Structure-Guided Discovery of Antitubercular Agents That Target the Gyrase ATPase Domain Variam U. Jeankumar, [a] Shalini Saxena, [a] Rahul Vats, [a] Rudraraju Srilakshmi Reshma, [a] Renuka Janupally, [a] Pushkar Kulkarni, [b, c] Perumal Yogeeswari, [a] and Dharmarajan Sriram* [a] Introduction The currently available arsenal of anti-tuberculosis (TB) drugs remains insufficient to combat the emergence of drug resist- ance, necessitating research into the development of novel agents that lack cross-resistance mediated by mutations in the bacterial targets. [1, 2] With drug discovery programs becoming more focused and rational, extending to the genomic level, the identification and selection of the appropriate target has become a fundamental prerequisite. Moxifloxacin, the most advanced drug for the treatment of TB, targets the GyrA segment of DNA gyrase—the sole type II topoisomerase present in Mycobacterium tuberculosis—which introduces negative supercoils in an ATP-dependent manner. Moxifloxacin has demonstrated excellent potency against the drug-sensitive and drug-resistant strains of M. tuberculosis, vali- dating the druggability of DNA gyrase as a potential antituber- cular drug target; however, the emergence of fluoroquinolone- resistant strains underscores the genuine urgency in the search for new therapeutic agents to combat TB. The ATPase domain (GyrB) of bacterial DNA gyrase remains pharmaceutically un- derexploited, despite being genetically demonstrated to be a bactericidal target in M. tuberculosis. [3] Inhibitors of the GyrB subunit competitively inhibit its ATPase activity, thereby de- priving bacteria of the energy source required for maintaining the topological state of DNA in the cell and affecting processes such as DNA replication. The development of new inhibitors that target the GyrB subunit offers an excellent opportunity to create an effective treatment for TB that is not affected by target-mediated cross-resistance associated with previously re- ported drugs. [3–10] Furthermore, the increasing amount of struc- tural information available for bacterial DNA gyrases has in- creased their potential as drug targets and has opened the possibility of using structure-based drug design (SBDD) in this context. [5, 6, 8] This study involved an SBDD approach to identify potential inhibitors of the M. tuberculosis gyrase ATPase domain. The ini- tial hit obtained by pharmacophore screening of a commercial chemical database was subsequently remodeled by using a me- dicinal chemistry approach to generate a lead molecule that shows a 10-fold enhancement in potency over the starting hit compound. The binding affinity at the enzyme level was fur- ther corroborated by biophysical characterization techniques. Early pharmacokinetic (PK) evaluations of the optimized ana- logue were encouraging, demonstrating the suitability of this compound class for development as leads in the fight against TB. Results and Discussion We took an e-pharmacophore approach, which uses energy- and ligand-based techniques to identify putative ligands that [a] Dr. V. U. Jeankumar, Dr. S. Saxena, Dr. R. Vats, R. S. Reshma, R. Janupally, Prof. P. Yogeeswari, Prof. D. Sriram Department of Pharmacy, Birla Institute of Technology & Science – Pilani Hyderabad Campus, Shameerpet, R.R. District Hyderabad 500078, Andhra Pradesh (India) E-mail : dsriram@hyderabad.bits-pilani.ac.in [b] Dr. P. Kulkarni Dr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus Gachibowli, Hyderabad 500046 (India) [c] Dr. P. Kulkarni Zephase Therapeutics (an incubated company at Dr. Reddy’s Institute of Life Sciences), University of Hyderabad Campus Gachibowli, Hyderabad 500046 (India) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201500556. In this study we explored the pharmaceutically underexploited ATPase domain of DNA gyrase (GyrB) as a potential platform for developing novel agents that target Mycobacterium tuber- culosis. In this effort a combination of ligand- and structure- based pharmacophore modeling was used to identify structur- ally diverse small-molecule inhibitors of the mycobacterial GyrB domain based on the crystal structure of the enzyme with a pyrrolamide inhibitor (PDB ID: 4BAE). Pharmacophore modeling and subsequent in vitro screening resulted in an ini- tial hit compound 5 [(E)-5-(5-(2-(1H-benzo[d]imidazol-2-yl)-2-cy- anovinyl)furan-2-yl)isophthalic acid; IC 50 = 4.6  0.1 mm], which was subsequently tailored through a combination of molecular modeling and synthetic chemistry to yield the optimized lead compound 24 [(E)-3-(5-(2-cyano-2-(5-methyl-1H-benzo[d]imida- zol-2-yl)vinyl)thiophen-2-yl)benzoic acid; IC 50 = 0.3  0.2 mm], which was found to display considerable in vitro efficacy against the purified GyrB enzyme and potency against the H 37 Rv strain of M. tuberculosis. Structural handles were also identified that will provide a suitable foundation for further op- timization of these potent analogues. ChemMedChem 2016, 11, 539 – 548 # 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 539 Full Papers DOI: 10.1002/cmdc.201500556