Specificity and mechanism of cytochrome P450s: Use of potent inhibitors and stoichiometric measurements to derive models for binding affinity and coupling efficiency

The biological oxidation of foreign chemicals, also known as xenobiotics, is carried out largely by a superfamily of enzymes called cytochromes P450 (CYP). These heme-containing enzymes constitute a superfamily of monooxygenases that are able to activate molecular oxygen to oxidize several kinds of organic functional groups. There is much interest in mammalian P450s in the pharmaceutical sciences because they determine the rate of clearance of every drug to some extent. Therefore, P450s determine how multiple drugs affect each other's pharmacokinetics and whether or not they could cause drug-drug interactions.; Yet of the 63 distinct human isoforms sequenced in the genome, only a subset oxidizes the majority of drugs. The ability of P450s to metabolize a wide variety of lipophilic substrates partly originates from their hydrophobic active sites. At the same time, each isoform displays obvious differences in substrate selectivity and regiospecificity that are driven by steric and electronic properties. This is the subject of chapters 2--4 which describe the use of potent inhibitors of varying steric features and charge states to probe P450s 2C9 and 2C19---two enzymes estimated to metabolize about 20% of all drugs. Therefore, with the first low nanomolar Ki inhibitors, new hypotheses for specific enzyme-ligand interactions have been drawn and tested by both metabolism and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies.; The rate at which a drug is metabolized by a P450 is also tied to its potential to interact with other drugs; however, the rate of P450-mediated drug metabolism is not always correlated with substrate affinity. Because CYPs activate molecular oxygen through a series of high energy oxo-iron intermediates, these intermediates often dissociate from the heme before oxidizing the substrate. By monitoring the ratio of these shunt pathway products (e.g. H2O 2) to the amount of oxidized substrate in vitro, the first 3D-QSAR for H2O2 was generated. Substrates were aligned using the Tripos SYBYL software. Hydrogen peroxide data was described by the alignment with a cross-validated q2 of 0.59 and a standard error of 0.29. This shows the in vitro system can lead to shunt pathway models that help predict the rate of substrate turnover.