Mutated IDH1 inhibitor as antitumor medicine
An anti-tumor drug, IDH1 technology, applied in the field of medicine
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Embodiment 1
[0036] The extraction and separation of embodiment one Physalis F
[0037] 7 kg of stems and leaves of Physalis physalis were extracted with 60% ethanol / water and concentrated to obtain total ethanol extract (PS, 1000 g). All the extracts were dispersed in water, extracted with equal volumes of cyclohexane and EtOAc, respectively, and then distilled under reduced pressure to obtain EtOAc extract (PSE, 180 g). with CHCl 3 – MeOH (100:1-0:1, v / v) was used as eluent to further separate the components of the EtOAc layer by SiO2 column chromatography (CC) to obtain a total of 12 fractions. Using cyclohexane-EtOAc (2:3, v / v) as eluent, SiO 2 CC further isolated the PSE-2 fraction, and recrystallized the resulting fraction from acetone, thereby preparing physalin F (1.0 g). By comparing its 1 H and 13 C NMR spectral data is consistent with the literature report (PhytotherapyResearch: 16 (5) (2002) 445-458), identified as Physalisin F (purity> 97.0%).
Embodiment 2
[0038] Example 2 Enzyme Activity Evaluation System Screening Physalis F Targeted to Inhibit Mutant IDH1 Enzyme Activity 1. Expression and Purification of IDH1 and Mutant IDH1 Protein
[0039] The 132-arginine (R) of the wild-type IDH1 plasmid with a C-terminal His tag was mutated to histidine (R132H) using a rapid site-directed mutagenesis kit. The target gene was constructed into pET28a vector, and the recombinant plasmid was transduced into Escherichia coli BL21(DE). Strains selected on a plate containing 60 mM kanamycin were picked, and then single clones were picked in Luria-Bertani (LB) medium. Culture at 37°C until the OD600 value is 0.8, then add 0.4mM isopropyl-β-D-thiogalactopyranoside (IPTG) and induce at 18°C for 18h to make the cells express the protein. Harvested cells were resuspended in lysis buffer supplemented with protease inhibitors (20 mM Tris, pH 8.0, 250 mM NaCl, 5 mM β-mercaptoethanol (β-ME), 0.1% TritonX-100, and 5% glycerol) and stored on ice. Lyse...
Embodiment 3
[0056] Example 3 Verification of the inhibitory activity of physidin F based on HT1080 cells
[0057] 1. Physalis F inhibits the production of R-2-HG in HT1080 cells
[0058] The hallmark of the carcinogenesis of cells carrying mutant IDH1 is the production and accumulation of large amounts of R-2-HG in cancer cells and peripheral blood. due to IDH1 R132H and IDH1R 132C With the same catalytic activity, α-KG can be reduced to R-2-HG by consuming NADPH. Therefore, the selection with IDH1 R132C The most common cell line HT1080 was mutated to further study the biological activity of phycidin F in vitro. After HT1080 cells were given different concentrations of phycidin F, the concentration of R-2-HG in the cell culture medium was determined by LC-MS / MS to reflect the inhibitory effect of physidin F on mutant IDH1. The result is as image 3 As shown, physalicin F can significantly reduce the concentration of R-2-HG in HT1080 cells in a dose-dependent manner. The results sho...
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