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Fluorescence polarization screening assays for small molecule allosteric modulators of ABL kinase function

Grover, P and Shi, H and Baumgartner, M and Camacho, CJ and Smithgall, TE (2015) Fluorescence polarization screening assays for small molecule allosteric modulators of ABL kinase function. PLoS ONE, 10 (7).

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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 noncatalytic 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.


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Item Type: Article
Status: Published
CreatorsEmailPitt UsernameORCID
Grover, Pprg32@pitt.eduPRG32
Shi, Hhas52@pitt.eduHAS52
Baumgartner, Mmpb21@pitt.eduMPB21
Camacho, CJccamacho@pitt.eduCCAMACHO
Smithgall, TEtsmithga@pitt.eduTSMITHGA
ContributionContributors NameEmailPitt UsernameORCID
Date: 29 July 2015
Date Type: Publication
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Journal or Publication Title: PLoS ONE
Volume: 10
Number: 7
DOI or Unique Handle: 10.1371/journal.pone.0133590
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Computational and Systems Biology
School of Medicine > Microbiology and Molecular Genetics
Refereed: Yes
Date Deposited: 23 Aug 2016 14:25
Last Modified: 30 Mar 2021 17:55


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