Differences in Functional Clustering of Endogenous and Exogenous Substrates Between Members of the CYP1A Subfamily

Abstract

The ability of four mammalian cytochromes P450 (CYP) of the CYP1A subfamily, human and mouse CYP1A1s and human and rabbit CYP1A2s, to metabolize a series of steroids and related compounds was investigated us- ing high throughput approaches. Oxidation rates and metabolite patterns for 16 steroid substrates and for 20 polycyclic aromatic hydrocarbon (PAH) substrates were determined in standardized automated conditions. Multivariate statistics of normalized activity data sets was used to sort out significant information and to compare functional signatures of assayed enzymes. Interestingly, for steroid substrates, rabbit CYP1A2 unambiguously aggregates with human and mouse CYP1A1s and appears functionally divergent from human CYP1A2. In contrast, the functional classification was found consistent with the sequence classification when exogenous PAH substrates were tested. The observed features rely on a large set of substrates, all presenting a similar chemical scaffold but decorated with different substituents similar to chemical series used in drug development. Differential functional clusters are thus evidenced for endogenous and exoge- nous substrates with CYP1A enzymes. A few residues on rabbit CYP1A2 that may account for its unusual 1A1-like speci- ficity toward steroids have been identified both within the active site and at the protein surface. These specific residues thus seem to play a controlling role for global substrate class discrimination, potentially by involving substrate bulkiness and shape sensing.