Comparison of the Substrate Specificities of Human Liver Cytochrome P450s 2C9 and 2C18: Application to the Design of a Specific Substrate of CYP 2C18 Claire Minoletti, Sylvie Dijols, Patrick M. Dansette, and Daniel Mansuy* Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, URA400 CNRS, UniVersite´ Paris V, 45 Rue des Saints-Pe` res, 75270 Paris Cedex 06, France ReceiVed February 10, 1999; ReVised Manuscript ReceiVed April 15, 1999 ABSTRACT: A series of 2-aroylthiophenes derived from tienilic acid by replacement of its OCH 2 COOH substituent with groups bearing various functions have been synthesized and studied as possible substrates of recombinant human liver cytochrome P450s 2C9 and 2C18 expressed in yeast. Whereas only compounds bearing a negative charge acted as substrates of CYP 2C9 and were hydroxylated at position 5 of their thiophene ring at a significant rate, many neutral 2-aroylthiophenes were 5-hydroxylated by CYP 2C18 with k cat values of >2 min -1 . Among the various compounds that were studied, those bearing an alcohol function were the best CYP 2C18 substrates. One of them, compound 3, which bears a terminal O(CH 2 ) 3 - OH function, appeared to be a particularly good substrate of CYP 2C18. It was regioselectively hydroxylated by CYP 2C18 at position 5 of its thiophene ring with a K M value of 9 ( 1 μM and a k cat value of 125 ( 25 min -1 , which are the highest described so far for a CYP 2C. A comparison of the oxidations of 3, by yeast-expressed CYP 1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5, showed that only CYP 2C8, 2C18, and 2C19 were able to catalyze the 5-hydroxylation of 3. However, the catalytic efficiency of CYP 2C18 for that reaction was considerably higher (k cat /K M value being 3-4 orders of magnitude larger than those found for CYP 2C8 and 2C19). Several human P450s exhibited small activities for the oxidative O-dealkylation of 3. The four recombinant CYP 2Cs were the best catalysts for that reaction (k cat between 1 and 5 min -1 ) when compared to all the P450s that were tested, even though it is a minor reaction in the case of CYP 2C18. All these results show that compound 3 is a new, selective, and highly efficient substrate for CYP 2C18 that should be useful for the study of this P450 in various organs and tissues. They also suggest some key differences between the active sites of CYP 2C9 and CYP 2C18 for substrate recognition. Cytochrome P450s play a key role in the metabolism of exogenous compounds such as drugs. Their broad substrate specificity is now well-understood in terms of enzyme specificity (1). Cytochrome P450s of the 3A and 2C subfamilies are the major isozymes in human liver (2, 3). To predict which P450 will be involved in the metabolism of a new biologically active molecule and to understand the structural basis that governs the specificity of a given P450 for a particular drug, it is important to determine the structure of the substrate binding sites of the main human liver P450s. In the absence of crystal structures for such membrane-bound mammalian P450s, the use of a series of indirect methods, combining biochemical results, spectroscopic studies, and theoretical models, is required. This has been successfully achieved for CYP 2D6 (see, for instance, ref 4 and references therein). In the case of CYP 2C9, a model for the interaction of this cytochrome with its substrates has been proposed on the basis of biochemical and UV-visible and 1 H NMR (5) results, as well as molecular modeling (5, 6). In that model, CYP 2C9 substrates, which most often are anionic at pH 7.4, would interact through their anionic site with a cationic residue of the protein (5-7). This model takes into account the major interactions that seem to explain the highly specific inhibitory effects of sulfaphenazole toward CYP 2C9 (7). Other models derived from molecular modeling have pro- posed similar “pharmacophores” for CYP 2C9 substrates; however, these models involve a hydrogen bond between a polar residue of substrates with a CYP 2C9 amino acid instead of an ionic interaction (8-10). Genetic analysis of the CYP 2C subfamily shows the presence of at least seven genes with several allelic variants. CYP 2C8, 2C9, and 2C19 are the isozymes of the 2C subfamily which are expressed at the highest level in human liver (2, 3, 11, 12). The fourth member of the CYP 2C subfamily, CYP 2C18, appears to be expressed at a very low level in human liver (13, 14). However, CYP 2C18 seems to be a major P450 2C in the skin and in the lung as judged from its mRNA levels (15, 16). Whereas specific substrates and inhibitors are known for CYP 2C8, 2C9, and 2C19, and the nature of substrate active sites of CYP 2C9 and 2C19 begins to be understood thanks to the aforementioned studies (5-10) and to recent results using CYP 2C9-2C19 chimeric proteins and mutants (17, 18), almost nothing is known about the substrate active site of CYP 2C18. Moreover, to date, no reaction has been shown to be specifically catalyzed by this enzyme (2). This paper reports the results of a comparative study of the substrate specificities of recombinant CYP 2C9 and CYP * To whom correspondence should be addressed. Fax: 33-1-42 86 83 87. E-mail: dmjccjcd@bisance.citi2.fr. 7828 Biochemistry 1999, 38, 7828-7836 10.1021/bi9903289 CCC: $18.00 © 1999 American Chemical Society Published on Web 05/26/1999