Limits...
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

Hit compound 142 interacts directly with the ABL N32L protein.A) Differential scanning fluorimetry assays were performed on the ABL N32L protein in the presence of the four confirmed hit compounds as described under Materials and Methods. The average change in the mid-point of the thermal melt profile (ΔTm) relative to the Tm obtained with the N32L protein in the presence of the DMSO carrier solvent is plotted on the Y-axis ± SE (n = 2). B) Surface plasmon resonance was performed with the ABL N32L protein immobilized on the biosensor chip and compound 142 as analyte. Responses were recorded for the four compound concentrations shown, and the flow path was switched back to buffer after 180 s to induce dissociation (arrow). The resulting sensorgrams (black lines) were fit by a 1:1 Langmuir binding model (red lines) to generate kinetic constants. C) Compound 142 inhibits p41 peptide binding to the ABL N32L and SH3 proteins in the FP assay. Compound 142 was added to N32L and SH3 FP assays over the range of concentrations shown, and the resulting FP signals are presented as the mean ± SE. Significant inhibition for both N32L and SH3 was observed at 3 and 10 μM (*p < 0.05 by 2-tailed t-test). D) Chemical structure of compound 142 (dipyridamole).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4519180&req=5

pone.0133590.g007: Hit compound 142 interacts directly with the ABL N32L protein.A) Differential scanning fluorimetry assays were performed on the ABL N32L protein in the presence of the four confirmed hit compounds as described under Materials and Methods. The average change in the mid-point of the thermal melt profile (ΔTm) relative to the Tm obtained with the N32L protein in the presence of the DMSO carrier solvent is plotted on the Y-axis ± SE (n = 2). B) Surface plasmon resonance was performed with the ABL N32L protein immobilized on the biosensor chip and compound 142 as analyte. Responses were recorded for the four compound concentrations shown, and the flow path was switched back to buffer after 180 s to induce dissociation (arrow). The resulting sensorgrams (black lines) were fit by a 1:1 Langmuir binding model (red lines) to generate kinetic constants. C) Compound 142 inhibits p41 peptide binding to the ABL N32L and SH3 proteins in the FP assay. Compound 142 was added to N32L and SH3 FP assays over the range of concentrations shown, and the resulting FP signals are presented as the mean ± SE. Significant inhibition for both N32L and SH3 was observed at 3 and 10 μM (*p < 0.05 by 2-tailed t-test). D) Chemical structure of compound 142 (dipyridamole).

Mentions: As an independent measure of hit compound interaction with the ABL N32L target protein, we performed differential scanning fluorimetry assays [35]. For these experiments, the ABL N32L protein was heated with a molar excess of each compound in the presence of the reporter dye, SYPRO orange. As the temperature rises and the N32L protein unfolds, the reporter dye accesses the hydrophobic interior of the protein, resulting in an increase in dye fluorescence. The resulting protein ‘melt curve’ is then fit by regression analysis to obtain a Tm value, the temperature at which half-maximal thermal denaturation is observed. Small molecule binding to a target protein can either increase or decrease the Tm value, depending upon the effect of the compound on protein stability. Differential scanning fluorimetry was performed with the wild type N32L protein in the presence of each of the hit compounds from the FP assay, and the change in Tm value (ΔTm) was determined compared to DMSO as the reference control. As shown in Fig 7A, all four compounds produced a significant decrease in the Tm value. Compound 142 had the largest impact on N32L thermal stability, producing a decrease of more than 2°C in the Tm, consistent with its effect in the FP assay (Fig 6B).


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)

Hit compound 142 interacts directly with the ABL N32L protein.A) Differential scanning fluorimetry assays were performed on the ABL N32L protein in the presence of the four confirmed hit compounds as described under Materials and Methods. The average change in the mid-point of the thermal melt profile (ΔTm) relative to the Tm obtained with the N32L protein in the presence of the DMSO carrier solvent is plotted on the Y-axis ± SE (n = 2). B) Surface plasmon resonance was performed with the ABL N32L protein immobilized on the biosensor chip and compound 142 as analyte. Responses were recorded for the four compound concentrations shown, and the flow path was switched back to buffer after 180 s to induce dissociation (arrow). The resulting sensorgrams (black lines) were fit by a 1:1 Langmuir binding model (red lines) to generate kinetic constants. C) Compound 142 inhibits p41 peptide binding to the ABL N32L and SH3 proteins in the FP assay. Compound 142 was added to N32L and SH3 FP assays over the range of concentrations shown, and the resulting FP signals are presented as the mean ± SE. Significant inhibition for both N32L and SH3 was observed at 3 and 10 μM (*p < 0.05 by 2-tailed t-test). D) Chemical structure of compound 142 (dipyridamole).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133590.g007: Hit compound 142 interacts directly with the ABL N32L protein.A) Differential scanning fluorimetry assays were performed on the ABL N32L protein in the presence of the four confirmed hit compounds as described under Materials and Methods. The average change in the mid-point of the thermal melt profile (ΔTm) relative to the Tm obtained with the N32L protein in the presence of the DMSO carrier solvent is plotted on the Y-axis ± SE (n = 2). B) Surface plasmon resonance was performed with the ABL N32L protein immobilized on the biosensor chip and compound 142 as analyte. Responses were recorded for the four compound concentrations shown, and the flow path was switched back to buffer after 180 s to induce dissociation (arrow). The resulting sensorgrams (black lines) were fit by a 1:1 Langmuir binding model (red lines) to generate kinetic constants. C) Compound 142 inhibits p41 peptide binding to the ABL N32L and SH3 proteins in the FP assay. Compound 142 was added to N32L and SH3 FP assays over the range of concentrations shown, and the resulting FP signals are presented as the mean ± SE. Significant inhibition for both N32L and SH3 was observed at 3 and 10 μM (*p < 0.05 by 2-tailed t-test). D) Chemical structure of compound 142 (dipyridamole).
Mentions: As an independent measure of hit compound interaction with the ABL N32L target protein, we performed differential scanning fluorimetry assays [35]. For these experiments, the ABL N32L protein was heated with a molar excess of each compound in the presence of the reporter dye, SYPRO orange. As the temperature rises and the N32L protein unfolds, the reporter dye accesses the hydrophobic interior of the protein, resulting in an increase in dye fluorescence. The resulting protein ‘melt curve’ is then fit by regression analysis to obtain a Tm value, the temperature at which half-maximal thermal denaturation is observed. Small molecule binding to a target protein can either increase or decrease the Tm value, depending upon the effect of the compound on protein stability. Differential scanning fluorimetry was performed with the wild type N32L protein in the presence of each of the hit compounds from the FP assay, and the change in Tm value (ΔTm) was determined compared to DMSO as the reference control. As shown in Fig 7A, all four compounds produced a significant decrease in the Tm value. Compound 142 had the largest impact on N32L thermal stability, producing a decrease of more than 2°C in the Tm, consistent with its effect in the FP assay (Fig 6B).

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