A colorimetric assay optimization for high-throughput screening of dihydroorotase by detecting ureido groups

作者：Rice Amy J; Truong Lena; Johnson Michael E^*; Lee Hyun

来源：Analytical Biochemistry, 2013, 441(1): 87-94.

DOI：10.1016/j.ab.2013.05.035

• Ureido
• Colorimetric assay
• Assay optimization
• High-throughput screening
• Dihydroorotase

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-mu l 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. Published by Elsevier Inc.

• 出版日期2013-10-1