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）感染的长期和复杂的多药治疗导致了医疗不依从性和新出现的耐多药（MDR）和广泛耐药（XDR）结核病问题，这在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。根除结核感染所需的长期治疗被认为反映了结核分枝杆菌（Mtb）在宿主坏死肉芽肿中以非复制状态持续存在的能力，其特征是抗生素对杀菌药物的耐受性，这些药物主要针对活跃分裂的结核杆菌。高磷酸化鸟苷(p)ppGpp是由严格反应酶RelA合成的，在细菌生长限制和抗生素耐受中起重要作用。RelA对结核分枝杆菌在各种体外和体内生长限制条件下的长期存活至关重要。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部表现出对异烟肼的敏感性增强。我们在葛兰素史克的发展中国家疾病开放实验室（GSK-DDW）的合作伙伴已经在rela抑制试验和全细胞筛选中筛选了超过200万种化合物。在代表18个独特支架的178个hit中，我们发现12个支架中有8个对营养匮乏的Mtb具有有效的rela特异性活性。本课题的目标是验证RelA在体内作为结核分枝杆菌持续存在的靶点，并开发针对持续存在杆菌的RelA抑制剂，最终目标是鉴定一种先导化合物，通过“唤醒”持续存在杆菌，使其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。具体来说，我们将使用各种体外试验筛选命中点以选择候选物，这些候选物将用于初步毒性研究并在体内验证靶点。除了提高医疗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合并感染艾滋病毒的患者特别有用，因为药物-药物相互作用和免疫重建可能使同时治疗两种感染复杂化。

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.