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

基于片段的筛选方法合理化结核分枝杆菌 P450 分子选择性

• 批准号: BB/I019669/1
• 负责人: Chris Abell
• 金额: $ 44.43万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI019669%2F1
• 关键词: fragment; based; screening; approach; rationalizing

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.

测定人类和其他生物的全DNA序列的方法的发展，在鉴定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方面，揭示了大量的新信息。从抗生素治疗和生物技术应用等角度来看，一旦这些新型酶的底物和催化反应建立起来，显然有许多机会。然而，如果对新发现的酶所起的生理作用没有初步的了解，通常需要相当大的努力来确定它的功能。此外，鉴定其功能的特定抑制剂也可能是费力的，并涉及筛选大量的化学物质库。然而，近年来出现了一种新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）技术涉及对更有限的小分子文库的结合进行分析，而不是在典型的化合物筛选中使用，例如在制药部门。FBS方法的初始“命中”可能只与目标酶结合相当弱，但在蛋白质结构方法的帮助下，它们的结合位置可以准确地确定。一旦获得了许多这样的命中点，它们结合位置的综合信息以及这些位点和周围环境的化学性质可以为“精心设计”分子结构提供基础，从而实现更紧密的结合，甚至可以化学“合并”在相邻位点结合的片段，从而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用于工业（如药物开发领域），作为涉及大量化学文库的更典型的“高通量筛选”技术的补充方法。在目前的提案中，我们将利用和发展FBS技术来研究一类称为细胞色素P450 （P450）的酶，这些酶来自于结核分枝杆菌（Mtb）。p450是一种酶，它将氧与与蛋白质结合的血红素组中的铁原子结合，然后“激活”氧，使氧原子能够插入它们的底物中，底物通常是与酶的活性位点结合的脂质分子，靠近血红素组。在结核分枝杆菌中发现了大量（20个）p450酶，人们认识到其中许多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缺乏。本项目将使用FBS方法确定与选定的参与胆固醇代谢和其他异常生物化学的Mtb p450结合的片段，然后使用结构生物学方法确定其结合模式。结合片段的身份和位置将用于进一步的工作，以化学方式连接片段，并为片段添加功能，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，我们将制作包含脂质、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来鉴定功能未知的“孤儿”Mtb p450的底物分子-再次使用化学合成和结构生物学的结合来“定位”这些p450的真正底物，从而为Mtb的复杂脂质生物化学提供新的知识。因此，该应用将为重要细菌的生理化学提供重要的新信息，并通过所使用方法的技术发展推动FBS在酶底物鉴定中的新应用。

项目成果

Effect of DMSO on Protein Structure and Interactions Assessed by Collision-Induced Dissociation and Unfolding

• DOI: 10.1021/acs.analchem.7b02329
• 发表时间: 2017-09-19
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Chan, Daniel S. -H.;Kavanagh, Madeline E.;Abell, Chris
• 通讯作者: Abell, Chris

Structural Characterization and Ligand/Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1.

结构表征和配体/抑制剂鉴定提供了对结核分枝杆菌细胞色素P450 CYP126A1的功能见解。

• DOI: 10.1074/jbc.m116.748822
• 发表时间: 2017-01-27
• 期刊: The Journal of biological chemistry
• 作者: Chenge JT;Duyet LV;Swami S;McLean KJ;Kavanagh ME;Coyne AG;Rigby SE;Cheesman MR;Girvan HM;Levy CW;Rupp B;von Kries JP;Abell C;Leys D;Munro AW
• 通讯作者: Munro AW

Overcoming the limitations of fragment merging: rescuing a strained merged fragment series targeting Mycobacterium tuberculosis CYP121.

• DOI: 10.1002/cmdc.201300219
• 发表时间: 2013-09
• 期刊: CHEMMEDCHEM
• 影响因子: 3.4
• 作者: Hudson, Sean A.;Surade, Sachin;Coyne, Anthony G.;McLean, Kirsty J.;Leys, David;Munro, Andrew W.;Abell, Chris
• 通讯作者: Abell, Chris

Structural Characterization and Ligand/Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1

结构表征和配体/抑制剂鉴定提供了对结核分枝杆菌细胞色素 P450 CYP126A1 的功能见解

• DOI: 10.17863/cam.8296
• 发表时间: 2017
• 作者: Chenge J

Structural characterization of CYP144A1 - a cytochrome P450 enzyme expressed from alternative transcripts in Mycobacterium tuberculosis.

• DOI: 10.1038/srep26628
• 发表时间: 2016-05-26
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Chenge J;Kavanagh ME;Driscoll MD;McLean KJ;Young DB;Cortes T;Matak-Vinkovic D;Levy CW;Rigby SE;Leys D;Abell C;Munro AW
• 通讯作者: Munro AW