has 80% or more sequence identity with the amino acid sequence represented by Sequence ID No.

2. The process involves selecting a compound capable of cleaving the bond between the first amino acid sequence and the second amino acid sequence due to interaction, based on at least one of the following pieces of information: method.

5. A first peptide containing a first binding region consisting of a part of the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, A second peptide containing a second binding region consisting of a portion of the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

2. Includes a base sequence that codes for Recombinant microorganisms or cells.

6. A cancer treatment drug containing a peptide that includes a portion of the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence that has 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

1.

7. A cancer treatment drug comprising mRNA encoding a peptide that includes a portion of the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence that has 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1.

Images