Development of a Mass Spectrometric Assay to Identify Compounds Forming Reactive Metabolites with La Protein in Microsomal Mixtures

摘要

Covalent binding of drug candidates to proteins poses potential liabilities for therapeutic advancement. Information regarding possible toxicity should be assessed for both the parent compound and possible metabolic products prior to in vivo studies. Glutathione is often used as a protein mimic and can serve as a trapping agent due to its ubiquity in the body and natural role in neutralizing free radicals. Bioactivation of compounds in hepatocytes and total covalent protein binding can be measured by radioactivity. In this study, the 12.7 kDa His-tagged construct of La protein was chosen because it contains reactive cysteines, can be easily purified from a microsomal reaction mixture, does not require radioactivity, and has the potential to inform upon both parent and metabolic product reactivity.