CN113577290B - Cell death inducing agent, cell proliferation inhibiting agent, and pharmaceutical composition for treating diseases caused by abnormal cell proliferation - Google Patents

Cell death inducing agent, cell proliferation inhibiting agent, and pharmaceutical composition for treating diseases caused by abnormal cell proliferation Download PDF

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CN113577290B
CN113577290B CN202110853954.7A CN202110853954A CN113577290B CN 113577290 B CN113577290 B CN 113577290B CN 202110853954 A CN202110853954 A CN 202110853954A CN 113577290 B CN113577290 B CN 113577290B
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gst
protein
related protein
sequence
base sequence
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CN113577290A (en
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田中洋行
味吞宪二郎
新津洋司郎
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Nitto Denko Corp
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    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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    • C12N15/09—Recombinant DNA-technology
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity

Abstract

The present application relates to a cell death inducing agent, a cell proliferation inhibiting agent, and a pharmaceutical composition for treating a disease caused by abnormal cell proliferation. The present application is directed to cancer cells that induce cell death and inhibit cell proliferation. The present application comprises, as active ingredients, a drug that inhibits GST-pi and a drug that inhibits a steady state maintenance related protein that exhibits synthetic lethality when inhibited simultaneously with GST-pi.

Description

Cell death inducing agent, cell proliferation inhibiting agent, and pharmaceutical composition for treating diseases caused by abnormal cell proliferation
The present application is a divisional application of chinese patent application No.201580076931.2 (PCT application No. PCT/JP 2015/085991) having a filing date of 2015, 12 months, 24, and a title of "a cell death inducing agent, a cell proliferation inhibiting agent, and a pharmaceutical composition for treating a disease caused by abnormal cell proliferation".
Technical Field
The present application relates to a cell death inducing agent, a cell proliferation inhibiting agent, and a pharmaceutical composition for treating a disease caused by abnormal cell proliferation, which are directed to cancer cells, and further relates to a method for screening a cell death inducing agent and/or a cell proliferation inhibiting agent.
Background
As a disease caused by abnormal cell proliferation, cancer is typically exemplified. Cancer is a disease in which cells proliferate uncontrolled due to mutation of genes, epigenetic abnormality, and the like. As genetic abnormalities in cancer, a variety of abnormalities have been reported (for example, non-patent document 1, etc.), most of which are thought to have some correlation with signal transduction associated with proliferation, differentiation, survival of cells. In addition, the abnormal gene causes abnormal intracellular signal transduction, which is composed of normal molecules, and this causes activation and inactivation of a specific signal cascade (cascades), which may eventually cause abnormal proliferation of cells.
The primary cancer treatment mainly focuses on the inhibition of cell proliferation itself, but the treatment also physiologically inhibits the proliferation of normal cells, and thus is accompanied by side effects such as alopecia, digestive organ disorders, bone marrow suppression, and the like. Therefore, in order to suppress such side effects, development of a therapeutic agent for cancer has been conducted based on a new concept such as a molecular targeting drug targeting an abnormality of a gene specific to cancer or an abnormality of signal transduction.
Cancer is thought to occur due to the accumulation of abnormalities in various oncogenes, cancer suppressor genes, DNA repair enzyme genes, and the like in the same cell. As oncogenes, RAS genes, FOS genes, MYC genes, BCL-2 genes and the like are known. Among the genetic abnormalities unique to cancer, the KRAS gene mutation was confirmed in about 95% of pancreatic cancers and about 45% of colorectal cancers, and also, the KRAS gene mutation was confirmed with high frequency in various cancers. KRAS proteins are G proteins that are locally present inside the cell membrane. KRAS et al RAS form the following cascade: it activates RAFs such as C-RAF, B-RAF, etc., which in turn activates MEK, which activates MAPK. If a point mutation occurs in KRAS, GTPase activity decreases, and GTP-bound active form is maintained, so that a downstream signal is constantly sustained, and as a result, abnormal cell proliferation occurs. The KRAS gene is used as a representative, and the cancer gene causes abnormal proliferation of cells, so that the cancer of the cells progresses, and then the cancer diseases are developed.
glutathione-S-transferase (GST) is one of enzymes catalyzing glutathione conjugation, and is known as an enzyme that conjugates a substance such as a drug with Glutathione (GSH) to form a water-soluble substance. GST is typically classified into 6 isozymes of α, μ, ω, pi, θ and ζ based on the amino acid sequence. Among them, in particular, expression of GST-pi (also called GSTP 1) is elevated in various cancer cells, which has been pointed out as one of the causes of resistance against some anticancer agents. In fact, it is known that when GST-pi inhibitor is allowed to act on a cancer cell line that overexpresses GST-pi and exhibits drug resistance, drug resistance is inhibited (non-patent documents 2 to 4). In addition, in recent reports, it has been reported that when an siRNA against GST-pi is allowed to act on an androgen-independent prostate cancer cell line that overexpresses GST-pi, proliferation thereof is inhibited and apoptosis thereof increases (non-patent document 5).
In addition, GST-. Pi.is known to form a complex with c-Jun N-terminal kinase (JNK) to inhibit the activity of JNK (non-patent document 6). Furthermore, GST-pi is known to be involved in S-glutathionylation of a protein involved in cellular stress reaction (non-patent document 7). Furthermore, GST-. Pi.is also known to bring about a protective effect against cell death induced by Reactive Oxygen Species (ROS) (non-patent document 8). It can be understood that GST-. Pi.in GST has various characteristics and functions.
It has been reported that when an siRNA against GST-pi is applied to a cancer cell line having a mutation in KRAS, activation of Akt is inhibited and autophagy is enhanced, but induction of apoptosis is moderate (non-patent document 9). Patent document 1 discloses that apoptosis of cancer cells can be induced by using a drug for inhibiting GST-pi and an autophagy inhibitor such as 3-methyladenine as active ingredients. Further, patent document 2 discloses that when expression of GST-pi, akt, etc. is simultaneously repressed, cell proliferation can be inhibited, cell death can be induced, and autophagy induced by expression repression of GST-pi is more significantly inhibited by simultaneously repressing expression of Akt, etc.
However, it has not been said that the relationship between GST-pi expression and cell proliferation or cell death, the effect of GST-pi on signal transduction, and the like in cancer cells have been fully elucidated.
Prior art literature
Patent literature
Patent document 1: WO 2012/176882
Patent document 2: WO2014/098210
Non-patent literature
Non-patent document 1: futreal et al, nat Rev cancer.2004;4 (3):177-83
Non-patent document 2: takahashi and Niitsu, gan To Kagaku ryoho.1994;21 (7):945-51
Non-patent document 3: ban et al, cancer Res.1996;56 (15):3577-82
Non-patent document 4: nakajima et al, J Pharmacol Exp Ther.2003;306 (3):861-9
Non-patent document 5: hokaiwado et al, carcinogensis.2008; 29 (6):1134-8
Non-patent document 6: adler et al, EMBO J.1999,18,1321-1334
Non-patent document 7: townsend et al, J.biol. Chem.2009,284,436-445
Non-patent document 8: YIn et al, cancer Res.2000, 4053-4057
Non-patent document 9: nishita et al, AACR 102nd Annual Meeting,Abstract No.1065
Disclosure of Invention
Problems to be solved by the invention
Accordingly, an object of the present invention is to provide an agent having a cell death inducing effect and/or a cell proliferation inhibiting effect on cancer cells, to provide a pharmaceutical composition for treating a disease caused by abnormal cell proliferation, and to provide a method for screening a cell death inducing agent and/or a cell proliferation inhibiting agent.
Means for solving the problems
In view of the above-mentioned object, the present inventors have conducted intensive studies and as a result, have found that, in cancer cells, when GST-pi is inhibited while inhibiting a steady-state maintenance-related protein which exhibits synthetic lethality (synthetic lethality) when inhibited simultaneously with GST-pi, cell death can be induced more strongly and cell proliferation can be inhibited more effectively than in the case of inhibiting either one, and have completed the present application. The present application includes the following.
(1) A cell death-inducing agent for cancer cells, which contains, as active ingredients, a drug that inhibits GST-pi and a drug that inhibits a steady-state maintenance-related protein that exhibits synthetic lethality when inhibited simultaneously with GST-pi.
(2) A cell proliferation inhibiting agent for cancer cells, which contains, as active ingredients, a drug that inhibits GST-pi and a drug that inhibits a steady state maintenance-related protein that exhibits synthetic lethality when inhibited simultaneously with GST-pi.
(3) The agent according to (1) or (2), wherein the steady-state maintenance-related protein exhibiting synthetic lethality while GST-PI is inhibited is a protein selected from the group consisting of a cell cycle regulatory protein, an anti-apoptosis-related protein and a PI3K signaling pathway-related protein.
(4) The agent according to (3), wherein the cell cycle regulatory protein exhibiting synthetic lethality while GST-pi is inhibited is at least 1 cell cycle regulatory protein selected from the group consisting of ATM, CDC25A, p, PRKDC, RBBP8, SKP2, MCM10, RNPC1, CCNL1, CENPH, BRSK1, MCM8, CCNB3, MCMDC1 and MYLK.
(5) The agent according to (3), wherein the cell cycle regulatory protein exhibiting synthetic lethality while GST-pi is inhibited is at least 1 protein selected from the group consisting of p21, RNPC1, CCNL1, MCM8, CCNB3 and MCMDC 1.
(6) The agent according to (3), wherein the anti-apoptosis-related protein exhibiting synthetic lethality while GST-pi is inhibited is at least 1 anti-apoptosis-related protein selected from the group consisting of AATF, ALOX12, ANXA1, ANXA4, API5, ATF5, AVEN, AZU1, BAG1, BCL2L1, BFAR, CFLAR, IL2, MALT1, MCL1, MKL1, MPO, MTL5, MYBL2 and MYO 18A.
(7) The agent according to (3), wherein the PI3K signaling pathway-related protein exhibiting synthetic lethality while GST-PI is inhibited is at least 1 PI3K signaling pathway-related protein selected from the group consisting of MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP1, PIK3CA, and SRF.
(8) The agent according to (1) or (2), wherein the drug is a substance selected from the group consisting of RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides and vectors expressing at least 1 of them.
(9) The agent according to (1) or (2), wherein the agent inhibiting the steady-state maintenance related protein is a compound acting on the steady-state maintenance related protein.
(10) The agent of claim (1), wherein the agent is capable of inducing apoptosis.
(11) The agent according to (1) or (2), wherein the cancer cell is a cancer cell expressing GST-pi at a high level.
(12) A pharmaceutical composition for treating a disease caused by abnormal cell proliferation, which comprises the agent according to any one of (1) to (11) above.
(13) The pharmaceutical composition of claim (12), wherein the disease is cancer.
(14) The pharmaceutical composition of (13), wherein the cancer is a cancer that expresses GST-pi at a high level.
(15) A method of screening for a cell death-inducing agent and/or a cell proliferation-inhibiting agent for cancer cells, the cell death-inducing agent and/or the cell proliferation-inhibiting agent being used simultaneously with a drug that inhibits GST-pi, the method comprising selecting a drug that inhibits a steady-state maintenance-related protein that exhibits synthetic lethality when inhibited simultaneously with GST-pi.
(16) The screening method according to (15), wherein the steady-state maintenance-related protein exhibiting synthetic lethality while GST-PI is inhibited is a protein selected from the group consisting of a cell cycle regulatory protein, an anti-apoptosis-related protein and a PI3K signaling pathway-related protein.
(17) The screening method according to (16), wherein the cell cycle regulatory protein exhibiting synthetic lethality while GST-pi is inhibited is at least 1 protein selected from the group consisting of ATM, CDC25A, p, PRKDC, RBBP8, SKP2, MCM10, RNPC1, CCNL1, CENPH, BRSK1, MCM8, CCNB3, MCMDC1 and MYLK.
(18) The screening method according to (16), wherein the cell cycle regulatory protein exhibiting synthetic lethality while GST-pi is inhibited is at least 1 protein selected from the group consisting of p21, RNPC1, CCNL1, MCM8, CCNB3 and MCMDC 1.
(19) The screening method according to (16), wherein the anti-apoptosis-related protein exhibiting synthetic lethality while GST-pi is inhibited is at least 1 anti-apoptosis-related protein selected from the group consisting of AATF, ALOX12, ANXA1, ANXA4, API5, ATF5, AVEN, AZU1, BAG1, BCL2L1, BFAR, CFLAR, IL2, MALT1, MCL1, MKL1, MPO, MTL5, MYBL2 and MYO 18A.
(20) The screening method according to (16), wherein the PI3K signaling pathway-related protein exhibiting synthetic lethality while GST-PI is inhibited is at least 1 PI3K signaling pathway-related protein selected from the group consisting of MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP1, PIK3CA, and SRF.
(21) The screening method of any one of (15) to (20), comprising the steps of: a step of contacting a cancer cell with a test substance; a step of measuring the expression level of the steady-state maintenance related protein in the cell; and a step of selecting the test substance as a drug for inhibiting the steady-state maintenance-related protein when the expression level is reduced, as compared with the case of measuring in the absence of the test substance.
(22) A method for screening a cell death-inducing agent and/or a cell proliferation-inhibiting agent for cancer cells, the cell death-inducing agent and/or the cell proliferation-inhibiting agent being used simultaneously with a drug that inhibits a steady-state maintenance-related protein that exhibits synthetic lethality when simultaneously inhibited with GST-pi, the method comprising selecting a drug that inhibits GST-pi.
(23) The screening method according to (22), wherein the steady-state maintenance-related protein exhibiting synthetic lethality while GST-PI is inhibited is a protein selected from the group consisting of a cell cycle regulatory protein, an anti-apoptosis-related protein and a PI3K signaling pathway-related protein.
(24) The screening method according to (23), wherein the cell cycle regulatory protein exhibiting synthetic lethality while GST-pi is inhibited is at least 1 protein selected from the group consisting of ATM, CDC25A, p, PRKDC, RBBP8, SKP2, MCM10, RNPC1, CCNL1, CENPH, BRSK1, MCM8, CCNB3, MCMDC1 and MYLK.
(25) The screening method according to (23), wherein the cell cycle regulatory protein exhibiting synthetic lethality while GST-pi is inhibited is at least 1 protein selected from the group consisting of p21, RNPC1, CCNL1, MCM8, CCNB3 and MCMDC 1.
(26) The screening method according to (23), wherein the anti-apoptosis-related protein exhibiting synthetic lethality while GST-pi is inhibited is at least 1 anti-apoptosis-related protein selected from the group consisting of AATF, ALOX12, ANXA1, ANXA4, API5, ATF5, AVEN, AZU1, BAG1, BCL2L1, BFAR, CFLAR, IL2, MALT1, MCL1, MKL1, MPO, MTL5, MYBL2 and MYO 18A.
(27) The screening method according to (23), wherein the PI3K signaling pathway-related protein exhibiting synthetic lethality while GST-PI is inhibited is at least 1 PI3K signaling pathway-related protein selected from the group consisting of MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP1, PIK3CA, and SRF.
(28) The screening method of any one of (22) to (27), comprising the steps of: a step of contacting a cancer cell with a test substance; a step of measuring the expression level of GST-pi in the cells; and a step of selecting the test substance as a GST-pi-inhibiting agent when the expression level is reduced, as compared with the case of measuring in the absence of the test substance.
(29) A method of screening for a cell death inducing agent and/or a cell proliferation inhibiting agent, the method comprising the step of selecting for an agent that inhibits GST-pi and a steady state maintenance related protein that exhibits synthetic lethality when inhibited simultaneously with GST-pi.
(30) The screening method according to (29), wherein the steady-state maintenance-related protein exhibiting synthetic lethality while GST-PI is inhibited is a protein selected from the group consisting of a cell cycle regulatory protein, an anti-apoptosis-related protein and a PI3K signaling pathway-related protein.
(31) The screening method according to (30), wherein the cell cycle regulatory protein exhibiting synthetic lethality while GST-pi is inhibited is at least 1 protein selected from the group consisting of ATM, CDC25A, p, PRKDC, RBBP8, SKP2, MCM10, RNPC1, CCNL1, CENPH, BRSK1, MCM8, CCNB3, MCMDC1 and MYLK.
(32) The screening method according to (30), wherein the cell cycle regulatory protein exhibiting synthetic lethality while GST-pi is inhibited is at least 1 protein selected from the group consisting of p21, RNPC1, CCNL1, MCM8, CCNB3 and MCMDC 1.
(33) The screening method according to (30), wherein the anti-apoptosis-related protein exhibiting synthetic lethality while GST-pi is inhibited is at least 1 anti-apoptosis-related protein selected from the group consisting of AATF, ALOX12, ANXA1, ANXA4, API5, ATF5, AVEN, AZU1, BAG1, BCL2L1, BFAR, CFLAR, IL2, MALT1, MCL1, MKL1, MPO, MTL5, MYBL2 and MYO 18A.
(34) The screening method according to (30), wherein the PI3K signaling pathway-related protein exhibiting synthetic lethality while GST-PI is inhibited is at least 1 PI3K signaling pathway-related protein selected from the group consisting of MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP1, PIK3CA, and SRF.
(35) The screening method of any one of (29) to (34), comprising the steps of: a step of contacting a cancer cell with a test substance; a step of measuring the expression level of GST-pi in the cell and the expression level of a steady-state maintenance-related protein exhibiting synthetic lethality when the GST-pi is simultaneously inhibited; and a step of selecting the test substance as a drug for inhibiting GST-pi and a steady-state maintenance related protein that exhibits synthetic lethality when inhibited simultaneously with GST-pi when the expression amount of GST-pi and the expression amount of the steady-state maintenance related protein that exhibits synthetic lethality when inhibited simultaneously with GST-pi are both reduced, as compared with the case of measuring in the absence of the test substance.
The present specification includes disclosures of Japanese patent application Nos. 2014-266198, 2015-135494 and 2015-247725, which are the basis of priority of the present application.
Effects of the application
The cell death inducing agent of the present application can induce cell death very effectively against cancer cells. Thus, the cell death inducing agent according to the present application can exhibit a very high efficacy as a pharmaceutical composition for treating a disease caused by abnormal proliferation of cancer cells.
In addition, the cell proliferation inhibitor according to the present invention can inhibit cell proliferation very effectively against cancer cells. Thus, the cell proliferation inhibitor according to the present invention can exhibit a very high efficacy as a pharmaceutical composition for treating a disease caused by abnormal proliferation of cancer cells.
In addition, by using the screening method according to the present invention, a drug that induces cell death and/or inhibits cell proliferation very strongly against cancer cells can be selected.
Drawings
FIG. 1 is a characteristic diagram showing the results of measurement of GST-pi mRNA and p21 mRNA in cells expressing mutant KRAS when an siRNA inhibiting GST-pi expression and/or an siRNA inhibiting p21 expression are acted.
FIG. 2 is a characteristic diagram showing the results of time-lapse quantification of p21 mRNA when GST-. Pi.was knocked down simultaneously.
FIG. 3 is a characteristic diagram showing the results of measurement of the number of cells when GST-. Pi.and p21 were knocked down simultaneously.
FIG. 4 is a characteristic diagram showing the results of measuring the number of cells when GST-. Pi.and p21 were knocked down 3 times simultaneously.
FIG. 5 is a characteristic diagram showing the results of measuring the number of cells when GST-. Pi.and p21 were knocked down 3 times simultaneously.
FIG. 6 is a photograph of a phase difference image of A549 cells obtained by knocking down GST-. Pi.and p21 simultaneously 3 times.
FIG. 7 is a photograph of a phase difference image of MIAPaCa-2 cells obtained by knocking down GST-pi and p21 simultaneously 3 times.
FIG. 8 is a photograph of a phase difference image of PANC-1 cells obtained by knocking down GST-. Pi.and p21 simultaneously 3 times.
FIG. 9 is a photograph of a phase difference image of HCT116 cells obtained by knocking down GST-. Pi.and p21 simultaneously 3 times.
FIG. 10 is a photograph of a phase difference image of M7609 cells obtained by knocking down GST-pi 3 times and staining with beta-galactosidase.
FIG. 11 is a characteristic diagram showing the results of quantifying the expression of the PUMA gene when GST-. Pi.and p21 were knocked down simultaneously.
FIG. 12 is a characteristic diagram showing the results of comparing the relative survival rates of GST-pi and candidate proteins exhibiting synthetic lethality (cyclin) when knocked down alone and when knockdown simultaneously, respectively.
FIG. 13 is a characteristic diagram showing the results of comparing the relative survival rates of GST-pi and candidate proteins exhibiting synthetic lethality (anti-apoptosis-related proteins) when knocked down alone and when knockdown simultaneously, respectively.
FIG. 14 is a characteristic diagram showing the results of comparing the relative survival rates of GST-PI and candidate proteins exhibiting synthetic lethality (PI 3K signaling pathway related proteins) when knocked down alone and when knockdown simultaneously, respectively.
FIG. 15 is a characteristic diagram showing the results of comparing the relative survival rates when GST-. Pi.and MYLK were knocked down separately and simultaneously.
Detailed Description
The cell death-inducing agent and the cell proliferation-inhibiting agent according to the present invention contain, as active ingredients, a GST-pi-inhibiting agent and a steady state maintenance-related protein that exhibits synthetic lethality when inhibited simultaneously with GST-pi. The cell death induction agent and the cell proliferation inhibition agent according to the present invention exhibit a cell death induction effect and a cell proliferation inhibition effect on cancer cells. Here, cancer cells are cells that exhibit abnormal proliferation due to genes (cancer-related genes).
For example, among cancer-related genes, KRAS genes, FOS genes, MYC genes, BCL-2 genes, SIS genes, and the like can be cited as oncogenes. Among the cancer-related genes, examples of the cancer suppressor gene include an RB gene, a p53 gene, a BRCA1 gene, an NF1 gene, and a p73 gene. However, the cancer cells are not limited to cancer cells related to these specific cancer-related genes, and can be widely used for cells exhibiting abnormal cell proliferation.
The cell death inducing agent and the cell proliferation inhibiting agent according to the present invention are particularly preferably used for cancer cells expressing GST-. Pi.at a high level. Here, the expression of GST-. Pi.at a high level means that the expression amount of GST-. Pi.is significantly higher than that of a normal cell in a cell exhibiting abnormal cell proliferation (so-called cancer cell). The GST-pi expression level can be measured by a conventional method such as RT-PCR or microarray.
In many cases, the cancer cells expressing GST-. Pi.at high levels include, for example, cancer cells expressing mutant KRAS. That is, the cell death inducing agent and the cell proliferation inhibiting agent according to the present invention are preferably applied to a cancer cell expressing a mutant KRAS.
The mutant KRAS refers to a protein having an amino acid sequence obtained by introducing a mutation such as deletion, substitution, addition, or insertion into the amino acid sequence of wild-type KRAS. Here, the mutation in the mutant KRAS is a so-called function-obtaining mutation (gain of function mutation). That is, cells expressing mutant KRAS have reduced GTPase activity due to these mutations, for example, to maintain GTP-bound active form, and thus constantly maintain downstream signaling, resulting in abnormal cell proliferation compared to cells expressing wild-type KRAS. Examples of the gene encoding mutant KRAS include genes having mutations at least at 1 of codon 12, codon 13 and codon 61 of the wild-type KRAS gene. In particular, as the mutant KRAS, mutations at codons 12 and 13 are preferred. Specifically, there may be mentioned a mutation in which glycine encoded by codon 12 of the KRAS gene is replaced with serine, aspartic acid, valine, cysteine, alanine or arginine, and a mutation in which glycine encoded by codon 13 of the KRAS gene is changed to aspartic acid.
GST-pi, as used in this specification, refers to an enzyme encoded by the GSTP1 gene that catalyzes glutathione conjugation. GST-pi exists in a variety of animals, including humans, and its sequence information is also known (e.g., human: nm_000852 (np_000843), rat: NM-012577 (NP-036709), mouse NM-013541 (NP-038569), etc. the numbers represent the accession numbers of the NCBI database, the base sequence numbers are outside the numbering, and the amino acid sequence numbers are in parentheses. As an example, the nucleotide sequence of the coding region of the human GST-pi gene registered in the database is shown in SEQ ID NO. 1, and the amino acid sequence of the human GST-pi protein encoded by the human GST-pi gene is shown in SEQ ID NO. 2.
As used herein, steady state maintenance related proteins that exhibit synthetic lethality when inhibited simultaneously with GST-pi are the following proteins: a protein which has a function related to the homeostasis (homoostasis) of a cell, and which has a significantly increased mortality rate when GST-pi is inhibited simultaneously with GST-pi in a cancer cell, compared to the mortality rate when GST-pi is inhibited alone in a cancer cell. Here, the synthetic lethality, also referred to as synthetic lethality, refers to the following phenomenon: the gene alone does not show low lethality or lethality against cells or individuals, but exhibits significantly increased lethality or lethality when a plurality of genes coexist. In particular, in the present specification, synthetic lethality refers to lethality against cancer cells.
In the present specification, examples of steady-state maintenance-related proteins that exhibit synthetic lethality when GST-PI is simultaneously inhibited include cell cycle regulatory proteins, anti-apoptosis-related proteins, and PI3K signal transduction pathway-related proteins. The cell cycle controlling protein is a protein having a function of controlling a cell cycle. The anti-apoptosis-related protein is a protein having a function related to the inhibition of apoptosis. The PI3K signaling pathway related protein is a protein other than AKT1 among proteins related to PI3K/AKT signaling pathway.
The term "protein having a function of regulating a cell cycle" means any protein related to a cell cycle consisting of a G1 phase (stationary phase before DNA replication), a S phase (DNA synthesis phase), a G2 phase (stationary phase before cell division) and an M phase (cell division phase). More specifically, the regulation of the cell cycle includes various events (events) such as regulation of a mechanism that smoothly progresses in the order of G1 phase to S phase to G2 phase to M phase, regulation of entry into S phase occurring in G1 phase, and regulation of entry into M phase occurring in G2 phase. Thus, the cell cycle controlling protein may be, for example, a protein involved in the progress of these events in the cell cycle or a protein that positively or negatively controls these events. More specifically, examples of the Cyclin-Dependent Kinase (CDK) and the like which are necessary for the initiation of the S phase and the M phase are mentioned. The activity of Cyclin-dependent kinases is positively regulated by the binding of Cyclin (Cyclin). In addition, the activity of Cyclin-dependent kinase is negatively controlled by Cyclin-dependent kinase inhibitors (Cyclin-Dependent Kinase Inhibitor, CKI) such as p21 (CIP 1/WAF 1) and tyrosine kinase. Thus, cyclin-dependent kinase inhibitors such as cyclin and p21, and tyrosine kinases that control the activity of cyclin-dependent kinases are also included in the cyclin-dependent proteins.
Specifically, examples of the cell cycle controlling protein exhibiting synthetic lethality when the protein is inhibited simultaneously with GST-pi include at least 1 cell cycle controlling protein selected from the group consisting of ATM, CDC25A, p, PRKDC, RBBP8, SKP2, MCM10, RNPC1, CCNL1, CENPH, BRSK1, MCM8, CCNB3, MCMDC1 and MYLK. Of these 15 cell cycle regulatory proteins, 1 cell cycle regulatory protein can be inhibited simultaneously with GST-pi, or more than 2 cell cycle regulatory proteins can be inhibited simultaneously with GST-pi.
In particular, as the cell cycle regulatory protein, it is preferable that at least 1 cell cycle regulatory protein selected from the group consisting of p21, RNPC1, CCNL1, MCM8, CCNB3 and MCMDC1 is inhibited simultaneously with GST-pi. When these 6 cell cycle regulatory proteins were each inhibited alone, the inhibition rate of cell proliferation was low, and only when GST-pi was inhibited simultaneously, a very high cell proliferation inhibition effect was exhibited. That is, it can be said that the drugs inhibiting these 6 cell cycle regulatory proteins are excellent in safety when used alone. Therefore, as cell cycle regulatory proteins exhibiting synthetic lethality when inhibited simultaneously with GST-pi, these 6 cell cycle regulatory proteins are preferable.
p21 is a cyclin-controlling protein belonging to the CIP/KIP family encoded by CDKN1A gene, and has an effect of repressing the cyclin-CDK complex by binding thereto, and an effect of repressing the progression of the cell cycle in the G1 phase and the G2/M phase. Specifically, p21 is reported to be a gene activated by p53 (one of the oncogenes), and when p53 is activated by DNA damage or the like, it activates p21, and the cell cycle is stopped in G1 and G2/M phases. In addition to this, p21 has also been reported to have a function of suppressing apoptosis, which has an effect of protecting cells from apoptosis induced by a chemotherapeutic agent or the like in vitro assays and animal assays (Gartel and Tyner,2002; abbs and Dutta, 2009). p21 is present in a variety of animals, including humans, and its sequence information is also known (e.g., human: NM_000389.4, NM_078467.2, NM_001291549.1, NM_001220778.1, NM_001220777.1 (NP_ 001207707.1, NP_001278478.1, NP_001207706.1, NP_510867.1, NP_ 000380.1), etc. the numbers represent NCBI database accession numbers, outside the numbering is a base sequence number, and the amino acid sequence numbers are in parentheses. As an example, the base sequence of the human CDKN1A gene registered as nm_000389.4 in the database is shown in sequence No. 3, and the amino acid sequence of the human p21 protein encoded by the human CDKN1A gene is shown in sequence No. 4. In the present specification, p21 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 4 encoded by the base sequence of SEQ ID NO. 3. As for p21, the sequence information is registered as a plurality of registration numbers as described above, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 3 shows the base sequence of one of the transcriptional variants.
RNPC1 is an RNA binding protein encoded by the RNPC1 gene, and refers to a target protein of p 53. RNPC1 is present in various animals including humans, and its sequence information is also known (for example, humans: NM_017495.5, NM_183425.2, NM_001291780.1, XM_005260446.1 (XP_ 005260503.1, NP_059965.2, NP_906270.1, NP_ 001278709.1) and the like. For example, the nucleotide sequence of the human RNPC1 gene registered as NM-017495.5 in the database is shown in SEQ ID NO. 5, and the amino acid sequence of the human RNPC1 protein encoded by the human RNPC1 gene is shown in SEQ ID NO. 6. In the present specification, RNPC1 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 6 encoded by the base sequence of SEQ ID NO. 5. The sequence information of RNPC1 is registered as a plurality of registration numbers as described above, and there are a plurality of transcription variants. The base sequence of SEQ ID NO. 5 shows the base sequence of one of the transcriptional variants.
CCNL1 refers to cyclin-L1 encoded by the CCNL1 gene. CCNL1 is present in a variety of animals, including humans, and its sequence information is also known (e.g., humans: nm_020307.2, xm_005247647.2, xm_005247648.1, xm_005247649.1, xm_005247650.1, xm_005247651.1, xm_006713710.1, xm_006713711.1 (xp_ 005247704.1, xp_005247705.1, xp_005247706.1, xp_005247707.1, xp_005247708.1, xp_006713773.1, np_ 064703.1), etc.. Numbering represents NCBI database accession numbers, base sequence numbers outside brackets, amino acid sequence numbers. As an example, the base sequence of the human CCNL1 gene registered as nm_020307.2 in the database is shown in sequence No. 7, and the amino acid sequence of the human CCNL1 protein encoded by the human CCNL1 gene is shown in sequence No. 8. In the present specification, CCNL1 is not limited to a protein composed of the amino acid sequence of sequence No. 8 encoded by the base sequence of sequence No. 7. As described above, sequence information of CCNL1 is registered as a plurality of registration numbers, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 7 shows the base sequence of one of the transcriptional variants.
MCM8 refers to the Mini-chromosome maintenance protein 8 (Mini-chromosome maintenance 8) encoded by the MCM8 gene. MCM8 is present in various animals including humans, and its sequence information is also known (e.g., humans: nm_032485.5, nm_182802.2, nm_001281520.1, nm_001281521.1, nm_001281522.1, xm_005260859.1 (xp_ 005260916.1, np_115874.3, np_001268449.1, np_877954.1, np_001268450.1, np_ 001268451.1), etc.. Numbering represents NCBI database accession numbers, base sequence numbers outside the numbering, amino acid sequence numbers within brackets). As an example, the base sequence of the human MCM8 gene registered as nm_032485.5 in the database is shown in sequence No. 9, and the amino acid sequence of the human MCM8 protein encoded by the human MCM8 gene is shown in sequence No. 10. In the present specification, MCM8 is not limited to a protein composed of the amino acid sequence of seq id No. 10 encoded by the base sequence of seq id No. 9. As described above, the sequence information of MCM8 is registered as a plurality of registration numbers, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 9 shows the base sequence of one of the transcriptional variants.
CCNB3 refers to cyclin-B3 encoded by the CCNB3 gene. CCNB3 is present in various animals including humans, and its sequence information is also known (e.g., humans: nm_033670.2, nm_033031.2, xm_006724610.1 (np_ 391990.1, np_149020.2, xp_ 006724673.1), etc.. Numbering represents NCBI database accession numbers, base sequence numbers are outside the numbering, amino acid sequence numbers are within brackets). As an example, the base sequence of the human CCNB3 gene registered as nm_033670.2 in the database is shown in sequence No. 11, and the amino acid sequence of the human CCNB3 protein encoded by the human CCNB3 gene is shown in sequence No. 12. In the present specification, CCNB3 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 12 encoded by the base sequence of SEQ ID NO. 11. The sequence information of CCNB3 is registered as a plurality of registration numbers as described above, and a plurality of transcriptional variants exist. The base sequence of SEQ ID NO. 11 shows the base sequence of one of the transcriptional variants.
MCMDC1 refers to the micro chromosome maintenance defect domain 1 (Mini-chromosome maintenance deficient domain containing 1) encoded by the MCMDC1 gene. MCMDC1 is present in various animals including humans, and its sequence information is also known (e.g., humans: NM-017696.2, NM-153255.4 (NP-060166.2, NP-694987.1), etc.. The numbers represent NCBI database accession numbers, outside the numbering are base sequence numbers, within brackets are amino acid sequence numbers). As an example, the base sequence of the human MCMDC1 gene registered as nm_017696.2 in the database is shown in sequence No. 13, and the amino acid sequence of the human MCMDC1 protein encoded by the human MCMDC1 gene is shown in sequence No. 14. In the present specification, MCMDC1 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 14 encoded by the base sequence of SEQ ID NO. 13. The sequence information of MCMDC1 is registered as a plurality of registration numbers as described above, and there are various transcription variants. The base sequence of SEQ ID NO. 13 shows the base sequence of one of the transcriptional variants.
ATM is an ATM Serine/Threonine oxidase (ATM Serine/Threonine Kinase) encoded by an ATM gene, and refers to a protein belonging to the PI3/PI4 phosphorylase family. ATM is present in a variety of animals including humans, and its sequence information is also known (e.g., humans: nm_000051.3, xm_005271561.2, xm_005271562.2, xm_005271564.2, xm_006718843.1, xm_006718844.1, xm_006718845.1 (np_ 000042.3, xp_005271618.2, xp_005271619.2, xp_005271621.2, xp_006718906.1, xp_006718907.1, xp_ 006718908.1), etc.. Numbering represents NCBI database accession numbers, outside of the accession numbers are base sequence numbers, within brackets are amino acid sequence numbers). As an example, the base sequence of the human ATM gene registered in the database as nm_000051.3 is shown in sequence No. 15, and the amino acid sequence of the human ATM protein encoded by the human ATM gene is shown in sequence No. 16. In the present specification, ATM is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 16 encoded by the base sequence of SEQ ID NO. 15. In ATM, sequence information is registered as a plurality of registration numbers as described above, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 15 shows the base sequence of one of the transcriptional variants.
CDC25A is a dephosphorylase belonging to the CDC25 family encoded by the CDC25A gene, and refers to a protein activated by dephosphorylating CDC 2. CDC25A exists in a variety of animals including humans, and its sequence information is also known (e.g., human: NM_001789.2, NM_201567.1, XM_006713434.1, XM_006713435.1, XM_006713436.1 (NP_ 001780.2, NP_963861.1, XP_006713497.1, XP_006713498.1, XP_ 006713499.1) and the like. As an example, the base sequence of the human CDC25A gene registered as nm_001789.2 in the database is shown in sequence No. 17, and the amino acid sequence of the human CDC25A protein encoded by the human CDC25A gene is shown in sequence No. 18. In the present specification, CDC25A is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 18 encoded by the base sequence of SEQ ID NO. 17. As described above, the sequence information of CDC25A is registered as a plurality of registration numbers, and a plurality of transcriptional variants exist. The base sequence of SEQ ID NO. 17 shows the base sequence of one of the transcriptional variants.
PRKDC is a catalytic subunit protein of a DNA-dependent protein phosphorylase (DNA-dependent Protein Kinase) encoded by the PRKDC gene, and refers to a protein belonging to the PI3/PI4 phosphorylase family. PRKDC is present in a variety of animals, including humans, and its sequence information is also known (e.g., humans: NM-006904.6, NM-001081640.1 (NP-008835.5, NP-001075109.1), etc.. The numbers represent NCBI database accession numbers, outside of the numbering are base sequence numbers, within brackets are amino acid sequence numbers). As an example, the base sequence of the human PRKDC gene registered in the database as nm_006904.6 is shown in sequence No. 19, and the amino acid sequence of the human PRKDC protein encoded by the human PRKDC gene is shown in sequence No. 20. In the present specification, PRKDC is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 20 encoded by the base sequence of SEQ ID NO. 19. The PRKDC has sequence information registered as a plurality of registration numbers as described above, and various transcriptional variants exist. The base sequence of SEQ ID NO. 19 shows the base sequence of one of the transcriptional variants.
RBBP8 is retinoblastoma binding protein 8 (Retinoblastoma Binding Protein) encoded by the RBBP8 gene, which refers to an intranuclear protein that binds directly to retinoblastoma protein. RBBP8 is present in a variety of animals, including humans, and its sequence information is also known (e.g., human: NM_002894.2, NM_203291.1, NM_203292.1, XM_005258325.1, XM_005258326.1, XM_006722519.1, XM_006722520.1, XM_006722521.1, XM_006722522.1 (NP_ 002885.1, NP_976036.1, NP_976037.1, XP_005258382.1, XP_005258383.1, XP_006722582.1, XP_006722583.1, XP_006722584.1, XP_ 006722585.1), etc. the numbers represent NCBI database accession numbers, outside of which are base sequence numbers, within brackets are amino acid sequence numbers. For example, the base sequence of the human RBBP8 gene registered as nm_002894.2 in the database is shown in SEQ ID NO. 21, and the amino acid sequence of the human RBBP8 protein encoded by the human RBBP8 gene is shown in SEQ ID NO. 22. In the present specification, RBBP8 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 22 encoded by the base sequence of SEQ ID NO. 21. As described above, sequence information of RBBP8 is registered as a plurality of registration numbers, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 21 shows the base sequence of one of the transcriptional variants.
SKP2 is an S-phase Kinase-associated protein (S-phase Kinase-associated Protein) encoded by the SKP2 gene, belongs to Fbox protein, and is one of four subunits of E3 ubiquitin protein ligase. SKP2 is present in various animals including humans, and its sequence information is also known (e.g., humans: nm_005983.3, nm_032637.3, nm_001243120.1, xm_006714487.1 (np_ 005974.2, np_116026.1, np_001230049.1, xp_ 006714550.1), etc.. Numbering represents NCBI database accession numbers, base sequence numbers outside the brackets, amino acid sequence numbers within the brackets). As an example, the nucleotide sequence of the coding region of the human SKP2 gene registered as NM-005983.3 in the database is shown in SEQ ID NO. 23, and the amino acid sequence of the human SKP2 protein encoded by the human SKP2 gene is shown in SEQ ID NO. 24. In the present specification, SKP2 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 24 encoded by the base sequence of SEQ ID NO. 23. The sequence information of SKP2 is registered as a plurality of registration numbers as described above, and there are a plurality of transcription variants. The base sequence of SEQ ID NO. 23 shows the base sequence of one of the transcriptional variants.
MCM10 refers to the minichromosome maintenance protein 10 (Mini-Chromosome Maintenance 10) encoded by the MCM10 gene. MCM10 is present in various animals, including humans, and its sequence information is also known (e.g., humans: NM-182751.2, NM-018518.4 (NP-877428.1, NP-060988.3), etc.. The numbers represent NCBI database accession numbers, outside the brackets are base sequence numbers, within brackets are amino acid sequence numbers). As an example, the base sequence of the human MCM10 gene registered as nm_182751.2 in the database is shown in sequence No. 25, and the amino acid sequence of the human MCM10 protein encoded by the human MCM10 gene is shown in sequence No. 26. In the present specification, MCM10 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 26 encoded by the base sequence of SEQ ID NO. 25. As described above, the sequence information of MCM10 is registered as a plurality of registration numbers, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 25 shows the base sequence of one of the transcriptional variants.
CENPH is centromere protein H (Centromere Protein H) encoded by CENPH gene, and is one of the proteins constituting activated centromere (kinetocore) disposed on centromere. CENPH is present in various animals including humans, and its sequence information is also known (e.g., human: NM-022909.3 (NP-075060.1), etc.. Numbering represents NCBI database accession numbers, including base sequence numbers outside brackets, amino acid sequence numbers within brackets). As an example, the base sequence of the human CENPH gene registered as NM-022909.3 in the database is shown in SEQ ID NO. 27, and the amino acid sequence of the human CENPH protein encoded by the human CENPH gene is shown in SEQ ID NO. 28. In the present specification, CENPH is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 28 encoded by the base sequence of SEQ ID NO. 27. For CENPH, there may be multiple transcriptional variants. The base sequence of SEQ ID NO. 27 shows a base sequence of a transcriptional variant.
BRSK1 is a serine/threonine oxidase encoded by the BRSK1 gene, and refers to a phosphorylase that functions at a cell cycle checkpoint in DNA damage. BRSK1 is present in a variety of animals, including humans, and its sequence information is also known (e.g., humans: NM-032430.1, XM-005259327.1, XR-430213.1 (NP-115806.1, XP-005259384.1), etc.. The numbers represent NCBI database accession numbers, base sequence numbers outside the brackets, amino acid sequence numbers within the brackets). As an example, the base sequence of the human BRSK1 gene registered in the database as nm_032430.1 is shown in sequence No. 29, and the amino acid sequence of the human BRSK1 protein encoded by the human BRSK1 gene is shown in sequence No. 30. In the present specification, BRSK1 is not limited to a protein composed of the amino acid sequence of seq id No. 30 encoded by the base sequence of seq id No. 29. As described above, sequence information of BRSK1 is registered as a plurality of registration numbers, and a plurality of transcriptional variants exist. The base sequence of SEQ ID NO. 29 shows the base sequence of one of the transcriptional variants.
MYLK (myosin light chain kinase) is a myosin light chain kinase as a calcium/calmodulin dependent enzyme that regulates the phosphorylation of the light chain by myosin, thereby promoting the interaction of myosin with actin filaments, resulting in contractile activity. The gene encoding MYLK encodes both smooth muscle and non-muscle subtypes. MYLK is present in a variety of animals, including humans, and its sequence information is also known (e.g., human: NM_053028.3, NM_053026.3, NM_053027.3, NM_053025.3, NM_053031.2, NM_053032.2, XM_01512862.1, XM_01512861.1, XM_01512860.1 (NP_444256.3, NP_444254.3, NP_444255.3, NP_444259.1, NP_444260.1, XP_01511164.1, XP_01511163.1, XP_01511162.1) and the like. For example, the base sequence of the human MYLK gene registered in the database as nm_053028.3 is shown in sequence number 41, and the amino acid sequence of the human MYLK protein encoded by the human MYLK gene is shown in sequence number 42. In the present specification, MYLK is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 42 encoded by the base sequence of SEQ ID NO. 41. As described above, sequence information of MYLK is registered as a plurality of registration numbers, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 41 shows the base sequence of one of the transcriptional variants.
On the other hand, the protein having a function related to apoptosis inhibition means a protein having the following functions: apoptosis is inhibited by inhibiting mechanisms such as nuclear aggregation, cell contraction, bubble formation, and fragmentation of DNA. The expression "apoptosis-inhibiting function" means either one of the function of inhibiting apoptosis and the function of inhibiting an apoptosis-promoting factor. Examples of factors that promote apoptosis include various factors such as caspase, fas, and TNFR.
Specifically, examples of the anti-apoptosis-related protein exhibiting synthetic lethality when the protein is inhibited simultaneously with GST-pi include at least 1 anti-apoptosis-related protein selected from the group consisting of AATF, ALOX12, ANXA1, ANXA4, API5, ATF5, AVEN, AZU1, BAG1, BCL2L1, BFAR, CFLAR, IL2, MALT1, MCL1, MKL1, MPO, MTL5, MYBL2 and MYO 18A. Of the 20 anti-apoptosis-related proteins, 1 anti-apoptosis-related protein can be inhibited simultaneously with GST-pi, and more than 2 anti-apoptosis-related proteins can be inhibited simultaneously with GST-pi.
AATF was identified as a protein that interacts with the protein kinase MAP3K12/DLK thought to be associated with apoptosis. For AATF, it is disclosed that it comprises a leucine zipper as a characteristic motif of transcription factor and that a potent transcriptional activation occurs upon fusion with the Gal4 DNA binding domain. In addition, it is known that apoptosis induced by MAP3K12 can be suppressed by overexpression of a gene encoding AATF. AATF is present in a variety of animals, including humans, and its sequence information is also known (e.g., human: NM_0121138.3, XM_0101956799.1, XM_01019510111.1, XR_959558.1, XR_934439.1 (NP-036270.1, XP_01955101.1, XP_01010101522913.1) etc. the numbers represent NCBI database accession numbers, base sequence numbers outside the numbers, amino acid sequence numbers within brackets. As an example, the base sequence of the human AATF gene registered in the database as nm_012138.3 is shown in sequence No. 39, and the amino acid sequence of the human AATF protein encoded by the human AATF gene is shown in sequence No. 40. In the present specification, AATF is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 40 encoded by the base sequence of SEQ ID NO. 39. As for AATF, sequence information is registered as a plurality of registration numbers as described above, and a plurality of transcriptional variants exist. The base sequence of SEQ ID NO. 39 shows the base sequence of one of the transcriptional variants.
ALOX12 is an arachidonic acid-12-lipoxygenase (arachidonate 12-lipoxygenase) known to be associated with atherosclerosis, osteoporosis, and the like. ALOX12 is known to participate in the apoptotic process by controlling the expression of vascular endothelial growth factor to positively control angiogenesis and promote survival of vascular smooth muscle cells and the like. ALOX12 is present in various animals including humans, and its sequence information is also known (e.g., human: NM-000697.2, XM-0101523780.1 (NP-000688.2, XP-0101522082.1, AAH 69557.1), etc.. The numbers represent NCBI database accession numbers, base sequence numbers outside the brackets, amino acid sequence numbers within the brackets). As an example, the base sequence of the human ALOX12 gene registered as nm_000697.2 in the database is shown in sequence No. 43, and the amino acid sequence of the human ALOX12 protein encoded by the human ALOX12 gene is shown in sequence No. 44. In the present specification, ALOX12 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 44 encoded by the base sequence of SEQ ID NO. 43. As described above, sequence information of ALOX12 is registered as a plurality of registration numbers, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 43 shows the base sequence of one of the transcriptional variants.
ANXA1 is a protein that binds to phospholipids and is present locally in the membrane. ANXA1 inhibits phospholipase A2 and has anti-inflammatory activity. ANXA1 is present in a variety of animals including humans, and its sequence information is also known (e.g., humans: nm_000700.2, xm_01518609.1, xm_01518608.1 (np_000691.1, aah 3457.1), etc.. Numbering represents NCBI database accession numbers, base sequence numbers outside the numbering, amino acid sequence numbers within brackets). As an example, the base sequence of the human ANXA1 gene registered as nm_000700.2 in the database is shown in sequence No. 45, and the amino acid sequence of the human ANXA1 protein encoded by the human ANXA1 gene is shown in sequence No. 46. In the present specification, ANXA1 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 46 encoded by the base sequence of SEQ ID NO. 45. The ANXA1 has sequence information registered as a plurality of registration numbers as described above, and various transcription variants exist. The base sequence of SEQ ID NO. 45 shows the base sequence of one of the transcriptional variants.
ANXA4 is a calcium dependent phospholipid binding protein belonging to the annexin family, which may interact with ATP, exhibiting anti-coagulant activity in vitro, inhibiting phospholipase A2 is known. ANXA4 is present in various animals including humans, and its sequence information is also known (e.g., humans: nm_001153.3, xm_01532805.1 (np_001144.1, xp_01531107.1, aah63672.1, aah00182.1, aah 11659.1), etc.) numbering represents NCBI database accession numbers, base sequence numbers outside the numbering, amino acid sequence numbers within brackets. As an example, the base sequence of the human ANXA4 gene registered as nm_001153.3 in the database is shown in sequence No. 47, and the amino acid sequence of the human ANXA4 protein encoded by the human ANXA4 gene is shown in sequence No. 48. In the present specification, ANXA4 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 48 encoded by the base sequence of SEQ ID NO. 47. The ANXA4 has sequence information registered as a plurality of registration numbers as described above, and various transcription variants exist. The base sequence of SEQ ID NO. 47 shows the base sequence of one of the transcriptional variants.
API5 is an apoptosis repressor protein by which it is known to stop apoptosis after growth factor deficiency. API5 inhibits transcription factor E2F 1-induced apoptosis and negatively controls it while interacting with Acinus, a nuclear factor associated with DNA fragmentation in apoptosis. API5 is present in a variety of animals including humans, and its sequence information is also known (e.g., human: NM_001142930.1, NM_006595.3, NM_001243747.1, NM_001142931.1, XM_006718359.2, NR_024525.1 (NP_001136402.1, NP_001136403.1, NP_001230676.1, NP_006586.1, XP_006718422.1) and the like. As an example, the base sequence of the human API5 gene registered as nm_001142930.1 in the database is shown in SEQ ID NO. 49, and the amino acid sequence of the human API5 protein encoded by the human API5 gene is shown in SEQ ID NO. 50. In the present specification, API5 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 50 encoded by the base sequence of SEQ ID NO. 49. The API5 has sequence information registered as a plurality of registration numbers as described above, and has a plurality of transcription variants. The base sequence of SEQ ID NO. 49 shows the base sequence of one of the transcriptional variants.
ATF5 is known to be associated with diseases caused by human T cell leukemia type 1 virus. ATF5 is a transcriptional activator that binds to CAMP Response Element (CRE) present in various viral promoters and the like, and it is known to suppress differentiation from neural precursor cells to neurons. ATF5 is present in a variety of animals, including humans, and its sequence information is also known (e.g., human: NM_012668.5, NM_001193646.1, NM_001290746.1, XM_01010110262029.1 (NP_036200.2, NP_001277675.1, NP_001180575.1, XP_01011114931.1) etc. the numbers represent NCBI database accession numbers, base sequence numbers outside the numbers, amino acid sequence numbers within brackets. For example, the base sequence of the human ATF5 gene registered as NM-012068.5 in the database is shown in SEQ ID NO. 51, and the amino acid sequence of the human ATF5 protein encoded by the human ATF5 gene is shown in SEQ ID NO. 52. In the present specification, ATF5 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 52 encoded by the base sequence of SEQ ID NO. 51. The sequence information of ATF5 is registered as a plurality of registration numbers as described above, and there are a plurality of transcription variants. The base sequence of SEQ ID NO. 51 shows the base sequence of one of the transcriptional variants.
AVEN is a protein known as a factor for inhibiting apoptosis and caspase activation, and is known to be associated with a split-like personality disorder (Schizoid Personality Disorder) and the affective disorder (alexitymia). AVEN is known to repress apoptosis mediated by Apaf 1. AVEN is present in various animals including humans, and its sequence information is also known (e.g., human: nm_020371.2, xm_01015218200.1, xm_005254563.2, xm_0101521819.1, xm_010101521818.1 (np_065104.1, xp_01015201010101010101122.1, xp_01010152010101120.1, xp_005254620.1, aah63533.1, aaf 91470.1), etc.) numbering represents NCBI database accession numbers, base sequence numbers outside brackets, amino acid sequence numbers. As an example, the base sequence of the human AVEN gene registered as nm_020371.2 in the database is shown in sequence number 53, and the amino acid sequence of the human AVEN protein encoded by the human AVEN gene is shown in sequence number 54. In the present specification, AVEN is not limited to a protein composed of the amino acid sequence of sequence No. 54 encoded by the base sequence of sequence No. 53. The sequence information of AVEN is registered as a plurality of registration numbers as described above, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 53 shows the base sequence of one of the transcriptional variants.
AZU1 is a protein contained in the azulene particles and has monocyte chemotactic activity and antibacterial activity. AZU1 is an important multifunctional inflammatory mediator. AZU1 is present in various animals including humans, and its sequence information is also known (e.g., human: NM-001700.3 (NP-001691.1, EAW69592.1, AAH93933.1, AAH93931.1, AAH 69495.1), etc.. The numbers represent NCBI database accession numbers, base sequence numbers outside the brackets, amino acid sequence numbers within the brackets). As an example, the base sequence of the human AZU1 gene registered as nm_001700.3 in the database is shown in sequence number 55, and the amino acid sequence of the human AZU1 protein encoded by the human AZU1 gene is shown in sequence number 56. In the present specification, AZU1 is not limited to a protein composed of the amino acid sequence of sequence No. 56 encoded by the base sequence of sequence No. 55. The sequence information of AZU1 is registered as a plurality of registration numbers as described above, and there are a plurality of transcription variants. The base sequence of SEQ ID NO. 55 shows the base sequence of one of the transcriptional variants.
BAG1 binds to the membrane protein BCL2, which represses pathways leading to apoptosis and apoptosis. BAG1 enhances the anti-apoptotic effect of BCL2, representing a link between the proliferation factor receptor and the anti-apoptotic mechanism. BAG1 is present in a variety of animals, including humans, and its sequence information is also known (e.g., humans: NM-004323.5, NM-001172415.1 (NP-004314.5, NP-001165886.1, AAH 14774.2), etc.. The numbers represent NCBI database accession numbers, base sequence numbers outside the brackets, amino acid sequence numbers within the brackets). As an example, the base sequence of the human BAG1 gene registered as nm_004323.5 in the database is shown in sequence number 57, and the amino acid sequence of the human BAG1 protein encoded by the human BAG1 gene is shown in sequence number 58. In the present specification, BAG1 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 58 encoded by the base sequence of SEQ ID NO. 57. As described above, the sequence information of BAG1 is registered as a plurality of registration numbers, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 57 shows the base sequence of one of the transcriptional variants.
BCL2L1 belongs to the BCL2 protein family, which forms heterodimers or homodimers, is widely associated with activity in the cytoplasm, and is a regulator of anti-apoptosis or promotion of apoptosis. BCL2L1 is disclosed to exist in the mitochondrial outer membrane and control the opening of mitochondrial outer membrane channels. BCL2L1 is present in various animals including humans, and its sequence information is also known (e.g., human: nm_138578.1, nm_001191.2, xm_0101528966.1, xm_0101528965.1, xm_01528961.1, xm_01528960.1, xm_01528964.1, xm_01528963.1, xm_01528962.1, xm_005260487.3, xm_005486.2 (np_612815.1, np_001182.1, aah19307.1, xp_0101527268.1, xp_0101527267.1, xp_01010152727266.1, xp_0152727265.1, xp_01527263.1, xp_527262.1, xp_005544.1, xp_0057262.1, and the like) and the base numbers are included in the amino acid sequence numbers of the base, and the base numbers are outside the base numbers. As an example, the base sequence of the human BCL2L1 gene registered as nm_138578.1 in the database is shown in sequence number 59, and the amino acid sequence of the human BCL2L1 protein encoded by the human BCL2L1 gene is shown in sequence number 60. In the present specification, BCL2L1 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 60 encoded by the base sequence of SEQ ID NO. 59. As described above, the sequence information of BCL2L1 is registered as a plurality of registration numbers, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 59 shows the base sequence of one of the transcriptional variants.
BFAR is a bifunctional apoptosis regulator with anti-apoptotic activity against both apoptosis mediated by cell death receptors and apoptosis mediated by mitochondrial factors. BFAR is present in a variety of animals, including humans, and its sequence information is also known (e.g., humans: NM_016561.2, XM_006725196.2, XM_01546704.1, XM_005255350.2, XM_011115500.1 (NP-057645.1, XP_01545006.1, XP_010101520822.1, XP_006725259.1, XP_005255407.1, AAH 03054.1), etc.. Numbering represents NCBI database accession numbers, outside of which are base sequence numbers, amino acid sequence numbers are placed in brackets. As an example, the base sequence of the human BFAR gene registered as nm_016561.2 in the database is shown in sequence number 61, and the amino acid sequence of the human BFAR protein encoded by the human BFAR gene is shown in sequence number 62. In the present specification, BFAR is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 62 encoded by the base sequence of SEQ ID NO. 61. The BFAR has sequence information registered as a plurality of registration numbers as described above, and various transcription variants exist. The base sequence of SEQ ID NO. 61 shows the base sequence of one of the transcriptional variants.
CFLAR is a regulator of apoptosis, which is known to be structurally similar to caspase 8. However, CFLAR does not have caspase activity and is broken down into 2 peptides by caspase 8. CFLAR is present in various animals including humans, and its sequence information is also known (e.g., humans: nm_003879.5, nm_001202519.1, nm_001202518.1, nm_001308043.1, nm_001308042.1, nm_001202517.1, nm_001202516.1, nm_001127184.2, nm_001202515.1, nm_001127183.2, xm_01512100.1 (np_003870.4, np_001294972.1, np_001294971.1, np_001189448.1, np_001189446.1, np_001189445.1, np_001189444.1, np_001120656.1, xp_01510402.1) and the like). As an example, the base sequence of the human CFLAR gene registered as nm_003879.5 in the database is shown in sequence number 63, and the amino acid sequence of the human CFLAR protein encoded by the human CFLAR gene is shown in sequence number 64. In the present specification, CFLAR is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 64 encoded by the base sequence of SEQ ID NO. 63. As for CFLAR, sequence information is registered as a plurality of registration numbers as described above, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 63 shows the base sequence of one of the transcriptional variants.
IL2 is interleukin 2, a secreted cytokine important for proliferation of T and B lymphocytes. IL2 is present in various animals including humans, and its sequence information is also known (e.g., human: NM-000586.3 (NP-000577.2), etc.. Numbering represents NCBI database accession numbers, outside of which are base sequence numbers, in brackets are amino acid sequence numbers). As an example, the base sequence of the human IL2 gene registered as nm_000586.3 in the database is shown in sequence number 65, and the amino acid sequence of the human IL2 protein encoded by the human IL2 gene is shown in sequence number 66. In the present specification, IL2 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 66 encoded by the base sequence of SEQ ID NO. 65. For IL2, there may be a variety of transcriptional variants. The base sequence of SEQ ID NO. 65 shows a base sequence of a transcriptional variant.
MALT1 is encoded by the following genes: in mucosa-associated lymphoid tissue lymphomas, the gene is obtained by rearrangement of the baculovirus IAP repeat sequence comprising a chromosomal translocation between protein 3 (apoptosis inhibitor 2) and the immunoglobulin heavy chain site. MALT1 is thought to activate nfkb. MALT1 is present in a variety of animals including humans, and its sequence information is also known (e.g., human: NM_173844.2, NM_006785.3, XM_011525794.1 (NP_ 776216.1, NP_006776.1, XP_ 011524096.1) and the like. As an example, the base sequence of the human MALT1 gene registered as nm_006785.3 in the database is shown in sequence number 67, and the amino acid sequence of the human MALT1 protein encoded by the human MALT1 gene is shown in sequence number 68. In the present specification, MALT1 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 68 encoded by the base sequence of SEQ ID NO. 67. The sequence information of MALT1 is registered as a plurality of registration numbers as described above, and there are a plurality of transcription variants. The base sequence of SEQ ID NO. 67 shows the base sequence of one of the transcriptional variants.
MCL1 is an anti-apoptotic protein that is a Bcl-2 family member. For the MCL1 gene, the longest variant among the variants resulting from alternative splicing represses apoptosis to increase cell survival, and the shorter variant induces apoptosis to induce cell death. MCL1 is present in various animals including humans, and its sequence information is also known (e.g., humans: nm_021960.4, nm_001197320.1, nm_182763.2 (np_ 068779.1, np_001184249.1, np_ 877495.1), etc.. Numbering represents NCBI database accession numbers, base sequence numbers outside the brackets, amino acid sequence numbers in brackets). As an example, the base sequence of the human MCL1 gene registered as nm_021960.4 in the database is shown in sequence number 69, and the amino acid sequence of the human MCL1 protein encoded by the human MCL1 gene is shown in sequence number 70. In the present specification, MCL1 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 70 encoded by the base sequence of SEQ ID NO. 69. The sequence information of MCL1 is registered as a plurality of registration numbers as described above, and a plurality of transcriptional variants exist. The base sequence of SEQ ID NO. 69 shows the base sequence of one of the transcriptional variants.
The interaction of MKL1 with the transcription factor cardiomyopathy (myogardin), which is a useful regulator for smooth muscle cell differentiation, is known. Most of MKL1 is present in the nucleus, aiding in signal transduction from the cytoskeleton to the nucleus. The MKL1 gene is associated with a translocation that results in its fusion with the RNA binding motif protein 15 gene. MKL1 is present in various animals including humans and its sequence information is also known (e.g., humans: NM_001282662.1, NM_001282660.1, NM_020831.4, NM_001282661.1, XM_011530287.1, XM_011530286.1, XM_011530285.1, XM_011530284.1, XM_011530283.1, XM_005261691.3 (NP_ 001269591.1, NP_001269589.1, NP_065882.1, NP_001269590.1, XP_011528589.1, XP_011528588.1, XP_011528587.1, XP_011528586.1, XP_011528585.1, XP_005261751.1, XP_005261749.1, XP_ 005261748.1) and the like. The numbers represent NCBI database accession numbers, outside of which are base sequence numbers, within brackets are amino acid sequence numbers). As an example, the base sequence of the human MKL1 gene registered as nm_001282662.1 in the database is shown in sequence number 71, and the amino acid sequence of the human MKL1 protein encoded by the human MKL1 gene is shown in sequence number 72. In the present specification, MKL1 is not limited to a protein consisting of the amino acid sequence of sequence No. 72 encoded by the base sequence of sequence No. 71. As described above, sequence information of MKL1 is registered as a plurality of accession numbers, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 71 shows the base sequence of one of the transcriptional variants.
MPO is a myeloperoxidase, which is a heme protein synthesized during bone marrow differentiation, constituting the main component of neutrophil eosinophil aniline blue particles. MPO produces hypohalous acids that play a central role in the bactericidal activity in neutrophils. MPO is present in various animals including humans, and its sequence information is also known (e.g., humans: NM-000250.1, XM-011524823.1, XM-011524822.1, XM-011524821.1 (NP-000241.1), etc.. The numbers represent NCBI database accession numbers, base sequence numbers outside the brackets, amino acid sequence numbers within the brackets). As an example, the base sequence of the human MPO gene registered as NM-000250.1 in the database is shown in SEQ ID NO. 73, and the amino acid sequence of the human MPO protein encoded by the human MPO gene is shown in SEQ ID NO. 74. In the present specification, MPO is not limited to a protein composed of the amino acid sequence of seq id No. 74 encoded by the base sequence of seq id No. 73. The sequence information of MPO is registered as a plurality of registration numbers as described above, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 73 shows the base sequence of one of the transcriptional variants.
MTL5 is a metallothionein-like protein, which has been disclosed to be specifically expressed in the testis and ovary of mice. It is considered that metallothionein plays a central role in the control of cell proliferation and differentiation, and is involved in the formation of sperm. MTL5 is present in a variety of animals, including humans, and its sequence information is also known (e.g., human: NM_004923.3, NM_001039656.1, XM_011545404.1, XM_011545403.1, XM_011545402.1 (NP_ 001034745.1, NP_004914.2, XP_011543706.1, XP_011543705.1, XP_ 011543704.1) and the like. As an example, the base sequence of the human MTL5 gene registered as nm_004923.3 in the database is shown in sequence number 75, and the amino acid sequence of the human MTL5 protein encoded by the human MTL5 gene is shown in sequence number 76. In the present specification, MTL5 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 76 encoded by the base sequence of SEQ ID NO. 75. The sequence information of MTL5 is registered as a plurality of registration numbers as described above, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 75 shows the base sequence of one of the transcriptional variants.
MYBL2 is a nuclear protein belonging to the MYB family as a transcription factor, and is involved in the progression of the cell cycle. MYBL2 is phosphorylated by cyclin A/cyclin dependent kinase 2 in S phase, and has both activities of activating factor and inhibiting factor. MYBL2 is present in various animals including humans, and its sequence information is also known (e.g., humans: NM-001278610.1, NM-002466.3 (NP-001265539.1, NP-002457.1), etc.. The numbers represent NCBI database accession numbers, outside the numbering are base sequence numbers, within brackets are amino acid sequence numbers). As an example, the base sequence of the human MYBL2 gene registered as nm_002466.3 in the database is shown in sequence No. 77, and the amino acid sequence of the human MYBL2 protein encoded by the human MYBL2 gene is shown in sequence No. 78. In the present specification, MYBL2 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 78 encoded by the base sequence of SEQ ID NO. 77. As described above, sequence information of MYBL2 is registered as a plurality of registration numbers, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 77 indicates the base sequence of one of the transcriptional variants.
MYO18A is myosin 18A, which is known to be associated with 8p11 myeloproliferative syndrome. MYO18A has motor activity (motoractivity) and ATPase activity. MYO18A is present in various animals including humans, and its sequence information is also known (e.g., humans: NM-203318.1, NM-078471.3 (NP-976063.1, NP-510880.2), etc.. The numbers represent NCBI database accession numbers, outside the numbering are base sequence numbers, within brackets are amino acid sequence numbers). As an example, the base sequence of the human MYO18A gene registered as nm_078471.3 in the database is shown in sequence No. 79, and the amino acid sequence of the human MYO18A protein encoded by the human MYO18A gene is shown in sequence No. 80. In the present specification, MYO18A is not limited to a protein composed of the amino acid sequence of sequence No. 80 encoded by the base sequence of sequence No. 79. The sequence information of MYO18A is registered as a plurality of registration numbers as described above, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 79 shows the base sequence of one of the transcriptional variants.
On the other hand, the PI3K signaling pathway related protein is a protein other than AKT1 among proteins related to PI3K/AKT signaling pathway, in other words, a PI3K/AKT signaling pathway related protein other than AKT 1. Note that PI3K/AKT signal transduction pathways are disclosed, for example, in Cell 2007 Jun 29;129 (7):1261-74.
Specifically, examples of PI3K signaling pathway-related proteins that exhibit synthetic lethality when simultaneously inhibited with GST-PI include at least 1 PI3K signaling pathway-related protein selected from the group consisting of MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP1, PIK3CA, and SRF. Of the 14 PI3K signal transduction pathway-related proteins, 1 PI3K signal transduction pathway-related protein can be inhibited simultaneously with GST-PI, and more than 2 PI3K signal transduction pathway-related proteins can be inhibited simultaneously with GST-PI.
In particular, as the PI3K signaling pathway-related protein, it is preferable that at least 1 PI3K signaling pathway-related protein selected from the group consisting of MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG and RAC1 is inhibited simultaneously with GST-PI. These 7 PI3K signaling pathway-related proteins exhibited a very high cell proliferation inhibitory effect compared to the case where they were inhibited alone or the case where GST-PI was inhibited alone, respectively. Therefore, it is preferable to select from among these 7 PI3K signal transduction pathway-related proteins, PI3K signal transduction pathway-related proteins that exhibit synthetic lethality when simultaneously inhibited with GST-PI.
MTOR (mechanical target of rapamycin, mechanistic target of rapamycin) is a target protein of rapamycin, which is known as one of serine/threonine kinases. MTOR is a phosphatidylinositol kinase related kinase and is known to mediate cellular responses to stimuli such as DNA damage and nutritional deficiencies. MTOR is known to act as a target for cell cycle arrest and immunosuppression effects by FKBP 12-rapamycin complexes. The MTOR is present in various animals including humans, and its sequence information is also known (e.g., human: NM_004958.3, XM_005263438.1, XM_01541166.1 (NP_004949.1, XP_005263495.1) etc. the numbers represent NCBI database accession numbers, base sequence numbers outside the brackets, amino acid sequence numbers inside the brackets. For example, the base sequence of the human MTOR gene registered as NM-004958.3 in the database is shown in SEQ ID NO. 81, and the amino acid sequence of the human MTOR protein encoded by the human MTOR gene is shown in SEQ ID NO. 82. In the present specification, MTOR is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 82 encoded by the base sequence of SEQ ID NO. 81. As for the MTOR, the sequence information is registered as a plurality of registration numbers as described above, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 81 shows the base sequence of one of the transcriptional variants.
IRAK1 (interlukin 1receptor associated kinase 1) is an interleukin 1 receptor-related kinase 1, and is one of serine/threonine kinases that act on the interleukin 1receptor by being stimulated. IRAK1 results in part in the induction up-regulation of IL1 associated with the transcription factor nfkb. IRAK1 is present in various animals including humans, and its sequence information is also known (e.g., humans: nm_001025243.1, nm_001025242.1, nm_001569.3, xm_01531158.1, xm_005274668.2 (np_001020414.1, np_001020413.1, np_001560.2, xp_011019460.1, xp_005274725.1), etc.. Numbering represents NCBI database accession numbers, base sequence numbers outside the numbering, amino acid sequence numbers within brackets). As an example, the base sequence of the human IRAK1 gene registered in the database as nm_001025243.1 is shown in sequence number 83, and the amino acid sequence of the human IRAK1 protein encoded by the human IRAK1 gene is shown in sequence number 84. In the present specification, IRAK1 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 84 encoded by the base sequence of SEQ ID NO. 83. As described above, sequence information of IRAK1 is registered as a plurality of registration numbers, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 83 indicates the base sequence of one of the transcriptional variants.
IRS1 (insulin receptor substrate 1) is known as a protein phosphorylated by insulin receptor tyrosine kinase. IRS1 is present in various animals including humans, and its sequence information is also known (e.g., human: NM-005544.2 (NP-005535.1), etc.. The numbers represent NCBI database accession numbers, base sequence numbers outside the numbering, amino acid sequence numbers within brackets). As an example, the base sequence of the human IRS1 gene registered in the database of nm_005544.2 is shown in sequence No. 85, and the amino acid sequence of the human IRS1 protein encoded by the human IRS1 gene is shown in sequence No. 86. In the present specification, IRS1 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 86 encoded by the base sequence of SEQ ID NO. 85. For IRS1, there may be multiple transcriptional variants. The base sequence of SEQ ID NO. 85 shows the base sequence of one of the transcriptional variants.
MYD88 (myeloid differentiation primary response 88) is a cytoplasmic junction (adapter) protein that plays a major role in both innate and adaptive immune responses. MYD88 functions as a necessary signal transduction material in interleukin 1 and Toll-like receptor signaling pathways. These signaling pathways control activation of a variety of inflammatory genes. MYD88 has an N-terminal death domain and a C-terminal Toll-interleukin 1 receptor domain. MYD88 is present in various animals including humans, and its sequence information is also known (e.g., humans: nm_001172567.1, nm_002468.4, nm_001172569.1, nm_001172568.1, nm_001172566.1, xm_005265172.1, xm_006713170.1 (np_001166038.1, np_002459.2, np_001166040.1, np_001166039.1, np_001166037.1, xp_005265229.1, xp_006713233.1), etc. numbering represents NCBI database accession numbers, outside of which are base sequence numbers, amino acid sequence numbers in brackets). As an example, the base sequence of the human MYD88 gene registered as nm_001172567.1 in the database is shown in sequence number 87, and the amino acid sequence of the human MYD88 protein encoded by the human MYD88 gene is shown in sequence number 88. In the present specification, MYD88 is not limited to a protein composed of the amino acid sequence of sequence No. 88 encoded by the base sequence of sequence No. 87. The sequence information of MYD88 is registered as a plurality of registration numbers as described above, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 87 shows the base sequence of one of the transcriptional variants.
The protein NFKB1 (nuclear factor κB1, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1) is 105kD and is cleaved into 50kD when translated via the 26S proteasome. The 105kD protein is a Rel protein specific transcription inhibitor, and the 50kD protein is a DNA binding subunit in the NFKB protein (NFKB) complex. NFKB1 exists in various animals including humans, and its sequence information is also known (e.g., humans: nm_003998.3, nm_001165412.1, xm_01532006.1, xm_01532007.1, xm_01532008.1, xm_01532009.1 (np_003989.2, np_001158884.1, xp_011015306309.1, xp_01101039.1, xp_0110103.1, xp_01101311.1), etc.) numbering represents NCBI database accession numbers, outside of which are base sequence numbers, in brackets amino acid sequence numbers. As an example, the base sequence of the human NFKB1 gene registered in the database as nm_003998.3 is shown in sequence No. 89, and the amino acid sequence of the human NFKB1 protein encoded by the human NFKB1 gene is shown in sequence No. 90. In the present specification, NFKB1 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 90 encoded by the base sequence of SEQ ID NO. 89. The sequence information of NFKB1 is registered as a plurality of registration numbers as described above, and there are a plurality of transcription variants. The base sequence of SEQ ID NO. 89 shows the base sequence of one of the transcriptional variants.
PIK3CG (phosphotidylinositol-4, 5-biphospholoth 3-kinase), which binds to p85 regulatory subunits to form PI3K, like other class 1 (class 1) catalytic subunits (p110_α, p110_β, and p110_δ), is known. PIK3CG is present in various animals including humans and its sequence information is also known (e.g., humans: nm_001282426.1, nm_001282427.1, nm_002649.3, xm_01516316.1, xm_01516317.1, xm_005250443.2 (np_001269355.1, np_001269356.1, np_002640.2, xp_01514618.1, xp_01514619.1, xp_005250500.1) and the like numbering represents NCBI database accession numbers, outside of which are base sequence numbers, within brackets, amino acid sequence numbers). As an example, the base sequence of the human PIK3CG gene registered in the database as nm_001282426.1 is shown in sequence number 91, and the amino acid sequence of the human PIK3CG protein encoded by the human PIK3CG gene is shown in sequence number 92. In the present specification, PIK3CG is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 92 encoded by the base sequence of SEQ ID NO. 91. The sequence information of PIK3CG is registered as a plurality of registration numbers as described above, and there are a plurality of transcription variants. The base sequence of SEQ ID NO. 91 shows the base sequence of one of the transcriptional variants.
RAC1 (Ras-related C3 botulinum toxin substrate 1, ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac 1)) is known as a GTPase belonging to the RAS superfamily of small GTP binding proteins. Factors belonging to this superfamily control a wide variety of intracellular events including growth, cytoskeletal remodeling and activation of protein kinases. RAC1 is present in various animals including humans, and its sequence information is also known (e.g., human: NM-018890.3, NM-006908.4 (NP-061485.1, NP-008839.2), etc. numbering represents NCBI database accession numbers, base sequence numbers outside the numbering, amino acid sequence numbers within brackets). As an example, the base sequence of the human RAC1 gene registered as NM-018890.3 in the database is shown in SEQ ID NO. 93, and the amino acid sequence of the human RAC1 protein encoded by the human RAC1 gene is shown in SEQ ID NO. 94. In the present specification, RAC1 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 94 encoded by the base sequence of SEQ ID NO. 93. As described above, the sequence information of RAC1 is registered as a plurality of registration numbers, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 93 shows the base sequence of one of the transcriptional variants.
AKT3 (V-AKT mouse thymoma virus oncogene homolog 3, V-AKT murine thymoma viral oncogene homolog 3) is also known as PKB, which is a member of the serine/threonine protein kinase family AKT. AKT kinase is known to be a regulator in the cellular signaling system in response to insulin and growth factors. The above-mentioned kinases are involved in various biological processes such as glycogen synthesis and glucose uptake, cell proliferation, differentiation, apoptosis, and tumor formation. The kinase has been disclosed to be stimulated by Platelet Derived Growth Factor (PDGF), insulin and insulin-like growth factor 1 (IGF 1). AKT3 is present in a variety of animals, including humans, and its sequence information is also known (e.g., human: NM_181690.2, NM_001206729.1, NM_005465.4, XM_00527995.2, XM_011105501.1, XM_005272994.3, XM_005272997.3, XM_011115404.1, XM_01111544012.1, XM_011114013.1, XM_006711726.2 (NP_859029.1, NP_001658.1, NP_005456.1, XP_005273052.1, XP_01542313.1, XP_005273054.1, XP_01542316.1, XP_01542314.1, XP_01111789.1) and the like represent NCBI database accession numbers, including base sequence numbers and amino acid sequence numbers. As an example, the base sequence of the human AKT3 gene registered as nm_181690.2 in the database is shown in sequence number 95, and the amino acid sequence of the human AKT3 protein encoded by the human AKT3 gene is shown in sequence number 96. In the present specification, AKT3 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 96 encoded by the base sequence of SEQ ID NO. 95. As described above, sequence information of AKT3 is registered as a plurality of registration numbers, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 95 shows the base sequence of one of the transcriptional variants.
EIF4B (eukaryotic translation initiation factor 4B) is known as a protein associated with the PI3K-Akt signaling pathway and signaling by GPCRs. EIF4B is essential for binding of mRNA to ribosomes, and has a function closely related to EIF4-F and EIF4-A, which binds to the vicinity of the 5' -terminal cap structure of mRNA in the presence of EIF4-F and ATP, and is known to promote ATPase activity and thus ATP-dependent RNA helicity of EIF4-A and EIF 4-F. EIF4B is present in various animals including humans, and its sequence information is also known (e.g., humans: nm_001300821.1, nm_001417.5, xm_00671974.1 (np_0017750.1, np_001408.2, xp_00671937.1), etc. numbering represents NCBI database accession numbers, base sequence numbers outside brackets, amino acid sequence numbers inside brackets). For example, the base sequence of the human EIF4B gene registered in the database as nm_001300821.1 is shown in SEQ ID NO. 97, and the amino acid sequence of the human EIF4B protein encoded by the human EIF4B gene is shown in SEQ ID NO. 98. In the present specification, EIF4B is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 98 encoded by the base sequence of SEQ ID NO. 97. The sequence information of EIF4B is registered as a plurality of registration numbers as described above, and there are a plurality of transcription variants. The base sequence of SEQ ID NO. 97 shows the base sequence of one of the transcriptional variants.
EIF4E (eukaryotic translation initiation factor 4E) is known to be a protein constituting eukaryotic translation initiation factor 4F complex (which recognizes the 7-methylguanosine cap structure at the 5' -end of mRNA). EIF4E assists in translation initiation by allowing ribosomes to accumulate at the 5' end cap structure. Binding of EIF4E to the 4F complex is the rate limiting step during translation initiation. EIF4E is present in a variety of animals, including humans, and its sequence information is also known (e.g., human: NM_001968.3, NM_001130679.1, NM_001130678.1, XM_006714126.2, XM_006714127.2 (NP_001959.1, NP_001124151.1, NP_001124150.1, XP_006714189.1, XP_006719190.1) and the like. As an example, the base sequence of the human EIF4E gene registered as NM-001968.3 in the database is shown in SEQ ID NO. 99, and the amino acid sequence of the human EIF4E protein encoded by the human EIF4E gene is shown in SEQ ID NO. 100. In the present specification, EIF4E is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 100 encoded by the base sequence of SEQ ID NO. 99. The sequence information of EIF4E is registered as a plurality of registration numbers as described above, and there are various transcription variants. The base sequence of SEQ ID NO. 99 indicates the base sequence of one of the transcriptional variants.
ILK (integrin linked kinase ) is a protein with a kinase-like domain and 4 ankyrin-like repeats, which is known to control integrin-mediated signal transduction by binding to the cytoplasmic domain of β integrin on the cell membrane. ILK is present in various animals including humans, and its sequence information is also known (e.g., humans: nm_004517.3, nm_001278441.1, nm_001014794.2, nm_001278442.1, nm_001014795.2, xm_005252904.3, xm_005252905.1, xm_01102065.1 (np_004508.1, np_001265370.1, np_001014794.1, np_001265371.1, np_001014795.1, xp_005252961.1, xp 005252962.1, xp_011018367.1) and the like. Numbers represent NCBI database accession numbers, outside of which are base sequence numbers and amino acid sequence numbers within brackets. As an example, the base sequence of the human ILK gene registered as nm_004517.3 in the database is shown in sequence number 101, and the amino acid sequence of the human ILK protein encoded by the human ILK gene is shown in sequence number 102. In the present specification, ILK is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 102 encoded by the base sequence of SEQ ID NO. 101. The ILK has sequence information registered as a plurality of accession numbers as described above, and various transcriptional variants exist. The base sequence of SEQ ID NO. 101 shows the base sequence of one of the transcriptional variants.
MTCP1 (T lymphocyte proliferation protein 1, material T-cell proliferation 1) was identified as being associated with a variety of T (X; 14) translocations associated with proliferation of mature T cells. The region has a complex structure with a common promoter (common promoter), and a 5 'exon spliced into 2 different 3' exons encoding 2 different proteins. The upstream 13kD protein belongs to the TCL1 family, which is believed to be involved in leukemia induction. MTCP1 is present in various animals including humans, and its sequence information is also known (e.g., human: NM-001018025.3 (NP-001018025.1) and the like. Numbering represents NCBI database accession numbers, base sequence numbers outside the numbering, amino acid sequence numbers within brackets). As an example, the base sequence of the human MTCP1 gene registered as nm_001018025.3 in the database is shown in sequence No. 103, and the amino acid sequence of the human MTCP1 protein encoded by the human MTCP1 gene is shown in sequence No. 104. In the present specification, MTCP1 is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 104 encoded by the base sequence of SEQ ID NO. 103. For MTCP1, there may be multiple transcriptional variants. The base sequence of SEQ ID NO. 103 shows the base sequence of one of the transcriptional variants.
PIK3CA (phosphotidylinositol-4, 5-biphosphite 3-kinase), catalytic subunit α (catalytic subunit alpha), is a 110kD catalytic subunit of phosphotidylinositol 3-kinase, and PtdIns, ptdIns P and PtdIns (4, 5) P2 are phosphorylated with ATP. PIK3CA is present in various animals including humans, and its sequence information is also known (e.g., human: nm_006218.2, xm_006713658.2, xm_01512894.1 (np_006209.2, xp_006713721.1, xp_01511196.1), etc.. Numbering represents NCBI database accession numbers, base sequence numbers outside the numbering, amino acid sequence numbers within brackets). As an example, the base sequence of the human PIK3CA gene registered in the database as nm_006218.2 is shown in sequence No. 105, and the amino acid sequence of the human PIK3CA protein encoded by the human PIK3CA gene is shown in sequence No. 106. In the present specification, PIK3CA is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 106 encoded by the base sequence of SEQ ID NO. 105. The sequence information of PIK3CA is registered as a plurality of registration numbers as described above, and there are a plurality of transcription variants. The base sequence of SEQ ID NO. 105 shows the base sequence of one of the transcriptional variants.
SRF (serum response factor ) is known as a ubiquitous nucleoprotein that promotes cell proliferation and differentiation. SRF belongs to the superfamily of transcription factors MADS (MCM 1, agamous, deficens, and SRF). SRF binds to serum response factors (SREs) in the promoter region of the target gene. SRF controls activation of various early genes such as c-fos, and thus is involved in cell cycle control, apoptosis, cell growth and cell differentiation. SRF are present in various animals, including humans, and their sequence information is also known (e.g., human: NM-003131.3, NM-001292001.1 (NP-003122.1, NP-001278930.1), etc. numbering represents NCBI database accession numbers, base sequence numbers outside the numbering, amino acid sequence numbers within brackets). As an example, the base sequence of the human SRF gene registered as nm_003131.3 in the database is shown in sequence No. 107, and the amino acid sequence of the human SRF protein encoded by the human SRF gene is shown in sequence No. 108. In the present specification, SRF is not limited to a protein composed of the amino acid sequence of SEQ ID NO. 108 encoded by the base sequence of SEQ ID NO. 107. As for SRF, sequence information is registered as a plurality of registration numbers as described above, and a plurality of transcription variants exist. The base sequence of SEQ ID NO. 107 shows the base sequence of one of the transcriptional variants.
As described above, GST-. Pi.s, ATMs, CDC25A, p, PRKDC, RBBP8, SKP2, MCM10, RNPC1, CCNL1, CENPH, BRSK1, MYLK, MCM8, CCNB3, MCMDC1, AATF, ALOX12, ANXA1, ANXA4, API5, ATF5, AVEN, AZU1, BAG1, BCL2L1, BFAR, CFLAR, IL2, MALT1, MCL1, MKL1, MPO, MTL5, MYBL2, MYO18A, MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP1, PIK3CA and SRF may be determined by specific base sequences and amino acid sequences, but the possibility of occurrence of sequences and mutation (including multiple types) between biological individuals must be considered.
That is, GST-. Pi.atm, CDC25A, p, PRKDC, RBBP8, SKP2, MCM10, RNPC1, CCNL1, CENPH, BRSK1, MYLK, MCM8, CCNB3, MCMDC1, AATF, ALOX12, ANXA1, ANXA4, API5, ATF5, AVEN, AZU1, BAG1, BCL2L1, BFAR, CFLAR, IL2, MALT1, MCL1, MKL1, MPO, MTL5, MYBL2, MYO18A, MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP1, PIK3CA and SRF are not limited to proteins having the same sequence as the amino acid sequence registered in the database, but also include proteins having 1 or more than 2 (typically 1 or more than 1, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids, and has equivalent functions to GST-pi, ATM, CDC25A, p, PRKDC, RBBP8, SKP2, MCM10, RNPC1, CCNL1, CENPH, BRSK1, MYLK, MCM8, CCNB3, MCMDC1, AATF, ALOX12, ANXA1, ANXA4, API5, ATF5, AVEN, AZU1, BAG1, BCL2L1, BFAR, CFLAR, IL2, MALT1, MCL1, MKL1, MPO, MTL5, MYBL2, MYO18A, MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP1, PIK3CA and SRF.
In addition, in the case of the optical fiber, GST-pi, ATM, CDC25A, p, PRKDC, RBBP8, SKP2, MCM10, RNPC1, CCNL1, CENPH, BRSK1, MYLK, MCM8, CCNB3, MCMDC1, AATF, ALOX12, ANXA1, ANXA4, API5, ATF5, AVEN, AZU1, BAG1, BCL2L1, BFAR, CFLAR, IL2, MALT1, MCL1, MKL1, MPO, MTL5, MYBL2, MYO18A, MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP1, PIK3CA and SRF are also included as a composition comprising 70% or more, 80% or more, 90% or more, a composition comprising a nucleotide sequence which is composed of a nucleotide sequence having a nucleotide sequence of 95% or more or 97% or more and which encodes a protein having a function equivalent to GST-. Pi.ATM, CDC25A, p, PRKDC, RBBP8, SKP2, MCM10, RNPC1, CCNL1, CENPH, BRSK1, MYLK, MCM8, CCNB3, MCMDC1, AATF, ALOX12, ANXA1, ANXA4, API5, ATF5, AVEN, AZU1, BAG1, BCL2L1, BFAR, CFLAR, IL2, MALT1, MCL1, MKL1, MPO, MTL5, MYBL2, MYO18A, MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP, PIK3CA and SRF.
The specific functions of GST-. Pi.ATM, CDC25A, p, PRKDC, RBBP8, SKP2, MCM10, RNPC1, CCNL1, CENPH, BRSK1, MYLK, MCM8, CCNB3, MCMDC1, AATF, ALOX12, ANXA1, ANXA4, API5, ATF5, AVEN, AZU1, BAG1, BCL2L1, BFAR, CFLAR, IL2, MALT1, MCL1, MKL1, MPO, MTL5, MYBL2, MYO18A, MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP1, PIK3CA and SRF are as described above.
Unless otherwise indicated, the terms "used in the present specification", "described in the present specification" and the like mean that all the inventions described in the present specification are described as follows. Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. All patents, publications, and other publications referred to in this specification are also incorporated in their entirety into this specification.
The "GST-pi-inhibiting agent" used in the present specification is not limited, and includes, for example, an agent that inhibits the production of GST-pi and/or the activity of GST-pi, an agent that promotes the decomposition and/or inactivation of GST-pi, and the like. Examples of the drug for suppressing the production of GST-pi include, but are not limited to, RNAi molecules against DNA encoding GST-pi, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, and vectors for expressing the same.
In addition, as a drug for inhibiting GST-pi, any compound that acts against GST-pi can be used. As such a compound, an organic compound (an amino acid, a polypeptide or a derivative thereof, a low molecular compound, a sugar, a high molecular compound, or the like), an inorganic compound, or the like can be used. In addition, such a compound may be either a natural substance or a non-natural substance. Examples of the derivative of the polypeptide include a modified polypeptide obtained by adding a modifying group, and a variant polypeptide obtained by changing an amino acid residue. Such a compound may be a single compound, or may be a compound library, an expression product of a gene library, a cell extract, a cell culture supernatant, a microbial fermentation product, a marine organism extract, a plant extract, or the like. That is, the "GST-pi-inhibiting agent" is not limited to nucleic acids such as RNAi molecules, but includes any compound.
Specifically, examples of the drug for inhibiting GST-pi activity include, but are not limited to, substances binding to GST-pi, for example, glutathione analogs (for example, substances described in WO 95/08563, WO 96/40205, WO 99/54346, non-patent document 4, etc.), ketoprofen (non-patent document 2), indomethacin (Hall et al, cancer Res.1989;49 (22): 6265-8), ethacrynic acid, pioglitazone (Tew et al, cancer Res.1988;48 (13): 3622-5), anti-GST-pi antibodies, dominant negative mutants of GST-pi, etc. These drugs may be commercially available or appropriately manufactured based on known techniques.
As a drug for inhibiting the production of GST-pi or the activity thereof, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides and vectors for expressing the same are preferred from the viewpoints of high specificity and low possibility of side effects.
Inhibition of GST-pi can be determined from the following phenomena: GST-pi expression and activity are inhibited in cells, as compared with the case where GST-pi inhibitor is not allowed to act. The expression of GST-pi can be evaluated by any known method, and examples thereof include, but are not limited to, immunoprecipitation using an anti-GST-pi antibody, EIA, ELISA, IRA, IRMA, western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, hybridization using a nucleic acid specifically hybridizing to a nucleic acid encoding GST-pi or a specific fragment thereof or a transcription product (e.g., mRNA) or a splice product of the nucleic acid, northern blotting, southern blotting, and various PCR methods.
In addition, the activity of GST-pi can be evaluated by analyzing the known activity of GST-pi, including, for example, binding to a protein such as Raf-1 (particularly phosphorylated Raf-1) or EGFR (particularly phosphorylated EGFR), etc., without limitation, and any known method such as immunoprecipitation, western blotting, mass spectrometry, downdraw method (pull-down), surface Plasmon Resonance (SPR), etc. can be used for the evaluation.
The term "drug for inhibiting a protein involved in maintenance of homeostasis which exhibits synthetic lethality when simultaneously inhibited with GST-. Pi." as used herein is not limited, but includes, for example, a drug for inhibiting the production and/or activity of the protein, a drug for promoting the decomposition and/or inactivation of the protein, and the like. Examples of the drug for inhibiting the production of the protein include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, and vectors for expressing the same, which are directed against the DNA encoding the protein involved in maintaining steady state. In addition, as a drug for inhibiting the activity of the protein related to the maintenance of the steady state, and a drug for promoting the decomposition and/or inactivation of the protein related to the maintenance of the steady state, any compound that acts on the protein can be used. As such a compound, an organic compound (an amino acid, a polypeptide or a derivative thereof, a low molecular compound, a sugar, a high molecular compound, or the like), an inorganic compound, or the like can be used. In addition, such a compound may be either a natural substance or a non-natural substance. Examples of the derivative of the polypeptide include a modified polypeptide obtained by adding a modifying group, and a variant polypeptide obtained by changing an amino acid residue. Such a compound may be a single compound, or may be a compound library, an expression product of a gene library, a cell extract, a cell culture supernatant, a microbial fermentation product, a marine organism extract, a plant extract, or the like. That is, the "drug for inhibiting a steady-state maintenance related protein that exhibits synthetic lethality when simultaneously inhibited with GST-. Pi." is not limited to nucleic acids such as RNAi molecules, but includes any compounds.
More specifically, examples of drugs that inhibit the activity of p21 in a Cell Cycle regulatory protein that exhibits synthetic lethality when simultaneously inhibited with GST-pi are not limited to the following, but include, for example, low molecular weight compounds butyrolactone I (Sax et al, cell Cycle, jan;1 (1): 90-6, 2002) that inhibit the enzymatic activities of CDC2, CDK2 and CDK5 while promoting the proteolytic cleavage of p21 protein; psychotropic drugs (Psychoactive drug) quetiapine (Kondo et al, transl. Psychiary, apr 2;3:e243, 2013) thought to specifically inhibit p21 expression in nerve cells, oligodendrocytes (oligodendrocyte) of CD-1 mice; sorafenib (Inoue et al, cancer Biology & Therapy,12:9,827-836,2011), a low molecular weight compound that specifically inhibits p21 and multiple kinases to Raf, VEGFR, PDGFR, without the involvement of p53, p27, akt; low molecular compound UC2288, which specifically inhibits p21 without the involvement of p53 or Akt (wetterseten et al, cancer Biology & Therapy,14 (3), 278-285, 2013); RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-p 21 antibodies, dominant negative mutants of p21, and the like, directed against the DNA encoding p 21. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the agent for inhibiting the activity of RNPC1 in a cell cycle regulatory protein exhibiting synthetic lethality when it is inhibited simultaneously with GST-pi include, but are not limited to, RNAi molecules against DNA encoding RNPC1, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-RNPC 1 antibodies, dominant negative mutants of RNPC1, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of drugs that inhibit CCNL1 activity in a cell cycle regulatory protein that exhibits synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-CCNL 1 antibodies, dominant negative mutants of CCNL1, and the like, directed against DNA encoding CCNL 1. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the agent for inhibiting the activity of MCM8 in a cell cycle regulatory protein exhibiting synthetic lethality when it is inhibited simultaneously with GST-pi include, but are not limited to, RNAi molecules against DNA encoding MCM8, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-MCM 8 antibodies, dominant negative mutants of MCM8, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of drugs that inhibit CCNB3 activity in a cell cycle regulatory protein that exhibits synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-CCNB 3 antibodies, dominant negative mutants of CCNB3, and the like, against DNA encoding CCNB 3. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of drugs that inhibit the activity of MCMDC1 in a cell cycle regulatory protein that exhibits synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-MCMDC 1 antibodies, dominant negative mutants of MCMDC1, and the like, directed against DNA encoding MCMDC 1. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of drugs that inhibit the activity of ATM in a cell cycle regulatory protein that exhibits synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, low molecular weight compounds CGK 733 (Won et al, nat. Chem. Biol.2,369, 2006) that selectively inhibit the kinase activity of ATM, low molecular weight compounds KU-55933 (Lau et al, nat. Cell Biol.7,493, 2005) that selectively inhibit the kinase activity of ATM, KU-60019 (Zirkin et al, J Biol chem. Jul 26;288 (30): 2170-83, 2013), CP-466722 (Rainey et al, cancer res. Sep 15;68 (18): 7466-74, 2008), RNAi molecules directed against DNA encoding ATM, ribozymes, antisense nucleic acids, DNA/RNA polynucleotides, chimeric vectors expressing them, anti-ATM antibodies, negative mutants of ATM, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drugs that inhibit the activity of CDC25A in a cell cycle regulatory protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, low molecular weight compounds NSC95397 that inhibit the activities of the dephosphorylating enzymes of human CDC25A, human CDC25B and human CDC25C (Lazo JS et al mol. Pharmacol.61:720-728, 2002), low molecular weight compounds SC alpha alpha delta that inhibit the activities of the dephosphorylating enzymes of human CDC25A, human CDC25B, human CDC25C and human tyrosine dephosphorylating enzyme PTB1B (Rice, R.L. et al, biochemistry 36 (50): 15965-15974,1997), RNAi molecules directed against DNA encoding CDC25A, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing these, anti-CDC 25A antibodies, dominant negative mutants of CDC25A, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of PRKDC in a cell cycle regulatory protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules against DNA encoding PRKDC, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-PRKDC antibodies, dominant negative mutants of PRKDC, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the agent for inhibiting the activity of RBBP8 in a cell cycle regulatory protein exhibiting synthetic lethality when it is inhibited simultaneously with GST-pi include, but are not limited to, RNAi molecules against DNA encoding PRBBP8, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors for expressing the same, anti-RBBP 8 antibodies, dominant negative mutants of RBBP8, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of drugs that inhibit the activity of SKP2 in a cell cycle regulatory protein that exhibits synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-SKP 2 antibodies, dominant negative mutants of SKP2, and the like, against DNA encoding SKP 2. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the agent for inhibiting the activity of MCM10 in a cell cycle regulatory protein exhibiting synthetic lethality when it is inhibited simultaneously with GST-pi include, but are not limited to, RNAi molecules against DNA encoding MCM10, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-MCM 10 antibodies, dominant negative mutants of MCM10, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of CENPH in a cell cycle regulatory protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules against DNA encoding CENPH, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-CENPH antibodies, dominant negative mutants of CENPH, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of drugs that inhibit the activity of BRSK1 in a cell cycle regulatory protein that exhibits synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-BRSK 1 antibodies, dominant negative mutants of BRSK1, and the like, against DNA encoding BRSK 1. These drugs may be commercially available or appropriately manufactured based on known techniques.
The drug for inhibiting the activity of MYLK in a cell cycle regulatory protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi is not limited to the following examples, and examples thereof include: the low molecular compound HA-100 dihydrochloride (Gerard et al, J Clin invest. Jan;77 (1): 61-5.1986), which inhibits MYLK activity in addition to the activities of various protein kinases (PKA, PKC, etc.); staurosporine (Tamaoki et al, biochem. Biophys. Res. Commun. 135:397-402.1986), a low molecular compound that inhibits MYLK activity in addition to the activities of various protein kinases (PKA, PKC, PKG, caMK, caMKII, phosphorylase kinase, etc.); a low molecular compound Calphostin C (Kobayashi et al, biochem. Biophys. Res. Commun. 159:548-553.1989) which is a selective inhibitor of PKC and also inhibits MYLK activity; the low molecular compound piceatannol (Oliver et al, J.biol. Chem.269: 29697-29703.1994), which is also a tyrosine kinase inhibitor, inhibits the activity of MYLK; low molecular compound a-3 hydrochloride which inhibits the activity of MYLK in addition to the activities of various casein kinases and protein kinases (Inagaki et al mol. Pharmacol.29, 577.1986); low molecular compound H-7 dihydrochloride, which also inhibits MYLK activity, as a cell permeable serine/threonine kinase inhibitor (Kawamoto et al, biochem. Biophys. Res. Commun. 125:258-264.1984); low molecular compound H-9 hydrochloride (Wolf et al, J.biol. Chem.260: 15718-15722.1985) which inhibits MYLK activity in addition to the activities of various protein kinases (PKA, PKC, etc.); a low molecular weight compound W-5 which also inhibits MYLK activity as a calmodulin (CaM) antagonist (Hidaka et al mol. Pharmacol. 20:571-578.1981); a low molecular weight compound W-7 which also inhibits MYLK activity as a calmodulin (CaM) antagonist (Hidaka et al, proc. Natl. Acad. Sci. U.S.A.78:4354-4357.1981); a low molecular weight compound W-13 isomer hydrochloride which inhibits MYLK activity in addition to various protein kinase activities (PKA, PKC, etc.) (Hidaka et al, proc. Natl. Acad. Sci. U.S.A.78:4354-4357.1981); low molecular compound ML-7 dihydrochloride (Saitoh et al, J. Biol. Chem. 262:7796-7801.1987) which inhibits MYLK activity in addition to the activities of various protein kinases (PKA, PKC, etc.); low molecular compounds ML-9 (Saitoh et al, biochem. Biophys. Res. Commun. 140:280-287.1986) which selectively inhibit MYLK and CaMK; myricetin, a low molecular compound that inhibits MYLK activity in addition to various casein kinase and protein kinase activities (Hagiwara et al, biochem. Phacol. 37:2987-2992.1988); a low molecular compound E6 berbamine which also inhibits MYLK activity as a cell-permeable calmodulin (CaM) antagonist (Hu et al, biochem. Pharmacol. Oct 20;44 (8): 1543-7.1992); a low molecular compound K-252a which inhibits MYLK activity in addition to the activity of various protein kinases (Kase et al J. Antiboot. 39:1059-1065.1986); a low molecular compound K-252b which inhibits MYLK activity in addition to the activity of various protein kinases (Nakanishi et al J. Antiboot. 39:1066-1071.1986); low molecular weight compounds, gramicin, which are c-Src inhibitors and also inhibit MYLK activity (Rosett et al, biochem. J.67:390-400.1957); RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing them, anti-MYLK antibodies, dominant negative mutants of MYLK, and the like, directed against DNA encoding MYLK. These drugs may be commercially available or appropriately manufactured based on known techniques.
On the other hand, examples of drugs that inhibit the activity of AATF in an anti-apoptosis-related protein that exhibits synthetic lethality when simultaneously inhibited with GST-pi are not limited to the following, but include RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-AATF antibodies, dominant negative mutants of AATF, and the like, for example. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the agent for inhibiting the activity of ALOX12 in an anti-apoptosis-related protein exhibiting synthetic lethality when the agent is inhibited simultaneously with GST-pi include, but are not limited to, RNAi molecules against DNA encoding ALOX12, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors for expressing the same, anti-ALOX 12 antibodies, dominant negative mutants of ALOX12, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of ANXA1 in an anti-apoptosis-related protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors for expressing them, anti-ANXA 1 antibodies, dominant negative mutants of ANXA1, and the like, against DNA encoding ANXA 1. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of ANXA4 in an anti-apoptosis-related protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors for expressing them, anti-ANXA 4 antibodies, dominant negative mutants of ANXA4, and the like, against DNA encoding ANXA 4. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of API5 in an anti-apoptosis-related protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules against DNA encoding API5, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-API 5 antibodies, dominant negative mutants of API5, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of ATF5 in an anti-apoptosis-related protein exhibiting synthetic lethality when the drug is inhibited simultaneously with GST-pi include, but are not limited to, RNAi molecules against DNA encoding ATF5, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors for expressing the same, anti-ATF 5 antibodies, dominant negative mutants of ATF5, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of AVEN in an anti-apoptosis-related protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules against DNA encoding AVEN, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-AVEN antibodies, dominant negative mutants of AVEN, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drugs that inhibit the activity of AZU1 in the anti-apoptosis-related protein that exhibits synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules against DNA encoding AZU1, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors for expressing them, anti-AZU 1 antibodies, dominant negative mutants of AZU1, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of drugs that inhibit the activity of BAG1 in an anti-apoptosis-related protein that exhibits synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-BAG 1 antibodies, dominant negative mutants of BAG1, and the like, against DNA encoding BAG 1. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of BCL2L1 in an anti-apoptosis-related protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-BCL 2L1 antibodies, dominant negative mutants of BCL2L1, and the like, against DNA encoding BCL2L 1. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the agent for inhibiting the activity of BFAR in an anti-apoptosis-related protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules against DNA encoding BFAR, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-BFAR antibodies, dominant negative mutants of BFAR, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of CFLAR in an anti-apoptosis-related protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules against DNA encoding CFLAR, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors for expressing the same, anti-CFLAR antibodies, dominant negative mutants of CFLAR, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of IL2 in an anti-apoptosis-related protein exhibiting synthetic lethality when the drug is inhibited simultaneously with GST-pi include, but are not limited to, RNAi molecules against DNA encoding IL2, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors for expressing the same, anti-IL 2 antibodies, dominant negative mutants of IL2, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of MALT1 in an anti-apoptosis-related protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules against DNA encoding MALT1, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors for expressing the same, anti-MALT 1 antibodies, dominant negative mutants of MALT1, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of drugs that inhibit the activity of MCL1 in an anti-apoptotic-related protein that exhibits synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, synribo (homoharringtonine), RNAi molecules against DNA encoding MCL1, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing these, anti-MCL 1 antibodies, dominant negative mutants of MCL1, and the like, which are recognized as therapeutic agents for chronic myelogenous leukemia. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of MKL1 in an anti-apoptosis-related protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-MKL 1 antibodies, dominant negative mutants of MKL1, and the like, against DNA encoding MKL 1. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of MPO in an anti-apoptosis-related protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules against DNA encoding MPO, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-MPO antibodies, dominant negative mutants of MPO, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of MTL5 in an anti-apoptosis-related protein exhibiting synthetic lethality when the drug is simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules against DNA encoding MTL5, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors for expressing the same, anti-MTL 5 antibodies, dominant negative mutants of MTL5, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of MYBL2 in an anti-apoptosis-related protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules against DNA encoding MYBL2, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors for expressing the same, anti-MYBL 2 antibodies, dominant negative mutants of MYBL2, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of MYO18A in an anti-apoptosis-related protein exhibiting synthetic lethality when simultaneously inhibited with GST-pi include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors for expressing the same, anti-MYO 18A antibodies, dominant negative mutants of MYO18A, and the like, against DNA encoding MYO 18A. These drugs may be commercially available or appropriately manufactured based on known techniques.
On the other hand, the drugs that inhibit the activity of MTOR in the PI3K signaling pathway related protein, which exhibits synthetic lethality when inhibited simultaneously with GST-. PI., are not limited to the following examples, and examples thereof include: rapamycin, a low molecular weight compound that inhibits MTOR activity, as an immunosuppressive agent for macrolides (Chang et al, trends Pharmacol. Sci.12:218-223.1991); everolimus (Everolimus) (Weisblum et al, br. Med. Bull. 40:47-53.1984), a low molecular weight compound that inhibits MTOR activity, as an immunosuppressive agent derived from the macrolide system of rapamycin; a low molecular compound BEZ235 (Maira et al mol. Cancer Ther.7 (7): 1851-1863.2008) which inhibits MTOR activity as a PI3K tyrosine kinase inhibitor; a low molecular weight compound AZD8055 (Huang et al, J Biol chem. Nov 18;286 (46): 40002-12.2011) which selectively inhibits the kinase activity of MTOR; low molecular compounds PI-103 (demyets et al 2010.Basic Res Cardiol.Mar;106 (2): 217-31.2011), niclosamide (Balgi et al PLoS One.Sep 22;4 (9): e 7124.2009), PP242 (Bao et al J.cell biol.210 (7): 1153-64.2015), timosamide AIII (King et al PLoS One.Sep 30;4 (9): e7283 2009), KU 0063794 (Garcia-Martinez et al, biochem.J.421 (1): 29-42.2009), AZD2014 (Guichard et al, mol. Cancer.Ther.14 (11): 2508-18.2015), tamarolimus (Temmole et al J.Clin.One.22: 2347.2004), and Paul (U.Xuer.37529) as a pyridine furopyrimidine (U.S.6-64.2015), and yellow paint (U.S. 35, U.35, U.S. 35, U.35) as pyridine furopyrimidine compounds; 31 (8): 1424-33 2010), GDC-0980 (Wallin et al, mol. Cancer Ther.10 (12): 2426-36.2011), SF1126 (Garlich et al, cancer Res.68 (1): 206-15.2008), CH5132799 (Tanaka et al, clin Cancer Res.17 (10): 3272-81.2011), WYE-354 (Yu et al, cancer Res.69 (15): 6232-40.2009), compound 401 (ball et al, J. Biol. Chem.282, 24463.2007), geothermal limus (Gadduccci et al: gyneccol. Endocrinol.24, 239.2008), K1059615 (Steven et al, ACS. Med. Lett.1 (1): 39-43 2010), PF 04691502 (Yun et al, mol. Car.89 (11): 2132-99.2011) PP121 (apse et al, nat. Chem. Biol.4 (11): 691-9.2008), OSI-027 (Bhaqwat et al Mol. Cancer Ther.10 (8): 1394-406.2011), WYE-125132 (Yu et al, cancer res.70 (2): 621-31.2010), umirolimus (Umirolimus) (Baldo et al, 2008.Curr.Cancer Drug Targets.8 (8): 647-65.2008), WAY-600 (Yu et al, cancer Res.69 (15): 6232-40.2009), WYE-687 (Yu et al, cancer Res.69 (15): 6232-40.2009), PKI-179 (Venkatesan et al, bioorg. Med. Lett.20 (19): 5869-73.2010), PF-05212384 (Akintunde et al, J. Hematol. Oncol.6: 88.2013), CAY10626 (Rameh et al, J. Biol. Chem. 274:8347-8350.1999), NVP-BGT226 (Chang et al, clin. Cancer Res.17 (22): 7116-26.2011), XL 147 derivative 1 (Akinde et al, J. Hematol. 88.2013), DNA encoding DNA molecules encoding DNA, RNA, DNA, RNA, encoding,. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the agent for inhibiting the activity of IRAK1 in PI3K signaling pathway-related proteins that exhibits synthetic lethality when simultaneously inhibited with GST-PI include, but are not limited to, the following: low molecular weight compounds which specifically inhibit the activity of IL-1 Kinase, as benzimidazole compounds, interleukin-1 Receptor-Associated Kinase-1/4 Inhibitor (Interleukin-1 Receptor-Associated-Kinase-1/4 Inhibitor) (bhattraceryya et al, am.j. Physiol. Gastro. Lever physiol.293, G429.2007), RNAi molecules directed against DNA encoding IRAK1, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing them, anti-IRAK 1 antibodies, dominant negative mutants of IRAK1, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of IRS1 in PI3K signaling pathway-related proteins that exhibit synthetic lethality when simultaneously inhibited with GST-PI include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-IRS 1 antibodies, dominant negative mutants of IRS1, and the like, against DNA encoding IRS 1. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting MYD88 activity in PI3K signaling pathway-related protein, which exhibits synthetic lethality when simultaneously inhibited with GST-PI, include, but are not limited to, the following: a peptide of 26 amino acids derived from MYD88 that represses homodimerization, namely the MYD88 repressor peptide peplh-MYD (derosssi et al, j.biol. Chem., 269:10444-50.1994); a low molecular compound TJ-M2010 (Li et al, transfer proc.45 (5): 1842-5.2013) which specifically inhibits MYD 88; RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing them, anti-MYD 88 antibodies, dominant negative mutants of MYD88, and the like, directed against DNA encoding MYD 88. These drugs may be commercially available or appropriately manufactured based on known techniques.
The drug that inhibits NFKB1 activity in PI3K signaling pathway-related proteins that exhibits synthetic lethality when simultaneously inhibited with GST-PI is not limited to the following examples, and examples thereof include: a low molecular weight compound BAY 11-7085 (Pierce et al J.biol. Chem.272: 21096-21103.1997) which inhibits both NF- κB activation and IκBα phosphorylation; a low molecular compound of Caragana (Helenalin) inhibiting NF- κB as an anti-cancer agent inducing apoptosis (Lyss et al J.biol. Chem.273 (50): 33508-16.1998); a low molecular weight compound, phenethyl caffeate (Sud' ina et al, FEBS Lett.Aug 23;329 (1-2): 21-4.1993), which is an inhibitor of HIV integrase and tyrosine kinase, specifically inhibits NF- κB activity; and NF kappa B activation inhibitor II, JSH-23 (Shin et al, FEBS Lett.571:50-54.2004), QNZ (Tobe et al, biorg. Med. Chem.11:3869-3878.2003), andrographolide (Yu et al, planta Med. Dec;69 (12): 1075-9.2003), curcumin (Asai et al, j.nutr.131: 2932-2935.2001), aspirin (Kopp. Et al, science. 265:956-959.1994), sulfasalazine (Liptay et al, br. J. Pharmacol.128,1361. 1999), chinaberry amide (Engelmeier et al, J. Agric. Food Chem. 48:1400-1404.2000), SM 7368 (Lee et al, biochem. Biophys. Res. Commun. 336:716-722.2005), sulindac sulfide (Meade et al, J. Biol. Chem. 268:6610-6614.1993), trichodin (Erkel et al, FEBS. 477,219, 2000), CHS-828 (Hovdi et al, clin. Memory Res.8 (2843-50.2002), Z-VRPR-50.2002), hailfin et al, proc. Nature. Acer. Sci. U. Sci. No. 35, str. 6) and (6:6-35), namex. 35), nafion. Nafion (e. 35), nafion. 6:6-35), nafion. Phi. 6:6-35, nafion. Phi. Nafion, nafion. 6-35, nafion. Phi. Nafion. Nafip. Et. Kp.,.,., BAY 11-7082 (Pierce et al, J.biol. Chem.272: 21096-21103.1997), RNAi molecules directed against DNA encoding NFKB1, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing them, anti-NFKB 1 antibodies, dominant negative mutants of NFKB1, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
The drug for inhibiting the activity of PIK3CG in PI3K signaling pathway-related proteins that exhibits synthetic lethality when simultaneously inhibited with GST-PI is not limited to the following examples, and examples thereof include: a low molecular compound CUDC-907 (Qian et al, clin Cancer Res.18 (15)) that specifically inhibits the activity of both PI3K and mTOR, a low molecular compound PKI-402 (Dehnhardt et al, J Med chem.Jan 28;53 (2): 798-810.2010), a low molecular compound PF-04691502 (Yuan et al, mol Cancer Ther,10 (11), 2189-2199.2011) that specifically inhibits the activity of both PI3K and mTOR, a low molecular compound NVP-BGT226 (Glienke et al, tumor Biology,33 (3): 757-765.2012), and IPI-145 (INK 1197) (Winkler et al, chem biol.2013 v 21 (20): 4-2), SAR 3K (3935) (763 i.762), and so on; 154 (3): 1247-592013), ZSTK474 (Toyama et al, arthritis Res Ther.12 (3): R92.2010), VS-5584 (SB 2343) (Hart et al, mol Cancer Ther.2013 Feb;12 (2): 151-61.2013), AS-605240 (Camps et al, nature media, 11 (9): 936-943.2005), PIK-90 (Van et al, J Cell biol.2006Jul 31;174 (3): 437-45.2006), PF-4989216 (Walls et al, clin Cancer Res.2014 Feb 1;20 (3): 631-43.2014), TG100-115 (Walls et al, proc Natl Acad Sci U S a. Dec 26;103 19866-71.2006), BKM120 (Bendell et al J.Clin. Oncol.30 (3): 282-90.2012), BEZ235 tosylate (Maira et al Mol Cancer Ther2008; 7:1851-1863.2008), LY294002 (Maira et al, biochem. Soc. Trans.37 (Pt 1): 265-72.2009), PI-103 (Raynaud et al, molecular Cancer Therapeutics,8 (7): 1725-1738.2009), XL147 (Shapiro et al, proc 97th Annu Meet AACR,14-18.2007), AS-252424 (Pomel et al, J Med chem. Jun 29); 49 (13) 3857-71.2006), AS-604850 (Camps et al, nature media, 11 (9) 936-943.2005), CAY10505 (Tyagi et al, can J Physiol Pharmacol. Jul;90 (7) 881-5.2012), CH5132799 (Ohwada et al, bioorganic & medicinal chemistry letters,21 (6): 1767-1772.2011), BAY 80-6946 (Copanlisib) (Patnaik et al, J Clin Oncol,29,2011), GDC-0032 (Ndekaku et al, J Med chem. Jun 13;56 4597-610.2013), GSK1059615 (Knight et al ACS Medicinal Chemistry Letters,1 (1): 39-43.2010), CAL-130 (Subramannian et al, cancer cell.21 (4): 459-72.2012), XL765 (Laird et al, R-NCI-EORTC International Conference on Molecular Targets and Cancer therapeutics.October p.B250 2007), RNAi molecules directed against DNA encoding PIK3CG, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing them, anti-PIK 3CG antibodies, dominant negative mutants of PIK3CG, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of drugs that inhibit the activity of RAC1 in PI3K signaling pathway-related proteins that exhibit synthetic lethality when simultaneously inhibited with GST-PI include, but are not limited to, low molecular weight compounds NSC 23766 (Gao et al, proc.Natl.Acad.Sci.U.S. A.101:7618-7623.2004) that specifically inhibit RAC GTPase activity, peptide W56 derived from the guanylate exchange factor recognition/activation site of RAC1 (Gao et al, j.biol.chem.276 47530.2001), RNAi molecules directed against DNA encoding RAC1, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing these, anti-RAC 1 antibodies, dominant mutants of RAC1, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
The drugs that inhibit the activity of AKT3 in PI3K signaling pathway-related proteins that exhibit synthetic lethality when simultaneously inhibited with GST-PI are not limited to the following examples, and examples thereof include: low molecular weight compound MK-2206 dihydrochloride (Hirai et al, mol. Cancer ther.9 (7): 1956-67.2010), which specifically inhibits the activity of Akt1, akt2 and Akt 3; the low molecular compound troxitabine, which specifically inhibits Akt activity (Moore et al, biochem. Pharmacol. 38:4037-4044.1989); low molecular weight compounds Akt inhibitory factor VIII (Barnett et al, biochem. J.385:399-408.2005) which are cell permeable quinoxaline compounds that specifically inhibit the activity of Akt1, akt2 and Akt 3; a low molecular compound GSK 690693 (Rhodes et al, cancer Res.68 (7): 2366-2374.2008) which is a compound derived from an amino furazan and specifically inhibits the activity of Akt1, akt2 and Akt 3; low molecular weight compounds AT7867 (Grimsshaw et al mol. Cancer Ther.9 (5): 1100-10.2010) which specifically inhibit the activity of Akt1, akt2 and Akt 3; RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-AKT 3 antibodies, dominant negative mutants of AKT3, and the like, directed against DNA encoding AKT 3. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the agent for inhibiting the activity of EIF4B in PI3K signaling pathway-related proteins that exhibit synthetic lethality when simultaneously inhibited with GST-PI include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-EIF 4B antibodies, dominant negative mutants of EIF4B, and the like, against DNA encoding EIF 4B. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of drugs that inhibit the activity of EIF4E in a protein involved in the synthetic lethal PI3K signaling pathway when simultaneously inhibited with GST-PI include, but are not limited to, low-molecular compound 4EGI-1 (Moerke et al, cell.128:257-267.2007), RNAi molecules against DNA encoding EIF4E, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-EIF 4E antibodies, dominant negative mutants of EIF4E, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of ILK in PI3K signaling pathway-related protein, which exhibits synthetic lethality when simultaneously inhibited with GST-PI, include, but are not limited to, the following: a low-molecular compound Cpd 22 (Lee et al, j.med.chem.54, 6364.2011) which specifically inhibits ILK as a pyrazole compound having cell permeability; low molecular compound QLT0267 (Younes et al Mol Cancer ter. Aug;4 (8): 1146-56.2005), RNAi molecules directed against DNA encoding ILK, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing them, anti-ILK antibodies, dominant negative mutants of ILK, and the like, which specifically inhibit the kinase activity of ILK. These drugs may be commercially available or appropriately manufactured based on known techniques.
Examples of the drug for inhibiting the activity of MTCP1 in a PI3K signaling pathway-related protein exhibiting synthetic lethality when simultaneously inhibited with GST-PI include, but are not limited to, RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing the same, anti-MTCP 1 antibodies, dominant negative mutants of MTCP1, and the like, against DNA encoding MTCP 1. These drugs may be commercially available or appropriately manufactured based on known techniques.
The drug for inhibiting the activity of PIK3CA in PI3K signaling pathway-related proteins that exhibits synthetic lethality when simultaneously inhibited with GST-PI is not limited to the following examples, and examples thereof include: low molecular weight compounds HS-173 (Lee et al, cancer Lett. Jan 1;328 (1): 152-9.2013) that specifically inhibit the activity of both PI3K, CUDC-907 (Qian et al, clin Cancer Res.18 (15): 4104-4113.2012) that specifically inhibit the activity of both PI3K and mTOR, PKI-402 (Dehnhardt et al, J Med Chem. Jan 28;53 (2): 798-810.2010), PF-04691502 (Yuan et al, mol Cancer Ther,10 (11), 2189-2199.2011) that specifically inhibit the activity of both PI3K and mTOR, NVP-T226 (Glienke et al, mol Biology,33 (3): 757-52), L (37-810.2010), and thereby specifically inhibiting the activity of both PI3K and mTOR (including SLO3, and mTOR) (Daol3: FIG. 7432; daol3: TV, daol3, 37: 7441; 154 (3): 1247-592013), ZSTK474 (Toyama et al, arthritis Res Ther.12 (3): R92.2010), VS-5584 (SB 2343) (Hart et al, mol Cancer Ther.2013Feb;12 (2): 151-61.2013), PIK-75 (Zheng et al, mol pharmacol.Oct;80 (4): 657-64.2011), PIK-90 (Van et al, J Cell biol.2006Jul 31;174 (3): 437-45.2006), A66 (Jamieson et al, biochem J.438:53-62.2011), CNX1351 (Nacht et al, J Med Chem), 56 712-721.2013), PF-4989216 (Walls et al Clin Cancer Res.2014 Feb 1;20 (3): 631-43.2014), BKM120 (Bendell et al j.clin. Oncol.30 (3): 282-90.2012), BEZ235 tosylate (maila et al Mol Cancer ter 2008; 1851-1863.2008), LY294002 (Maira et al, biochem. Soc. Trans.37 (Pt 1): 265-72.2009), PI-103 (Raynaud et al, molecular Cancer Therapeutics,8 (7): 1725-1738.2009), XL147 (Shapiro et al, proc 97th Annu Meet AACR,14-18.2007), CH5132799 (Ohwada et al, bioorganic & medicinal chemistry letters,21 (6): 1767-1772.2011), BAY 80-6946 (Copanlisib) (Patnaik et al, J Clin Oncol,29,2011), GDC-0032 (Ndeaku et al, J Med chem. Jun 13); 56 4597-610.2013), XL765 (Laird et al, AACR-NCI-EORTC International Conference on Molecular Targets and Cancer therapeutics, october p.B250 2007), RNAi molecules directed against DNA encoding PIK3CA, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing them, anti-PIK 3CA antibodies, dominant negative mutants of PIK3CA, and the like. These drugs may be commercially available or appropriately manufactured based on known techniques.
The drug for inhibiting the activity of SRF in PI3K signaling pathway-related protein that exhibits synthetic lethality when simultaneously inhibited with GST-PI is not limited to the following examples, and examples thereof include: a low molecular compound CCG-1423 (Evelyn et al mol. Cancer ter.6, 2249.2007) which is a cell-permeable benzamide compound and specifically blocks the RHO signaling pathway and activation of SRF; a low molecular compound CCG-100602 (Evelyn et al, bioorg Med Chem Lett 20 665-72.2010) which is an analog of CCG-1423 and specifically blocks activation of the RHO signaling pathway and SRF; RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, vectors expressing them, anti-SRF antibodies, dominant negative mutants of SRF, and the like, directed against DNA encoding SRF. These drugs may be commercially available or appropriately manufactured based on known techniques.
In particular, as a drug for inhibiting the production of a cell cycle regulatory protein or an anti-apoptosis-related protein or a PI3K signal transduction pathway-related protein, which exhibits synthetic lethality when simultaneously inhibited with GST-PI, such as p21, or the like, or the activity thereof, an RNAi molecule, a ribozyme, an antisense nucleic acid, a DNA/RNA chimeric polynucleotide, and a vector for expressing the same are preferable from the viewpoints of high specificity and low possibility of side effects.
Inhibition of the steady state maintenance related protein may be determined according to the following phenomena: the expression and activity of the protein are inhibited in the cells, compared with the case where the protein is not acted on by the inhibitor. The expression of the protein can be evaluated by any known method, and examples thereof include, but are not limited to, immunoprecipitation using an antibody, EIA, ELISA, IRA, IRMA, western blotting, immunohistochemical methods, immunocytochemical methods, flow cytometry, hybridization methods using various nucleic acids specifically hybridizing with a nucleic acid encoding the protein or a specific fragment thereof or a transcription product (e.g., mRNA) or a splice product of the nucleic acid, northern blotting, southern blotting, and various PCR methods.
In addition, the activity of p21 can be evaluated by known activities of p21 (e.g., binding to cyclin-CDK 2 or cyclin-CDK 1 complex, etc.), without limitation, and any known method such as immunoprecipitation, western blotting, mass spectrometry, pulldown, surface Plasmon Resonance (SPR), etc. can be used for such evaluation.
As used herein, RNAi molecules refer to any molecule that causes RNA interference, and include, without limitation, siRNA (small interfering RNA), miRNA (microrna), shRNA (short hairpin RNA), ddRNA (DNA-directed RNA), piRNA (Piwi-interacting RNA), or rasiRNA (repeat-related siRNA), and modifications thereof, and the like. These RNAi molecules may be commercially available or may be designed and prepared based on known sequence information (i.e., the base sequences and/or amino acid sequences shown in SEQ ID Nos. 1 to 30, 39 to 108).
In addition, in the present specification, antisense nucleic acid includes RNA, DNA, PNA, or a complex thereof.
The DNA/RNA chimeric polynucleotide, when used in the present specification, is not limited, but includes, for example, a double-stranded polynucleotide composed of DNA and RNA for repressing the expression of a target gene as described in Japanese patent application laid-open No. 2003-219893.
The agent for inhibiting GST-pi and the agent for inhibiting a protein involved in maintaining homeostasis which exhibits synthetic lethality when simultaneously inhibited with GST-pi may be contained in a single formulation or may be contained in 2 or more formulations, respectively. In the latter case, each formulation may be administered simultaneously or at intervals. In the case of administration at time intervals, the preparation containing the GST-pi-inhibiting drug may be administered before or after the preparation containing the drug inhibiting the steady-state maintenance related protein which exhibits synthetic lethality when simultaneously inhibited with GST-pi.
In the cell death inducing agent and the cell proliferation inhibiting agent according to the present invention, the number of the drugs for inhibiting the steady-state maintenance related protein may be 1 or 2 or more. For example, as the agent for inhibiting a steady-state maintenance related protein contained in the cell death induction agent and the cell proliferation inhibition agent according to the present invention, 2 or more agents for inhibiting a cell cycle related protein, 2 or more agents for inhibiting an anti-apoptosis related protein, 1 or more agents for inhibiting a cell cycle related protein, and 1 or more agents for inhibiting an anti-apoptosis related protein may be used.
In addition, ATM, CDC25A, p, PRKDC, RBBP8, SKP2, MCM10, RNPC1, CCNL1, CENPH, BRSK1, MCM8, CCNB3, MCMDC1, AATF, ALOX12, ANXA1, ANXA4, API5, ATF5, AVEN, AZU1, BAG1, BCL2L1, BFAR, CFLAR, IL2, MALT1, MCL1, MKL1, MPO, MTL5, MYBL2, MYO18A, MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP1, PIK3CA, and SRF are steady-state maintenance-related proteins that exhibit a synergistic lethality to cancer cells when inhibited simultaneously with GST-pi. Thus, the agent that inhibits the protein becomes an active ingredient of an agent or composition that enhances induction of cell death and/or inhibition of cell proliferation (hereinafter, also referred to as "cell death induction enhancer", "cell proliferation inhibition enhancer", "cell death induction enhancing composition" or "cell proliferation inhibition enhancing composition") caused by the agent that inhibits GST-pi. In other words, by administering an effective amount of the agent that inhibits the protein, induction of cell death and/or inhibition of cell proliferation due to administration of the agent that inhibits GST-pi can be enhanced.
When the agent or composition is administered, the amount of the active ingredient to be blended in the agent or composition of the present invention may be an amount capable of inducing cell death such as apoptosis and/or inhibiting cell proliferation. In addition, it is preferable that the amount does not cause adverse effects greater than the benefit of administration. The amount may be known or may be determined appropriately by in vitro tests using cultured cells or the like, tests in model animals such as mice, rats, dogs or pigs, and such test methods are well known to those skilled in the art. Induction of apoptosis can be evaluated by various known methods, for example, detection of phenomena unique to apoptosis such as DNA fragmentation, binding of annexin V to cell membrane, change in mitochondrial membrane potential, activation of caspase, TUNEL staining, and the like. The inhibition of cell proliferation can be evaluated by various known methods, for example, measurement of the number of living cells with time, measurement of the size, volume or weight of a tumor, measurement of DNA synthesis, WST-1 method, brdU (bromodeoxyuridine) method, 3H thymidine incorporation method, and the like. The amount of active ingredient may vary depending on the agent or the mode of administration of the composition. For example, in the case where a plurality of units of the composition are used for 1 administration, the amount of the active ingredient blended in each 1 unit of the composition may be set to one of plural parts of the amount of the active ingredient necessary for 1 administration. The amount to be blended can be appropriately adjusted by those skilled in the art.
In addition, a cell death-inducing agent, a cell proliferation-inhibiting agent, a cell death-inducing composition or a cell proliferation-inhibiting composition can be produced by mixing a drug for inhibiting GST-pi with a drug for inhibiting a steady-state maintenance-related protein that exhibits synthetic lethality when simultaneously inhibited with GST-pi as an active ingredient.
In addition, a combination for cell death induction or cell proliferation inhibition, which is a combination of an agent that inhibits GST-pi and an agent that inhibits a steady state maintenance-related protein that exhibits synthetic lethality when simultaneously inhibited with GST-pi, can be provided. In addition, there can be provided a method of inducing cell death or a method of inhibiting cell proliferation, which comprises administering an effective amount of an agent that inhibits GST-pi and an agent that inhibits a steady state maintenance-related protein that exhibits synthetic lethality when inhibited simultaneously with GST-pi.
The method of inducing apoptosis and the like and the method of inhibiting cell proliferation may be either in vitro or in vivo. In addition, for the drug in the method, as described above, an effective amount of the drug may be an amount that induces cell death, or inhibits cell proliferation, in the cells administered. In addition, it is preferable that the amount does not cause adverse effects greater than the benefit of administration. The amount may be known or may be appropriately determined by an in vitro test using cultured cells or the like, such test methods being well known to those skilled in the art. Induction of cell death or inhibition of cell proliferation can be evaluated by various known methods including the above-described methods. For such effective amounts, it is not necessary that all cells in the same cell population undergo cell death or proliferation inhibition when the drug is administered to a prescribed cancer cell population. For example, the effective amount may be an amount that inhibits apoptosis or proliferation of 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 8% or more, 10% or more, 12% or more, 15% or more, 20% or more, 25% or more of the cells in the cell population.
The cell death inducing agent or the cell proliferation inhibiting agent of the present invention is effective as a component of a pharmaceutical composition for a disease caused by abnormal cell proliferation because it can induce cell death or inhibit cell proliferation effectively even in cancer cells. In addition, a pharmaceutical composition for a disease caused by abnormal cell proliferation can be produced by combining a drug that inhibits GST-pi and a drug that inhibits a steady-state maintenance-related protein that exhibits synthetic lethality when simultaneously inhibited with GST-pi as active ingredients. In addition, a disease caused by abnormal cell proliferation can be treated, including the step of administering the manufactured pharmaceutical composition to a subject in need thereof in an effective amount.
The pharmaceutical composition is effective for the treatment of diseases caused by abnormal cell proliferation, particularly diseases in which abnormal cell death or cell proliferation occurs due to the expression of mutant KRAS.
Examples of diseases caused by cells expressing mutant KRAS include, but are not limited to, benign or malignant tumors (also referred to as cancer, malignant neoplasms), hyperplasia, keloids, cushing's syndrome, primary aldosteronism, erythema (erythroplakia), polycythemia vera (polycythemia vera), leukoplakia (leukoplakia), hypertrophic scars (hyperplastic scar), lichen planus, and melanosis (lentiginosis).
Examples of the cancer in the present invention include cancer, cancer expressing GST-pi at a high level, cancer caused by cells expressing mutant KRAS (sometimes simply referred to as KRAS cancer), and the like, and KRAS cancer is included in many cases in cancer expressing GST-pi at a high level. Examples of the cancers include, but are not limited to, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, and the like; cancers such as brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, duodenal cancer, appendiceal cancer, carcinoma of large intestine, rectal cancer, liver cancer, pancreatic cancer, gall bladder cancer, bile duct cancer, anus cancer, kidney cancer, ureter cancer, bladder cancer, prostate cancer, penile cancer, testis cancer, uterine cancer, ovarian cancer, vulval cancer, vaginal cancer, and skin cancer, leukemia, and malignant lymphoma. In the present invention, "cancer" includes epithelial malignant tumor and non-epithelial malignant tumor. Cancers in the present invention may exist in any part of the body, for example, brain, head and neck, chest, limbs, lung, heart, thymus, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (colon, cecum, appendix, rectum), liver, pancreas, gall bladder, anus, kidney, ureter, bladder, prostate, penis, testis, uterus, ovary, vulva, vagina, skin, striated muscle, smooth muscle, synovium, cartilage, bone, thyroid, adrenal gland, peritoneum, mesentery, bone marrow, blood, vascular system, lymph system such as lymph node, lymph fluid, and the like.
The pharmaceutical composition may contain, in addition to a drug for inhibiting GST-pi and a drug for inhibiting a protein involved in steady-state maintenance that exhibits synthetic lethality when GST-pi is simultaneously inhibited, other active ingredients. Here, the term "combination" includes, for example, the following cases: is administered as a separate preparation from the other active ingredients; and administering the other active ingredient as a mixture with at least 1 other drug. When administered as a separate formulation, the formulation containing the other active ingredient may be administered before, simultaneously with, or after the other formulation.
Examples of the other active ingredient include an ingredient effective for the treatment of a disease to be treated. For example, in the case where the disease to be treated is cancer, an anticancer agent may be used in combination. Examples of anticancer agents include alkylating agents such as ifosfamide, nimustine hydrochloride, cyclophosphamide, dacarbazine, melphalan, and ramustine (ranimustine), antitumor antibiotics such as gemcitabine hydrochloride, enocitabine hydrochloride, sodium stearyl phosphate (cytarabine ocfosfate), cytarabine sulfate, tegafur/uracil (tegafur uracil), tegafur/gemustine/potassium oxazinate complex (tegafur-gimeracil-oteracil potassium combination drugs) (for example, TS-1), metabolic antagonists such as doxifluridine, hydroxyurea, fluorouracil, methotrexate, mercaptopurine, nordaunorubicin hydrochloride, epirubicin hydrochloride, daunorubicin citrate, doxorubicin hydrochloride, pirarubicin hydrochloride, bleomycin hydrochloride, mitomycin C, etoposide, irinotecan hydrochloride, vinblastine, tarabine, fluvoxamine, dacarbazine, dp, and other antitumor antibiotics such as fluvoxamine, and cisplatin sulfate, and other therapeutic agents such as fluvoxamine, and cisplatin, and other therapeutic agents.
In the case where the active ingredient in the various reagents, compositions, methods of treatment and the like of the present invention described in the present specification is a nucleic acid, for example, an RNAi molecule, a ribozyme, an antisense nucleic acid, a DNA/RNA chimeric polynucleotide and the like, the above nucleic acid may be used as a naked nucleic acid directly or may be carried on various vectors. As the vector, any known vector such as a plasmid vector, a phage vector, a phagemid vector, a cosmid vector, a viral vector and the like can be used. The vector preferably contains at least a promoter (promoter) which enhances the expression of the carried nucleic acid, in which case the nucleic acid is preferably operably linked to said promoter. By operably linked to a promoter is meant that the nucleic acid and promoter are configured such that the protein encoded by the nucleic acid is correctly produced by the action of the promoter. The vector may be a vector capable of replication in a host cell or a vector incapable of replication in a host cell, and transcription of the gene may be performed outside the nucleus of the host cell or within the nucleus. In the latter case, the nucleic acid may also be incorporated into the genome of the host cell.
In addition, the active ingredient may be carried on various nonviral lipid or protein carriers. Examples of the carrier include, but are not limited to, cholesterol, liposome, antibody precursor, cyclodextrin nanoparticle, fusion peptide, aptamer, biodegradable polylactic acid copolymer, polymer, etc., whereby the uptake efficiency into cells can be improved (see, for example, pirollo and Chang, cancer res.2008;68 (5): 1247-50, etc.). Cationic liposomes, polymers (e.g., polyethylenimine, etc.), are particularly useful. Further examples of the polymer that can be used as the support include supports described in, for example, US 2008/0207553, US 2008/0312174, and the like.
In the various pharmaceutical compositions of the present invention described in the present specification, the active ingredient may be combined with other optional ingredients as long as the effect of the active ingredient is not impaired. Examples of such optional components include other chemotherapeutic agents, pharmaceutically acceptable carriers, excipients, diluents, and the like. The composition may be coated with a suitable material, for example, an enteric coating or a time-lapse disintegrable material, depending on the route of administration and the form of drug release, or may be incorporated into a suitable drug release device.
The various agents and compositions of the present invention described in the present specification (including various pharmaceutical compositions) can be administered by various routes including oral and non-oral routes (for example, oral, intravenous, intramuscular, subcutaneous, topical, intratumoral, rectal, intraarterial, portal intravenous, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, intrauterine, etc., without limitation), and can be prepared into dosage forms suitable for each route of administration. The dosage form and the preparation method may be appropriately any known method (see, for example, standard pharmaceutical science, du-on-side happiness et al, south Jiang Tang, 2003, etc.).
For example, the dosage form suitable for oral administration is not limited, and examples thereof include powders, granules, tablets, capsules, liquids, suspensions, emulsions, gels, syrups, and the like, and examples thereof suitable for parenteral administration include injections such as solution injections, suspension injections, emulsion injections, and pre-use formulation injections. Formulations for non-oral administration may be in the form of aqueous or non-aqueous isotonic sterile solutions or suspensions.
The various agents or compositions of the invention described in this specification (including various pharmaceutical compositions) can be targeted to specific tissues, cells. Targeting can be achieved by any known method. In the case where delivery to cancer is desired, for example, the following methods and the like can be used without limitation: passive targeting is carried out by making the formulation into a size suitable for exerting EPR (high permeability and retention, enhanced permeability and retention) effect with a diameter of 50 to 200 μm (in particular 75 to 150 μm etc.); active targeting, which uses ligands such as CD19, HER2, transferrin receptor, folate receptor, VIP receptor, EGFR (Torulin, AAPS J.2007;9 (2): E128-47), RAAG10 (Japanese patent Table 2005-532050), PIPA (Japanese patent Table 2006-506071), KID3 (Japanese patent Table 2007-52997), peptides having RGD motif, NGR motif, F3, lyP-1 (Ruoslahti et al, J Cell biol.2010;188 (6): 759-68) and the like as targeting agents. In addition, retinoids (retinoids) or derivatives thereof are also known to be useful as targeting agents for cancer cells (WO 2008/120815), and thus carriers containing retinoids as targeting agents can also be utilized. In addition to the above documents, the carriers are described in WO 2009/036368, WO 2010/014117, WO 2012/170952, and the like.
The various reagents and compositions of the invention described in the present specification (including various pharmaceutical compositions) may be supplied in any form, and from the viewpoint of storage stability, may be supplied in a form that can be prepared before use, for example, in a form that can be prepared by a doctor and/or pharmacist, nurse, or other auxiliary medical staff at or near a medical site. This form is particularly useful when the reagent or composition of the present invention contains a component such as a lipid, a protein, or a nucleic acid, which is difficult to stably preserve. In this case, the agent or composition of the present invention is provided in the form of 1 or 2 or more containers containing at least 1 of the above-mentioned essential components, and can be formulated before use, for example, within 24 hours, preferably within 3 hours, and more preferably immediately before use. In the preparation, reagents, solvents, dispensing devices, etc. generally available at the preparation site can be used as appropriate.
Thus, the invention also relates to: a kit for formulating a composition comprising 1 or 2 or more containers containing, either alone or in combination, the active ingredients that may be contained in the various agents or compositions of the invention; and, necessary components of various reagents or compositions provided in the form of such kits. The kit of the present invention may contain instructions describing the preparation method, administration method, etc. of the various reagents or compositions of the present invention, for example, instructions, electronic recording media such as a CD, DVD, etc., in addition to the above. In addition, the kit of the present invention may contain all the components of the various reagents or compositions for carrying out the present invention, but not necessarily all the components. Therefore, the kit of the present invention may not contain reagents, solvents, such as sterile water, physiological saline, glucose solution, etc., which are generally available at medical sites, experimental institutions, etc.
The effective amount in the various treatment methods of the present invention described in the present specification is, for example, an amount that alleviates symptoms of a disease or delays or stops the progression of a disease, and is preferably an amount that inhibits or cures a disease. In addition, it is preferable that the amount does not cause adverse effects greater than the benefit of administration. The amount can be suitably determined by in vitro tests using cultured cells or the like, tests in model animals such as mice, rats, dogs or pigs, and such test methods are well known to those skilled in the art. In addition, the amount of the drug to be used in the treatment method of the present invention is known to those skilled in the art, or can be appropriately determined by the above-described test or the like.
The specific amount of active ingredient administered in the treatment method of the present invention described in the present specification can be determined in consideration of the following various conditions related to the subject to be treated: for example, the severity of the symptoms, the general health of the subject, age, weight, sex of the subject, diet, period and frequency of administration, the medicines used, responsiveness to treatment, dosage form, and compliance with treatment, etc.
The administration route includes: various routes including both oral and non-oral, e.g., oral, intravenous, intramuscular, subcutaneous, topical, intratumoral, rectal, intraarterial, portal intravenous, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, intrauterine, and the like.
The administration frequency may vary depending on the agent used, the nature of the composition, and the conditions of the subject including the above conditions, and may be, for example, 1 time per day (i.e., 2, 3, 4, or 5 times per day), 1 time per day (i.e., 2, 3, 4, 5, 6, 7 days, etc.), 1 time per week, and 1 time per several weeks (i.e., 2, 3, 4, etc.).
As used herein, the term "subject" refers to any biological individual, preferably an animal, more preferably a mammal, even more preferably a human. In the present invention, the subject may be a healthy subject or a subject suffering from a disease. Where it is desired to treat a particular disease, typically it is intended to refer to a subject suffering from, or at risk of suffering from, the disease.
In addition, as used in the present specification, the term "treatment" includes all kinds of preventive and/or therapeutic interventions that are medically allowed for the purpose of cure, temporary relief, prevention, and the like of a disease. For example, the term "treatment" includes medically approved interventions for various purposes including delay or cessation of disease progression, reduction or disappearance of lesions, prevention of disease onset or prevention of recurrence, and the like.
Further, ATM, CDC25A, p, PRKDC, RBBP8, SKP2, MCM10, RNPC1, CCNL1, CENPH, BRSK1, MCM8, CCNB3, MCMDC1, AATF, ALOX12, ANXA1, ANXA4, API5, ATF5, AVEN, AZU1, BAG1, BCL2L1, BFAR, CFLAR, IL2, MALT1, MCL1, MKL1, MPO, MTL5, MYBL2, MYO18A, MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF E, ILK, MTCP1, PIK3CA and SRF are proteins that exhibit synergistic lethality to cancer cells when inhibited simultaneously with GST-pi, as described above. Therefore, it is possible to screen a cancer cell death-inducing agent and/or a cell proliferation-inhibiting agent for use in combination with a GST-pi-inhibiting drug, using inhibition of these steady state maintenance-related proteins as an index. That is, a substance capable of inhibiting these steady-state maintenance related proteins can be used as a candidate substance for a cancer cell death induction agent and/or a cell proliferation inhibition agent, which is used together with a GST-pi-inhibiting drug.
For example, as an example of cancer cells, cells expressing mutant KRAS are contacted with a test substance, and the expression level of the above-mentioned steady-state maintenance-related protein exhibiting synthetic lethality to the cells expressing mutant KRAS when the cells are simultaneously inhibited with GST-pi is measured. In the case where the expression level upon contact with the test substance is reduced as compared with the expression level measured in the absence of the test substance, the test substance may be selected as a candidate substance for a drug inhibiting the above-mentioned steady-state maintenance-related protein.
On the other hand, a drug that inhibits GST-pi is a protein that exhibits synthetic lethality against cancer cells when simultaneously inhibited with a drug that inhibits the above-described steady state maintenance-related protein, which exhibits synthetic lethality against cancer cells when simultaneously inhibited with GST-pi. Therefore, a cancer cell death induction reagent and/or a cell proliferation inhibition reagent which are used together with a drug for inhibiting the above-mentioned steady-state maintenance related protein can be selected using inhibition of GST-pi as an index. That is, a substance capable of inhibiting GST-pi can be used as a candidate substance for a cancer cell death induction agent and/or a cell proliferation inhibition agent, which are used together with a drug for inhibiting the above-mentioned steady-state maintenance related protein.
For example, as an example of cancer cells, cells expressing mutant KRAS are contacted with a test substance, and the GST-. Pi.expression level in the cells is measured. In the case where the expression level upon contact with the test substance is reduced as compared with the expression level measured in the absence of the test substance, the test substance may be selected as a candidate substance for the GST-pi-inhibiting drug.
Similarly, a reagent for inducing cell death and/or a reagent for inhibiting cell proliferation of cancer cells can be selected using inhibition of GST-pi and inhibition of a protein involved in steady state maintenance that exhibits synthetic lethality to cancer cells when GST-pi is simultaneously inhibited as an index. That is, a substance capable of inhibiting GST-pi and inhibiting the above-mentioned steady-state maintenance related protein can be used as a candidate substance for a cell death inducing agent and/or a cell proliferation inhibiting agent for cancer cells.
For example, as an example of cancer cells, cells expressing mutant KRAS are contacted with a test substance, and the expression level of GST-. Pi.and the expression level of the steady-state maintenance-related protein in the cells are measured. In the case where the respective expression amounts upon contact with the test substance are reduced as compared with the respective expression amounts measured in the absence of the test substance, the test substance can be selected as a candidate substance for a drug that inhibits GST-pi and inhibits a steady-state maintenance-related protein that exhibits synthetic lethality against cancer cells when simultaneously inhibited with GST-pi.
The substance to be measured is not limited to any particular substance, and may be any substance. The substance to be measured may be a single substance or a mixture containing a plurality of constituent components. The substance to be tested may be a composition containing an unidentified substance such as an extract from a microorganism or a culture medium, or may be a composition containing a known composition at a predetermined composition ratio. The test substance may be any of proteins, nucleic acids, lipids, polysaccharides, organic compounds, and inorganic compounds.
Examples
The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to the following examples.
[ experiment 1] GST-pi and P21 knockdown by siRNA
As an example of cancer cells, 1X 10 will be 5 The M7609 cells (KRAS mutant human colorectal cancer cells) and the PANC-1 cells (KRAS mutant human pancreatic cancer cells) were inoculated into 6cm dishes and cultured in Roswell Park Memorial Institute 1640 (RPMI 1640, sigma) supplemented with 10% fetal bovine serum (Fetal bovine serum, FBS) and 0.5% L-glutamine for 18 hours. Unless otherwise specified, the culture conditions are as follows37℃、5％CO 2 Is carried out under the condition of (2). In addition, as an example of cancer cells, 0.5X10 5 Individual a549 cells (KRAS mutant human lung cancer cells) were inoculated into 6cm dishes and cultured in Dulbecco's modified Eagle medium (DMEM, sigma) supplemented with 10% fbs and 1% l-glutamine for 18 hours. In addition, as an example of cancer cells, 1×10 will be 5 The individual MIA PaCa-2 cells (KRAS mutant human pancreatic cancer cells) were seeded in 6cm dishes and cultured in DMEM supplemented with 10% FBS and 1% L-glutamine for 18 hours. In addition, as an example of cancer cells, 0.5X10 5 Each HCT116 cell (KRAS mutant human colorectal cancer cell) was inoculated into a 6cm dish and cultured in McCoy's 5A medium (McCoy, sigma Co.) supplemented with 10% FBS and 0.5% L-glutamine for 18 hours.
In this experiment, first, GST-. Pi.siRNA and/or P21siRNA were transfected into PANC-1, A549 or MIA PaCa-2 cells reaching 20 to 30% confluence (conflux) as described below using Lipofectamine RNA iMAX (Life Technologies).
Lipofectamine/siRNA mixed solution for transfection was prepared as follows. First, 15. Mu.L of Lipofectamine RNAiMAX and 485. Mu.L of OPTI-MEM (Sigma Co.) were mixed to prepare a Lipofectamine solution. Next, a prescribed amount of 50. Mu.M siRNA was fixed to 500. Mu.L with OPTI-MEM to prepare an siRNA solution (for example, in the case of preparing an siRNA solution having a final concentration of 50nM to be used, 6. Mu.L of 50. Mu.M siRNA was mixed with 494. Mu.L of OPTI-MEM), and the resulting solution was mixed with the lipofectamine solution and allowed to stand at room temperature for 15 minutes. The following siRNA was used as the siRNA. In the following description, capital letters denote RNA and lowercase letters denote DNA.
GST-πsiRNA：
Sense strand: GGGAGGCAAGACCUUCAUUtt (sequence number 31)
Antisense strand: AAUGAAGGUCUUGCCUCCCtg (sequence number 32)
P21 siRNA：
Sense strand: UCCUAAGAGUGCUGGGCAUtt (sequence number 33)
Antisense strand: AUGCCCAGCACUCUUAGGAtt (sequence number 34)
Control siRNA:
sense strand: ACGUGACACGUUCGGAGAAtt (sequence number 35)
Antisense strand: UUCUCCGAACGUGUCACGUtt (sequence number 36)
GST-πsiRNA-2：
Sense strand: UCUCCCUCAUCUACACCAAtt (sequence number 37)
Antisense strand: UUGGUGUAGAUGAGGGAGAtg (sequence number 38)
To dishes containing PANC-1, MIA PaCa-2 cells or a549 cells, the following solutions were added, respectively: GST-pi siRNA with a final concentration of 50nM and P21 siRNA, GST-pi siRNA with a final concentration of 50nM or P21 siRNA (with the addition of control siRNA with a final concentration of 50 nM), and GST-pi siRNA with a final concentration of 100nM (without the addition of control siRNA). As a control, dishes with control siRNA added at a final concentration of 100nM were used. After 1 day of culture without medium replacement, the GST-. Pi.mRNA amount and the P21 mRNA amount were measured by the quantitative PCR method using a 7300 real-time PCR apparatus (Applied Bio Systems Co.). The results are shown in FIG. 1. As shown in FIG. 1, it was found that the amount of P21 mRNA was increased by knocking down GST-. Pi.with siRNA.
In addition, in the case where GST-. Pi.siRNA or control siRNA was added to dishes containing A549 cells or MIA PaCa-2 cells at a final concentration of 50nM, the amount of P21 mRNA was measured in the same manner every day from the day of addition of GST-. Pi.siRNA or control siRNA until the 4 th day. The results are shown in FIG. 2. As shown in FIG. 2, it was found that the expression level of P21 mRNA increased with the passage of time when GST-. Pi.was knocked down by siRNA.
In another aspect, the effect of GST-pi siRNA and/or P21 siRNA on cell number was examined. First, the following solutions were added to dishes containing PANC-1, MIA PaCa-2 cells or a549 cells, respectively: GST-pi siRNA with a final concentration of 50nM, P21 siRNA, GST-pi siRNA with a final concentration of 50nM or P21 siRNA (while adding control siRNA with a final concentration of 50 nM). As a control, dishes with control siRNA added at a final concentration of 100nM were used. After 5 days of culture without medium exchange, cells were peeled from the dish by trypsin treatment and collected, and the number of cells was counted. The results are shown in FIG. 3. As shown in FIG. 3, it was found that when GST-. Pi.siRNA or P21 siRNA was used to knock down GST-. Pi.and P21, respectively, the proliferation of cells was suppressed, but the number of cells could not be made smaller than the number of inoculated cells. However, when GST-pi and P21 siRNA are knocked down simultaneously, the proliferation of PANC-1 cells and MIA PaCa-2 cells expressing mutant KRAS can be inhibited and the death of the cells can be induced.
It is to be noted that, according to fig. 3, it is considered that, with respect to a549 cells expressing mutant KRAS, cell death was not induced by the above-described treatment. Therefore, the number of transfection times of GST-pi siRNA and P21 siRNA was increased, and the effect of GST-pi siRNA and/or P21 siRNA on the cell number was examined for A549 cells and HCT116 cells expressing mutant KRAS.
First, for PANC-1 cells, MIA PaCa-2 cells, HCT116 cells, the following solutions were added to the dishes: GST-pi siRNA with final concentration of 25nM and P21 siRNA, GST-pi siRNA with final concentration of 25nM or P21 siRNA (control siRNA with final concentration of 25nM is added in either case); for a549 cells, the following solutions were added to the dishes separately: GST-pi siRNA with final concentration of 50nM, P21 siRNA, GST-pi siRNA with final concentration of 50nM or P21 siRNA (control siRNA was added with final concentration of 50 nM), respectively. As controls, control siRNA was added at a final concentration of 50nM for PANC-1 cells, MIA PaCa-2 cells, HCT116 cells, and 100nM for A549 cells. After 2 days and after 4 days, the replacement medium (PANC-1 cells were RPMI 1640 supplemented with 10% fbs, a549 cells and MIA PaCa-2 cells were DMEM supplemented with 10% fbs, HCT116 cells were McCoy supplemented with 10% fbs), GST-pi siRNA or P21 siRNA (both added with a final concentration of 25 nM) was again added to the dishes for PANC-1 cells, MIA PaCa-2 cells, HCT116 cells, respectively, and GST-pi siRNA or P21 siRNA (both added with a final concentration of 25 nM) was again added to the dishes for a549 cells, respectively, with a final concentration of 50 nM. At this time, also as a control, control siRNA was added to PANC-1, MIA PaCa-2 cells at a final concentration of 50 nM; for a549 cells, control siRNA was added at a final concentration of 100 nM. After that, the cells were cultured without changing the medium, and after 7 days from the cell inoculation, the cells were peeled off from the dish by trypsin treatment and collected, and the number of cells was counted. In this example, a phase difference image (phase difference image) of the cells was captured.
The results of cell number measurement for A549 cells, PANC-1 cells, and MIA PaCa-2 cells are shown in FIG. 4, and the results of cell number measurement for HCT116 cells are shown in FIG. 5. The phase difference image obtained by photographing a549 cells is shown in fig. 6, the phase difference image obtained by photographing MIA PaCa-2 cells is shown in fig. 7, the phase difference image obtained by photographing PANC-1 cells is shown in fig. 8, and the phase difference image obtained by photographing HCT116 cells is shown in fig. 9.
As shown in FIGS. 4 and 5, when GST-pi and P21 were knocked down simultaneously 3 times using GST-pi siRNA and P21 siRNA, it was found that cell death could be induced in cancer cells expressing mutant KRAS (A549 cells, MIA PaCa-2 cells, PANC-1 cells, HCT116 cells) by a cell number of less than the number of cells initially inoculated 7 days after cell inoculation.
As shown in FIGS. 6 to 9, it was assumed that cell aging was induced by knocking down the cell types of GST-pi-expressing mutant KRAS cells (A549 cells, MIA PaCa-2 cells, PANC-1 cells, and HCT116 cells) with GST-pi siRNA into flat and large cells. Furthermore, it was found that when GST-pi and P21 were knocked down simultaneously using GST-pi siRNA and P21 siRNA, the aging phenotype of the cells observed in the GST-pi knockdown state disappeared. Based on the results, it was considered that when GST-pi and P21 were knocked down simultaneously using GST-pi siRNA and P21 siRNA, cell aging induced by the knockdown of GST-pi was suppressed by the knockdown of P21.
It was confirmed that GST-. Pi.was able to induce cell aging as shown in FIGS. 6 to 9 by knocking down GST-. Pi.with M7609 cells. First, GST-pi siRNA was added to dishes containing M7609 cells at a final concentration of 30 nM. After 1 day and after 2 days, the medium (RPMI 1640 supplemented with 10% fbs) was replaced and GST-pi siRNA was again added to dishes containing M7609 cells at a final concentration of 30 nM. Then, the culture medium was changed every 1 day, and after 13 days from the Cell inoculation, the cells were stained according to the recommended protocol using the Cell aging β -galactosidase staining kit (Cell Signaling company), and phase difference images were taken. The results are shown in FIG. 10. As shown in FIG. 10, M7609 cells with GST-pi knockdown became flat and large cells, and blue color development by beta galactosidase was observed in cells of this phenotype, and it was found that cell aging was induced.
In addition, in cancer cells expressing mutant KRAS, it was verified whether or not cell death induced by simultaneous knocking down of GST-. Pi.and P21 was apoptosis by measuring the expression level of PUMA gene as an apoptosis inducer.
First, the following solutions were added to dishes containing a549 cells and MIA PaCa-2 cells, respectively: GST-pi siRNA with final concentration of 50nM, P21 siRNA, GST-pi siRNA with final concentration of 50nM or P21 siRNA (control siRNA was added at the final concentration of 50 nM). As a control, control siRNA was added at a final concentration of 100 nM. After 1 day of culture without medium replacement, the amount of PUMA mRNA was measured by quantitative PCR using a 7300 real-time PCR apparatus (Applied Bio Systems).
The results are shown in FIG. 11. As shown in FIG. 11, it was found that the mRNA amount of PUMA, which is an apoptosis-promoting factor, was greatly increased by using GST-pi siRNA and P21 siRNA and knocking down GST-pi and P21 simultaneously. From the results, it was found that cell death induced by simultaneous knocking down of GST-. Pi.and P21 was apoptosis.
It should be noted that apoptosis-related protein groups are present in cells. Apoptosis-related proteins are roughly divided into 2 groups, an apoptosis-inhibiting protein group and an apoptosis-inducing protein group. The apoptosis inhibitor protein group comprises Bcl-2, bcl-XL, bcl-W, MCL-1, bcl-B, etc. In addition, the apoptosis-inducing protein group includes Bax, bak, BOK, BIM, BID, BAD, NOXA, PUMA, and the like. In general, apoptosis-inhibiting proteins such as Bcl-2, bcl-XL and MCL-1 are present on the outer wall of mitochondria to inhibit the release of cytochrome C, thereby inhibiting apoptosis. On the other hand, apoptosis-inducing proteins such as Bax, BIM, BID and BAD exist in cytoplasm, migrate to the outer wall of mitochondria in response to death signals, promote release of cytochrome C, and induce apoptosis.
In addition, when p53 is activated due to DNA damage or the like, transcription of Bax, NOXA, PUMA is promoted, and apoptosis is induced. In particular, PUMA is a protein isolated as an apoptosis-inducing protein activated by p53, and by binding directly to Bcl-2, PUMA suppresses the apoptosis-inhibiting effect of Bcl-2, thereby inducing apoptosis.
As described above, the results of experiment 1 show that when a drug for inhibiting GST-pi and a drug for inhibiting P21 are caused to act on cancer cells, cell proliferation can be significantly inhibited, and cell death can be induced with high efficiency. It should be noted that, even when a GST-. Pi.inhibiting drug is allowed to act on cancer cells alone, cell proliferation is inhibited, but cell death cannot be induced, and even when a P21 inhibiting drug is allowed to act on the cells alone, cell proliferation is inhibited only to a weak extent. Thus, it can be said that it is an unexpected effect that cell death can be induced against cancer cells by allowing these drugs to act simultaneously.
[ experiment 2]
The synthetic lethality against cancer cells was confirmed in experiment 1 using GST-pi siRNA and P21 siRNA. In this experiment 2, a cell cycle regulatory protein exhibiting synthetic lethality by being inhibited simultaneously with GST-. Pi.was selected.
First, 1X 10 was prepared with DMEM supplemented with 10% FBS and 1% L-glutamine 4 Each/mL of MIA PaCa-2 cell suspension was inoculated into each well of a 96-well plate at 100. Mu.L, and then cultured in DMEM supplemented with 10% FBS and 1% L-glutamine for 18 hours. GST-pi siRNA-2 and/or siRNA against the target gene were transfected into MIA PaCa-2 cells reaching 20-30% confluence as described below using Lipofectamine RNAiMAX.
Lipofectamine/siRNA mixed solution for transfection was prepared as follows. First, 51. Mu.L of DNase-free water (Ambion) was added to each siRNA (0.1 nmol) contained in Human siGENOME siRNA Library-Cell Cycle Regulation-SMARTpool (Dharacon), and allowed to stand at room temperature for 90 minutes. To this aqueous siRNA solution, 19.9. Mu.L of OPTI-MEM was added to prepare an siRNA solution (solution A). Next, 50. Mu.M of GST-pi siRNA-2 aqueous solution and 50. Mu.M of control siRNA aqueous solution were diluted 10-fold with OPTI-MEM to prepare 5. Mu.M of GST-pi siRNA-2 diluted solution and 5. Mu.M of control siRNA diluted solution (solution B), and 31.2. Mu.L of solution A was mixed with 8.8. Mu.L of solution B (solution C). Subsequently, 150. Mu.L of Lipofectamine RNAiMAX was mixed with 2.35mL of OPTI-MEM to prepare a Lipofectamine solution (solution D). Then, 37.5. Mu.L of solution C was mixed with 37.5. Mu.L of solution D, and the mixture was allowed to stand at room temperature for 15 minutes (solution E). To each well of a 96-well plate in which MIA-PaCa-2 cells were cultured, 10. Mu.L of solution E was added, respectively.
Another 50. Mu.M control siRNA aqueous solution (5.5. Mu.L) was mixed with OPTI-MEM (189.5. Mu.L) to prepare a solution (solution F). Next, 50. Mu.M of the aqueous control siRNA solution was released 10-fold with OPTI-MEM to prepare a 5. Mu.M diluted solution of control siRNA (solution G), and 31.2. Mu.L of solution F was mixed with 8.8. Mu.L of solution G (solution H). Subsequently, 150. Mu.L of Lipofectamine RNAiMAX was mixed with 2.35mL of OPTI-MEM to prepare a Lipofectamine solution (solution I). Then, 37.5. Mu.L of solution H was mixed with 37.5. Mu.L of solution I, and the mixture was allowed to stand at room temperature for 15 minutes (solution J). To each well of a 96-well plate in which MIA-PaCa-2 cells were cultured, 10. Mu.L of solution J was added, respectively, as a control. Then, the cells were cultured in DMEM supplemented with 10% FBS and 1% L-glutamine. After 5 days, proliferation evaluation test was performed using CyQUANT NF cell proliferation assay kit (Invitrogen).
First, a 1 XHBSS buffer was added to 22. Mu.L of Cyquant NF staining reagent to prepare a staining reaction solution for cell proliferation assay of Cyquant NF. The medium of the transfected cells was aspirated, and 50. Mu.L of the staining reaction solution was added. After standing at 37℃for 30 minutes, the fluorescent light at a wavelength of 520nm was observed when excited at an excitation wavelength of 480 nm.
The results are shown in FIG. 12. As shown in FIG. 12, the 170 genes encoding cyclin were screened for synthetic lethality with GST-pi, and as a result, ATM, CDC25A, PRKDC, RBBP, SKP2, MCM10, RNPC1, CCNL1, CENPH, BRSK1, MCM8, CCNB3 and MCMDC1 were successfully screened as cyclin that exhibit synthetic lethality by being inhibited simultaneously with GST-pi, in addition to P21 confirmed by experiment 1. Among them, P21, RNPC1, CCNL1, MCM8, CCNB3 and MCMDC1 were selected as cell cycle regulatory proteins that only weakly inhibited cell proliferation (proliferation inhibition rate less than 20%) when inhibited alone, but exhibited synthetic lethality when inhibited simultaneously with GST-pi. From this, it can be said that the drugs inhibiting the cyclin selected from the group consisting of P21, RNPC1, CCNL1, MCM8, CCNB3 and MCMDC1 have very low toxicity and excellent safety when used alone.
[ experiment 3]
In experiment 2, a cell cycle regulatory protein exhibiting synthetic lethality by being inhibited simultaneously with GST-. Pi.was screened. In this experiment 3, a protein having an anti-apoptotic function, which exhibits synthetic lethality by being inhibited simultaneously with GST-. Pi.was screened.
First, 1X 10 was prepared with DMEM supplemented with 10% FBS and 1% L-glutamine 4 Each/mL of MIA PaCa-2 cell suspension was inoculated into each well of a 96-well plate at 100. Mu.L, and then cultured in DMEM supplemented with 10% FBS and 1% L-glutamine for 18 hours. GST-pi siRNA-2 and/or siRNA against the target gene were transfected into MIA PaCa-2 cells reaching 20-30% confluence as described below using Lipofectamine RNAiMAX.
Lipofectamine/siRNA mixed solution for transfection was prepared as follows. First, 51. Mu.L of DNase-free water (Ambion) was added to each siRNA (0.1 nmol) contained in a custom siRNA Library (siGENOME SMARTpool Cherry-pick Library, dharmacon) in which 140 genes considered to have an anti-apoptotic function were independently selected, and the mixture was allowed to stand at room temperature for 90 minutes. To this aqueous siRNA solution, 19.9. Mu.L of OPTI-MEM was added to prepare an siRNA solution (solution A). Next, 50. Mu.M of GST-pi siRNA-2 aqueous solution and 50. Mu.M of control siRNA aqueous solution were diluted 10-fold with OPTI-MEM to prepare 5. Mu.M of GST-pi siRNA-2 solution and 5. Mu.M of control siRNA solution (solution B), and 31.2. Mu.L of solution A was mixed with 8.8. Mu.L of solution B (solution C), respectively. Subsequently, 150. Mu.L of Lipofectamine RNAiMAX was mixed with 2.35mL of OPTI-MEM to prepare a Lipofectamine solution (solution D). Then, 37.5. Mu.L of solution C was mixed with 37.5. Mu.L of solution D, and the mixture was allowed to stand at room temperature for 15 minutes (solution E). To each well of a 96-well plate in which MIA-PaCa-2 cells were cultured, 10. Mu.L of solution E was added, respectively.
Another 50. Mu.M control siRNA aqueous solution (5.5. Mu.L) was mixed with OPTI-MEM (189.5. Mu.L) to prepare a solution (solution F). Next, 50. Mu.M of the aqueous control siRNA solution was released 10-fold with OPTI-MEM to prepare a 5. Mu.M diluted solution of control siRNA (solution G), and 31.2. Mu.L of solution F was mixed with 8.8. Mu.L of solution G (solution H). Subsequently, 150. Mu.L of Lipofectamine RNAiMAX was mixed with 2.35mL of OPTI-MEM to prepare a Lipofectamine solution (solution I). Then, 37.5. Mu.L of solution H was mixed with 37.5. Mu.L of solution I, and the mixture was allowed to stand at room temperature for 15 minutes (solution J). To each well of a 96-well plate in which MIA-PaCa-2 cells were cultured, 10. Mu.L of solution J was added, respectively, as a control. Then, the cells were cultured in DMEM supplemented with 10% FBS and 1% L-glutamine. After 5 days, proliferation evaluation test was performed using CyQUANT NF cell proliferation assay kit (Invitrogen).
First, 11mL of 1 XHBSS buffer was added to 22. Mu.L of Cyquant NF staining reagent to prepare a staining reaction solution for cell proliferation assay of Cyquant NF. The medium of the transfected cells was aspirated, and 50. Mu.L of the staining reaction solution was added. After standing at 37℃for 30 minutes, the fluorescent light at a wavelength of 520nm was observed when excited at an excitation wavelength of 480 nm.
The results are shown in FIG. 13. As shown in FIG. 13, as a result of screening for 140 genes encoding anti-apoptosis-related proteins for synthetic lethality with GST-pi, AATF, ALOX12, ANXA1, ANXA4, API5, ATF5, AVEN, AZU1, BAG1, BCL2L1, BFAR, CFLAR, IL2, MALT1, MCL1, MKL1, MPO, MTL5, MYBL2 and MYO18A were successfully screened as anti-apoptosis-related proteins exhibiting synthetic lethality by being inhibited simultaneously with GST-pi.
[ experiment 4]
In experiment 2, a cell cycle regulatory protein exhibiting synthetic lethality by being inhibited simultaneously with GST-pi was selected, and in experiment 3, a protein exhibiting synthetic lethality by being inhibited simultaneously with GST-pi was selected. In this experiment 4, PI3K signaling pathway-related proteins exhibiting synthetic lethality by being inhibited simultaneously with GST-PI were screened.
First, DMEM supplemented with 10% FBS and 1% L-glutamine was usedMaking 1X 10 4 Each/mL of MIA PaCa-2 cell suspension was inoculated into each well of a 96-well plate at 100. Mu.L, and then cultured in DMEM supplemented with 10% FBS and 1% L-glutamine for 18 hours. GST-pi siRNA-2 and/or siRNA against the target gene were transfected into MIA PaCa-2 cells reaching 20-30% confluence as described below using Lipofectamine RNAiMAX.
Lipofectamine/siRNA mixed solution for transfection was prepared as follows. First, 51. Mu.L of DNase-free water (Ambion) was added to each siRNA (0.1 nmol) contained in a custom siRNA Library (siGENOME SMARTpool Cherry-pick Library, dharmacon) in which 80 genes thought to be related to the PI3K signaling pathway were independently selected, and the mixture was allowed to stand at room temperature for 90 minutes. To this aqueous siRNA solution, 19.9. Mu.L of OPTI-MEM was added to prepare an siRNA solution (solution A). Next, 50. Mu.M of GST-pi siRNA-2 aqueous solution or 50. Mu.M of control siRNA aqueous solution was diluted 10-fold with OPTI-MEM to prepare 5. Mu.M of GST-pi siRNA-2 solution or 5. Mu.M of control siRNA solution (solution B), respectively, and 31.2. Mu.L of solution A was mixed with 8.8. Mu.L of solution B (solution C). Subsequently, 150. Mu.L of Lipofectamine RNAiMAX was mixed with 2.35mL of OPTI-MEM to prepare a Lipofectamine solution (solution D). Then, 37.5. Mu.L of solution C was mixed with 37.5. Mu.L of solution D, and the mixture was allowed to stand at room temperature for 15 minutes (solution E). To each well of a 96-well plate in which MIA-PaCa-2 cells were cultured, 10. Mu.L of solution E was added, respectively.
Another 50. Mu.M control siRNA aqueous solution (5.5. Mu.L) was mixed with OPTI-MEM (189.5. Mu.L) to prepare a solution (solution F). Next, 50. Mu.M of the aqueous control siRNA solution was released 10-fold with OPTI-MEM to prepare a 5. Mu.M diluted solution of control siRNA (solution G), and 31.2. Mu.L of solution F was mixed with 8.8. Mu.L of solution G (solution H). Subsequently, 150. Mu.L of Lipofectamine RNAiMAX was mixed with 2.35mL of OPTI-MEM to prepare a Lipofectamine solution (solution I). Then, 37.5. Mu.L of solution H was mixed with 37.5. Mu.L of solution I, and the mixture was allowed to stand at room temperature for 15 minutes (solution J). To each well of a 96-well plate in which MIA-PaCa-2 cells were cultured, 10. Mu.L of solution J was added, respectively, as a control. Then, the cells were cultured in DMEM supplemented with 10% FBS and 1% L-glutamine. After 5 days, proliferation evaluation test was performed using CyQUANT NF cell proliferation assay kit (Invitrogen).
First, 11mL of 1 XHBSS buffer was added to 22. Mu.L of Cyquant NF staining reagent to prepare a staining reaction solution for cell proliferation assay of Cyquant NF. The medium of the transfected cells was aspirated, and 50. Mu.L of the staining reaction solution was added. After standing at 37℃for 30 minutes, the fluorescent light at a wavelength of 520nm was observed when excited at an excitation wavelength of 480 nm.
The results are shown in FIG. 14. As shown in FIG. 14, as a result of screening for synthetic lethality with GST-PI for 80 genes encoding proteins associated with PI3K signaling pathway, MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP1, PIK3CA and SRF were successfully screened as PI3K signaling pathway-related proteins exhibiting synthetic lethality by being inhibited simultaneously with GST-PI. Among them, MTOR, IRAK1, IRS1, MYD88, NFKB1, PIK3CG and RAC1 were confirmed to have a very high cell proliferation inhibition effect by inhibiting these proteins simultaneously with GST-pi.
[ experiment 5]
In this experiment 5, synthetic lethality was studied for the cell cycle regulatory protein MYLK (HEGATOLOGY, vol.44, no.1,2006, 152-163) against A549 cells (KRAS mutant human lung cancer cells) when inhibited simultaneously with GST-pi.
First, 1 day before siRNA transfection, A549 cells were inoculated into each well together with 2.25mL of DMEM (without antibiotics) supplemented with 10% FBS, so that the cells became 0.25X10 5 And/or holes. In this experiment, three replicates (assays) were performed for each sample using a 6-well plate. GST-pi siRNA-3 and/or siRNA against MYLK (using 2 of MYLKa and MYLKb) were transfected into the cultured A549 cells using Lipofectamine RNAiMAX as follows.
Lipofectamine/siRNA mixed solution for transfection was prepared as follows. First, 50nM of GST-pi siRNA-3 solution and the siRNA solution against MYLK (0.5. Mu.L each) were mixed, and 124. Mu.L of OPTI-MEM was added to prepare an siRNA solution. The siRNA solutions were prepared so that the concentration of the siRNA became the same for only the control siRNA solution, the combination of the control siRNA solution and GST-. Pi.siRNA-3 solution, and the combination of the control siRNA solution and the siRNA solution against MYLK. In addition, 7.5. Mu.L of Lipofectamine RNAiMAX was mixed with 117.5. Mu.L of OPTI-MEM to prepare a Lipofectamine solution. Next, the prepared siRNA solution was mixed with Lipofectamine solution and allowed to stand at room temperature for 5 minutes.
The resulting mixed solution was dropped dropwise into wells, and each well was finally made to be 2.5mL (final concentration of siRNA: 20 nM). Then, the mixture was gently shaken at 37℃with 5% CO 2 Is cultured for 75 hours under the condition of (2). After completion of the culture, a proliferation evaluation test was performed in the same manner as in experiments 1 to 4. The results are shown in fig. 15. As shown in FIG. 15, it was found that MYLK, which is one of the cell cycle regulatory proteins, exhibited synthetic lethality against cancer cells by simultaneous inhibition of GST-pi. That is, as is clear from the results shown in FIG. 15, the effect of inhibiting cell proliferation was weak when GST-pi-inhibiting drugs and MYLK-inhibiting drugs were each individually applied to cancer cells, but cell proliferation could be inhibited very strongly by simultaneously applying GST-pi-inhibiting drugs and MYLK-inhibiting drugs to cancer cells.
The following siRNA was used as the siRNA. In the following description, capital letters denote RNA and lowercase letters denote DNA.
GST-πsiRNA-3：
Sense strand: CCUUUUGAGACCCUGCUGUtt (sequence number 109)
Antisense strand: ACAGCAGGGUCUCAAAAGGtt (sequence number 110)
MYLKa：
Sense strand: CUGGGGAAGAAGGUGAGUAtt (sequence number 111)
Antisense strand: UACUCACCUUCUUCCCCAGtt (Serial No. 112)
MYLKb：
Sense strand: CAAGAUAGCCAGAGUUUAAtt (sequence No. 113)
Antisense strand: UUAAACUCUGGCUAUCUUGtt (sequence number 114)
All publications, patents, and patent applications cited in this specification are incorporated herein by reference.

Claims (8)

1. A cancer cell death-inducing agent comprising, as active ingredients, a GST-pi-inhibiting agent and a steady state maintenance-related protein which exhibits synthetic lethality when inhibited simultaneously with GST-pi,
the cancer cells are cancer cells expressing mutant KRAS,
the steady state maintenance related protein exhibiting synthetic lethality simultaneously with the inhibition of GST-pi is at least 1 protein selected from the group consisting of IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP1 and SRF,
the GST-pi-inhibiting agent is a substance selected from the group consisting of RNAi molecules against DNA encoding GST-pi, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, and vectors expressing at least 1 of them,
the drug that inhibits a steady-state maintenance-related protein that exhibits synthetic lethality when inhibited simultaneously with GST-pi is
A substance selected from the group consisting of RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides and vectors expressing at least 1 of them against DNA encoding the steady state maintenance related protein, or
An antibody to the steady state maintenance related protein or a dominant negative mutant of the steady state maintenance related protein.
2. A cancer cell proliferation inhibitor comprising, as active ingredients, a GST-pi-inhibiting agent and a steady state maintenance-related protein which exhibits synthetic lethality when inhibited simultaneously with GST-pi,
the cancer cells are cancer cells expressing mutant KRAS,
the steady state maintenance related protein exhibiting synthetic lethality simultaneously with the inhibition of GST-pi is at least 1 protein selected from the group consisting of IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP1 and SRF,
the GST-pi-inhibiting agent is a substance selected from the group consisting of RNAi molecules against DNA encoding GST-pi, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, and vectors expressing at least 1 of them,
the drug that inhibits a steady-state maintenance-related protein that exhibits synthetic lethality when inhibited simultaneously with GST-pi is
A substance selected from the group consisting of RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides and vectors expressing at least 1 of them against DNA encoding the steady state maintenance related protein, or
An antibody to the steady state maintenance related protein or a dominant negative mutant of the steady state maintenance related protein.
3. The agent of claim 1, wherein the agent is capable of inducing apoptosis.
4. The agent of claim 1 or 2, wherein the cancer cell is a cancer cell that expresses GST-pi at a high level.
5. A pharmaceutical composition for treating a disease caused by abnormal proliferation of cancer cells, the pharmaceutical composition comprising the agent of any one of claims 1 to 4.
6. The pharmaceutical composition of claim 5, wherein the cancer cell is a cancer cell that expresses GST-pi at a high level.
7. A method for screening a cell death-inducing agent and/or a cell proliferation-inhibiting agent for cancer cells, the cell death-inducing agent and/or the cell proliferation-inhibiting agent being used simultaneously with a GST-pi-inhibiting agent, the method comprising selecting an agent that inhibits a steady state maintenance-related protein that exhibits synthetic lethality when inhibited simultaneously with GST-pi,
the cancer cells are cancer cells expressing mutant KRAS,
the steady state maintenance related protein exhibiting synthetic lethality simultaneously with the inhibition of GST-pi is at least 1 protein selected from the group consisting of IRAK1, IRS1, MYD88, NFKB1, PIK3CG, RAC1, AKT3, EIF4B, EIF4E, ILK, MTCP1 and SRF,
The drug that inhibits a steady-state maintenance-related protein that exhibits synthetic lethality when inhibited simultaneously with GST-pi is
A substance selected from the group consisting of RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides and vectors expressing at least 1 of them against DNA encoding the steady state maintenance related protein, or
An antibody to the steady state maintenance related protein or a dominant negative mutant of the steady state maintenance related protein.
8. The screening method of claim 7, comprising the steps of: a step of contacting a cancer cell with a test substance; a step of measuring the expression level of the steady-state maintenance related protein in the cell; and a step of selecting the test substance as a drug for inhibiting the steady-state maintenance-related protein when the expression level is reduced, as compared with the case of measuring in the absence of the test substance.
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