Development of novel inhibitors of cholesterol oxidases in Mycobacterium tuberculosis

结核分枝杆菌（Mtb，结核病的病原体）是对全世界人类生命的巨大威胁。世界上三分之一的人口被这种细菌感染。许多Mtb菌株已经对一些或所有一线抗生素产生了耐药性，这些耐药、多药甚至完全耐药菌株对结核病（TB）的成功治疗构成了重大挑战。迫切需要新的策略和细菌中的新靶酶来提供可以提供有效结核病治疗的新型抗生素。在这个项目中，目的是开发关键Mtb细胞色素P450酶（P450）的新抑制剂，这些酶对于细菌在人类巨噬细胞中吞噬时的生存至关重要。P450的CYP 125 A1、CYP 142 A1和（在较小程度上）CYP 124 A1催化宿主胆固醇催化的主要步骤，这是巨噬细胞中Mtb的主要能量来源。这些酶的抑制提供了一种新的途径来杀死巨噬细胞中的Mtb细菌，否则它们可能会在休眠状态下存活数年。所使用的策略将是基于片段的筛选-一种相对较新的方法，其中靶蛋白被筛选用于与小化合物（“片段”）结合，使用高通量NMR和/或量热法来鉴定“命中”分子。然后使用X射线晶体学解析与这些不同分子复合的靶P450的结构，然后通过例如化学连接/合并分子以形成更大和更紧密结合的抑制剂来指导化合物开发，或者通过化学方法加工原始命中物以延伸它们的结构，从而填充酶活性位点的相邻部分，有效和特异的抑制剂。这一策略在我们正在进行的其他Mtb P450的研究中证明是成功的，包括对Mtb生存能力至关重要的CYP 121 A1，以及其他Mtb P450的研究。本课题组已成功表达并纯化了胆固醇羟化酶，并测定了其晶体结构。这将有助于这些P450的片段筛选，以允许开发新的抑制剂和测试这些化合物对Mtb细菌的有效性。该项目将涉及在分子生物学，蛋白质表达，结构生物学，酶学/生物催化，光谱技术和抑制剂开发等领域对学生进行培训。该项目还提供了一个与我们在剑桥的研究合作者合作的机会，以获得片段筛选技术的进一步技能。该项目符合BBSRC在优先领域的职权范围，如“生物科学技术开发”（通过使用片段筛选和抑制剂开发方法，以及结构生物学来产生有效对抗关键Mtb酶的新化合物）和“对抗抗菌素耐药性”（通过从片段筛选方法产生新的先导化合物，并证明它们对宿主中细菌存活至关重要的Mtb P450酶的活性）。该项目还与DTP主题“世界级基础生物科学”保持一致（通过应用新的片段筛选技术，为开发有效的Mtb酶抑制剂提供新的结构导向策略，并通过与克里克研究所的合作者合作进行验证。

Mycobacterium tuberculosis (Mtb, the causative agent of tuberculosis) represents a huge threat to human life worldwide. A third of the world's population is infected by the bacterium. Numerous strains of Mtb have developed resistance to some or all of the frontline antibiotics, and these drug-, multidrug- and even totally drug-resistant strains pose major challenges for the successful treatment of tuberculosis (TB). New strategies and new target enzymes in the bacterium are desperately needed to provide novel antibiotics that can deliver effective TB treatments. In this project, the aim is to develop new inhibitors of key Mtb cytochrome P450 enzymes (P450s) that are crucial for the bacterium to survive while engulfed in the human macrophage. The P450s CYP125A1, CYP142A1 and (to a lesser extent) CYP124A1 catalyse the primary step in the catabolism of host cholesterol, which is a major energy source for Mtb in the macrophage. Inhibition of these enzymes provides a new route to killing Mtb bacteria in the macrophage, where they otherwise might survive in a dormant state for several years. The strategy used will be fragment based screening - a relatively new method in which target proteins are screened for binding to small compounds ("fragments"), using high throughput NMR and/or calorimetry methods to identify "hit" molecules. The structures of the target P450s in complex with these various molecules are then solved using X-ray crystallography, which then informs modelling work to guide compound development through e.g. chemically linking/merging molecules together to form larger and tighter-binding inhibitors, or by chemically elaborating the original hits to extend their structures in order to fill adjacent parts of the enzyme active site in order to produce more effective and specific inhibitors. This strategy has proved successful in our ongoing work on other Mtb P450s, including CYP121A1, which is essential for Mtb viability, as well as in studies of other Mtb P450s. The targeted cholesterol hydroxylases have been successfully expressed and purified in large yield in our group, and we have determined the crystal structures of these enzymes. This will facilitate fragment screening for these P450s to allow development of novel inhibitors and testing of these compounds for their effectiveness against Mtb bacteria. The project will involve training of the student in areas including molecular biology, protein expression, structural biology, enzymology/biocatalysis, spectroscopic techniques and inhibitor development. The project also provides an opportunity to work with our research collaborators at Cambridge to gain further skills in fragment screening technology. The project aligns with BBSRC remit in priority areas such as "Technology Development for the Biosciences" (through the use of fragment screening and inhibitor development methods, together with structural biology to produce new compounds effective against key Mtb enzymes) and "Combatting Antimicrobial Resistance" (through producing new lead compounds derived from fragment screening approaches, and demonstrating their activity against Mtb P450 enzymes crucial for bacterial survival in the host). The project also aligns with the DTP theme of "World Class Underpinning Biosciences" (through application of novel fragment screening techniques in order to provide new structure-led strategies for the development of potent Mtb enzyme inhibitors, and their validation through collaboration with collaborators at the Crick Institute.