Probing a novel allosteric binding site in Mycobacterium DNA gyrase to tackle TB and antimicrobial resistance

背景抗菌素耐药性（antimicrobial resistance，AMR）破坏了抗生素的有效性。氟喹诺酮类药物是通过抑制DNA促旋酶来治疗细菌感染的一线抗生素。一个最近表征的变构位点上的金黄色葡萄球菌DNA旋转酶提供了潜在的设计变构抑制剂的旋转酶。由于变构位点与氟喹诺酮结合位点不同，所以变构抑制剂可以克服临床上观察到的氟喹诺酮耐药性（促旋酶中的点突变）。该项目旨在研究结核分枝杆菌（Mtb）中的这种变构位点，该位点迄今尚未探索，但与S.目的1.使用计算技术和同源建模来建立结核分枝杆菌变构位点的结构模型.使用计算药物化学来设计Mtb.3的有效抑制剂。确定变构抑制剂的作用模式：它们是与有切口的还是切割的DNA复合物结合？4. 2019年有140万人死于结核病，牛结核病是牛的主要传染病。多药耐药性（XDR）-结核病是对几乎所有一线抗生素具有耐药性的菌株。因此，需要对可行的治疗靶点进行新的研究，例如这种变构位点。及时性这项工作利用了我们最近发现的具有抗Mtb细胞活性的大肠杆菌促旋酶变构抑制剂（MIC 90 8 μ M）。监督团队在研究细菌DNA促旋酶的小分子抑制方面有着既定的记录。实验方法学生将接受计算药物化学（了解蛋白质结构，配体-蛋白质相互作用，计算机辅助药物设计）和合成化学的培训。将使用生物化学和晶体学的组合来表征合成的分子，以确定其Mtb促旋酶抑制模式。

BackgroundAntimicrobial resistance (AMR) undermines the effectiveness of antibiotics. Fluoroquinolones are frontline antibiotics that treat bacterial infections via the inhibition of DNA gyrase. A recently characterised allosteric site on Staphylococcus aureus DNA gyrase offers the potential for the design of allosteric inhibitors of gyrase. Since the allosteric site is distinct from the fluoroquinolone-binding site, allosteric inhibitors can overcome fluoroquinolone resistance observed in the clinic (point mutations in gyrase). The project aims to investigate this allosteric site in Mycobacterium tuberculosis (Mtb) which has been unexplored to date, but has high amino acid conservation compared to the sequence of S. aureus gyrase.Objectives1. Use computational techniques and homology modelling to build a structural model of the Mtb allosteric site.2. Use computational medicinal chemistry to design potent inhibitors of Mtb.3. Ascertain the mode of action of the allosteric inhibitors: do they bind to the nicked or cleaved DNA complex?4. Establish synergy of allosteric inhibitors with fluoroquinolones.Novelty1.4 million people died of TB in 2019 and bovine TB is a major infectious disease in cattle. Multi-drug resistance (XDR)-TB are strains resistance to nearly all frontline antibiotics. Therefore, new investigations into viable therapeutic targets is required such as this allosteric site.TimelinessThis work capitalises on our recent discovery of Escherichia coli gyrase allosteric inhibitors with cellular activity against Mtb (MIC90 8 uM). The supervisory team have an established track record in investigating small molecule inhibition of bacterial DNA gyrase. All the methodologies for these studies have been established.Experimental ApproachThe student will be trained in computational medicinal chemistry (to understand protein structure, ligand-protein interactions, computer-aided drug design) and synthetic chemistry. Synthesised molecules will be characterized using a combination of biochemistry and crystallography to ascertain their mode of Mtb gyrase inhibition.