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Mycobacterium tuberculosis DNA gyrase manipulates the DNA topology using controlled breakage and religation of DNA driven by ATP hydrolysis. DNA gyrase has been validated as the enzyme target of fluoroquinolones (FQs), second-line antibiotics used for the treatment of multidrug-resistant tuberculosis. Mutations around the DNA gyrase DNA-binding site result in the emergence of FQ resistance in M. tuberculosis; inhibition of DNA gyrase ATPase activity is one strategy to overcome this. Here, virtual screening, subsequently validated by biological assays, was applied to select candidate inhibitors of the M. tuberculosis DNA gyrase ATPase activity from the Specs compound library (www.specs.net). Thirty compounds were identified and selected as hits for in vitro biological assays, of which two compounds, G24 and G26, inhibited the growth of M. tuberculosis H37Rv with a minimal inhibitory concentration of 12.5 μg/mL. The two compounds inhibited DNA gyrase ATPase activity with IC50 values of 2.69 and 2.46 μM, respectively, suggesting this to be the likely basis of their antitubercular activity. Models of complexes of compounds G24 and G26 bound to the M. tuberculosis DNA gyrase ATP-binding site, generated by molecular dynamics simulations followed by pharmacophore mapping analysis, showed hydrophobic interactions of inhibitor hydrophobic headgroups and electrostatic and hydrogen bond interactions of the polar tails, which are likely to be important for their inhibition. Decreasing compound lipophilicity by increasing the polarity of these tails then presents a likely route to improving the solubility and activity. Thus, compounds G24 and G26 provide attractive starting templates for the optimization of antitubercular agents that act by targeting DNA gyrase.

2-trans enoyl-acyl carrier protein reductase (InhA) is a promising target for developing novel chemotherapy agents for tuberculosis, and their inhibitory effects on InhA activity were widely investigated by the physicochemical experiments. However, the reason for the wide range of their inhibitory effects induced by similar agents was not explained by only the difference in their chemical structures. In our previous molecular simulations, a series of heteroaryl benzamide derivatives were selected as candidate inhibitors against InhA, and their binding properties with InhA were investigated to propose novel derivatives with higher binding affinity to InhA. In the present study, we extended the simulations for a series of 4-hydroxy-2-pyridone derivatives to search widely for more potent inhibitors against InhA. Using ab initio fragment molecular orbital (FMO) calculations, we elucidated the specific interactions between InhA residues and the derivatives at an electronic level and highlighted key interactions between InhA and the derivatives. The FMO results clearly indicated that the most potent inhibitor has strong hydrogen bonds with the backbones of Tyr158, Thr196, and NADH of InhA. This finding may provide informative structural concepts for designing novel 4-hydroxy-2-pyridone derivatives with higher binding affinity to InhA. Our previous and present molecular simulations could provide important guidelines for the rational design of more potent InhA inhibitors.

The enoyl-acyl carrier protein reductase InhA of Mycobacterium tuberculosis is an attractive, validated target for antituberculosis drug development. Moreover, direct inhibitors of InhA remain effective against InhA variants with mutations associated with isoniazid resistance, offering the potential for activity against MDR isolates. Here, structure-based virtual screening supported by biological assays was applied to identify novel InhA inhibitors as potential antituberculosis agents. High-speed Glide SP docking was initially performed against two conformations of InhA differing in the orientation of the active site Tyr158. The resulting hits were filtered for drug-likeness based on Lipinski’s rule and avoidance of PAINS-like properties and finally subjected to Glide XP docking to improve accuracy. Sixteen compounds were identified and selected for in vitro biological assays, of which two (compounds 1 and 7) showed MIC of 12.5 and 25 μg/mL against M. tuberculosis H37Rv, respectively. Inhibition assays against purified recombinant InhA determined IC50 values for these compounds of 0.38 and 0.22 μM, respectively. A crystal structure of the most potent compound, compound 7, bound to InhA revealed the inhibitor to occupy a hydrophobic pocket implicated in binding the aliphatic portions of InhA substrates but distant from the NADH cofactor, i.e., in a site distinct from those occupied by the great majority of known InhA inhibitors. This compound provides an attractive starting template for ligand optimization aimed at discovery of new and effective compounds against M. tuberculosis that act by targeting InhA.

2-trans enoyl-acyl carrier protein reductase (InhA) has been identified as a promising target for the development of novel chemotherapy for tuberculosis. In the present study, a series of heteroaryl benzamide derivatives were selected as potent inhibitors against InhA, and their binding properties with InhA were investigated at atomic and electronic levels by ab initio molecular simulations based on protein-ligand docking, classical molecular mechanics optimizations and ab initio fragment molecular orbital (FMO) calculations. The results evaluated by FMO highlight some key interactions between InhA and the derivatives, indicating that the most potent derivative has strong hydrogen bonds with the Met98 side chain of InhA and strong electrostatic interactions with the nicotinamide adenine dinucleotide cofactor. These findings provide informative structural concepts for designing novel heteroaryl benzamide derivatives with higher binding affinity to InhA.