hPGDS-IN-1 is a hPGDS inhibitor.

HPGDS:12 nM

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).