Clinically observed InhA mutations rewire cofactor-site dynamics to evade isoniazid–NAD inhibition

Among the main drugs used for tuberculosis treatment, isoniazid (INH) is one of the first-line drugs, and its molecular target is the enzyme InhA, an enoyl-ACP reductase encoded by the inhA gene. Several missense mutations in inhA have been found in clinical isolates, and multiple routes of resistance have been proposed, but how these mutations affect the isoniazid target remains under discussion. Herein, we integrated multiple biophysical and structural techniques to investigate the effects of missense mutations in the inhA coding region of isoniazid-resistant clinical isolates. Calorimetric studies using isothermal titration calorimetry (ITC) were performed to estimate the affinity of NADH against wild-type and mutant enzymes; The thermal stability of the enzymes was also investigated using differential scanning fluorimetry (DSF); Crystal or Cryo-EM structures were obtained for APO, NADH or INH-NAD bound states; and molecular dynamics experiments using the protein structures of the wild type and mutants were performed in APO, NADH, or INH-NAD bound states. Although the decreased affinity for NADH was expected, we show that resistance might arise from closure of the cofactor-binding site driven by changes in protein dynamics, and, alternatively, we provide evidence that the resistance may result from a decrease in affinity for the INH-NAD adduct without altering NADH affinity. The thermodynamic and structural association studies indicate that specific mutants exhibit significant entropic-enthalpic compensation, contributing to resistance. Finally, this study provides a novel classification of missense mutations based on their effects, guiding future treatment recommendations.