Use of auranofin in the preparation of medicaments for treating diseases of USP6 gene rearrangement and abnormal activation

Abstract

The application provides an application of auro-thio-pan in preparation of a drug for treating a USP6 gene rearrangement and abnormal activation disease, and the auro-thio-pan is a lipophilic gold-based compound. Researches of the application show that in addition to the USP6 gene rearrangement disease, tumors such as osteosarcoma, RUNX1 gene rearrangement and CBFbeta gene rearrangement leukemia, synovial sarcoma and the like also have abnormal activation of USP6, and are clinical application scenarios of USP6 inhibitors. It is further found that the compound auro-thio-pan can effectively combine with a catalytic domain of USP6 and inhibit the deubiquitination enzyme activity of USP6, and inhibit the growth of RUNX1 rearrangement tumors at the cell and animal levels. The application opens up a new direction for the development of a drug for treating the USP6 gene rearrangement and abnormal activation tumor and the like, and expands the application range of auro-thio-pan in the treatment of clinical diseases, and provides a possibility for improving the prognosis and survival of corresponding clinical disease patients.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of compound application, and relates to application of auranofin in preparation of a drug for treating USP6 gene rearrangement and abnormal activation diseases, and is application of a lipophilic gold-based compound USP6 inhibitor in targeted treatment of a drug for treating USP6 gene rearrangement and abnormal activation diseases, wherein the compound is auranofin. BACKGROUND

[0002] USP6 protein, also known as TRE17, TRE2 or HRP1, belongs to the member of ubiquitin-specific proteases (USPs) family, and can catalyze specific hydrolysis and fall-off of ubiquitin molecules from a substrate protein connected with ubiquitin. However, abnormal activation of USP6 in a disease state can promote occurrence and development of tumors and other diseases through multiple signal pathways such as JAK1 / STAT3, Wnt / beta-catenin, NF-kappa B and MAPK / ERK. At present, abnormal activation forms of USP6 include gene rearrangement and gene overexpression. In diseases such as primary aneurysmal bone cyst, nodular fasciitis, tendon sheath fibroma and ossifying myositis, USP6 is prone to gene rearrangement, mainly fusion of the promoter segment of CDH11, MYH9 and the coding segment of USP6 (Mod Pathol. 2017; 30(11): 1577-1588), and then directly causes abnormal expression and activation of USP6. Unfortunately, there is no clear and effective targeted treatment scheme for USP6 abnormal activation related tumors and other diseases at present, and further research is urgently needed.

[0003] The lipophilic gold-based compound auranofin is an oral anti-inflammatory drug, and is clinically used for treating rheumatoid arthritis. In recent years, it has been found that auranofin has various clinical biological activities, including anti-parasite, anti-virus, anti-diabetes and anti-tumor, etc. In the aspect of anti-tumor, it is reported that auranofin can enhance the treatment effect of chemotherapy drugs on non-small cell lung cancer and pancreatic ductal carcinoma by inducing oxidative stress (J Exp Clin Cancer Res. 2024 Mar 22; 43(1): 88.). In addition, the United States has successively carried out multiple clinical trials on auranofin for anti-tumor, such as glioblastoma (NCT02770378), ovarian cancer (NCT03456700) and the like. However, whether auranofin can directly bind to USP6 and inhibit the activity thereof, and whether it can be used for targeted treatment of USP6 rearrangement and other abnormal activation related tumors and other diseases to achieve the purpose of treatment have not been reported. SUMMARY

[0004] The application aims to provide an application of a lipophilic gold-based compound in the preparation of a treatment for a USP6 gene rearrangement and abnormal activation disease, wherein the lipophilic gold-based compound is Auranofin, which is a chemical name of 2,3,4,6-tetraacetyl-1-thio-β-D-glucopyranose and triethylphosphine gold complex, and a molecular formula is C 20 H 34 AuO9PS. The drug is prepared from the lipophilic gold-based compound and a pharmaceutical excipient.

[0005] The application firstly analyzes a database to find that, in addition to the classic known USP6 gene rearrangement in primary aneurysmal bone cyst, nodular fasciitis, tendon sheath fibroma, ossifying myositis and other diseases, USP6 abnormal activation exists in a variety of tumors. USP6 is highly expressed in osteosarcoma, RUNX1 gene rearrangement and CBFβ gene rearrangement leukemia, and is closely related to low response to chemotherapy and poor prognosis. In addition, based on Crispr sensitivity analysis, it is found that synovial sarcoma has the highest dependence on USP6 in a pan-cancer. These results show that the USP6 gene rearrangement disease, osteosarcoma, RUNX1 gene rearrangement and CBFβ gene rearrangement leukemia, synovial sarcoma and the like are clinical indications of the USP6 inhibitor.

[0006] Further, the application finds that the compound Auranofin can be directly combined with the purified USP6 catalytic domain (T532-Q1406) in vitro. Further combined with the Ub-AMC experiment, it is found that the compound Auranofin inhibits the deubiquitinase activity of USP6 in vitro in a dose-dependent manner. The application provides evidence for the application of Auranofin in the USP6 gene rearrangement and abnormal activation tumor and the like.

[0007] Further, the application finds that the compound Auranofin can selectively inhibit the primary patient-derived USP6 high-expression RUNX1 rearrangement leukemia cells, and the killing effect on the primary RUNX1 rearrangement leukemia cells is obviously stronger than that on the non-RUNX1 rearrangement leukemia primary cells. In addition, the compound Auranofin can significantly inhibit the growth of the RUNX1 rearrangement leukemia cells in the axillary transplanted tumor in the NSG mouse in vivo.

[0008] The application firstly discloses that the lipophilic gold-based compound Auranofin can be used as the USP6 inhibitor, and is used for the treatment of the USP6 gene rearrangement disease, osteosarcoma, RUNX1 gene rearrangement and CBFβ gene rearrangement leukemia, synovial sarcoma and the like USP6 abnormal activation related tumor and the like disease.

[0009] Further, the medicament comprises a pharmaceutically effective amount of auro-thioic acid and a pharmaceutically acceptable carrier. The effective amount refers to the amount of the compound sufficient to have an effect on the treatment of the disease when administered to a subject. The effective amount can vary depending on the severity of the disease, and the physical condition, age, weight, gender, and the like of the subject to be treated.

[0010] The carrier is any formulation or carrier medium capable of delivering an effective amount of the active substance without interfering with the biological activity of the active substance and without toxic side effects to the host or subject. Further, the carrier comprises at least one of a filler, a wetting agent, a binder, a disintegrant, a lubricant.

[0011] The medicament can be prepared into a formulation according to the preparation method of the formulation described in pharmacy. The formulation of the medicament described above is a liquid formulation or a solid formulation.

[0012] Further, the dosage form of the medicament can be, but is not limited to, an oral formulation, an injection solution, a freeze-dried powder injection, or a large infusion.

[0013] The present application has the beneficial effects that the present application first proposes that lipophilic gold-based compound auro-thioic acid can be used as a USP6 inhibitor, and the use of auro-thioic acid in the treatment of diseases related to USP6 gene rearrangement and abnormal activation, such as human primary aneurysmal bone cyst, nodular fasciitis, osteosarcoma, and RUNX1-rearranged leukemia. Auro-thioic acid can significantly inhibit the deubiquitinase activity of USP6, expand the application range of auro-thioic acid in the treatment of clinical diseases, provide new insights for the development of USP6 targeted inhibitors, and provide the possibility for further improving the clinical efficacy, prognosis, and survival of USP6 abnormal activation related tumors such as USP6 gene rearrangement diseases, osteosarcoma, leukemia, and synovial sarcoma. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Based on the Gene Expression Omnibus database established by the National Center for Biotechnology Information, the important biological significance of USP6 in osteosarcoma was analyzed. The analysis results show that USP6 is highly expressed in osteosarcoma and osteosarcoma lung metastasis tissue samples compared with non-tumor tissues, and the expression amount of USP6 in osteosarcoma patients who are not sensitive to chemotherapy is higher than that in patients who are sensitive to chemotherapy.

[0015] Figure 2is based on TARGET database, which is a high-throughput database of childhood tumors established by the National Center for Biotechnology Information. The analysis of USP6 in acute myeloid leukemia shows that the expression of USP6 in t(8;21) subtype (i.e. RUNX1 gene rearrangement) and inv(16) subtype (i.e. CBFβ gene rearrangement) is significantly higher than that in other AML subtypes, and in RUNX1 rearranged AML, the overall survival rate of patients with high expression of USP6 is significantly worse than that of patients with low expression of USP6.

[0016] Figure 3 is based on DepMap database established by Broad Institute in cooperation with other research institutions. The analysis of Crispr dependence on USP6 gene in pan-tumor types shows that synovial sarcoma has the highest dependence on USP6.

[0017] Figure 4 is the affinity binding ability and deubiquitinase activity inhibition of auranofin to the catalytic domain (T532-Q1406) of USP6.

[0018] Figure 5 is the tumor inhibitory effect of auranofin on RUNX1 rearranged AML primary cells and axillary transplanted tumor animals.

[0019] In the figure, the expression level of USP6 in the "Normal" subtype is compared with that in the t(8;21) subtype and the inv(16) subtype, and the two-factor variance analysis and independent sample t test are used for comparison between the control group and the auranofin administration group: n.s. represents p>0.05; * represents p<0.05; ** represents p<0.01; and *** represents p<0.001. DETAILED DESCRIPTION

[0020] The application will be further described in detail below in combination with the accompanying drawings and examples. The following examples are only used to illustrate the application and are not used to limit the scope of the application.

[0021] The experimental methods not specified in the examples are generally carried out according to the conventional conditions or the conditions recommended by the manufacturers.

[0022] The application of the compound auranofin described in the application can refer to the conventional drug preparation method and actual development. The drug dosage form and biological preparation are any dosage form recognized in medicine, such as powder, injection, capsule, tablet or oral liquid.

[0023] Example 1

[0024] Based on the NCBI-GEO database website (https: / / www.ncbi.nlm.nih.gov / ), the clinical classification, chemotherapy sensitivity, and USP6 gene expression data of osteosarcoma patients (GSE14359 and GSE14827 datasets) were obtained to analyze the differences in USP6 expression in patients with different clinical classifications and chemotherapy sensitivity. The results showed that USP6 was highly expressed in osteosarcoma and osteosarcoma lung metastasis tissue samples compared to non-tumor tissue, and the expression of USP6 in osteosarcoma patients with chemotherapy resistance was higher than that in patients with chemotherapy sensitivity. See Figure 1 .

[0025] Example 2

[0026] Based on the TARGET database website (https: / / www.cancer.gov / ccg / research / genome-sequencing / target), the survival time, molecular subtype, and USP6 gene expression information of TARGET-AML patients were obtained to analyze the differences in USP6 expression in different AML molecular subtypes. The results showed that the expression of USP6 in t(8;21) subtype (i.e., RUNX1 gene rearrangement) and inv(16) subtype (i.e., CBFβ gene rearrangement) was significantly higher than that in other AML subtypes, and in RUNX1 rearranged AML, the overall survival rate of patients with high expression of USP6 was significantly worse than that of patients with low expression of USP6. See Figure 2 .

[0027] Example 3

[0028] Based on the DepMap database website (https: / / depmap.org / portal / ), the Crispr dependency information of USP6 gene in pan-cancer cell lines was obtained, and the Crispr dependency of different tumor types on USP6 gene was compared. The lower the value, the higher the dependence of the cancer on USP6 gene. The results showed that the average ± standard deviation of synovial sarcoma was significantly lower than that of other cancers, indicating that synovial sarcoma had the highest dependence on USP6 gene. See Figure 3 .

[0029] Example 4

[0030] The in vitro recombinant protein of USP6 catalytic domain (T532-Q1406) labeled with green fluorescent signal was incubated with 16 gradient concentrations of compound auranofin at room temperature. Based on the micro-thermal fluctuation technology, the affinity of the interaction between USP6 protein and compound auranofin was quantitatively analyzed. After the in vitro recombinant protein of USP6 was incubated with 7 concentrations of compound auranofin at room temperature, Ub-AMC probe (purchased from Hefei Keshengjing Peptide Biotechnology Co., Ltd.) was added, and TECAN microplate reader was used to continuously detect at 25°C, excitation wavelength 355 nm, and emission wavelength 445 nm for 120 minutes. The inhibitory effect of different concentrations of compound auranofin on USP6 deubiquitinating enzyme activity was plotted. It was found that auranofin could bind to the catalytic domain of USP6 and inhibit the deubiquitinating enzyme activity of USP6 in a concentration-dependent manner. See Figure 4 .

[0031] Example 5

[0032] Hematopoietic stem cells, non-RUNX1 rearranged AML primary cells and RUNX1 rearranged AML primary cells were inoculated in cell culture dishes and treated with different concentrations of compound auranofin (0-4 μmol / L) for 3 days. The survival rate of cells in each group was calculated by CTG system detection. RUNX1 rearranged leukemia cells (KASUMI-1) were inoculated into NSG mice by subaxillary injection. The mice received normal saline (control group; intraperitoneal injection) and auranofin (experimental group, 2.5 or 5 mg / kg; intraperitoneal injection) treatment for 15 days. The volume of the tumor was tracked and recorded. It was found that the killing effect of compound auranofin on RUNX1 rearranged leukemia primary cells was significantly stronger than that on non-RUNX1 rearranged leukemia primary cells, and the killing effect on normal HSC hematopoietic stem cells was not strong. At the same time, compound auranofin could significantly inhibit the growth of RUNX1 rearranged leukemia cells in subaxillary transplanted tumors in NSG mice. See Figure 5 .

Claims

1. Aurinophene in the preparation of therapeutic... USP6 Its application in abnormally activated leukemia drugs is characterized by, The chemical name of the aurinophene is: 2,3,4,6-tetraacetyl-1-thio-β-D-pyranose and triethylphosphine gold complex, with the molecular formula C. 20 H 34 AuO9PS; the aurinophene inhibits USP6 Deubiquitinating enzyme activity, thereby inhibiting USP6 Gene rearrangement and abnormal activation, the leukemia mentioned is RUNX1 Rearrangement of leukemia.

2. The application according to claim 1, characterized in that, The drug is made from aurinophene and a pharmaceutically acceptable carrier.

3. The application according to claim 1, characterized in that, The drug dosage forms are gels, oral preparations, injections, and lyophilized powder injections.

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