仓鼠糜酶2抑制剂高通量筛选模型的建立及其应用

High-throughput screening for hamster chymase 2 inhibitors

摘要:
本研究拟建立重组仓鼠糜酶2 (chymase 2) 抑制剂的体外高通量筛选模型, 寻找新型的糜酶2抑制剂。首先, 利用大肠杆菌表达、制备成熟的重组仓鼠糜酶2蛋白, 以384孔微板为媒介, 建立基于荧光方法测定酶活性的抑制剂筛选模型, 结果表明所建立的糜酶2抑制剂高通量筛选模型具有灵敏、稳定、重复性好的特点 (Z' = 0.84)。利用建立的模型对40 080种样品 (包括28 060种化合物和12 020种天然产物提取物) 进行抑制活性筛选, 取抑制率大于90%的613种样品进行复筛。最终确定化合物J16647和J16648具有较强的抑制活性, 其IC₅₀值分别为0.823和0.690 μmol·L⁻¹。

Abstract:
To screen potential hamster chymase 2 inhibitors, a high-throughput screening (HTS) model was established. Recombinant hamster chymase 2 with active form was cloned and expressed in E. coli. The HTS model with total volume of 50 μL in 384-well microplate was based on fluorescence analysis and was proved sensitive as well as specific (Z' = 0.84). A total of 40 080 samples (including 28 060 compounds and 12 020 natural products) were screened, and 613 samples with inhibition greater than 90% were selected for further rescreening. Finally, compounds J16647 and J16648 were identified with high inhibitory activity on chymase 2, and whose IC₅₀ values were 0.823 and 0.690 μmol·L⁻¹, respectively.