Addressing antimicrobial resistance in Mycobacterium tuberculosis - a fragment screening-led approach

抗生素耐药性对人类健康构成严重威胁，是抗生素过度使用的必然结果。一个主要威胁是结核分枝杆菌（Mtb）菌株对几十年来成功治疗结核病的主要药物（如利福平、链霉素和异烟肼）产生了耐药性。耐主要药物（多药耐药）或这些药物加上其他结核病抗生素（广泛耐药，XDR；甚至完全耐药，TDR菌株）的结核分枝杆菌菌株广泛存在。这促使世界卫生组织宣布进入“全球紧急状态”，以推动研究开发具有新作用方式和攻击不同结核分枝杆菌目标的新型抗生素。在初步工作中，我们的团队和合作者通过研究毒力菌株Mtb H37Rv2的细胞色素P450 （P450）酶来解决这一挑战。Mtb H37Rv基因组编码20种不同的p450酶，初步工作表明，其中几种酶对细菌的生存能力和/或其感染和维持人类宿主感染的能力至关重要3。在与剑桥大学的研究人员合作中，我们利用了相对较新的基于片段筛选（FBS）的方法，以识别与重要的Mtb P450酶结合的小配体（使用量热法和基于核磁共振的方法），并使用x射线晶体学确定它们如何与P450结合。通过利用合成化学对片段进行化学“加工”，并在结构信息的指导下，已经开发出几种与结核分枝杆菌p450紧密结合的新分子，包括遗传必需的CYP121A1 -一种环二肽氧化酶，以及宿主胆固醇氧化CYP125A1和CYP124A1 p450，它们在结核分枝杆菌被人类巨噬细胞吞噬时对维持感染至关重要。我们已经有了fbs衍生的几种关键MtbP450s4,5的抑制剂，现在需要优化它们的结构，以有效地抑制靶标，并获得有利于Mtb细胞良好渗透的化学性质。该学生将通过开发基于HPLC /GC-MS的分析方法来识别关键Mtb P450形成的产品，并通过演示和量化fbs衍生化合物对P450的抑制作用来推进该项目。研究还将包括新型结核分枝杆菌P450的表达/纯化，通过片段筛选鉴定这些P450的新配体/抑制剂，以及在生产新型结核分枝杆菌抗生素的过程中，关键结核分枝杆菌P450与新抑制剂复合物的结构表征。

Resistance to antibiotics poses a grave threat to human health worldwide, but is an inevitable consequence of antibiotic overuse. A major threat is posed by strains of Mycobacterium tuberculosis (Mtb) that have developed resistance to leading drugs (e.g. rifampicin, streptomycin and isoniazid) that have been the backbone of successful TB treatments for decades1. Mtb strains resistant to the leading drugs (multidrug resistant, MDR) or to these plus other TB antibiotics (extensively drug resistant, XDR; and even totally drug resistant, TDR strains) are widespread. This prompted the World Health Organization to declare a "global emergency" in a drive to promote research into development of new types of antibiotics that have new modes of action and that attack different Mtb targets. In preliminary work, our group and collaborators have addressed this challenge through studies of cytochrome P450 (P450) enzymes from the virulent strain Mtb H37Rv2. The Mtb H37Rv genome encodes 20 different P450s, and preliminary work revealed that several of these enzymes are crucial to the viability of the bacterium and/or its ability to infect and to sustain infection in the human host3. In collaboration with researchers at the University of Cambridge, we have exploited the relatively new approach of fragment based screening (FBS) in order to identify small ligands that bind to important Mtb P450 enzymes (using calorimetric and NMR-based methods), and to determine how they bind to their P450s using X-ray crystallography. Through chemical "elaboration" of fragments using synthetic chemistry and guided by structural information, several new molecules have been developed that bind tightly to Mtb P450s, including the genetically essential CYP121A1 - a cyclic dipeptide oxidase, and the host cholesterol oxidizing CYP125A1 and CYP124A1 P450s that are crucial for Mtb to sustain infection when engulfed by the human macrophage3. We have reached a point where we have FBS-derived inhibitors of several key MtbP450s4,5, and now require to optimize their structures for both effective target inhibition and for chemical properties conducive to good Mtb cell penetration. The student will progress this project through development of HPLC-/GC-MS based assays to identify products formed by key Mtb P450s and through demonstrating and quantifying P450 inhibition by FBS-derived compounds. Research will also include expression/purification of novel Mtb P450s, identification of new ligands/inhibitors of these P450 by fragment screening, and structural characterization of key Mtb P450s in complex with new inhibitors en route to production of novel classes of Mtb antibiotics.