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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

Molecular dynamics (MD) and molecular docking predict binding of compound 142 to the ABL SH3:linker interface.Top: The lowest energy pose of the ligand (compound 142; carbon atoms rendered in green) is shown docked to a snapshot of an MD simulation of the ABL N32L structure. SH3 domain residues predicted to contribute to ligand binding include Asn97, Thr98, Asn115, Trp118, and Trp129 (carbons in red). The backbone of the linker is shown as an orange ribbon, with Gly246, Val247, Pro249 and Trp254 predicted to contribute to the binding pocket. One of the piperidine groups of compound 142 makes hydrophobic contacts with linker Pro249 and Trp254, while the pyrimido-pyrimidine scaffold of compound 142 is π-stacking with Trp118. Middle panel: Model of the SH3:linker interface in the N32L region based on the crystal structure of the downregulated ABL core (PDB:2FO0), highlighting the interaction of linker Pro249 with SH3 Trp118 and Trp129. Ligand binding (top panel) is predicted to displace this regulatory interaction, leading to kinase activation. Lower panel: The lowest energy pose of compound 142 is shown docked to a snapshot of an MD simulation of the SH3 domain in the absence of the linker. The position of the 142 ligand is similar (within 1.5 Å RMSD) to that in the SH3 domain of N32L (top), except that the ligand contacts Glu117 rather than linker residues Gly246 and Val247. Without the linker, the potential hydrophobic stabilization of the 142 piperidine group is also lost.
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pone.0133590.g010: Molecular dynamics (MD) and molecular docking predict binding of compound 142 to the ABL SH3:linker interface.Top: The lowest energy pose of the ligand (compound 142; carbon atoms rendered in green) is shown docked to a snapshot of an MD simulation of the ABL N32L structure. SH3 domain residues predicted to contribute to ligand binding include Asn97, Thr98, Asn115, Trp118, and Trp129 (carbons in red). The backbone of the linker is shown as an orange ribbon, with Gly246, Val247, Pro249 and Trp254 predicted to contribute to the binding pocket. One of the piperidine groups of compound 142 makes hydrophobic contacts with linker Pro249 and Trp254, while the pyrimido-pyrimidine scaffold of compound 142 is π-stacking with Trp118. Middle panel: Model of the SH3:linker interface in the N32L region based on the crystal structure of the downregulated ABL core (PDB:2FO0), highlighting the interaction of linker Pro249 with SH3 Trp118 and Trp129. Ligand binding (top panel) is predicted to displace this regulatory interaction, leading to kinase activation. Lower panel: The lowest energy pose of compound 142 is shown docked to a snapshot of an MD simulation of the SH3 domain in the absence of the linker. The position of the 142 ligand is similar (within 1.5 Å RMSD) to that in the SH3 domain of N32L (top), except that the ligand contacts Glu117 rather than linker residues Gly246 and Val247. Without the linker, the potential hydrophobic stabilization of the 142 piperidine group is also lost.

Mentions: Data presented in the previous sections demonstrate that compound 142 interacts with the regulatory N32L region of ABL, resulting in a decrease in thermal stability and a concomitant increase in kinase activity. We used molecular dynamics (MD) simulations to explore the dynamics of the N32L region used in the assays. To model the effect of the linker being displaced from the SH3 domain, we manually pulled the linker a short distance away from the SH3 domain prior to the simulation (see Materials and Methods). After approximately 20 ns, the linker reconnected with the SH3 domain through the interaction of linker Pro249 and SH3 Trp118. To explore possible binding sites for this compound on the N32L region of the ABL kinase core, we used the computational docking tool smina [32] to dock 142 to snapshots of the simulation prior to the reconnection of the SH3:linker interface. As shown in Fig 10 (top panel), compound 142 fits into a surface pocket defined by the SH3:linker interface in the N32L protein. This predicted binding site involves an aromatic interaction between the pyrimido-pyrimidine moiety of 142 and the indole side chain of SH3 Trp118, as well as polar contacts involving all four hydroxyl groups on the ligand. This aromatic interaction is consistent with probe peptide displacement as well as the observed decrease in the FP signal produced by the W118A mutation (Fig 5C). Two of the hydroxyl groups of 142 make potential hydrogen bonds with the side and main chains of SH3 Asn97 as well as the side chain of Thr98. The other two hydroxyl groups of 142 form hydrogen bonds with the main chain carbonyls of linker Gly246 and Val247 as well as the side chain of SH3 Asn115. In addition, one of the piperidine groups of compound 142 makes hydrophobic contacts with SH3 Trp129, while the other approaches the side chains of linker residues Pro249 and Trp254. Note that in the crystal structure of the fully assembled, downregulated conformation of the ABL core, linker Pro249 inserts between SH3 Trp118 and Trp129 (Fig 10, middle panel); displacement of this regulatory contact by compound 142 binding may contribute to kinase activation.


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)

Molecular dynamics (MD) and molecular docking predict binding of compound 142 to the ABL SH3:linker interface.Top: The lowest energy pose of the ligand (compound 142; carbon atoms rendered in green) is shown docked to a snapshot of an MD simulation of the ABL N32L structure. SH3 domain residues predicted to contribute to ligand binding include Asn97, Thr98, Asn115, Trp118, and Trp129 (carbons in red). The backbone of the linker is shown as an orange ribbon, with Gly246, Val247, Pro249 and Trp254 predicted to contribute to the binding pocket. One of the piperidine groups of compound 142 makes hydrophobic contacts with linker Pro249 and Trp254, while the pyrimido-pyrimidine scaffold of compound 142 is π-stacking with Trp118. Middle panel: Model of the SH3:linker interface in the N32L region based on the crystal structure of the downregulated ABL core (PDB:2FO0), highlighting the interaction of linker Pro249 with SH3 Trp118 and Trp129. Ligand binding (top panel) is predicted to displace this regulatory interaction, leading to kinase activation. Lower panel: The lowest energy pose of compound 142 is shown docked to a snapshot of an MD simulation of the SH3 domain in the absence of the linker. The position of the 142 ligand is similar (within 1.5 Å RMSD) to that in the SH3 domain of N32L (top), except that the ligand contacts Glu117 rather than linker residues Gly246 and Val247. Without the linker, the potential hydrophobic stabilization of the 142 piperidine group is also lost.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133590.g010: Molecular dynamics (MD) and molecular docking predict binding of compound 142 to the ABL SH3:linker interface.Top: The lowest energy pose of the ligand (compound 142; carbon atoms rendered in green) is shown docked to a snapshot of an MD simulation of the ABL N32L structure. SH3 domain residues predicted to contribute to ligand binding include Asn97, Thr98, Asn115, Trp118, and Trp129 (carbons in red). The backbone of the linker is shown as an orange ribbon, with Gly246, Val247, Pro249 and Trp254 predicted to contribute to the binding pocket. One of the piperidine groups of compound 142 makes hydrophobic contacts with linker Pro249 and Trp254, while the pyrimido-pyrimidine scaffold of compound 142 is π-stacking with Trp118. Middle panel: Model of the SH3:linker interface in the N32L region based on the crystal structure of the downregulated ABL core (PDB:2FO0), highlighting the interaction of linker Pro249 with SH3 Trp118 and Trp129. Ligand binding (top panel) is predicted to displace this regulatory interaction, leading to kinase activation. Lower panel: The lowest energy pose of compound 142 is shown docked to a snapshot of an MD simulation of the SH3 domain in the absence of the linker. The position of the 142 ligand is similar (within 1.5 Å RMSD) to that in the SH3 domain of N32L (top), except that the ligand contacts Glu117 rather than linker residues Gly246 and Val247. Without the linker, the potential hydrophobic stabilization of the 142 piperidine group is also lost.
Mentions: Data presented in the previous sections demonstrate that compound 142 interacts with the regulatory N32L region of ABL, resulting in a decrease in thermal stability and a concomitant increase in kinase activity. We used molecular dynamics (MD) simulations to explore the dynamics of the N32L region used in the assays. To model the effect of the linker being displaced from the SH3 domain, we manually pulled the linker a short distance away from the SH3 domain prior to the simulation (see Materials and Methods). After approximately 20 ns, the linker reconnected with the SH3 domain through the interaction of linker Pro249 and SH3 Trp118. To explore possible binding sites for this compound on the N32L region of the ABL kinase core, we used the computational docking tool smina [32] to dock 142 to snapshots of the simulation prior to the reconnection of the SH3:linker interface. As shown in Fig 10 (top panel), compound 142 fits into a surface pocket defined by the SH3:linker interface in the N32L protein. This predicted binding site involves an aromatic interaction between the pyrimido-pyrimidine moiety of 142 and the indole side chain of SH3 Trp118, as well as polar contacts involving all four hydroxyl groups on the ligand. This aromatic interaction is consistent with probe peptide displacement as well as the observed decrease in the FP signal produced by the W118A mutation (Fig 5C). Two of the hydroxyl groups of 142 make potential hydrogen bonds with the side and main chains of SH3 Asn97 as well as the side chain of Thr98. The other two hydroxyl groups of 142 form hydrogen bonds with the main chain carbonyls of linker Gly246 and Val247 as well as the side chain of SH3 Asn115. In addition, one of the piperidine groups of compound 142 makes hydrophobic contacts with SH3 Trp129, while the other approaches the side chains of linker residues Pro249 and Trp254. Note that in the crystal structure of the fully assembled, downregulated conformation of the ABL core, linker Pro249 inserts between SH3 Trp118 and Trp129 (Fig 10, middle panel); displacement of this regulatory contact by compound 142 binding may contribute to kinase activation.

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