Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, United States.
Program in Biochemistry and Structural Biology, Stony Brook University, Stony Brook, New York 11794-5215, United States.
ACS Infect Dis. 2023 Oct 13;9(10):1993-2004. doi: 10.1021/acsinfecdis.3c00296. Epub 2023 Sep 29.
By illuminating key 6-azasteroid-protein interactions in both () and the closely related model organism (), we sought to improve the antimycobacterial potency of 6-azasteroids and further our understanding of the mechanisms responsible for their potentiation of the antituberculosis drug bedaquiline. We selected a newly developed 6-azasteroid analog and an analog reported previously ( , (7), 1239-1251) to study their phenotypic effects on and , both alone and in combination with bedaquiline. The 6-azasteroid analog, 17β-[-(4-trifluoromethoxy-diphenylmethyl)carbamoyl]-6-propyl-azaandrostan-3-one, robustly potentiated bedaquiline-mediated antimycobacterial activity, with a nearly 8-fold reduction in bedaquiline minimal inhibitory concentration (85 nM alone versus 11 nM with 20 μM 6-azasteroid). This analog displayed minimal inhibitory activity against recombinant mycobacterial 3β-hydroxysteroid dehydrogenase, a previously identified target of several 6-azasteroids. Dose-dependent potentiation of bedaquiline by this analog reduced mycobacterial intracellular ATP levels and impeded the ability of to neutralize exogenous oxidative stress in culture. We developed two 6-azasteroid photoaffinity probes to investigate azasteroid-protein interactions in whole cells. Using bottom-up mass spectrometric profiling of the cross-linked proteins, we identified eight potential / protein targets for 6-azasteroids. The nature of these potential targets indicates that proteins related to oxidative stress resistance play a key role in the BDQ-potentiating activity of azasteroids and highlights the potential impact of inhibition of these targets on the generation of drug sensitivity.
通过阐明 （）和密切相关的模式生物 （）中关键的 6-氮杂甾体蛋白相互作用，我们旨在提高 6-氮杂甾体的抗分枝杆菌效力，并进一步了解其增强抗结核药物贝达喹啉作用的机制。我们选择了一种新开发的 6-氮杂甾体类似物和一种以前报道的类似物（，（7），1239-1251）来研究它们对 （）和 （）的表型影响，单独使用和与贝达喹啉联合使用。6-氮杂甾体类似物 17β-[-(4-三氟甲氧基二苯甲基)氨甲酰基]-6-丙基-氮杂雄甾烷-3-酮，强力增强了贝达喹啉介导的抗分枝杆菌活性，使贝达喹啉最小抑菌浓度（85 nM 单独使用，而与 20 μM 6-氮杂甾体联用则为 11 nM）降低近 8 倍。该类似物对重组分枝杆菌 3β-羟甾脱氢酶的抑制活性最小，这是几种 6-氮杂甾体的先前鉴定的靶标。该类似物对贝达喹啉的剂量依赖性增强作用降低了分枝杆菌细胞内的 ATP 水平，并阻碍了在培养中对的清除外源性氧化应激的能力。我们开发了两种 6-氮杂甾体光亲和探针来研究全细胞中氮杂甾体-蛋白相互作用。通过对交联蛋白的自上而下的质谱分析，我们鉴定出了 6-氮杂甾体的八个潜在 / 蛋白靶标。这些潜在靶标的性质表明，与氧化应激抗性相关的蛋白质在 6-氮杂甾体增强贝达喹啉的活性中起着关键作用，并突出了抑制这些靶标对产生药物敏感性的潜在影响。
