Applications of amino methylene cyclohexane 1, 3-dione compound
A technology of compounds and uses, applied in the field of biomedicine
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Embodiment 1
[0047] Example 1 Molecular level experiments of compounds or their salts targeting LC3B
[0048] By constructing a prokaryotic expression system to express and purify LC3B protein, a preliminary screening and verification platform was established by fluorescence polarization experiment, and the activity of the purchased and synthesized small compound library was determined.
[0049] Recombinant protein GST-LC3B (final concentration 180nM, SEQ ID NO:1) and N-terminal FITC-labeled peptide (SEQ ID NO:2, final concentration 18nM) were placed in FP buffer (50mM HEPES pH7.5, 0.1mg / mL BSA , 1 mM DTT), to which were added serially diluted compounds using FP buffer, and then the above mixture was incubated at 25° C. in the dark. Monitor the fluorescence polarization value (PerkinElmer Envision, emission wavelength 480nm; absorption wavelength 535nm), and calculate IC with GraphPadPrism 6.0 program 50 value.
[0050] The test results are shown in Table 1, wherein the IC of the compoun...
Embodiment 2
[0053] Example 2 Regulatory effect of compound or its salt on autophagy
[0054] Western blot detection of LC3-I / LC3-II protein
[0055] Hela cells were inoculated into 6-well plates, cultured overnight, treated with a certain concentration of compounds for 12 hours, and then starved for 24 hours with serum-free medium. Aspirate the culture medium, wash once with PBS, add SDS-PAGE and use 2× loading buffer to lyse the cells. The sample was boiled at 99°C for 10 minutes, separated by SDS-PAGE, and then detected by LC3B antibody (Novus) for LC3-I / LC3-II. The detection results are as follows: figure 2 As shown in A.
[0056] Depend on figure 2 A It can be seen that LC3-II accumulates with the prolongation of compound treatment time.
[0057] Immunofluorescence staining and fluorescence microscope photography
[0058] Hela cells were seeded on glass coverslips in a 6-well plate, cultured until the cells were in good condition, treated with a certain concentration of compoun...
Embodiment 3
[0060] Example 3 Inhibitory effects of compounds or their salts in combination with some marketed drugs on the proliferation of different types of tumor cells
[0061] Tumor cell lines: large B lymphoma cells (DB, Toledo, Pfeiffer, SU-DHL6, WSU-DLCL2, OCI-Ly19, SU-DHL2, SU-DHL8), mantle cell lymphoma cell lines (REC-1, Z- 138, Jeko-1, Maver-1), pancreatic cancer cell lines (AsPC-1, BxPC-3, MIAPaCa-2, Panc-3.014, CaPan-1, Panc-1), colon cancer cell line HCT116, non-small cell Lung cancer cell lines (PC9, HCC827, NCI-H1975) and multiple myeloma cell lines (H929, KMS26, RPMI-8226).
[0062] Listed drugs used in combination: ibrutinib, imatinib, gemcitabine, erlotinib, pemetrexed disodium, AZD3759 and lenalidomide.
[0063] Experimental method: use complete medium containing 10% FBS, after cell counting, inoculate ~10000 cells / 100 μL per well in 96-well plate, at the same time give the marketed drug and compound 38 treatment, the drug is diluted in a concentration gradient of 1:3...
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