Enzyme-Based Amperometric Platform to Determine the Polymorphic Response in Drug Metabolism by Cytochromes P450

Abstract

"Personalized medicine" is a new concept in health care, one aspect of which defines the specificity and dosage of drugs according to effectiveness and safety for each patient. Dosage strongly depends from the rate of metabolism which is primarily regulated by the activity of cytochrome P450. In addition to the need for a genetic characterization of the patients, there is also the necessity to determine the drug-clearance properties of the polymorphic P450 enzyme. To address this issue, human P450 2D6 and 2C9 were engineered and covalently linked to an electrode surface allowing fast, accurate, and reliable measurements of the kinetic parameters of these phase-1 drug metabolizing polymorphic enzymes. In particular, the catalytic activity of P450 2C9 on the electrode surface was found to be improved when expressed from a gene-fusion with flavodoxin from Desulfovibrio vulgaris (2C9/FLD). The results are validated using marker drugs for these enzymes, bufuralol for 2D6, and warfarin for 2C9/FLD. The platform is able to measure the same small differences in K(M), and it allows a fast and reproducible mean to generated the product identified by HPLC from which the k(cat) is calculated.

Engineering Macaca fascicularis cytochrome P450 2C20 to reduce animal testing for new drugs

“…Bioelectrochemical methods lead not only to the acquisition of fundamental knowledge on the redox properties of P450 enzymes [31] but also open opportunities for technological and commercial applications [32,33]. To this end, the purified P450 2C20 was initially immobilized on both carbon and gold surfaces and its redox properties acquired and compared.…”

Surface-modified electrodes in the mimicry of oxidative drug metabolism

“…A CYP450-based amperometric platform was proposed by Gilardi et al for measuring the individually variable, polymorphic response in Phase-I drug metabolism depending on drug therapy [56]. Since no catalytically active protein was observed after direct immobilization of CYP450 enzymes on the electrode surface, chemical spacers were used to form a SAM by covalently binding to the gold electrode on one side, while the other side bound the protein (Fig.…”

“…3. Two strategies for the immobilization of CYP2D6 on a gold electrode via SH groups (left side) and via NH2 groups (right side) [56]. polycrystalline gold working electrode surface, as depicted in Fig.…”

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

“…Large-scale expression (4 liters) of P450 2D15 in E. coli DH5α cells transformed with pCW-2D15 was carried out as described previously for other P450 cytochromes [35] decreasing the postinduction expression time and temperature to 24 hours and 24 °C, respectively. Purification of P450 2D15 was carried out starting from the isolated membrane fraction using an anion exchange DEAE sepharose column (GE-healthcare, Italy) and followed by a nickel ion affinity chromatography step (GE-healthcare, Italy) where the protein remained bound through the engineered His-tag and subsequently eluted using a 0-40 mM linear gradient of histidine.…”