Elsevier

Bioelectrochemistry

Volume 105, October 2015, Pages 110-116
Bioelectrochemistry

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

https://doi.org/10.1016/j.bioelechem.2015.03.012Get rights and content

Highlights

  • Cytochrome P450 2D15 is entrapped on glassy carbon electrodes with PDDA.

  • Immobilization of cytochrome P450 2D15 in the presence of AuNps is achieved.

  • Electrocatalysis in the presence of AuNps with metoprolol as substrate is shown.

  • Electrochemically produced alpha-hydroxy-metaprolol by P450 2D15 is detected.

  • In vitro electrochemical platform for bypassing in vivo animal testing is proposed.

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 vitro methods for a more detailed study of animal P450 enzymes' metabolism, reducing the number of animals sacrificed in preclinical tests.

Introduction

In view of the huge numbers of animals used during preclinical tests in drug discovery [1], both the European Union (European Commission and European Parliament, Directive 2010/63/EU) [2], [3] and the US Food and Drug Administration (FDA) [4] are strongly supporting the development of alternative in vitro methods to implement the three Rs, that is “reduce, refine and replace” animal models [5].

Hepatic cytochromes P450 are central to toxicological studies due to their primary role in phase I metabolism of more than 80% of marketed drugs [6]. However to date, very little data is published on comparative in vivo interspecies studies with even less data available on the different animal P450 expression and/or their metabolic profile [7], [8], [9], [10]. To this end, a fast and reliable in vitro method would offer the possibility of a more detailed study of the interaction of new drugs with the animal P450 enzymes therefore increasing the predictive value of these preclinical trials.

Canis familiaris is one of the most widely studied animal models used in safety determination of new pharmaceuticals [11], [12] and, although the major isoforms of the human cytochromes P450 2D6, 3A4, 2E1, 2C19 and 1A2 have been identified in C. familiaris [13], [14], [15], [16], there is still a lack of knowledge on their pharmacogenomic/metabolic diversity [9].

Electrochemical techniques already developed in our lab for human hepatic monooxygenases including cytochromes P450 [17], [18], [19], [20], [21], [22] represent the ideal approach for a sensitive, accurate and rapid evaluation of animal P450–drug interactions obviating both the requirement for a redox partner and the addition of NADPH cofactor as already reported for some animal P450 enzymes recombinantly expressed in a soluble form [23], [24], [25].

In this work, C. familiaris P450 2D15 was chosen as a model for the investigation of canine cytochromes P450 by adopting electrochemical approaches to provide a method for the screening of the safety of new chemical entities/drugs. This enzyme shares 75% identity with the human cytochrome P450 2D6 which alone is responsible for the metabolism of 20–25% of commonly used therapeutic drugs including antiarrhythmics, adrenoceptor antagonists, and tricyclic antidepressants [6], [26]. The high P450 2D6 polymorphism profile is strictly related to either adverse drug reactions or no drug response [26], [27] and to date six different variants have been already identified in the corresponding homologous C. familiaris P450 2D15 [28], [29], [30].

Generally, the study of the catalytic properties of canine P450 2D enzymes is hampered by the difficulty in their preparation from liver microsomes [14] or by their recombinant expression and purification in a stable form when not associated to the membranes [14], [15], [28], [30]. To this end, in this work we report for the first time the recombinant expression and purification of the C. familiaris P450 2D15 in an N-terminally modified form maintaining its structural integrity and function. Furthermore, the electrocatalytic functionality of the purified enzyme is tested whilst immobilized on glassy carbon electrodes (GC).

Two different immobilization strategies were adopted: a) entrapment within the cationic polymer polydiallyldimethylammonium chloride (PDDA) (GC/PDDA/2D15); b) immobilization in presence of gold nanoparticles (AuNPs) stabilized with didodecyldimethylammonium bromide (DDAB) (GC/AuNPs/2D15). Gold nanoparticles are widely used in electrochemical applications [31] especially because they act as excellent electron transfer relays by enhancing the electron transfer from the electrode to the protein [32].

Electrochemical properties of C. familiaris P450 2D15 immobilized on glassy carbon electrodes were characterized by cyclic voltammetry and the activity of this enzyme in the presence of metoprolol, a selective β1 receptor blocker used in treatment of cardiovascular disease and a marker for human P450 2D6 activity, assayed by chronoamperometry. The separation and identification of the product formed was carried out by HPLC leading to the calculation of the KM value of metoprolol for the first time through the electrochemical system GC/AuNPs/2D15.

Section snippets

Reagents

Kits for plasmid and gene purification were purchased from Sigma Aldrich (Italy). Restriction enzymes, T4 DNA ligase, Vent Polymerase and dNTPS were from New England Biolabs (UK). Chromatographic resins were purchased from GE healthcare (Italy). Quinidine ((S)-[(2R,4S,5R)-5-ethenyl-1-azabicyclo[2.2.2]octan-2-yl](6-methoxyquinolin-4-yl)methanol) and (±)-Metoprolol ({2-hydroxy-3-[4-(2-methoxyethyl)phenoxy]propyl}(propan-2-yl)amine) (+)-tartrate salt, racemic mixture, were purchased from Sigma

Recombinant P450 2D15

The gene coding for the full length cytochrome P450 2D15 was amplified from C. familiaris liver cDNA and three mutations were identified by full DNA sequencing. A silent substitution of an adenine in guanine has found 325 bases after the 2D15 start with two other mutations leading to the amino acid substitutions of Ile109Val and Phe115Leu in the full-length protein (g345c and g738a). Cytochrome P450 variants in C. familiaris 2D15 are not unusual and have been previously reported [28], [29], [30]

Conclusions

The development of an electrochemical platform for the screening of animal cytochromes P450 represents an interesting and feasible solution to the ever increasing demand to reduce animal testing, maintaining a high level of predictivity of the animal models used in preclinical tests and providing the opportunity to study the P450 metabolism of new drugs and chemical entities.

To this end, the cloning of a new C. familiaris P450 2D15 variant and its expression in a pure form for its

Acknowledgments

The authors wish to acknowledge financial support from the Regione Piemonte CIPE 2006 (CYP-TECH 35/2005) (CYPTECH-project, Italy) and Progetto Ateneo-San Paolo 2012 (SADJATEN12) (awarded to S. Sadeghi).

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