Development and Validation of Semiautomated 96-Well Transport Assay Using LLC-PK1 Cells Transfected with Human P-Glycoprotein for High-Throughput Screening

Abstract

Transport assays using P-gp-expressing cell lines are commonly used to identify P-gp substrates and inhibitors in drug discovery. The P-gp cell-based assay is performed manually in 12- or 24-well plates and requires improvement for high-throughput screening. In this study, we established an efficient semiautomated 96-well transport assay using LLC-PK1 cells transfected with human P-gp. The protocol was optimized with a microplate washer for exchanging media and buffer to enhance throughput. P-gp substrates and inhibitors, and the paracellular marker Dextran Texas Red® were used to validate the 96-well transport assay. Cell monolayer integrity after washing by a microplate washer was confirmed by measuring paracellular permeability of Dextran Texas Red. Permeability and net flux ratio of the P-gp substrates and the inhibitory potency of the P-gp inhibitors were comparable in 24- and 96-well plates. The regression value of net flux ratio of P-gp substrates was high between the two formats (r²=0.99). The optimized 96-well transport assay using the microplate washer was found to be an efficient high-throughput screening tool that provided the same quality data as the 24-well plate for the identification of P-gp substrates and inhibitors in drug discovery.

Highly Conformationally Restricted Cyclopropane Tethers with Three‐Dimensional Structural Diversity Drastically Enhance the Cell Permeability of Cyclic Peptides

“…The results of a cell‐membrane permeability assay using pig kidney epithelial cells (LLC‐PK1 cells) for cyclic peptides are summarized in Figure . The control peptide P1 was almost impermeable (permeation coefficient (PE)=0.09), and peptides P2 and P3 containing turn‐inducing amino acids, l ‐ or d ‐proline, respectively, also had very low permeability.…”