The Substrate Binding Site of Human Liver Cytochrome P450 2C9: An Approach Using Designed Tienilic Acid Derivatives and Molecular Modeling

Abstract

Biochemical experiments, using the well-defined human liver CYP2C9 expressed in yeast, and molecular modeling techniques were used to derive a predictive model for substrates of CYP2C9. The ability of 10 2-aroylthiophenes related to tienilic acid to act as substrates for CYP2C9 was studied. Four of them were original compounds that were synthesized and completely characterized by several spectroscopic techniques. In these 10 compounds various chemical functions, such as ester, amide, alcohol, phenol, ether or tetrazole functions, replaced the OCH2COOH function of tienilic acid. Among them, only the derivatives containing an acidic function (carboxylic acids, phenol, and tetrazole whose pKaS are 4.8, 6.3, and 3.8, respectively) underwent a 5-hydroxylation of their thiophene ring like tienilic acid. Despite their close structural analogy with tienilic acid, all of the other compounds not only did not undergo any 5-hydroxylation of their thiophene ring but also failed to act as inhibitors of CYP2C9. These results strongly suggested that the presence, at pH 7.4, of a negative charge on the substrate is a very important feature in its recognition by CYP2C9. In fact, the four new substrates of CYP2C9 described in this study, a carboxylic acid, phenol, and tetrazole derivative, each of which is related to tienilic acid, and the antiinflammatory drug, suprofen (with Km between 12 and 130 microM and kcat between 0.2 and 1.3 min-1), as well as almost all CYP2C9 substrates reported in the literature, exhibit a pKa below 7 (except phenytoin whose pKa is 8.1). They mainly exist as anions at physiological pH. By using molecular modeling techniques, 12 CYP2C9 substrates were superimposed with respect to their hydroxylation site and fitted onto templates, which were rigid molecules such as (S)-warfarin and phenytoin. It was thus possible to arrange them in order that all their anionic sites were at a distance around 4 A from a common point (a putative cationic site of the protein) in space. These results provide a model of the substrate binding site of CYP2C9, in which substrates interact through their anionic site A- with a cationic residue of the CYP2C9 protein C+. In that model, the distance between the hydroxylation site (Hy) and the anionic site (A-) is 7.8 +/- 1.6 A, and the <HyA-C+ angle is 82 +/- 15 degrees.

Oxidation of Tienilic Acid by Human Yeast‐Expressed Cytochromes P‐450 2C8, 2C9, 2C18 and 2C19

“…In fact, the catalytic efficiency of CYP 2C9 (kcat: K,,, = 0.3) was 33-fold and 300-fold better than those of CYP 2C18 and CYP 2C8, respectively. This great efficiency of CYP 2C9 is due, for the most part, to the nature of its active site, which seems to recognize anionic substrates well, presumably because of the presence of a cationic amino acid residue from the protein located close to the heme (Mancy et al, 1995). Interestingly, tienilic acid is a mechanismbased inhibitor very selective for CYP 2C9, since CYP 2C18 and CYP 2C8, which catalyze the 5-hydroxylation of tienilic acid, are not inactivated at a significant level during that reaction (Figs 2 and 4).…”

A New Class of Cyp2c9 Inhibitors: Probing 2c9 Specificity With High-Affinity Benzbromarone Derivatives

“…As such, many studies have attempted to define the determinants of 2C9-binding molecules with analogs of known binders to examine specific chemical properties like tautomeric structure (He et al, 1999), aromaticity (Rao et al, 2000), pK a values (Mancy et al, 1995), and hydrogen bond donors (Jones et al, 1996a) and acceptors (Ekins et al, 2000). Such studies are still important even with the crystal structure of rabbit 2C5 (83% identity) and probable proprietary human 2C9 structures.…”

Assessment of Arginine 97 and Lysine 72 as Determinants of Substrate Specificity in Cytochrome P450 2c9 (Cyp2c9)

“…CYP2C9, one of the most important forms in overall drug metabolism, is distinguished from other human CYP2C forms by a preference for substrates bearing a negative charge at physiological pH (Smith and Jones, 1992;Mancy et al, 1995); however, neutral or positively charged substrates may also be substrates. Various models have been developed to explain the preference for anionic substrates.…”

“…Mansuy and colleagues originally proposed a pharmacophore model based on examination of tienilic acid derivatives and other CYP2C9 substrates (Mancy et al, 1995). In this model, a positively charged residue bordering the substrate binding site was proposed to interact electrostatically with the negative center on the substrate.…”

“…In humans, approximately 15 enzymes from the CYP1-3 families are responsible for the metabolic clearance of most lipophilic chemicals. Among this group, forms from the same subfamily show discrete but often overlapping substrate specificities.CYP2C9, one of the most important forms in overall drug metabolism, is distinguished from other human CYP2C forms by a preference for substrates bearing a negative charge at physiological pH (Smith and Jones, 1992;Mancy et al, 1995); however, neutral or positively charged substrates may also be substrates. Various models have been developed to explain the preference for anionic substrates.…”