Validation of RelA as a Target for Mycobacterium Tuberculosis Persisters

描述（由申请人提供）：目前用于治疗活动性结核病（TB）感染的冗长而复杂的多药治疗导致了药物不依从性和新出现的多药耐药（MDR）和广泛耐药（XDR）TB问题，这些问题在HIV合并感染的情况下尤其致命。此外，艾滋病毒感染者即使成功完成抗结核治疗，复发的风险也会增加。根除TB感染所需的延长治疗被认为反映了结核分枝杆菌（Mtb）以非复制状态在宿主坏死性肉芽肿内持续存在的能力，所述非复制状态的特征在于对杀菌药物的抗生素耐受性，所述杀菌药物主要靶向活跃分裂的结核杆菌。由严格反应酶RelA合成的过磷酸化鸟苷（（p）ppGpp）在细菌生长限制和抗生素耐受中起重要作用。RelA对于在各种体外和体内生长限制条件期间的长期Mtb存活是必需的。最近，我们发现relA缺陷型结核分枝杆菌在营养饥饿期间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）的合作者已经在RelA抑制试验和全细胞筛选中筛选了超过200万种化合物的库。在代表18个独特支架的178个命中中，我们发现12个测试的支架中有8个对营养缺乏的Mtb具有有效的RelA特异性活性。该提案的目标是验证RelA作为体内Mtb持续者的靶点，并开发针对持续性杆菌的RelA抑制剂，最终目标是确定一种先导化合物，该化合物可以通过“唤醒”持续者并使其更容易被标准抗结核药物杀死来缩短结核病的治疗时间。具体而言，我们将使用各种体外试验筛选命中以选择候选物，这些候选物将用于初步毒性研究并在体内验证靶标。除了提高医疗依从性和减少耐药性的可能性外，治疗活动性结核病的简化药物方案在HIV合并感染患者中特别有用，因为药物-药物相互作用和免疫重建可能使两种感染的并发管理复杂化。

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.