title: Application of a high-throughput fluorescent acetyltransferase assay to identify inhibitors of homocitrate synthase.
abstract: 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.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Stacie L
authors.initials: SL
authors.lastname: Bulfer
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: McQuade
authors.forename: Martha J
authors.initials: MJ
authors.lastname: Larsen
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Trievel
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D002614:Chelating Agents / Q000737:chemistry / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D051548:Histone Acetyltransferases / Q000378:metabolism*; D008670:Metals / Q000737:chemistry; D009281:Naphthalenes / Q000737:chemistry; D007652:Oxo-Acid-Lyases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D011758:Pyrroles / Q000737:chemistry; D015203:Reproducibility of Results; D012568:Schizosaccharomyces / Q000201:enzymology; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D000214:Acyl Coenzyme A; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D008670:Metals; D009281:Naphthalenes; D011758:Pyrroles; D054852:Small Molecule Libraries; D013438:Sulfhydryl Compounds; C498335:ThioGlo-1; D051548:Histone Acetyltransferases; C021365:homocitrate synthase; D007652:Oxo-Acid-Lyases