The TR-FRET-based
LANCE Ultra assay (Perkin Elmer) was used to determine
IC50 values for various CSF1R inhibitors. Kinase activity
and inhibition in this assay were measured as recommended by the manufacturer.
Briefly, a specific Ultra ULight GT peptide substrate (50 nM final
concentration) was allowed to get phosphorylated by CSF1R (0.5 nM
final concentration) in enzymatic buffer (50 mM HEPES pH 7.5, 10 mM
MgCl2, 1 mM EGTA, 0.01% Tween 20, 2 mM dithiothreitol,
1% DMSO) containing ATP at the concentration of the Km value (25 μM) of the kinase for 1 h at room temperature.
All compounds were tested in an 8-point dose–response curve
up to a final concentration of 10 μM. The compound transfer
was facilitated via acoustic dispensing with the Echo 520 (Beckman
Labcyte) using the Echo Dose–Response software package. Subsequently,
phosphorylation or inhibition was detected by the addition of specific
europium (Eu)-labeled anti-phospho antibodies (2 nM), which upon binding
to the phospho-peptide gives rise to a FRET signal. The FRET signal
was recorded in a time-resolved manner in a Perkin Elmer EnVision
reader. All assays were performed in a final volume of 20 μL
in low-volume white 384-well plates from Corning (4513). All assay
data were analyzed with the Quattro Workflow software package from
Quattro Research.
LANCE Ultra assay (Perkin Elmer) was used to determine
IC50 values for various CSF1R inhibitors. Kinase activity
and inhibition in this assay were measured as recommended by the manufacturer.
Briefly, a specific Ultra ULight GT peptide substrate (50 nM final
concentration) was allowed to get phosphorylated by CSF1R (0.5 nM
final concentration) in enzymatic buffer (50 mM HEPES pH 7.5, 10 mM
MgCl2, 1 mM EGTA, 0.01% Tween 20, 2 mM dithiothreitol,
1% DMSO) containing ATP at the concentration of the Km value (25 μM) of the kinase for 1 h at room temperature.
All compounds were tested in an 8-point dose–response curve
up to a final concentration of 10 μM. The compound transfer
was facilitated via acoustic dispensing with the Echo 520 (Beckman
Labcyte) using the Echo Dose–Response software package. Subsequently,
phosphorylation or inhibition was detected by the addition of specific
europium (Eu)-labeled anti-phospho antibodies (2 nM), which upon binding
to the phospho-peptide gives rise to a FRET signal. The FRET signal
was recorded in a time-resolved manner in a Perkin Elmer EnVision
reader. All assays were performed in a final volume of 20 μL
in low-volume white 384-well plates from Corning (4513). All assay
data were analyzed with the Quattro Workflow software package from
Quattro Research.