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

Compound 142 fails to activate downregulated HCK in vitro.Top panels: Recombinant, downregulated wild type ABL core and near full-length HCK proteins were assayed in the presence of compound 142 (1 μM) or with DMSO alone 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. Bottom: Amino acid sequence alignment of the ABL and HCK SH3 domains and SH2-kinase linkers. SH3 and linker residues predicted to contribute to compound 142 binding are highlighted in red (see Fig 10). The type of interaction is indicated as side chain (s), main chain (m), or aromatic (a).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133590.g011: Compound 142 fails to activate downregulated HCK in vitro.Top panels: Recombinant, downregulated wild type ABL core and near full-length HCK proteins were assayed in the presence of compound 142 (1 μM) or with DMSO alone 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. Bottom: Amino acid sequence alignment of the ABL and HCK SH3 domains and SH2-kinase linkers. SH3 and linker residues predicted to contribute to compound 142 binding are highlighted in red (see Fig 10). The type of interaction is indicated as side chain (s), main chain (m), or aromatic (a).

Mentions: Like ABL, SRC-family kinases exhibit a very similar arrangement of SH3, SH2, and kinase domains in the downregulated conformation, and are also susceptible to activation by mutations and binding partners that disrupt intramolecular SH3:linker interaction [37,38]. To determine whether compound 142 also activates SRC-family kinases by a similar SH3:linker displacement mechanism, we performed kinetic kinase assays on the SRC-family member HCK in the presence and absence of this ABL activator. As shown in Fig 11, addition of compound 142 to recombinant near-full-length HCK had no effect the reaction velocity, suggesting that it is selective for ABL. Alignment of the SH3 domain and SH2-kinase linker sequences supports this view (Fig 11, bottom). Five of the seven residues predicted by the docking model to participate in compound 142 binding to the ABL SH3:linker surface are either substituted with different amino acids or are missing from the HCK sequence. These observations suggest that even subtle differences in the SH3 and linker sequences of ABL and SRC-family kinases can be exploited for the development of selective agonists or antagonists.


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 fails to activate downregulated HCK in vitro.Top panels: Recombinant, downregulated wild type ABL core and near full-length HCK proteins were assayed in the presence of compound 142 (1 μM) or with DMSO alone 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. Bottom: Amino acid sequence alignment of the ABL and HCK SH3 domains and SH2-kinase linkers. SH3 and linker residues predicted to contribute to compound 142 binding are highlighted in red (see Fig 10). The type of interaction is indicated as side chain (s), main chain (m), or aromatic (a).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133590.g011: Compound 142 fails to activate downregulated HCK in vitro.Top panels: Recombinant, downregulated wild type ABL core and near full-length HCK proteins were assayed in the presence of compound 142 (1 μM) or with DMSO alone 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. Bottom: Amino acid sequence alignment of the ABL and HCK SH3 domains and SH2-kinase linkers. SH3 and linker residues predicted to contribute to compound 142 binding are highlighted in red (see Fig 10). The type of interaction is indicated as side chain (s), main chain (m), or aromatic (a).
Mentions: Like ABL, SRC-family kinases exhibit a very similar arrangement of SH3, SH2, and kinase domains in the downregulated conformation, and are also susceptible to activation by mutations and binding partners that disrupt intramolecular SH3:linker interaction [37,38]. To determine whether compound 142 also activates SRC-family kinases by a similar SH3:linker displacement mechanism, we performed kinetic kinase assays on the SRC-family member HCK in the presence and absence of this ABL activator. As shown in Fig 11, addition of compound 142 to recombinant near-full-length HCK had no effect the reaction velocity, suggesting that it is selective for ABL. Alignment of the SH3 domain and SH2-kinase linker sequences supports this view (Fig 11, bottom). Five of the seven residues predicted by the docking model to participate in compound 142 binding to the ABL SH3:linker surface are either substituted with different amino acids or are missing from the HCK sequence. These observations suggest that even subtle differences in the SH3 and linker sequences of ABL and SRC-family kinases can be exploited for the development of selective agonists or antagonists.

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