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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

Compound 142 cooperates with DPH to stimulate ABL core autophosphorylation in cells.Human embryonic kidney 293T cells were transfected with an expression vector for the wild type ABL core followed by treatment with compound 142 overnight over the range of concentrations shown. The experiment was performed in the absence or presence of the myristic acid binding pocket agonist DPH at a final concentration of 10 μM. ABL proteins were immunoprecipitated from clarified cell lysates, and analyzed by immunoblotting with phosphospecific antibodies to three regulatory sites: pTyr412 in the activation loop, pTyr89 in the SH3 domain, and pTyr245 in the SH2-kinase linker, as well as an ABL antibody to control for protein recovery. This experiment was repeated twice with comparable results.
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pone.0133590.g012: Compound 142 cooperates with DPH to stimulate ABL core autophosphorylation in cells.Human embryonic kidney 293T cells were transfected with an expression vector for the wild type ABL core followed by treatment with compound 142 overnight over the range of concentrations shown. The experiment was performed in the absence or presence of the myristic acid binding pocket agonist DPH at a final concentration of 10 μM. ABL proteins were immunoprecipitated from clarified cell lysates, and analyzed by immunoblotting with phosphospecific antibodies to three regulatory sites: pTyr412 in the activation loop, pTyr89 in the SH3 domain, and pTyr245 in the SH2-kinase linker, as well as an ABL antibody to control for protein recovery. This experiment was repeated twice with comparable results.

Mentions: In a final series of studies, we evaluated the effect of compound 142 on the activity of the ABL core in cells. For these studies, we expressed the wild type ABL core in 293T cells, and then treated the cells over a range of compound 142 concentrations (1–10 μM). We then immunoprecipitated ABL and analyzed the level of phosphorylation at three important regulatory tyrosine sites by immunoblotting with phosphospecific antibodies [8,39]: pY412 (activation loop), pY245 (SH2-kinase linker), and pY89 (SH3 domain). As shown in Fig 12, treatment with compound 142 alone did not affect phosphorylation of these sites. We then repeated this experiment in the presence of DPH, the myristic acid binding pocket activator described above, at a concentration of 10 μM. In the absence of compound 142, DPH had a very small effect on ABL phosphorylation. However, the combination of compound 142 and DPH led to remarkable enhancement of phosphorylation on all three regulatory tyrosines, providing strong evidence for an activating effect of compound 142 on ABL in cells. These observations suggest that in the cellular environment, unlike in vitro, compound 142 alone may be not sufficient to disturb SH3:linker interaction and stimulate ABL activity. However, when the regulatory interaction of the myristoylated Ncap with the kinase domain C-lobe is perturbed by DPH, then the activating effect of SH3:linker displacement becomes readily apparent.


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)

Compound 142 cooperates with DPH to stimulate ABL core autophosphorylation in cells.Human embryonic kidney 293T cells were transfected with an expression vector for the wild type ABL core followed by treatment with compound 142 overnight over the range of concentrations shown. The experiment was performed in the absence or presence of the myristic acid binding pocket agonist DPH at a final concentration of 10 μM. ABL proteins were immunoprecipitated from clarified cell lysates, and analyzed by immunoblotting with phosphospecific antibodies to three regulatory sites: pTyr412 in the activation loop, pTyr89 in the SH3 domain, and pTyr245 in the SH2-kinase linker, as well as an ABL antibody to control for protein recovery. This experiment was repeated twice with comparable results.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133590.g012: Compound 142 cooperates with DPH to stimulate ABL core autophosphorylation in cells.Human embryonic kidney 293T cells were transfected with an expression vector for the wild type ABL core followed by treatment with compound 142 overnight over the range of concentrations shown. The experiment was performed in the absence or presence of the myristic acid binding pocket agonist DPH at a final concentration of 10 μM. ABL proteins were immunoprecipitated from clarified cell lysates, and analyzed by immunoblotting with phosphospecific antibodies to three regulatory sites: pTyr412 in the activation loop, pTyr89 in the SH3 domain, and pTyr245 in the SH2-kinase linker, as well as an ABL antibody to control for protein recovery. This experiment was repeated twice with comparable results.
Mentions: In a final series of studies, we evaluated the effect of compound 142 on the activity of the ABL core in cells. For these studies, we expressed the wild type ABL core in 293T cells, and then treated the cells over a range of compound 142 concentrations (1–10 μM). We then immunoprecipitated ABL and analyzed the level of phosphorylation at three important regulatory tyrosine sites by immunoblotting with phosphospecific antibodies [8,39]: pY412 (activation loop), pY245 (SH2-kinase linker), and pY89 (SH3 domain). As shown in Fig 12, treatment with compound 142 alone did not affect phosphorylation of these sites. We then repeated this experiment in the presence of DPH, the myristic acid binding pocket activator described above, at a concentration of 10 μM. In the absence of compound 142, DPH had a very small effect on ABL phosphorylation. However, the combination of compound 142 and DPH led to remarkable enhancement of phosphorylation on all three regulatory tyrosines, providing strong evidence for an activating effect of compound 142 on ABL in cells. These observations suggest that in the cellular environment, unlike in vitro, compound 142 alone may be not sufficient to disturb SH3:linker interaction and stimulate ABL activity. However, when the regulatory interaction of the myristoylated Ncap with the kinase domain C-lobe is perturbed by DPH, then the activating effect of SH3:linker displacement becomes readily apparent.

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