Use of an RNA helicase dhx33 inhibitor in the preparation of a medicament for treating kidney cancer

Abstract

The application belongs to the field of biological medicine, and discloses application of an RNA helicase DHX33 inhibitor in preparation of a medicine for treating or assisting in treating kidney cancer. The application establishes an important role of DHX33 protein in development of kidney cancer, and the provided inhibitor has an effect of inhibiting DHX33 helicase activity, and further causes glutamine metabolism reduction mediated by DHX33 function loss. The inhibitor can induce kidney cancer cell growth inhibition caused by abnormal glutamine metabolism, increase an expression amount of IL-24, and inhibit occurrence and development of kidney cancer, and therefore has important medical development value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to RNA helicase DHX33 inhibitors and their application in the preparation of drugs for the treatment of renal cell carcinoma. Background Technology

[0002] Kidney cancer originates from malignant tumors of the epithelial cells of the renal tubules. Clinically, the most common type is clear cell renal cell carcinoma (ccRCC), accounting for approximately 60-80% of all kidney cancers; followed by papillary renal cell carcinoma (PRCC), accounting for approximately 8-15%; and then chromophobe renal cell carcinoma (ChRCC), accounting for approximately 6-10%. Kidney cancer commonly occurs in smokers, obese individuals, those with a past or family history of the disease, those frequently exposed to radioactive materials, and those undergoing long-term dialysis. Common hereditary kidney cancer mutations occur in genes such as VHL, MET, and FLCN. The risk of developing kidney cancer can be reduced by quitting smoking and alcohol and engaging in appropriate exercise.

[0003] Renal cell carcinoma typically occurs on one side of the kidney, presenting as a spherical tumor with a pseudocapsule. Under a light microscope, renal cell carcinoma cells are often arranged in acinar pattern, but can also be tubular or papillary. Most are solid nests, with a few being cystic. The three main symptoms are hematuria (blood in the urine), kidney pain, and an abdominal mass. Some patients may experience other symptoms such as hypertension, unexplained fever, and anemia. When the triad of symptoms (hematuria, kidney pain, and an abdominal mass) is present, the disease is usually in its middle or late stages.

[0004] Due to the kidney's hidden location, it is difficult to detect cancerous changes, and it is generally discovered in the middle or late stages. However, with the development of medical imaging, the early detection rate is gradually increasing. Currently, kidney cancer diagnosis relies on routine laboratory tests, imaging examinations (chest X-ray, ultrasound), CT scans, and MRI scans for clinical diagnosis and to determine treatment options. CT scans can qualitatively identify most types of kidney cancer. Routine laboratory tests primarily assess the patient's general condition, kidney and liver function, and serve as a reference for prognosis.

[0005] Early-stage localized renal cell carcinoma can be treated with partial nephrectomy (PN) and radical nephrectomy (RN), while intermediate and advanced stages can be treated with immunotherapy and targeted therapy.

[0006] Medical treatment for metastatic renal cell carcinoma includes anti-VEGF / VEGFR (vascular endothelial growth factor or vascular endothelial growth factor receptor) pathways, with representative drugs including sorafenib and sunitinib; and inhibition of the mTOR (mammalian target of rapamycin) pathway, with representative drugs including everolimus and tesiromol.

[0007] For metastatic renal cell carcinoma, anti-tumor angiogenesis and immune checkpoint inhibitors can be used, such as nivolumab in combination with ipilimumab for the treatment of intermediate- to high-risk advanced renal cell carcinoma, and pembrolizumab in combination with axitinib for the treatment of advanced clear cell renal cell carcinoma.

[0008] In summary, there are various treatment options for renal cell carcinoma in clinical practice, but most of them are not perfect and have a high possibility of recurrence. The low prognosis and survival rate of clinical treatment need to be addressed. Overall, we still look forward to the development of safe and more effective drugs for the treatment of renal cell carcinoma. Summary of the Invention

[0009] In view of this, the object of the present invention is to provide an RNA helicase DHX33 inhibitor and its use in the preparation of a medicament or composition for the treatment or adjuvant treatment of renal cell carcinoma.

[0010] To achieve the objectives of this invention, in a first aspect, the present invention provides an RNA helicase DHX33 inhibitor for the treatment or adjuvant treatment of renal cell carcinoma. In embodiments of the present invention, the RNA helicase DHX33 inhibitor (i.e., a DHX33 protein inhibitor) is compound A or a pharmaceutically acceptable salt or prodrug thereof:

[0011]

[0012] This invention discloses for the first time that DHX33 protein can be used as a therapeutic target for renal cell carcinoma. Therefore, in a second aspect, this invention provides the application of the above-mentioned RNA helicase DHX33 as a novel therapeutic target for renal cell carcinoma.

[0013] The reference sequence number of the DHX33 gene on NCBI is: NM_020162.4.

[0014] Thirdly, this invention provides a targeted drug for the treatment or adjuvant treatment of renal cell carcinoma, wherein the target of the drug is RNA helicase DHX33. This targeted drug can inhibit the activity of DHX33 helicase, induce the expression of interleukin-24 (IL-24), and thereby inhibit the growth process of cancer. The active ingredient of this targeted drug is compound A or a pharmaceutically acceptable salt or prodrug thereof. Therefore, this invention also provides the effect of an RNA helicase DHX33 inhibitor in inducing high expression of IL-24, thereby inhibiting the growth of renal cell carcinoma cells, wherein the RNA helicase DHX33 inhibitor is compound A or a pharmaceutically acceptable salt or prodrug thereof.

[0015] Fourthly, the present invention provides the use of the above-mentioned RNA helicase DHX33 inhibitor in the treatment or adjuvant treatment of renal cell carcinoma, wherein the inhibitor is selected from compound A or its pharmaceutically acceptable salt or prodrug.

[0016] Fifthly, the present invention provides the use of the above-mentioned RNA helicase DHX33 inhibitor in the preparation of a medicament or pharmaceutical composition for the treatment or adjuvant treatment of renal cell carcinoma, wherein the inhibitor is selected from compound A or a pharmaceutically acceptable salt or prodrug thereof.

[0017] In embodiments of the present invention, renal cell carcinoma is characterized by positive expression of the DHX33 protein, including but not limited to advanced, unresectable, and standard treatment failure.

[0018] In a sixth aspect, the present invention provides the above-mentioned RNA helicase DHX33 inhibitor for inhibiting glutamine metabolism and thereby inhibiting the growth of renal cell carcinoma cells, wherein the inhibitor is selected from compound A or its pharmaceutically acceptable salt or prodrug.

[0019] In embodiments of the present invention, the frequency or dosage of the RNA helicase DHX33 inhibitor intake can be determined by a physician based on individual physical condition, age, sex, weight, and other factors. In specific embodiments, the intake frequency can range from once to three times per day. In embodiments of the present invention, the intake dose of the RNA helicase DHX33 inhibitor needs to ensure an effective drug exposure of 4000-7500 ng·h / mL per day. In specific embodiments, in mice, the oral dose of the RNA helicase DHX33 inhibitor can be 25 mg-300 mg / kg once, and the intravenous dose can be 2.5 mg-25 mg / kg per injection. In a specific implementation plan, in mice, the oral dose of the RNA helicase DHX33 inhibitor can be, for example, 35 mg-290 mg / kg, 45 mg-280 mg / kg, 55 mg-270 mg / kg, 65 mg-260 mg / kg, 75 mg-250 mg / kg, 85 mg-240 mg / kg, 95 mg-230 mg / kg, 105 mg-220 mg / kg, 115 mg-210 mg / kg, 125 mg-200 mg / kg, 135 mg-190 mg / kg, 145 mg-180 mg / kg, or 155 mg-170 mg / kg per dose. When administered intravenously in mice, the single injection dose of the RNA helicase DHX33 inhibitor can be, for example, 3.0 mg-24.5 mg / kg, 3.5 mg-24 mg / kg, 4.0 mg-23.5 mg / kg, 4.5 mg-23 mg / kg, 5.0 mg-22.5 mg / kg, 5.5 mg-22 mg / kg, 6.0 mg-21.5 mg / kg, 6.5 mg-21 mg / kg, 7.0 mg-20.5 mg / kg, 7.5 mg- 20mg / kg, 8.0mg-19.5mg / kg, 8.5mg-19mg / kg, 9.0mg-18.5mg / kg, 9.5mg-18mg / kg, 10.0mg-17.5mg / kg, 10.5mg- 17.0mg / kg, 11.0mg-16.5mg / kg, 11.5mg-16mg / kg, 12.0mg-15.5mg / kg, 12.5mg-15.0mg / kg or 13.0mg-14.5mg / kg.

[0020] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0021] This invention establishes the important role of DHX33 protein in the development and progression of renal cell carcinoma. The provided RNA helicase DHX33 inhibitor inhibits DHX33 helicase activity, thereby inducing high expression of IL-24 and inhibiting glutamine metabolism, thus suppressing the growth of renal cell carcinoma. This RNA helicase DHX33 inhibitor can significantly inhibit the growth of renal cell carcinoma, thereby achieving the goal of treating renal cell carcinoma and thus has significant pharmaceutical development value. Attached Figure Description

[0022] Figure 1 In a preferred embodiment of the present invention, the expression level of DHX33 protein in representative human renal cell carcinoma tissue was significantly increased compared with that in normal renal tissue.

[0023] Figure 2 To analyze the expression level of DHX33 protein in renal cell carcinoma cells compared with normal HSF (human skin fibroblast) cells.

[0024] Figure 3 To analyze the expression level of DHX33 protein in renal cell carcinoma cells compared with lung cancer cell lines that highly express DHX33.

[0025] Figure 4 In a preferred embodiment of the present invention, the half-maximal inhibitory concentration (IC50) of A498 cells treated with RNA helicase DHX33 inhibitor compound A was analyzed. 50 The value is 30 nM.

[0026] Figure 5 In a preferred embodiment of the present invention, the half-inhibitory concentration (IC50) of Caki cells treated with RNA helicase DHX33 inhibitor compound A was analyzed. 50 The value is 30 nM.

[0027] Figure 6 This is an analysis of the clonal growth of A498 cells treated with compound A, an RNA helicase DHX33 inhibitor, in a preferred embodiment of the present invention. The results show the clonal growth of cells treated with DMSO and compound A (30 nM).

[0028] Figure 7 This invention provides a preferred embodiment of the study analyzing the changes in transcriptional levels of genes involved in glutamine in renal cancer cells A498 under different treatment times with RNA helicase DHX33 inhibitor compound A.

[0029] Figure 8 This invention provides a preferred embodiment for analyzing the IL24-ELISA quantitative analysis of A498 cells treated with RNA helicase DHX33 inhibitor compound A for 16 hours.

[0030] Figure 9 In a preferred embodiment of the present invention, the changes in gene transcription levels involved in the glutamine metabolism pathway in renal cancer cells A498 after treatment with a lentivirus that knocks down the expression level of DHX33 protein for 72 hours were described. Detailed Implementation

[0031] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0032] 1. Cell Culture

[0033] Human renal cell carcinoma lines A498 and Caki-2 were purchased from Wuhan Procell Biotechnology Co., Ltd. A498 cells were cultured in Minimum Essential Medium (containing NEAA) complete medium, which contained 10% fetal bovine serum (FBS), 2 mM L-glutamine, non-essential amino acids, streptomycin, and penicillin. Caki-2 cells were cultured in McCoy's 5A complete medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, non-essential amino acids, streptomycin, and penicillin. Human skin fibroblasts (HSF) were purchased from Shanghai Bosen Biotechnology Co., Ltd., cultured in Dulbecco's Modified Eagle Medium (high glucose) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, non-essential amino acids, streptomycin, and penicillin.

[0034] 2. Western blot analysis

[0035] Total protein was extracted from renal cell carcinoma cells A498 and Caki-2, and normal HSF cells after reaching 80% confluence. The specific extraction method involved suspending cells in a cell lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton-X-100, 1% SDS, supplemented with protease and phosphatase inhibitors). After incubation on ice for 5 minutes, the cell lysates were further disrupted by sonication. The supernatant was collected by centrifugation, and 5 μL of the sample was used for protein content determination using the BCA method. The sample was then loaded with appropriate loading buffer and PBS buffer and boiled at 95°C for 5 minutes. Equal volumes of each sample were used for protein analysis (each sample loaded with 50 μg of total protein). The protein was then transferred to a polyvinylidene fluoride (PVDF) membrane. Electroporation was performed for 1 h 30 min, followed by blocking for 30 min. The primary antibody was diluted with 1×TBST and incubated with the membrane overnight at 4°C. The membrane was then washed several times with 1×TBST buffer and incubated at room temperature for 2 hours with a secondary antibody (Anti-rabbit IgG) diluted in 1×TBST. The blot was visualized using an ECL kit (Thermo Fisher). Antibodies used were: anti-GAPDH, Absin (abs830030); anti-DHX33, Bethy1 (AA300-800A).

[0036] 3. Real-time quantitative PCR

[0037] To analyze the molecular mechanism by which DHX33 protein promotes the growth of renal cell carcinoma cells, quantitative PCR (SYBR greensupermix (Bio-Rad)) was used to analyze the expression changes of important genes in renal cell carcinoma cells. Cells were seeded at an appropriate density in 6-well plates. The next day, an appropriate concentration of the compound was added to Minimum Essential Medium (containing NEAA) complete medium. The compound was used to treat cells for 0 hours and 6 hours, after which cells were harvested and RNA was extracted. Quantitative PCR analysis was then performed on the RNA samples. The target genes analyzed were: SLC10A5, SLC12A2, SLC2A1, GLUL, SLC3A2, SLC7A5, ASNS, GPT2, PAST1, PYCR1, SLC1A5, GOT1, IL-24, and IL-7. Primers were designed using the online "realtime PCR tool" of IDT (http: / / sg.idtdna.com / site) and purchased from BGI (Shenzhen) Co., Ltd.

[0038] The primer sequences for the genes involved in glutamine metabolism and interleukin in human cells are as follows (all primers are from 5'-3'):

[0039]

[0040] 4. Half-inhibitory concentration (IC50) 50 ) Measurement

[0041] Renal cancer cells were divided into 1x10 4 Seed 100 μL / well of cells onto a 96-well plate and allow the cells to adhere completely. Add the compound to Minimum Essential Medium (containing NEAA) at concentrations of 19 nM, 39 nM, 78 nM, 156 nM, 312 nM, 625 nM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM, and mix thoroughly using a multichannel pipette. After 72 hours of incubation, add the compound to the Minimum Essential Medium (containing NEAA) in the 96-well plate using CCK-8 reagent (Shanghai Yisheng Biotechnology Co., Ltd.) according to standard procedures. Incubate for 1 hour, then read the plate using an ELISA reader (OD). 450nm The experiment was repeated three times, and inhibition curves of the compound at different concentrations were plotted (e.g., Figure 2 (As shown), calculate the half-inhibitory concentration (IC50) of the compound. 50 ).

[0042] 5. Immunohistochemical analysis

[0043] Renal cell carcinoma tissue microarrays were purchased from Shanghai Weiao Biotechnology Co., Ltd. The microarray contained 80 columns of papillary renal cell carcinoma tissue, with non-tumor tissue areas used as controls. Immunohistochemical (IHC) staining was performed according to the manufacturer's instructions. The antibody used was anti-DHX33 (Santa Cruz Biotech, Inc.). Experimental results ( Figure 1 The dark areas stained in the middle show that DHX33 protein is highly expressed in various human renal cell carcinoma tissues (especially in the cell nucleus).

[0044] 6. IL24-ELISA detection

[0045] Cancer cells were seeded onto 6-well plates and allowed to adhere completely. Compound A was added to Minimum Essential Medium (containing NEAA) at concentrations of 0 nM, 20 nM, and 40 nM, and mixed thoroughly. After 16 hours of incubation with the compound and cells, the cell supernatant was collected. The IL24-ELISA kit is used to detect IL-24 levels. Two replicates are set up. After a series of chemical reactions involving biotinylate antibody and enzyme, the IL-24 levels at different compound concentrations are plotted using an ELISA reader at OD (450nm). The IL-24 levels in cancer cells are analyzed by measuring the OD (450nm) values.

[0046] 7. Clonal growth of cells (Foci)

[0047] 2.4×10 3 Each cell was cultured in 10.0 mL of Minimum Essential Medium (containing NEAA) complete medium (with or without inhibitors) in a 100 mm cell culture dish at 37°C in a CO2 incubator, with the medium being changed weekly. Cell clone growth was observed. After 2-3 weeks, when the cell clones had grown to a sufficient size, Geimsa staining was performed, and photographs were taken for statistical analysis.

[0048] 8. Data Statistical Analysis

[0049] Data are expressed as mean + SD. Statistical significance was determined using the Student's t-test. A p-value < 0.05 was indicated by *; a p-value < 0.01 was indicated by **; and a p-value < 0.001 was indicated by ***.

[0050] Example 1. High-efficiency expression of DHX33 protein in papillary renal cell carcinoma

[0051] In this embodiment, immunohistochemistry was used to analyze the expression of DHX33 protein in human papillary renal cell carcinoma tissue.

[0052] We analyzed microarrays of paraffin-embedded tissue sections from human papillary renal cell carcinoma (HCC), including 80 different HCC tissues, with non-tumor tissue regions serving as normal tissue controls. The paraffin-embedded tissue microarrays were first incubated at 60°C for 30 min, then rapidly dewaxed in tissue clearing and dewaxing solution, and gradually hydrated in a series of solutions with progressively decreasing ethanol concentrations (100%, 95%, 70%, 50%, and 25%) (gently agitated for 5 min each time, repeated once for each concentration). Finally, hydration was continued for 10 min in distilled water. Antigens were then presented in a steam oven with 50 mM Tris-buffered saline (pH 9.0) for 40 min, followed by cooling to room temperature. The tissues were then incubated in a methanol solution containing 1% H₂O₂ to inactivate endogenous peroxidase. After blocking with 10% FBS at room temperature for 1 h, the tissues were incubated with primary antibody overnight at 4°C. Then, a standard protocol was performed using the DAKO kit (DAKO, Denmark) as recommended by the manufacturer. The antibody used was anti-DHX33 (Santa Cruz Biotech, Inc.). Experimental results ( Figure 1 The dark, circular areas shown in the image indicate high expression of DHX33 protein in various human papillary renal cell carcinoma tissues, particularly in the cell nucleus. Table 2 below provides data for 80 cancerous and non-tumor tissue regions, showing that approximately 44% of pathological tissues from papillary renal cell carcinoma patients exhibit high DHX33 protein expression. Analysis of pathological sections revealed that DHX33 expression was negative in non-tumor tissue regions.

[0053] Table 2: Pathological information and immunohistochemical analysis of DHX33 protein in 80 types of human papillary renal cell carcinoma tissues

[0054]

[0055]

[0056]

[0057] Note: For the DHX33 protein staining score in this table, a score of 4 or higher indicates positive expression, and a score of 3 or lower indicates negative expression.

[0058] Example 2. DHX33 protein is highly expressed in renal cell carcinoma cells and lowly expressed in normal cells.

[0059] In addition to analyzing DHX33 protein expression at the tissue level, expression analysis of DHX33 protein was also performed at the cell line level. HSF cells were selected as a control, and the expression of DHX33 protein in two cancer cell lines, A498 and Caki-2, was analyzed. The results are as follows: Figure 2 As shown, after adjusting for the internal control GAPDH content, protein expression in cancer cells was significantly higher than in normal cells. The DHX33 protein content in A549 cells was 0.9 times higher than that in HSF, while the DHX33 protein content in Caki cells was 0.5 times higher than that in HSF. This result validates the previous histological analysis at the cellular level. Further analysis of the DHX33 protein content in A498 cells and the known DHX33-highly-expressing lung cancer cell lines H1299 and A549 yielded the following results: Figure 3 As shown, the DHX33 protein content in A498 cells is comparable to that in A549 and H1299 cells, indicating that the DHX33 protein content in A498 cells is relatively high.

[0060] Example 3. DHX33 inhibitors can effectively inhibit the growth and proliferation of renal cell carcinoma cells, but have no inhibitory effect on normal cells.

[0061] To analyze the inhibitory effect of DHX33 inhibitors on renal cell carcinoma, we selected two representative renal cell carcinoma strains, A498 and Caki, and performed three independent half-maximal inhibitory concentration (IC50) tests on compound A. 50 The inhibition curve of the cells was detected as follows: Figure 4 and Figure 5 As shown, the measured half-maximal inhibitory concentration (HMC) was approximately 30 nM. To compare the sensitivity of normal cells to the DHX33 inhibitor, we also analyzed the inhibitory effect of compound A in HSF, and the results are as follows: Figure 6 As shown, compound A had virtually no significant inhibitory effect on HSF. In addition to the half-maximal inhibitory concentration (WMC), cell clonal growth analysis was also performed, using the methods described above. We selected a representative renal cancer cell line, A498, for the experiment. At a concentration of 30 nM, we found that the DHX33 inhibitor compound A significantly inhibited the growth of renal cancer cells. Figure 7 ).

[0062] Example 4. DHX33 inhibitor induces IL-24 expression in renal cell carcinoma cells.

[0063] Interleukin IL-24, derived from melanoma cells and megakaryocytes, belongs to the IL-10 family and can inhibit tumor cell proliferation and reduce tumorigenicity by regulating the cell cycle and inducing apoptosis. To analyze whether the DHX33 inhibitor compound A induces increased IL-24 expression in renal cancer cells, thereby inhibiting cancer cell growth, we used an ELISA kit to analyze the changes in IL-24 levels in A498 cancer cells after treatment with different doses of compound A (including 0 nM, 30 nM, and 60 nM). The results are as follows: Figure 8 As shown, the expression level of IL-24 increased with increasing dosage of compound A. This experiment was conducted using... The IL-24-ELISA assay kit showed that the expression level of IL-24 was increasing.

[0064] Example 5. DHX33 inhibitors can regulate the expression of genes involved in glutamine metabolism enzymes in renal cell carcinoma cells.

[0065] Tumor cells can obtain nutrients and energy from glutamine, which also supports intracellular homeostasis and promotes tumor growth. The expression and activity of key enzymes in the glutamine metabolic pathway can alter tumor cell survival. Among them, glutamine synthase can promote tumor cell proliferation in different species and types of tumor cells. Tumor cell growth can be inhibited by inhibiting glutamine metabolism. Genes related to glutamine include SLC10A5, SLC12A2, SLC2A1, GLUL, SLC3A2, SLC7A5, ASNS, GPT2, PAST1, PYCR1, SLC1A5, and GOT1. To determine whether the DHX33 protein is an important factor affecting lipid glutamine metabolism, we used real-time quantitative PCR to convert the DHX33 protein into its complementary DNA molecules using reverse transcriptase, and then used these DNA molecules as templates to analyze the transcript levels of the aforementioned genes. Figure 9 As shown, we knocked down the DHX33 protein (shDHX33) and established a control group (SCR). We then used real-time quantitative PCR to analyze the transcriptional changes of these transcripts in glutamine metabolic enzymes. Figure 9 As shown, except for SLC12A2, the other SLC10A5, SLC2A1, GLUL, SLC3A2, SLC7A5, ASNS, GPT2, PAST1, PYCR1, SLC1A5, and GOT1 all showed significant changes.

Claims

1. The use of an RNA helicase DHX33 inhibitor in the preparation of a medicament or pharmaceutical composition for the treatment or adjuvant treatment of clear cell renal cell carcinoma, characterized in that, The inhibitor is selected from compound A or a pharmaceutically acceptable salt thereof: Compound A.

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