Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Anal Biochem. 2011 Mar 1;410(1):133-40. doi: 10.1016/j.ab.2010.11.004. Epub 2010 Nov 10.
Homocitrate synthase (HCS) catalyzes the first step of l-lysine biosynthesis in fungi by condensing acetyl-coenzyme A and 2-oxoglutarate to form 3R-homocitrate and coenzyme A. Due to its conservation in pathogenic fungi, HCS has been proposed as a candidate for antifungal drug design. Here we report the development and validation of a robust fluorescent assay for HCS that is amenable to high-throughput screening for inhibitors in vitro. Using this assay, Schizosaccharomyces pombe HCS was screened against a diverse library of approximately 41,000 small molecules. Following confirmation, counter screens, and dose-response analysis, we prioritized more than 100 compounds for further in vitro and in vivo analysis. This assay can be readily adapted to screen for small molecule modulators of other acyl-CoA-dependent acyltransferases or enzymes that generate a product with a free sulfhydryl group, including histone acetyltransferases, aminoglycoside N-acetyltransferases, thioesterases, and enzymes involved in lipid metabolism.
同型柠檬酸合酶（HCS）通过缩合乙酰辅酶 A 和 2-氧代戊二酸催化真菌中 l-赖氨酸生物合成的第一步，形成 3R-同型柠檬酸和辅酶 A。由于其在病原真菌中的保守性，HCS 已被提议作为抗真菌药物设计的候选物。在这里，我们报告了一种用于 HCS 的稳健荧光测定法的开发和验证，该测定法适用于体外抑制剂的高通量筛选。使用该测定法，对酿酒酵母 HCS 进行了约 41,000 种小分子的多样化文库筛选。经过确认、反向筛选和剂量反应分析，我们为进一步的体外和体内分析确定了 100 多种化合物的优先级。该测定法可以很容易地适应筛选其他酰基辅酶 A 依赖性酰基转移酶或生成具有游离巯基产物的酶的小分子调节剂，包括组蛋白乙酰转移酶、氨基糖苷 N-乙酰转移酶、硫酯酶和参与脂质代谢的酶。
