Ligand-Based Virtual Screening for Discovery of Indole Derivatives as Potent DNA Gyrase ATPase Inhibitors Active against Mycobacterium tuberculosis and Hit Validation by Biological Assays.

Mycobacterium tuberculosis is the single most important global infectious disease killer and a World Health Organization critical priority pathogen for development of new antimicrobials. M. tuberculosis DNA gyrase is a validated target for anti-TB agents, but those in current use target DNA breakage-reunion, rather than the ATPase activity of the GyrB subunit. Here, virtual screening, subsequently validated by whole-cell and enzyme inhibition assays, was applied to identify candidate compounds that inhibit M. tuberculosis GyrB ATPase activity from the Specs compound library. This approach yielded six compounds: four carbazole derivatives (1, 2, 3, and 8), the benzoindole derivative 11, and the indole derivative 14. Carbazole derivatives can be considered a new scaffold for M. tuberculosis DNA gyrase ATPase inhibitors. IC50 values of compounds 8, 11, and 14 (0.26, 0.56, and 0.08 µM, respectively) for inhibition of M. tuberculosis DNA gyrase ATPase activity are 5-fold, 2-fold, and 16-fold better than the known DNA gyrase ATPase inhibitor novobiocin. MIC values of these compounds against growth of M. tuberculosis H37Ra are 25.0, 3.1, and 6.2 µg/mL, respectively, superior to novobiocin (MIC > 100.0 µg/mL). Molecular dynamics simulations of models of docked GyrB:inhibitor complexes suggest that hydrogen bond interactions with GyrB Asp79 are crucial for high-affinity binding of compounds 8, 11, and 14 to M. tuberculosis GyrB for inhibition of ATPase activity. These data demonstrate that virtual screening can identify known and new scaffolds that inhibit both M. tuberculosis DNA gyrase ATPase activity in vitro and growth of M. tuberculosis bacteria.

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.