Functional characterization of pharmacogenetic influences on CYP450 enzyme activtiy and kinetics

Cytochrome P450 enzymes are part of a superfamily of enzymes that are classified as oxidoreductases and are the main enzymes in hepatic phase I drug metabolism. They exhibit one of the most diverse and precise reaction cycles, catalyzing the challenging incorporation of oxygen into inactive hydrocarbons and heteroatoms resulting in various reactions such as substrate oxidation, hydroxylation, epoxidation, oxidative N-dealkylation, and oxidative demethylation. This process typically involves the incorporation of single electrons and the generation of reactive intermediates. Under certain conditions, this highly specific and tightly regulated reaction cycles uncouples, resulting in a reaction abort, the accumulation of reactive intermediates and ultimately increase reactive oxygen species (ROS) formation. This process is termed uncoupling and under normal conditions contributes to the important cellular oxidative homeostasis. Increased uncoupling leads to an imbalance and increases the oxidative load to a toxic extent. While to date the triggers for uncoupling are mostly unknown, it has been shown that the uncoupling susceptibility varies from one CYP450 enzyme to another. Pharmacogenetic alterations in CYP450 genes are commonly associated with altered enzyme function and mostly characterized through a decreased or increased enzyme activity and hence drug metabolism. These effects are mostly compensated through drug dosage adjustments or substitution. Pharmacogenetic alterations however have also been postulated to alter the reaction mechanism and coupling efficiencies of the enzyme and therefore increase its uncoupling susceptibility. With the aim to functionally characterize CYP2B6 and its variants (CYP2B6*6 and CYP2B6*34) as well as CYP2C8 and its variants (CYP2C8*2 and CYP2C8*3) in vitro and ex vivo, it was necessary to first stabilize the enzymes due to their generally low in vitro stability. Therefore, based on a systematic literature search, 45 substances were screened for their stabilizing potential in a biocatalyisis engineering approach, revealing that the combination of cysteine, dithiothreitol and phosphocholine results in the highest activity increase among the main drug metabolizing enzymes CYP3A4, CYP2B6, CYP2C19, CYP2D6 and CYP2J2. Using the optimized biocatalysis buffer with stabilizing potential, recombinant human CYP2B6 and CYP2C8 genetic variants were studied in terms of their reaction pathway mechanisms in vitro and ex vivo. Both enzyme’s pharmacogenetic variants have been associated with clinical manifestations in extrahepatic tissues. These manifestations were not explainable by the location of the single nucleotide polymorphisms or the activity of the enzyme variants. Functional assessment of the three possible reaction pathways including the normal coupled cycle, the uncoupled cycle and its counterreaction – the peroxide shunt, revealed that a substrate specific increase in peroxide shunt and a variant specific increase in reaction uncoupling can be observed in CYP2B6 and CYP2C8 variants. The knowledge obtained from this study indicates that pharmacogenetic effects extend beyond drug clearance, influencing enzyme function, which can subsequently impact ROS formation and contribute to inflammatory symptoms and diseases. Such functional pharmacogenetic variations cannot be mitigated solely through dosing adjustments and are hardly predictable due to the apparent substrate and variant specificity.