Open AccessA Versatile Method to Determine the Cellular Bioavailability of Small-Molecule Inhibitors
Author(s) -
Kevin B. Teuscher,
Min Zhang,
Haitao Ji
Publication year - 2017
Publication title -
journal of medicinal chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.01
H-Index - 261
eISSN - 1520-4804
pISSN - 0022-2623
DOI - 10.1021/acs.jmedchem.6b00923
Subject(s) - chemistry , intracellular , bioavailability , bromodomain , extracellular , small molecule , cell , membrane permeability , pharmacology , biochemistry , membrane , epigenetics , biology , gene
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.