In MKN45 and EBC1 cells, tivantinib at 1 or 10 μM did not suppress MET, AKT or ERK phosphorylation after 6 h, while 1 μM crizotinib suppressed these signals. These findings limit use of tivantinib as a selective cellular MET probe under the tested conditions. [2]
Enantiomer-controlled binding to unphosphorylated MET
Biochemical assay · Unphosphorylated human MET kinase domain; inactive FGFR2 control [1]
Indirect affinity mass spectrometry detected ARQ 197 bound to unphosphorylated MET, but not FGFR2. The ARQ 198 enantiomer did not bind either protein. This was a relative binding experiment, not a quantitative affinity determination.
Literature-Reported Treatment Conditions
- Treatment concentration
- 20 μM inhibitor; 14 μM protein
- Exposure duration
- 1 h at room temperature
- Method
- Binding followed by separation of protein-bound from free inhibitor and LC/MS
- Vehicle
- 2% final DMSO
- Co-treatment
- Single-agent tivantinib; comparator compounds tested separately.
- Reported limits
- No Kd or binding stoichiometry established; replicate number and uncertainty definition not specified in the selected binding text/caption.
Methods and source locations
Compound reported in the paper: ARQ 197 (tivantinib); Compound label in the paper: ARQ 197
Evidence concerns the named experimental compound; no Adooq supplier use, lot equivalence, salt conversion or independent stereochemical lot validation is established.
Figure 1A–B; Experimental Procedures: Indirect Affinity Mass Spectrometry; Results: ARQ 197 Shows Enantioselective Binding
Protein-bound inhibitor detected by LC/MS: Indirect affinity mass spectrometry with protein-free control. — Figure 1B and corresponding Results paragraph
Time- and ATP-dependent inhibition of MET autophosphorylation
Biochemical assay · Initially unphosphorylated recombinant human MET kinase domain [1]
ARQ 197 delayed and inhibited MET autophosphorylation, with potency changing as the reaction proceeded and ATP concentration increased. ARQ 198 was a weak/inactive comparator under these conditions.
Literature-Reported Treatment Conditions
- Treatment concentration
- ARQ 197 concentration titration shown in Figure 1C–F; 1 μM MET; 0.1 mM ATP for the early IC50
- Exposure duration
- 30 min compound/enzyme preincubation at 4°C; assay at 30°C; early rate window and later 1 h reaction interval
- Method
- ATP consumption coupled to NADH oxidation; no-inhibitor progress curves and ARQ 198 comparator
- Vehicle
- 2% DMSO
- Co-treatment
- Single-agent tivantinib; comparator compounds tested separately.
- Reported limits
- The ± statistic and number of independent determinations for 548 ± 120 nM are not defined in the selected text/caption.
Methods and source locations
Compound reported in the paper: ARQ 197 (tivantinib); Compound label in the paper: ARQ 197
Evidence concerns the named experimental compound; no Adooq supplier use, lot equivalence, salt conversion or independent stereochemical lot validation is established.
Figure 1C–F and caption; Experimental Procedures: c-Met Autophosphorylation Assay; Results: ARQ 197 Inhibits the Catalytic Activity of c-Met
Initial-rate MET autophosphorylation inhibition at 0.1 mM ATP: Continuous coupled spectrophotometric assay, absorbance at 340 nm; initial rate after lag phase. — Results: ARQ 197 Inhibits the Catalytic Activity of c-Met, first paragraph; Figure 1C–D
MET autophosphorylation inhibition after reaction continued for 1 h: Same autophosphorylation assay; later reaction interval. — Same Results paragraph; Figure 1F
Early MET autophosphorylation inhibition at 0.5 mM ATP: Early rate within 20 min following lag phase; comparison with 0.1 mM ATP. — Results: ARQ 197 Inhibits the Catalytic Activity of c-Met, second paragraph; Figure 1E–F
Six-hour cellular MET-pathway comparison
Cell-based study · MKN45 gastric cancer, EBC1 lung cancer and A549 lung cancer cells [2]
At 6 h, tivantinib at 1 or 10 μM did not suppress MET, AKT or ERK phosphorylation in MKN45/EBC1 cells; 1 μM crizotinib suppressed these signals. A549 had no detectable phospho-MET under these conditions. These results constrain extrapolation from isolated MET assays to cellular pathway inhibition.
Literature-Reported Treatment Conditions
- Treatment concentration
- Tivantinib 1 and 10 μM; separate crizotinib comparator 1 μM
- Exposure duration
- 6 h
- Method
- Immunoblotting for phosphorylation, total proteins and β-actin
- Vehicle
- Compounds prepared in DMSO; final DMSO fraction for Figure 3A not specified.
- Co-treatment
- Single-agent tivantinib; comparator compounds tested separately.
- Reported limits
- Repeated experiments are described as similar; independent replicate number and densitometric effect size are not given in Figure 3A caption.
Methods and source locations
Compound reported in the paper: ARQ 197 (tivantinib); Compound label in the paper: Tivantinib (ARQ 197)
Evidence concerns the named experimental compound; no Adooq supplier use, lot equivalence, salt conversion or independent stereochemical lot validation is established.
Figure 3A and caption; Results: Tivantinib does not suppress c-MET phosphorylation and downstream signaling; Methods: Cell lines and reagents, Immunoblot analysis
MET Tyr1234/1235, AKT Ser473 and ERK Thr202/Tyr204 phosphorylation: Immunoblots with total MET/AKT/ERK and β-actin controls; no-drug lanes. — Figure 3A image/caption and corresponding Results paragraph
Cell-free tubulin polymerization comparison
Biochemical assay · Purified porcine brain tubulin polymerization assay [2]
Tivantinib reduced the polymerization-associated fluorescence increase relative to the no-drug control. Crizotinib and PHA-665752 did not produce a comparable inhibition; vincristine inhibited and paclitaxel enhanced polymerization.
Literature-Reported Treatment Conditions
- Treatment concentration
- Tivantinib 10 μM in Figure 5B
- Exposure duration
- 60 min plotted time course; fluorescence recorded every minute
- Method
- Cell-free tubulin polymerization assay with no-drug, inhibiting and enhancing controls
- Vehicle
- Compounds dissolved in DMSO stocks; final assay DMSO concentration not specified.
- Co-treatment
- Single-agent tivantinib; comparator compounds tested separately.
- Reported limits
- Caption reports triplicate, without defining independent versus technical replication. No IC50 or binding affinity is reported for the adopted comparison.
Methods and source locations
Compound reported in the paper: ARQ 197 (tivantinib); Compound label in the paper: Tivantinib (ARQ 197)
Evidence concerns the named experimental compound; no Adooq supplier use, lot equivalence, salt conversion or independent stereochemical lot validation is established.
Figure 5B and caption; Methods: Microtubule polymerization assay; Results: Tivantinib disrupts microtubule by inhibiting tubulin polymerization
Tubulin polymerization-associated fluorescence over time: Fluorescence assay at 30°C, excitation 355 nm/emission 460 nm; plotted triplicates summarized as average ± SEM. — Figure 5B image, legend and Methods: Microtubule polymerization assay
