In Vitro Profiling of Antitubercular Compounds by Rapid, Efficient, and Nondestructive Assays Using Autoluminescent Mycobacterium tuberculosis.
In Vitro Profiling of Antitubercular Compounds by Rapid, Efficient, and Nondestructive Assays Using Autoluminescent Mycobacterium tuberculosis.
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DOI：
10.1128/aac.00282-21
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发表时间：
2021-07-16
影响因子：
4.9
通讯作者：
Cho S
中科院分区：
文献类型：
--
作者：
Shetye GS;Choi KB;Kim CY;Franzblau SG;Cho S
抗感染药物的发现极大地促进了体外测试的可用性，这些测试在预测HITS的临床前成功方面更加熟练。由于结核分枝杆菌的生长速度相对较慢，以及基于全细胞的体外分析固有的耗时，阻碍了结核病药物的开发，因此，基于快速、非侵入性的生物发光分析方法已被广泛应用于抗结核药物的发现和开发。在这项研究中，利用自发光技术对体外试验进行了优化，以确定最低抑菌浓度(MIC)、最低杀菌浓度(MBC)、时间-杀伤曲线、抗巨噬细胞内化结核分枝杆菌的活性(90%有效浓度[EC90])和抗生素后效应(PAE)，以提供快速和动态的生物学信息。基于发光的MIC、MBC、Time-Kill、EC90和PAE分析的标准化是通过将已建立的结核病药物和两种以ClpC1为靶标的TB引线的结果与常规分析和/或先前研究的结果进行比较而实现的。累积起来，各种简化的基于发光的体外分析方法的使用已经将全面的体外分析(最低抑菌浓度、最低抑菌浓度、时间-杀死时间、EC90和PAE)的时间减少了2 个月。基于发光的体外MBC和EC90检测产生时间和浓度依赖的杀伤信息，可用于药代动力学-药效学(PK-PD)建模。MBC和EC90时间-杀灭曲线显示，Eumicin的杀菌活性明显快于红霉素。优降素和鲁福霉素的PAEs与一线结核病药物利福平相当。几种无损的、基于发光的结核分析方法的优化有助于高效地进行结核药物引线的体外图谱分析。
Anti-infective drug discovery is greatly facilitated by the availability of in vitro assays that are more proficient at predicting the preclinical success of screening hits. Tuberculosis (TB) drug discovery is hindered by the relatively slow growth rate of Mycobacterium tuberculosis and the use of whole-cell-based in vitro assays that are inherently time-consuming, and for these reasons, rapid, noninvasive bioluminescence-based assays have been widely used in anti-TB drug discovery and development. In this study, in vitro assays that employ autoluminescent M. tuberculosis were optimized to determine MIC, minimum bactericidal concentration (MBC), time-kill curves, activity against macrophage internalized M. tuberculosis (90% effective concentration [EC90]), and postantibiotic effect (PAE) to provide rapid and dynamic biological information. Standardization of the luminescence-based MIC, MBC, time-kill, EC90, and PAE assays was accomplished by comparing results of established TB drugs and two ClpC1-targeting TB leads, ecumicin and rufomycin, to those obtained from conventional assays and/or to previous studies. Cumulatively, the use of the various streamlined luminescence-based in vitro assays has reduced the time for comprehensive in vitro profiling (MIC, MBC, time-kill, EC90, and PAE) by 2 months. The luminescence-based in vitro MBC and EC90 assays yield time and concentration-dependent kill information that can be used for pharmacokinetic-pharmacodynamic (PK-PD) modeling. The MBC and EC90 time-kill graphs revealed a significantly more rapid bactericidal activity for ecumicin than rufomycin. The PAEs of both ecumicin and rufomycin were comparable to that of the first-line TB drug rifampin. The optimization of several nondestructive, luminescence-based TB assays facilitates the in vitro profiling of TB drug leads in an efficient manner.