Fluorescence-based assay for polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) and identification of novel antimycobacterial WecA inhibitors.
Fluorescence-based assay for polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) and identification of novel antimycobacterial WecA inhibitors.
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DOI：
10.1016/j.ab.2016.08.008
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发表时间：
2016-11-01
影响因子：
2.9
通讯作者：
Kurosu M
中科院分区：
文献类型：
--
作者：
Mitachi K;Siricilla S;Yang D;Kong Y;Skorupinska-Tudek K;Swiezewska E;Franzblau SG;Kurosu M
聚异戊二烯磷酸-GlcNAc-1-磷酸转移酶 (WecA) 是结核分枝杆菌 (Mtb) 和其他一些细菌生长的必需酶。 Mtb WecA 催化从 UDP-GlcNAc 到十异戊二烯基-P-P-GlcNAc 的转化，十异丙烯基-P-P-GlcNAc 是第一种膜锚定糖磷脂，负责 Mtb 中霉糖阿拉伯半乳聚糖的生物合成。抑制 WecA 将阻断复制和非复制状态下 Mtb 必需细胞壁成分的整个生物合成，使该酶成为新药开发的目标。在这里，我们报告了一种基于荧光的 WecA 测定方法，使用改良的 UDP-GlcNAc、UDP-葡萄糖胺-C6-FITC (1)、由耻垢分枝杆菌菌株制备的膜组分和大肠杆菌 B21WecA。在优化条件下，UDP-葡萄糖胺-C6-FITC(1)可以转化为相应的十异戊二烯基-P-P-葡萄糖胺-C6-FITC(3)，收率61.5%。十异戊二烯基-P-P-葡萄糖胺-C6-FITC 很容易用正丁醇萃取，并且可以通过紫外-可见 (UV-Vis) 光谱法进行定量。通过筛选针对细菌磷酸转移酶设计的化合物库，发现了一种选择性 WecA 抑制剂 UT-01320 (12)，它可以在低浓度下杀死复制型和非复制型 Mtb。 UT-01320 (12) 还可杀死巨噬细胞中的细胞内 Mtb。我们的结论是，本文报道的 WecA 测定法适合中通量和高通量筛选，从而促进新型 WecA 抑制剂的发现。
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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