Mechanistic Studies of the Cationic Binding Pocket of CYP2C9 In Vitro and In Silico: Metabolism of Nonionizable Analogs of Tienilic Acid

Abstract

Tienilic acid (TA) is selectively oxidized at the C-5 position of the thiophene ring by the human liver enzyme cytochrome P450 2C9 (CYP2C9). This oxidation is mediated by the proximal positioning of the thiophene over the heme iron, which is proposed to be coordinated by an interaction of the TA carboxylic acid to a cationic binding pocket in the enzyme active site. In this study, we investigated how chemical modification of TA influences the bioactivation by CYP2C9. For this investigation, nine analogs of TA were chosen with substitutions on either side of the molecule. We tested three parameters, including CYP2C9 inhibition, metabolic profiling, and in silico docking. Of the 10 compounds tested, only two (TA and a noncarboxyl analog) resulted in competitive and time-dependent inhibition of CYP2C9. Metabolic profiling revealed a trend in which substitution of the carboxylate with nonionizable functional groups resulted in metabolic switching from oxidation of the aromatic ring to dealkylation reactions at the opposite side of the structure. The in silico modeling predicted an opposite binding orientation to that of TA for many analogs, including the 3-thenoyl regio-isomer analog, which contradicts previous models. Together these data show that disrupting interactions with the cationic binding pocket of CYP2C9 will impact the sites of metabolism and inhibition of the enzyme.

Transitioning from Idiopathic to Explainable Autoimmune Hepatitis

“…Furthermore, druginduced immune-mediated liver injury has been associated with genetic predispositions [188][189][190][191][192][193][194] and molecular mimicries [27,185,195] that are reminiscent of the pathogenic mechanisms associated with idiopathic autoimmune hepatitis. Drug-metabolizing enzymes of the P450 cytochrome family have been the principal autoantigens implicated in type 2 autoimmune hepatitis (CYP2D6) [119], autoimmune-like hepatitis associated with tienilic acid (CYP2C9) [196,197], and autoimmune polyglandular syndrome type 1 which includes autoimmune hepatitis (CYP1A2, CYP2A6) [142,198,199]. Reactive drug metabolites that bind to proteins on the hepatocyte surface or to the enzymes that produce them can serve as haptens that generate antigenic complexes [195,[200][201][202].…”

Mechanistic Approaches for Modifying the Coordination Sphere of Metal–Amyloid-β Complexes: Covalent Adduct Formation and Oxidation

“…In addition, TM bound to the Cu(II) center in the transient ternary complex could be first attacked by a nucleophilic amino acid residue in Aβ (e.g., His; vide infra) producing a covalent bond between Aβ and the 2-methylthiophene moiety with the dissociation of Cu(II) from Cu(II)-Aβ. A second nucleophilic attack on thiophene, after possible epoxidation of thiophene in the Aβ covalently bound to 2-methylthiophene, 37,38 could afford a covalent adduct with Aβ as the final product with the addition of 94 Da to native Aβ [Scheme 1 (i)]. 39 To support whether the fragmentation of L1 to TM is necessary for its covalent linkage Ag/AgCl in H2O).…”