title: A Versatile Method to Determine the Cellular Bioavailability of Small-Molecule Inhibitors.
abstract: 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.
authors.affiliation: Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute , 12902 Magnolia Drive, Tampa, Florida 33612-9416, United States.|Department of Chemistry, University of Utah , Salt Lake City, Utah 84112, United States.
authors.forename: Kevin B
authors.initials: KB
authors.lastname: Teuscher
authors.affiliation: Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute , 12902 Magnolia Drive, Tampa, Florida 33612-9416, United States.
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute , 12902 Magnolia Drive, Tampa, Florida 33612-9416, United States.|Department of Oncologic Sciences, University of South Florida College of Medicine , Tampa, Florida 33612, United States.|Department of Chemistry, University of Utah , Salt Lake City, Utah 84112, United States.
authors.forename: Haitao
authors.identifier: 0000-0001-5526-4503
authors.initials: H
authors.lastname: Ji
mesh_terms: D001682:Biological Availability; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D013058:Mass Spectrometry; D054852:Small Molecule Libraries
chemical_list: D054852:Small Molecule Libraries