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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

Pilot screen identifies inhibitors of p41 interaction with the ABL N32L protein.A) A library of 1200 FDA-approved compounds was screened using the ABL N32L protein (25 μg/well) and the p41 probe peptide (50 nM) in the FP assay. The solid lines correspond to the mean FP signals for the wild type (WT) and SH3 mutant (W118A) control N32L proteins across all assay plates, with the dotted lines indicating three standard deviations from the means (± 3σ). Compounds were screened at 10 μM and each FP signal is represented as an individual circle. Five putative hit compounds were identified (green circles). B) The five potential hit compounds were re-tested in quadruplicate at 10 μM vs. the DMSO control under FP screening assay conditions, and the mean FP values are shown ± SE. Four of these compounds significantly inhibited the FP signal relative to the DMSO control as indicated by the asterisk (p < 0.05; 2-tailed t-test). The dotted line shows the FP value three standard deviations below the DMSO control FP signal (-3σ).
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pone.0133590.g006: Pilot screen identifies inhibitors of p41 interaction with the ABL N32L protein.A) A library of 1200 FDA-approved compounds was screened using the ABL N32L protein (25 μg/well) and the p41 probe peptide (50 nM) in the FP assay. The solid lines correspond to the mean FP signals for the wild type (WT) and SH3 mutant (W118A) control N32L proteins across all assay plates, with the dotted lines indicating three standard deviations from the means (± 3σ). Compounds were screened at 10 μM and each FP signal is represented as an individual circle. Five putative hit compounds were identified (green circles). B) The five potential hit compounds were re-tested in quadruplicate at 10 μM vs. the DMSO control under FP screening assay conditions, and the mean FP values are shown ± SE. Four of these compounds significantly inhibited the FP signal relative to the DMSO control as indicated by the asterisk (p < 0.05; 2-tailed t-test). The dotted line shows the FP value three standard deviations below the DMSO control FP signal (-3σ).

Mentions: To test the performance of the ABL N32L FP assay under screening conditions, we performed a pilot screen of 1200 FDA-approved compounds. The wild type ABL N32L protein (25 μg) was added to each well together with the p41 probe peptide (50 nM). The compounds were then added to a final concentration of 10 μM in 1% DMSO. Each plate contained twenty-eight wells with the wild type N32L target protein plus DMSO as positive controls, and twenty-eight wells with the non-binding W118A mutant protein plus DMSO as negative controls. The overall Z factor for the pilot screen was 0.57, indicative of a reliable screening assay [34]. The average FP signals observed with the controls as well as the readings observed with each of the test compounds are presented in Fig 6A.


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)

Pilot screen identifies inhibitors of p41 interaction with the ABL N32L protein.A) A library of 1200 FDA-approved compounds was screened using the ABL N32L protein (25 μg/well) and the p41 probe peptide (50 nM) in the FP assay. The solid lines correspond to the mean FP signals for the wild type (WT) and SH3 mutant (W118A) control N32L proteins across all assay plates, with the dotted lines indicating three standard deviations from the means (± 3σ). Compounds were screened at 10 μM and each FP signal is represented as an individual circle. Five putative hit compounds were identified (green circles). B) The five potential hit compounds were re-tested in quadruplicate at 10 μM vs. the DMSO control under FP screening assay conditions, and the mean FP values are shown ± SE. Four of these compounds significantly inhibited the FP signal relative to the DMSO control as indicated by the asterisk (p < 0.05; 2-tailed t-test). The dotted line shows the FP value three standard deviations below the DMSO control FP signal (-3σ).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519180&req=5

pone.0133590.g006: Pilot screen identifies inhibitors of p41 interaction with the ABL N32L protein.A) A library of 1200 FDA-approved compounds was screened using the ABL N32L protein (25 μg/well) and the p41 probe peptide (50 nM) in the FP assay. The solid lines correspond to the mean FP signals for the wild type (WT) and SH3 mutant (W118A) control N32L proteins across all assay plates, with the dotted lines indicating three standard deviations from the means (± 3σ). Compounds were screened at 10 μM and each FP signal is represented as an individual circle. Five putative hit compounds were identified (green circles). B) The five potential hit compounds were re-tested in quadruplicate at 10 μM vs. the DMSO control under FP screening assay conditions, and the mean FP values are shown ± SE. Four of these compounds significantly inhibited the FP signal relative to the DMSO control as indicated by the asterisk (p < 0.05; 2-tailed t-test). The dotted line shows the FP value three standard deviations below the DMSO control FP signal (-3σ).
Mentions: To test the performance of the ABL N32L FP assay under screening conditions, we performed a pilot screen of 1200 FDA-approved compounds. The wild type ABL N32L protein (25 μg) was added to each well together with the p41 probe peptide (50 nM). The compounds were then added to a final concentration of 10 μM in 1% DMSO. Each plate contained twenty-eight wells with the wild type N32L target protein plus DMSO as positive controls, and twenty-eight wells with the non-binding W118A mutant protein plus DMSO as negative controls. The overall Z factor for the pilot screen was 0.57, indicative of a reliable screening assay [34]. The average FP signals observed with the controls as well as the readings observed with each of the test compounds are presented in Fig 6A.

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