| Assay Method Information | |
| | Inhibition of c-Kit, PDGFRα, PDGFRβ, CSFIR and FLT3 Kinase Activity Assay |
| Description: | On the day of the assay, compounds were solubilized in DMSO and dispensed into a 384-well white Opti-plate (PerkinElmer, catalog #6007290) to generate a 22 point 1:2 titration. Enzyme solution was prepared for each of the tyrosine kinases in 50 mM HEPES, pH 7.4, 5 mM MgCl2 for c-Kit or 10 mM MgCl2 for FLT3, CSFIR, PDGFRa and PDGFRβ, 2 mM MnCl2, 0.01% Brij-35 and 0.01% BSA. Working enzyme concentrations were prepared for c-Kit at 2 nM (2×), PDGFRa and PDGFRß at 10 nM (2×), CSFIR at 2 nM (2×) and FLT3 at 0.4 nM (2×). Five microliters of enzyme dilution of each tyrosine kinase were added to their respective 384-well white Opti-plate pre-dispensed with compound and allowed to incubate for 1 h at rt. Utilizing the same enzyme buffer recipe, 2x substrate mixes were prepared for each enzyme as follows: For c-Kit, 1.6 μM (2×) TK substrate and 16 μM (2×) ATP (Promega, catalog #V915). For PDGFRα, 3.2 μM TK substrate and 1 μM ATP. For PDGFRβ, 3.2 μM TK substrate and 40 μM ATP. For CSFIR, 3.2 M TK substrate and 20 μM ATP. For FLT3, 3.2 μM TK substrate and 100 μM ATP. Reactions were initiated by addition of 5 μL of the respective 2x substrate mix to each well of the plates containing the respective tyrosine kinase enzymes and were allowed to proceed for 120 minutes at rt, except for FLT3, the reaction time is 45 min. Following the reaction, 10 μL of detection mix consisting of 0.2 μM (2×) of Streptavidin-XL665 and 2 μM (2×) of TK-Antibody-Cryptate prepared in detection buffer (Part 62SDBRDF of KinEASE assay kit) was added and then allowed to incubate for 60 min. The TR-FRET signal was quantified by measuring the ratio of emission at 665 nm to 620 nm after excitation at 320 nm by reading on a PerkinElmer Envision multimode reader. Compound potencies (IC50 values) were determined using a standard 4-parameter non-linear regression fit. |
| Affinity data for this assay | |
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