Species selective dipeptide inhibitors for Mtb proteasome

描述（由申请人提供）：体外活性并不总是转化为体内活性，在结核分枝杆菌（Mtb）治疗中也是如此。例如，两种一线抗结核药物异烟肼和利福平在体外能迅速杀死结核分枝杆菌，但在体内的杀菌能力减慢和降低。结核分枝杆菌群体的异质性导致了对药物的不同易感性。特别是，缓慢复制或非复制（统称“NR”）结核分枝杆菌对大多数一线抗结核药物具有非遗传性耐药。我们的长期目标是开发能够杀死NR结核分枝杆菌的抗结核药物，以补充杀死复制结核分枝杆菌的药物。在过去的八年中，发现了原核泛素样蛋白(Pup)-蛋白酶体系统的组成部分。虽然Mtb蛋白酶体在标准生长条件下是可有可无的，但遗传证据表明它对Mtb在小鼠体内存活至关重要。我们还建立了一个概念，即尽管蛋白酶体在哺乳动物中起着至关重要的作用，但可以发现小的化学分子对结核分枝杆菌蛋白酶体的抑制作用比人类蛋白酶体具有广泛的物种选择性，这种抑制作用导致NR结核分枝杆菌的死亡。在其他细菌中，另一种受调节的蛋白质降解机器Clp的破坏导致了死亡。因此，抑制或强制激活腔室蛋白酶可能是一种新的抗感染策略。在我们最近对1600个带帽二肽的筛选中，我们发现了一个先导化合物，它可以有效地和物种选择性地抑制结核分枝杆菌蛋白酶体，而不是人类蛋白酶体。抑制剂与一氧化氮协同作用对非复制型结核分枝杆菌具有杀菌作用。通过竞争试验，我证实了二肽能穿透分枝杆菌并抑制其中的蛋白酶体。在这个应用程序中，我们将把我们的先导化合物扩展成一个小的集中化合物库，其设计和合成是由底物分析、结构分析、分枝杆菌活性和代谢稳定性指导的。

DESCRIPTION (provided by applicant): In vitro activities do not always translate into in vivo activities, and this is true in Mycobacterium tuberculosis (Mtb) therapeutics. For example, the two first line anti-Mtb drugs isoniazid and rifampicin kill Mtb rapidly in vitro, but their steriliation abilities are slowed and reduced in vivo. Heterogeneity of Mtb populations imparts varied susceptibility to the drugs. In particular, slowly-replicating or non-replicating (collectively "NR) Mtb is non-heritably resistant to most first line anti-TB drugs. Our long-term goal is to develop anti-Mtb drugs that kill NR Mtb populations to complement drugs that kill replicating Mtb populations. During the past eight years, components of a prokaryotic ubiquitin-like protein (Pup)-proteasome system have been discovered. Although the Mtb proteasome is dispensable under standard growth conditions, genetic evidence demonstrates its essentiality for Mtb to survive in mice. We also established the concept that despite the essential role of the proteasome in mammals, small chemical molecules can be discovered with extensive (>1000-fold) species selectivity for inhibiting the Mtb proteasome over the human proteasome, and that such inhibition leads to killing of NR Mtb. In other bacteria, disruption of another regulated protein degradation machine, Clp, has led to killing. Thus, either inhibition or forced activation f chambered proteases may represent a new anti-infective strategy. In our recent screening of 1600 capped dipeptides, we identified a lead compound that potently and species-selectively inhibited the Mtb proteasome over the human proteasome. The inhibitors were bactericidal for non-replicating Mtb in synergy with nitric oxide. Using a competition assay, I confirmed that the dipeptides penetrate the mycobacteria and inhibit the proteasome within them. In this application, we will expand our lead compounds into a small focused compound library whose design and synthesis are guided by substrate-profiling, structural analysis, mycobactericidal activity, and metabolic stability.