IBB PAS Repository

Fluorescence-based assay for polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) and identification of novel antimycobacterial WecA inhibitors.

Mitachi, Katsuhiko and Siricilla, Shajila and Yang, Dong and Kong, Ying and Skorupinska-Tudek, Karolina and Swiezewska, Ewa and Franzblau, Scott G. and Kurosu, Michio (2016) Fluorescence-based assay for polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) and identification of novel antimycobacterial WecA inhibitors. Analytical Biochemistry, 512 . pp. 78-90.

[img] Microsoft Word
46kB

Official URL: http://linkinghub.elsevier.com/retrieve/pii/S0003-...

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 fluorescencebased 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 nbutanol and can be quantified by ultravioletevisible (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.

Item Type:Article
Subjects:Q Science > QH Natural history > QH301 Biology
Divisions:Department of Lipid Biochemistry
ID Code:1242
Deposited By: Ewa Swiezewska
Deposited On:16 Nov 2016 08:55
Last Modified:16 Nov 2016 08:55

Repository Staff Only: item control page