Date of Graduation

2015

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Grazyna D Szklarz

Committee Co-Chair

David Lederman

Committee Member

William P Petros

Committee Member

Yon Rojanasakul

Committee Member

Timothy S Tracy

Abstract

Cytochrome P450s (P450s) are a large family (>11,000) of heme thiolated proteins that are responsible for ~ 75% of the metabolism of pharmaceuticals on the market. Understanding P450 mediated metabolism is crucial for accurate in vitro predictions of drug metabolism. P450 protein-protein interactions have been shown to alter enzyme catalytic activity. Furthermore, these interactions are isoform specific, and can elicit activation, inhibition, or no effect on enzymatic activity. Studies show these effects are also dependent on the protein binding partner cytochrome P450 reductase (CPR), and the order of protein addition to purified reconstituted enzyme systems. In the current work, we use controlled immobilization of P450s to a gold surface to gain a better understanding of P450-P450 interactions between three key drug-metabolizing isoforms (CYP2C9, CYP3A4, and CYP2D6). Molecular modeling was used to assess the favorability of homo/heteromeric P450 complex formation. P450 complex formation in vitro was analyzed in real-time utilizing surface plasmon resonance (SPR). Lastly, the effects of P450 complex formation were investigated utilizing our immobilized platform and reconstituted enzyme systems.;Molecular modeling shows favorable binding of CYP2C9-CPR, CYP2C9-CYP2D6, CYP2C9-CYP2C9, and CYP2C9-CYP3A4 in rank order. KD values obtained via SPR show strong binding, in the nanomolar range, of the above pairs, with CYP2D6 yielding the lowest KD, followed by CYP2C9, CPR, and CYP3A4. Metabolic incubations show immobilized CYP2C9 metabolism was activated by homomeric complex formation. CYP2C9 metabolism was not affected by the presence of CYP3A4 with saturating CPR concentrations. CYP2C9 metabolism was activated by CYP2D6 in solution, but inhibited when CYP2C9 was immobilized, both at saturating and sub-saturating CPR concentrations. Order of addition of proteins (CYP2C9, CYP2D6, CYP3A4, and CPR) influenced magnitude of inhibition for CYP3A4, but not CYP2D6. These results indicate isoform specific P450 interactions and effects on P450 mediated-metabolism. These findings are important in evaluating how in vitro results are obtained for measuring P450 kinetics, and provide a better mechanistic understanding of P450-P450 interactions to allow for better prediction of in vivo metabolism from in vitro data.;We also demonstrate that gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized P450s without aggregation. Given that transfer of the 1st electron to the P450 heme group acts as the gating step for the catalytic cycle, understanding electron transfer in P450s could shed light on metabolism kinetics. Conductance measurements of nanopillars with immobilized CYP2C9 using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme.;The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II ligands may decrease the ease of electron transport processes in the enzyme, in addition to occupying the active site. These findings further our understanding of how P450 metabolism is mediated through substrates, and provides an important technological advancement for studying P450s that avoids complications found in current methodologies. These two studies demonstrate the ability of an immobilized P450 platform to provide information on protein-protein interactions, substrate protein interactions, and atypical enzyme kinetics.

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