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

项目摘要/摘要 MTB中的胆固醇代谢是基础研究和新药开发努力的一个有吸引力的靶点。 结核分枝杆菌胆固醇代谢途径是针对细菌的，而在该途径上有缺陷的突变株是 在不同的感染模式下减毒。这表明，化学抑制这一途径也会减弱 结核分枝杆菌体内毒力。我们最近发现了一系列化合物，可以阻止Mtb和Mtb的胆固醇吸收 刺激细菌中cAMP的过量产生。这一点很重要，因为cAMP调节分枝杆菌中枢 代谢、转录、致病性、休眠和应激反应。因此，化学刺激性营地 除胆固醇利用外，结核分枝杆菌的生产还将扰乱细菌生理的多个不同方面。 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。在 肉芽肿结核分枝杆菌被隔离在富含胆固醇和低氧的微环境中，这被认为有利于 细菌的持久性。在感染过程中，MΦ‘S产生肿瘤坏死因子-α，驱动结核组织病理，并被要求 形成并维持肉芽肿。目前认为，肿瘤坏死因子-α耗竭可干扰肉芽肿的结构和功能。 促进抗生素的可获得性或破坏肉芽肿微环境，使结核病抗生素更多 有效。很早就知道，Mα的S产生的肿瘤坏死因子-Φ可以被下调，以响应高水平的 胞内cAMP水平以及MTB衍生的cAMP下调肿瘤坏死因子-α的产生也被认为是MΦ‘S。 我们的化合物系列刺激足够多的结核分枝杆菌衍生的cAMP，以下调感染者的肿瘤坏死因子-α的产生。 细胞水平。因此，刺激mtb中cAMP的过量生产可能是降低肿瘤坏死因子-α水平的新策略。 具体地说，就是在感染MΦ的S中，增强了目前抗结核药物的活性。 在这里，我们建议表征这些探针如何阻止胆固醇摄取的分子机制。 并刺激Mtb的cAMP生产过剩(目标1)。我们还将确定结核分枝杆菌衍生的cAMP 调节：(I)宿主免疫反应，(Ii)MΦ功能，以及(Iii)评估V-59单独和在 在体内与已知的结核病抗生素结合，目的是评估V-59的治疗潜力(目标2)。 因为我们已经有一种化合物，它具有极好的效力，并通过口服证明在体内有效 在这条路线上，我们处于有利地位，可以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.