Molecular modelling of the human cytochrome P450 isoform CYP2A6 and investigations of CYP2A substrate selectivity

Abstract

(1) The generation of a homology model of CYP2A6, the major catalyst of human hepatic coumarin 7-hydroxylase activity, involves the use of the recently published substrate-bound CYP102 crystal structure as a template. (2) A substantial number of structurally diverse CYP2A6 substrates are found to dock satisfactorily within the putative active site of the enzyme, leading to the formulation of a structural template (or pharmacophore) for CYP2A6 specificity/selectivity. (3) The CYP2A6 model is consistent with available evidence from site-directed mutagenesis studies carried out on CYP2A subfamily isoforms, and enables some explanation of species differences in CYP2A-mediated metabolism of certain substrates. (4) Quantitative structure–activity relationship (QSAR) analysis of CYP2A5 (the mouse orthologue) mutants yields statistically significant correlations between various properties of amino acid residues and coumarin 7-hydroxylase activity.

Introduction

Cytochrome P4502A6 (CYP2A6) represents a relatively minor component (∼4% on average) of the human hepatic cytochrome P450 (CYP) complement (Shimada et al., 1994, Guengerich, 1995, Rendic and Di Carlo, 1997). The levels of CYP2A6 expressed in human liver are quite variable, however, and their concordance with CYP2B6 levels has led to the suggestion that these CYP2 isoforms may be coordinately regulated in Homo sapiens, especially as their respective genes occupy similar chromosomal locations (Miles et al., 1990, Forrester et al., 1992, Gonzalez, 1992).

Two allelic variants of CYP2A6 have been characterized (Fernandez-Salguero et al., 1995, Hadidi et al., 1997) which appear to be present at varying levels depending on the ethnogeographical population. For example, CYP2A6*2 is found in only about 2% of individuals from the African–American ethnic group (Guengerich, 1995) although relatively high allelic frequencies of both CYP2A6*1 and CYP2A6*2 have been reported in Japanese (Hadidi et al., 1997): moreover, a related member of the CYP2A subfamily, namely CYP2A7, has been isolated from a human liver library (Ding et al., 1995, Chang and Waxman, 1996). Despite bearing a 94% similarity to CYP2A6, cDNA-expressed CYP2A7 is unable to bind haem effectively and is, consequently, catalytically inactive (Yamano et al., 1990, Ding et al., 1995). It is possible that this may be due to the replacement of Arg128 in CYP2A6 by leucine in CYP2A7 because Arg128 represents one of the key binding residues which forms a salt bridge with a haem propionate moiety in CYP2A6, according to a previous modelling study (Lewis and Lake, 1995). Although it is possible that allelic variants of CYP2A6 may play a role in the inter-individual variations in metabolic competence attributable to CYP2A in man, a comprehensive study of this genetic polymorphism remains to be performed, however (Fernandez-Salguero et al., 1995, Raunio and Pelkonen, 1995, Gullsten et al., 1997, Hadidi et al., 1997, Yokoi and Kamataki, 1998).

A significant number of CYP2A6 substrates are known (Honkakoski and Negishi, 1997, Rendic and Di Carlo, 1997) and a representative list is presented in Table 1, together with the reaction which is CYP2A6-catalyzed in each case. It should be appreciated, however, that some of these compounds are also metabolized by other P450 isoforms and, in some instances, at the same site in the substrate molecule. Consequently, relatively few drug oxidations can be regarded as CYP2A6-specific, although coumarin is generally accepted as representing a marker substrate for CYP2A6 where the specifically catalyzed reaction involves 7-hydroxylation (Miles et al., 1990, Fentem and Fry, 1992, Lake et al., 1992a, Pearce et al., 1992, Forrester et al., 1992, van Iersel et al., 1994a, Steensma et al., 1994, Lake, 1996, Shimada et al., 1996). Interestingly, the CYP2A6*1 allelic variant, which exhibits a change from leucine to histidine at position 160 in the protein sequence, appears to metabolize coumarin preferentially via 3,4-epoxidation (Hadidi et al., 1997) and thus shows some degree of similarity with CYP2A isoforms in other mammalian species (such as the rat, for example) with respect to coumarin metabolism. In fact, there are certain species differences between rat, mouse and other primates in the metabolism of this substrate, some of which may be attributable to CYP2A isoforms (Lewis and Lake, 1995, Lake, 1996).

The metabolism (and, in some cases, toxicity) of coumarin has been extensively studied in several mammalian species, including: rat (Lake et al., 1989, Lake et al., 1992a, Lake et al., 1992b, Lake et al., 1994, Fentem and Fry, 1992, Pearce et al., 1992, Steensma et al., 1994, Lake and Grasso, 1996), mouse (Lindberg and Negishi, 1989, Juvonen et al., 1991, Lindberg et al., 1992, van Iersel et al., 1994b, Salonpaa et al., 1994, Lake and Grasso, 1996, Lovell et al., 1998), hamster (Lake et al., 1992a, Lake and Grasso, 1996) and human (Lake et al., 1989, Fentem and Fry, 1992, Pearce et al., 1992, Steensma et al., 1994, van Iersel et al., 1994a), and a recent review compares and contrasts coumarin metabolism in different mammalian species (Lake, 1996).

In particular, it is found that there are marked differences in coumarin 7-hydroxylase activity between various species of primate (Lake, 1996) where New World primates, such as the marmoset and squirrel monkey, exhibit extremely low metabolism of coumarin via this pathway compared with extensive 7-hydroxylation in Old World primates, such as the baboon and cynomolgus monkey, together with the situation in man (Lake, 1996). It can be argued that the levels of CYP2A orthologues may exhibit marked differences between Old and New World primates, such that the observed variations in coumarin 7-hydroxylase activity could be one of the effects of primate evolution, where continental drift-induced separation of prosimian populations and resulting dietary/habitat pressures produced profound alterations in the hepatic P450 complements in each case (Lewis and Lake, 1995).

Furthermore, pathways of coumarin metabolism differ between the typical experimental rodent species, rat and mouse, compared with the situation in Homo sapiens (Lake, 1996). For example, in the rat the primary route of coumarin clearance is via a 3,4-epoxidation pathway although, because other CYP isoforms such as CYP1A1 and CYP2B1 also catalyze this reaction, it is not clear whether this species selectivity is entirely due to CYP2A participation. Nevertheless, such marked differences between the metabolism of coumarin in an experimental animal species relative to that in man have important implications for the safety evaluation and risk assessment of coumarin and related compounds due to the fact that 3,4-epoxidation constitutes a route of metabolic activation, whereas 7-hydroxylation represents a major detoxification pathway of coumarin metabolism (reviewed in Lake, 1996). The situation in the mouse is somewhat complicated by the altered regiospecificity and substrate selectivity of the two mouse CYP2A isoforms, CYP2A4 and CYP2A5, which also exhibit sexual dimorphism in relation to testosterone 15α-hydroxylase activity (Burkhart et al., 1985, Squires and Negishi, 1986, Squires and Negishi, 1988, Gonzalez et al., 1989, Lindberg et al., 1989, Iwasaki et al., 1993a, Iwasaki et al., 1993b). Apparently, the differences in substrate selectivity and testosterone regiospecificity of these two mouse CYP2A isoforms are largely determined by a single amino acid change (leucine to phenylalanine at position 209) in the enzyme (Lindberg and Negishi, 1989) although mutagenesis at a small number of other sites appears to augment this effect. Table 2 summarizes the reported information on site-specific mutations carried out on CYP2A subfamily proteins and it has been shown, for example, that position 209 corresponds to a putative active site residue in CYP2A orthologues (Lewis and Lake, 1995, Lewis, 1996).

In our previous study, we focused on the use of three-dimensional models of CYP2A1, CYP2A4, CYP2A5 and CYP2A6 to explain the species differences in coumarin and testosterone metabolism in terms of specific amino acid residue contacts within the putative haem environment (Lewis and Lake, 1995). We now present material on substrate metabolism mediated by CYP2A6 which uses putative active site modelling to yield a structural template for CYP2A6-selective oxidations of drugs and other chemicals.