Bioactivation of α,β-Unsaturated Carboxylic Acids Through Acyl Glucuronidation.

Following oral administration of [14C]GDC-0810, an α,β-unsaturated carboxylic acid, to monkeys, unchanged parent and its acyl glucuronide metabolite, M6, were the major circulating drug-related components. In addition, greater than 50% of circulating radioactivity in plasma was found to be non-extractable 12 hours post-dose, suggesting possible covalent binding to plasma proteins. In the same study, one of the minor metabolites was a cysteine conjugate of M6 (M11) that was detected in plasma and excreta (urine and bile). The potential mechanism for the covalent binding was further investigated using in vitro methods. In incubations with GSH or cysteine (5mM), GSH- and cysteine-conjugates of M6 were identified. The cysteine reaction was efficient with a half-life of 58.6 min (kreact = 0.04 1/M/sec). Loss of 176 Da (glucuronic acid) followed by 129 Da (glutamate) in mass fragmentation analysis of the GSH-adduct of M6 (M13) suggested that glucuronic acid was not modified. The conjugation of N-glucuronide M4 with cysteine in buffer was >1000-fold slower than with M6. Incubations of GDC-0810, M4, or M6 with monkey or human liver microsomes in the presence of NADPH and GSH did not produce any oxidative GSH adducts, and the respective substrates were qualitatively recovered. In silico analysis quantified the inherent reactivity differences between the glucuronide and its acid precursor. Collectively, these results show that acyl-glucuronidation of α,β-unsaturated carboxylic acids can activate the compound towards reactivity with GSH, cysteine or thiol in proteins, and should be considered during the course of drug discovery. Significance Statement Acyl glucuronidation of the α,β-unsaturated carboxylic acid in GDC-0810 activates the conjugated alkene towards nucleophilic addition by GSH or other reactive thiols. This is the first example that a bioactivation mechanism could lead to protein covalent binding to α,β-unsaturated carboxylic acid compounds.