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.

Figure 1.

Workflow for determination of inhibitor cellular bioavailability.

Figure 2.

(A–C) The workflow for determination of inhibitor intracellular concentration and the workflows for two control experiments that evaluate the efficiency of solvent extraction and compound nonspecific binding. (D) Common solvents and additives used to extract small-molecule inhibitors from the studied cells.

Figure 3.

Compounds 1–4. For 1, the K_D values for BRD9 and BRD7 were determined by isothermal titration calorimetry (ITC) studies and reported by the Structural Genomics Consortium (SGC, www.thesgc.org/). The IC₅₀ values of 2 for BRD1 and TAF1 were determined by BROMOscan and also reported by the SGC. The biochemical K_i values of 3 and 4 for the β-catenin/BCL9 interaction were determined by the AlphaScreen assay.^, The cell-based IC₅₀ values were determined using the MTs tetrazolium assay to monitor the inhibitory effects on growth of triple negative breast cancer MDA-MB-231 cells. Each set of data is expressed as mean ± standard deviation (SD) (n = 3).

Figure 4.

Stability of 1–4 in DMEM media over a period of 72 h with or without 10% FBS. The HPLC chromatograms at the starting time point in 5 mL of DMEM media with 10% FBS are shown in Supporting Information, Figure S1. Each set of data is expressed as mean ± standard deviation (SD) (n = 3).

Figure 5.

HPLC/DAD chromatograms of 1 (A) and 2 (B) and HPLC/VWD chromatograms of 3 (C) and 4 (D) in MDA-MB-231 cells under various control conditions. The retention time for 1–4 is 16.4, 15.9, 15.8, and 19.0 min, respectively. The MS data for pure and intracellular 1–4 are shown in Supporting Information, Figures S3-S6.

Figure 6.

Dose dependence of the cellular uptake of 1–4. Compounds 1 and 2 was incubated in DMEM media containing 10% FBS for 6 h. Compounds 3 and 4 were incubated in DMEM media containing 5% FBS for 6 h. Each set of data is expressed as mean ± SD (n = 3).

Figure 7.

Time-dependent stability of 1–4 in MDA-MB-231 cells. The input concentrations were 10, 20, 2, and 20 μM for 1–4, respectively. Each set of data is expressed as mean ± SD (n = 3).

Figure 8.

Cellular bioavailability data for 1–4.