title: A colorimetric assay optimization for high-throughput screening of dihydroorotase by detecting ureido groups.
abstract: Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine biosynthesis pathway and is a potential new antibacterial drug target. No target-based high-throughput screening (HTS) assay for this enzyme has been reported to date. Here, we optimized two colorimetric-based enzymatic assays that detect the ureido moiety of the DHOase substrate, carbamyl-aspartate (Ca-asp). Each assay was developed in a 40-μl assay volume using 384-well plates with a different color mix, diacetylmonoxime (DAMO)-thiosemicarbazide (TSC) or DAMO-antipyrine. The sensitivity and color interference of both color mixes were compared in the presence of common HTS buffer additives, including dimethyl sulfoxide, reducing agents, detergents, and bovine serum albumin. DAMO-TSC (Z'-factors 0.7-0.8) was determined to be superior to DAMO-antipyrine (Z'-factors 0.5-0.6) with significantly less variability within replicates. An HTS pilot screening with 29,552 compounds from four structurally diverse libraries confirmed the quality of our newly optimized colorimetric assay with DAMO-TSC. This robust method has no heating requirement, which was the main obstacle to applying previous assays to HTS. More important, this well-optimized HTS assay for DHOase, the first of its kind, should make it possible to screen large-scale compound libraries to develop new inhibitors against any enzymes that produce ureido functional groups.
authors.affiliation: Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL 60607, USA.
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Rice
authors.forename: Lena
authors.initials: L
authors.lastname: Truong
authors.forename: Michael E
authors.initials: ME
authors.lastname: Johnson
authors.forename: Hyun
authors.initials: H
authors.lastname: Lee
mesh_terms: D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D001408:Bacillus anthracis / Q000201:enzymology; D003124:Colorimetry / Q000379:methods*; D004080:Dihydroorotase / Q000032:analysis* / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*
chemical_list: D001224:Aspartic Acid; D004080:Dihydroorotase; C006893:ureidosuccinic acid
keywords: Assay optimization; Colorimetric assay; Dihydroorotase; High-throughput screening; Ureido