کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10532662 | 961686 | 2013 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A colorimetric assay optimization for high-throughput screening of dihydroorotase by detecting ureido groups
ترجمه فارسی عنوان
بهینه سازی آزمون رنگ سنجی برای غربالگری دی هیدرووراتاز با تشخیص گروه های اوریدو
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کلمات کلیدی
اوریدو، تست رنگ سنجی، بهینه سازی آزمون، غربالگری بالا، دی هیدروورتاز،
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
چکیده انگلیسی
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 (Zâ²-factors 0.7-0.8) was determined to be superior to DAMO-antipyrine (Zâ²-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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 441, Issue 1, 1 October 2013, Pages 87-94
Journal: Analytical Biochemistry - Volume 441, Issue 1, 1 October 2013, Pages 87-94
نویسندگان
Amy J. Rice, Lena Truong, Michael E. Johnson, Hyun Lee,