Fluorescence-based assay for polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) and identification of novel antimycobacterial WecA inhibitors.
PBN-AR
Instytucja
Instytut Biochemii i Biofizyki Polskiej Akademii Nauk
Informacje podstawowe
Główny język publikacji
EN
Czasopismo
Analytical biochemistry
ISSN
0003-2697
EISSN
1096-0309
Wydawca
Elsevier BV
DOI
Rok publikacji
2016
Numer zeszytu
Strony od-do
78-90
Numer tomu
512
Identyfikator DOI
Liczba arkuszy
Autorzy
Pozostali autorzy
+ 6
Słowa kluczowe
EN
Bacterial phosphotransferases;
Fluorescence-based assay;
HTS;
Mycobacterium tuberculosis;
Prenyl-phosphate-GlcNAc-1-phosphate transferase;
WecA;
WecA inhibitors
Streszczenia
Język
EN
Treść
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.
Inne
System-identifier
5288
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