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.
Highlights
The ABL protein-tyrosine kinase plays diverse roles in the regulation of cell proliferation, survival, adhesion, migration and the genotoxic stress response [1,2,3]
We developed a fluorescence polarization (FP) assay based on the Nterminal region of ABL consisting of the Ncap, SH3 and SH2 domains, and the SH2-kinase linker (ABL Ncap-SH3-SH2-linker region (N32L) protein)
A small molecule that binds to the ABL N32L protein and enhances SH3 interaction with the linker in cis is predicted to prevent probe peptide binding, resulting in a decrease in the FP signal
Summary
The ABL protein-tyrosine kinase plays diverse roles in the regulation of cell proliferation, survival, adhesion, migration and the genotoxic stress response [1,2,3]. Chronic use of kinase inhibitors often leads to drug resistance due to selection for mutations that disrupt drug binding or allosterically influence the conformation of the drug binding pocket [7]. The growing problem of imatininb resistance in BCR-ABL has fueled efforts to identify compounds that work outside of the kinase active site. Such compounds offer advantages in terms of enhanced specificity, because they have the potential to exploit non-conserved regulatory features unique to ABL that persist to some extent in BCR-ABL as well [8]. Small molecules that occupy the myristic acid binding site in the Clobe of the kinase domain have proven to be effective allosteric inhibitors of BCR-ABL function [14,15]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
