Article Figures & Data
Figures
- Fig. 1.
Viability and pMET potency shifts after washout differ between MET inhibitors. EBC-1 cells were treated with selected MET inhibitors for 1 hour, before media were replaced three times to remove the compound from the supernatant, and 72-hour viability was measured. Dose-response curves for tepotinib (A) and capmatinib (B) show activity after washout compared with nonwashed control treatment. (C) Mean IC50 ± S.D. was calculated using Genedata screener. Multiple unpaired Student t tests, corrected for multiple comparisons using the Holm-Šidák method; N = 6; ***P < 0.001; **P < 0.01; *P < 0.05. MET phosphorylation 24 hours after washout for tepotinib (D) and for capmatinib (E) was measured with 0.5 µM tepotinib as inhibitor control and DMSO as vehicle. (F) Differences in mean IC50 ± S.D. of MET inhibitors were analyzed by multiple unpaired Student t tests; N = 3; ***P < 0.001; **P < 0.01; *P < 0.05. MET amp: MET amplification; NSCLC: non–small cell lung cancer; wt: wild-type.
- Fig. 2.
Time-dependent target engagement studied by SPR and NanoBRET. (A) Dissociation kinetics of inhibitor-target complexes observed over time on a Biacore 8K+. Starting point in each case is a saturated MET surface. (B) Recovery of surface activities determined over a period of 3 days on a Biacore 4000. The MET-inhibitor complexes are continuously flooded with inhibitor-free buffer. At indicated timepoints, the recovery of free binding sites was determined with a reference molecule and converted to remaining target occupancies. (C) Cellular target engagement of MET was determined by NanoBRET assay. Cells were treated for 1 hour with the respective MET inhibitors. For washout samples, IC50 determination was performed after 4 hours of incubation following washout. DMSO and tracer were used as 100% occupancy control. Shift factors were calculated using the ratio of washout and control IC50 values. Mean IC50 and S.D. are based on 3–6 independent experiments. BRET, bioluminescence resonance energy transfer; RT, residence time time; SPR: surface plasmon resonance.
- Fig. 3.
Sustained effects after washout correlate with increased compound concentration in cell lysates. (A) Concentrations from EBC-1 cell lysates after 1-hour compound incubation and washing steps normalized to 10,000 cells per well for different treatment concentrations and MET inhibitors; N = 3 with six replicates each [data shown as scatter dot plot with arithmetic means (indicated with -) and S.D. expressed with error bars]. (B) A closeup shows compound partition between cell lysates and incubation media after treatment with 5 µM MET inhibitor. The stacked bars showing the means of recovered compound amounts from cell lysates (bottom) and cell supernatant (top), with S.D. expressed with error bars.
- Fig. 4.
Physicochemical profiles of tepotinib and crizotinib translate into lysosomal compound reservoirs. EBC-1 cells were cotreated with tepotinib and chloroquine for 1 hour, before media were replaced three times to remove the compound from the supernatant, and MET phosphorylation was measured after 24 hours. (A) Activity of cotreatments compared with tepotinib monotreatment. Mean ± S.E.M.; N = 3; two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. LysoTracker staining of A549 cells (green) and nuclear counterstain using DAPI (blue). Imaging was performed using the high-content imager Micro Confocal (Molecular Devices) and a 20x objective. Scale bars, 50 µM. (B) A549 cells treated for 1 hour with 0.1 µM and 10 µM tepotinib and 0.5% DMSO (control). (C) Images quantified counting vesicles using MetaXpress software. Two-point normalization using 0.5% DMSO (100%) and 50 µM chloroquine (0%). (D) Treatment with 12.5 µM tepotinib control and washout. DAPI: 4′,6-diamidino-2-phenylindole; ns, not significant.
- Fig. 5.
Schematic display of kinetics for the intracellular distribution of tepotinib and the charged form of the molecule (tepotinib+) as a lysosomotropic compound. Compound distribution within a cell is a complex, dynamic process influenced by multiple factors. The highlighted aspects are the duration of the drug-target complex and the dissociation rate constant (koff) (A) and compound lysosomal reservoir (B). Additional factors influencing the equilibrium include protein binding and transport across the membrane.
Tables
Compound Structure MW [g/mol] HAc [N] HDon [N] TPSA [Å2] logP pKa1 pKa2 Tepotinib 
492.58 8 0 95 3.99 9.25 — Crizotinib 450.34 6 3 78 3.87 9.21 5.29 Capmatinib 412.43 7 1 85 2.36 4.57 2.18 Savolitinib 345.37 9 0 92 1.28 5.80 — HAc, hydrogen bond acceptors; HDon, hydrogen bond donators; MW, molecular weight; TPSA, topological polar surface area.
Data Supplement
- 590_supp_figs.pdf -
Supplementary Figure S1. EBC-1 cells were treated with selected MET inhibitors for 1 hour, before media were replaced three times to remove the compound from the supernatant, and 72-hour viability was measured.
Supplementary Figure S2. EBC-1 cells were treated with selected MET inhibitors for 1 hour, before media were replaced three times to remove the compound from the supernatant, and 72-hour viability was measured.
Supplementary Figure S3. Cellular target engagement of MET was determined by NanoBRET assay.
Supplementary Figure S4. Cellular target engagement of MET was determined by NanoBRET assay.
Supplementary Figure S5. EBC-1 cells were co-treated with selected MET inhibitors and chloroquine for 1 hour, before media were replaced three times to remove the compound from the supernatant, and MET phosphorylation was measured after 24 hours.
Supplementary Figure S6. LysoTracker staining (green) and nuclear counterstain using DAPI (blue). Imaging was performed using the high content imager ImageXpressULTRA (Molecular Devices) and a 20x objective.
- 590_supp_figs.pdf -
