Discovery of New Inhibitors of Mycobacterium tuberculosis InhA Enzyme Using Virtual Screening and a 3D-Pharmacophore-Based Approach Ivani Pauli, ‡,+,§,† Ricardo N. dos Santos, ° Diana C. Rostirolla, + Leonardo K. Martinelli, +,§ Rodrigo G. Ducati, + Luís F. S. M. Timmers, ‡,+,§ Luiz A. Basso, +,§ Dió genes S. Santos, +,§ Rafael V. C. Guido, ° Adriano D. Andricopulo,* ,° and Osmar Norberto de Souza* ,‡,+,§,• ‡ Laborató rio de Bioinforma ́ tica, Modelagem e Simulaç ã o de Biossistemas - LABIO, Faculdade de Informa ́ tica, PUCRS, Brazil + Centro de Pesquisas em Biologia Molecular e Funcional - CPBMF, Instituto Nacional de Ciê ncia e Tecnologia em Tuberculose - INCT-TB, PUCRS, Brazil § Programa de Pó s-Graduaç ã o em Biologia Celular e Molecular - PPGBCM, Brazil • Programa de Pó s-Graduaç ã o em Ciê ncia da Computaç ã o - PPGCC, Brazil ° Laborató rio de Química Medicinal e Computacional - LQMC, Instituto de Física de Sã o Carlos - IFSC-USP, Brazil ABSTRACT: Mycobacterium tuberculosis InhA (MtInhA) is an attractive enzyme to drug discovery efforts due to its validation as an effective biological target for tuberculosis therapy. In this work, two different virtual-ligand-screening approaches were applied in order to identify new InhA inhibitors’ candidates from a library of ligands selected from the ZINC database. First, a 3-D pharmacophore model was built based on 36 available MtInhA crystal structures. By combining structure-based and ligand-based information, four pharmacophoric points were designed to select molecules able to satisfy the binding features of MtInhA substrate-binding cavity. The second approach consisted of using four well established docking programs, with different search algorithms, to compare the binding mode and score of the selected molecules from the aforementioned library. After detailed analyses of the results, six ligands were selected for in vitro analysis. Three of these molecules presented a satisfactory inhibitory activity with IC 50 values ranging from 24 (±2) μM to 83 (±5) μM. The best compound presented an uncompetitive inhibition mode to NADH and 2-trans-dodecenoyl-CoA substrates, with K i values of 24 (±3) μM and 20 (±2) μM, respectively. These molecules were not yet described as antituberculars or as InhA inhibitors, making its novelty interesting to start efforts on ligand optimization in order to identify new effective drugs against tuberculosis having InhA as a target. More studies are underway to dissect the discovered uncompetitive inhibitor interactions with MtInhA. ■ INTRODUCTION Tuberculosis (TB) is more prevalent in the world today than at any other time in human history. 1 The resurgence of TB as a public health threat was mainly due to the arising of multi (MDR- TB), extensively (XDR-TB), 2 and recently, totally (TDR-TB) 3 drug resistant Mycobacterium tuberculosis strains. TB coinfections in HIV/AIDS patients, also contributed significantly to increase this problem. 4,5 Besides, M. tuberculosis, the TB etiological agent, is known to use diverse strategies to survive in a variety of host lesions and to evade immune surveillance. 1 In this context the urgency to pursue novel alternative treatments to TB infected patients is clear. To achieve global control of this epidemic, there is a need for new TB drugs, which can (1) shorten treatment duration; (2) target drug resistant strains; (3) simplify treatment by reducing the daily pill burden; (4) lower dosing frequency; and (5) be coadministered with HIV medications. 1 Among the most attractive molecular targets to the design of novel antibacterial agents are the Fatty Acid Synthase (FAS) pathway enzymes. 6,7 The Mycobacterium tuberculosis InhA (MtInhA) or 2-trans-enoyl-ACP (CoA) reductase (E.C.1.3.1.9), the fourth enzyme of the type II fatty acid synthase system (FAS II), is one of the key enzymes involved in the elongation cycle of fatty acids in M. tuberculosis. Its biological role includes the preferentially reduction of long chain enoyl thioester substrates (e.g., containing 16 or more carbon atoms) yielding the long carbon chain of the meromycolate branch of mycolic acids (C 40-60 ), α-branched fatty acids, the hallmark of mycobacteria. 8 Previously, it has been shown that InhA is essential to the mycolic acid biosynthesis in Mycobacterium. 9 Its inactivation induces the Received: April 4, 2013 Published: July 27, 2013 Article pubs.acs.org/jcim © 2013 American Chemical Society 2390 dx.doi.org/10.1021/ci400202t | J. Chem. Inf. Model. 2013, 53, 2390-2401