Whole Cell Target Engagement Identifies Novel Inhibitors of Mycobacterium tuberculosis Decaprenylphosphoryl-β-D-ribose Oxidase
Whole Cell Target Engagement Identifies Novel Inhibitors of Mycobacterium tuberculosis Decaprenylphosphoryl-β-D-ribose Oxidase
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DOI：
10.1021/acsinfecdis.5b00065
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发表时间：
2015-12-01
影响因子：
5.3
通讯作者：
Argyrou, Argyrides
中科院分区：
文献类型：
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作者：
Batt, Sarah M.;Cacho Izquierdo, Monica;Argyrou, Argyrides
我们已经针对结核分枝杆菌十异戊二烯磷酸-β-D-核糖氧化酶（Mt-DprE 1）的潜在的结核病化疗干预。在M.使用牛BCG来分析可在临床上获得的GlaxoSmithKline抗分枝杆菌化合物组，并且鉴定出一种化合物（GSK 710），其显示出相对于对照菌株高8倍的最小抑制浓度。GSK 710的类似物显示出使用重组Mt-DprE 1的酶测定法的全细胞效力和体外活性之间的明确关系，其中通过酶的黄素辅因子的荧光猝灭测量结合亲和力。M.牛BCG对GSK 710和密切相关的类似物的自发抗性突变体被分离，并且对10个这样的突变体的测序揭示了在DprE 1内的两个位点E221 Q或G248 S处的单点突变，提供了DprE 1是这些化合物的主要靶标的进一步证据。最后，延时显微镜实验表明，暴露于M。结核杆菌对这一系列化合物的反应迅速阻止细菌生长，随后是较慢的细胞溶解期。
We have targeted the Mycobacterium tuberculosis decaprenylphosphoryl-beta-D-ribose oxidase (Mt-DprE1) for potential chemotherapeutic intervention of tuberculosis. A multicopy suppression strategy that overexpressed Mt-DprEl in M. bovis BCG was used to profile the publically available GlaxoSmithKline antimycobacterial compound set, and one compound (GSK710) was identified that showed an 8-fold higher minimum inhibitory concentration relative to the control strain. Analogues of GSK710 show a clear relationship between whole cell potency and in vitro activity using an enzymatic assay employing recombinant Mt-DprE1, with binding affinity measured by fluorescence quenching of the flavin cofactor of the enzyme. M. bovis BCG spontaneous resistant mutants to GSK710 and a closely related analogue were isolated and sequencing of ten such mutants revealed a single point mutation at two sites, E221Q or G248S within DprE1, providing further evidence that DprEl is the main target of these compounds. Finally, time-lapse microscopy experiments showed that exposure of M. tuberculosis to a compound of this series arrests bacterial growth rapidly followed by a slower cytolysis phase.