Application of salicylanilide compounds
A salicylanilide and compound technology, applied in the application field of small molecule compounds, can solve the problems of high toxicity and side effects, poor specificity, etc.
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Embodiment 1
[0024] Optimizing the self-ubiquitination screening system of Nedd4 small molecule inhibitors in vitro
[0025] Optimize the self-ubiquitination system in vitro, including the protein content, reaction time and drug concentration of UBE1, Ubch5c and Nedd4 (aa421-900).
[0026] 1. Determining the Concentration of UBE1
[0027] Add components according to in vitro ubiquitination reaction (solution final concentration 0.1M MgCl 2 3 μL, 0.5M Tris-Cl (pH7.4) + 40mM DTT mixed solution 3μL, 100mM ATP 1μL, 0.5μg / μL Ub 2μL, 0.35μM Nedd4 (WW4-HECT), 500nM Ubch5c, total volume 30μL, filled with water, 30°C water bath, react for 1h), add 5*SDS, cook at 95°C for 5min to fully denature the protein, perform SDS-PAGE electrophoresis, and then detect by silver staining. The concentration gradient of UBE1 was set at 0, 5, 10, 20, 40, 80, 100, and 160 (nM). Final selection of the concentration of 20nM, the results see figure 1 .
[0028] 2. Determining the Concentration of Ubch5c
[0029]...
Embodiment 2
[0045] Screening of small molecule inhibitors of Nedd4
[0046] Add each component according to the optimized reaction system of in vitro self-ubiquitination (final concentration of solution 0.1M 3 μL MgCl 23μL, 0.5M Tris-Cl (pH 7.4) + 40mM DTT mixed solution 3μL, 100mM ATP 1μL, 0.5μg / μL Ub 2μL, 70nM Ubch5c, 80nM UBE1, 160nM Nedd4 (WW4-HECT), total volume 30μL, filled with water, The concentration gradient of the drug was 20 μM, and the reaction was carried out in a constant temperature water bath at 30° C. for 40 minutes. After the reaction is complete, add 7.5 μL 5*SDS, mix well, and cook at 95°C for 5 minutes to fully denature the protein. After boiling, homogenize with a suspension shaker, and centrifuge briefly, and store the sample at -20°C. Finally, silver staining was performed.
[0047] For screening results, see Figure 5 and Figure 6 , S4105 can effectively inhibit the autoubiquitination of Nedd4.
Embodiment 3
[0049] Effect of S4105 on HeLa cell viability detected by MTT assay
[0050] ① 96-well plate plating: collect HeLa cervical cancer cells in the log phase, adjust the concentration of the cell suspension, add 200 μL of medium to each well, and plate to make the cell density to be tested 7000 / well.
[0051] ②5% CO 2 , and incubated at 37°C for 24h, adding the drug numbered S4105 is the drug of the present invention. The drug was dissolved in DMSO, and 5 concentration gradients (80 μM, 100 μM, 120 μM, 140 μM) were set up, and 3 replicate wells were set up at the same time. Add 1 μL of drug to each well. Add 1 μL DMSO to the control group.
[0052] ③5% CO 2 , incubate at 37°C for 48 hours, add 20 μL of MTT (ready-to-use, and use PBS to make a 5 mg / mL MTT solution) into each well and continue to incubate for 4 hours.
[0053] ④ Carefully wash away the liquid in the hole, and be sure not to suck away the sediment.
[0054] ⑤ Add 200 μL dimethyl sulfoxide to each well to dissol...
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