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[目的]构建人源胱硫醚β合酶CBS△516-525体外高通量筛选模型,在基础酶活水平,筛选天然产物库,发现新型、高效且特异的CBS天然产物抑制剂。[方法]在192串联双孔板内,构建纯酶CBS△516-525的高通量筛选模型,以200!mol/L初筛5 206个天然产物,经50!mol/L和5!mol/L复测验证后,选择抑制效果好的新型抑制剂进行剂量依赖性测试并检测其对同源酶胱硫醚γ裂解酶(CSE)的抑制效果。[结果]原核表达纯化出具有生物活性的CBS△516-525酶,经高通量筛选发现2个IC50约1!mol/L的CBS天然产物抑制剂,且对同源酶CSE均有180倍选择性。[结论]在基础酶活条件下,高通量筛选发现了选择性大于180倍的CBS高效天然产物抑制剂,这可为CBS的分子机制和功能研究提供特异的分子工具。
[Objective] To construct a high-throughput screening model of human cystathionine β-synthase CBS △ 516-525 in vitro and screen the natural product library at the level of basic enzyme activity to find novel, efficient and specific CBS natural product inhibitors. [Method] A high-throughput screening model of pure CBS △ 516-525 was constructed in 192 tandem two-well plates. A total of 5 206 natural products were screened with 200 mol / L and 50 mol / L and 5 mol / L retest test, select a good inhibitor of the new inhibitor dose-dependent test and test its homologous enzyme cystathionine γ-lyase (CSE) inhibitory effect. [Result] The purified CBS △ 516-525 enzyme with biological activity was prokaryotic expressed. Two high-throughput screening of two CBS natural product inhibitors with IC50 of about 1 mol / L and 180-fold higher homologous enzyme CSE Selective. [Conclusion] CBS high efficiency natural product inhibitors with a selectivity more than 180 times were found under the conditions of basic enzyme activity, which could provide specific molecular tools for the study of molecular mechanism and function of CBS.