A fragment-based screening approach to rationalizing M. tuberculosis P450 molecular selectivity

人类和其他生物整个DNA序列测定方法的发展揭示了大量新的信息，包括识别新的蛋白质和酶，以及细胞内发生的不寻常和意想不到的化学过程。从抗生素治疗和生物技术应用等角度来看，一旦这些新型酶催化的底物和反应建立起来，显然就有无数的机会。然而，如果对一种新鉴定的酶的生理作用没有初步了解，则通常需要付出相当大的努力才能确定它的功能。此外，识别其功能的特定抑制物也可能是费力的，涉及筛选巨大的化学物库。然而，近年来出现了一种新型的方法来定义与纯化酶结合的分子。这种基于片段的筛选(FBS)技术包括分析比在典型化合物筛选中使用的更有限的小分子文库的结合，例如在制药部门中使用的。FBS方法最初的HITS可能只与目标酶结合得很弱，但借助蛋白质结构方法可以准确地确定它们的结合位置。一旦获得了许多这样的点击，关于它们的结合位置以及这些位置和周围环境中的化学性质的组合信息可以提供一个基础，用于‘阐述’分子结构，从而实现更紧密的结合，或者甚至通过化学‘合并’结合在相邻位置的片段，从而能够构建更紧密的结合分子，作为目标酶的有效抑制剂。这种FBS方法现在越来越多地作为一种补充方法在工业中使用(在药物开发等领域)，作为更典型的涉及巨大化学库的高通量筛选技术的补充。在目前的方案中，我们将利用和发展FBS技术来研究引起结核病的结核分枝杆菌(Mtb)中一种称为细胞色素P450(P450s)的酶。P450是一种酶，它将氧与结合到蛋白质上的血红素基团中的铁原子结合，然后激活氧，使氧原子能够插入到它们的底物中，底物通常是结合在酶的活性部位的脂分子，靠近血红素基团。在结核分枝杆菌中发现了大量的P450，其中许多被认为是细菌生存和感染宿主所必需的酶。然而，关于大多数P450的底物和生理功能的信息很少。该项目将使用FBS方法定义与选定的Mtb P450结合的片段，这些片段参与胆固醇代谢和其他不寻常的生物化学，然后使用结构生物学方法定义它们的结合模式。然后，片段的结合的身份和位置将被用于进一步的工作，以化学方式连接片段并向片段添加官能团，以创建更紧密的结合分子，作为P450催化功能的特定抑制剂，并作为其机制的探针。此外，我们将制作包含脂类、类固醇和其他分子片段的新型文库，这些分子是P450的典型底物，并以一种新颖的方式使用FBS来识别功能未知的“孤儿”Mtb P450的底物分子-再次使用化学合成和结构生物学的组合来“定位”这些P450的真实底物，从而提供关于Mtb复杂的脂质生物化学的新知识。因此，这一应用将提供关于一种重要细菌的生理化学的重要新信息，并通过所用方法的技术发展推动FBS在酶底物鉴定中的新应用。

The development of methods for the determination of entire DNA sequences of humans and other organisms has revealed huge amounts of new information in terms of identification of novel proteins and enzymes and unusual and unexpected chemical processes that take place within cells. From perspectives such as antibiotic therapy and biotechnological applications, there are obviously numerous opportunities once the substrates and reactions catalyzed by these novel enzymes are established. However, considerable efforts are often required to establish the function of a newly identified enzyme if there is no initial knowledge of the physiological role it plays. In addition, identification of specific inhibitors of its function can also be laborious and involve screening of enormous libraries of chemicals. However, recent years have seen the advent of a novel type of approach to defining molecules that bind to purified enzymes. This fragment based screening (FBS) technology involves analysis of binding of much more limited libraries of small molecules than would be used in typical compound screens as used, for example, in the Pharmaceutical sector. Initial 'hits' from the FBS approach may bind the target enzyme only quite weakly, but with the help of protein structural methods their position of binding can be determined accurately. Once a number of such hits are obtained, the combined information on their binding locations and the nature of the chemistry in these sites and the surrounding environment can provide a basis for 'elaborating' the structure of the molecules such that tighter binding is achieved, or even chemically 'merging' fragments bound at adjacent sites to enable construction of much tighter binding molecules as potent inhibitors of the target enzyme. This FBS method is now increasingly used in industry (in areas such as drug development) as a complementary method to the more typical 'high throughput screening' techniques that involved huge chemical libraries. In the current proposal, we will exploit and develop the FBS technology in studies of a class of enzyme called cytochromes P450 (P450s) from the TB-causing bacterium Mycobacterium tuberculosis (Mtb). The P450s are enzymes that bind oxygen to an iron atom in a heme group bound to the protein, and then 'activate' the oxygen to enable the insertion of an oxygen atom into their substrates, which are typically lipid molecules that bind in the active site of the enzyme, close to the heme group. A large number (20) of P450s are found in Mtb, and it is recognized that many of these are essential enzymes for bacterial survival and for their infection of the host. However, there is a paucity of information on the substrates and physiological functions of the majority of the P450s. This project will use FBS methods to define fragments that bind to selected Mtb P450s involved in cholesterol metabolism and in other unusual biochemistry, and then define their binding mode using structural biology methods. The identities and positions of binding of the fragments will then be used in further work to chemically join fragments and to add functionalities to fragments in order to create much tighter binding molecules as specific inhibitors of catalytic functions of the P450s, and as probes of their mechanism. Further, we will make new types of libraries that contain fragments of lipids, steroids and other molecules that are typical substrates for P450s, and use FBS in a novel way to identify substrate molecules for 'orphan' Mtb P450s with unknown function - again using a combination of chemical synthesis and structural biology to 'home in' on true substrates for these P450s, and so provide new knowledge on Mtb's complex lipid biochemistry. This application will thus provide important new information on the physiological chemistry of an important bacterium, and drive new applications of FBS in enzyme substrate identification through technical developments in the methodologies used.