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

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.

Stereocontrolled Total Synthesis of Muraymycin D1 Having a Dual Mode of Action against Mycobacterium tuberculosis

“…Extensive bacterial growth inhibitory assays of 1 , 2 , and 3 against Gram-positive and -negative bacteria including Mycobacterium spp . revealed that 1 – 3 exhibited bacteriostatic activity against M. tuberculosis ; the MIC 50 values are comparable to UT-01320 (a selective WecA inhibitor) 24,25 , capuramycin (a selective MurX inhibitor), and tunicamycin (a non-selective phosphotransferase inhibitor). However, 1 – 3 did not show antibacterial activity against Gram-negative bacteria ( E. coli, K. pneumoniae, P. aeruginosa and A. baumannii ) and Gram-positive bacteria ( S. aureus , C. difficile , and E. faecium ) even at >100 μg/mL concentrations.…”

“…Inhibition of WecA blocks the entire biosynthesis of essential cell wall components of M. tuberculosis in both replicating and non-replicating states, making this enzyme a target for development of novel TB drugs. 24 The synthetic molecules ( 1 , 2 , and 3 ) were evaluated in MurX and WecA assays (Table 1). Muraymycin D1 ( 1 ) and muraymycin D1-amide ( 2 ) exhibited equal enzyme inhibitory activities against the bacterial phosphotransferases (MurX and WecA) and their IC 50 values were in low μM range.…”

N -Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

“…Since dual activity of an inhibitor is a highly favorable property (45,48,49), we developed simple assays to evaluate the inhibition of both mycobacterial WecA and translocase I. While numerous methods were reported for monitoring WecA and/or translocase I activities in different bacteria (33,50,51), to the best of our knowledge, a parallel evaluation of inhibitory activities of antibiotics on mycobacterial WecA and translocase I was reported for the first time only recently (47). Similar to our and numerous other assays, Mitachi et al (47) employed crude mycobacterial membranes as a source of the tested enzymes.…”

Inhibition of N-Glycosylation towards Novel Anti-Cancer Chemotherapeutics

“…lipopolysaccharide in Gram-negative and arabinogalactan-mycolic acid in Mycobacterium spp . ), respectively [15]. It is interesting to note that tunicamycin shows >10 times stronger inhibitory activity against WecA (IC 50 0.120 ± 7.80 µg/mL) than that of MraY (IC 50 2.73 ± 0.138 µg/mL).…”

“…A sufficient amount of DPAGT1 enzyme purification for compound screening is not feasible due to lack of chemical stability. On the other hand, a high-throughput screening (HTS) against WecA enzyme has been established recently (Mitachi et al, 2016) [15]. Chemical synthesis of a library of complex nucleoside analogs is practical via modern synthetic method.…”