Schepetkin Igor A, Kirpotina Liliya N, Khlebnikov Andrei I, Quinn Mark T
Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717, USA.
Mol Pharmacol. 2007 Apr;71(4):1061-74. doi: 10.1124/mol.106.033100. Epub 2007 Jan 17.
We screened a chemolibrary of drug-like molecules for their ability to activate reactive oxygen species (ROS) production in murine phagocytes, and we identified 26 novel compounds with potent neutrophil activating properties. We used substructure screening, fragment-focusing, and structure-activity relationship analyses to further probe the parent library and defined at least two groups of activators of ROS production in murine neutrophils: t-butyl benzene and thiophene-2-amide-3-carboxylic ester derivatives. Further studies of the active compounds revealed 11 compounds that activated ROS production in human neutrophils, and six of these compounds also activated intercellular Ca(2+) mobilization and chemotaxis in human neutrophils. Of the latter compounds, compound 14 (1,3-benzodioxolane-5-carboxylic acid 4'-benzyloxy-3'-methoxybenzylidene-hydrazide) activated neutrophils at nanomolar concentrations, and Ca(2+) mobilization was inhibited by pertussis toxin and N-t-butoxycarbonyl-Phe-Leu-Phe-Leu-Phe (Boc-2), an antagonist of formyl peptide receptors (FPR/FPRL1). Likewise, activation by compound 14 was desensitized after N-formyl-Met-Leu-Phe pretreatment. Similar biological activities were found for compound 104 (1,3-benzodioxolane-5-carboxylic acid 3'-bromo-5'-ethoxy-4'-hydroxybenzylidene-hydrazide), an analog of compound 14. Furthermore, conformational analysis of the activators of chemotaxis and Ca(2+) mobilization showed a high degree of similarity in distances between pharmacophore points of compounds 14 and 104 with a model of FPR published by Edwards et al. (Mol Pharmacol 68:1301-1310, 2005), indicating that conformational features of the agonists identified here are structurally compatible with steric constraints of the ligand-binding pocket of the receptor. Based on these results, we conclude that compounds 14 and 104 represent novel small-molecule agonists of FPR. These studies enhance our understanding of FPR ligand/receptor interactions and structure/activity relationships of phagocyte agonists.
我们筛选了一个类药物分子化学文库，以检测其激活小鼠吞噬细胞中活性氧（ROS）产生的能力，并且我们鉴定出了26种具有有效嗜中性粒细胞激活特性的新型化合物。我们使用子结构筛选、片段聚焦和构效关系分析进一步探究母体文库，并确定了至少两组小鼠嗜中性粒细胞中ROS产生的激活剂：叔丁基苯和噻吩-2-酰胺-3-羧酸酯衍生物。对活性化合物的进一步研究发现，有11种化合物可激活人嗜中性粒细胞中的ROS产生，其中6种化合物还可激活人嗜中性粒细胞中的细胞内Ca(2+)动员和趋化性。在后者这些化合物中，化合物14（1,3-苯并二氧戊环-5-羧酸4'-苄氧基-3'-甲氧基苄叉酰肼）在纳摩尔浓度下即可激活嗜中性粒细胞，并且百日咳毒素和N-叔丁氧羰基-Phe-Leu-Phe-Leu-Phe（Boc-2，一种甲酰肽受体（FPR/FPRL1）拮抗剂）可抑制Ca(2+)动员。同样，在N-甲酰基-Met-Leu-Phe预处理后，化合物14的激活作用会脱敏。化合物104（1,3-苯并二氧戊环-5-羧酸3'-溴-5'-乙氧基-4'-羟基苄叉酰肼），即化合物14的类似物，也具有类似的生物学活性。此外，趋化性和Ca(2+)动员激活剂的构象分析表明，化合物14和104的药效基团点之间的距离与Edwards等人发表的FPR模型（《分子药理学》68:1301-1310, 2005）具有高度相似性，这表明此处鉴定出的激动剂的构象特征在结构上与受体配体结合口袋的空间限制相容。基于这些结果，我们得出结论，化合物14和104代表FPR的新型小分子激动剂。这些研究增进了我们对FPR配体/受体相互作用以及吞噬细胞激动剂的结构/活性关系的理解。
