4 research outputs found
Development of gold-immobilized P450 platform for exploring the effect of oligomer formation on P450-mediated metabolism for in vitro to in vivo drug metabolism predictions.
The cytochrome P450 (P450) enzyme family is responsible for the biotransformation of a wide range of endogenous and xenobiotic compounds, as well as being the major metabolic enzyme in first pass drug metabolism. In vivo drug metabolism for P450 enzymes is predicted using in vitro data obtained from a reconstituted expressed P450 system, but these systems have not always been proven to accurately represent in vivo enzyme kinetics, due to interactions caused by oligomer formation. These in vitro systems use soluble P450 enzymes prone to oligomer formation and studies have shown that increased states of protein aggregation directly affect the P450 enzyme kinetics. We have developed an immobilized enzyme system that isolates the enzyme and can be used to elucidate the effect of P450 aggregation on metabolism kinetics. The long term goal of my research is to develop a tool that will help improve the assessment of pharmaceuticals by better predicting in vivo kinetics in an in vitro system. The central hypothesis of this research is that P450-mediated kinetics measured in vitro is dependent on oligomer formation and that the accurate prediction of in vivo P450-mediated kinetics requires elucidation of the effect of oligomer formation. The rationale is that the development of a P450 bound to a Au platform can be used to control the aggregation of enzymes and bonding to Au may also permit replacement of the natural redox partners with an electrode capable of supplying a constant flow of electrons. This dissertation explains the details of the enzyme attachment, monitoring substrate binding, and metabolism using physiological and electrochemical methods, determination of enzyme kinetics, and the development of an immobilized-P450 enzyme bioreactor. This work provides alternative approaches to studying P450-mediated kinetics, a platform for controlling enzyme aggregation, electrochemically-driven P450 metabolism, and for investigating the effect of protein-protein interactions on drug metabolism
Electrocatalytic Drug Metabolism by CYP2C9 Bonded to A Self-Assembled Monolayer-Modified Electrode
Cytochrome P450 (P450) enzymes typically require the presence of at least
cytochrome P450 reductase (CPR) and NADPH to carry out the metabolism of
xenobiotics. To address whether the need for redox transfer proteins and the
NADPH cofactor protein could be obviated, CYP2C9 was bonded to a gold
electrode through an 11-mercaptoundecanoic acid and octanethiol self-assembled
monolayer (SAM) through which a current could be applied. Cyclic voltammetry
demonstrated direct electrochemistry of the CYP2C9 enzyme bonded to the
electrode and fast electron transfer between the heme iron and the gold
electrode. To determine whether this system could metabolize warfarin
analogous to microsomal or expressed enzyme systems containing CYP2C9,
warfarin was incubated with the CYP2C9-SAM-gold electrode and a controlled
potential was applied. The expected 7-hydroxywarfarin metabolite was observed,
analogous to expressed CYP2C9 systems, wherein this is the predominant
metabolite. Current-concentration data generated with increasing
concentrations of warfarin were used to determine the Michaelis-Menten
constant (Km) for the hydroxylation of warfarin (3 μM),
which is in good agreement with previous literature regarding
Km values for this reaction. In summary, the
CYP2C9-SAM-gold electrode system was able to carry out the metabolism of
warfarin only after application of an electrical potential, but in the absence
of either CPR or NADPH. Furthermore, this system may provide a unique platform
for both studying P450 enzyme electrochemistry and as a bioreactor to produce
metabolites without the need for expensive redox transfer proteins and
cofactors