Development of a Column-Based High-Throughput Assay for Assessment of Small Molecules Binding to alpha-1-Acid Glycoprotein Using LC/MS

摘要

Protein binding is a very important determinant of drug disposition and action. Albumin is the most abundant component of plasma, while alpha-1-acid glycoprotein (AGP) is is known to be the main carrier of basically charged lipophilic compounds. AGP binding can become even more important under disease conditions, as AGP levels can elevate significantly in response to infection or inflammatory events. For these reasons, it is increasingly important to characterize AGP-drug binding in the drug discovery and development process. The most commonly used method for measuring AGP binding is equilibrium dialysis and ultracentrifugation using purified AGP. However, these traditional methodologies are time consuming and labor intensive. To better support drug discovery, we developed a column-based assay to characterize drug-AGP binding. The heart of this assay is an HPLC column with an AGP stationary phase. In this format, multiple compounds can be loaded onto AGP-coated HPLC column. Compounds are then separated by the strength of their interaction with the stationary phase bound AGP molecules, and monitored by tandem mass spectrometry. We characterized >20 commercial compounds and observed correlation between the logarithm of binding constant (published value) and AGP column retention time. We also characterized several Abbott compounds using both AGP column and equilibrium dialysis. Results showed that retention time is a good indicator for AGP binding. The capacity of this assay is up to 50 compounds per LC run (60min), which is suitable for drug discovery support.