Differences in Sustained Cellular Effects of MET inhibitors Are Driven by Prolonged Target Engagement and Lysosomal Retention

Intracellular distribution of drug compounds is dependent on phys-icochemical 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 inhibi-tors capmatinib, crizotinib, savolitinib, and tepotinib and their ef-fects on cell viability and MET phosphorylation under steady-state and washout conditions (to mimic an open organic system) in a hu-man lung cancer cell line. To examine the differences of the under-lying molecular mechanisms at the receptor level, we investigated the residence time at the kinase domain and the cellular target en-gagement. 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. Postwashout effects were corre-lated with the partitioning of the drug into acidic subcellular com-partments 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 mecha-nism for MET inhibitors, enabling prolonged receptor occupancy in dynamic, open-physiologic 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 such as sustained target inhibition. This study has 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 rethink their assay designs in the future.