Inhibition of Indigoidine Synthesis as a High-Throughput Colourimetric Screen for Antibiotics Targeting the Essential Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT.

IF 5.4 3区 材料科学 Q2 CHEMISTRY, PHYSICAL
Alistair S Brown, Jeremy G Owen, James Jung, Edward N Baker, David F Ackerley
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引用次数: 3

Abstract

A recently-validated and underexplored drug target in Mycobacterium tuberculosis is PptT, an essential phosphopantetheinyl transferase (PPTase) that plays a critical role in activating enzymes for both primary and secondary metabolism. PptT possesses a deep binding pocket that does not readily accept labelled coenzyme A analogues that have previously been used to screen for PPTase inhibitors. Here we report on the development of a high throughput, colourimetric screen that monitors the PptT-mediated activation of the non-ribosomal peptide synthetase BpsA to a blue pigment (indigoidine) synthesising form in vitro. This screen uses unadulterated coenzyme A, avoiding analogues that may interfere with inhibitor binding, and requires only a single-endpoint measurement. We benchmark the screen using the well-characterised Library of Pharmaceutically Active Compounds (LOPAC1280) collection and show that it is both sensitive and able to distinguish weak from strong inhibitors. We further show that the BpsA assay can be applied to quantify the level of inhibition and generate consistent EC50 data. We anticipate these tools will facilitate both the screening of established chemical collections to identify new anti-mycobacterial drug leads and to guide the exploration of structure-activity landscapes to improve existing PPTase inhibitors.

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抑制靛蓝苷合成的高通量比色法筛选抗生素靶向结核分枝杆菌必需磷酸甲肽转移酶PptT。
在结核分枝杆菌中,一个最近被证实但尚未被充分开发的药物靶点是PptT,它是一种必需的磷酸甲肽基转移酶(PPTase),在激活初级和次级代谢的酶中起着关键作用。PptT具有一个深结合袋,不容易接受标记的辅酶a类似物,这些类似物以前用于筛选PPTase抑制剂。在这里,我们报道了一种高通量、比色筛选的发展,该筛选可以监测pptt介导的非核糖体肽合成酶BpsA在体外合成蓝色色素(靛蓝素)的激活。该筛选使用未掺假的辅酶A,避免了可能干扰抑制剂结合的类似物,并且只需要单终点测量。我们使用表征良好的药物活性化合物库(LOPAC1280)集合对筛选进行基准测试,并表明它既敏感又能够区分弱抑制剂和强抑制剂。我们进一步表明,BpsA测定可以用于量化抑制水平,并产生一致的EC50数据。我们预计这些工具将有助于筛选已建立的化学集合,以确定新的抗分枝杆菌药物先导,并指导探索结构-活性景观,以改进现有的PPTase抑制剂。
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来源期刊
ACS Applied Energy Materials
ACS Applied Energy Materials Materials Science-Materials Chemistry
CiteScore
10.30
自引率
6.20%
发文量
1368
期刊介绍: ACS Applied Energy Materials is an interdisciplinary journal publishing original research covering all aspects of materials, engineering, chemistry, physics and biology relevant to energy conversion and storage. The journal is devoted to reports of new and original experimental and theoretical research of an applied nature that integrate knowledge in the areas of materials, engineering, physics, bioscience, and chemistry into important energy applications.
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