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.

Progress in biopharmaceutical development

“…Owing to P450 BM3 characteristics, multiple protein engineering studies have been performed on this enzyme to widen its catalytic abilities . Moreover, several constructions have been already reported including different fusion human P450 enzymes, engineered by connecting the P450 BM3 reductase domain with human cytochromes P450 3A4, 2C9, 2C19 , 2A6, CYP2C6, and CYP4F11 , monkey 2C20 , and dog CYP2D15 . Also the catalytic performance of one of the created chimeric proteins was improved in terms of coupling efficiency and enzyme turnover by engineering the loop connecting the two domains .…”

Human Cytochrome P450 3A4 as a Biocatalyst: Effects of the Engineered Linker in Modulation of Coupling Efficiency in 3A4-BMR Chimeras

“…For this reason we already reported the construction of different fusion human P450 enzymes, engineered by connecting the reductase domain BMR with human cytochromes P450 2E1(Fairhead et al, 2005), 3A4, 2C9, 2C19 (Dodhia et al, 2006), 2A6, CYP2C6, and CYP4F11 (Ortolani, 2012; Rua, 2012a; Castrignanò et al, 2014), monkey 2C20 (Rua et al, 2012b) and dog CYP2D15 (Sadeghi and Gilardi, 2013; Rua et al, 2015). …”

“…One factor that makes P450 BM3 a well-coupled biocatalyst resides in the productive interaction between the heme catalytic domain (BMP) and its flavin-containing reductase domain (BMR) that, unlike in other cytochromes P450, are all part of the same polypetide chain. For this reason we already reported the construction of different fusion human P450 enzymes, engineered by connecting the reductase domain BMR with human cytochromes P450 2E1(Fairhead et al, 2005 ), 3A4, 2C9, 2C19 (Dodhia et al, 2006 ), 2A6, CYP2C6, and CYP4F11 (Ortolani, 2012 ; Rua, 2012a ; Castrignanò et al, 2014 ), monkey 2C20 (Rua et al, 2012b ) and dog CYP2D15 (Sadeghi and Gilardi, 2013 ; Rua et al, 2015 ).…”

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

“…For this reason we already reported the construction of different fusion human P450 enzymes, engineered by connecting the reductase domain BMR with human cytochromes P450 2E1 (Fairhead et al, 2005), 3A4, 2C9, 2C19 (Dodhia et al, 2006), 2A6, CYP2C6, and CYP4F11 (Ortolani, 2012;Rua, 2012a;Castrignanò et al, 2014), monkey 2C20 (Rua et al, 2012b) and dog CYP2D15 (Sadeghi and Gilardi, 2013;Rua et al, 2015).…”