Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck
The eight mammalian Src-family tyrosine kinases are dynamic, multi-domain structures, which adopt distinct “open” and “closed” conformations. In the closed conformation, the regulatory SH3 and SH2 domains pack against the back of the kinase domain, providing allosteric control of kinase activity. Sm...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2019-11-01
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| Series: | Frontiers in Chemistry |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fchem.2019.00822/full |
| _version_ | 1811339755527340032 |
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| author | Heather R. Dorman David Close Bentley M. Wingert Carlos J. Camacho Paul A. Johnston Thomas E. Smithgall |
| author_facet | Heather R. Dorman David Close Bentley M. Wingert Carlos J. Camacho Paul A. Johnston Thomas E. Smithgall |
| author_sort | Heather R. Dorman |
| collection | DOAJ |
| description | The eight mammalian Src-family tyrosine kinases are dynamic, multi-domain structures, which adopt distinct “open” and “closed” conformations. In the closed conformation, the regulatory SH3 and SH2 domains pack against the back of the kinase domain, providing allosteric control of kinase activity. Small molecule ligands that engage the regulatory SH3-SH2 region have the potential to modulate Src-family kinase activity for therapeutic advantage. Here we describe an HTS-compatible fluorescence polarization assay to identify small molecules that interact with the unique-SH3-SH2-linker (U32L) region of Hck, a Src-family member expressed exclusively in cells of myeloid lineage. Hck has significant potential as a drug target in acute myeloid leukemia, an aggressive form of cancer with substantial unmet clinical need. The assay combines recombinant Hck U32L protein with a fluorescent probe peptide that binds to the SH3 domain in U32L, resulting in an increased FP signal. Library compounds that interact with the U32L protein and interfere with probe binding reduce the FP signal, scoring as hits. Automated 384-well high-throughput screening of 60,000 compounds yielded Z'-factor coefficients > 0.7 across nearly 200 assay plates, and identified a series of hit compounds with a shared pyrimidine diamine substructure. Surface plasmon resonance assays confirmed direct binding of hit compounds to the Hck U32L target protein as well as near-full-length Hck. Binding was not observed with the individual SH3 and SH2 domains, demonstrating that these compounds recognize a specific three-dimensional conformation of the regulatory regions. This conclusion is supported by computational docking studies, which predict ligand contacts with a pocket formed by the juxtaposition of the SH3 domain, the SH3-SH2 domain connector, and the SH2-kinase linker. Each of the four validated hits stimulated recombinant, near-full-length Hck activity in vitro, providing evidence for allosteric effects on the kinase domain. These results provide a path to discovery and development of chemical scaffolds to target the regulatory regions of Hck and other Src family kinases as a new approach to pharmacological kinase control. |
| first_indexed | 2024-04-13T18:31:19Z |
| format | Article |
| id | doaj.art-3a76ff033d804132b1fb8dd3497204eb |
| institution | Directory Open Access Journal |
| issn | 2296-2646 |
| language | English |
| last_indexed | 2024-04-13T18:31:19Z |
| publishDate | 2019-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Chemistry |
| spelling | doaj.art-3a76ff033d804132b1fb8dd3497204eb2022-12-22T02:35:05ZengFrontiers Media S.A.Frontiers in Chemistry2296-26462019-11-01710.3389/fchem.2019.00822487009Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, HckHeather R. Dorman0David Close1Bentley M. Wingert2Carlos J. Camacho3Paul A. Johnston4Thomas E. Smithgall5Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, United StatesDepartment of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA, United StatesDepartment of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United StatesDepartment of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United StatesDepartment of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA, United StatesDepartment of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, United StatesThe eight mammalian Src-family tyrosine kinases are dynamic, multi-domain structures, which adopt distinct “open” and “closed” conformations. In the closed conformation, the regulatory SH3 and SH2 domains pack against the back of the kinase domain, providing allosteric control of kinase activity. Small molecule ligands that engage the regulatory SH3-SH2 region have the potential to modulate Src-family kinase activity for therapeutic advantage. Here we describe an HTS-compatible fluorescence polarization assay to identify small molecules that interact with the unique-SH3-SH2-linker (U32L) region of Hck, a Src-family member expressed exclusively in cells of myeloid lineage. Hck has significant potential as a drug target in acute myeloid leukemia, an aggressive form of cancer with substantial unmet clinical need. The assay combines recombinant Hck U32L protein with a fluorescent probe peptide that binds to the SH3 domain in U32L, resulting in an increased FP signal. Library compounds that interact with the U32L protein and interfere with probe binding reduce the FP signal, scoring as hits. Automated 384-well high-throughput screening of 60,000 compounds yielded Z'-factor coefficients > 0.7 across nearly 200 assay plates, and identified a series of hit compounds with a shared pyrimidine diamine substructure. Surface plasmon resonance assays confirmed direct binding of hit compounds to the Hck U32L target protein as well as near-full-length Hck. Binding was not observed with the individual SH3 and SH2 domains, demonstrating that these compounds recognize a specific three-dimensional conformation of the regulatory regions. This conclusion is supported by computational docking studies, which predict ligand contacts with a pocket formed by the juxtaposition of the SH3 domain, the SH3-SH2 domain connector, and the SH2-kinase linker. Each of the four validated hits stimulated recombinant, near-full-length Hck activity in vitro, providing evidence for allosteric effects on the kinase domain. These results provide a path to discovery and development of chemical scaffolds to target the regulatory regions of Hck and other Src family kinases as a new approach to pharmacological kinase control.https://www.frontiersin.org/article/10.3389/fchem.2019.00822/fullSrc-family kinaseallosteric actionSH2 and SH3 domainsHck kinasehigh throughput screeningfluorescence polarization assay |
| spellingShingle | Heather R. Dorman David Close Bentley M. Wingert Carlos J. Camacho Paul A. Johnston Thomas E. Smithgall Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck Frontiers in Chemistry Src-family kinase allosteric action SH2 and SH3 domains Hck kinase high throughput screening fluorescence polarization assay |
| title | Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck |
| title_full | Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck |
| title_fullStr | Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck |
| title_full_unstemmed | Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck |
| title_short | Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck |
| title_sort | discovery of non peptide small molecule allosteric modulators of the src family kinase hck |
| topic | Src-family kinase allosteric action SH2 and SH3 domains Hck kinase high throughput screening fluorescence polarization assay |
| url | https://www.frontiersin.org/article/10.3389/fchem.2019.00822/full |
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