Abstract 1019: The MET inhibitor tepotinib shows sustained effects on MET phosphorylation and viability in cellular washout experiments
Authors: Berges Nina , Klug Jan Henrik , Eicher Anna , Loehr Jennifer , Schwarz Daniel , Bomke Joerg , Leuthner Birgitta , Perrin Dominique , Schadt Oliver 
Published: July 2021

Abstract
It is well accepted that a drug's efficacy is modulated by the affinity of the drug to the target of interest and the stability of the protein/inhibitor complex. Additionally, drug efficacy is affected by pharmacokinetic properties. For example, lipophilic weak bases are able to easily permeate membranes at neutral pH in a non-protonated form, but also tend to accumulate in acidic compartments such as lysosomes. Consequently, lysosomes can act as a drug reservoir. The degree of lysosomal sequestration generally correlates with the pKa value of the basic function, and although the process is considered to be fully reversible, the degree and accessibility of the lysosomal reservoir heavily depends on compound-specific physicochemical properties. MET inhibitors such as crizotinib, capmatinib, savolitinib and tepotinib are providing the opportunity to study how differences in the physicochemical profiles of MET inhibitors affect the interplay of drug-target residence time and lysosomal storage, and how these factors translate into target inhibition and cell viability effects under washout conditions. Human lung cancer cell lines were treated with MET inhibitors across a range of concentrations for 1 h, cells were washed to remove compounds in the supernatant, and remaining compound activity was analyzed by measuring MET phosphorylation and lung cancer cell viability. Tepotinib showed persistent effects in both assays with significant differences observed between the tested MET inhibitors. In the viability assay comparing standard and washout conditions, the half-maximal inhibitory concentrations of tepotinib and crizotinib increased by a factor ranging from 10 to 20. In contrast, those of capmatinib and savolitinib increased by a factor &gt;250, indicating a more sustained efficacy for tepotinib and crizotinib under washout conditions. Here, we present data on the underlying mechanisms affecting cellular efficacy under washout conditions, including residence time via surface plasmon resonance analysis, compound-partition coefficients via liquid chromatography with tandem mass spectrometry quantification, and lysosomal storage via a competitive dye displacement assay. This comparative analysis provides strong evidence that tepotinib demonstrates persistent efficacy in dynamic cellular systems, exceeding that of several other MET inhibitors. The underlying reason for this observation is probably the consequence of a long residence time of tepotinib (~20 h) combined with an optimal physicochemical profile allowing for a beneficial contribution of lysosomal retention. These in vitro findings may also explain the sustained pharmacokinetic and pharmacodynamic in vivo characteristics of tepotinib.
Citation Format: Nina Berges, Jan Henrik Klug, Anna Eicher, Jennifer Loehr, Daniel Schwarz, Joerg Bomke, Birgitta Leuthner, Dominique Perrin, Oliver Schadt. The MET inhibitor tepotinib shows sustained effects on MET phosphorylation and viability in cellular washout experiments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1019.