Immobilized Cytochrome P450 2C9 (CYP2C9): Applications for Metabolite Generation, Monitoring Protein-Protein Interactions, and Improving In-vivo Predictions Using Enhanced In-vitro Models

Abstract

Development of Au-Immobilized P450 Platform for Exploring the Effect of Oligomer Formation on P450-Mediated Metabolism for In Vitro to In Vivo Drug Metabolism Predictions Lance A. Wollenberg Cytochrome P450 (P450) enzymes are a family of oxoferroreductase enzymes containing a heme moiety and are well known to be involved in the metabolism of a wide variety of endogenous and xenobiotic materials. It is estimated that roughly 75% of all pharmaceutical compounds are metabolized by these enzymes. Traditional reconstituted in-vitro incubation studies using recombinant P450 enzymes are often used to predict in-vivo kinetic parameters of a drug early in development. However, in many cases, these reconstituted incubations are prone to aggregation which has been shown to affect the catalytic activity of an enzyme. Moreover, the presence of other isoforms of P450 enzymes present in a metabolic incubation, as is the case with microsomal systems, may affect the catalytic activity of an enzyme through isoformspecific protein-protein interactions. Both of these effects may result in inaccurate prediction of in-vivo drug metabolism using in-vitro experiments. Here we described the development of immobilized P450 constructs designed to elucidate the effects of aggregation and protein-protein interactions between P450 isoforms on catalytic activities. The long term objective of this project is to develop a system to control the oligomeric state of Cytochrome P450 enzymes to accurately elucidate discrepancies between in vitro reconstituted systems and actual in vivo drug metabolism for the precise prediction of metabolic activity. This approach will serve as a system to better draw correlations between invivo and in-vitro drug metabolism data. The central hypothesis is that Cytochrome P450 enzymes catalytic activity can be altered by protein-protein interactions occurring between Cytochrome P450 enzymes involved in drug metabolism, and is dependent on varying states of protein aggregation. This dissertation explains the details of the construction and characterization of a nanostructure device designed to control the state of aggregation of a P450 enzyme. Moreover, applications of immobilized P450 enzyme constructs will also be used for monitoring proteinprotein interaction and metabolite production with the use of immobilized-P450 bioreactor constructs. This work provides insight into the effect on catalytic activity caused by both P450 aggregation as well as isoform-specific protein-protein interactions and provides insight in the production of biosynthetically produced drug metabolites.

Protein bioelectronics: a review of what we do and do not know

“…381 However, Raman shifts were also observed, indicative of the inactive form, and it was not possible to reduce the protein heme group. This result is interesting given recent work, which demonstrated that biologically active CYP2D6 can be adsorbed via a SAM on Au, 382 and may reveal surface-specific differences in immobilized P450 function. Surface plasmon resonance (SPR) spectroscopy has been successfully used to study multi-layer /aggregation formation during immobilization (Figure 16).…”

“…However, Raman shifts were also observed, indicative of the inactive form, and it was not possible to reduce the protein's heme group. This result is interesting given recent work, which demonstrated that biologically active CYP2D6 can be adsorbed via a SAM on Au [377], and may reveal surface-specific differences in immobilized P450 function.…”