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Electrochemistry of Canis familiaris cytochrome P450 2D15 with gold nanoparticles: An alternative to animal testing in drug discovery.

Authors
  • Rua, Francesco1
  • Sadeghi, Sheila J2
  • Castrignanò, Silvia1
  • Valetti, Francesca1
  • Gilardi, Gianfranco3
  • 1 Department of Life Sciences and Systems Biology, University of Torino, Italy. , (Italy)
  • 2 Department of Life Sciences and Systems Biology, University of Torino, Italy; Centre for Nanostructured Interfaces and Surfaces, University of Torino, Italy. , (Italy)
  • 3 Department of Life Sciences and Systems Biology, University of Torino, Italy; Centre for Nanostructured Interfaces and Surfaces, University of Torino, Italy. Electronic address: [email protected] , (Italy)
Type
Published Article
Journal
Bioelectrochemistry (Amsterdam, Netherlands)
Publication Date
Oct 01, 2015
Volume
105
Pages
110–116
Identifiers
DOI: 10.1016/j.bioelechem.2015.03.012
PMID: 26092534
Source
Medline
Keywords
License
Unknown

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.

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