Modeling of Phase 1 Metabolism Cytochromes P450

Cytochrome P450 is the most important phase 1 enzyme, and belongs to a superfamily containing over 70 families of membrane-bound heme proteins that catalyze the oxidative metabolism of structurally diverse chemicals.3,4 All members of a particular cytochrome P450 subfamily have a 55% amino acid sequence homology with one another, while members of the same family show an identity of more than 40%.7 The isoenzymes differ from each other in amino acid sequence, in control by inhibitors and inducing agents, in the substrates they act upon, and the reactions they catalyze. Some isoenzymes have overlapping substrate specificities, acting on the same substrates as each other but at differing rates. Each cytochrome P450 has a unique system of substrate recognition, leading to the observed substrate specificities; for example, CYP2C9 prefers acidic and neutral but not basic substrates, CYP2D6 prefers basic substrates, CYP3A4 metabolizes both acidic and basic substrates, but log P plays a role, and CYP1A2 metabolism is governed by size.

Seven of the 57 known human isoforms of cytochromes P450 (CYP1A2, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) are responsible for more than 90% of the metabolism of all drugs in current clinical use.8 Some of these enzymes (especially CYP2D6 and CYP2C9) show polymorphisms, which can either result in rapid or very poor metabolism.9-11 For example, debrisoquine is extensively metabolized in normal individuals to 4-hydroxydebrisoquine, but poor metabolizers can develop high levels of the parent compound, which may be toxic.12 The impaired metabolic oxidation of at least 30 drugs with diverse structures and pharmacological actions has been associated with the phenotype of poor debrisoquine metabolism. In a few cases, poor metabolizers may be unable to bioactivate a parent drug, such as encaimide, to its therapeutically active metabolites.13,14 Binding of any drug to these cytochromes P450 can cause drug-drug interactions, which can lead to severe side effects. This has in the last few years resulted in early termination of development, refusal of approval, severe prescribing restrictions, and withdrawal of drugs from the market. Regulators, including the US Food and Drug Administration, have therefore issued guidance for in vitro and in vivo drug interaction studies to be conducted during development.15 Structural Modeling of Cytochromes P450

The major human cytochromes P450 involved in xenobiotic transformations are from families 1, 2, and 3, with CYP3A4 representing the most important enzyme for phase 1 drug metabolism, which is broadly commensurate with its average percentage of the human hepatic microsomal cytochrome P450 complement.16 Table 1 summarizes the more important human cytochromes P450 involved in exogenous metabolism, and indicates typical substrates, inducers, and inhibitors of these enzymes, showing the structural diversity of such compounds. Apart from the determination of structural characteristics of these cytochromes P450 via x-ray crystallography,17 where six mammalian structures (rabbit 2B4,18 rabbit 2C5,19-21 human 2C8,22 human 2C9,23,24 human CYP2D6,25 and human 3A426,27) have been resolved, the challenges for computational chemistry in this area have focused on homology modeling, requirements for substrate selectivity, estimation of binding affinity, and the search for descriptors that may correlate with kinetic data (rates or clearance) for cytochrome P450-mediated metabolism and other absorption, distribution, metabolism, excretion, and toxicology properties.28,29

Recently published x-ray crystal structures30 of human cytochromes P450 (Table 2) provide an opportunity for comparing previously reported models of these enzymes, and to establish a degree of validity for homology modeling. For CYP2C8 and CYP2C9, the models have been shown to exhibit a-carbon root mean square distances of 1.2 A and 1.5 aA, respectively, thus demonstrating the utility of, in this case, the CYP2C5 crystal structure as a template for generating three-dimensional models for other CYP2C subfamily enzymes.31-33 An example of a CYP2C9 homology model is shown in Figure 1. Furthermore, there may be wider applicability of such crystallographic templates toward the CYP2 family in general, although the recent inhibitor-bound CYP2B4 crystal structure can be expected to represent an ideal template for the homology modeling of CYP2B6. Information from the human cytochrome P450 crystal structures is proving useful for the rationalization of substrate selectivity, which can lead to predictive tools for the high-throughput screening of new chemical entities (NCEs). For example, the active-site volumes of CYP2C8, CYP2C9, and CYP3A4 indicate the likely maximum sizes of substrates and their superimposed templates; also, the topography of these active sites helps to explain, in part, substrate structural preferences (summarized in Table 3). For example, there are clear topographical differences between CYP2C8 and CYP3A4 active sites that are in agreement with the known substrate selectivity between these two enzymes, despite evidence for some degree of overlap in this respect. More details with regard to the field of cytochrome P450 modeling can be found in a recent review. 34

Table 1

Human cytochromes P450 from families CYP1, CYP2, CYP3, and CYP416-123"

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