Bioisosteric Design Identifies Inhibitors of Mycobacterium tuberculosis DNA Gyrase ATPase Activity

Mutations in DNA gyrase confer resistance to fluoroquinolones, second-line antibiotics for Mycobacterium tuberculosis infections. Identification of new agents that inhibit M. tuberculosis DNA gyrase ATPase activity is one strategy to overcome this. Here, bioisosteric designs using known inhibitors as templates were employed to define novel inhibitors of M. tuberculosis DNA gyrase ATPase activity. This yielded the modified compound R3-13 with improved drug-likeness compared to the template inhibitor that acted as a promising ATPase inhibitor against M. tuberculosis DNA gyrase. Utilization of compound R3-13 as a virtual screening template, supported by subsequent biological assays, identified seven further M. tuberculosis DNA gyrase ATPase inhibitors with IC50 values in the range of 0.42–3.59 μM. The most active compound 1 showed an IC50 value of 0.42 μM, 3-fold better than the comparator ATPase inhibitor novobiocin (1.27 μM). Compound 1 showed noncytotoxicity to Caco-2 cells at concentrations up to 76-fold higher than its IC50 value. Molecular dynamics simulations followed by decomposition energy calculations identified that compound 1 occupies the binding pocket utilized by the adenosine group of the ATP analogue AMPPNP in the M. tuberculosis DNA gyrase GyrB subunit. The most prominent contribution to the binding of compound 1 to M. tuberculosis GyrB subunit is made by residue Asp79, which forms two hydrogen bonds with the OH group of this compound and also participates in the binding of AMPPNP. Compound 1 represents a potential new scaffold for further exploration and optimization as a M. tuberculosis DNA gyrase ATPase inhibitor and candidate anti-tuberculosis agent.

DNA旋转酶（DNA gyrase）的突变可导致对氟喹诺酮类（fluoroquinolones）的耐药性，而氟喹诺酮类是治疗结核分枝杆菌（Mycobacterium tuberculosis）感染的二线抗生素。筛选可抑制结核分枝杆菌DNA旋转酶ATP酶活性的新型化合物，是克服该耐药性的策略之一。本研究以已知抑制剂为模板，通过生物电子等排体设计，获得了靶向结核分枝杆菌DNA旋转酶ATP酶活性的新型抑制剂。该策略得到了修饰后的化合物R3-13，与模板抑制剂相比，其药物相似性得到优化，且作为靶向结核分枝杆菌DNA旋转酶的ATP酶抑制剂表现出良好潜力。以化合物R3-13作为虚拟筛选模板，结合后续生物学实验验证，共筛选得到7种新型结核分枝杆菌DNA旋转酶ATP酶抑制剂，其半数抑制浓度（IC50）范围为0.42~3.59 μM。活性最强的化合物1的IC50值为0.42 μM，其活性比对照ATP酶抑制剂新生霉素（novobiocin，1.27 μM）高出3倍。化合物1在浓度高达其IC50值76倍的条件下，对Caco-2细胞未表现出细胞毒性。通过分子动力学模拟结合能量分解计算，发现化合物1可结合于结核分枝杆菌DNA旋转酶GyrB亚基中ATP类似物AMPPNP的腺苷基团结合口袋。化合物1与结核分枝杆菌GyrB亚基的结合主要由残基Asp79（天冬氨酸残基79）介导：该残基可与化合物1的羟基形成两个氢键，同时也参与AMPPNP的结合过程。化合物1可作为新型母核结构，为靶向结核分枝杆菌DNA旋转酶ATP酶抑制剂的进一步开发与优化，以及抗结核候选药物的研发提供潜在方向。