Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function
Top: The recombinant ABL core protein, consisting of the Ncap, SH3, SH2 and kinase domains, was assayed in the presence of compound 142 (10 μM), the known ABL activator DPH (10 μM), and imatinib (1 μM) or with DMSO as control (Con) using a kinetic kinase assay (see Materials and Methods). Data are plotted as pmol ADP produced per ng kinase as a function of time. The cartoon (right) depicts the domain organization of the wild type ABL core, and indicates the binding site for DPH (myristic acid binding pocket) as well as the predicted binding site for compound 142 (SH3 domain). Bottom: Kinase assays were performed using an ABL core protein with a high-affinity linker (HAL) in the presence of the same three compounds; the cartoon indicates the position of the modified linker (HAL). In both cases, the ATP and peptide substrate concentrations were set to their respective Km values (wild type ABL: 9.78 ± 0.14 μM for ATP and 144.65 ± 1.64 μM for substrate; ABL HAL: 21.24 ± 1.6 μM for ATP and 150.25 ± 5.35 μM for substrate).