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

Abstract

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. Postwashout 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 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 statementGenerally, 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. 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.

Effective treatment of MET exon 14 skipping mutation-positive non-small cell lung cancer using capmatinib following serious maculopapular rash caused by two MET inhibitors: a case report

“…In contrast, capmatinib was applied only after pembrolizumab treatment, a 25% dose reduction, and an increased dose of dexamethasone may have avoided serious TRAEs. Recent studies have reported alterations in metabolic pathways induced by CYP3A ( 9 ) and the ability of capmatinib and tepotinib to access lysosomes ( 10 ), but none of these features for the MET-inhibitory investigational drug; these differences may also have influenced TRAEs. In addition, the tumor progressed after the first-line MET inhibitor treatment but responded to capmatinib.…”

The Preclinical Pharmacology of Tepotinib—A Highly Selective MET Inhibitor with Activity in Tumors Harboring MET Alterations

“…The 45-minute incubation with tepotinib was sufficient to completely inhibit HGF-induced MET phosphorylation 14 hours after withdrawal of tepotinib, with an IC 50 value of 5.3 nmol/L ( 30, 33 ), demonstrating rapid uptake and cellular retention of tepotinib, with long-lasting MET inhibition. Persistence of tepotinib inhibition under washout conditions may be explained by the long residence time of tepotinib and its retention in the lysosomal compartment to provide a local drug reservoir ( 41 ). Furthermore, 10% (v/v) serum only moderately affected tepotinib inhibition of HGF-induced MET phosphorylation in A549 cells (average IC 50 values of 21 and 23 nmol/L with murine and human serum, respectively; ref.…”