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

Alexey V. Kuzikov, Rami A. Masamrekh, Tatiana A. Filippova, Yaraslau I. Haurychenka, Andrei A. Gilep, Tatsiana V. Shkel, Natallia V. Strushkevich, Sergey A. Usanov, Victoria V. Shumyantseva

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7 Citations (Scopus)

Abstract

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 β-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 Eox = 497 ± 14 mV (vs. Ag/AgCl) for estrone and Eox = 483 ± 17 mV (vs. Ag/AgCl) for β-estradiol, respectively. Sensitivity values for estrone and β-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 β-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 – Vmax and Michaelis constant – KM) 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 (kinact) and half-life of inactivation (t1/2) 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 (KI) 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.

Original languageEnglish
Article number135539
JournalElectrochimica Acta
Volume333
DOIs
Publication statusPublished - 10 Feb 2020
Externally publishedYes

Keywords

  • CYP19A1
  • Enzyme electrodes
  • Estrogens
  • Exemestane
  • Mechanism-based inhibition

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