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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 activates the ABL kinase core in vitro.Top: The recombinant ABL core protein, consisting of the Ncap, SH3, SH2 and kinase domains, was assayed in the presence of compound 142 (10 μM), the known ABL activator DPH (10 μM), and imatinib (1 μM) or with DMSO as control (Con) using a kinetic kinase assay (see Materials and Methods). Data are plotted as pmol ADP produced per ng kinase as a function of time. The cartoon (right) depicts the domain organization of the wild type ABL core, and indicates the binding site for DPH (myristic acid binding pocket) as well as the predicted binding site for compound 142 (SH3 domain). Bottom: Kinase assays were performed using an ABL core protein with a high-affinity linker (HAL) in the presence of the same three compounds; the cartoon indicates the position of the modified linker (HAL). In both cases, the ATP and peptide substrate concentrations were set to their respective Km values (wild type ABL: 9.78 ± 0.14 μM for ATP and 144.65 ± 1.64 μM for substrate; ABL HAL: 21.24 ± 1.6 μM for ATP and 150.25 ± 5.35 μM for substrate).
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pone.0133590.g008: Compound 142 activates the ABL kinase core in vitro.Top: The recombinant ABL core protein, consisting of the Ncap, SH3, SH2 and kinase domains, was assayed in the presence of compound 142 (10 μM), the known ABL activator DPH (10 μM), and imatinib (1 μM) or with DMSO as control (Con) using a kinetic kinase assay (see Materials and Methods). Data are plotted as pmol ADP produced per ng kinase as a function of time. The cartoon (right) depicts the domain organization of the wild type ABL core, and indicates the binding site for DPH (myristic acid binding pocket) as well as the predicted binding site for compound 142 (SH3 domain). Bottom: Kinase assays were performed using an ABL core protein with a high-affinity linker (HAL) in the presence of the same three compounds; the cartoon indicates the position of the modified linker (HAL). In both cases, the ATP and peptide substrate concentrations were set to their respective Km values (wild type ABL: 9.78 ± 0.14 μM for ATP and 144.65 ± 1.64 μM for substrate; ABL HAL: 21.24 ± 1.6 μM for ATP and 150.25 ± 5.35 μM for substrate).

Mentions: We first examined the effect of compound 142 on the activity of the wild type ABL kinase core protein. As shown in Fig 8, compound 142 stimulated wild type ABL kinase activity by about 40% at a concentration of 10 μM relative to the DMSO control in this assay. As a positive control, we also assayed ABL core activity in the presence of the same concentration of a previously described ABL activator, DPH, and observed a similar degree of activation. DPH, unlike compound 142, stimulates ABL through the kinase domain via the myristic acid binding pocket in the C-lobe [27].


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 activates the ABL kinase core in vitro.Top: The recombinant ABL core protein, consisting of the Ncap, SH3, SH2 and kinase domains, was assayed in the presence of compound 142 (10 μM), the known ABL activator DPH (10 μM), and imatinib (1 μM) or with DMSO as control (Con) using a kinetic kinase assay (see Materials and Methods). Data are plotted as pmol ADP produced per ng kinase as a function of time. The cartoon (right) depicts the domain organization of the wild type ABL core, and indicates the binding site for DPH (myristic acid binding pocket) as well as the predicted binding site for compound 142 (SH3 domain). Bottom: Kinase assays were performed using an ABL core protein with a high-affinity linker (HAL) in the presence of the same three compounds; the cartoon indicates the position of the modified linker (HAL). In both cases, the ATP and peptide substrate concentrations were set to their respective Km values (wild type ABL: 9.78 ± 0.14 μM for ATP and 144.65 ± 1.64 μM for substrate; ABL HAL: 21.24 ± 1.6 μM for ATP and 150.25 ± 5.35 μM for substrate).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133590.g008: Compound 142 activates the ABL kinase core in vitro.Top: The recombinant ABL core protein, consisting of the Ncap, SH3, SH2 and kinase domains, was assayed in the presence of compound 142 (10 μM), the known ABL activator DPH (10 μM), and imatinib (1 μM) or with DMSO as control (Con) using a kinetic kinase assay (see Materials and Methods). Data are plotted as pmol ADP produced per ng kinase as a function of time. The cartoon (right) depicts the domain organization of the wild type ABL core, and indicates the binding site for DPH (myristic acid binding pocket) as well as the predicted binding site for compound 142 (SH3 domain). Bottom: Kinase assays were performed using an ABL core protein with a high-affinity linker (HAL) in the presence of the same three compounds; the cartoon indicates the position of the modified linker (HAL). In both cases, the ATP and peptide substrate concentrations were set to their respective Km values (wild type ABL: 9.78 ± 0.14 μM for ATP and 144.65 ± 1.64 μM for substrate; ABL HAL: 21.24 ± 1.6 μM for ATP and 150.25 ± 5.35 μM for substrate).
Mentions: We first examined the effect of compound 142 on the activity of the wild type ABL kinase core protein. As shown in Fig 8, compound 142 stimulated wild type ABL kinase activity by about 40% at a concentration of 10 μM relative to the DMSO control in this assay. As a positive control, we also assayed ABL core activity in the presence of the same concentration of a previously described ABL activator, DPH, and observed a similar degree of activation. DPH, unlike compound 142, stimulates ABL through the kinase domain via the myristic acid binding pocket in the C-lobe [27].

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