Characterizing Metabolic Inhibition Using Electrochemical Enzyme/DNA Biosensors

Abstract

Studies of metabolic enzyme inhibition are necessary in drug development and toxicity investigations as potential tools to limit or prevent appearance of deleterious metabolites formed, for example by cytochrome (cyt) P450 enzymes. In this paper, we evaluate the use of enzyme/DNA toxicity biosensors as tools to investigate enzyme inhibition. We have examined DNA damage due to cyt P450cam metabolism of styrene using DNA/enzyme films on pyrolytic graphite (PG) electro*des monitored via Ru(bpy)32+–mediated DNA oxidation. Styrene metabolism initiated by hydrogen peroxide was evaluated with and without the inhibitors, imidazole, imidazole-4-acetic acid and sulconazole (in micromolar range) to monitor DNA damage inhibition. The initial rates of DNA damage decreased with increased inhibitor concentrations. Linear and nonlinear fits of Michaelis-Menten inhibition models were used to determine apparent inhibition constants (KI*) for the inhibitors. Elucidation of the best fitting inhibition model was achieved by comparing correlation coefficients and the sum of the square of the errors (SSE) from each inhibition model. Results confirmed the utility of the enzyme/DNA biosensor for metabolic inhibition studies. A simple competitive inhibition model best approximated the data for imidazole, imidazole-4-acetic acid and sulconazole with KI* of 268.2, 142.3 and 204.2 µM, respectively.

Electrochemical Methods for the In Vitro Assessment of Drug Metabolism

“…Styrene metabolism initiated by hydrogen peroxide was evaluated with and without the following inhibitors: imidazole, imidazole-4-acetic acid and sulconazole. The initial rates of DNA damage decreased with increasing inhibitor concentrations, and the calculated inhibition constants were directly related to the changes in styrene metabolism and DNA adduct formation in the presence of the inhibitor, which confirmed the usefulness of this enzyme/DNA biosensor as a rapid screening tool for metabolic inhibition studies of pharmaceutical candidates (Hull et al, 2009). …”

The molecular action of the novel insecticide, Pyridalyl

“…The inhibition of essentially all P450s prevents Pyridalyl metabolism and the formation of the active metabolite, resulting in cell survival. The more specific P450 inhibitor, imidazole (Hull et al, 2009) proved ineffective at preventing the metabolism of Pyridalyl (Fig. 4).…”

“…In view of inhibition of the action of Pyridalyl by the cytochrome P450 inhibitor, 1-ABT, the effect (at 10 mM) of this inhibitor, as well as of PBO and the p450 inhibitor, imidazole (Hull et al, 2009) on [ 3 H]Pyridalyl metabolism in BM36 cells was evaluated by HPLC analysis of metabolic products.…”

Experimental and theoretical investigation effect of flavonols antioxidants on DNA damage

“…Fe(CN) 6 3−/4-, as a redox probe, was used to evaluate the screen printed carbon electrode (SPCE) and SPGE (Fig. 1b).…”