Electrochemical oxidation of estrogens as a method for CYP19A1 (aromatase) electrocatalytic activity determination

In this study we present an original approach for determination of electrocatalytic activity of CYP19A1 (aromatase), immobilized on screen-printed graphite electrodes modified by didodecyldimethylammonium bromide (DDAB). Using square wave voltammetry we have shown that estrone and beta-estradiol produced during CYP19A1-dependent electrocatalytic reactions towards androstenedione and testosterone, respectively, can be determined by their direct electrochemical oxidation on the surface of graphite electrode at E-ox = 497 +/- 14 mV (vs. Ag/AgCl) for estrone and E-ox = 483 +/- 17 mV (vs. Ag/AgCl) for b-estradiol, respectively. Sensitivity values for estrone and beta-estradiol were determined as 0.1 A/M and 0.12 A/M, respectively. Limits of detection (S/N = 3) were calculated as 11 nM and 3.4 nM for estrone and beta-estradiol, respectively. We used estrogen electrochemical oxidation for determination of immobilized on screen-printed graphite electrodes CYP19A1 steady-state kinetic parameters (maximal rate of the reaction - V-max and Michaelis constant - K-M) towards androstenedione and testosterone. Furthermore, we evaluated the applicability of the approach for studying of CYP19A1 inhibitors, using exemestane as an example, which is a known mechanism-based aromatase inhibitor and a drug for breast cancer treatment. The maximal inactivation rate constant (k(inact)) and half-life of inactivation (t(1/2)) values for exemestane mechanism-based inhibition towards CYP19A1 in the electrochemical system were estimated as 0.038 +/- 0.003 min(-1) and 18.2 +/- 1.4 min, respectively, while the concentration of inhibitor required for half-maximal inactivation (K-I) was calculated to be in a range of 0.3-5.7 nM. The parameters correlate to those obtained previously in CYP19A1-dependent reconstituted systems. The approach is promising for CYP19A1 inhibitor screening during drug development for breast cancer treatment. (C) 2019 Elsevier Ltd. All rights reserved.