Rational, structure-based inhibitor design, synthesis and evaluation as a first step towards the discovery of new anti-tuberculosis drugs

背景：结核分枝杆菌和M.牛结核病是人类和动物结核病的主要病因。世界卫生组织（WHO）报告称，2018年有150万人（包括25.1万艾滋病毒感染者）死于结核病，1000万人患病，是所有传染病中最高的。已经观察到对用于治疗结核病的药物的耐药性的发生率增加，包括显示极端耐药性（XDR-TB）的菌株。DEFRA报告说，在截至2018年9月的12个月内，英国有2，805群牛的牛结核病检测呈阳性，在过去十年中，有30.5万头牛不得不在英国屠宰，成本为5亿英镑。因此，治疗或根除结核病的方法对人类和动物的健康至关重要。分枝杆菌具有比其他革兰氏阳性菌更不寻常和更复杂的细胞壁结构。细菌的外表面由分枝菌酸（~C60长链脂肪酸）的疏水蜡状包衣形成（Alderwick等人，Cold Spring Harb Perspect Med.2015，5，a021113），其充当假外膜。它位于多糖结构上，阿拉伯半乳聚糖含有几种罕见的糖（半乳呋喃糖/阿拉伯呋喃糖/鼠李糖），然后附着在肽聚糖层上。这种“隐形”的外衣掩盖了细菌对其宿主的防御，使它们能够在哺乳动物组织中定居并形成生物膜，而不会引发宿主的反应。我们可以用抑制剂/药物选择性地靶向分枝杆菌特有的几种酶，降低脱靶毒性的机会。研究项目：这将涉及新化合物的设计、合成和评价，作为选定靶酶的推定抑制剂：InhA（一种烯酰还原酶）参与分枝菌酸生物合成; UGM和GlfT 2参与半乳聚糖生物合成。有高分辨率的X射线结构可用于这些酶，允许新的抑制剂设计在合成前使用蛋白质-配体对接程序（包括GOLD或OE对接）进行计算机测试。对于InhA，已知引起耐药性的临床重要突变，可以针对这些突变体对接新化合物，以测试它们的结合是否会受到影响。大多数结核病病例发生在第三世界，因此新药需要易于生产，并具有口服活性，以尽量降低成本。研究将集中在上述一种/两种酶上。学生将从小组中可用的载体中表达酶，并根据需要合成其测定底物。初始测试化合物将通过对接现有的小分子/片段文库（大小

Background: Mycobacterium tuberculosis and M. bovis are the main causes of tuberculosis (TB) in humans and animals respectively. The World Health Organisation (WHO) reported that 1.5M people (including 251K with HIV) died from TB and 10M fell ill in 2018, the highest of any infectious disease. Increasing occurrences of drug resistance to the drugs used to treat TB include strains showing extreme drug resistance (XDR-TB) have been observed. DEFRA reported that 2,805 herds of cattle in Great Britain tested positive for bovine TB in the 12 months to Sept 2018, with 305K cattle having to be slaughtered in GB over the last decade at the cost of £500M. Methods of treating or eradicating TB are therefore crucial to both human and animal health. Mycobacteria have an unusual and more complex cell wall structure than other Gram-positive bacteria. The exterior surface of the bacterium is formed from a hydrophobic, waxy coat of mycolic acids (~C60 long chain fatty acids) (Alderwick et al. Cold Spring Harb Perspect Med. 2015, 5, a021113) that acts as a pseudo-outer membrane. This sits on a polysaccharide structure, arabinogalactan that contains several rare sugars (galactofuranose/arabinofuranose/rhamnose) which is then attached to the peptidoglycan layer. This 'stealth' coat masks the bacteria from its host's defences and allows them to colonize mammalian tissues and form biofilms without triggering a host response. We can selectively target several enzymes unique to Mycobacteria with inhibitors/drugs with a reduced chance of off-target toxicity. Research Project: This will involve the design, synthesis and evaluation of new compounds as putative inhibitors of selected target enzymes: InhA (an enoyl reductase) involved in mycolic acidbiosynthesis; UGM and GlfT2 involved in galactan biosynthesis. There are high resolution X-ray structures available for these enzymes allowing new inhibitor designs to be tested in silico using protein-ligand docking programs including GOLD or OEDocking prior to their synthesis. For InhA, the clinically important mutations that cause resistance are known and new compounds can be docked against these mutants to test if their binding will be impacted. The majority of TB cases are in the third world, therefore new drugs need to be easy to produce and be orally active to minimise their cost.The research will focus on one/two of the enzymes mentioned above. The student will express the enzyme(s) from vectors available in the group and synthesize their assay substrates as necessary. Initial test compounds will be designed by docking existing small molecule/fragment libraries (size