Читать книгу Transporters and Drug-Metabolizing Enzymes in Drug Toxicity - Albert P. Li - Страница 30

The earliest approach for assessing protein covalent binding is using radiolabeled compounds with human liver microsomal or hepatocyte incubations. It is the gold‐standard approach for measuring RM formation and can quantify the extent of RMs covalently binding to proteins. The original experimental protocol for determining covalent binding properties of radioactive drugs was developed by Evans et al. [20]. In their protocol in vitro human liver microsomes are used as the metabolizing enzyme source and incubated with radioactive drug and cofactors for oxidative metabolism. Quantification of covalent binding is measured through counting the radioactivity of the protein pellet.

Although the covalent binding assay is reliable and widely used by pharmaceutical companies, the use of radiolabeled drugs makes running high throughput screening difficult, and it is an expensive option for assessing whether a compound is likely to undergo bioactivation. A more common and economical screening approach is using electrophile trapping experiments that do not require radiolabeled drugs.