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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

Concentration-dependent activation of the ABL kinase core protein by compound 142.The wild type ABL kinase core was assayed in the presence of compound 142 and DPH at the indicated concentrations using a kinetic kinase assay (see Materials and Methods). Reaction velocities are plotted as a function of compound concentrations. The resulting data were curve-fit to determine the EC50, Kact and Vmax for each activator as described under Materials and Methods. Each of these parameters was determined in triplicate, and the mean values ± SE are presented in the table below the graph. The table also provides the ratio Vmax/Kact as a measure of overall enhancement of catalytic efficiency in the presence of each of these two ABL agonists.
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pone.0133590.g009: Concentration-dependent activation of the ABL kinase core protein by compound 142.The wild type ABL kinase core was assayed in the presence of compound 142 and DPH at the indicated concentrations using a kinetic kinase assay (see Materials and Methods). Reaction velocities are plotted as a function of compound concentrations. The resulting data were curve-fit to determine the EC50, Kact and Vmax for each activator as described under Materials and Methods. Each of these parameters was determined in triplicate, and the mean values ± SE are presented in the table below the graph. The table also provides the ratio Vmax/Kact as a measure of overall enhancement of catalytic efficiency in the presence of each of these two ABL agonists.

Mentions: To further characterize ABL activation by compound 142, we repeated kinetic kinase assays with the wild type ABL core over a range of compound concentrations. As shown in Fig 9, compound 142 activates the ABL core in a concentration-dependent manner, with an EC50 value of 0.63 ± 0.07 μM. This value compares favorably to that obtained with DPH, the myristic acid binding pocket agonist (EC50 = 1.11 ± 0.5 μM). We also calculated the activation constant (Kact) for each compound from these data, which is defined as the concentration at which the reaction rate reaches half-maximum velocity (Vact). For compound 142, the Kact was calculated as 0.4 ± 0.02 μM, while DPH yielded a value of 1.02 ± 0.07 μM. This analysis also showed that the extent of ABL activation by compound 142 was higher than that observed for DPH, based on the Vmax.


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)

Concentration-dependent activation of the ABL kinase core protein by compound 142.The wild type ABL kinase core was assayed in the presence of compound 142 and DPH at the indicated concentrations using a kinetic kinase assay (see Materials and Methods). Reaction velocities are plotted as a function of compound concentrations. The resulting data were curve-fit to determine the EC50, Kact and Vmax for each activator as described under Materials and Methods. Each of these parameters was determined in triplicate, and the mean values ± SE are presented in the table below the graph. The table also provides the ratio Vmax/Kact as a measure of overall enhancement of catalytic efficiency in the presence of each of these two ABL agonists.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133590.g009: Concentration-dependent activation of the ABL kinase core protein by compound 142.The wild type ABL kinase core was assayed in the presence of compound 142 and DPH at the indicated concentrations using a kinetic kinase assay (see Materials and Methods). Reaction velocities are plotted as a function of compound concentrations. The resulting data were curve-fit to determine the EC50, Kact and Vmax for each activator as described under Materials and Methods. Each of these parameters was determined in triplicate, and the mean values ± SE are presented in the table below the graph. The table also provides the ratio Vmax/Kact as a measure of overall enhancement of catalytic efficiency in the presence of each of these two ABL agonists.
Mentions: To further characterize ABL activation by compound 142, we repeated kinetic kinase assays with the wild type ABL core over a range of compound concentrations. As shown in Fig 9, compound 142 activates the ABL core in a concentration-dependent manner, with an EC50 value of 0.63 ± 0.07 μM. This value compares favorably to that obtained with DPH, the myristic acid binding pocket agonist (EC50 = 1.11 ± 0.5 μM). We also calculated the activation constant (Kact) for each compound from these data, which is defined as the concentration at which the reaction rate reaches half-maximum velocity (Vact). For compound 142, the Kact was calculated as 0.4 ± 0.02 μM, while DPH yielded a value of 1.02 ± 0.07 μM. This analysis also showed that the extent of ABL activation by compound 142 was higher than that observed for DPH, based on the Vmax.

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