br Materials and methods br Results br

Materials and methods
Results
Discussion Isoniazid (INH) is together with rifampicin the corner stone of the TB treatment. It has been administrated to >40 million people saving millions of lives. Its outstanding cidal effect rapidly blocks bacterial spread and significantly improves patient\'s conditions after a few weeks of treatment. On the basis of INH mode of action, it has been proposed that direct inhibitors of the enoyl-ACP-reductase of Mtb can retain the outstanding antitubercular profile of INH overcoming most of the issues associated with the drug, such us drug resistance and toxicological effects. Additionally, compounds inhibiting InhA without requiring activation by KatG could be active under Anti-diabetic Compound Library conditions (Vilcheze et al., 2011), where catalase-mediated activation is suppressed by the lack of oxygen (Karakousis et al., 2008). Following the above-mentioned rational, a biochemical assay based on NADH consumption in the presence of docecenoyl-CoA by purified InhA enzyme has allowed the identification of different direct inhibitors of Mtb enoyl-ACP reductase. All the compounds showing IC50 values in the micromolar range were clustered and tested for antitubercular activity against wild-type and InhA overexpressor strains to identify five chemical series of bona-fide inhibitors (whole-cell activity clearly linked to enzymatic inhibition). Enzymatic and whole-cell activities will provide the desired efficacy at an acceptable dose, but a drug also needs to have the right physicochemical properties to increase the probability of success (Hann and Keseru, 2012). The drug-like chemical properties of the selected InhA inhibitors were evaluated to select thiadiazole series as the family with the best balance between potency and physicochemical properties. GSK613 and GSK625 are the most attractive representative compounds of the thiadiazole series. Both exhibited a single-digit nanomolar activity in the enzyme assay and micromolar in the whole-cell assay and showed a selective antibacterial activity against M. tuberculosis. The series does not required KatG activation and retain the antitubercular activity against sensitive, MDR, and XDR clinical isolates. The in vitro frequency of spontaneous resistance is three orders of magnitude better than the one obtained for isoniazid. The four different point mutations identified conferring resistance were found in inhA coding region, producing a single amino acid change at either of two positions: Gly96 or Met103. These two amino acids map to the active site of the enzyme and have not been reported to be involved in resistance to InhA neither for isoniazid or ethionamide. Like other InhA direct inhibitors, thiadiazoles bind to the enzyme–NADH complex, but in contrast to previously crystallized InhA inhibitors such as pyrrolidine carboxamides (He et al., 2006), Genzyme10850, and triclosan (Kuo et al., 2003), thiadiazoles do not establish any direct interaction with Tyr158, and unlike the isoniazid-NADH adduct, GSK625 does not cause the flipping of the Phe149 side chain and thus there is no interaction with the isonicotinic acid binding pocket. Recently, it was proposed that high-affinity slow binding inhibition of InhA is related to the ordering of the substrate-binding loop, brought about by the long residence time of inhibitors on the enzyme (Lu et al., 2010). Importantly, in all of the co-crystal structures with thiadiazoles, the substrate-binding loop (residues 197–226) was ordered and well resolved in the crystal structure, as previously seen with high-affinity slow onset FabI inhibitor structures, implying that the compounds may also be slow, tight binding inhibitors, which generally enhances the in vivo activity and increases their potential as antitubercular drugs.
Conclusion Standard antitubercular treatment consists of a combination of four compounds (isoniazid, rifampicin, ethambutol, and pyrazinamide) during 2months that eliminate all the fast growing bacteria and 4 additional months of isoniazid and rifampicin that complete patient cure.