Application of Jinnofen in preparation of medicine for treating USP6 gene rearrangement and abnormal activation diseases

Abstract

The invention provides an application of Jinnofen in preparation of a medicine for treating USP6 gene rearrangement and abnormal activation diseases. The Jinnofen is a lipophilic gold-based compound. The research shows that besides USP6 gene rearrangement diseases, tumors such as osteosarcoma, RUNX1 gene rearrangement and CBF beta gene rearrangement leukemia, synovial sarcoma and the like have USP6 abnormal activation, and the USP6 inhibitor is a clinical application scene of the USP6 inhibitor. It is further found that the compound Jinnofen can be effectively combined with a USP6 catalytic structural domain, inhibit USP6 deubiquitination enzyme activity and inhibit growth of RUNX1 rearrangement type tumors in the cell and animal level. The invention opens up a new direction for research and development of drugs for treating diseases such as USP6 gene rearrangement and abnormally activated tumors at present, also expands the application range of Jinnofen in clinical disease treatment, and provides possibility for improvement of prognosis and survival of patients with corresponding clinical diseases.

Description

Technical Field

[0001] The present invention belongs to the application field of compounds, and relates to the application of auranofin in the preparation of drugs for treating diseases caused by USP6 gene rearrangement and abnormal activation. It is the application of a lipophilic gold-based compound USP6 inhibitor in the preparation of drugs for targeted treatment of diseases caused by USP6 gene rearrangement and abnormal activation. The compound is auranofin. Background Art

[0002] The USP6 protein, also known as TRE17, TRE2 or HRP1, belongs to the member of the Ubiquitin-specific proteases (USPs) family and can specifically catalyze the hydrolysis and detachment of ubiquitin molecules from substrate proteins conjugated with ubiquitin. However, the abnormal activation of USP6 in disease states can promote the occurrence and development of diseases such as tumors through multiple signaling pathways such as JAK1 / STAT3, Wnt / β-catenin, NF-κB, and MAPK / ERK. Currently, the abnormal activation forms of USP6 include gene rearrangement and gene overexpression and other types. Among them, in diseases such as primary aneurysmal bone cyst, nodular fasciitis, tenosynovial fibroma, and myositis ossificans, USP6 is extremely prone to gene rearrangement, mainly manifested as the fusion of the promoter regions of genes such as CDH11 and MYH9 and the coding region of USP6 (Mod Pathol. 2017; 30(11): 1577-1588), which directly leads to the abnormal expression and activation of USP6. Unfortunately, there is currently no clear and effective targeted treatment plan for diseases such as tumors related to USP6 abnormal activation, and further research is urgently needed.

[0003] The lipophilic gold-based compound auranofin is an oral anti-inflammatory drug and is clinically used to treat rheumatoid arthritis. In recent years, studies have found that auranofin also has a variety of clinical biological activities, including anti-parasitic, anti-viral, anti-diabetic and anti-tumor, etc. In terms of anti-tumor, it has been reported that it can enhance the therapeutic effect of chemotherapeutic drugs on non-small cell lung cancer and pancreatic ductal cancer 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 the anti-tumor efficacy of auranofin, such as the secondary development of the efficacy of glioblastoma (NCT02770378), ovarian cancer (NCT03456700), etc. However, whether auranofin can directly bind to USP6 and inhibit its activity, and whether it can be used for targeted treatment of diseases such as tumors related to USP6 rearrangement and other abnormal activation to achieve the purpose of treatment, etc., have not been reported yet. Summary of the Invention

[0001] The object of the present invention is to provide the use of a lipophilic gold-based compound in the preparation of a medicament for treating diseases caused by USP6 gene rearrangement and abnormal activation. The lipophilic gold-based compound is auranofin, the chemical name of which is 2,3,4,6-tetraacetyl-1-thio-β-D-glucopyranose triethylphosphine gold complex, and the molecular formula is C 20 H 34 AuO 9 PS. The medicament is prepared from a lipophilic gold-based compound and pharmaceutical excipients.

[0002] Firstly, through database analysis, the present invention finds that in addition to the classical known rearrangement of USP6 gene in diseases such as primary aneurysmal bone cyst, nodular fasciitis, tenosynovial fibroma, myositis ossificans, etc., USP6 is abnormally activated in a variety of tumors. USP6 is highly expressed in osteosarcoma compared with non-tumor tissues, highly expressed in leukemia with RUNX1 gene rearrangement and CBFβ gene rearrangement, and the high expression 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 among pan-cancer types. These results indicate that diseases caused by USP6 gene rearrangement, osteosarcoma, leukemia with RUNX1 gene rearrangement and CBFβ gene rearrangement, synovial sarcoma, etc. are clinical indications for USP6 inhibitors.

[0003] Furthermore, the present invention finds that the compound auranofin can directly bind to the in vitro purified catalytic domain (T532-Q1406) of USP6. Further combined with the Ub-AMC experiment, it is found that the compound auranofin inhibits the in vitro deubiquitinase activity of USP6 in a dose-dependent manner. The present invention provides evidence for the application of auranofin in diseases such as USP6 gene rearrangement and abnormally activated tumors.

[0004] Furthermore, the present invention finds that the compound auranofin can selectively inhibit primary patient-derived RUNX1-rearranged leukemia cells with high USP6 expression, and has a significantly stronger killing effect on primary RUNX1-rearranged leukemia cells than non-RUNX1-rearranged primary leukemia cells. In addition, the compound auranofin can significantly inhibit the growth of RUNX1-rearranged leukemia cells in axillary xenograft tumors in NSG mice.

[0005] The present invention discloses for the first time that the lipophilic gold-based compound auranofin can be used as a USP6 inhibitor for the treatment of diseases such as diseases caused by USP6 gene rearrangement, osteosarcoma, leukemia with RUNX1 gene rearrangement and CBFβ gene rearrangement, synovial sarcoma and other tumors related to abnormal activation of USP6.

[0005] Furthermore, the drug comprises a pharmaceutically effective amount of auranofin and a pharmaceutically acceptable carrier. The effective amount refers to the amount of the compound that is sufficient to be effective in treating a disease when administered to a subject. The effective dose may vary depending on the severity of the disease, as well as the physical condition, age, weight, gender, etc. of the subject to be treated.

[0006] The carrier is any preparation or carrier medium that can deliver an effective dose of the active substance, does not interfere with the biological activity of the active substance, and is non-toxic and has no side effects on the host or subject. Further, the carrier includes at least one of a filler, a wetting agent, a binder, a disintegrant, and a lubricant.

[0007] The drug can be formulated into a preparation according to the preparation methods described in pharmacy. The preparation form of the above-mentioned drug is a liquid preparation or a solid preparation.

[0008] Furthermore, the dosage form of the drug can be, but is not limited to, an oral preparation, an injection freeze-dried powder injection, or a large volume infusion.

[0009] The beneficial effects of the present invention: The present invention firstly proposes that the lipophilic gold-based compound auranofin can be used as a USP6 inhibitor in the treatment of USP6 gene rearrangement and abnormal activation-related diseases such as human primary aneurysmal bone cyst, nodular fasciitis, osteosarcoma, and RUNX1 rearrangement leukemia. Auranofin can significantly inhibit the deubiquitinase activity of USP6, expanding the application scope of auranofin in the treatment of clinical diseases, providing new insights for the development of USP6-targeted inhibitors, and at the same time providing the possibility of further improving the clinical efficacy of USP6 gene rearrangement diseases, osteosarcoma, leukemia, synovial sarcoma and other USP6 abnormal activation-related tumors, and improving their prognosis and survival. BRIEF DESCRIPTION OF THE DRAWINGS

[0001] Figure 1 Based on the Gene Expression Omnibus database established by the National Center for Biotechnology Information in the United States, 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 level of USP6 in osteosarcoma patients with chemotherapy resistance is higher than that in chemotherapy-sensitive patients.

[0002] Figure 2Based on the high-throughput database for childhood cancers (TARGET database) established by the National Center for Biotechnology Information of the United States, the important biological significance of USP6 in acute myeloid leukemia was analyzed. The analysis results showed that the expression level of USP6 in the 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 rearrangement-type AML, the overall survival rate of patients with high USP6 expression was significantly worse than that of patients with low USP6 expression.

[0003] Figure 3 Based on the DepMap database established jointly by the Broad Institute and other research institutions, the Crispr dependence of the USP6 gene in pan-cancer types was analyzed. The analysis results showed that synovial sarcoma had the highest dependence on USP6.

[0004] Figure 4 It is the affinity binding ability and deubiquitinase activity inhibition of auranofin on the catalytic domain (T532-Q1406) of USP6.

[0005] Figure 5 It is the tumor inhibitory effect of auranofin on primary cells of RUNX1 rearrangement AML and tumor xenografts in axilla of animals.

[0006] In the figure, when comparing the USP6 expression level in the "Normal" subtype with that in the t(8;21) subtype and inv(16) subtype, and when comparing between the control group and the auranofin administration group, two-way ANOVA and independent samples t-test were used: n.s. indicates p > 0.05; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001. Detailed implementation mode

[0001] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0002] For the experimental methods without specific conditions noted in the embodiments, they are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer.

[0003] For the application of the compound auranofin described in the present invention, conventional drug formulation methods and actual development can be referred to. The drug dosage form and biological preparation are any dosage form recognized in medicine, such as powder, injection, capsule, tablet or oral liquid.

[0004] Example 1 Based on the official website of the NCBI-GEO database (https: / / www.ncbi.nlm.nih.gov / ), clinical information such as the clinical classification and chemosensitivity of osteosarcoma patients and USP6 gene expression data (GSE14359 and GSE14827 datasets) were obtained, and the differences in the expression levels of USP6 in patients with different clinical classifications and chemosensitivities were analyzed. The results showed that USP6 was highly expressed in osteosarcoma and osteosarcoma lung metastasis tissue samples compared with non-tumor tissues, and the expression level of USP6 in osteosarcoma patients with chemoresistance was higher than that in chemosensitive patients. The results are shown in Figure 1 。

[0005] Example 2 Based on the official website of the TARGET database (https: / / www.cancer.gov / ccg / research / genome-sequencing / target), clinical information such as the survival time and molecular subtypes of TARGET-AML patients and USP6 gene expression information were obtained, and the differences in the expression levels of USP6 in different AML molecular subtypes were analyzed. The results showed that the expression levels of USP6 in the t(8;21) subtype (i.e., RUNX1 gene rearrangement) and inv(16) subtype (i.e., CBFβ gene rearrangement) were significantly higher than those in other AML subtypes, and in RUNX1 rearrangement-type AML, the overall survival rate of patients with high USP6 expression was significantly worse than that of patients with low USP6 expression. The results are shown in Figure 2 。

[0006] Example 3 Based on the official website of the DepMap database (https: / / depmap.org / portal / ), the Crispr dependency information of USP6 gene in pan-cancer cell lines was obtained, classified according to different tumor types, and the Crispr dependency of USP6 gene in different tumor types was compared. The lower the assigned value, the higher the dependency of the cancer type on the USP6 gene. The results showed that the mean ± standard deviation of synovial sarcoma was significantly lower than that of other cancer types, indicating that synovial sarcoma had the highest dependency on the USP6 gene. The results are shown in Figure 3 。

[0007] Example 4 The in vitro recombinant protein of USP6 catalytic domain (T532-Q1406) labeled with green fluorescent signal was co-incubated with 16 gradient concentrations of aurothioglucose at room temperature, and then the affinity of the interaction between USP6 protein and aurothioglucose was quantitatively analyzed based on the microcalorimetric surging technology. After the in vitro recombinant USP6 protein was co-incubated with 7 gradient concentrations of aurothioglucose at room temperature, Ub-AMC probe (purchased from Hefei Kesen Jingtai Biotechnology Co., Ltd.) was added, and the inhibition of USP6 deubiquitinase activity under the action of different concentrations of aurothioglucose was continuously detected for 120 minutes at 25 °C, excitation wavelength of 355 nm, and emission wavelength of 445 nm using a TECAN microplate reader. The results showed that aurothioglucose could bind to the USP6 catalytic domain and inhibit the USP6 deubiquitinase activity in a concentration-dependent manner. The results are shown in Figure 4 .

[0008] Example 5 Hematopoietic stem cells, non-RUNX1 rearranged AML primary cells and RUNX1 rearranged AML primary cells were seeded in cell culture dishes and treated with different concentrations of aurothioglucose (0 - 4 μmol / L) for 3 days respectively, and then the survival rate of each group of cells was detected and calculated using the CTG system. RUNX1 rearranged leukemia cells (KASUMI-1) were inoculated into NSG mice by axillary injection, and the mice were treated with normal saline (control group; intraperitoneal injection) and aurothioglucose (experimental group, 2.5 or 5 mg / kg; intraperitoneal injection) for 15 days, and the tumor volume was tracked and recorded. The results showed that the killing effect of aurothioglucose on RUNX1 rearranged leukemia primary cells was significantly stronger than that on non-RUNX1 rearranged leukemia primary cells, and at the same time, the killing effect on normal HSC hematopoietic stem cells was not strong. At the same time, aurothioglucose could significantly inhibit the growth of RUNX1 rearranged leukemia cells in axillary transplanted tumors in NSG mice. The results are shown in Figure 5 .

Claims

1. The use of auranofin in the preparation of a drug for treating USP6 gene rearrangement and abnormal activation diseases, characterized in that: The chemical name of the auranofin is: 2,3,4,6-tetraacetyl-1-thio-β-D-pyranose glucose and triethylphosphine gold complex, with a molecular formula of C 20 H 34 AuO9PS.

2. The use according to claim 1, characterized in that: The diseases include USP6 gene rearrangement diseases such as primary aneurysmal bone cyst, nodular fasciitis, tenosynovial fibroma, and myositis ossificans, as well as osteosarcoma, RUNX1 gene rearrangement and CBFβ gene rearrangement leukemia, and synovial sarcoma, which are tumors related to abnormal USP6 activation.

3. The use according to claim 1, characterized in that: Auranofin inhibits USP6 gene rearrangement and abnormal activation by inhibiting USP6 deubiquitinase activity.

4. The use according to claim 1, characterized in that: The medicine is prepared from auranofin and a pharmaceutically acceptable carrier.

5. The use according to claim 4, characterized in that: The carrier is a pharmaceutical excipient, physiological saline or distilled water.

6. The use according to claim 1, characterized in that: The drug dosage form is a gel, soft capsule, oral preparation, injection, lyophilized powder injection or large infusion dosage form.

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