Data_Sheet_1_Novel Screen to Assess Bactericidal Activity of Compounds Against Non-replicating Mycobacterium abscessus.PDF

Mycobacterium abscessus infections are increasing worldwide. Current drug regimens are largely ineffective, yet the current development pipeline for M. abscessus is alarmingly sparse. Traditional discovery efforts for M. abscessus assess the capability of a new drug to inhibit bacterial growth under nutrient-rich growth conditions, but this does not predict the impact when used in the clinic. The disconnect between in vitro and in vivo activity is likely due to the genetic and physiological adaptation of the bacteria to the environmental conditions encountered during infection; these include low oxygen tension and nutrient starvation. We sought to fill a gap in the drug discovery pipeline by establishing an assay to identify novel compounds with bactericidal activity against M. abscessus under non-replicating conditions. We developed and validated a novel screen using nutrient starvation to generate a non-replicating state. We used alamarBlue® to measure metabolic activity and demonstrated this correlates with bacterial viability under these conditions. We optimized key parameters and demonstrated reproducibility. Using this assay, we determined that niclosamide was bactericidal against non-replicating bacilli, highlighting its potential to be included in M. abscessus regimens. In contrast, most other drugs currently used in the clinic for M. abscessus infections, were completely inactive, potentially explaining their poor efficacy. Thus, our assay allows for rapid identification of bactericidal compounds in a model using conditions that are more relevant in vivo. This screen can be used in a high-throughput way to identify novel agents with properties that promise an increase in efficacy, while also shortening treatment times.

全球范围内，结核分枝杆菌感染病例呈上升趋势。目前的治疗方案大多无效，而针对结核分枝杆菌的研发管线却令人担忧地稀疏。传统上对结核分枝杆菌的新药发现工作主要评估新药在营养丰富的生长条件下抑制细菌生长的能力，但这并不能预测其在临床应用中的影响。体外与体内活性之间的脱节很可能是由于细菌在感染过程中遇到的环境条件（包括低氧张力及营养剥夺）导致的遗传和生理适应性；这些条件包括低氧张力及营养剥夺。我们旨在填补药物发现流程中的空白，通过建立一种检测在非复制条件下对结核分枝杆菌具有杀菌活性的新型化合物的检测方法。我们开发并验证了一种利用营养剥夺产生非复制状态的全新筛选方法。我们使用alamarBlue®来测量代谢活性，并证明这与其在非复制条件下细菌存活率的相关性。我们优化了关键参数，并展示了其可重复性。利用此检测方法，我们发现尼可刹米对非复制菌具有杀菌作用，突显了其被纳入结核分枝杆菌治疗方案中的潜力。相比之下，目前大多数用于治疗结核分枝杆菌感染的药物在临床应用中均完全无效，这或许可以解释它们疗效不佳的原因。因此，我们的检测方法允许在更接近体内条件的环境中快速识别杀菌化合物。这种筛选方法可以以高通量的方式进行，以识别具有提高疗效和缩短治疗时间潜力的新型药物。