| Chronic inflammation forms the basis of chronic degenerative diseases, atherosclerosis, cancer, osteoporosis, asthma,autoimmune diseases etc. The first committed step in the synthesis of leukotrienes is the conversion of arachidonic acid to leukotrienes in two successive reactions catalyzed by 5-lipoxygenase (5-LOX). So 5-LOX has become the important drug target for anti-inflammation drugs discovery.To discover novel 5-LOX inhibitors, in my study, using arachidonic acid as substrates and 2,7-dichlorodihydro diacetate (H2DCFDA) as flourescence reactant, a fluorescence-based high-throughput screening (HTS) assay for screening non iron-chelation and iron-chelation 5-LOX inhibitors was developed. Through modify enzyme concentration,reaction time,et al,the screening model is sensitivity and stability.In my studies, using non iron-chelation high-throughput screening (HTS) assay 15200 metabolites from fungi were screened,35 strains were test positive. 16702 metabolites from actinomycetes were screened,23 strains were test positive. In my studies,9 compounds from 2833 compounds were identified as 5-LOX inhibitors. Compound F01WB287C is presumed to inhibit the 5-LOX through chelation of the active site iron of 5-LOX with IC50 values of 2μM. Compound F02ZA2396B showed similar inhibitory activities in the reaction buffer with or without iron, the IC50 values is 5.36μM,so it inhibitory mechanism may be not through chelation of iron of 5-LOX but other mechanism. Compound F02ZA2554A, 1833-7, 1833-27, F01WA960A, F01WB1695B,1914-1 and 868 had higher inhibitory activities in the reaction buffer without iron than in that with iron. The inhibitory activities of F02ZA2554A, F01WA960A and F01WB1695B to 5-LOX correspond to zileuton. The inhibitory activities of 9 compounds have never been reported before.This study provides a new HTS assay for detecting new 5-LOX inhibitors and the 9 new 5-LOX inhibitors may have potential therapy in anti-inflammatory diseases.
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