Pharmaceutical application of a targeting CD36 inhibitor

Abstract

The present invention discloses a pharmaceutical application of a CD36 inhibitor. Based on the CD36 protein structure, through the Virtual Flow virtual drug screening platform, the top 100 small molecules are screened from the drug target library for actual screening to screen for active small molecules that inhibit the function of the CD36 protein, and the effectiveness of the active small molecules of CD36 is characterized by inhibiting the oxLDL uptake level. Six potential active compounds including F449-4678 are screened out from the active small molecules. Through MST small molecule and protein binding affinity test, Dil-oxLDL flow cytometry is used to detect the IC50 of the small molecule inhibitor, and CCK-8 is used to detect cell proliferation; in addition, it is verified on LLC-LUC xenograft tumor mice that when the active small molecule is administered to the mice, it can effectively inhibit the growth of tumors, thereby achieving an anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to the screening of an inhibitor targeting cluster of differentiation 36 (CD36), and the preparation of a drug for inhibiting various malignant tumors and its application. Background Art

[0002] Cluster of differentiation 36 (CD36) is a membrane protein receptor located on the cell surface that can bind and transport fatty acids. CD36, a type II transmembrane protein with an extracellular loop, includes two transmembrane domains, a large extracellular domain containing a ligand-binding site, and two short cytoplasmic tails at the N-terminus and C-terminus. The motif in the N-terminal transmembrane region is responsible for the dimerization of the CD36 molecule, and the short cytoplasmic tail at the C-terminus can bind to Src-family protein tyrosine kinase (SFK) to initiate most CD36-mediated signal transduction. The stability of the extracellular domain is closely related to three pairs of disulfide bridges. The two hydrophobic pockets formed by the long extracellular loop are the main sites where CD36 binds different ligands to exert different biological functions. The hydrophobic pocket of entrance 1 is the main transport channel for ligands, including oxidized low-density lipoprotein (Ox-LDL) and advanced glycation end products (AGEs). The hydrophobic pocket of entrance 2 is considered to potentially provide a channel for fatty acid (FA) transport in the crystal structure study of CD36. The main binding sites of CD36 with FA, thrombospondin-1 (TSP-1), Ox-LDL, and the erythrocyte surface adhesion protein PfEMP1 of Plasmodium-infected erythrocytes are amino acids at positions 127-297, 93-120 (CLESH domain), 157-171, and 97-110, respectively.

[0003] In a normal human body, CD36 is mainly distributed in tissues such as fat, myocardium, skeletal muscle, breast, colon, duodenum, and bone marrow [8]. Existing studies have found that CD36 is highly expressed in tumor tissues such as lung cancer, liver cancer, and breast cancer. The expression of CD36 is not only tissue-specific but also cell-specific. CD36 in a normal human body is expressed on the surface of a variety of immune cells and non-immune cells. Immune cells mainly include macrophages, monocytes, dendritic cells, T cells, and B cell subsets. Non-immune cells mainly include platelets, immature red blood cells, podocytes, skeletal muscle cells, adipocytes, cardiomyocytes, endothelial cells, and certain special epithelial cells. Even in the same tissue, the expression of CD36 in different cell types is different. For example, in tumor tissues, the expression of CD36 on the surface of cancer cells is higher than that of endothelial cells in the tumor stromal tissue. In addition, CD36 may also be present in endosomes, endoplasmic reticulum, and mitochondria and may shuttle between these organelles through vesicle trafficking.

[0004] Currently, there is little research on CD36 in tumors, and there is no inhibitor of CD36 with ideal effects on the market. We found that the expression level of CD36 in tumor tissues is significantly higher than that in adjacent tissues, and it is significantly increased in tumor tissues and is closely related to the survival rate of patients. Therefore, it has become an urgent problem to be solved to develop a highly effective and specific small molecule inhibitor of CD36. Summary of the Invention

[0005] To solve the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide some potential active small molecules with CD36 activity inhibition.

[0006] The object of the present invention in the field is to provide the applications of these compounds.

[0007] The object of the present invention can be achieved by the following technical solutions:

[0008] Technical solution: The six potential active small molecules F449-4678, Y501-4688, P132-0554, G748-0096, D294-4966, L977-0025 with CD36 activity inhibition described in this application have the following structures:

[0009]

[0010] The CD36 inhibitor was obtained by downloading from the PDB database based on the crystal complex structure of CD36 (PDB ID: 5LGD); the co-crystallized small molecule site in its extracellular domain (Gly 30–Asn 439) was defined as the molecular docking active site; the Glide docking algorithm in the Schrödinger software package was used for virtual screening of the ChemDiv commercial compound database (including approximately 2.5 million compounds after preparation); after screening, the top 1000 compound molecules were screened based on Lipinski's rule of five, cluster analysis, etc., and the top 100 diverse molecules were obtained for subsequent experimental detection.

[0011] This application also discloses the use of the CD36 inhibitor in the preparation of anti-tumor drugs.

[0012] This application further discloses the use of the CD36 inhibitor in the preparation of drugs for preventing and / or treating tumors.

[0013] As a preference of the present invention, the tumor is selected from non-small cell lung cancer, pancreatic cancer, liver cancer, and breast cancer.

[0014] Furthermore, the drug can inhibit the uptake of oxLDL, and at the same time inhibit the proliferation of tumor cells, thereby achieving the effect of treating tumors.

[0015] Beneficial effects: Based on the CD36 protein structure, through the Virtual Flow virtual drug screening platform, the top 100 small molecules were screened from the drug target library (Chemdiv) for actual screening to screen out active small molecules that inhibit the function of CD36 protein, and the effectiveness of the active small molecules of CD36 was characterized by inhibiting the oxLDL uptake level. Six potential active compounds, chemdiv ID Y501-4688, P132-0554, G748-0096, D294-4966, L977-0025, F449-4678, were screened out from the active small molecules. Through the MST small molecule and protein binding affinity test, the IC50 of the small molecule inhibitor was detected by Dil-oxLDL flow cytometry, and cell proliferation was detected by CCK-8; in addition, through verification on LLC-LUC xenograft tumor mice, when the active small molecule F449-4678 was given to the mice, it could effectively inhibit the growth of tumors, thereby achieving the anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Schematic diagram of building a virtual drug screening platform and screening CD36 inhibitors by combining Dil-oxLDL uptake

[0017] Figure 2: Screening of small molecules binding to CD36 by Dil-oxLDL uptake experiments in Raw264.7 cells and CD36-overexpressing 293T cells

[0018] Figure 3 : Affinity of bioactive small molecules for CD36 protein

[0019] Figure 4 : Verification of the IC50 of bioactive small molecules by cell uptake Dil-oxLDL experiments

[0020] Figure 5 : Effect of CD36 inhibitors on tumor cell proliferation

[0021] Figure 6 : CD36 inhibitors inhibit the growth of LLC-LUC xenograft tumor mice Specific implementation manners

[0022] The technical solution of the present invention will be further described in detail below in combination with specific implementation manners:

[0023] Experimental examples:

[0024] I. Experimental materials

[0025] 1. Cell lines: 293T, 293F, MCF-7, A549, HepG2, and Miapaca cells were purchased from American Type Culture Collection (ATCC).

[0026] 2. Main experimental reagents

[0027] ChemDiv small molecules (purchased from ChemDiv), Human DiI-Oxidized Low Density Lipoprotein (Human DiI-Ox-LDL) human red fluorescent-labeled oxidized low density lipoprotein (purchased from Shanghai Yeasen), premium fetal bovine serum [South American origin] (purchased from Genial), DEME medium (purchased from ThermoFisher), II. Experimental methods

[0028] 1. Computer-aided virtual screening of small molecule compounds

[0029] The CD36 inhibitor was obtained by downloading from the PDB database based on the crystal complex structure of CD36 (PDB ID: 5LGD); the co-crystallized small molecule site in its extracellular domain (Gly 30–Asn 439) was defined as the molecular docking active site; the Glide docking algorithm in the Schrödinger software package was used for virtual screening of the ChemDiv commercial compound database (including approximately 2.5 million compounds after preparation); after screening, the top 1000 compound molecules were screened based on Lipinski's rule of five, clustering analysis, etc., and the top 100 diverse molecules were obtained for subsequent experimental detection.

[0030] 2. CD36 Protein Expression and Purification

[0031] 2.1 The expression vector of the CD36 protein is the eukaryotic expression vector pCDNA3.1-CMV, with Amp resistance, and it is expressed and purified using 293F cells.

[0032] 2.2 293F Cell Culture and Protein Expression

[0033] 2.2.1 Cells were inoculated into 300 ml of medium in a 1 L shake flask at an inoculation density of 0.5*10 6 cells / ml. Note: The volume of medium that can be filled in a 1 L shake flask is 150 ml - 300 ml.

[0034] 2.2.2 Incubate in a shaker incubator at 37 °C, 120 rpm, and 5% carbon dioxide concentration for 24 h until the cell density reaches 1X10 6 cells / ml (the cells need to double in number every 24 h).

[0035] 2.2.3 Pipette 300 μg of plasmid (filtered and sterilized) into 30 ml of PBS, then vortex for 3 seconds to mix well.

[0036] 2.2.4 Add 1.2 ml of filtered and sterilized PEI solution (0.5 mg / ml) to the PBS / DNA mixture.

[0037] 2.2.5 Let the PEI-DNA mixture stand at room temperature for 20 min.

[0038] 2.2.6 Add the DNA / PEI mixture to the cells, and the cell density must reach 1X10 6 cells / ml.

[0039] 2.2.7 After transfection, incubate in a shaker incubator at 37 °C, 120 rpm, and 5% carbon dioxide concentration for 48 h.

[0040] 2.2.8 Harvest the intracellular protein by centrifugation at 3000 g for 5 min and store it at -80 °C

[0041] 2.3 Purification of CD36 (with flag tag) protein

[0042] 2.3.1 After 48 h of transfection, centrifuge to collect cell precipitate at 3000 rpm, and resuspend the cells with NP-40 lysis buffer. NP40 lysis buffer: 50 mM Hepes, 150 mM NaCl, 1% NP40 plus PMSF protease inhibitor, pH 7.4

[0043] 2.3.2 Grind the cell suspension with a Dounce homogenizer 20 times to break the cells, lyse on ice for 40 min, and centrifuge at 14000 rpm for 10 min

[0044] 2.3.3 Take 1 ml of Flag Resin packing material, wash it 3 times with HEPES buffer (50 mM Hepes, 150 mM NaCl, pH 7.4), 10 times the bed volume each time, centrifuge at 3000 rpm for 5 min, and carefully discard the supernatant

[0045] 2.3.4 Binding of protein to Flag packing material: Incubate at 4 °C with a rotator for 4 h or overnight, and under room temperature conditions, rotate and mix end to end with a tube rotator for at least 1 hour

[0046] Note: To achieve the best elution effect, the incubation time can be extended or the incubation temperature can be reduced

[0047] 2.3.5 Centrifuge at 3000 rpm for 5 min, carefully remove the supernatant, add Hepes buffer (plus PMSF), and wash the packing material three times. Remove as much supernatant as possible without disturbing the packing material and enter the elution step

[0048] 2.3.6 Elute with competitive elution buffer solution: Add 2 mL of 100 μg / ml DYKDDDDK octapeptide elution buffer solution to the washed packing material, and gently resuspend the packing material with the tip of a wide-bore pipette. Incubate at 4 °C for 2 h, and gently tap the test tube 1-2 times during incubation. After incubation, centrifuge at 3000 rpm for 5 min. Carefully transfer the supernatant to a new vial for further use

[0049] 2.3.7 Run a gel to verify the quality of protein purification

[0050] 2.4 Separation of CD36 (with flag tag) protein by AKTA protein purification system

[0051] Purify and obtain CD36 (with flag tag) protein by molecular sieve

[0052] 3. Microscale thermophoresis

[0053] Use the MST experiment to screen whether small molecules bind to the CD36 protein. The specific experimental steps are as follows:

[0054] 3.1 Buffer exchange

[0055] For the labeling operation, the protein needs to be dissolved in a labeling buffer with an appropriate pH. The buffer for dissolving the protein should not contain primary amine compounds (such as ammonium ions, Tris, glycine, ethanolamine, triethylamine, glutathione) or imidazole, as these compounds will significantly reduce the protein labeling efficiency. Low protein purity or the presence of carrier proteins such as BSA in the protein sample

[0056] will all affect the protein labeling. The specific operation is as follows:

[0057] 3.1.1 Add 3 ml of double-distilled water to dissolve the buffer salt in the vial.

[0058] 3.1.2 Invert and mix column A well, twist off the small cap at the bottom of column A, and unscrew the column cap.

[0059] 3.1.3 Place the column in a 1.5 - 2 ml EP tube, centrifuge at 3000 rpm for 1 min to remove the excess liquid in column A, add 300 μl of labeling buffer, centrifuge at 3000 rpm for 1 min, and wash 3 times. Add 40 - 100 μl of the protein solution to column A, place column A in a new EP tube, centrifuge at 3000 rpm at 4°C for 2 min to obtain the protein in the exchanged buffer.

[0060] 3.2 Protein labeling

[0061] 3.2.1 Adjust the protein concentration to 2 - 20 μM with the labeling buffer.

[0062] 3.2.2 Add 50 μl of DMSO to dissolve the solid dye (the concentration of the dye is approximately 650 μM at this time).

[0063] 3.2.3 Mix well to fully dissolve the dye, and dilute the dye concentration to 2 - 3 times the protein concentration with the labeling buffer.

[0064] 3.2.4 Mix the dye and protein in a 1:1 volume ratio, incubate at room temperature in the dark for 30 min, and simultaneously prepare step 3.3

[0065] 3.3 Protein purification

[0066] To optimize the MST experimental results, the unreacted excess free dye needs to be removed by passing through the column. The purity of the labeled protein can be obtained by measuring the ratio of the protein to the dye (for example, the ratio of the two can be measured by measuring the absorbance values of the protein at 280 nm and the dye at 650 nm, with a molar absorbance of 250 M-1cm-1).

[0067] 3.3.1 Empty the storage solution in column B and equilibrate column B with protein storage buffer or the test solution for the final MST test (a total of 8 ml is required. Rely on gravity to let the buffer flow out of column B to equilibrate the column).

[0068] 3.3.2 Add 200 μl of the labeling reaction solution to the middle position of column B, let the reaction solution completely immerse column B, and discard the outflowing liquid.

[0069] 3.3.3 Add another 300 μl of the rinsing solution to column B and repeat operation 3.3.2.

[0070] 3.3.4 Add 600 μl of the rinsing solution, collect the eluted liquid (the first two drops of the outflowing liquid can be discarded). Measure the ratio of protein to dye by spectroscopy and aliquot the protein.

[0071] 3.4 MST reaction

[0072] Take the small molecule mother liquor, dilute it successively by 2-fold for a total of 16 PCR tubes. Add the labeled protein in 3.3.4 to each tube in a volume ratio of 1:1, mix well, and aspirate to the top of the capillary using a capillary, avoiding air bubbles inside the capillary. Place the capillary into the Nano Temper MST instrument to read the fluorescence value, and use the instrument software program to calculate the binding constant KD value.

[0073] 4. Raw264.7 cell Dil-oxLDL uptake experiment

[0074] Dissolve the small molecule: The mother liquor concentration is 50 mM, dissolve it with DMSO, and dilute it to 10 mM for standby

[0075] 4.1 Revive and plate Raw264.7 cells

[0076] 4.2 Co-incubate the small molecule drug and Dil-Ox-LDL in Raw264.7 cells

[0077] Divide the cells into 3 groups: one group is the control,

[0078] one group (+5 μg / ml Dil-Ox-LDL)

[0079] one group added with (the small molecule drug and 5 μg / ml Dil-Ox-LDL), note to add the positive control

[0080] 4.3 Seed cells in a 24-well plate, then after culturing at 37 °C for 24 h, discard the serum-containing medium, add 500 μl of fresh serum-free medium overnight and co-culture with the small molecule inhibitor for starvation treatment

[0081] 4.4 Incubation with Dil-Ox-LDL: Add 200 μl of 5 μg / ml Dil-Ox-LDL to each well and incubate for 2 h at 37°C.

[0082] 4.5 Wash 3 times with 200 μl of PBS for 5 min each time, digest with trypsin, terminate digestion with DMEM containing 2% FBS, centrifuge at 3000 rpm, resuspend with 400 μl of PBS, and detect the FL2 channel by flow cytometry.

[0083] 5. Dil-oxLDL uptake experiment in 293T cells overexpressing CD36

[0084] Dissolution of small molecule: The concentration of the stock solution is 50 mM, dissolved in DMSO and diluted to 10 mM for standby.

[0085] 5.1 Construction of 293T cell line expressing CD36

[0086] Transfect CD36 into 293T cells and obtain transfected cells after 48 h.

[0087] 5.2 Co-incubation of small molecule drug and Dil-Ox-LDL in HEK-293T-CD36 cells

[0088] Divide the cells into 3 groups: one group is the control,

[0089] one group (+5 μg / ml Dil-Ox-LDL)

[0090] one group added with (small molecule drug and 5 μg / ml Dil-Ox-LDL), note to add positive control 5.3 Seed the cells in a 96-well plate, then after culturing at 37°C for 24 h, discard the serum-containing medium, add fresh serum-free medium and incubate overnight for starvation treatment.

[0091] 5.4 Incubation with Dil-Ox-LDL: Add 200 μl of 5 μg / ml Dil-Ox-LDL to each well and incubate for 2 h at 37°C.

[0092] 5.5 Wash 3 times with 200 μl of PBS for 5 min each time, digest with trypsin, terminate digestion with DMEM containing 2% FBS, centrifuge at 3000 rpm, resuspend with 400 μl of PBS, and detect the FL2 channel by flow cytometry.

[0093] 6. Cell resuscitation and culture

[0094] 6.1 Medium preparation:

[0095] Cell medium: DMEM medium containing 10% FBS

[0096] 6.2 Cell resuscitation:

[0097] Take out the cryopreservation tube from the liquid nitrogen container and directly immerse it in warm water at 37°C, shaking it from time to time to make it melt as soon as possible. Take out the cryopreservation tube from the 37°C water bath, open the lid, suck out the cell suspension with a pipette, add it to a centrifuge tube and add more than 10 times the culture medium, and mix well. Centrifuge at 1000 rpm for 5 min. Discard the supernatant, add complete medium to resuspend the cells, count, adjust the cell density, inoculate into a culture flask, and incubate statically in a 37°C incubator. Replace the culture medium once the next day and continue culturing.

[0098] 6.3 Cell passage and culture:

[0099] When the cells grow to 80% of the culture dish, passage the cells, digest with trypsin for 5 min, add complete medium to terminate the digestion, centrifuge at 1000 rpm for 5 min. Discard the supernatant, add complete medium to resuspend the cells, count, adjust the cell density, inoculate into a culture flask, and incubate statically in a 37°C incubator. Replace the culture medium once the next day and continue culturing.

[0100] 7. Cell proliferation (Cell Counting Kit-8, CCK-8) experiment:

[0101] 7.1 Cell plating: When the cells grow to 80% of the culture dish, digest with trypsin for 5 min, add complete medium to terminate the digestion, centrifuge at 1000 rpm for 5 min. Discard the supernatant, resuspend the cells with complete medium, count, adjust the cell density to 5.0*10 5 cells / mL, inoculate the cells into a 96-well plate, add 100 ul to each well, and incubate statically in a 37°C incubator.

[0102] 7.2 Cell drug administration: In A549 cells, change the medium and administer different concentrations of CD36 inhibitor (0, 1, 2, 5, 10 uM) respectively.

[0103] 7.3 CCK-8 experiment: At different time points (0, 6, 12, 24 h) respectively, change the medium after cell drug administration, add CCK-8 solution (containing 10% CCK-8), and incubate the 96-well plate in the incubator for 1 hour. Measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0104] 8. IC50 experiment for small molecule inhibitor Dil-oxLDL uptake

[0105] 8.1 Construction of 293T cell line expressing CD36

[0106] Transfect CD36 into 293T cells, and obtain the transfected cells 48 h later

[0107] 8.2 Co-incubation of small molecule drug and Dil-Ox-LDL in HEK-293T-CD36 cells

[0108] Drug concentration setting (uM): 100, 25, 6.25, 1.56, 0.39, 0.097, 0.024, 0

[0109] 8.3 Seed cells in a 96-well plate, then incubate at 37 °C for 24 h. Discard the serum-containing medium, add fresh serum-free medium, and incubate overnight for starvation treatment.

[0110] 8.4 Dil-Ox-LDL incubation: Add 200 μl of 5 μg / ml Dil-Ox-LDL to each well and incubate at 37 °C for 2 h.

[0111] 8.5 Wash 3 times with 200 μl of PBS for 5 min each time. Digest with trypsin and terminate digestion with DMEM containing 2% FBS. Centrifuge at 3000 rpm, resuspend with 400 μl of PBS, and detect the FL2 channel by flow cytometry.

[0112] 9. Inhibitory effect of CD36 inhibitor F449-4678 on the growth of A549 cell xenografts

[0113] 9.1 Establishment of animal model:

[0114] Use 6-week-old SPF-grade nude mice, male, with a body weight of about 20 g. Digest A549 cells. Pay attention to observation. Sometimes the cells in the corner cannot be digested, which will affect the inoculation amount. Then centrifuge to obtain the cell pellet, add PBS to resuspend, and prepare a cell suspension of 3×10 7 mL -1 . Suspend in Matrigel (3:1) in an ice bath. Mix well and place in an ice bath. Take it to the animal room and start tumor inoculation. Inject 0.1 ml (about 2×10 6 / mouse) subcutaneously into the right axilla of each nude mouse. After inoculation, closely observe the growth of the mice and the changes of the xenografts. When the long diameter of the xenograft is greater than 5.0 mm, it is determined as tumor formation. A total of 20 mice are implanted with tumors.

[0115] 9.2 Animal administration and experimental records:

[0116] After tumor formation in nude mice, intraperitoneally inject two doses of 5 mg / kg and 10 mg / kg of the active small molecule F449-4678 every two days. Measure the tumor size with a vernier caliper, continuously administer the drug for 21 days, and observe and record the tumor size after drug withdrawal.

[0117] III. Results

[0118] 1. In silico virtual screening of small molecule compounds

[0119] The CD36 inhibitor was obtained by downloading from the PDB database based on the crystal complex structure of CD36 (PDB ID: 5LGD); the co-crystallized small molecule site in its extracellular domain (Gly 30–Asn 439) was defined as the molecular docking active site; the Glide docking algorithm in the Schrödinger software package was used for virtual screening of the ChemDiv commercial compound database (including approximately 2.5 million compounds after preparation); after screening, the top 1000 compound molecules were screened based on Lipinski's rule of five, clustering analysis, etc., and the top 1100 diverse molecules were obtained for subsequent experimental detection( Figure 1 ), (Table 1).

[0120] Table 1. Numbers and names of the top 100 small molecule compounds screened by computer virtual screening based on the CD36 structure

[0121]

[0122]

[0123] 2. Screening by Dil-oxLDL uptake experiment

[0124] The above 100 small molecules screened by virtual screening were subjected to Dil-oxLDL uptake experiments to screen for the inhibitory effect of CD36 small molecule inhibitors on the uptake of Dil-oxLDL by CD36. On Raw264.7 cells and 293T cells overexpressing human CD36, the above potential active small molecules were given as stimuli to screen for candidate small molecules that could inhibit the uptake of oxLDL by CD36 (Tables 2 and 3). In addition, by analyzing the inhibition rate of small molecule inhibitors on the uptake of oxLDL by CD36 in the two types of cells, it was found that there were a total of 6 candidate active small molecules that could simultaneously affect the function of oxLDL uptake, and their Chemdiv IDs were Y501-4688, P132-0554, G748-0096, D294-4966, L977-0025, F449-4678( Figure 2 ).

[0125] Table 2. Inhibition rate of different small molecules on the uptake of Dil-oxLDL by CD36 detected by flow cytometry in Raw264.7 cells

[0126]

[0127]

[0128] Table 3. Inhibition rate of different small molecules on the uptake of Dil-oxLDL by CD36 detected by flow cytometry in CD36-overexpressing 293T cells

[0129]

[0130]

[0131]

[0132] 3. Detection of the Affinity between Small Molecules and CD36 Protein by Microscale Thermophoresis

[0133] The above six candidate active small molecules were subjected to microscale thermophoresis (MST) experiments for protein-small molecule interaction screening. The Kd curve was further fitted through Binding Affinity to investigate the binding ability of the six candidate small molecules to CD36 (Table 4). The results showed that all six small molecules had strong affinity for CD36( Figure 3 ).

[0134] Table 4 Preliminary Screening of Candidate Small Molecules by MST Binding Affinity

[0135]

[0136] 3. Inhibition of CD36 Protein Uptake by Small Molecule Inhibitors IC 50 Experiment

[0137] According to the above six potential CD36 small molecule inhibitors, we overexpressed CD36 protein in 293T cells and stimulated the cells with active small molecules at different concentrations (100, 25, 6.25, 1.56, 0.39, 0.097, 0.024, 0 uM). The inhibition rate of CD36 protein uptake by small molecules at different concentrations was analyzed by flow cytometry, and the IC 50 of different active small molecules was analyzed. We found that the active small molecule F449-4678 had the best inhibitory effect on oxLDL uptake, and the other five potential small molecules could effectively inhibit the ability of CD36 to uptake oxLDL( Figure 4 ).

[0138] 5. Active Small Molecules Inhibit Tumor Cell Proliferation and Induce Apoptosis.

[0139] Based on the above IC 50 , further study was carried out on whether active small molecules could inhibit tumor cell proliferation. First, on lung cancer cells A549 and breast cancer cells MCF7, the CCK-8 cell proliferation experiment was used to investigate the effect of different active small molecules on tumor cell proliferation. Through CCK8, it was verified that the active small molecule F449-4678 had the best inhibitory effect on tumor cell proliferation, and the remaining small molecules could also inhibit tumor cell proliferation( Figure 5 A-B). Second, the effect of different concentrations of the active small molecule F449-4678 on cell proliferation was investigated( Figure 5C-D). Compared with the control group, F449-4678 at 5 μM and 10 μM could effectively inhibit the proliferation of tumor cells. The above results indicate that F449-4678 can inhibit cell proliferation.

[0140] 6. Inhibitory effect of CD36 inhibitor No. F449-4678 on tumor growth in LLC-LUC xenograft tumor mice

[0141] Based on the above results, we intraperitoneally injected two doses of F449-4678 (5 mg / kg and 10 mg / kg) in a mouse model of LLC-LUC xenograft tumors to investigate its inhibitory effect on tumor growth. The experiments showed that, analyzed from tumor volume, tumor weight, and in vivo imaging results, the CD36 inhibitor F449-4678 could effectively inhibit the tumor growth in LLC-LUC xenograft tumor mice at a dose of 10 mg / kg ( Figure 6 A-6C). The results analysis showed that the small molecule F449-4678 could inhibit tumor growth and the proliferation of tumor cells.

[0142] In summary, we established a Virtual Flow virtual drug screening platform, screened the top 100 small molecules from a drug target library (Chemdiv) for actual screening, screened active small molecules that inhibit the function of CD36 protein, and characterized the effectiveness of active small molecules of CD36 by inhibiting the oxLDL uptake level. Six potential active compounds were screened out from the active small molecules: chemdiv ID: Y501-4688, P132-0554, G748-0096, D294-4966, L977-0025, F449-4678. Through MST to test the binding affinity between small molecules and proteins, Dil-oxLDL flow cytometry to detect the IC50 of small molecule inhibitors, and CCK-8 to detect cell proliferation; in addition, through verification on LLC-LUC xenograft tumor mice, administering the active small molecule F449-4678 to mice could effectively inhibit tumor growth, thereby achieving an anti-tumor effect.

Claims

1. Use of any one of the following compounds in the preparation of an anti-tumor drug, wherein the tumor is selected from non-small cell lung cancer or breast cancer, 。

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