Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function
A) Crystal structure of the auto-inhibited ABL core (PDB: 2FO0) . Key features include the N-cap, SH3 and SH2 domains, the SH2-kinase linker, and the kinase domain. The disordered portion of the N-cap is indicated by the dotted line. The N-terminal portion of the N-cap is myristoylated and engages a deep pocket in the kinase domain C-lobe. B) Cartoon depiction of the intramolecular interactions regulating assembly of the downregulated ABL core. Note that the linker forms a polyproline helix that binds in cis to the SH3 domain. C) Fluorescence polarization (FP) assay. The FP assay combines a recombinant ABL Ncap-SH3-SH2-linker (N32L) protein and a SH3-binding peptide probe labeled with a fluorescent moiety (F). The probe peptide binds the SH3 domain in the ABL N32L protein, resulting in an FP signal. Small molecules (S) may bind to the SH3 domain and block probe peptide binding directly; such molecules would be expected to disrupt SH3:linker interaction (case 1). Alternatively, small molecules may stabilize SH3:linker interaction, making the SH3 domain inaccessible to the probe peptide (case 2). In either case, small molecule binding is predicted to result in a loss of the FP signal.