A Versatile Method to Determine the Cellular Bioavailability of Small-Molecule Inhibitors

The
determination of the cellular bioavailability of small-molecule inhibitors
is a critical step for interpreting cell-based data and guiding inhibitor
optimization. Herein, a HPLC-MS based protocol was developed to determine
inhibitor cellular bioavailability. This generalizable protocol allows
determination of the accurate intracellular concentrations and characterization
of various properties of inhibitors including the extra- and intracellular
stability, the dose- and time-dependence of the intracellular concentrations,
the cell permeability, and the nonspecific binding with the cell culture
plates, the extracellular matrices, and the cell membrane. The inhibitors
of the protein–protein interactions, bromodomains, and the
β-catenin/B-cell lymphoma 9 (BCL9) interaction were used to
examine the protocol, and the cellular bioavailability of the inhibitors
in cancer cells was determined. High nonspecific binding and low cellular
uptake were observed for two bromodomain inhibitors. The two β-catenin/BCL9
inhibitors had low nonspecific binding but different cellular uptake.
These inhibitors exhibited different stability kinetics in cells.