NVP-BVU972 1185763-69-2

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Molecular Weight:

340.38 NVP-BVU972 is a selective and potent Met inhibitor with IC50 of 14 nM.


Biological Activity


NVP-BVU972 potently inhibits MET kinase but displays low inhibition
against other kinases including the most closely related kinase RON with
IC50 values of more than 1000 nM. NVP-BVU972 also suppresses
constitutive MET phosphorylation in GTL-16 cells or HGF-stimulated MET
phosphorylation in A549 cells with IC50 values of 7.3 nM and 22 nM,
respectively. NVP-BVU972 potently prevents the growth of the MET gene
amplified cell lines GTL-16, MKN-45 and EBC-1 with IC50 values of 66 nM,
82 nM and 32 nM, respectively.


In line with their high frequency in the
NVP-BVU972 screen, Y1230 and D1228 mutations give rise to dramatic
shifts in the measured IC50 values for NVP-BVU972 in BaF3 cell line.
Resistance triggered by V1155L is more limited to NVP-BVU972. A
dose-dependent reduction in TPR-MET phosphorylation when applying
NVP-BVU972 to BaF3 cells expressing wild-type TPR-MET. Both Y1230H and
D1228A mutations abrogated the effect of NVP-BVU972 but not AMG 458.
However, F1200I and L1195V interferes with the potency of NVP-BVU972 to
prevent TPR-MET phosphorylation.






TR-FRET biochemical assay with MET wild type and mutants


Enzyme activity is measured in a time resolved fluorescence resonance
energy transfer (TR-FRET) assay, detecting tyrosine phosphorylation with
a Eu-labelled anti-phospho-tyrosine antibody (fluorescence donor) and
Allophycocyanin conjugated to Streptavidin (fluorescence acceptor) which
binds to a biotin on the substrate peptide. For each variant, Km concentrations for ATP are determined in the absence of NVP-BVU972, and
the ATP concentration in the kinase reaction is set to Km (4
μM for MET wt, 1 μM for MET Y1230H and MET F1200I).


NVP-BVU972 is
dissolved and diluted in DMSO and assayed in quadruplicate. Kinase
reactions are carried out in 50 mM Tris-HCl pH 7.5, 8 mM MgCl2, 4 mM MnCl2, 0.05 % Tween 20, 0.05% bovine serum albumin, 0.1 mM EDTA, 1 mM DTT, 0.1 mM Na3VO4,
in white 1536 well plates at room temperature. NVP-BVU972 and enzyme
are incubated in a volume of 2 μL for 20 min, followed by the addition
of 1 μL ATP and 1 μL biotinylated peptide substrate (PTK1) to final
concentrations of Km and 1 μM, respectively.


Enzyme
concentrations in the reactions are 5 nM for MET wt, and 4 nM for the
F1200I and Y1230H variants. After 90 min, reactions are stopped by
addition of 1 μL stop/detection solution to reach final concentrations
of 10 mM EDTA, 3.5 nM Eu-labelled antiphospho-tyrosine antibody PY20,
and 10 nM Streptavidin Allophycocyanin. Time resolved fluorescence
resonance energy transfer is measured in an Envision plate reader
(excitation 320 nm, emission 615 nm and 665 nm).






Method


BaF3 cells containing TPR-MET or various mutants thereof are grown in
RPMI 1640 medium containing 10% fetal calf serum. For maintenance of
parental BaF3 cells the medium is additionally supplemented with 10
ng/mL interleukin-3 (IL-3). For proliferation assays, BaF3 cells are
seeded on 96-well-plates in triplicates at 104 cells per well
and incubated with various concentrations of NVP-BVU972 for 72 hours
followed by quantification of viable cells using a resazurin sodium salt
dye reduction readout. IC50 values are determined with the XLFit Excel
Add-In using a 4-parameter dose response model.

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