不同作用机制的5-脂氧化酶抑制剂筛选方法的建立和应用

Development and application of a screening system for distinct me chanism 5-LOX inhibitors

目的拟建立体外高通量的用于筛选不同作用机制的5-脂氧化酶(5-lipoxygenase,5-LOX)抑制剂的方法。方法以花生四烯酸为底物,2,7-二氯二氢荧光素乙酰乙酸为荧光反应物,建立荧光测活方法,并通过不加Fe3+和外加10mmol/LFe3+的方法建立两种不同的5-LOX酶反应体系。结果 5-脂氧化酶在不加Fe3+和外加10mmol/LFe^(3+)的反应体系中的信号/本底(S/B)值分别为5.02和37.81,两种反应的Z'因子值均约为0.7。5-脂氧化酶铁离子螯合抑制剂齐留通在未加Fe^(3+)的反应体系对酶有强的抑制活性,其IC_(50)值为0.21μm,在外加10mmol/L Fe^(3+)的反应体系对酶无抑制作用。非铁离子螯合抑制剂去甲二氢愈创木酸在两种反应体系对酶均有抑制作用,其IC_(50)值分别为0.52和0.93μM。利用该方法从微生物代谢产物中筛选到两个活性化合物WF-5239和Trivaric acid,其中化合物WF-5239在未加Fe^(3+)的反应体系对酶有强的抑制活性,其IC_(50)值为2μmol/L,在外加10mmol/L Fe^(3+)的反应体系对酶无抑制作用,推测可能为铁离子螯合型抑制剂。化合物Trivaric acid在两个筛选体系对酶的抑制作用基本一致,其IC_(50)值均约为5.36μmol/L,推测可能为非铁离子螯合型抑制剂。结论本研究建立了灵敏、稳定的可用于筛选不同作用机制5-LOX抑制剂的方法。

Objective To develop two high-throughput screening assays for screening 5-1ipoxygenase （5- LOX） inhibitors with distinct inhibitoy mechanism. Methods Using arachidonic acid （AA） as substrate and 2,7-dichlorodihydro diacetate （H2DCFDA） as fluorescence reagent, a fluorescence-based 5-LOX assay was developed, and two reaction buffers with different concentration of Fe3＋ were used for screening iron-chelation or non-ironchelation inhibitors. Results Two assays, with or without additional Fe3＋, produced signal/background （S/B） value of 37.81 and 5.02 respectively, and the Z-factor were 0.7. Zileuton showed strong inhibitory activity against 5-LOX in the buffer without additional Fe3＋ with the IC5o value of 0.21μmol/L, but did not showed any inhbitory activity in the buffer with additional Fe3＋ at 20μmol/L. Nordihydroguaiaretic acid （NDGA）, a 5-LOX non-iron-chelation inhibitor, showed the similar strong inhibitory activity in both buffer systems with the ICs0 0.52mol/L and 0.93mol/ L respectively. By screening, WF-5239 and Trivaric acid, two microorganism metabolites, were identified as 5-LOX inhibitors. WF-5239 exhibited an ICs0 value of 2.0＋tmol/L in buffer without additional Fe3＋, but had no inhibitory activity in assay with additional Fe3＋, which suggests that it may inhibit 5-LOX through chelation of the active site Fe3＋.Trivaric acid showed similar inhibitory activities in both assay buffers with an IC50 value of about 5.36μmol/L, suggesting it may be a 5-LOX non iron-chelation inhibitor. Conclusion were established for identification the iron-chelation and non iron-chelation Two sensitive and robust screening assays 5-LOX inhibitors.