PT - JOURNAL ARTICLE AU - Nina Berges AU - Jan Henrik Klug AU - Anna Eicher AU - Jennifer Loehr AU - Daniel Schwarz AU - Joerg Bomke AU - Birgitta Leuthner AU - Dominique Perrin AU - Oliver Schadt TI - Differences in sustained cellular effects of MET inhibitors are driven by prolonged target engagement and lysosomal retention AID - 10.1124/molpharm.122.000590 DP - 2022 Jan 01 TA - Molecular Pharmacology PG - MOLPHARM-AR-2022-000590 4099 - http://molpharm.aspetjournals.org/content/early/2022/11/18/molpharm.122.000590.short 4100 - http://molpharm.aspetjournals.org/content/early/2022/11/18/molpharm.122.000590.full AB - Intracellular distribution of drug compounds is dependent on physicochemical characteristics and may have a significant bearing on the extent of target occupancy and, ultimately, drug efficacy. We assessed differences in the physicochemical profiles of MET inhibitors capmatinib, crizotinib, savolitinib, and tepotinib and their effects on cell viability and MET phosphorylation under steady-state and washout conditions (to mimic an open organic system) in a human lung cancer cell line. To examine the differences of the underlying molecular mechanisms at the receptor level, we investigated the residence time at the kinase domain and the cellular target engagement. We found that the ranking of the drugs for cell viability was different under steady-state and washout conditions, and that under washout conditions, tepotinib displayed the most potent inhibition of phosphorylated MET. Post-washout effects were correlated with the partitioning of the drug into acidic subcellular compartments such as lysosomes, and the tested MET inhibitors were grouped according to their ability to access lysosomes (crizotinib and tepotinib) or not (capmatinib and savolitinib). Reversible lysosomal retention may represent a valuable intracellular storage mechanism for MET inhibitors, enabling prolonged receptor occupancy in dynamic, open physiological systems and may act as a local drug reservoir. The use of washout conditions to simulate open systems and investigate intracellular drug distribution is a useful characterization step that deserves further investigation. Significance Statement Generally, determination of potency and receptor occupancy is performed under steady-state conditions. In vivo conditions are more complex due to concentration differences between compartments and equilibrium processes. Experiments under steady-state cannot explore effects like sustained target inhibition. In our study, we have shown that differences between MET inhibitors are observable by applying washout conditions to in vitro assays. This important finding applies to most compound classes and may inspire readers to re-think their assay designs in the future.