Small-molecule inhibitor CTP13 targeting SLC25A1 and application of small-molecule inhibitor CTP13

Abstract

The invention belongs to the field of molecular targeted therapy, and discloses a small-molecule inhibitor CTPI3 of a targeted mitochondrial citric acid transporter SLC25A1 and application of the small-molecule inhibitor CTPI3 in tumor therapy. On the basis of an existing inhibitor CTPI2 structure, molecular design is carried out through an induced fit docking (IFD) module of Schrdinger software, and CTPI3 is obtained. Experiments show that the median inhibitory concentration (IC50) of CTPI3 on acute myelogenous leukemia (AML) cell lines (such as Kasumi-1 and THP1) and primary AML cells is obviously lower than that of CTPI2, the synergistic effect of CTPI3 and Venetoclax is enhanced, the mitochondrial ATP yield is reduced, and the TCA cycle metabolite level is affected. Animal experiments prove that CTPI3 can significantly prolong the lifetime of AML model mice and reduce leukemia load (spleen weight is reduced, and bone marrow and peripheral blood GFPcells are reduced), and has no significant toxicity (liver, kidney and brain histopathology is normal). The invention provides an efficient and safe new candidate drug for SLC25A1 targeted therapy.

Description

Technical Field

[0001] The present invention belongs to the field of molecular targeted therapy, and specifically relates to a small molecule inhibitor CTPI3 of the SLC family-related protein SLC25A1 and its application in treating tumors. Background Art

[0002] Acute myeloid leukemia (AML) is a hematopoietic tumor characterized by clonal proliferation, differentiation arrest, and inhibition of apoptosis of malignant stem or progenitor cells. Unlike solid tumors, AML is unresectable and has a poorer clinical prognosis, with an overall 5-year survival rate of less than 50%. To overcome relapse and drug resistance, a variety of small molecule inhibitors targeting aberrantly expressed proteins, such as gilteritinib and sorafenib, have been marketed. These small molecule inhibitors have made significant contributions to improving the prognosis of AML patients.

[0003] Membrane transporters encoded by SLC family genes can transport substrates such as amino acids, nucleotides, glucose, various ions, and drugs through both facilitated transport and secondary active transport. Due to the crucial role of these substrates in cells, SLC transporters are closely linked to cancer. Numerous studies have demonstrated that metabolic reprogramming plays a crucial role in the pathogenesis and drug resistance of AML. The transporter encoded by SLC25A1 is located on the mitochondrial membrane and mediates the entry of citrate from mitochondria into the cytoplasm. Citrate participates in lipid synthesis in the cytoplasm and participates in the tricarboxylic acid cycle within the mitochondria, promoting mitochondrial respiration. Studies have confirmed that SLC25A1 plays an oncogenic role in solid tumors, including lung and breast cancer. Therefore, SLC25A1 is a potential therapeutic target for both hematologic and solid tumors.

[0004] Currently, the only known inhibitor of SLC25A1 is citrate transporter inhibitor 2 (CTPI2). It is derived by replacing the Z group within the Y group of CTPI1, identified in yeast, with a nitro substituent containing a chlorine atom. However, the affinity of CTPI2 for SLC25A1 in AML cell lines remains low. Previous experiments by the team confirmed that CTPI2 cannot be used in animal models due to high concentrations and strong toxicity. Therefore, for better clinical translation, it is necessary to develop novel derivatives with improved affinity for SLC25A1 in hematological malignancies. Currently, there are no marketed small molecule inhibitors targeting SLC25A1.

[0005] Therefore, the development and research of small molecule inhibitors targeting SLC25A1 has become an urgent issue to be addressed. Summary of the Invention

[0006] In response to the problems existing in the prior art, the present invention provides a small molecule inhibitor targeting SLC25A1 and its application. The induced fit docking (IFD) module in the Schrödinger drug design software package was used to flexibly dock the compound into the active site of the target protein, ultimately obtaining the structural formula of the next-generation SLC25A1 inhibitor CTPI3. The IC50 value of the new SLC25A1 inhibitor on leukemia cells and patient specimens was determined using a CCK8 assay. The effect of the new SLC25A1 inhibitor on the degree of apoptosis in leukemia cells was determined using a flow cytometry apoptosis assay. The efficacy and safety of the new SLC25A1 inhibitor in vivo were determined using animal experiments. Furthermore, its inhibitory effect on leukemia cells was determined using an in vitro synergistic assay with the drug venetoclax, thereby effectively studying the value and significance of small molecule inhibitors targeting SLC25A1.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0008] The present invention discloses a small molecule compound CTPI3 having SLC25A1 inhibitory activity, characterized in that the chemical structure of the small molecule compound CTPI3 is as follows: .

[0009] The present invention also discloses that the above-mentioned small molecule compound CTPI3 is used to target the mitochondrial citrate transporter SLC25A1.

[0010] The present invention also discloses the use of the above-mentioned small molecule compound CTPI3 in the preparation of a drug for treating acute myeloid leukemia (AML).

[0011] The present invention also discloses the use of the above-mentioned small molecule compound CTPI3 in the preparation of a drug for treating blood tumors with high SLC25A1 expression.

[0012] The present invention also discloses a method for preparing the compound CTPI3, comprising the following steps: (1) 3-Nitro-4-chlorobenzenesulfonamide, o-bromobenzoic acid, anhydrous potassium carbonate, cuprous iodide and BFMO (bio-iron manganese oxide) were added to an ethanol solution, refluxed at 100°C for 6 h under nitrogen protection, and purified to obtain an intermediate product; (2) The intermediate product was reacted with thionyl chloride, and then isopropanol and triethylamine were added to purify and obtain CTPI3.

[0013] Furthermore, the eluent for column chromatography purification in step (1) is dichloromethane:methanol = 20:1 (containing 1 / 1000 acetic acid), and the eluent for column chromatography purification in step (2) is petroleum ether:ethyl acetate = 5:1.

[0014] The present invention also discloses an anti-tumor pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned compound CTPI3 and a pharmaceutically acceptable carrier.

[0015] The present invention also discloses a synergistic anti-leukemia drug combination, comprising the above-mentioned compound CTPI3 and Venetoclax.

[0016] Compared with the prior art, the present invention has the following beneficial effects.

[0017] A pharmacophore model based on the structure of the original inhibitor CTPI2 was automatically generated using the Receptor-ligandPharmacophores Create protocol under CreatePharmacophores Automatically in DS 3.0. A receiver operating characteristic (ROC) curve was constructed to evaluate candidate pharmacophore models. The selected pharmacophore model with the highest resolution was used to screen the FDA database for specific SLC25A1 inhibitors. Compounds were screened and ranked based on their "Fit value" scores, and small molecules with a "Fit value" greater than 1 were retained for further molecular docking. The compounds were flexibly docked into the active site of the target protein using the induced fit docking (IFD) module in the Schrödinger Drug Design software package. Ultimately, the structural formula of CTPI3, a next-generation SLC25A1 inhibitor, was determined.

[0018] CTPI3 has demonstrated promising efficacy and safety in both in vitro and in vivo models of acute myeloid leukemia, and has demonstrated synergistic effects with venetoclax. CTPI3 demonstrates efficacy and safety not only in mice but also in patient specimens. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1: CTPI3 has certain efficacy and safety in acute myeloid leukemia in vitro and in vivo experiments and can produce synergistic effects with venetoclax. A: The binding mode of CTPI3 and the SLC25A1 active site predicted based on molecular docking analysis; B: Dose-response curve of the inhibitory effect of CTPI3 on ​​AML cell lines (Kasumi-1, THP1, AE9a, MLL-AF9), primary AML blasts and healthy mononuclear cells (MNCs); C: AML model carrying AE-9a treated with CTPI3 Kaplan–Meier survival curves of mice (n=7 per group); D: In vivo therapeutic effect of CTPI3 in the AE9a model: compared with the control group, spleen weight decreased, and GFP⁺ leukemia burden in bone marrow (BM) and peripheral blood (PB) was reduced; E: Body weight monitoring of mice after CTPI3 treatment (left) and hematoxylin-eosin staining of liver, kidney and brain tissues (right), showing no obvious toxicity; F: CTPI3 enhances the sensitivity of AML cells to venetoclax, left: Cell viability and synergy index of Kasumi-1 cells under venetoclax ± CTPI3 treatment; middle: CTPI3 or combined treatment reduces mitochondrial ATP production; right: Heat map showing changes in key TCA intermediates and fatty acid levels after combined treatment. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments, as all technologies implemented based on the present invention fall within the scope of the present invention.

[0021] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0022] Example 1. Preparation of compounds.

[0023] .

[0024] The pharmacophore model based on the structure of the original inhibitor CTPI2 was automatically generated using the Receptor-ligand Pharmacophores Create pharmacophore generation protocol under Create Pharmacophores Automatically in DS 3.0. The ROC curve was used to evaluate the candidate pharmacophore model. The FDA database was screened based on the selected highest resolution pharmacophore model to find specific SLC25A1 inhibitors. According to the score of "Fit value", the compounds were screened and sorted, and the small molecule drugs with "Fit Value" greater than 1 were retained for further molecular docking. We used the induced fit docking (IFD) module in the Schrödinger drug design software package to dock the compounds flexibly into the active site of the target protein. Finally, the structure of the new generation of SLC25A1 inhibitor CTPI3 was obtained.

[0025] Into a 50 mL three-necked flask, 3-nitro-4-chlorobenzenesulfonamide (2.0 g, 0.008 mol), o-bromobenzoic acid (3.2 g, 0.016 mol), anhydrous potassium carbonate (3.5 g, 0.025 mol), cuprous iodide (0.15 g, 0.0008 mol), BFMO (biogenic iron-manganese oxide) (0.5 g, 0.002 mol) were added in turn, followed by 30 mL of ethanol solution. Under nitrogen protection, the suspension was transferred to a 100°C oil bath and stirred for 6h under reflux. The reaction liquid turned gray-green and solid precipitated. After TLC monitoring, the reaction was completed and the reaction liquid was cooled to room temperature. The solid precipitate in the reaction liquid was removed by suction filtration, and the filter cake was discarded. The filtrate was concentrated under reduced pressure to obtain a gray-green oil. Purification was performed by silica gel column chromatography (DCM:MeOH=20:1, with one thousandth of acetic acid added) to obtain 1.8 g of yellow oil. After overnight storage at low temperature, white crystals were precipitated.

[0026] Compound CTPI2 (0.5 g) was added to a 100 mL flask, 20 mL of thionyl chloride was added and refluxed for 1 h. After monitoring the completion of the reaction, the concentrated product was concentrated by rotary evaporation, and 10 mL of isopropanol and 1 mL of triethylamine were added. After stirring for half an hour, column chromatography (PE:EA=5:1) was used for purification to obtain 0.4 g of light yellow solid CTPI3.

[0027] The structure of CTPI3 is: .

[0028] Example two, compound effect experiment.

[0029] 1. Cell line.

[0030] Human acute myeloid leukemia cell lines KASUMI-1 and THP-1 were obtained from Shanghai Cell Bank of Chinese Academy of Sciences. Murine leukemia cell lines AE-9a, MLL-AF9 were gifted by Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences.

[0031] 2. Experimental reagents.

[0032] RPMI-1640 medium, fetal bovine serum (Clark), CCK8 reagent (Bi Yun Tian), apoptosis detection kit (BD), penicillin streptomycin double antibody (BI), CTPI2 (Enamine), Venetoclax (MCE).

[0033] 3. Experimental methods.

[0034] Drug design: The pharmacophore model based on the structure of the original inhibitor CTPI2 was automatically generated using the Receptor-ligand Pharmacophores Create pharmacophore generation protocol under CreatePharmacophores Automatically in DS3.0. The ROC curve was used to evaluate the candidate pharmacophore model. Based on the selected highest resolution pharmacophore model, the FDA database was screened to find specific SLC25A1 inhibitors. According to the score of "Fit value", the compounds were screened and sorted, and the small molecules with "Fit Value" value greater than 1 were retained for further molecular docking. We used the induced fit docking (IFD) module in the Schrödinger drug design software package to dock the compounds flexibly to the active site of the target protein. Finally, the structure of the new generation of SLC25A1 inhibitor CTPI3 was obtained.

[0035] Cell culture: KASUMI-1, HL60, THP-1, U937 and AE-9a cell lines were cultured in 1640 medium + 10% fetal bovine serum + 1% double antibody under the condition of 5% CO2.

[0036] Apoptosis detection: Take the cells that need to be detected, centrifuge to obtain the precipitate, wash with PBS for 2 times, resuspend the cells with 100µL 1×buffer, add 5µL PI dye and 5µL FITC dye, mix evenly, incubate in the flow tube for 15-60min, add volume to 500µL, and then use flow cytometry to detect.

[0037] CCK8 assay for cell proliferation: Count the cells and plate them into a 96-well plate, with 5,000-20,000 cells per well in a 100 µL volume. After 24-120 hours, add 10 µL of CCK8 reagent to each well. Incubate at 37°C for 1-2 hours, and then measure the absorbance at 450 nm.

[0038] Mouse tumor formation experiment: The C57 mice used in this experiment were 6-8 weeks old female mice. After irradiation with 4.75 Gy, the AE-9a cells were washed twice with sterile PBS, centrifuged again, resuspended in 1 mL of PBS, and counted. Based on the count, the cell suspension was prepared with empty 1640 medium, and the final concentration of 1×10 cells per 200 µL of suspension was 1×10 cells. 4 Using a 1 mL sterile syringe in a biosafety cabinet, 200 µL of cell suspension was injected through the tail vein to complete the mouse tumor-bearing experiment.

[0039] Mouse Dosing: 48-72 hours after tumor formation, administration was initiated at 15 mg / kg per mouse, dissolved in 100 µL corn oil, vortexed, and injected intraperitoneally. Administration was continued every other day until the mice developed disease.

[0040] ATP content detection.

[0041] 1. Sample Preparation: (Note: Sample lysis must be performed at 4°C or on ice) Pellet cells by centrifugation in a centrifuge tube, discard the supernatant, and gently flick the cells to disperse. Add 200 µL of lysis buffer per well of a 6-well plate to lyse the cells. To ensure thorough lysis, tap the bottom of the centrifuge tube or use a vortex to ensure that the lysis buffer fully contacts and lyses the cells. Cells typically lyse immediately upon contact with the lysis buffer. After lysis, centrifuge at 12,000 g for 5 minutes at 4°C. Remove the supernatant for subsequent analysis.

[0042] 2. Prepare the standard curve: Thaw the reagents in an ice bath. Dilute the ATP standard solution with ATP lysis buffer to an appropriate concentration gradient. The specific concentration will depend on the ATP concentration in the sample. For initial testing, use concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µM. In subsequent experiments, adjust the standard concentration range based on the ATP concentration in the sample.

[0043] 3. Preparation of ATP Assay Working Solution: Prepare an appropriate amount of ATP Assay Working Solution, using a ratio of 100 µL per sample or standard. Thaw the reagents in an ice bath. Take an appropriate amount of ATP Assay Reagent and dilute it with ATP Assay Reagent Diluent at a ratio of 1:4. This diluted ATP Assay Working Solution is used in subsequent experiments. The ATP Assay Working Solution can be temporarily stored in an ice bath.

[0044] 4. Determination of ATP Concentration: a. Add 100µL of ATP Assay Working Solution to the assay wells or tubes. Incubate at room temperature for 3-5 minutes to deplete the background ATP and reduce the background. Add 100µL of ATP Assay Working Solution to each of 10-20 assay wells or tubes at once to save time. b. Add 20µL of sample or standard to the assay wells or tubes and quickly mix using a pipette. After at least 2 seconds, measure the RLU or CPM using a chemiluminometer or liquid scintillation analyzer. c. Calculate the ATP concentration in the sample based on the standard curve. d. To eliminate errors caused by differences in protein amounts during sample preparation, determine the protein concentration in the sample. Then, convert the ATP concentration to nmol / mg protein.

[0045] Targeted metabolomics testing: Metabolomics testing was commissioned to Novogene.

[0046] 4. Experimental results.

[0047] We screened compounds with a Fit Value > 1 from the FDA-approved drug library based on the "Fit Value" score and further optimized their structures. Subsequently, we used the Induced Fit Docking (IFD) module in the Schrödinger software suite to perform flexible molecular docking of the candidate compounds with the SLC25A1 active site, thereby screening a new generation of inhibitors of CTPI3. This compound has a relatively good docking score and predicted binding conformation ( Figure 1 A). CTPI3 can significantly reduce the survival rate of multiple AML cell lines including Kasumi-1, THP1, AE9a and MLL-AF9, while having low toxicity to normal mononuclear cells ( Figure 1 B). In AE9a and MLL-AF9 mouse leukemia models, CTPI3 treatment significantly prolonged the overall survival of mice ( Figure 1 C). In vivo experiments showed that CTPI3 can effectively reduce spleen size, destroy spleen tissue structure, and reduce GFP in bone marrow, spleen, and peripheral blood. + Proportion( Figure 1D). The body weight of mice remained stable during treatment, and H&E staining of the liver, kidney, and brain showed no obvious histopathological toxicity ( Figure 1 E). In addition, the combination of CTPI3 and venetoclax can achieve a stronger synergistic anti-leukemia effect, manifested by more significant proliferation inhibition and reduced ATP production ( Figure 1 F). Targeted metabolomics further showed that the levels of TCA cycle intermediates and fatty acids were significantly reduced after combined treatment, suggesting that the combined effect of CTPI3 and venetoclax may exceed the sum of their individual effects ( Figure 1 F). Combination Figure 1 The results show that CTPI3 has certain effectiveness and safety, and can produce synergistic effects with venetoclax, a commonly used drug for acute myeloid leukemia at this stage, and has good clinical translation potential.

[0048] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A small molecule compound CTPI3 having SLC25A1 inhibitory activity, characterized in that: The chemical structure of the small molecule compound CTPI3 is shown below: 。 2. The small molecule compound CTPI3 according to claim 1 is used to target the mitochondrial citrate transporter SLC25A1.

3. Use of the small molecule compound CTPI3 according to claim 1 in the preparation of a drug for treating acute myeloid leukemia (AML).

4. Use of the small molecule compound CTPI3 according to claim 1 in the preparation of a drug for treating hematological tumors with high SLC25A1 expression.

5. A method for preparing the compound CTPI3 according to claim 1, comprising the following steps: (1) 3-Nitro-4-chlorobenzenesulfonamide, o-bromobenzoic acid, anhydrous potassium carbonate, cuprous iodide, and BFMO (bio-iron manganese oxide) were added to an ethanol solution, refluxed at 100°C for 6 h under nitrogen protection, and the intermediate product was purified; (2) The intermediate product is reacted with thionyl chloride, and then isopropanol and triethylamine are added to purify CTPI3.

6. The method according to claim 5, characterized in that The eluent for column chromatography purification in step (1) is dichloromethane:methanol = 20:1 (containing 1 / 1000 acetic acid), and the eluent for column chromatography purification in step (2) is petroleum ether:ethyl acetate = 5:

1.

7. An anti-tumor pharmaceutical composition comprising a therapeutically effective amount of the compound CTPI3 according to claim 1 and a pharmaceutically acceptable carrier.

8. A synergistic anti-leukemia drug combination comprising the compound CTPI3 according to claim 1 and Venetoclax.

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