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

Katsuhiko Mitachi, Shajila Siricilla, Dong Yang, Ying Kong, Karolina Skorupinska-Tudek, Ewa Swiezewska, Scott G. Franzblau, Michio Kurosu

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)78-90
Number of pages13
JournalAnalytical Biochemistry
Volume512
DOIs
StatePublished - Nov 1 2016

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Fluorescein-5-isothiocyanate
Transferases
Mycobacterium tuberculosis
Assays
Phosphates
Fluorescence
Uridine Diphosphate
Glucosamine
Biosynthesis
Screening
Membranes
1-Butanol
Macrophages
Enzymes
Smegma
Escherichia coli
Spectrometry
Bacteria
Phosphotransferases
Cells

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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

In: Analytical Biochemistry, Vol. 512, 01.11.2016, p. 78-90.

Research output: Contribution to journalArticle

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

AU - Skorupinska-Tudek, Karolina

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AU - Franzblau, Scott G.

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