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

FP assay for small molecule modulators of ABL kinase function.A) Crystal structure of the auto-inhibited ABL core (PDB: 2FO0) [11]. Key features include the N-cap, SH3 and SH2 domains, the SH2-kinase linker, and the kinase domain. The disordered portion of the N-cap is indicated by the dotted line. The N-terminal portion of the N-cap is myristoylated and engages a deep pocket in the kinase domain C-lobe. B) Cartoon depiction of the intramolecular interactions regulating assembly of the downregulated ABL core. Note that the linker forms a polyproline helix that binds in cis to the SH3 domain. C) Fluorescence polarization (FP) assay. The FP assay combines a recombinant ABL Ncap-SH3-SH2-linker (N32L) protein and a SH3-binding peptide probe labeled with a fluorescent moiety (F). The probe peptide binds the SH3 domain in the ABL N32L protein, resulting in an FP signal. Small molecules (S) may bind to the SH3 domain and block probe peptide binding directly; such molecules would be expected to disrupt SH3:linker interaction (case 1). Alternatively, small molecules may stabilize SH3:linker interaction, making the SH3 domain inaccessible to the probe peptide (case 2). In either case, small molecule binding is predicted to result in a loss of the FP signal.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133590.g001: FP assay for small molecule modulators of ABL kinase function.A) Crystal structure of the auto-inhibited ABL core (PDB: 2FO0) [11]. Key features include the N-cap, SH3 and SH2 domains, the SH2-kinase linker, and the kinase domain. The disordered portion of the N-cap is indicated by the dotted line. The N-terminal portion of the N-cap is myristoylated and engages a deep pocket in the kinase domain C-lobe. B) Cartoon depiction of the intramolecular interactions regulating assembly of the downregulated ABL core. Note that the linker forms a polyproline helix that binds in cis to the SH3 domain. C) Fluorescence polarization (FP) assay. The FP assay combines a recombinant ABL Ncap-SH3-SH2-linker (N32L) protein and a SH3-binding peptide probe labeled with a fluorescent moiety (F). The probe peptide binds the SH3 domain in the ABL N32L protein, resulting in an FP signal. Small molecules (S) may bind to the SH3 domain and block probe peptide binding directly; such molecules would be expected to disrupt SH3:linker interaction (case 1). Alternatively, small molecules may stabilize SH3:linker interaction, making the SH3 domain inaccessible to the probe peptide (case 2). In either case, small molecule binding is predicted to result in a loss of the FP signal.

Mentions: The growing problem of imatininb resistance in BCR-ABL has fueled efforts to identify compounds that work outside of the kinase active site. Such compounds offer advantages in terms of enhanced specificity, because they have the potential to exploit non-conserved regulatory features unique to ABL that persist to some extent in BCR-ABL as well [8]. The kinase activity of ABL is tightly regulated in vivo by an auto-inhibitory mechanism. The ABL ‘core’ region, which includes a myristoylated N-terminal ‘cap’ (N-cap), SH3 and SH2 domains, an SH2-kinase linker and the kinase domain, is both necessary and sufficient for ABL auto-inhibition [9]. Subsequent X-ray crystal structures of the ABL core revealed three critical intramolecular interactions that regulate kinase activity [10–12] (Fig 1A and 1B). First, the SH2-kinase linker forms a polyproline type II helix that binds to the SH3 domain, forming an interface between the SH3 domain and the N-lobe of the kinase domain. Second, the SH2 domain interacts with the back of the kinase domain C-lobe through an extensive network of hydrogen bonds. Aromatic interactions between the side chains of SH2 Tyr158 and kinase domain Tyr361 also help to stabilize this interaction (see Panjarian et al. for an explanation of the ABL amino acid numbering scheme [13]). Finally, the myristoylated N-cap binds a deep hydrophobic pocket in the C-lobe of the kinase domain, clamping the SH3 and SH2 domains against the back of the kinase domain. Small molecules that occupy the myristic acid binding site in the C-lobe of the kinase domain have proven to be effective allosteric inhibitors of BCR-ABL function [14,15].


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)

FP assay for small molecule modulators of ABL kinase function.A) Crystal structure of the auto-inhibited ABL core (PDB: 2FO0) [11]. Key features include the N-cap, SH3 and SH2 domains, the SH2-kinase linker, and the kinase domain. The disordered portion of the N-cap is indicated by the dotted line. The N-terminal portion of the N-cap is myristoylated and engages a deep pocket in the kinase domain C-lobe. B) Cartoon depiction of the intramolecular interactions regulating assembly of the downregulated ABL core. Note that the linker forms a polyproline helix that binds in cis to the SH3 domain. C) Fluorescence polarization (FP) assay. The FP assay combines a recombinant ABL Ncap-SH3-SH2-linker (N32L) protein and a SH3-binding peptide probe labeled with a fluorescent moiety (F). The probe peptide binds the SH3 domain in the ABL N32L protein, resulting in an FP signal. Small molecules (S) may bind to the SH3 domain and block probe peptide binding directly; such molecules would be expected to disrupt SH3:linker interaction (case 1). Alternatively, small molecules may stabilize SH3:linker interaction, making the SH3 domain inaccessible to the probe peptide (case 2). In either case, small molecule binding is predicted to result in a loss of the FP signal.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133590.g001: FP assay for small molecule modulators of ABL kinase function.A) Crystal structure of the auto-inhibited ABL core (PDB: 2FO0) [11]. Key features include the N-cap, SH3 and SH2 domains, the SH2-kinase linker, and the kinase domain. The disordered portion of the N-cap is indicated by the dotted line. The N-terminal portion of the N-cap is myristoylated and engages a deep pocket in the kinase domain C-lobe. B) Cartoon depiction of the intramolecular interactions regulating assembly of the downregulated ABL core. Note that the linker forms a polyproline helix that binds in cis to the SH3 domain. C) Fluorescence polarization (FP) assay. The FP assay combines a recombinant ABL Ncap-SH3-SH2-linker (N32L) protein and a SH3-binding peptide probe labeled with a fluorescent moiety (F). The probe peptide binds the SH3 domain in the ABL N32L protein, resulting in an FP signal. Small molecules (S) may bind to the SH3 domain and block probe peptide binding directly; such molecules would be expected to disrupt SH3:linker interaction (case 1). Alternatively, small molecules may stabilize SH3:linker interaction, making the SH3 domain inaccessible to the probe peptide (case 2). In either case, small molecule binding is predicted to result in a loss of the FP signal.
Mentions: The growing problem of imatininb resistance in BCR-ABL has fueled efforts to identify compounds that work outside of the kinase active site. Such compounds offer advantages in terms of enhanced specificity, because they have the potential to exploit non-conserved regulatory features unique to ABL that persist to some extent in BCR-ABL as well [8]. The kinase activity of ABL is tightly regulated in vivo by an auto-inhibitory mechanism. The ABL ‘core’ region, which includes a myristoylated N-terminal ‘cap’ (N-cap), SH3 and SH2 domains, an SH2-kinase linker and the kinase domain, is both necessary and sufficient for ABL auto-inhibition [9]. Subsequent X-ray crystal structures of the ABL core revealed three critical intramolecular interactions that regulate kinase activity [10–12] (Fig 1A and 1B). First, the SH2-kinase linker forms a polyproline type II helix that binds to the SH3 domain, forming an interface between the SH3 domain and the N-lobe of the kinase domain. Second, the SH2 domain interacts with the back of the kinase domain C-lobe through an extensive network of hydrogen bonds. Aromatic interactions between the side chains of SH2 Tyr158 and kinase domain Tyr361 also help to stabilize this interaction (see Panjarian et al. for an explanation of the ABL amino acid numbering scheme [13]). Finally, the myristoylated N-cap binds a deep hydrophobic pocket in the C-lobe of the kinase domain, clamping the SH3 and SH2 domains against the back of the kinase domain. Small molecules that occupy the myristic acid binding site in the C-lobe of the kinase domain have proven to be effective allosteric inhibitors of BCR-ABL function [14,15].

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