Characterisation of naturally occurring isothiocyanates as glutathione reductase inhibitors

Glutathione reductase (GR), an essential antioxidant enzyme against oxidative stress, has become an attractive drug target for the development of anticancer and antimalarial drugs. In this regard, we evaluated the naturally occurring isothiocyanates as promising GR inhibitors and elucidated the mechanism of action. It was found that benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) inhibited yeast GR (yGR) and human GR (hGR) in a time- and concentration-dependent manner. The K(i) and k(inact) of BITC against yGR were determined to be 259.87 µM and 0.0266 min(−1), respectively. The GR inhibition occurred only in the presence of NADPH and persisted after extensive dialysis. The tandem mass spectrometric analysis revealed that Cys(61) rather than Cys(66) at the active site of yGR was mono-benzyl thiocarbamoylated by BITC. Inhibition of intracellular GR by BITC and PEITC in cultured cancer cells was also observed. BITC and PEITC were evaluated as competitive and irreversible inhibitors of GR.