Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function
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{"title"=>"Fluorescence polarization screening assays for small molecule allosteric modulators of ABL kinase function", "type"=>"journal", "authors"=>[{"first_name"=>"Prerna", "last_name"=>"Grover", "scopus_author_id"=>"56800069700"}, {"first_name"=>"Haibin", "last_name"=>"Shi", "scopus_author_id"=>"56844154000"}, {"first_name"=>"Matthew", "last_name"=>"Baumgartner", "scopus_author_id"=>"55840148500"}, {"first_name"=>"Carlos J.", "last_name"=>"Camacho", "scopus_author_id"=>"7006303658"}, {"first_name"=>"Thomas E.", "last_name"=>"Smithgall", "scopus_author_id"=>"7006513776"}], "year"=>2015, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "pui"=>"605997166", "doi"=>"10.1371/journal.pone.0133590", "sgr"=>"84941710722", "scopus"=>"2-s2.0-84941710722", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"26222440"}, "id"=>"91db9648-ac96-38a4-8f57-6b564c6a9de3", "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.", "link"=>"http://www.mendeley.com/research/fluorescence-polarization-screening-assays-small-molecule-allosteric-modulators-abl-kinase-function", "reader_count"=>13, "reader_count_by_academic_status"=>{"Student > Doctoral Student"=>1, "Researcher"=>3, "Student > Ph. D. Student"=>6, "Student > Postgraduate"=>1, "Other"=>1, "Student > Bachelor"=>1}, "reader_count_by_user_role"=>{"Student > Doctoral Student"=>1, "Researcher"=>3, "Student > Ph. D. Student"=>6, "Student > Postgraduate"=>1, "Other"=>1, "Student > Bachelor"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>3, "Medicine and Dentistry"=>1, "Agricultural and Biological Sciences"=>2, "Chemistry"=>7}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Chemistry"=>{"Chemistry"=>7}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>2}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2193816"], "description"=>"<p>A) A library of 1200 FDA-approved compounds was screened using the ABL N32L protein (25 μg/well) and the p41 probe peptide (50 nM) in the FP assay. The solid lines correspond to the mean FP signals for the wild type (WT) and SH3 mutant (W118A) control N32L proteins across all assay plates, with the dotted lines indicating three standard deviations from the means (± 3σ). Compounds were screened at 10 μM and each FP signal is represented as an individual circle. Five putative hit compounds were identified (green circles). B) The five potential hit compounds were re-tested in quadruplicate at 10 μM vs. the DMSO control under FP screening assay conditions, and the mean FP values are shown ± SE. Four of these compounds significantly inhibited the FP signal relative to the DMSO control as indicated by the asterisk (p < 0.05; 2-tailed t-test). The dotted line shows the FP value three standard deviations below the DMSO control FP signal (-3σ).</p>", "links"=>[], "tags"=>["control N 32L proteins", "ABL N 32L protein", "ABL Kinase Function", "SH 3 domain", "Fluorescence Polarization Screening Assays", "probe peptide", "ABL kinase activity", "assay development experiments", "Small Molecule Allosteric Modulators", "SH 2 domains", "antithrombotic drug dipyridamole", "N 32L protein", "SH 3", "dph", "FP signal"], "article_id"=>1497121, "categories"=>["Biological Sciences"], "users"=>["Prerna Grover", "Haibin Shi", "Matthew Baumgartner", "Carlos J. Camacho", "Thomas E. Smithgall"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0133590.g006", "stats"=>{"downloads"=>0, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Pilot_screen_identifies_inhibitors_of_p41_interaction_with_the_ABL_N32L_protein_/1497121", "title"=>"Pilot screen identifies inhibitors of p41 interaction with the ABL N32L protein.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-29 03:40:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2193817"], "description"=>"<p>A) Differential scanning fluorimetry assays were performed on the ABL N32L protein in the presence of the four confirmed hit compounds as described under Materials and Methods. The average change in the mid-point of the thermal melt profile (ΔT<sub>m</sub>) relative to the T<sub>m</sub> obtained with the N32L protein in the presence of the DMSO carrier solvent is plotted on the Y-axis ± SE (n = 2). B) Surface plasmon resonance was performed with the ABL N32L protein immobilized on the biosensor chip and compound 142 as analyte. Responses were recorded for the four compound concentrations shown, and the flow path was switched back to buffer after 180 s to induce dissociation (<i>arrow</i>). The resulting sensorgrams (black lines) were fit by a 1:1 Langmuir binding model (red lines) to generate kinetic constants. C) Compound 142 inhibits p41 peptide binding to the ABL N32L and SH3 proteins in the FP assay. Compound 142 was added to N32L and SH3 FP assays over the range of concentrations shown, and the resulting FP signals are presented as the mean ± SE. Significant inhibition for both N32L and SH3 was observed at 3 and 10 μM (*p < 0.05 by 2-tailed t-test). D) Chemical structure of compound 142 (dipyridamole).</p>", "links"=>[], "tags"=>["control N 32L proteins", "ABL N 32L protein", "ABL Kinase Function", "SH 3 domain", "Fluorescence Polarization Screening Assays", "probe peptide", "ABL kinase activity", "assay development experiments", "Small Molecule Allosteric Modulators", "SH 2 domains", "antithrombotic drug dipyridamole", "N 32L protein", "SH 3", "dph", "FP signal"], "article_id"=>1497122, "categories"=>["Biological Sciences"], "users"=>["Prerna Grover", "Haibin Shi", "Matthew Baumgartner", "Carlos J. Camacho", "Thomas E. Smithgall"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0133590.g007", "stats"=>{"downloads"=>3, "page_views"=>25, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Hit_compound_142_interacts_directly_with_the_ABL_N32L_protein_/1497122", "title"=>"Hit compound 142 interacts directly with the ABL N32L protein.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-29 03:40:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2193820"], "description"=>"<p><i>Top</i>: 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 <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133590#sec002\" target=\"_blank\">Materials and Methods</a>). 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). <i>Bottom</i>: 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 K<sub>m</sub> 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).</p>", "links"=>[], "tags"=>["control N 32L proteins", "ABL N 32L protein", "ABL Kinase Function", "SH 3 domain", "Fluorescence Polarization Screening Assays", "probe peptide", "ABL kinase activity", "assay development experiments", "Small Molecule Allosteric Modulators", "SH 2 domains", "antithrombotic drug dipyridamole", "N 32L protein", "SH 3", "dph", "FP signal"], "article_id"=>1497125, "categories"=>["Biological Sciences"], "users"=>["Prerna Grover", "Haibin Shi", "Matthew Baumgartner", "Carlos J. Camacho", "Thomas E. Smithgall"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0133590.g008", "stats"=>{"downloads"=>0, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Compound_142_activates_the_ABL_kinase_core_in_vitro_/1497125", "title"=>"Compound 142 activates the ABL kinase core in vitro.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-29 03:40:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2193821"], "description"=>"<p>The wild type ABL kinase core was assayed in the presence of compound 142 and DPH at the indicated concentrations using a kinetic kinase assay (see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133590#sec002\" target=\"_blank\">Materials and Methods</a>). Reaction velocities are plotted as a function of compound concentrations. The resulting data were curve-fit to determine the EC<sub>50</sub>, K<sub>act</sub> and V<sub>max</sub> for each activator as described under Materials and Methods. Each of these parameters was determined in triplicate, and the mean values ± SE are presented in the table below the graph. The table also provides the ratio V<sub>max</sub>/K<sub>act</sub> as a measure of overall enhancement of catalytic efficiency in the presence of each of these two ABL agonists.</p>", "links"=>[], "tags"=>["control N 32L proteins", "ABL N 32L protein", "ABL Kinase Function", "SH 3 domain", "Fluorescence Polarization Screening Assays", "probe peptide", "ABL kinase activity", "assay development experiments", "Small Molecule Allosteric Modulators", "SH 2 domains", "antithrombotic drug dipyridamole", "N 32L protein", "SH 3", "dph", "FP signal"], "article_id"=>1497126, "categories"=>["Biological Sciences"], "users"=>["Prerna Grover", "Haibin Shi", "Matthew Baumgartner", "Carlos J. Camacho", "Thomas E. Smithgall"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0133590.g009", "stats"=>{"downloads"=>0, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Concentration_dependent_activation_of_the_ABL_kinase_core_protein_by_compound_142_/1497126", "title"=>"Concentration-dependent activation of the ABL kinase core protein by compound 142.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-29 03:40:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2193827"], "description"=>"<p><i>Top</i>: 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. <i>Middle panel</i>: 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. <i>Lower panel</i>: 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.</p>", "links"=>[], "tags"=>["control N 32L proteins", "ABL N 32L protein", "ABL Kinase Function", "SH 3 domain", "Fluorescence Polarization Screening Assays", "probe peptide", "ABL kinase activity", "assay development experiments", "Small Molecule Allosteric Modulators", "SH 2 domains", "antithrombotic drug dipyridamole", "N 32L protein", "SH 3", "dph", "FP signal"], "article_id"=>1497132, "categories"=>["Biological Sciences"], "users"=>["Prerna Grover", "Haibin Shi", "Matthew Baumgartner", "Carlos J. Camacho", "Thomas E. Smithgall"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0133590.g010", "stats"=>{"downloads"=>1, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Molecular_dynamics_MD_and_molecular_docking_predict_binding_of_compound_142_to_the_ABL_SH3_linker_interface_/1497132", "title"=>"Molecular dynamics (MD) and molecular docking predict binding of compound 142 to the ABL SH3:linker interface.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-29 03:40:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2193829"], "description"=>"<p><i>Top panels</i>: 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 <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133590#sec002\" target=\"_blank\">Materials and Methods</a>). Data are plotted as pmol ADP produced per ng kinase as a function of time. <i>Bottom</i>: 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 <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133590#pone.0133590.g010\" target=\"_blank\">Fig 10</a>). The type of interaction is indicated as side chain (s), main chain (m), or aromatic (a).</p>", "links"=>[], "tags"=>["control N 32L proteins", "ABL N 32L protein", "ABL Kinase Function", "SH 3 domain", "Fluorescence Polarization Screening Assays", "probe peptide", "ABL kinase activity", "assay development experiments", "Small Molecule Allosteric Modulators", "SH 2 domains", "antithrombotic drug dipyridamole", "N 32L protein", "SH 3", "dph", "FP signal"], "article_id"=>1497134, "categories"=>["Biological Sciences"], "users"=>["Prerna Grover", "Haibin Shi", "Matthew Baumgartner", "Carlos J. Camacho", "Thomas E. Smithgall"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0133590.g011", "stats"=>{"downloads"=>7, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Compound_142_fails_to_activate_downregulated_HCK_in_vitro_/1497134", "title"=>"Compound 142 fails to activate downregulated HCK in vitro.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-29 03:40:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2193830"], "description"=>"<p>Human embryonic kidney 293T cells were transfected with an expression vector for the wild type ABL core followed by treatment with compound 142 overnight over the range of concentrations shown. The experiment was performed in the absence or presence of the myristic acid binding pocket agonist DPH at a final concentration of 10 μM. ABL proteins were immunoprecipitated from clarified cell lysates, and analyzed by immunoblotting with phosphospecific antibodies to three regulatory sites: pTyr412 in the activation loop, pTyr89 in the SH3 domain, and pTyr245 in the SH2-kinase linker, as well as an ABL antibody to control for protein recovery. This experiment was repeated twice with comparable results.</p>", "links"=>[], "tags"=>["control N 32L proteins", "ABL N 32L protein", "ABL Kinase Function", "SH 3 domain", "Fluorescence Polarization Screening Assays", "probe peptide", "ABL kinase activity", "assay development experiments", "Small Molecule Allosteric Modulators", "SH 2 domains", "antithrombotic drug dipyridamole", "N 32L protein", "SH 3", "dph", "FP signal"], "article_id"=>1497135, "categories"=>["Biological Sciences"], "users"=>["Prerna Grover", "Haibin Shi", "Matthew Baumgartner", "Carlos J. Camacho", "Thomas E. Smithgall"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0133590.g012", "stats"=>{"downloads"=>12, "page_views"=>25, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Compound_142_cooperates_with_DPH_to_stimulate_ABL_core_autophosphorylation_in_cells_/1497135", "title"=>"Compound 142 cooperates with DPH to stimulate ABL core autophosphorylation in cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-29 03:40:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2193797"], "description"=>"<p>A) Crystal structure of the auto-inhibited ABL core (PDB: 2FO0) [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133590#pone.0133590.ref011\" target=\"_blank\">11</a>]. 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 <i>cis</i> 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.</p>", "links"=>[], "tags"=>["control N 32L proteins", "ABL N 32L protein", "ABL Kinase Function", "SH 3 domain", "Fluorescence Polarization Screening Assays", "probe peptide", "ABL kinase activity", "assay development experiments", "Small Molecule Allosteric Modulators", "SH 2 domains", "antithrombotic drug dipyridamole", "N 32L protein", "SH 3", "dph", "FP signal"], "article_id"=>1497109, "categories"=>["Biological Sciences"], "users"=>["Prerna Grover", "Haibin Shi", "Matthew Baumgartner", "Carlos J. Camacho", "Thomas E. Smithgall"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0133590.g001", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_FP_assay_for_small_molecule_modulators_of_ABL_kinase_function_/1497109", "title"=>"FP assay for small molecule modulators of ABL kinase function.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-29 03:40:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2193798"], "description"=>"<p>Wild type ABL N32L protein and the corresponding high-affinity linker (HAL) and W118A mutants were expressed in <i>E</i>. <i>coli</i> using the pET system [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133590#pone.0133590.ref040\" target=\"_blank\">40</a>] and purified by immobilized metal affinity chromatography. Protein purity and mass were verified by SDS polyacrylamide gel electrophoresis (A) and mass spectrometry (B).</p>", "links"=>[], "tags"=>["control N 32L proteins", "ABL N 32L protein", "ABL Kinase Function", "SH 3 domain", "Fluorescence Polarization Screening Assays", "probe peptide", "ABL kinase activity", "assay development experiments", "Small Molecule Allosteric Modulators", "SH 2 domains", "antithrombotic drug dipyridamole", "N 32L protein", "SH 3", "dph", "FP signal"], "article_id"=>1497110, "categories"=>["Biological Sciences"], "users"=>["Prerna Grover", "Haibin Shi", "Matthew Baumgartner", "Carlos J. Camacho", "Thomas E. Smithgall"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0133590.g002", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Recombinant_ABL_Ncap_SH3_SH2_linker_N32L_proteins_/1497110", "title"=>"Recombinant ABL Ncap-SH3-SH2-linker (N32L) proteins.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-29 03:40:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2193805"], "description"=>"<p>A) Sequences of the ABL SH3 binding peptides, p41, p40, p8, and 3BP-1, and their published binding affinities for the ABL SH3 domain [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133590#pone.0133590.ref020\" target=\"_blank\">20</a>,<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133590#pone.0133590.ref021\" target=\"_blank\">21</a>]. 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) [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133590#pone.0133590.ref021\" target=\"_blank\">21</a>]. 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) [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133590#pone.0133590.ref011\" target=\"_blank\">11</a>]. 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.</p>", "links"=>[], "tags"=>["control N 32L proteins", "ABL N 32L protein", "ABL Kinase Function", "SH 3 domain", "Fluorescence Polarization Screening Assays", "probe peptide", "ABL kinase activity", "assay development experiments", "Small Molecule Allosteric Modulators", "SH 2 domains", "antithrombotic drug dipyridamole", "N 32L protein", "SH 3", "dph", "FP signal"], "article_id"=>1497112, "categories"=>["Biological Sciences"], "users"=>["Prerna Grover", "Haibin Shi", "Matthew Baumgartner", "Carlos J. Camacho", "Thomas E. Smithgall"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0133590.g003", "stats"=>{"downloads"=>3, "page_views"=>23, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Peptide_and_linker_interactions_with_the_ABL_SH3_domain_/1497112", "title"=>"Peptide and linker interactions with the ABL SH3 domain.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-29 03:40:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2193809"], "description"=>"<p>A) To characterize the baseline FP signal, the 6-carboxy-fluorescein labeled probe peptides p41 (red), p40 (green), p8 (blue), and 3BP-1 (black) were serially diluted in the concentration range of 1–1000 nM. The FP signals (solid lines, left Y axis) and corresponding fluorescence intensities (dashed lines, right Y axis) were measured and plotted as a function of peptide concentration. Average values are shown ± SE from four measurements per condition. B) To test for probe peptide interaction with ABL N32L by FP, each peptide (50 nM) was incubated with the ABL N32L protein over the range of 0.08–25 μg/well. The resulting FP signals were corrected for baseline FP signal recorded in the absence of the N32L protein and plotted against the N32L protein concentration. Average FP values are shown ± SE from four measurements per condition; error bars are smaller than the diameter of some data points.</p>", "links"=>[], "tags"=>["control N 32L proteins", "ABL N 32L protein", "ABL Kinase Function", "SH 3 domain", "Fluorescence Polarization Screening Assays", "probe peptide", "ABL kinase activity", "assay development experiments", "Small Molecule Allosteric Modulators", "SH 2 domains", "antithrombotic drug dipyridamole", "N 32L protein", "SH 3", "dph", "FP signal"], "article_id"=>1497114, "categories"=>["Biological Sciences"], "users"=>["Prerna Grover", "Haibin Shi", "Matthew Baumgartner", "Carlos J. Camacho", "Thomas E. Smithgall"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0133590.g004", "stats"=>{"downloads"=>0, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Identification_of_p41_as_optimal_probe_peptide_for_the_ABL_N32L_FP_assay_/1497114", "title"=>"Identification of p41 as optimal probe peptide for the ABL N32L FP assay.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-29 03:40:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2193812"], "description"=>"<p>A) <i>The p41 FP probe binds the ABL N32L protein through the SH3 domain</i>. The p41 probe peptide (50 nM) was combined with wild type, HAL, and W118A ABL N32L proteins over the range of concentrations shown. The resulting FP signals were measured and plotted as a function of N32L protein concentration. B) <i>FP assay stability</i>. The p41 probe peptide (50 nM) was combined with the three ABL N32L proteins (12.8 μg/well) and FP signals were recorded over the time course shown. C) <i>DMSO tolerance</i>. FP assays consisting of the p41 probe peptide (50 nM) and each ABL N32L protein (25 μg/well) were incubated with the DMSO concentrations shown, and FP signals were recorded 1 h later. D) <i>Unlabeled peptide competition</i>. For the competition assay, the p41 probe peptide (50 nM) was mixed with unlabeled p41 peptide or a negative control peptide of unrelated sequence (QKEGERALPSIP) and similar length (Con) over the range of concentrations shown. The ABL N32L protein (20 μg/well) was then added, and FP signals were recorded. FP signals were corrected for the background p41 peptide FP signal and plotted as a function of the unlabeled peptide concentration. In all experiments (A through D), average FP values are shown ± SE from four measurements per condition.</p>", "links"=>[], "tags"=>["control N 32L proteins", "ABL N 32L protein", "ABL Kinase Function", "SH 3 domain", "Fluorescence Polarization Screening Assays", "probe peptide", "ABL kinase activity", "assay development experiments", "Small Molecule Allosteric Modulators", "SH 2 domains", "antithrombotic drug dipyridamole", "N 32L protein", "SH 3", "dph", "FP signal"], "article_id"=>1497117, "categories"=>["Biological Sciences"], "users"=>["Prerna Grover", "Haibin Shi", "Matthew Baumgartner", "Carlos J. Camacho", "Thomas E. Smithgall"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0133590.g005", "stats"=>{"downloads"=>2, "page_views"=>32, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ABL_N32L_FP_assay_development_and_optimization_/1497117", "title"=>"ABL N32L FP assay development and optimization.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-29 03:40:24"}

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{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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