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Tuberculosis remains one of the top causes of death worldwide, and combating its spread has been severely complicated by the emergence of drug-resistance mutations, highlighting the need for more effective drugs. Despite the resistance to isoniazid (INH) arising from mutations in the katGgene encoding the catalase-peroxidase KatG, most notably the S315T mutation, this compound is still one of the most powerful first-line antitubercular drugs, suggesting further pursuit of the development of tailored INH derivatives. The N′-acylated INH derivative with a long alkyl chain (INH-C10) has been shown to be more effective than INH against the S315T variant of Mycobacterium tuberculosis, but the molecular details of this activity enhancement are still unknown. In this work, we show that INH N′-acylation significantly reduces the rate of production of both isonicotinoyl radical and isonicotinyl–NAD by wild type KatG, but not by the S315T variant of KatG mirroring the in vivoeffectiveness of the compound. Restrained and unrestrained MD simulations of INH and its derivatives at the water/membrane interface were performed and showed a higher preference of INH-C10for the lipidic phase combined with a significantly higher membrane permeability rate (27.9 cm s–1), compared with INH-C2or INH (3.8 and 1.3 cm s–1, respectively). Thus, we propose that INH-C10is able to exhibit better minimum inhibitory concentration (MIC) values against certain variants because of its better ability to permeate through the lipid membrane, enhancing its availability inside the cell. MIC values of INH and INH-C10against two additional KatG mutations (S315N and D735A) revealed that some KatG variants are able to process INH faster than INH-C10into an effective antitubercular form (wtand S315N), while others show similar reaction rates (S315T and D735A). Altogether, our results highlight the potential of increased INH lipophilicity for treating INH-resistant strains.

Isoniazid (INH) is still one of the two most effective antitubercular drugs and is included in all recommended multitherapeutic regimens. Because of the increasing resistance of Mycobacterium tuberculosisto INH, mainly associated with mutations in the katGgene, new INH-based compounds have been proposed to circumvent this problem. In this work, we present a detailed comparative study of the molecular determinants of the interactions between wtKatG or its S315T mutant form and either INH or INH-C10, a new acylated INH derivative. MD simulations were used to explore the conformational space of both proteins, and results indicate that the S315T mutation did not have a significant impact on the average size of the access tunnel in the vicinity of these residues. Our simulations also indicate that the steric hindrance role assigned to Asp137 is transient and that electrostatic changes can be important in understanding the enzyme activity data of mutations in KatG. Additionally, molecular docking studies were used to determine the preferred modes of binding of the two substrates. Upon mutation, the apparently less favored docking solution for reaction became the most abundant, suggesting that S315T mutation favors less optimal binding modes. Moreover, the aliphatic tail in INH-C10seems to bring the hydrazine group closer to the heme, thus favoring the apparent most reactive binding mode, regardless of the enzyme form. The ITC data is in agreement with our interpretation of the C10alkyl chain role and helped to rationalize the significantly lower experimental MIC value observed for INH-C10. This compound seems to be able to counterbalance most of the conformational restrictions introduced by the mutation, which are thought to be responsible for the decrease in INH activity in the mutated strain. Therefore, INH-C10appears to be a very promising lead compound for drug development.