Assessment of electrocatalytic hydroxylase activity of cytochrome P450 3A4 (CYP3A4) by means of derivatization of 6β-hydroxycortisol by sulfuric acid for fluorimetric assay

Publication date: 1 May 2019

Source: Talanta, Volume 196

Author(s): Alexey Kuzikov, Rami Masamrekh, Tatsiana Shkel, Natallia Strushkevich, Andrei Gilep, Sergey Usanov, Alexander Archakov, Victoria Shumyantseva

Abstract

We used rapid one-step derivatization of 6β-hydroxylated hydrocortisone by sulfuric acid for fluorimetric determination of CYP3A4-dependent hydroxylase reaction in the electrochemical system. We have shown that CYP3A4 substrate – hydrocortisone – and its 6β-hydroxylated product have different emission wavelengths at an excitation λ_ex = 365 nm after treatment with sulfuric acid:ethanol (3:1) mixture (λ_em = 525 ± 2 nm and λ_em = 427 ± 2 nm, respectively). The detection limit for 6β-hydroxycortisol was estimated to be 0.32 μM (corresponding to 0.095 nmol in 300 μL sample) (S/N = 3). Using the fluorimetric method of 6β-hydroxycortisol detection following the electrolysis of hydrocortisone with CYP3A4 immobilized on a screen-printed graphite electrode modified by didodecyldimethylammonium bromide we have calculated the steady-state kinetic parameters of CYP3A4 for hydrocortisone: the maximal rate of the reaction (V_max) as 89 ± 5 pmol of product per min per pmol of electroactive enzyme and the Michaelis constant (K_M) as 10 ± 2 μM. In our system, ketoconazole inhibited hydroxylase activity of CYP3A4 towards hydrocortisone with the IC₅₀ value of 70 ± 5 nM. The approach proposed for determination of the CYP3A4 electrocatalytic activity can be used for throughput screening of different modulators of this cytochrome P450 isozyme during drug development.

Graphical abstract

CYP3A4-dependent electrocatalytic 6β-hydroxylation of hydrocortisone was studied using difference in fluorescence at the excitation wavelength of 365 nm of the substrate (emission wavelength 525 ± 2 nm) and the product (emission wavelength 427 ± 2 nm) after treatment with sulfuric acid:ethanol mixture.

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