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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

Peptide and linker interactions with the ABL SH3 domain.A) Sequences of the ABL SH3 binding peptides, p41, p40, p8, and 3BP-1, and their published binding affinities for the ABL SH3 domain [20,21]. Sequences of the wild type (WT) and high-affinity (HAL) SH2-kinase linker sequences are also shown at the bottom. The peptide sequences are presented in the C- to N-terminal orientation to align with those of the linkers. B) Crystal structure of the p41 peptide (cyan) bound to the ABL SH3 domain (PDB: 1BBZ) [21]. The SH3 surface is shown as a space filling model (red) and side chains of residues that interact with the p41 peptide are shown as sticks. C) Crystal structure of the SH2-kinase linker (orange) bound to the ABL SH3 domain (red) from the ABL core (PDB: 2FO0) [11]. Side chains of SH3 domain residues that interact with the p41 peptide as per panel B are shown as sticks. Note the lack of hydrophobic interactions and hydrogen bonds between the SH3 domain and the linker in comparison to the p41 peptide.
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pone.0133590.g003: Peptide and linker interactions with the ABL SH3 domain.A) Sequences of the ABL SH3 binding peptides, p41, p40, p8, and 3BP-1, and their published binding affinities for the ABL SH3 domain [20,21]. Sequences of the wild type (WT) and high-affinity (HAL) SH2-kinase linker sequences are also shown at the bottom. The peptide sequences are presented in the C- to N-terminal orientation to align with those of the linkers. B) Crystal structure of the p41 peptide (cyan) bound to the ABL SH3 domain (PDB: 1BBZ) [21]. The SH3 surface is shown as a space filling model (red) and side chains of residues that interact with the p41 peptide are shown as sticks. C) Crystal structure of the SH2-kinase linker (orange) bound to the ABL SH3 domain (red) from the ABL core (PDB: 2FO0) [11]. Side chains of SH3 domain residues that interact with the p41 peptide as per panel B are shown as sticks. Note the lack of hydrophobic interactions and hydrogen bonds between the SH3 domain and the linker in comparison to the p41 peptide.

Mentions: The target protein for the ABL FP assay consists of the first 255 residues of ABL (isoform 1b), and encompasses the Ncap, the SH3 and SH2 domains, as well as the SH2-kinase linker as described above. This ABL N32L protein was expressed in bacteria in soluble form, purified to homogeneity, and its purity and identity were confirmed by SDS-polyacrylamide gel electrophoresis and mass spectrometry, respectively (Fig 2). Previous studies have established that regulatory SH3:linker interaction is maintained in this construct, despite the absence of the kinase domain [8,33]. In addition to the wild type protein, two mutant forms of N32L were produced for use as controls. The first of these has an alanine substitution for a conserved tryptophan on the SH3 domain binding surface (W118A mutant; see Fig 3 for SH3 domain structure), which renders it unable to bind to the probe peptide and thus serves as a negative control. In the second mutant, five linker residues were replaced with prolines to enhance interaction with the SH3 domain [8]. This high-affinity linker (HAL) substitution suppresses the activating effects of kinase domain mutations and influences the conformation of the kinase domain, enhancing both imatinib and allosteric inhibitor action (see Introduction). The HAL protein therefore represents a second negative control for probe peptide binding to the SH3 domain. Both the W118A and HAL forms of the ABL N32L protein were also expressed and purified from bacteria, and yielded soluble purified proteins of the expected mass (Fig 2).


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)

Peptide and linker interactions with the ABL SH3 domain.A) Sequences of the ABL SH3 binding peptides, p41, p40, p8, and 3BP-1, and their published binding affinities for the ABL SH3 domain [20,21]. Sequences of the wild type (WT) and high-affinity (HAL) SH2-kinase linker sequences are also shown at the bottom. The peptide sequences are presented in the C- to N-terminal orientation to align with those of the linkers. B) Crystal structure of the p41 peptide (cyan) bound to the ABL SH3 domain (PDB: 1BBZ) [21]. The SH3 surface is shown as a space filling model (red) and side chains of residues that interact with the p41 peptide are shown as sticks. C) Crystal structure of the SH2-kinase linker (orange) bound to the ABL SH3 domain (red) from the ABL core (PDB: 2FO0) [11]. Side chains of SH3 domain residues that interact with the p41 peptide as per panel B are shown as sticks. Note the lack of hydrophobic interactions and hydrogen bonds between the SH3 domain and the linker in comparison to the p41 peptide.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133590.g003: Peptide and linker interactions with the ABL SH3 domain.A) Sequences of the ABL SH3 binding peptides, p41, p40, p8, and 3BP-1, and their published binding affinities for the ABL SH3 domain [20,21]. Sequences of the wild type (WT) and high-affinity (HAL) SH2-kinase linker sequences are also shown at the bottom. The peptide sequences are presented in the C- to N-terminal orientation to align with those of the linkers. B) Crystal structure of the p41 peptide (cyan) bound to the ABL SH3 domain (PDB: 1BBZ) [21]. The SH3 surface is shown as a space filling model (red) and side chains of residues that interact with the p41 peptide are shown as sticks. C) Crystal structure of the SH2-kinase linker (orange) bound to the ABL SH3 domain (red) from the ABL core (PDB: 2FO0) [11]. Side chains of SH3 domain residues that interact with the p41 peptide as per panel B are shown as sticks. Note the lack of hydrophobic interactions and hydrogen bonds between the SH3 domain and the linker in comparison to the p41 peptide.
Mentions: The target protein for the ABL FP assay consists of the first 255 residues of ABL (isoform 1b), and encompasses the Ncap, the SH3 and SH2 domains, as well as the SH2-kinase linker as described above. This ABL N32L protein was expressed in bacteria in soluble form, purified to homogeneity, and its purity and identity were confirmed by SDS-polyacrylamide gel electrophoresis and mass spectrometry, respectively (Fig 2). Previous studies have established that regulatory SH3:linker interaction is maintained in this construct, despite the absence of the kinase domain [8,33]. In addition to the wild type protein, two mutant forms of N32L were produced for use as controls. The first of these has an alanine substitution for a conserved tryptophan on the SH3 domain binding surface (W118A mutant; see Fig 3 for SH3 domain structure), which renders it unable to bind to the probe peptide and thus serves as a negative control. In the second mutant, five linker residues were replaced with prolines to enhance interaction with the SH3 domain [8]. This high-affinity linker (HAL) substitution suppresses the activating effects of kinase domain mutations and influences the conformation of the kinase domain, enhancing both imatinib and allosteric inhibitor action (see Introduction). The HAL protein therefore represents a second negative control for probe peptide binding to the SH3 domain. Both the W118A and HAL forms of the ABL N32L protein were also expressed and purified from bacteria, and yielded soluble purified proteins of the expected mass (Fig 2).

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