Structural basis of phosphoinositide biosynthesis in Mycobacterium tuberculosis

 描述（由申请人提供）：结核病（TB）是世界上最致命的疾病之一。目前，世界上三分之一的人口感染结核分枝杆菌（MyTB），结核病的病原体。仅在2012年，就有近900万人患上结核病，导致130万人死亡。目前的抗生素疗法具有有限的功效，并且通常必须联合使用并且持续延长的时间段。此外，耐药型MyTB越来越普遍，这突显了确定新靶点和开发抗结核新药的紧迫性。所有MyTB-宿主相互作用的关键决定因素是病原体细胞壁中的糖脂，它们都共享一个共同的磷脂酰肌醇（PI）锚到膜上，实际上PI是MyTB存活所必需的。分枝杆菌PI生物合成的关键第一步是由一种称为磷脂酰肌醇磷酸合酶（PIPS）的酶进行的。PIPS属于称为CDP-醇磷酸转移酶（CDP-AP）的酶的大家族。CDP-AP在所有生命界的所有甘油磷脂的生物合成中起关键作用。我们已经通过X射线晶体学确定了第一个代表性CDP-AP的原子分辨率结构，并且我们现在已经解决了与MyTB酶40%相同的PIPS的第二个结构。在此，我们建议：（i）使用该PIPS模型来研究MyTB中PI合成的分子细节（目标1）;（ii）利用我们对CDP-AP的专业知识来确定MyTB PIPS的结构（目标2）;（iii）利用我们产生大量功能性重组MyTB PIPS的能力来测量其活性，并建立高-MyTB PIPS抑制剂的通量筛选测定，同时通过虚拟筛选平行探索化学空间以鉴定潜在的先导物。我们的研究不仅将提供前所未有的洞察PI是如何在MyTB中产生的，而且还将使我们能够启动针对这种必需脂质合成的结构指导药物设计工作。PIPS代表了开发窄谱抗分枝杆菌疗法的关键靶标。事实上，MyTB PIPS的底物是唯一的，包括分枝杆菌在内的几种细菌。相比之下，真核生物的PI合酶不加工磷酸肌醇，并且大多数细菌完全缺乏磷脂酰肌醇。鉴于我们在CDP-AP和PIPS结构方面的成果和专业知识，我们在制定这一框架方面处于独特的地位。

 DESCRIPTION (provided by applicant): Tuberculosis (TB) is one of the world's deadliest diseases. Currently a third of the world's population is infected by Mycobacterium tuberculosis (MyTB), the causative agent for TB. In 2012 alone almost 9 million people developed TB resulting in 1.3 million deaths. Current antibiotic therapies have limited efficacy and must typically be used in combination and for prolonged periods of time. Furthermore, drug-resistant forms of MyTB are increasingly prevalent, underscoring the urgency to identify new targets and develop novel drugs against TB. The key determinants of all MyTB-host interactions are glycolipids in the cell wall of the pathogen that all share a common phosphatidylinositol (PI) anchor to the membrane and indeed PI is essential for MyTB survival. The crucial first step in biosynthesis of mycobacterial PI is carried out by an enzyme called phosphatidylinositolphosphate synthase (PIPS). PIPS belongs to a large family of enzymes called the CDP-alcohol phosphotransferases (CDP-AP). CDP-APs play the key role in the biosynthesis of all glycerophospholipids across all kingdoms of life. We have determined the atomic resolution structure by X-ray crystallography of the first representative CDP-AP and we now have solved a second structure of a PIPS which is 40% identical to the MyTB enzyme. Here we propose: (i) to use this PIPS model to study in molecular detail PI synthesis in MyTB (Aim 1); (ii) to build upon our expertise with CDP-APs to determine the structure of MyTB PIPS (Aim 2); (iii) to capitalize on our capability to generate abundant quantities of functional recombinant MyTB PIPS to measure its activity and set up a high-throughput screening assay for MyTB PIPS inhibitors whilst in parallel exploring chemical space by virtual screening to identify potential leads. Our research will not only offer unprecedented insight into how PI is made in MyTB, but it will also allow us to initiate structure-guided drug design efforts targeting the synthesis of this essential lipid. PIPS represents a key target for development of narrow spectrum anti- mycobacterial therapeutics. Indeed, the substrate for MyTB PIPS is unique to Archaea and few bacteria including Mycobacteria. In contrast, eukaryotic PI-synthases do not process inositol phosphate and most bacteria lack phosphatidylinositol entirely. We are in a unique position to set this framework given our results and expertise on the structures of CDP-APs and PIPS.