Bioreductive activation of a series of indolequinones by human DT-diaphorase: Structure-activity relationships

A series of indolequinones including derivatives of EO9 bearing various fun ctional groups and related indole-2-carboxamides have been studied with a v iew to identifying molecular features which confer substrate specificity fo r purified human NAD(P)H:quinone oxidoreductase (DT-diaphorase), bioreducti ve activation to DNA-damaging species, and selectivity for DT-diaphorase-ri ch cells in vitro. A broad spectrum of substrate specificity exists, but mi nor changes to the indolequinone nucleus have a significant effect upon sub strate specificity. Modifications at the 2-position are favorable in terms of substrate specificity as these positions are located at the binding site entrance as determined by molecular modeling studies. In contrast, substit utions at the (indol-3-yl)methyl position with bulky leaving groups or a gr oup containing a chlorine atom result in compounds which are poor substrate s, some of which inactivate DT-diaphorase. Modeling studies demonstrate tha t these groups sit close to the mechanistically important amino acids Tyr 1 56 and His 162 possibly resulting in either alkylation within the active si te or disruption of charge-relay mechanisms. An aziridinyl group at the 5-p osition is essential for potency and selectivity to DT-diaphorase-rich cell s under aerobic conditions. The most efficient substrates induced qualitati vely greater single-strand DNA breaks in cell-free assays via a redox mecha nism involving the production of hydrogen peroxide (catalase inhibitable). This damage is unlikely to form a major part of their mechanism of action i n cells since potency does not correlate with extent of DNA damage. In term s of hypoxia selectivity, modifications at the 3-position generate compound s which are poor substrates for DT-diaphorase but have high hypoxic cytotox icity ratios.