P-glycoprotein Substrate Assessment in Drug Discovery: Application of Modeling to Bridge Differential Protein Expression Across In Vitro Tools

P-glycoprotein (P-gp) efflux assay is an integral part of discovery screening, especially for drugs requiring brain penetration as P-gp ratio (ER) inversely correlates with exposure. However, significant variability in ER generated across cell lines can lead to misclassification a substrate and subsequently disconnect exposure data. We hypothesized that the depends on protein expression level vitro assay. Quantitative proteomics immunofluorescence staining were utilized characterize localization four recombinant lines, over-expressing human or mouse isoforms, followed by functional evaluation. Efflux data each line was compared against available rodent distribution The results suggested highest (hMDCK-MDR1 sourced from NIH) led greatest dynamic range efflux; thus, proving be most sensitive model predict penetration. Cell lower exhibited tendency compound-dependent saturation leading false negative results. Ultimately, kinetics characterized using compartmental generate system-independent parameters resolve such discrepancy. This study highlights need careful choice well tools utility modeling techniques enable appropriate interpretation