Structural basis of phosphoinositide biosynthesis in Mycobacterium tuberculosis

 描述(申请人提供)：结核病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)，这是结核病的病原体。仅在2012年，就有近900万人患上结核病，导致130万人死亡。目前的抗生素治疗效果有限，通常必须联合使用并延长时间。此外，耐药形式的结核分枝杆菌日益流行，突显了确定新的靶点和开发治疗结核病的新药的紧迫性。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)锚定在膜上，确实PI是MyTB生存所必需的。分枝杆菌PI生物合成的关键第一步是由一种名为磷脂酰肌醇磷酸合成酶(PIPS)的酶执行的。PIPS属于CDP-乙醇磷酸转移酶(CDP-AP)的一个大家族。CDP-AP在所有生命王国中所有甘油磷脂的生物合成中起着关键作用。我们已经用X射线结晶学测定了第一个具有代表性的CDP-AP的原子分辨结构，现在我们解决了PIPS的第二个结构，它与MyTB酶的同源性为40%。在这里，我们建议：(I)利用这一PIPS模型来详细研究MyTB中PI的合成(目标1)；(Ii)利用我们在CDP-AP方面的专业知识来确定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，并建立高通量筛选方法来检测MyTB PIPS抑制剂，同时通过虚拟筛选来平行探索化学空间，以确定潜在的先导。我们的研究不仅将提供前所未有的洞察力，了解PI是如何在MyTB中产生的，而且它还将使我们能够启动以结构为导向的药物设计工作，以合成这种重要的脂质。PIPS是发展窄谱抗分枝杆菌治疗药物的关键靶点。事实上，结核分枝杆菌颗粒的底物是古生菌和包括分枝杆菌在内的少数细菌所独有的。相比之下，真核细胞的PI-合成酶不处理肌醇磷酸，大多数细菌完全缺乏磷脂酰肌醇。鉴于我们在CDP-AP和PIP结构方面的成果和专业知识，我们处于制定这一框架的独特地位。

 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.