Direct Electrochemistry of Drug Metabolizing Human Flavin-Containing Monooxygenase: Electrochemical Turnover of Benzydamine and Tamoxifen

Abstract

This communication reports on the first electrochemical study of the human flavin-containing monooxygenase 3 (hFMO3) either absorbed or covalently linked to different electrode surfaces. Glassy carbon and gold electrodes gave reversible electrochemical signals of an active hFMO3. The midpoint potential measured for the immobilized enzyme on a glassy carbon electrode was -445 +/- 8 mV (versus Ag/AgCl). A monolayer coverage was obtained on gold functionalized with dithio-bismaleimidoethane that covalently linked surface accessible cysteines of hFMO3. A structural model of the enzyme was generated to rationalize electrochemistry results. The turnover of the active enzyme was measured with two specific drugs: tamoxifen and benzydamine. For tamoxifen, 1.7 and 8.0 microM of its N-oxide product were formed by the enzyme immobilized on glassy carbon and gold electrodes, respectively. In the case of benzydamine, a K(M) of 44 +/- 5 microM was measured upon application of a -600 mV bias to the enzyme immobilized on the glassy carbon electrode that is in good agreement with the values published for microsomal hFMO3 where NADPH is the electron donor.

Identification of human flavin-containing monooxygenase 3 substrates by a colorimetric screening assay

“…All steps were of expression and purification were performed according to previously published protocols [8,9]. Wild type hFMO3 was expressed in E. coli JM109 cells and grown 24 h post--induction.…”

Prospects on the Hyphenated Electrochemistry and Mass Spectrometry as a Practical Analytical Technique in the Assessment of Oxidative Drug Metabolism

“…Using isolated Cytochrome P450s requires additional reductase cofactor. A protein engineering strategy might also help to obtain sufficient interaction of the enzyme with electrode surfaces using an enzyme reductase fused protein [31]. This is due to the fact that the enzyme immobilization should not inactivate or change the enzyme behavior.…”

Bioelectrochemical profiling of two common polymorphic variants of human FMO3 in presence of graphene oxide

“…In light of this, much attention has been concentrated on the development of new methods useful for pharmacological research relevant to hFMO3 catalysis. One such technique which is applicable to these enzymes is electrochemistry where the direct electrochemical response of hFMO3 on gold and carbon surfaces has been previously demonstrated by our group [14][15][16]. The advantage of direct immobilization of hFMO3 on electrode surface is mostly related to the possibility of using reducing equivalents provided by the electrode surface instead of those of NADPH which is its natural redox partner, thus improving experimental conditions in terms of costs, reliability and efficiency.…”

“…These two hFMO3 variants were expressed in a recombinant system and immobilised on didodecyldimethylammonium bromide (DDAB)/GO glassy carbon electrodes and their activity tested with three hFMO3 marker drugs: benzydamine, a non-steroidal anti-inflammatory drug that is metabolised to its N-oxide by hFMO3 [15][16][17]21,25], tamoxifen, a breast cancer drug with antiestrogenic effect, also Noxygenated by hFMO3 [15][16][17]21,26,27],and sulindac sulfide, a nonsteroidal antiinflammatory drug [11] that is selectively re-oxidised to sulindac by hFMO3 S-oxygenation [2,25].…”