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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

ABL N32L FP assay development and optimization.A) The p41 FP probe binds the ABL N32L protein through the SH3 domain. The p41 probe peptide (50 nM) was combined with wild type, HAL, and W118A ABL N32L proteins over the range of concentrations shown. The resulting FP signals were measured and plotted as a function of N32L protein concentration. B) FP assay stability. The p41 probe peptide (50 nM) was combined with the three ABL N32L proteins (12.8 μg/well) and FP signals were recorded over the time course shown. C) DMSO tolerance. FP assays consisting of the p41 probe peptide (50 nM) and each ABL N32L protein (25 μg/well) were incubated with the DMSO concentrations shown, and FP signals were recorded 1 h later. D) Unlabeled peptide competition. For the competition assay, the p41 probe peptide (50 nM) was mixed with unlabeled p41 peptide or a negative control peptide of unrelated sequence (QKEGERALPSIP) and similar length (Con) over the range of concentrations shown. The ABL N32L protein (20 μg/well) was then added, and FP signals were recorded. FP signals were corrected for the background p41 peptide FP signal and plotted as a function of the unlabeled peptide concentration. In all experiments (A through D), average FP values are shown ± SE from four measurements per condition.
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pone.0133590.g005: ABL N32L FP assay development and optimization.A) The p41 FP probe binds the ABL N32L protein through the SH3 domain. The p41 probe peptide (50 nM) was combined with wild type, HAL, and W118A ABL N32L proteins over the range of concentrations shown. The resulting FP signals were measured and plotted as a function of N32L protein concentration. B) FP assay stability. The p41 probe peptide (50 nM) was combined with the three ABL N32L proteins (12.8 μg/well) and FP signals were recorded over the time course shown. C) DMSO tolerance. FP assays consisting of the p41 probe peptide (50 nM) and each ABL N32L protein (25 μg/well) were incubated with the DMSO concentrations shown, and FP signals were recorded 1 h later. D) Unlabeled peptide competition. For the competition assay, the p41 probe peptide (50 nM) was mixed with unlabeled p41 peptide or a negative control peptide of unrelated sequence (QKEGERALPSIP) and similar length (Con) over the range of concentrations shown. The ABL N32L protein (20 μg/well) was then added, and FP signals were recorded. FP signals were corrected for the background p41 peptide FP signal and plotted as a function of the unlabeled peptide concentration. In all experiments (A through D), average FP values are shown ± SE from four measurements per condition.

Mentions: We next investigated whether the FP signal obtained with the p41 probe peptide was due to interaction with SH3 domain of the recombinant ABL N32L target protein. For these experiments, we compared the FP signal produced from the wild type ABL N32L protein with the SH3 domain mutant (W118A) as well as the high-affinity linker (HAL) protein. As shown in Fig 5A, the wild type ABL N32L protein produced a concentration-dependent increase in the FP signal as observed previously. In contrast, the N32L W118A mutant failed to produce an FP signal with the p41 peptide over the same concentration range, indicating that the peptide requires this conserved SH3 domain tryptophan residue for binding as predicted from the crystal structure (see Fig 3). On the other hand, the ABL N32L HAL protein showed a greatly reduced FP signal in comparison to the wild type protein with the p41 probe. This result is consistent with enhanced cis-interaction of the linker with SH3 domain in this protein as a result of the higher linker proline content (see Fig 3A for HAL sequence). Results with these control proteins demonstrate that the p41 probe peptide interacts with the ABL N32L target protein through its SH3 domain. FP experiments with the recombinant purified ABL SH3 domain alone also produced a very similar FP response, supporting this conclusion (data not shown). Findings with these ABL N32L mutants support the idea that small molecules that disrupt or stabilize intramolecular interaction between the SH3 domain and linker will also reduce probe peptide binding and loss of the FP signal.


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)

ABL N32L FP assay development and optimization.A) The p41 FP probe binds the ABL N32L protein through the SH3 domain. The p41 probe peptide (50 nM) was combined with wild type, HAL, and W118A ABL N32L proteins over the range of concentrations shown. The resulting FP signals were measured and plotted as a function of N32L protein concentration. B) FP assay stability. The p41 probe peptide (50 nM) was combined with the three ABL N32L proteins (12.8 μg/well) and FP signals were recorded over the time course shown. C) DMSO tolerance. FP assays consisting of the p41 probe peptide (50 nM) and each ABL N32L protein (25 μg/well) were incubated with the DMSO concentrations shown, and FP signals were recorded 1 h later. D) Unlabeled peptide competition. For the competition assay, the p41 probe peptide (50 nM) was mixed with unlabeled p41 peptide or a negative control peptide of unrelated sequence (QKEGERALPSIP) and similar length (Con) over the range of concentrations shown. The ABL N32L protein (20 μg/well) was then added, and FP signals were recorded. FP signals were corrected for the background p41 peptide FP signal and plotted as a function of the unlabeled peptide concentration. In all experiments (A through D), average FP values are shown ± SE from four measurements per condition.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519180&req=5

pone.0133590.g005: ABL N32L FP assay development and optimization.A) The p41 FP probe binds the ABL N32L protein through the SH3 domain. The p41 probe peptide (50 nM) was combined with wild type, HAL, and W118A ABL N32L proteins over the range of concentrations shown. The resulting FP signals were measured and plotted as a function of N32L protein concentration. B) FP assay stability. The p41 probe peptide (50 nM) was combined with the three ABL N32L proteins (12.8 μg/well) and FP signals were recorded over the time course shown. C) DMSO tolerance. FP assays consisting of the p41 probe peptide (50 nM) and each ABL N32L protein (25 μg/well) were incubated with the DMSO concentrations shown, and FP signals were recorded 1 h later. D) Unlabeled peptide competition. For the competition assay, the p41 probe peptide (50 nM) was mixed with unlabeled p41 peptide or a negative control peptide of unrelated sequence (QKEGERALPSIP) and similar length (Con) over the range of concentrations shown. The ABL N32L protein (20 μg/well) was then added, and FP signals were recorded. FP signals were corrected for the background p41 peptide FP signal and plotted as a function of the unlabeled peptide concentration. In all experiments (A through D), average FP values are shown ± SE from four measurements per condition.
Mentions: We next investigated whether the FP signal obtained with the p41 probe peptide was due to interaction with SH3 domain of the recombinant ABL N32L target protein. For these experiments, we compared the FP signal produced from the wild type ABL N32L protein with the SH3 domain mutant (W118A) as well as the high-affinity linker (HAL) protein. As shown in Fig 5A, the wild type ABL N32L protein produced a concentration-dependent increase in the FP signal as observed previously. In contrast, the N32L W118A mutant failed to produce an FP signal with the p41 peptide over the same concentration range, indicating that the peptide requires this conserved SH3 domain tryptophan residue for binding as predicted from the crystal structure (see Fig 3). On the other hand, the ABL N32L HAL protein showed a greatly reduced FP signal in comparison to the wild type protein with the p41 probe. This result is consistent with enhanced cis-interaction of the linker with SH3 domain in this protein as a result of the higher linker proline content (see Fig 3A for HAL sequence). Results with these control proteins demonstrate that the p41 probe peptide interacts with the ABL N32L target protein through its SH3 domain. FP experiments with the recombinant purified ABL SH3 domain alone also produced a very similar FP response, supporting this conclusion (data not shown). Findings with these ABL N32L mutants support the idea that small molecules that disrupt or stabilize intramolecular interaction between the SH3 domain and linker will also reduce probe peptide binding and loss of the FP signal.

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