Use of small molecule compound SHIN1 in the preparation of drugs for treating uveal melanoma

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses application of a small-molecule compound SHIN1 in preparation of a drug for treating uveal melanoma (UM). The application proves that the small-molecule compound SHIN1 can significantly inhibit the proliferation and clonogenicity of UM cells through a cell experiment, and proves that SHIN1 can inhibit UM tumorigenesis in vivo through an animal experiment. Based on this, the application opens up a new field of SHIN1 for UM treatment, provides a new treatment drug for UM, solves the predicament that there is no available drug for UM in clinic, and prolongs the life of patients and improves the life quality of patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of the small molecule compound SHIN1 in the preparation of drugs for treating uveal melanoma. Background Technology

[0002] Uveal melanoma (UM) is the most common primary malignant tumor of the eye in adults, characterized by its high malignancy. Clinical data shows that approximately 50% of UM patients eventually develop distant metastases, and the prognosis for metastatic UM patients is extremely poor, with a median survival of less than one year. Currently, the main treatments for UM include surgical resection, radiotherapy, chemotherapy, and immunotherapy. However, UM has low sensitivity to conventional radiotherapy and chemotherapy, and after metastasis, it responds almost entirely to immune checkpoint inhibitors, resulting in extremely limited treatment efficacy. Therefore, developing novel targeted therapies for UM to reduce patient mortality and improve eye preservation rates has become an urgent clinical challenge.

[0003] One-carbon metabolism plays a crucial role in the metabolic reprogramming of tumor cells, providing essential intermediates for nucleotide biosynthesis, methylation, and redox balance by integrating the folate and methionine cycles. Serine hydroxymethyltransferases (SHMTs) are key rate-limiting enzymes in one-carbon metabolism, catalyzing the conversion of serine to glycine while simultaneously providing one-carbon units to the folate cycle. Studies have shown that SHMTs are upregulated in various tumor tissues and are closely associated with poor patient prognosis; therefore, the development of selective inhibitors has become a hot topic in cancer treatment research. SHIN1 is a highly effective dual SHMT inhibitor that has demonstrated antitumor activity in various tumor models. Studies have shown that lung adenocarcinoma cells are significantly more sensitive to SHIN1 than normal human bronchial epithelial cells; in a gastric cancer model, SHIN1 synergistically induces DNA damage with 5-fluorouracil; and in bladder cancer, SHIN1 induces apoptosis by promoting the accumulation of intracellular reactive oxygen species.

[0004] Although SHIN1 has shown significant anti-tumor activity in a variety of tumors, its efficacy and mechanism of action in UM have not yet been reported. Summary of the Invention

[0005] To address the lack of effective treatments for umbilical malignancy (UM) in existing technologies, this invention aims to provide a novel application of the small molecule compound SHIN1 in the preparation of anti-UM drugs. Based on the metabolic characteristics of UM, this invention proposes for the first time the application of the SHMT inhibitor SHIN1 in UM treatment, aiming to provide a new clinical treatment strategy for UM, improve treatment efficacy, prolong patient survival, and improve patients' quality of life.

[0006] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0007] In a first aspect, this invention provides the use of the small molecule compound SHIN1 in the preparation of a drug for treating uveal melanoma. The CAS registry number for the small molecule compound SHIN1 is 2146095-85-2, and its molecular formula is C1. 24 H 24 N4O2, molecular weight 400.47, molecular structure formula is as follows:

[0008] .

[0009] Furthermore, the small molecule compound SHIN1 inhibits the proliferation and colony formation of uveal melanoma cells and inhibits tumorigenesis of uveal melanoma in vivo, confirming the therapeutic effect of SHIN1 on uveal melanoma.

[0010] Further, the drug comprises the active ingredient SHIN1 and pharmaceutically acceptable excipients. "Pharmaceutically acceptable" means that when the substance is properly administered to animals or humans, it will not produce adverse, allergic, or other adverse reactions. The pharmaceutically acceptable excipients may be selected from one or more of diluents, fillers, surfactants, absorption enhancers, disintegrants, wetting agents, dispersants, etc. In a preferred embodiment, the pharmaceutically acceptable excipients include the following components: DMSO, PEG300, Tween 80, and ddH2O.

[0011] Furthermore, the administration methods of the drug include, but are not limited to, intraperitoneal injection, oral administration, intravenous injection, subcutaneous injection, and intravitreal injection.

[0012] In a second aspect, the present invention provides a pharmaceutical composition for treating uveal melanoma, the pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, said active ingredient being SHIN1.

[0013] Furthermore, the pharmaceutically acceptable excipients include the following components: DMSO, PEG300, Tween 80, and ddH2O.

[0014] Thirdly, the present invention provides the application of the small molecule compound SHIN1 in the preparation of products having one or more of the following functions:

[0015] a. Inhibits the proliferation of uveal melanoma cells;

[0016] b. Inhibits the clonogenic ability of uveal melanoma cells;

[0017] c. Inhibits the in vivo tumorigenesis ability of uveal melanoma.

[0018] Furthermore, the product is a drug or reagent that can be used in various in vivo and in vitro experimental studies related to UM, providing a basis for further exploration of the relevant mechanisms.

[0019] Furthermore, the uveal melanoma cells include human UM cell lines 92.1 and OMM2.3 cells.

[0020] The present invention has the following beneficial effects:

[0021] This invention demonstrates through cell experiments that the small molecule compound SHIN1 can significantly inhibit the proliferation and colony formation of UM cells, and through animal experiments, it confirms that SHIN1 can inhibit UM tumorigenesis in vivo. Based on this, this invention opens up a new field for the use of SHIN1 in UM treatment, providing a new therapeutic drug for UM, resolving the predicament of having no available drugs for UM clinically, and prolonging patient life and improving their quality of life. Attached Figure Description

[0022] Figure 1 Example 1 of this invention shows the proliferation curve results of SHIN1 significantly inhibiting the proliferation of UM cells. In the figure, A and B are the test results of two UM cell lines, 92.1 and OMM2.3, respectively (**P < 0.01; ***P < 0.001).

[0023] Figure 2 Example 2 of this invention shows the plate colony formation results of SHIN1 significantly inhibiting the colony formation ability of UM cells (92.1 and OMM2.3). In the figure, A is the cell crystal violet staining image, and B is the corresponding staining colony count result (***P <0.001).

[0024] Figure 3 The image shows the results of SHIN1 inhibiting UM tumor formation in vivo in Example 3 of this invention. In the image, A is a bioluminescence imaging image of mice, and B is a quantitative result of bioluminescence intensity of UM tumors in mice (*P < 0.05). Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents used in the following embodiments are all commercially available conventional reagents, and the experimental procedures involved are all conventional procedures in the art unless otherwise specified.

[0026] Example 1: SHIN1 Inhibition of UM Cell Proliferation in In Vitro

[0027] Experimental materials: Human UM cell lines 92.1 and OMM2.3. SHIN1 was purchased from Selleck (USA), DMSO from Sigma (USA), CCK8 from Dojindo (Japan), trypsin from Sinemet (China), and FBS, RPMI 1640, and PBS from Gibco (USA).

[0028] Experimental Procedure: Human UM cell lines 92.1 and OMM2.3 were cultured in RPMI 1640 medium (containing 10% FBS), respectively. When the cells were in the logarithmic growth phase, they were washed with PBS and then digested with trypsin. After centrifugation at 800 rpm for 5 min, the supernatant was discarded, and the cell pellet was resuspended in 2 mL of complete culture medium. After counting, a cell suspension of 2,000 cells / 100 μL was prepared. Drug and control groups were set up. SHIN1 was added to each well in the drug group to a final concentration of 10 μM, while the control group received the same concentration of DMSO. The cells were incubated at 37℃ in a 5% CO2 incubator. At 0, 24, 48, and 72 hours, 10 μL of LCK8 reagent was added to the corresponding wells, and the cells were incubated for another 2 h. The absorbance of the solutions at 450 nm was measured using a microplate reader, and cell proliferation curves were plotted based on the experimental results.

[0029] Experimental results: such as Figure 1 As shown in AB, in the two UM cell proliferation experiments of 92.1 and OMM2.3, the cell proliferation capacity of the drug group was significantly reduced compared with the control group, indicating that SHIN1 can effectively inhibit the proliferation of UM cells in vitro.

[0030] Example 2: SHIN1 Inhibition of UM Cell Colony Formation in In Vitro

[0031] Experimental materials: Human UM cell lines 92.1 and OMM2.3. SHIN1 was purchased from Selleck (USA), DMSO from Sigma (USA), trypsin from Neo-Synephrine (China), FBS, RPMI 1640 and PBS from Gibco (USA), and 4% paraformaldehyde and crystal violet staining solution from Sangon Biotech (China).

[0032] Experimental Procedure: Human UM cell lines 92.1 and OMM2.3 were cultured in RPMI 1640 medium (containing 10% FBS), respectively. When the cells were in the logarithmic growth phase, they were washed with PBS, digested with trypsin, centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 2 mL of complete culture medium. After counting, a cell suspension was prepared at a concentration of 800 cells / 100 μL. 800 cells were seeded into each well of a 12-well plate, and an additional 900 μL of complete culture medium was added to each well for further culture. After observing cell colony formation under a microscope, drug and control groups were set up. The drug group was treated with 10 μM SHIN1 in each well, and the control group was treated with the same concentration of DMSO. The cells were incubated at 37℃ in a 5% CO2 incubator. The culture medium containing the drug or DMSO solvent was replaced periodically. After reaching the experimental endpoint, the culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 15 min, followed by staining with crystal violet for 15 min. Wash with PBS until the background is colorless, photograph and count cell clone clusters, and perform statistical analysis.

[0033] Experimental results: such as Figure 2 As shown in AB, in the two UM cell clone formation experiments of 92.1 and OMM2.3, the number of clones formed in the drug group was significantly reduced compared with the control group, indicating that SHIN1 significantly inhibited the clone formation ability of UM cells.

[0034] Example 3: SHIN1 inhibition of UM xenograft tumors experiment

[0035] Experimental materials: Human UM cell line 92.1, Luciferase lentiviral vector purchased from Jimon Biotech (China), SHIN1, Tween 80, and PEG300 purchased from Selleck (USA), DMSO purchased from Sigma (USA), trypsin purchased from Sinemet (China), FBS, RPMI 1640, and PBS purchased from Gibco (USA), and BALB / c nude immunodeficient mice purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd. (China).

[0036] Experimental Procedure: A luciferase-labeled 92.1-Luc cell line (for subsequent luminescence detection) was constructed using a luciferase lentiviral vector. 1×10⁻⁶ cells were... 592.1-Luc cells were injected subretinally into mice to induce in situ tumor formation in the choroid, establishing a UM mouse model. Fourteen days post-injection, UM mice were randomly assigned to treatment or control groups and treated with either drug or solvent: 1) Control group: Intraperitoneal injection of 250 μL solvent (solvent formulation: 5% DMSO + 40% PEG300 + 5% Tween 80 + 50% ddH2O, "%" represents volume fraction), with the same injection frequency and timing as the treatment group; 2) Treatment group: Intraperitoneal injection of 50 mg / kg SHIN1 (solvent formulation: 5% DMSO + 40% PEG300 + 5% Tween 80 + 50% ddH2O, "%" represents volume fraction) every two days starting from day 14, for a total of 14 days. At the end of observation on day 30, the bioluminescence intensity of the tumors was measured using the IVIS Spectrum small animal in vivo optical imaging system to assess tumor activity.

[0037] Experimental results: such as Figure 3 As shown in AB, SHIN1 can inhibit UM tumorigenesis in vivo, manifested by a significant reduction in tumor bioluminescence intensity and activity. Therefore, animal experiments demonstrate that SHIN1 provides a novel drug for the treatment of UM.

[0038] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.

Claims

1. Application of the small molecule compound SHIN1 in the preparation of drugs for treating uveal melanoma.

2. The application according to claim 1, characterized in that, The small molecule compound SHIN1 inhibits the proliferation and colony formation of uveal melanoma cells and inhibits tumorigenesis of uveal melanoma in vivo.

3. The application according to claim 1, characterized in that, The drug includes the active ingredient SHIN1 and pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that, The pharmaceutically acceptable excipients include the following components: DMSO, PEG300, Tween 80, and ddH2O.

5. The application according to claim 1, characterized in that, The drug can be administered via intraperitoneal injection, oral administration, intravenous injection, subcutaneous injection, or intravitreal injection.

6. Application of the small molecule compound SHIN1 in the preparation of products with one or more of the following functions: a. Inhibits the proliferation of uveal melanoma cells; b. Inhibits the clonogenic ability of uveal melanoma cells; c. Inhibits the in vivo tumorigenesis ability of uveal melanoma.

7. The application according to claim 6, characterized in that, The product is a drug or reagent.

8. The application according to claim 6, characterized in that, The uveal melanoma cells include human UM cell lines 92.1 and OMM2.3.

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