Validation of RelA as a Target for Mycobacterium Tuberculosis Persisters

描述(申请人提供)：目前可用于治疗活动性结核病(TB)感染的漫长而复杂的多药疗法导致了医学上的不遵守和新出现的多药耐药(MDR)和广泛耐药(XDR)结核病问题，在艾滋病毒合并感染的情况下，这些问题尤其致命。此外，艾滋病毒携带者即使在成功完成抗结核治疗后，复发的风险也会增加。根除结核病感染所需的长期治疗被认为反映了结核分枝杆菌(Mtb)在宿主坏死性肉芽肿内以非复制状态持续存在的能力，其特点是对杀菌药物具有抗生素耐受性，杀菌药物主要针对主动分裂的结核杆菌。过度磷酸化鸟苷((P)ppGpp)是由严格反应酶relA合成的，在细菌生长抑制和抗生素耐受中发挥着重要作用。在不同的体外和体内生长限制条件下，RELA对于结核分枝杆菌的长期存活是必不可少的。最近，我们发现缺乏RelA的结核分枝杆菌在营养饥饿和慢性感染小鼠的肺中表现出对异烟肼的敏感性增强。我们在葛兰素史克的Tres Cantos发展中国家疾病开放实验室(GSK-DDW)的合作者通过重复抑制分析和全细胞筛选筛选了一个包含200多万种化合物的文库。在代表18种独特支架的178次点击中，我们发现12种支架中有8种被测试具有有效的、RelA特异性的抗营养缺乏的结核分枝杆菌活性。这项提案的目标是在体内验证rela作为结核分枝杆菌顽固者的靶标，并开发针对持久性杆菌的relA抑制剂，最终目标是确定一种先导化合物，这种化合物可以通过“唤醒”顽固者并使他们更容易被标准抗结核药物杀死，从而缩短结核病的治疗时间。具体地说，我们将使用各种体外测试来筛选命中，以选择候选药物，这些候选药物将用于初步毒性研究，并在体内验证靶点。除了改善服药依从性和减少产生耐药性的可能性外，治疗活动性结核病的简化药物方案对艾滋病毒合并感染的患者将特别有用，因为药物-药物相互作用和免疫重建可能会使这两种感染的同时管理复杂化。

DESCRIPTION (provided by applicant): The lengthy and complicated multidrug therapy currently available to treat active tuberculosis (TB) infection has contributed to medical non-adherence and the emerging problems of multidrug-resistant (MDR)- and extensively drug-resistant (XDR)-TB, which are particularly deadly in the setting of HIV co-infection. In addition, persons with HIV are at increased risk for relapse even upon successful completion of anti-TB treatment. The prolonged therapy required to eradicate TB infection is believed to reflect the ability of Mycobacterium tuberculosis (Mtb) to persist within host necrotic granulomas in a non-replicating state characterized by antibiotic tolerance to bactericidal drugs, which predominantly target actively dividing tubercle bacilli. Hyperphosphorylated guanosine ((p)ppGpp), which is synthesized by the stringent response enzyme RelA, plays an important role in bacterial growth restriction and antibiotic tolerance. RelA is essential for long-term Mtb survival during various i vitro and in vivo growth-limiting conditions. Recently, we found that relA-deficient Mtb exhibits enhanced susceptibility to isoniazid during nutrient starvation and in the lungs of chronically infected mice. Our collaborators at GlaxoSmithKline's Tres Cantos Open Lab for Diseases of the Developing World (GSK-DDW) have screened a library of over 2 million compounds in RelA-inhibition assays and whole- cell screens. Of 178 hits representing 18 unique scaffolds, we have found 8 of 12 scaffolds tested to have potent, RelA-specific activity against nutrient-starved Mtb. The goal of this proposal is to validate RelA as a target for Mtb persisters in vivo and develop RelA inhibitors for targeting persistent bacilli, with the ultimate goal of identifyinga lead compound, which can shorten the duration of treatment for TB by "awakening" persisters and rendering them more susceptible to killing by standard anti-TB drugs. Specifically, we will screen hits using various in vitro assays to select candidates, which will be used for preliminary toxicity studies and to validate the target in vivo. In addition to improving medical adherence and reducing the potential for the development of drug resistance, an abbreviated drug regimen to treat active TB would be particularly useful in HIV co-infected patients, since drug-drug interactions and immune reconstitution may complicate the concurrent management of both infections.