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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 establish a high-throughput in vitro screening method for 5-lipoxygenase (5-LOX) inhibitors with different mechanism of action. Methods Arachidonic acid was used as substrate, and 2,7-dichlorodihydrofluorescein acetoacetate was used as fluorescent reagent to establish a fluorescence assay. Two different methods were established by adding Fe3 + and 10 mmol / L Fe3 + 5-LOX enzyme reaction system. Results The signal / background (S / B) values of 5-lipoxygenase in the reaction system without Fe3 + and 10 mmol / L Fe3 + were 5.02 and 37.81, respectively. The values of Z ’ About 0.7.5-lipoxygenase ferric ion chelation inhibitor zileuton in the absence of Fe ~ (3 +) reaction system has a strong inhibitory activity of the enzyme, the IC 50 value of 0.21μm, in addition The reaction system of 10mmol / L Fe ~ (3 +) did not inhibit the enzyme. The non-ferrous ion chelating inhibitor nordihydroguajumic acid inhibited the enzyme in both reaction systems with IC 50 values of 0.52 and 0.93 μM, respectively. Two active compounds, WF-5239 and Trivaric acid, were screened from microbial metabolites by this method. Compound WF-5239 had a strong inhibitory activity on the enzyme in the reaction system without Fe 3+. The IC 50 ) Value of 2μmol / L, the addition of 10mmol / L Fe ~ (3 +) reaction system did not inhibit the enzyme, presumably iron chelate inhibitors. The inhibitory effect of the compound Trivaric acid on the two screening systems was basically the same, the IC 50 value was about 5.36μmol / L, suggesting that it may be a non-ferrous ion chelate inhibitor. Conclusion This study established a sensitive and stable method for the screening of 5-LOX inhibitors with different mechanisms of action.