Preclinical evaluation of compounds that inhibit cholesterol uptake in M. tuberculosis.

项目总结/摘要 结核分枝杆菌的胆固醇代谢是基础研究和新药开发工作的一个有吸引力的目标。 Mtb胆固醇代谢途径对细菌是特异性的，并且在该途径中有缺陷的突变体是 在各种感染模型中减弱。这表明，化学抑制这一途径也将削弱 结核分枝杆菌体内毒力。我们最近发现了一种化合物系列，可以阻止结核分枝杆菌和 刺激细菌中cAMP的过量产生。这是重要的，因为cAMP调节分枝杆菌中枢神经系统， 代谢、转录、致病性、休眠和胁迫反应。因此，化学刺激cAMP 除了胆固醇利用之外，Mtb的产生将扰乱细菌生理学的多个不同方面。 肉芽肿是限制免疫细胞募集和抗生素扩散的主要病理屏障。内 肉芽肿结核分枝杆菌被隔离在富含胆固醇和缺氧的微环境中， 细菌持久性在感染过程中，MΦ产生TNF-α，其驱动TB组织病理学，并需要 形成并维持肉芽肿。认为TNF-α耗竭可干扰肉芽肿结构， 提高抗生素的利用率或破坏肉芽肿微环境，使结核病抗生素 有效很久以前就知道MΦ的TNF-α的产生可以在高浓度的TNF-α刺激下下调。 细胞溶质cAMP水平，并且还已知Mtb衍生的cAMP下调MΦ中的TNF-α产生。 我们的化合物系列刺激足够的结核分枝杆菌衍生的cAMP，以下调TNF-α的产生，在感染 细胞水平。因此，刺激cAMP在Mtb中的过量产生可能是降低TNF-α水平的新策略 特别是在感染的MΦ中，以增强当前结核病药物的活性。 在这里，我们提出的特点，这些探针如何阻止胆固醇摄取的分子机制 刺激结核分枝杆菌cAMP的过量产生（目的1）。我们还将确定结核分枝杆菌衍生的cAMP 调节：（i）宿主免疫应答，（ii）MΦ功能，和（iii）评估单独的V-59和V-59在免疫应答中的功效。 与已知的TB抗生素组合，目的是评估V-59的治疗潜力（目的2）。 因为我们已经有了一种化合物，它具有优异的效力，并通过口服给药证明了体内功效。 因此，我们有能力在潜在的结核病候选药物和新的治疗策略方面取得进展。

Project Summary / Abstract Cholesterol metabolism in Mtb is an attractive target for basic research and new drug development efforts. The Mtb cholesterol metabolic pathway is specific to the bacteria and mutants defective in this pathway are attenuated in various infection models. This suggests that chemically inhibiting this pathway will also attenuate Mtb virulence in vivo. We recently discovered a compound series that blocks cholesterol uptake in Mtb and stimulates cAMP overproduction in the bacteria. This is important since cAMP regulates mycobacterial central metabolism, transcription, pathogenicity, dormancy, and stress responses. Thus, chemically stimulating cAMP production in Mtb will perturb multiple different aspects of bacterial physiology in addition to cholesterol utilization. Granulomas are a major pathologic barrier that limits immune cell recruitment and antibiotic diffusion. Within a granuloma Mtb is sequestered in a cholesterol rich and hypoxic microenvironment that is thought to favor bacterial persistence. During infection, MΦ’s produce TNF-α which drives TB tissue pathology and is required to form and maintain granulomas. It is thought that TNF-α depletion can disturb the granuloma architecture and promote enhanced antibiotic availability or disrupt the granuloma microenvironment making TB antibiotics more effective. It has long been known that TNF-α production by MΦ’s can be down regulated in response to high levels of cytosolic cAMP and it is also known that Mtb-derived cAMP down regulates TNF-α production in MΦ’s. Our compound series stimulates enough Mtb-derived cAMP to down regulate TNF-α production at the infected cell level. Thus, stimulating cAMP overproduction in Mtb could be a novel strategy to reduce TNF-α levels specifically in infected MΦ’s to enhance the activity of current TB drugs. Here we propose to characterize the molecular mechanisms of how these probes block cholesterol uptake and stimulate the overproduction of cAMP in Mtb (Aim 1). We also will determine how Mtb-derived cAMP modulates: (i) the host immune response, (ii) MΦ function, and (iii) evaluate the efficacy of V-59 alone and in combination with known TB antibiotics in vivo with a goal of evaluating the therapeutic potential of V-59 (Aim 2). Because we already have a compound that has excellent potency and demonstrates in vivo efficacy via the oral route, we are well positioned to make progress with a potential TB drug candidate and novel treatment strategy.