Application of medicine in inhibiting activity of YAP-TEAD transcription complex

Abstract

The invention discloses an application of a medicine cobimetinib (cobimetinib) in inhibiting the activity of a YAP-TEAD transcription complex, and relates to the field of medicine application. The invention relates to an application of cobimetinib (cobimetinib) and sorafenib (sorafenib) in the prevention of the drug tolerance of the liver cancer to the sorafenib (sorafenib) through the combined use of the cobimetinib and the sorafenib (sorafenib). According to the cobimetinib disclosed by the invention, the cobimetinib can be used for inhibiting the occurrence and the development of YAP highly activated tumors by targeting a YAP-TEAD transcription complex, so that the adaptation disease range of the cobimetinib is expanded; according to the present invention, the activity of the YAP is inhibited through the cobimetinib in the targeting manner, such that the drug tolerance of the liver cancer on the sorafenib can be prevented;

Description

Technical Field

[0001] The present invention relates to the field of drug application, and in particular to the application of a drug, cobimetinib, in inhibiting the activity of a YAP-TEAD transcription complex. Background Art

[0002] The Hippo signaling pathway has been listed as one of the signaling pathways with high frequency mutations in eight cancers in the results of the Cancer Genome Atlas analysis. For example, NF2 inactivation can lead to the occurrence of multiple neurofibromas in the nervous system, which mainly include schwannomas, meningiomas and ependymomas; in addition, NF2 deletion mutations can be seen in 19%-50% of malignant mesothelioma, 18%-22% of renal epithelial cell carcinoma and cervical squamous cell carcinoma; in addition to NF2, LATS1 / 2 mutations can be seen in 11% of malignant mesothelioma; LATS2 mutations can be seen in cervical squamous cell carcinoma; SAV1 frequent mutations can be seen in renal epithelial cell carcinoma and cervical squamous cell carcinoma; PTPN14 mutations can be seen in about 31% of a rare renal epithelial cell carcinoma-mucinous tubular and spindle cell carcinoma, and in about 23% of a common malignant skin tumor-basal cell carcinoma. In addition, mutations in regulatory factors upstream of the members of this pathway can also lead to abnormalities in the Hippo signaling pathway. For example, activating mutations in the Gαq / 11 encoding gene GNAQ / GNA11, a member of the G protein-coupled receptor family, can lead to the occurrence of uveal melanoma; APC (Adenomatous Polyposis Coli), an important member of the β-catenin degradation complex in the Wnt signaling pathway, can participate in the activation of the Hippo signaling pathway by interacting with SAV1 and LATS1, and mutations in its encoding gene APC often lead to the occurrence of various human colorectal cancers; LKB1 kinase promotes the correct assembly of the polarity complex upstream of the Hippo signaling pathway through the MARKs family kinases, and LKB1 inactivation mutations lead to the occurrence of lung cancer and pancreatic cancer. As the main effector of the negative regulation of the Hippo signaling pathway, the inactivation of the Hippo signaling pathway will directly activate the YAP-TEAD transcription complex and lead to cancer. Therefore, targeted inhibition of the assembly of the YAP-TEAD transcription complex is of great practical significance for cancer treatment. Although there have been many reports on YAP-TEAD targeted inhibitors, most of them are still in the clinical trial stage, and there are currently no drugs on the market that specifically target and inhibit YAP highly activated tumors.

[0003] Therefore, technicians in this field are committed to developing a drug that specifically targets and inhibits YAP highly activated tumors. Summary of the invention

[0004] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is a drug that specifically targets and inhibits YAP highly activated tumors.

[0005] To achieve the above objectives, the present invention provides an application of a drug, cobimetinib, in inhibiting the activity of the YAP-TEAD transcription complex.

[0006] Furthermore, it also includes a method for evaluating the anti-tumor effect of cobimetinib, including nude mouse tumor formation and drug administration experiments.

[0007] Further, the following steps are included:

[0008] Step 1, select the solvent DMSO and another MEK1 / 2 inhibitor PD0325901 as negative controls;

[0009] Step 2, obtaining nude mouse tumors of NCI-H226 cells containing NF2 protein inactivation mutations, i.e., NCI-H226 transplanted tumors;

[0010] Step 3, respectively, the solvents DMSO, PD0325901 and cobimetini prepared in step 1 were used to perform a drug administration experiment on nude mice bearing the NCI-H226 transplanted tumor obtained in step 2;

[0011] Step 4: Evaluate the ability to inhibit the growth of NCI-H226 nude mouse xenograft tumors under different dosing conditions.

[0012] Furthermore, the method further comprises the following steps:

[0013] Step 1, select the solvent DMSO and another MEK1 / 2 inhibitor PD0325901 as negative controls;

[0014] Step 2, obtaining nude mouse tumors containing HeLa cells with LATS1 / 2 kinase knockout, i.e., HeLa-LATS1 / 2K0 transplanted tumors;

[0015] Step 3, respectively, the solvents DMSO, PD0325901 and cobimetini prepared in step 1 were used to perform a drug administration experiment on nude mice bearing the HeLa-LATS1 / 2K0 transplanted tumor obtained in step 2;

[0016] Step 4: Evaluate the ability to inhibit the growth of HeLa-LATS1 / 2K0 nude mouse transplanted tumors under different administration conditions.

[0017] The present invention further provides an application of cobimetinib and sorafenib in combination to prevent liver cancer from developing drug resistance to sorafenib.

[0018] Furthermore, a method for evaluating the effect of combined medication is also provided, comprising the following steps:

[0019] Step (1) inducing liver cancer in nude mice by overexpressing Myc and knocking out p53 (Myc; sg-p53) to obtain a Myc; sg-p53 liver cancer model;

[0020] Step (2) conducting a drug administration experiment on the Myc;sg-p53 liver cancer model obtained in step (1) using cobimetinib alone, sorafenib alone, and cobimetinib and sorafenib in combination;

[0021] Step (3) evaluates the tumor size and cell proliferation ability within the lesion of the Myc;sg-p53 liver cancer model under different drug administration conditions.

[0022] Furthermore, cobimetinib is currently believed to function mainly through targeted inhibition of MEK1 / 2, and cobimetinib can target and disrupt YAP-TEAD transcription complex assembly.

[0023] Furthermore, cobimetinib is currently used to treat melanoma and breast cancer containing KRAS mutations mainly by targeting and inhibiting MEK1 / 2, an important kinase downstream of KRAS; cobimetinib can inhibit the occurrence and development of YAP-highly activated tumors.

[0024] Furthermore, drug resistance to sorafenib, a first-line chemotherapy drug for liver cancer, often develops during liver cancer treatment. The combination of cobimetinib and sorafenib can greatly improve drug resistance to sorafenib during liver cancer treatment.

[0025] In the preferred embodiment 1 of the present invention, the verification test process of the drug screening results is described in detail;

[0026] In another preferred embodiment 2 of the present invention, the process of evaluating the anti-tumor effect of cobimetinib by a nude mouse tumor formation experiment is described in detail;

[0027] In another preferred embodiment 3 of the present invention, the anti-tumor effect of cobimetinib is evaluated by a liver cancer model in detail;

[0028] In another preferred embodiment 4 of the present invention, the experimental process of the combined use of cobimetinib and sorafenib is described in detail.

[0029] The beneficial technical effects of the present invention are as follows:

[0030] The MEK1 / 2 inhibitor cobimetinib screened and identified by the present invention is a drug that has been approved by the FDA for marketing. It can be used as a safe and reliable drug for targeted treatment of patients with YAP-highly activated tumors, filling the gap of no drugs available for YAP-highly activated tumors.

[0031] Cobimetinib inhibits the palmitoylation modification of TEAD protein by binding to the palmitoylation modification pocket of TEAD, thereby inhibiting the YAP-TEAD interaction. Therefore, the present invention identifies a new target of cobimetinib.

[0032] Cobimetinib can inhibit the occurrence and development of YAP-highly activated tumors by targeting the YAP-TEAD transcription complex, expanding the scope of indications of cobimetinib.

[0033] Drug resistance to sorafenib during liver cancer treatment is often related to the expression of drug resistance-related genes mediated by YAP activation. Targeted inhibition of YAP activity by cobimetinib can prevent liver cancer from developing drug resistance to sorafenib, thereby improving the efficacy of sorafenib in liver cancer treatment.

[0034] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a diagram of drug screening result verification experiment results of a preferred embodiment 1 of the present invention;

[0036] Figure 2 This is a diagram showing the results of a verification experiment that cobimetinib according to a preferred embodiment 1 of the present invention inhibits YAP-TEAD interaction by directly binding to TEAD protein;

[0037] Figure 3 This is a diagram showing the experimental results of evaluating the anti-tumor effect of cobimetinib through a nude mouse tumor formation experiment according to a preferred embodiment 2 of the present invention;

[0038] Figure 4 This is a graph showing the experimental results of evaluating the anti-tumor effect of cobimetinib in a liver cancer occurrence model according to a preferred embodiment 3 of the present invention;

[0039] Figure 5 This is a diagram showing the experimental results of the combined use of cobimetinib and sorafenib according to a preferred embodiment 4 of the present invention. DETAILED DESCRIPTION

[0040] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0041] Example 1 Verification test of screening results

[0042] Through the YAP-TEAD targeted inhibitor screening system based on the phase change principle established in the early stage, the present invention conducted high-throughput screening of 6365 compounds including synthetic chemicals, natural products and microbial secondary metabolites. By analyzing the screening results, it was found that a known MEK1 / 2 inhibitor cobimetinib can effectively destroy the YAP-TEAD interaction in the phase change screening system.

[0043] In order to further verify the screening results, the verification results are as follows Figure 1As shown, Part A is an in vitro GST-pulldown experiment, and the results show that cobimetinib can destroy the YAP-TEAD interaction in a concentration gradient-dependent manner; Part B is a MicroScale Thermophoresis (MST) experiment to determine the changes in YAP-TEAD affinity before and after cobimetinib treatment; Part C is a cell-level determination of the difference in the strength of endogenous YAP-TEAD4 interaction before and after cobimetinib treatment; Part D is a cell-level determination of the difference in the strength of exogenous YAP-TEAD4 interaction before and after cobimetinib treatment; Part E shows that cobimetinib can inhibit the expression of YAP-TEAD downstream genes CYR61 and CTGF in a concentration-dependent manner; Part F shows that cobimetinib can inhibit the activity of the YAP-TEAD luciferase reporter gene in a concentration-dependent manner. This indicates that cobimetinib can disrupt the YAP-TEAD interaction and thus inhibit the transcriptional activity of the YAP-TEAD complex. Figure 1 Part A shows that cobimetinib can inhibit YAP-TEAD binding in a concentration gradient-dependent manner through GST-pulldown experiments; Part B shows that the results of MicroScale Thermophoresis (MST) experiments show that cobimetinib treatment can reduce the affinity between YAP-TEAD proteins to about 1 / 3 of the original; Parts C and D show that the results of protein immunoprecipitation experiments at the cellular level also show that cobimetinib can inhibit YAP-TEAD interaction. The transcriptional activity of the YAP-TEAD transcription complex is an important indicator of the YAP-TEAD interaction. The transcriptional complex YAP-TEAD binding can promote the expression of many genes that are important for cell proliferation, migration and tissue growth, among which CYR61 and CTGF are the most classic. Figure 1 Part E shows the results of RT-qPCR experiments, showing that cobimetinib can inhibit the expression of CYR61 and CTGF in a concentration-dependent manner; Part F shows that the luciferase reporter gene system also shows that cobimetinib can inhibit YAP-TEAD activity in a concentration-dependent manner.

[0044] YAP protein has poor natural folding properties. Except for a small amount of stable secondary structures, most of it is irregular and unstable curling, so it does not have drug-making ability. On the contrary, in addition to two relatively shallow drug-making "pockets" at Interface 2 and Interface 3 where TEAD protein interacts with YAP, there is also a relatively deep drug-making "pocket" in the center of the TEAD protein for TEAD to undergo palmitoylation modification. Therefore, we speculate that cobimetinib is likely to inhibit the YAP-TEAD interaction by directly binding to the TEAD protein. Further verification was carried out, and the verification results are as follows: Figure 2 As shown, Part A is the Biotin-pulldown experiment; Part B is the MicroScale Thermophoresis (MST) experiment, which determined that the affinity between cobimetinib and TEAD protein is approximately 1.67μM; Part C is the result of the cobimetinib-TEAD2 cocrystal complex, indicating that cobimetinib can bind to the central pocket of the TEAD protein; Parts D and E are comparisons of the spatial occupancy of cobimetinib and palmitate in the central pocket of the TEAD protein; Part F is a flowchart of the in vitro palmitoylation experiment of TEAD protein. The results of the in vitro palmitoylation experiment show that 3B8 can inhibit the palmitoylation modification of TEAD4 in a concentration-dependent manner. This indicates that cobimetinib can directly bind to the central pocket of the TEAD protein and inhibit the palmitoylation modification of the TEAD protein. Among them, Figure 2 Part A shows the results of the Biotin-pulldown experiment, indicating that cobimetinib can bind to the TEAD protein; Part B shows the results of the microthermophoresis (MST) experiment, indicating that the affinity between cobimetinib and TEAD is about 1.67μM. Part C shows that through co-crystallization and structural analysis of the cobimetinib-TEAD complex, it was found that cobimetinib can bind to the central pocket of the TEAD protein; Parts D and E show that by comparing with the known palmitate-TEAD complex structure, cobimetinib and palmitate have similar spatial occupancy in the central pocket of the TEAD protein. This suggests that there may be a competitive relationship between the two. Further results of in vitro palmitoylation experiments are shown in Figure 2. Figure 2As shown in parts F and G of , 3B8 can inhibit the palmitoylation modification of TEAD4 in a concentration-dependent manner. Studies have shown that palmitoylation modification occurring in the central pocket of TEAD is essential for the YAP-TEAD interaction, so we speculate that cobimetinib disrupts the YAP-TEAD interaction by inhibiting the palmitoylation modification of TEAD protein.

[0045] Example 2 Nude mouse experiment to evaluate the anti-tumor effect of cobimetinib

[0046] Previous studies have shown that the YAP-TEAD transcription complex plays an important role in tumor cell proliferation and tumor growth. In order to clarify from a functional perspective that cobimetinib can disrupt the YAP-TEAD interaction and thus inhibit the transcriptional activity of the YAP-TEAD complex, the anti-tumor effect of cobimetinib was evaluated by a nude mouse tumor formation experiment. The experimental results are as follows: Figure 3 As shown, Part A is a flowchart of cell inoculation and dosing for the NCI-H226 cell nude mouse tumor formation experiment; Part B is the growth curve of the NCI-H226 cell nude mouse transplant tumor under different dosing conditions; Parts C and D are the final photos and tumor weight ratios of the NCI-H226 cell nude mouse transplant tumor under different dosing conditions; Part E is a flowchart of cell inoculation and dosing for the HeLa-LATS1 / 2DKO cell nude mouse tumor formation experiment; Part F is the growth curve of the HeLa-LATS1 / 2DKO cell nude mouse transplant tumor under different dosing conditions; Parts G and H are the final photos and tumor weight ratios of the HeLa-LATS1 / 2DKO cell nude mouse transplant tumor under different dosing conditions. In order to fully illustrate the problem, the solvent DMSO and another MEK1 / 2 inhibitor PD0325901 were selected as negative controls. Among them, Figure 3 Part A of Figure 1 shows the results of nude mouse tumor formation and drug administration experiments on NCI-H226 cells containing NF2 protein inactivation mutations. NF2 is an important inhibitory factor upstream of YAP, and its inactivation will cause YAP activation. Therefore, NCI-H226 transplanted tumors are a type of tumor that is highly dependent on YAP activity in terms of growth. Figure 3 Parts B, C, and D of the results show that cobimetinib treatment significantly inhibited the growth of NCI-H226 nude mouse xenograft tumors compared with the vehicle group DMSO and another MEK1 / 2 inhibitor PD0325901 group. Secondly, we conducted nude mouse tumor formation and drug administration experiments with HeLa cells containing LATS1 / 2 kinase knockout (HeLa-LATS1 / 2KO cells). Figure 3As shown in the E part of Figure 1, LATS1 / 2 kinase is an important inhibitory factor upstream of YAP. Its inactivation directly activates YAP. Therefore, HeLa-LATS1 / 2KO transplanted tumor is a tumor that is also highly dependent on YAP activity in growth. In the nude mouse tumor formation and drug administration experiment results, Figure 3 F, G, and H show that cobimetinib treatment can significantly inhibit the growth of HeLa-LATS1 / 2KO nude mouse transplanted tumors compared with the vehicle group DMSO and another MEK1 / 2 inhibitor PD0325901 group. The above experimental results show that cobimetinib can inhibit the growth of YAP highly activated tumors.

[0047] Example 3 Evaluation of the anti-tumor effect of cobimetinib in a liver cancer model

[0048] Previous studies have shown that the YAP-TEAD transcription complex plays an important role in tumorigenesis. In order to functionally clarify that cobimetinib can disrupt the YAP-TEAD interaction and thus inhibit the transcriptional activity of the YAP-TEAD complex, the anti-tumor effect of cobimetinib was evaluated in a liver cancer model. The experimental results are as follows: Figure 4 As shown, Part A is the liver cancer model induced by Myc overexpression and p53 knockout (Myc; sg-p53) and the dosing pattern diagram, Parts B and C are the number of tumor foci in the Myc; sg-p53 liver cancer model under different dosing conditions; Parts E and F are the tumor foci size and cell proliferation ability evaluation in the lesions of the Myc; sg-p53 liver cancer model under different dosing conditions; Part G is the liver cancer model induced by MST1 / 2 knockout (MST1 / 2DKO) and the dosing pattern diagram. In order to fully illustrate the problem, the solvent DMSO and another MEK1 / 2 inhibitor PD0325901 were also selected as negative controls here. Studies have shown that both Myc protein overexpression and p53 protein deficiency can activate YAP. Therefore, the liver cancer induced by Myc overexpression and p53 knockout (Myc; sg-p53) is highly dependent on YAP activity, such as Figure 4 As shown in Part A. Figure 4 As shown in parts B and C of the study, the results of the Myc;sg-p53 liver cancer occurrence and drug administration experiments showed that cobimetinib treatment could significantly inhibit the number of liver cancer lesions compared with the vehicle group DMSO and another MEK1 / 2 inhibitor PD0325901 group. The lesion size was shown in Figure 2. Figure 4 As shown in part D, and the proliferation ability of tumor cells in the lesion is shown in Figure 4As shown in parts D and E of the figure, MST1 / 2 kinases are important inhibitors upstream of YAP, and their inactivation directly activates YAP. Therefore, liver-specific knockout of MST1 / 2-induced liver cancer is highly dependent on YAP activity, such as Figure 4 As shown in the F part of the figure. The results of the MST1 / 2DKO liver cancer occurrence and drug administration experiments showed that cobimetinib treatment could significantly inhibit the number of liver cancer lesions compared with the vehicle group DMSO and another MEK1 / 2 inhibitor PD0325901 group. Figure 4 As shown in parts G and H, the size of the lesion is Figure 4 As shown in Figure 1, and the proliferation capacity of tumor cells in the lesion, as Figure 4 As shown in parts H and I.

[0049] Example 4 Combination of cobimetinib and sorafenib

[0050] Sorafenib is a broad-spectrum tyrosine kinase inhibitor (TKI). It exerts a powerful anti-angiogenic effect mainly by targeting VEGFR1-3 and PDGFR, and has been the standard first-line systemic treatment for advanced hepatocellular carcinoma (HCC) for more than a decade. However, the clinical response rate of sorafenib is less than 20%, and the overall survival (OS) benefit of patients is very limited due to primary or secondary resistance. Previous studies have shown that YAP activation is involved in sorafenib resistance in patients with liver cancer. Based on this, we conducted a combination trial of cobimetinib and sorafenib, and the results showed that Figure 5 As shown, part A is the liver cancer model induced by Myc overexpression and p53 knockout (Myc; sg-p53) and the dosing pattern diagram; parts B and C are the number of tumor foci in the Myc; sg-p53 liver cancer model under different dosing conditions; parts E and F are the tumor foci size and cell proliferation ability evaluation in the Myc; sg-p53 liver cancer model under different dosing conditions. The results of the combined drug experiment in the Myc; sg-p53 liver cancer model showed that the combination of cobimetinib and sorafenib can significantly inhibit the number of liver cancer foci, lesion size, and tumor cell proliferation ability in the lesions of mice.

[0051] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. Use of a drug, cobimetinib, in inhibiting the activity of the YAP-TEAD transcription complex.

2. The use according to claim 1, characterized in that The application also includes a method for evaluating the anti-tumor effect of cobimetinib, including nude mouse tumor formation and drug administration experiments.

3. The use according to claim 2, characterized in that The application comprises the following steps: Step 1, select the solvent DMSO and another MEK1 / 2 inhibitor PD0325901 as negative controls; Step 2, obtaining nude mouse tumors of NCI-H226 cells containing NF2 protein inactivation mutations, i.e., NCI-H226 transplanted tumors; Step 3, respectively conducting a drug administration experiment on nude mice with NCI-H226 transplanted tumors obtained in step 2 with the solvent DMSO, the PD0325901 and the cobimetinib in step 1; Step 4: Evaluate the ability to inhibit the growth of the NCI-H226 nude mouse xenograft tumor under different administration conditions.

4. The use according to claim 2, characterized in that The application further comprises the following steps: Step 1, select the solvent DMSO and another MEK1 / 2 inhibitor PD0325901 as negative controls; Step 2, nude mice were inoculated with HeLa cells containing LATS1 / 2 kinase knockout to obtain HeLa-LATS1 / 2K0 transplanted tumors; Step 3, performing a drug administration experiment on nude mice bearing the HeLa-LATS1 / 2K0 transplanted tumor obtained in step 2 with the solvent DMSO, the PD0325901 and the cobimetinib respectively; Step 4: Evaluate the ability to inhibit the growth of the HeLa-LATS1 / 2K0 nude mouse transplanted tumor under different administration conditions.

5. A use of cobimetinib in preventing liver cancer from developing drug resistance to sorafenib, characterized in that: The cobimetinib and sorafenib are used in combination.

6. The use according to claim 5, characterized in that The application also includes a method for evaluating the effect of combined medication, comprising the following steps: Step (1) inducing liver cancer in nude mice by overexpressing Myc and knocking out p53 to obtain a Myc;sg-p53 liver cancer model; Step (2) conducting a drug administration experiment on the Myc;sg-p53 liver cancer model obtained in step (1) using the cobimetinib alone, the sorafenib alone, and the cobimetinib and sorafenib combination; Step (3) evaluates the tumor size and cell proliferation ability within the lesion of the Myc;sg-p53 liver cancer model under different administration conditions.

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