title: Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.
abstract: The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Prerna
authors.initials: P
authors.lastname: Grover
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Haibin
authors.initials: H
authors.lastname: Shi
authors.affiliation: Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Matthew
authors.initials: M
authors.lastname: Baumgartner
authors.affiliation: Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Carlos J
authors.initials: CJ
authors.lastname: Camacho
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Smithgall
mesh_terms: D000494:Allosteric Regulation; D020650:Combinatorial Chemistry Techniques; D005454:Fluorescence Polarization / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D016315:Proto-Oncogene Proteins c-abl / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology*; D018909:src Homology Domains
chemical_list: D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D016315:Proto-Oncogene Proteins c-abl