Bioluminescent Reporters for Rapid Mechanism of Action Assessment in Tuberculosis Drug Discovery
Bioluminescent Reporters for Rapid Mechanism of Action Assessment in Tuberculosis Drug Discovery
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DOI：
10.1128/aac.01178-16
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发表时间：
2016-11-01
影响因子：
4.9
通讯作者：
Warner, Digby F.
中科院分区：
文献类型：
--
作者：
Naran, Krupa;Moosa, Atica;Warner, Digby F.
结核病（TB）药物发现管道是由全细胞筛选中针对病原体结核分枝杆菌（Mycobacterium tuberculosis）鉴定的化合物推动的。表型筛选使得能够选择抑制在选定的体外条件下生长的活的完整杆菌中的基本细胞功能的分子。然而，推导作用机制（MOA），这是重要的，以避免混杂的目标，往往需要大量的生物资源，在一个漫长的过程中，有可能脱钩药物化学和生物学的努力。因此，需要开发能够快速MOA评估推定的“活性物”的方法以用于分诊决策。在这里，我们描述了一个修改后的版本的生物发光报告分析，允许非破坏性检测化合物的目标是两个大分子过程中的M。结核病：细胞壁生物合成或维持DNA完整性。将来自发光杆菌的luxCDABE操纵子偶联至驱动iniBAC操纵子（PiniB-LUX）或DNA损伤诱导基因recA（PrecA-LUX）或radA（Pradaptus X）表达的分枝杆菌启动子，提供了在延长的10至12天孵育期间触发这些启动子中任一种表达的化合物的真实的定量检测。针对已知抗TB剂的测试证实了每个报告基因在M中登记所应用抗生素的MOA的特异性。结核病，独立于杀菌或抑菌活性。此外，实验化合物获得的配置文件表明，有可能推断复杂的MOA中，多个细胞过程被破坏。这些结果证明了报告的效用的化合物的早期分类的基础上临时MOA，并建议其应用程序，以调查已知的和实验性的抗B剂的多药理学。
The tuberculosis (TB) drug discovery pipeline is fueled by compounds identified in whole-cell screens against the causative agent, Mycobacterium tuberculosis. Phenotypic screening enables the selection of molecules that inhibit essential cellular functions in live, intact bacilli grown under a chosen in vitro condition. However, deducing the mechanism of action (MOA), which is important to avoid promiscuous targets, often requires significant biological resources in a lengthy process that risks decoupling medicinal chemistry and biology efforts. Therefore, there is a need to develop methods enabling rapid MOA assessment of putative "actives" for triage decisions. Here, we describe a modified version of a bioluminescence reporter assay that allows non-destructive detection of compounds targeting either of two macromolecular processes in M. tuberculosis: cell wall biosynthesis or maintenance of DNA integrity. Coupling the luxCDABE operon from Photorhabdus luminescens to mycobacterial promoters driving expression of the iniBAC operon (PiniB-LUX) or the DNA damage-inducible genes, recA (PrecA-LUX) or radA (PradALUX), provided quantitative detection in real time of compounds triggering expression of any of these promoters over an extended 10- to 12-day incubation. Testing against known anti-TB agents confirmed the specificity of each reporter in registering the MOA of the applied antibiotic in M. tuberculosis, independent of bactericidal or bacteriostatic activity. Moreover, profiles obtained for experimental compounds indicated the potential to infer complex MOAs in which multiple cellular processes are disrupted. These results demonstrate the utility of the reporters for early triage of compounds based on the provisional MOA and suggest their application to investigate polypharmacology in known and experimental anti-B agents.