Structure-Based Design of a Novel Class of Potent Inhibitors of InhA, the Enoyl Acyl Carrier Protein Reductase from Mycobacterium Tuberculosis: A Computer Modelling Approach Gita Subba Rao*, Rajakrishnan Vijayakrishnan and Manoj Kumar Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India *Corresponding author: G. Subba Rao, e-mail: gitasubba@gmail.com The NADH-dependent Enoyl-ACP reductase (InhA) of Mycobacterium tuberculosis has been shown to be the primary target of the frontline drug isonia- zid (INH). However, INH must be first activated by katG gene, mutations in which have mediated resistance to INH. Recently, direct inhibitors of InhA have been reported. Using a structure-based approach, we have identified a tripeptide inhibitor with the sequence WYW, which is 100 times more potent than the existing inhibitors. It is therefore, a potential lead compound for the development of new anti-TB drugs. Key words: docking, InhA, molecular dynamics, Mycobacterium tuberculosis, structure-based drug design Received 29 May 2008, revised 21 August 2008 and accepted for publication 15 September 2008 The alarming increase in tuberculosis (TB) and the emergence of multi-drug resistant tuberculosis (MDR-TB) strains, and the fact that no novel TB drugs have been discovered during the past four dec- ades, has led to a high priority worldwide initiative for new anti-TB drugs and TB drug targets. Mycobacterium tuberculosis (M. tuberculosis) contains mycolic acids which are the main constituents of the mycobacterial cell wall (1). The NADH-dependent enoyl-ACP reductase, InhA, which is encoded by the mycobacterium gene InhA, is a key enzyme in the biosynthe- sis of mycolic acids. It is now well established that InhA is the pri- mary molecular target of isoniazid (INH) (2), the drug that has been the frontline agent for the treatment of TB. INH is a prodrug, which must first be activated by KatG, a catalase-peroxidase, to an acyl radical which covalently binds to the co-substrate for InhA, NADH (3). The covalent complex acts as a potent inhibitor of InhA (K i  1nM) (4). Resistance to INH is mediated mainly through muta- tions in the katG gene (5). Therefore, direct inhibitors of InhA, which do not require activation, could be suitable candidates for the development of new drugs against TB. Several direct inhibitors of InhA have been reported to have both in vitro and in vivo activity (6–8). These include pyrazole derivatives and indole-5-amides (7) with IC 50 values in sub-micromolar range. A novel series of pyrrolidine carboxamide compounds has been recently identified as potent, direct inhibitors of InhA, with IC 50 val- ues in sub-nanomolar range (8). With a view to identify potential lead compounds with higher potencies, we have used a structure- based approach for designing small peptides as direct inhibitors of InhA using the available crystal structure of the complexes of InhA with the series of pyrrolidine carboxamide compounds with known activity (8). Although peptides are generally known to have undesir- able pharmacokinetic properties, yet they have provided novel lead compounds and, in several cases, modified peptide analogues have been developed as drugs. Also, with recent advances in the drug delivery techniques, the opportunities for peptide drug development have been significantly enhanced. Based on the available X-ray structures of the complexes of InhA with a series of pyrrolidine carboxamide compounds with known activity (8) we have used a structure-based approach for designing potent inhibitors of InhA. Materials and Methods The starting point for the computational studies was the X-ray crys- tal structure of InhA complex with the cofactor NAD + and the com- pound S1 (PDB ID: 2H7I) (8,9). All computations were performed using Discovery Studio 1.7 (Accelrys Software Inc., San Diego, CA, USA) (10). Receptor setup The target protein [PDB ID 2H7I] was taken, the ligand S1 was extracted, hydrogens were added and their positions were optimized using the all atom CHARMm (version-c32b1) forcefield (11,12) and the Adopted Basis set Newton Raphson (ABNR) method available in D.S 1.7 protocol until the root mean deviation (RMS) gradient was < 0.05 kcal ⁄ mol ⁄  2 . The minimized protein was defined as the receptor using the binding site module of DS 1.7. The binding site was defined from the cavity finding method which was modified to 444 Chem Biol Drug Des 2008; 72: 444–449 Research Letter ª 2008 The Authors Journal compilation ª 2008 Blackwell Munksgaard doi: 10.1111/j.1747-0285.2008.00722.x