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

Grover P, Shi H, Baumgartner M, Camacho CJ, Smithgall TE - PLoS ONE (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Identification of p41 as optimal probe peptide for the ABL N32L FP assay.A) To characterize the baseline FP signal, the 6-carboxy-fluorescein labeled probe peptides p41 (red), p40 (green), p8 (blue), and 3BP-1 (black) were serially diluted in the concentration range of 1–1000 nM. The FP signals (solid lines, left Y axis) and corresponding fluorescence intensities (dashed lines, right Y axis) were measured and plotted as a function of peptide concentration. Average values are shown ± SE from four measurements per condition. B) To test for probe peptide interaction with ABL N32L by FP, each peptide (50 nM) was incubated with the ABL N32L protein over the range of 0.08–25 μg/well. The resulting FP signals were corrected for baseline FP signal recorded in the absence of the N32L protein and plotted against the N32L protein concentration. Average FP values are shown ± SE from four measurements per condition; error bars are smaller than the diameter of some data points.
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pone.0133590.g004: Identification of p41 as optimal probe peptide for the ABL N32L FP assay.A) To characterize the baseline FP signal, the 6-carboxy-fluorescein labeled probe peptides p41 (red), p40 (green), p8 (blue), and 3BP-1 (black) were serially diluted in the concentration range of 1–1000 nM. The FP signals (solid lines, left Y axis) and corresponding fluorescence intensities (dashed lines, right Y axis) were measured and plotted as a function of peptide concentration. Average values are shown ± SE from four measurements per condition. B) To test for probe peptide interaction with ABL N32L by FP, each peptide (50 nM) was incubated with the ABL N32L protein over the range of 0.08–25 μg/well. The resulting FP signals were corrected for baseline FP signal recorded in the absence of the N32L protein and plotted against the N32L protein concentration. Average FP values are shown ± SE from four measurements per condition; error bars are smaller than the diameter of some data points.

Mentions: To evaluate the suitability of the four ABL SH3 peptide ligands (p41, p40, p8, 3BP-1; Fig 3A) as FP probes, each peptide was synthesized and labeled with 6-carboxyfluorescein on its N-terminus. We first examined the baseline FP signal as well as the fluorescence intensity exhibited by each labeled peptide over a broad concentration range (1–1,000 nM) in the absence of the ABL N32L target protein. As shown in Fig 4A, probe peptide concentrations greater than 50 nM exhibited stable baseline FP readings with minimal well-to-well variation.


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

Grover P, Shi H, Baumgartner M, Camacho CJ, Smithgall TE - PLoS ONE (2015)

Identification of p41 as optimal probe peptide for the ABL N32L FP assay.A) To characterize the baseline FP signal, the 6-carboxy-fluorescein labeled probe peptides p41 (red), p40 (green), p8 (blue), and 3BP-1 (black) were serially diluted in the concentration range of 1–1000 nM. The FP signals (solid lines, left Y axis) and corresponding fluorescence intensities (dashed lines, right Y axis) were measured and plotted as a function of peptide concentration. Average values are shown ± SE from four measurements per condition. B) To test for probe peptide interaction with ABL N32L by FP, each peptide (50 nM) was incubated with the ABL N32L protein over the range of 0.08–25 μg/well. The resulting FP signals were corrected for baseline FP signal recorded in the absence of the N32L protein and plotted against the N32L protein concentration. Average FP values are shown ± SE from four measurements per condition; error bars are smaller than the diameter of some data points.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4519180&req=5

pone.0133590.g004: Identification of p41 as optimal probe peptide for the ABL N32L FP assay.A) To characterize the baseline FP signal, the 6-carboxy-fluorescein labeled probe peptides p41 (red), p40 (green), p8 (blue), and 3BP-1 (black) were serially diluted in the concentration range of 1–1000 nM. The FP signals (solid lines, left Y axis) and corresponding fluorescence intensities (dashed lines, right Y axis) were measured and plotted as a function of peptide concentration. Average values are shown ± SE from four measurements per condition. B) To test for probe peptide interaction with ABL N32L by FP, each peptide (50 nM) was incubated with the ABL N32L protein over the range of 0.08–25 μg/well. The resulting FP signals were corrected for baseline FP signal recorded in the absence of the N32L protein and plotted against the N32L protein concentration. Average FP values are shown ± SE from four measurements per condition; error bars are smaller than the diameter of some data points.
Mentions: To evaluate the suitability of the four ABL SH3 peptide ligands (p41, p40, p8, 3BP-1; Fig 3A) as FP probes, each peptide was synthesized and labeled with 6-carboxyfluorescein on its N-terminus. We first examined the baseline FP signal as well as the fluorescence intensity exhibited by each labeled peptide over a broad concentration range (1–1,000 nM) in the absence of the ABL N32L target protein. As shown in Fig 4A, probe peptide concentrations greater than 50 nM exhibited stable baseline FP readings with minimal well-to-well variation.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus