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Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

Fig 7

Hit compound 142 interacts directly with the ABL N32L protein.

A) Differential scanning fluorimetry assays were performed on the ABL N32L protein in the presence of the four confirmed hit compounds as described under Materials and Methods. The average change in the mid-point of the thermal melt profile (ΔTm) relative to the Tm obtained with the N32L protein in the presence of the DMSO carrier solvent is plotted on the Y-axis ± SE (n = 2). B) Surface plasmon resonance was performed with the ABL N32L protein immobilized on the biosensor chip and compound 142 as analyte. Responses were recorded for the four compound concentrations shown, and the flow path was switched back to buffer after 180 s to induce dissociation (arrow). The resulting sensorgrams (black lines) were fit by a 1:1 Langmuir binding model (red lines) to generate kinetic constants. C) Compound 142 inhibits p41 peptide binding to the ABL N32L and SH3 proteins in the FP assay. Compound 142 was added to N32L and SH3 FP assays over the range of concentrations shown, and the resulting FP signals are presented as the mean ± SE. Significant inhibition for both N32L and SH3 was observed at 3 and 10 μM (*p < 0.05 by 2-tailed t-test). D) Chemical structure of compound 142 (dipyridamole).

Fig 7