基于表面等离子体共振技术筛选STAT3小分子抑制剂的研究

Screening small molecular inhibitors of STAT3 based on surface plasmon resonance technology

目的基于表面等离子体共振(SPR)技术,筛选能与信号转导和转录激活因子3(STAT3)特异性结合并抑制其活性的小分子化合物。方法使用基于SPR技术的Biacore T200生物分子互作分析系统,在最优pH富集条件下将纯化蛋白STAT3偶联到SPR系统的CM5芯片上,从50个中药单体中筛选出能够与STAT3结合且响应值较高的化合物,并对其结合特异性进行确认,然后运用生物学相关实验确证筛选所得化合物对STAT3的抑制作用,最后采用分子对接技术拟合化合物与STAT3的结合模式,明确其可能的作用位点。结果初步筛选获得10多个高响应的候选分子,通过动力学测定发现其中仅有1个分子芹黄素显示特异性结合。Western-blot实验结果表明,芹黄素能够剂量依赖地抑制STAT3的磷酸化;双荧光素酶报告基因结果显示,芹黄素能够剂量依赖地抑制IL-6诱导的STAT3的转录活性。分子对接结果表明,芹黄素与STAT3蛋白的SH2结构域结合,与关键残基Glu638、Gln644、Gly656、Lys658形成氢键相互作用,与Tyr657残基形成π-π相互作用。结论基于SPR技术筛选,发现芹黄素是STAT3的抑制剂。

Objective To find small molecules binding specifically to signal transducer and activator of transcription3(STAT3)based on surface plasmon resonance(SPR)technology and confirm their inhibitory activities to STAT3.Methods The biomolecular interaction analysis T200 system based on SPR technology was used to couple the purified protein STAT3 to CM5 chip under the optimal pH conditions.The compounds with high binding response value were screened out from 50 candidate compounds derived from traditional Chinese medicines and the binding specificity was then confirmed.Biological experiments were performed to confirm the inhibitory effects of the screened compounds on STAT3.The binding pattern of STAT3 and the compound was fitted by molecular docking technique.Results More than 10 candidate molecules exhibited binding activities to STAT3 and kinetics assays revealed that only one candidate molecule,apigenin,showed specific binding.Western-blot analysis exhibited that apigenin inhibited the phosphorylation of STAT3 dose-dependently.Luciferase reporter gene assays demonstrated that apigenin also inhibited IL-6-induced STAT3 transcriptional activity in a dose-dependent manner.Molecular docking results showed that apigenin binds to the SH2 domain of STAT3,and interacts with key residues Glu638,Gln644,Gly656 and Lys658 by hydrogen bonds and with Tyr657 throughπ-πinteractions.Conclusion Apigenin was a direct inhibitor of STAT3.