title: Electrochemistry of Canis familiaris cytochrome P450 2D15 with gold nanoparticles: An alternative to animal testing in drug discovery.
abstract: This work reports for the first time the direct electron transfer of the Canis familiaris cytochrome P450 2D15 on glassy carbon electrodes to provide an analytical tool as an alternative to P450 animal testing in the drug discovery process. Cytochrome P450 2D15, that corresponds to the human homologue P450 2D6, was recombinantly expressed in Escherichia coli and entrapped on glassy carbon electrodes (GC) either with the cationic polymer polydiallyldimethylammonium chloride (PDDA) or in the presence of gold nanoparticles (AuNPs). Reversible electrochemical signals of P450 2D15 were observed with calculated midpoint potentials (E1/2) of −191 ± 5 and −233 ± 4 mV vs. Ag/AgCl for GC/PDDA/2D15 and GC/AuNPs/2D15, respectively. These experiments were then followed by the electro-catalytic activity of the immobilized enzyme in the presence of metoprolol. The latter drug is a beta-blocker used for the treatment of hypertension and is a specific marker of the human P450 2D6 activity. Electrocatalysis data showed that only in the presence of AuNps the expected α-hydroxy-metoprolol product was present as shown by HPLC. The successful immobilization of the electroactive C. familiaris cytochrome P450 2D15 on electrode surfaces addresses the ever increasing demand of developing alternative in vitromethods for amore detailed study of animal P450 enzymes' metabolism, reducing the number of animals sacrificed in preclinical tests.
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Rua
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Italy; Centre for Nanostructured Interfaces and Surfaces, University of Torino, Italy.
authors.forename: Sheila J
authors.initials: SJ
authors.lastname: Sadeghi
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Castrignanò
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Valetti
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Italy; Centre for Nanostructured Interfaces and Surfaces, University of Torino, Italy. Electronic address: gianfranco.gilardi@unito.it.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
mesh_terms: D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004285:Dogs; D055808:Drug Discovery; D055664:Electrochemical Techniques; D006046:Gold / Q000378:metabolism*; D053768:Metal Nanoparticles
chemical_list: D006046:Gold; D003577:Cytochrome P-450 Enzyme System
keywords: Animal testing; CYP; Carbon electrode; Gold nanoparticle; Immobilization; Metoprolol