Transcriptional regulator protein degradation-promoting composition and cancer therapeutic agent containing the same

Abstract

To provide a transcriptional regulator protein degradation-promoting composition that is capable of directly facilitating the induction of transcription regulator protein degradation and also capable of enhancing the degradation-inducing effect through the combined administration of multiple components, wherein the transcriptional regulator protein degradation-promoting composition serves as a highly effective cancer therapeutic agent, as well as to provide a therapeutic agent comprising the same.SOLUTION: The present invention provides a transcriptional regulator protein degradation-promoting composition that facilitates the degradation of the transcription regulator protein, comprising a PARG inhibitor, a PERK inhibitor, an HSP90 inhibitor, or a histone acetylation enzyme inhibitor, as well as a therapeutic agent comprising the same.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition for promoting the degradation of transcriptional regulator proteins, which comprises a component that promotes the degradation of transcriptional regulator proteins such as BRD protein, and a therapeutic agent comprising the same. [Background technology]

[0002] There are proteins in cells that cause diseases, and it is expected that specific degradation and removal of the disease-causing proteins within the cells can be applied to therapeutic drugs for the disease. The principle of targeted protein degradation is thought to be the use of the ubiquitin-proteasome system that is inherently present in cells. In other words, there is a mechanism in which proteins that are no longer needed within the cell are tagged with multiple small molecular weight ubiquitin proteins. Proteins tagged by ubiquitination are degraded by the proteasome, a large protein complex that is a proteolytic enzyme. Therefore, if it is possible to promote specific ubiquitination of the target protein or specific degradation by the proteasome of the target protein, it is thought that it is possible to promote the specific degradation of the target protein. For example, by using a hybrid compound that links a substance that binds to a target protein with a drug that binds to a ubiquitination enzyme (also called a tagging enzyme or ubiquitin ligase), it is possible to bring the target protein and the ubiquitination enzyme into close proximity, forcibly causing ubiquitination and inducing degradation.

[0003] Here, BRD4 has attracted attention as a target protein for cancer therapeutic drugs. BRD4, together with BRD2, BRD3, and BRDT, is known as a BET family protein, and is known to control gene expression as a transcription factor in cancer cells and to be involved in inflammation, cancer cell proliferation, tumor formation, etc. For this reason, BRD4 has also attracted attention as a target for the treatment of cancer, inflammatory diseases, and lifestyle-related diseases. As a PROTAC (targeted degradation inducer), a hybrid compound using the ubiquitination enzyme complex CRL2 (VHL) consisting of the ubiquitination enzyme CRL2 and the E3 ubiquitin ligase substrate recognition subunit VHL is known. As a PROTAC compound targeting BRD4, a compound MZ1 has been reported that links a ligand that recognizes VHL and a ligand that recognizes BRD4.

[0004] On the other hand, in Non-Patent Document 1 by the present inventors, the inventors have identified TRIP12, a ubiquitin-adding enzyme different from CRL2 (VHL) known so far, through a search for a protein that binds to BRD4 as a target protein when cells are treated with a degradation inducer. It was found that TRIP12 binds to a target protein after CRL2 (VHL) adds ubiquitin to the target protein. In addition, in order to clarify the role of TRIP12 in the action of the degradation inducer, cancer cells that do not have TRIP12 have been created. It was found that in cancer cells that do not have TRIP12, the degradation of the target protein is delayed when treated with a degradation inducer, and further, the cell death of the cancer cells caused by this degradation is also suppressed. The molecular mechanism by which TRIP12 promotes the action of the degradation inducer was further investigated, and it was found that the shape of the ubiquitin chain to which multiple ubiquitins are bound is different between CRL2 (VHL) and TRIP12, and that a chain of a special shape is synthesized by the action of both CRL2 (VHL) and TRIP12. In other words, we found that TRIP12 is an enzyme that assists in the synthesis of ubiquitin chains in a form suitable for protein degradation. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kaiho-Soma et al., 2021, Molecular Cell 81, 1411-1424. Summary of the Invention [Problem to be solved by the invention]

[0006] In conventional technology, research has been conducted to enhance the activity of individual decomposition inducers, such as MZ1, which is involved in decomposition induction, but little is known about factors or compounds that promote decomposition induction itself. For example, if factors that promote ubiquitination of specific target proteins such as BRD4 or decomposition by the proteasome, or factors that promote decomposition induction by acting on and inhibiting inhibitors, are found, they are thought to be effective in decomposing target proteins, such as transcription factors and chromatin regulators that are deeply involved in diseases such as cancer. For example, adding these factors to factors used in known PROTACs is expected to greatly improve the therapeutic effect. They are also expected to be an option to be used in place of those known factors.

[0007] The present invention has been made in consideration of the above-mentioned circumstances, and aims to provide a transcription control factor protein degradation-promoting composition that can promote the degradation induction of transcription control factor proteins themselves, the effect of inducing degradation can be enhanced by multiple co-administration, and which is useful as a cancer treatment drug with high therapeutic effect, and a treatment drug using the same. [Means for solving the problem]

[0008] In order to solve the above problems, the present invention has the following aspects. The first aspect of the present invention is The composition for promoting the degradation of transcription factor proteins comprises a PARG inhibitor, a PERK inhibitor, an HSP90 inhibitor or a histone acetyltransferase inhibitor, and promotes the degradation of transcription factor proteins.

[0009] A second aspect of the present invention is This is the composition for promoting degradation of a transcription regulator protein according to the first aspect, wherein the transcription regulator protein is BRD, BRG1, BRD9, ESR1 or CDK9.

[0010] A third aspect of the present invention is The composition for promoting degradation of a transcription regulator protein according to the first or second aspect promotes degradation of the transcription regulator protein via ubiquitination of the transcription regulator protein.

[0011] A fourth aspect of the present invention is The protein degradation-promoting composition according to any one of the first to third aspects, which promotes protein degradation mediated by MZ1, ARV771, dBET6, ACBI1, dBRD9-A or ARV471.

[0012] A fifth aspect of the present invention is The composition for promoting degradation of a transcription regulator protein according to any one of the first to fourth aspects, wherein the PARG inhibitor is PDD00017273, the PERK inhibitor is GSK2606414, GSK2656157 or AMG-PERK, the HSP90 inhibitor is Luminespib or 17-AAG, or the histone acetyltransferase inhibitor is A485 or SGC-CBP30.

[0013] A sixth aspect of the present invention is a method for producing a composition comprising the steps of: A therapeutic agent comprising the transcription control factor protein degradation-promoting composition described in any one of claims 1 to 5.

[0014] A seventh aspect of the present invention is A sixth aspect of the therapeutic agent is a cancer therapeutic agent. Effect of the Invention

[0015] According to the present invention, a transcription regulator protein degradation-promoting composition and a therapeutic drug using the same can be obtained, which can promote the degradation induction of transcription regulator proteins themselves, and the effect of inducing degradation can be enhanced by multiple co-administration, and which is useful as a cancer therapeutic drug with high therapeutic effect. [Brief description of the drawings]

[0016] [Figure 1]FIG. 1 is a graph showing the amount of HiBiT-BRD4 per added amount of MZ1 protein in HiBiT-BRD4-HCT116 cells used in this example. [Diagram 2] FIG. 2 is a schematic diagram showing the results of compound screening in this example. [Diagram 3] FIG. 2 is a graph showing the decomposition effect of the compound of this example on HiBiT-BRD4. [Figure 4] FIG. 1 is a photograph showing the results of Western blotting of a cell extract to which the PERG inhibitor of this example was added. [Diagram 5] FIG. 2 is a graph showing the quantification of the protein amount of BRD4 in the cell extract of this Example. [Figure 6] FIG. 1 is a graph showing the results of cell viability that can be measured by luminescence intensity in this example. [Figure 7] FIG. 1 is a graph showing the results of the apoptotic response that can be measured by the activity intensity of caspase 3 / 7 in this example. [Figure 8] FIG. 1 is a photograph showing the results of Western blotting of ubiquitinated BRD4,2 in this example. [Figure 9] FIG. 1 is a photograph showing the results of Western blotting of CUL2 in this example. [Figure 10] FIG. 1 is a photograph showing the results of Western blotting using the PERK inhibitor and HSP90 inhibitor of this example. [Figure 11] FIG. 1 is a photograph showing the results of Western blotting using the PERK inhibitor and HSP90 inhibitor of this example. [Figure 12] FIG. 1 is a photograph showing the results of Western blot analysis showing the degradation of BRD4 and BRD2 by the addition of GSK2606414 and SIM1 in this example. [Figure 13] FIG. 1 is a graph showing the results of cell viability measurements in this example. [Figure 14] FIG. 1 is a photograph showing the results of Western blotting using each of the histone acetylase inhibitors and histone deacetylase inhibitors of this example. [Figure 15]FIG. 1 is a photograph showing the results of Western blotting using the histone deacetylase inhibitor Vorinostat of this example. [Figure 16] FIG. 1 is a photograph showing the results of Western blotting using the histone acetyltransferase inhibitor SGC-CBP of this example. [Figure 17] FIG. 1 is a graph showing the results of cell survival using the histone acetyltransferase inhibitor SGC-CBP of this example. [Figure 18] FIG. 1 is a photograph showing the results of Western blot analysis investigating the relationship between BRD4-MZ1-VHL using the histone deacetylase inhibitor Vorinostat of this example. [Figure 19] FIG. 1 is a photograph showing the results of Western blot analysis investigating the relationship between BRD4-MZ1-VHL using the histone acetyltransferase inhibitor SGC-CBP of this example. [Figure 20] FIG. 1 is a photograph showing the results of Western blot analysis investigating the localization of BRD4 using the histone deacetylase inhibitor Vorinostat of this example. [Figure 21] FIG. 1 is a photograph showing the results of Western blot in which the localization of BRD4 was investigated using the histone acetyltransferase inhibitor SGC-CBP of this example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The transcription control factor protein degradation-promoting composition and therapeutic agent according to the present invention will be described below with reference to embodiments, although the present invention is not limited to the following embodiments.

[0018] (Composition for promoting the degradation of transcriptional regulator proteins) The composition for promoting the degradation of a transcriptional regulator protein of this embodiment contains a PARG inhibitor, a PERK inhibitor, an HSP90 inhibitor or a histone acetylase inhibitor.

[0019] The composition for promoting the degradation of transcriptional regulator proteins of the present embodiment promotes the degradation of transcriptional regulator proteins. The transcriptional regulator proteins targeted by the composition for promoting the degradation of transcriptional regulator proteins of the present embodiment broadly refer to proteins involved in the function of controlling transcription, and can mainly target BRD, BRG1, BRD9, ESR1 (ER-α), CDK9, etc. The BRD family of proteins, BRG1, BRD9, and CDK9, are known as chromatin regulators. ESR1 is known as a transcription factor.

[0020] Among these, the target BRD protein whose degradation is promoted can be selected from BRD, BRD2, BRD3, BRD4, BRDT, etc., and preferably, BRD2 and BRD4, whose degradation is promoted by MZ1 described later, can be targeted. As the BRD protein, BRD4 is more preferable. The BRD protein may be from various animals, and in this embodiment, human BRD, particularly human BRD4 (Genebank ID: 23476), is preferred.

[0021] The composition for promoting the degradation of a transcription control factor protein of this embodiment preferably promotes the degradation of the transcription control factor protein via ubiquitination of the transcription control factor protein. Therefore, in a system using the transcriptional regulator protein degradation-promoting composition of this embodiment, it is preferable to contain other components necessary for protein degradation in the ubiquitin-proteasome system. For example, it is preferable that the system contains ubiquitin, proteasome, MZ1, or other degradation-inducing drugs described below, or other degradation-promoting factors, etc. Examples of systems containing these include the environment within a living organism, for example, within a cell. Alternatively, an in vitro environment can be used in which the ubiquitin-proteasome system is functional in the presence of other components. The transcriptional regulator protein degradation-promoting composition of the present embodiment may be used together with other components involved in protein degradation, including other substances involved in the degradation of target proteins by the ubiquitin-proteasome system and components involved in other protein degradation systems.

[0022] When the transcription control factor protein degradation-promoting composition of this embodiment targets BRD, it is preferable that it contains a PARG inhibitor, a PERK inhibitor or a histone acetyltransferase inhibitor and promotes the degradation of the transcription control factor protein via MZ1. As mentioned above, the degradation of BRD, especially BRD2 and BRD4, is promoted by the degradation inducer MZ1. PARG inhibitors, PERK inhibitors, or histone acetyltransferase inhibitors can effectively promote protein degradation by MZ1. Conventionally, PROTACs using the degradation inducer MZ1 are known.

[0023] It is also preferable that the composition for promoting the degradation of a transcription control factor protein promotes protein degradation mediated by ARV771, dBET6, ACBI1, dBRD9-A or ARV471. Factors that promote the degradation of transcriptional regulator proteins, i.e., degradation-inducing drugs, may be, for example, reactions mediated by ARV771, which targets CRL2 (VHL) like MZ1, or dBET6, which targets CRL4 (CRBN). Furthermore, when the transcription factor protein to be degraded is BRG1, ACBI1 can be suitably used as the PROTAC degrader. When the transcription factor protein to be degraded is BRD9, dBRD9-A can be suitably used as the PROTAC degrader. When the transcription factor protein to be degraded is ESR1, ARV471 can be suitably used as the PROTAC degrader. When the transcription factor protein to be degraded is CDK9, Thal-SNS can be suitably used as the PROTAC degrader.

[0024] The composition for promoting the degradation of a transcriptional regulator protein of this embodiment may contain any one of a PARG inhibitor, a PERK inhibitor, an HSP90 inhibitor, and a histone acetyltransferase inhibitor, or may contain a plurality of them. It is particularly preferred that the composition for promoting the degradation of a transcriptional regulator protein of this embodiment contains two or more of the above, and that the above components are co-administered. For example, coadministration of the PARG inhibitor PDD00012273 or the PERK inhibitor GSK2606414 with SGC-CBP30, an inhibitor of the histone acetyltransferase CBP, further promotes BRD4 degradation.

[0025] In addition, the composition for promoting the degradation of a transcription control factor protein of this embodiment is (A) one or more of a PARG inhibitor, a PERK inhibitor, or a histone acetyltransferase inhibitor, (B) an HSP90 inhibitor; It is also preferred to include: That is, while (A) is considered to promote the binding of BRD to MZ1 via MZ1 and the ubiquitination of BRD, (B) the HSP90 inhibitor is considered to promote the proteasome-dependent degradation of ubiquitinated BRD. Therefore, a composition for promoting the degradation of a transcription regulator protein that contains both (A) and (B) is expected to be more effective in promoting the degradation of a transcription regulator protein, particularly when the target is BRD, since it promotes the binding of BRD to MZ1, the ubiquitination of BRD, and the proteasome-dependent degradation of BRD.

[0026] The PARG inhibitor that may be contained in the composition for promoting degradation of transcriptional regulator proteins of the present embodiment broadly refers to a compound that inhibits PARG (poly(ADP-ribose) glycohydrolase). PARG is known to degrade poly(ADP-ribose) upon DNA damage and to be involved in DNA repair. Known examples of PARG inhibitors include PDD00017272, PDD00017273, PDD00017278, PDD00031705, COH, and JA2131. In this embodiment, it is more preferable to use PDD00017273 (CAS: 1945950-21-9) as the PARG inhibitor.

[0027] The PERK inhibitor that may be contained in the composition for promoting degradation of transcriptional regulator proteins of this embodiment broadly refers to a compound that inhibits PERK (pancreatic ER kinase or PKR-like ER kinase). PERK is one of the sensor proteins for endoplasmic reticulum stress, and it is known that activation of PERK suppresses translation and induces transcription of abnormal protein response-related genes. Known PERK inhibitors include, for example, GSK2606414, GSK2656157, AMG-PERK, and ISRIB (fusion stress response inhibitor) transisomer. In this embodiment, it is more preferable to use GSK2606414 (CAS: 1337531-36-8), GSK2656157 (CAS: 1337532-29-2) or AMG-PERK44 (CAS: 1883548-84-2) as the PERK inhibitor.

[0028] The HSP90 inhibitor that may be contained in the composition for promoting the degradation of transcriptional regulator proteins of this embodiment broadly refers to a compound that inhibits HSP90 (a heat shock protein with a molecular weight of 90 kDa). HSP90 is one of the heat shock proteins, and is known to control cellular homeostasis, function as a molecular chaperone, and be involved in protein stabilization. Known HSP90 inhibitors include CH5138303, HSP990, BIIB021, KW-2478, Ganetespib (STA-9090), Tanespimycin (17-AAG), NMS-E973, Luminespib (NVP-AUY922, VER-52296), Onalespib (AT13387), VER-58589, Pimitespib (TAS-116), XL888, SNX-2112, SNX-5422, VER-49009, PU-H71, NVP-BEP800, Alvespimycin, and Galdenamycin. In this embodiment, it is more preferable to use Luminespib (CAS: 747412-49-3) or 17-AAG (CAS: 75747-14-7) as the HSP90 inhibitor.

[0029] The histone acetyltransferase (HAT) inhibitor that may be contained in the composition for promoting the degradation of transcriptional regulator proteins of the present embodiment broadly refers to a compound that inhibits histone acetyltransferase. Histone acetyltransferase acetylates a complex of DNA and histone, and in terms of its role in the transcriptional regulation of BRD, it is known that the binding of the BD of BRD4 with the acetylated histone promotes the transcriptional elongation by RNA polymerase II. Known histone acetyltransferase inhibitors include C646, SGC-CBP30, MG149, Ginkgolic Acid, A-485, WM-8014, CPTH2, HAT inhibitor II, and NicCur. In this embodiment, it is more preferable to use A-485 (CAS: 1889279-16-6) or SGC-CBP30 (CAS: 1613695-14-9) as the histone acetyltransferase inhibitor.

[0030] Furthermore, when the transcription control factor protein degradation-promoting composition of the present embodiment targets BRG1, BRD9, or ESR1, it is preferable that the composition contains the SGC-CBP, since the degradation of these targets is effectively promoted by SGC-CBP. When CDK9 is the target, it is preferable to include any one of the four types of SGC-CBP, PDD, GSK, and Luminespib, and more preferably to include two or more types. These effectively promote the degradation of CDK9.

[0031] (Therapeutic drug) The therapeutic agent of this embodiment contains the transcription regulator protein degradation-promoting composition. The therapeutic drug can be used for any disease as long as it can exert an effect of treatment, prevention, or amelioration of pathology by degrading a transcription factor protein. Specifically, the transcription factor protein targeted in this embodiment, particularly BRD4, is considered to be involved in the control of gene expression, inflammation, cancer cell proliferation, tumor formation, etc., and therefore can be used as a therapeutic drug for diseases related to these factors, such as cancer, inflammatory diseases, and lifestyle-related diseases.

[0032] The therapeutic agent is preferably a cancer therapeutic agent, as cancer treatments are generally in high demand.

[0033] (Effects of this embodiment) According to this embodiment, the degradation induction of transcription control factor proteins themselves can be promoted, the effect of degradation induction can be enhanced by multiple co-administration, and a transcription control factor protein degradation-promoting composition and a therapeutic drug using the same can be obtained, which are useful as cancer therapeutic drugs with high therapeutic effects.

[0034] The inventors screened for factors involved in the degradation of BRD proteins, particularly BRD4 protein, in the ubiquitin-proteasome system among cancer-related transcriptional regulator proteins, and discovered inhibitors with already known properties, such as PARG inhibitors, PERK inhibitors, HSP90 inhibitors, and histone acetyltransferase inhibitors. From these results, we have found a mechanism by which inhibitors with known properties can promote target degradation as a new use of inhibitors. We have also found a composition that promotes the degradation of BRD proteins such as BRD4, i.e., transcription factor proteins, containing these inhibitors. This transcription factor protein degradation promoting composition can be used in combination with a composition having a conventionally known transcription factor protein degradation mechanism, in addition to directly degrading the target protein. Therefore, when a conventionally known application method of protein degradation, such as a treatment method using PROTAC to degrade a target protein, is put into practical use, it can be used as a promoter by administering it together.

[0035] (Another aspect of this embodiment) This embodiment also includes the following aspects. Another aspect of this embodiment is a method for producing a therapeutic agent using the transcriptional regulator protein degradation-promoting composition. Another aspect of this embodiment is the use of the transcription regulator protein degradation-promoting composition for the manufacture of a therapeutic agent. Another aspect of this embodiment is the use of the transcription regulator protein degradation-promoting composition for the treatment of cancer. Another aspect of this embodiment is the use of the transcription regulator protein degradation-promoting composition for use in cancer treatment. Another aspect of this embodiment is a method for treating cancer using the composition for promoting the degradation of a transcriptional regulator protein. EXAMPLES

[0036] Examples will be given below, but the present invention is not limited to the Examples.

[0037] (Test Example) (Cell lines for screening) To screen for factors involved in the degradation of the transcription factor protein BRD4, we established a cell line to be used. The cells used were HiBiT-BRD4-HCT116 cells, in which a HiBiT tag (11 amino acids) was inserted into the BRD4 locus of HCT116 cells by genome editing. When these cells were lysed and LgBiT was added, the amount of BRD4 fusion protein could be detected by luminescence. FIG. 1 is a graph showing the amount of HiBiT-BRD4 per amount of added MZ1 protein in HiBiT-BRD4-HCT116 cells. It can be confirmed that MZ1, which is known to be involved in the degradation of BRD4, reduces the luminescence intensity of NaNoLuc, i.e., the amount of HiBiT-BRD4 protein. In other words, degradation of the BRD4 fusion protein was detected in an MZ1 concentration-dependent manner.

[0038] (Compound Screening) A part of the known compound library of Selleck was used as the target compound group for screening. Each compound (10 μM) was added to a 96-well plate for 4 hours, and then 30 nM MZ1 was added for 2 hours, and the luminescence was measured using a luminescence plate reader (n=3). The results of the screening are shown in Figure 2. (a) is a schematic diagram showing the distribution of compounds to be screened, with the degree of degradation enhancement (Enhancers) / repressors expressed in Log2, with the absence of enhancing compounds (DMSO added) used as the standard, in the absence of MZ1. Compounds that showed little change in BRD4 amount in the absence of MZ1 and changed BRD4 degradation in the presence of MZ1 (compounds in the vicinity of Enhancers in the figure) were selected. (b) is a plot diagram showing the results in the absence (-) and presence (+) of MZ. A known proteasome inhibitor was identified in the upper right of (b) (degradation suppression compound), confirming the construction of this experimental system.

[0039] Three compounds that were particularly successful in this screening were the PERG inhibitor PDD17273, the PERK inhibitor GSK2606414, and the HSP90 inhibitor Luminespib. These three types of compounds were added to the HiBiT-BRD4-HCT116 cells described above at three different concentrations, 1 μM, 3 μM, and 10 μM, and the decrease in luminescence, i.e., the amount of HiBiT-BRD4, was measured. FIG. 3 is a graph showing the decomposition effects of these three compounds on HiBiT-BRD4. In the presence of MZ1 (MZ+), all three inhibitors reduced HiBiT-BRD4 in a concentration-dependent manner, indicating that these three compounds promote the degradation of BRD4.

[0040] Furthermore, based on these results, the inventors predicted that in addition to these three types of inhibitors, PERG inhibitors, PERK inhibitors and HSP90 inhibitors also include compounds that promote the degradation of BRD, and reflected this in subsequent tests.

[0041] (Activity study of PERG inhibitor PDD17273) The PERG inhibitor PDD17273 (PDD) was added to HeLa cells at 3 μM PDD and 100 nM MZ1 for the times shown in the figure, and total cell extracts were prepared and subjected to Western blotting. Note that the conditions for Western blotting, including the following, were appropriately adjusted to allow for appropriate signal comparison, such as antibody concentration. Figure 4 is a photograph showing the results of Western blotting of cell extracts to which a PERG inhibitor had been added. The left axis shows the name of the antibody for each protein. The numbers on the MZ1 axis indicate the time after addition of MZ1, while - and + on PDD indicate the presence or absence of the addition of the PERG inhibitor PDD17273. Figure 5 shows the quantification of the protein amount of BRD4 in the cell extract. The protein amount was determined by quantifying the band intensity in the Western blot in Figure 4. The amount before the addition of MZ1 was set as 1, and the band intensity was measured as a multiple of the increase. As shown in the figure, the amount of BRD4 protein was reduced by MZ1 even without the addition of PDD. However, the amount of BRD4 protein was further reduced by the addition of PDD, and a significant difference was observed 4 hours after the addition of MZ1 (inside the box in the figure). Addition of PDD promoted the degradation of BRD4, BRD2, and BRD3, but did not affect the amount of ubiquitin ligases involved in the pathway. Here, it is known that BRD2 and BRD3 are only weakly degraded by MZ1, while BRD4 is a target protein of MZ1. The above results demonstrated that the PARG inhibitor PDD17273 promotes the degradation of BRD4, BRD2, and BRD3.

[0042] Next, the promotion of cell death induction by the PDD was examined. HeLa cells were added with 3 μM PDD, and then MZ1 was added 6 hours later. Cell viability was measured 3 days later using the CellTiter-Glo(R) 2.0 Cell Viability Assay (Promega). A graph of the cell viability measured by luminescence intensity is shown in Figure 6. It was revealed that the addition of PDD promoted cell death induced by MZ1.

[0043] Next, the promotion of apoptosis by the PDD was examined. HeLa cells were treated with 3 μM PDD, and 6 hours later, MZ1 was added at the indicated concentrations. 24 hours later, the apoptotic response was measured using the ApoLive-Glo™ Multiplex Assay (Promega). A graph showing the results of the apoptosis response that can be measured by the activity intensity of caspase 3 / 7 is shown in Figure 7. It was revealed that the addition of PDD promoted apoptosis induced by MZ1.

[0044] Next, the effect of the PDD on ubiquitin chain formation was examined. HeLa cells were treated with 3μM PDD, MZ1 was added 5 hours later, and harvested 1 hour later. Ubiquitin chains were harvested using TUBE2-agarose and subjected to Western blotting with antibodies against BRD2 and BRD4. Figure 8 shows a photograph of the results of Western blotting of ubiquitinated BRD4,2. In the TUBE lane (sample from which ubiquitin chains were recovered), the bands with increased molecular weights reacting with antibodies to BRD4 and BRD2 became stronger with the addition of PDD, indicating that ubiquitin chains were bound to BRD4 and BRD2. A decrease in unbound bands was observed. In other words, it was revealed that the addition of PDD promotes ubiquitination by MZ1.

[0045] We next investigated the effect of PDD on the interaction between BRD4, BRD2 and CUL2 proteins. After binding to MZ1, BRD4,2 binds to VHL protein via MZ1. It is known that CUL2 protein binds to VHL protein, promoting ubiquitination and leading to degradation by the proteasome. 293T cells were expressing FLAG-tagged BRD4 (FLAG-BRD4) and HA-tagged VHL (HA-VHL), and cells were harvested 45 min after addition of PDD and MZ1, and immunoprecipitated with anti-FLAG antibody. Interacting CUL2 was detected by Western blotting. Figure 9 shows a photograph of the results of Western blotting of CUL2. Since it was precipitated with anti-FLAG antibody, FLAG-BRD4 and the proteins bound to it are shown. For example, the lane reacting to all antibodies for BRD4, CUL2, and VHL is a lane in which proteins bound to these three species were precipitated. Bands of the complex of BRD4, CUL2, and VHL were seen even in cells to which only MZ1 was added, but the bands of CUL2 and VHL were further enhanced when PDD was added. These results suggest that PDD promotes not only the formation of a complex between MZ1 and BRD4,2, but also the formation of a complex between BRD4,2 and VHL and CUL2, thereby promoting ubiquitination.

[0046] (Study of the activity of PERK inhibitors and HSP90 inhibitors) We investigated the activity of GSK2606414, another PERK inhibitor, and the HSP90 inhibitor Luminespib using HCT116 cells. In HCT116 cells, 10 μM of the compounds PERK inhibitor GSK2606414 (GSK), PERK inhibitor GSK2656157, PERK inhibitor AMG-PERK, HSP90 inhibitor Luminespib (Lumi), and HSP90 inhibitor 17-AAG were added, MZ1 was added after 4 hours, and the cells were harvested 1 to 2 hours later, and Western blotting was performed using anti-BRD4 and anti-actin antibodies. The results of Western blotting using PERK inhibitors and HSP90 inhibitors are shown in Figure 10. Note that BRD4 (JESS) on the left axis was obtained using a quantitative Western blot device (Protein Simple), and Ponceau is protein staining using Ponceau. The results in the figure show that the addition of other PERK inhibitors, such as GSK2606414 and Luminespib, and HSP90 inhibitors, also promoted BRD4 degradation more strongly than the addition of MZ1 alone. This suggests that many PERK and HSP90 inhibitors may promote BRD4 degradation.

[0047] We also investigated the ubiquitination activity of PERK inhibitors and HSP90 inhibitors. Using HCT116 cells treated with the drugs, we recovered ubiquitin chains using TUBE. Figure 11 shows the results of Western blotting using a PERK inhibitor and an HSP90 inhibitor. Ubiquitin chains were detected using the B4D1 antibody. It was revealed that the addition of each inhibitor promoted ubiquitination.

[0048] (Examination of the activity of PERK inhibitor GSK2606414 and SIM1) We further examined the BRD degradation-promoting activity of the PERK inhibitor GSK2606414. We investigated the degradation of BRD2 and 4 by GSK (GSK2606414) and SIM1, which is known to bind to BRD and VHL (Nature Chemical Biology, 17 (2021), pp1157-1167). 12 is a photograph showing the results of Western blot analysis demonstrating the degradation of BRD4 and BRD2 by the addition of GSK2606414 and SIM1. As shown in the figure, BRD4 and BRD2 are degraded depending on the addition of GSK and the concentration of SIM1. Next, GSK2606414 and SIM1 were added to the HCT116 cells, and cell survival was measured using CellTiter Glo after 3 days. 13 is a graph showing the results of measuring cell viability. A decrease in cell viability was observed depending on the SIM1 concentration.

[0049] (Effect of histone acetyltransferase inhibitors and histone deacetylase inhibitors on BRD degradation) It is known that the binding of BRD4 to acetylated histones promotes transcription. Therefore, the inventors predicted that histone acetylation and histone deacetylation enzyme inhibitors would be involved in BRD4 and related to the effects of PROTACs. Therefore, they investigated the involvement of histone acetylation enzyme inhibitors and histone deacetylase inhibitors in BRD4 degradation. The histone acetyltransferase inhibitors used were A485 and SGC-CBP, and the histone deacetylase inhibitor used was vorinostat.

[0050] Figure 14 is a photograph showing the results of Western blotting using each of the inhibitors. Each inhibitor was added to the HCT116 cells at the concentration and time shown in the figure, and a total cell extract was prepared. Western blotting was then performed in the same manner as in the test example. The results in the figure show that A485 and SGC-CBP (histone acetyltransferase inhibitors) promote BRD4 degradation, and that Vorinostat (histone deacetylase inhibitor, HDAC) inhibits BRD4 degradation.

[0051] Next, we investigated the relationship between histone deacetylase inhibitors and ubiquitin chain formation. FIG. 15 is a photograph showing the results of Western blotting using the histone deacetylase inhibitor Vorinostat. Vorinostat was added to the HCT116 cells for 6 hours, and MZ1 and MG132 were added for 1 hour to prepare cell extracts. Western blotting was performed on samples in which ubiquitin chains were pulled down and on samples before pull-down (input) using the same TUBE-agarose complex that binds ubiquitin chains as described above. As shown in the figure, TUBE used is K29TUBE, K48TUBE, and K63TUBE, which react with ubiquitin chains bound via lysines 29, 48, and 63, respectively. As shown in the figure, the histone deacetylase inhibitor Vorinostat was found to reduce the ubiquitin chains mediated by MZ1 at K29, K48, and K63 of BRD4, suggesting that histone deacetylase inhibitors inhibit the degradation of BRD4 via the ubiquitin-proteasome system.

[0052] Next, we investigated the relationship between histone acetyltransferase inhibitors and ubiquitin chain formation. Figure 16 is a photograph showing the results of Western blotting using the histone acetyltransferase inhibitor SGC-CBP. Vorinostat was added to the HCT116 cells for 6 hours, and MZ1 and MG132 were added for 1 hour to prepare cell extracts. Western blotting was performed on the samples in which ubiquitin chains were pulled down and the samples before pull-down (input) using TUBE-agarose complex (TUBE is the same as above). As shown in the figure, the histone acetyltransferase inhibitor SGC-CBP increased the ubiquitin chains of BRD4 via K29, K48, and K63 by MZ1, suggesting that histone deacetylase inhibitors promote the degradation of BRD4 via the ubiquitin-proteasome system.

[0053] 17 is a graph showing the results of cell survival using the histone acetyltransferase inhibitor SGC-CBP. In HCT116 cells, cell survival was measured using CellTiter Glo after 3 days with (+) or without (-) the addition of MZ1 and SGC-CBP at the concentrations shown on the horizontal axis, and the vertical axis is plotted as luminescence intensity. As shown in the figure, it was revealed that CGC-CBP promotes MZ1-induced cell death.

[0054] We next investigated the relationship between the histone deacetylase inhibitor vorinostat and the formation of the BRD4-VHL complex. Figure 18 is a photograph showing the results of Western blot to investigate the relationship between BRD4-MZ1-VHL using the histone deacetylase inhibitor Vorinostat. FLAG-BRD4 and HA-VHL were expressed in 293T cells, and the drugs shown in the figure were added. IP:FLAG is a sample immunoprecipitated with FLAG antibody. Input indicates the sample before immunoprecipitation. As shown in the figure, the addition of Vorinostat reduced the signal of the complex compared to when Vorinostat was not added. It was revealed that Vorinostat reduces the formation of the BRD4-VHL complex by MZ1. In other words, it is thought that the histone deacetylase inhibitor Vorinostat inhibits ubiquitination due to the formation of the BRD4-VHL complex by MZ1.

[0055] Next, we investigated the relationship between the histone acetyltransferase inhibitor SGC-CBP and the formation of the BRD4-VHL complex. Figure 19 is a photograph showing the results of Western blot to investigate the relationship between BRD4-MZ1-VHL using the histone acetyltransferase inhibitor SGC-CBP. As in the previous figure, FLAG-BRD4 and HA-VHL were expressed in 293T cells, and the drugs shown in the figure were added. IP:FLAG is a sample immunoprecipitated with FLAG antibody. Input indicates the sample before immunoprecipitation. As shown in the figure, the addition of SGC-CBP increased the signal of the complex compared to when SGC-CBP was not added. The increase in the expression level of CUL2 in the TUBE sample was remarkable. SGC-CBP was found to increase the formation of the BRD4-VHL complex by MZ1, suggesting that the histone acetyltransferase inhibitor SGC-CBP promotes ubiquitination via the formation of the BRD4-VHL complex by MZ1.

[0056] Histone deacetylase inhibitors and histone acetylase inhibitors are thought to be involved in the binding of BRD4 to chromatin, so we investigated the effects of these inhibitors on the binding and dissociation of BRD4 from chromatin (translocation to the nucleus). First, we investigated the localization of BRD4 in response to the histone deacetylase inhibitor vorinostat. Figure 20 is a photograph showing the results of Western blotting to investigate the localization of BRD4 using the histone deacetylase inhibitor Vorinostat. The above-mentioned HCT116 cells were fractionated using a cell fractionation kit (Subcellular Protein Fractionation Kit for Cultured Cells, Thermo), and Western blotting was performed for the nucleus and chromatin. Detection was also performed using an antibody against the transcription factor ETS, which is involved in nucleic acid repair. As shown in the figure, the addition of Vorinostat decreased the BRD4 signal in the nucleus, whereas the addition of Vorinostat increased the BRD4 signal in the chromatin. Vorinostat shifted the localization of BRD4 to chromatin, and BRD binding to chromatin inhibited degradation.

[0057] Figure 21 is a photograph showing the results of Western blot to investigate the localization of BRD4 using the histone acetyltransferase inhibitor SGC-CBP. Cell fractionation and Western blot were performed as in the previous figure. Detection with an antibody against the transcription factor p53 was also performed. As shown in the figure, the BRD4 signal increased in the nucleus upon addition of SGC-CBP, whereas the BRD4 signal decreased in the chromatin. This indicates that SGC-CBP localizes BRD4 to the nucleus and inhibits its degradation by dissociating BRD from chromatin.

[0058] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included in the scope of the invention and its equivalents described in the claims, as well as in the scope and spirit of the invention. [Industrial Applicability]

[0059] According to the present invention, a transcription regulator protein degradation-promoting composition and a therapeutic drug using the same can be obtained, which can promote the degradation induction of transcription regulator proteins themselves, and the effect of inducing degradation can be enhanced by multiple co-administration, and which is useful as a cancer therapeutic drug with high therapeutic effect.

Claims

1. A composition for promoting the degradation of a transcription control factor protein, comprising a PARG inhibitor, a PERK inhibitor, an HSP90 inhibitor or a histone acetylase inhibitor, and promoting the degradation of a transcription control factor protein.

2. The composition for promoting the degradation of a transcription regulator protein according to claim 1 , wherein the transcription regulator protein is BRD, BRG1, BRD9, ESR1 or CDK9.

3. A transcription control factor protein degradation-promoting composition according to claim 1 or 2, which promotes the degradation of the transcription control factor protein via ubiquitination of the transcription control factor protein.

4. The composition for promoting the degradation of a transcription control factor protein according to claim 1 or 2, which promotes protein degradation mediated by MZ1, ARV771, dBET6, ACBI1, dBRD9-A or ARV471.

5. The composition for promoting degradation of a transcription control factor protein according to claim 1 or 2, wherein the PARG inhibitor is PDD00017273, the PERK inhibitor is GSK2606414, GSK2656157 or AMG-PERK, the HSP90 inhibitor is Luminespib or 17-AAG, or the histone acetyltransferase inhibitor is A485 or SGC-CBP30.

6. A therapeutic agent comprising the composition for promoting the degradation of a transcription control factor protein according to claim 1 or 2.

7. The therapeutic agent according to claim 6, which is a cancer therapeutic agent.

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