Fluorescence-based assay for polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) and identification of novel antimycobacterial WecA inhibitors |
| |
Authors: | Katsuhiko Mitachi Shajila Siricilla Dong Yang Ying Kong Karolina Skorupinska-Tudek Ewa Swiezewska Scott G. Franzblau Michio Kurosu |
| |
Affiliation: | 1. Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, 881 Madison Avenue, Memphis, TN 38163-0001, United States;2. Department of Microbiology, Immunology & Biochemistry, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163-0001, United Sates;3. Department of Lipid Biochemistry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warszawa, Poland;4. Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, IL 60612, United States |
| |
Abstract: | Polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) is an essential enzyme for the growth of Mycobacterium tuberculosis (Mtb) and some other bacteria. Mtb WecA catalyzes the transformation from UDP-GlcNAc to decaprenyl-P-P-GlcNAc, the first membrane-anchored glycophospholipid that is responsible for the biosynthesis of mycolylarabinogalactan in Mtb. Inhibition of WecA will block the entire biosynthesis of essential cell wall components of Mtb in both replicating and non-replicating states, making this enzyme a target for development of novel drugs. Here, we report a fluorescence-based method for the assay of WecA using a modified UDP-GlcNAc, UDP-Glucosamine-C6-FITC (1), a membrane fraction prepared from an M. smegmatis strain, and the E. coli B21WecA. Under the optimized conditions, UDP-Glucosamine-C6-FITC (1) can be converted to the corresponding decaprenyl-P-P-Glucosamine-C6-FITC (3) in 61.5% yield. Decaprenyl-P-P-Glucosamine-C6-FITC is readily extracted with n-butanol and can be quantified by ultraviolet–visible (UV–vis) spectrometry. Screening of the compound libraries designed for bacterial phosphotransferases resulted in the discovery of a selective WecA inhibitor, UT-01320 (12) that kills both replicating and non-replicating Mtb at low concentration. UT-01320 (12) also kills the intracellular Mtb in macrophages. We conclude that the WecA assay reported here is amenable to medium- and high-throughput screening, thus facilitating the discovery of novel WecA inhibitors. |
| |
Keywords: | Prenyl-phosphate-GlcNAc-1-phosphate transferase WecA Bacterial phosphotransferases Mycobacterium tuberculosis Fluorescence-based assay HTS WecA inhibitors |
本文献已被 ScienceDirect 等数据库收录! |
|