48 - Non-Enzymatic Radical Activation of the Potent Anti-Tubercular Isoniazid by Cu(II)

Abstract

Isoniazid (INH) is one of the most potent drugs available for tuberculosis treatment. As a pro-drug it requires activation by catalase/peroxidase to produce the reactive isonicotinic acyl radical intermediate responsible for its anti-tuberculosis activity, However, it is not clear whether INH can be activated non-enzymatically by Cu(II). Here we found that INH and Cu(II) together could induce synergistic DNA damage (strand breakage and 8-oxodG formation), while neither of them alone has any effect. DNA damage by INH/Cu(II) could be inhibited by Cu(I)-specific chelator and catalase, but not by SOD and the typical ●OH radical scavengers. Interestingly, ESR spin-trapping method showed that ●OH, ●H and a C-centered radical were produced during Cu(II)-catalyzed oxidation of INH. We proposed that the synergistic DNA damage induced by INH/Cu(II) might be due to the synergistic and site-specific production of ●OH near the binding site of copper and DNA. The C-centered radical was further unequivocally identified by ESR and HPLC-MS as isonicotinic acyl radical, which can react with nicotinamide coenzyme NADH to form the critical isonicotinic acyl-NAD adduct. The adduct can effectively inhibit in vitro activity of the enoyl-acyl carrier protein reductase InhA, an INH target in the biosynthetic pathway of mycolic acids. This study provided a new insight on non-enzymatic activation of INH by Cu(II) to produce the reactive isonicotinic acyl radical, ●OH and ●H, which may have important biological implications for future research on INH.