A Discovery Strategy for Selective Inhibitors of c‐Src in Complex with the Focal Adhesion Kinase SH3/SH2‐binding Region

Moroco, Jamie A.; Baumgartner, Matthew P.; Rust, Heather L.; Choi, Hwan Geun; Hur, Wooyoung; Gray, Nathanael S.; Camacho, Carlos J.; Smithgall, Thomas E.

doi:10.1111/cbdd.12473

Cited by 9 publications

(12 citation statements)

References 54 publications

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“…As positive controls, we also monitored the interaction of peptide ligands for the SH3 and SH2 domains. These included the VSL12 peptide for SH3 (same sequence as the peptide probe used for the FP assay), and a tyrosine phosphopeptide known to bind to the Hck SH2 domain (“pYEEI” peptide) (Moroco et al, 2015).…”

Section: Resultsmentioning

confidence: 99%

“…Each of the small molecule ligands shown in Figure 4 were docked onto the X-ray crystal structure of near-full-length Hck (PDB: 1QCF) using the application Smina (Koes et al, 2013), a refinement of AutoDock Vina previously used by our group to interrogate binding sites for competitive inhibitors and allosteric ligands for other non-receptor protein-tyrosine kinases (Grover et al, 2015; Moroco et al, 2015). Docking of all four compounds revealed a predicted binding pocket formed by the juxtaposition of the SH3 domain, the SH3-SH2 connector, and the SH2-kinase linker that is distinct from the ATP-binding site in the kinase domain (Figure 6, top).…”

Section: Resultsmentioning

confidence: 99%

“…As illustrated in Figure 1, small molecules that interact with this region of the kinase may have several impacts on overall kinase structure and function. One possibility is that the ligand may destabilize the closed, inactive conformation, resulting in kinase activation; previous studies have demonstrated this outcome using short peptide ligands that bind to the SH3 or SH2 domains or to both simultaneously (Moroco et al, 2014, 2015). Alternatively, the ligand may stabilize the regulatory interactions observed in the crystal structures of near-full-length Hck and Src, thus representing allosteric kinase inhibitors.…”

Section: Introductionmentioning

confidence: 99%

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Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck

Dorman

Wingert

et al. 2019

Front. Chem.

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck

Dorman

Wingert

et al. 2019

Front. Chem.

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“…The discovery that the SH2 domain is indispensable for SRC FA targeting is surprising, because both the SH2 domain and the SH3 domain have been considered to act as SRC protein interaction modules 55 that are able to direct SRC transiently to FAs. In cells expressing SRC-family kinases, a point mutation in the SH2 domain (R175L) of SRC251 (with a deletion mutation in kinase domain of SRC to avoid intramolecular interaction) still allows localization to within FAs 12 , which suggests that there may be a contribution of the SH3 domain or some other residues within the SH2 domain to SRC FA recruitment.…”

Section: Discussionmentioning

confidence: 99%

Focal adhesion kinase-dependent focal adhesion recruitment of SH2 domains directs SRC into focal adhesions to regulate cell adhesion and migration

Wu¹,

Chen²,

Kuo³

et al. 2015

Sci Rep

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Directed cell migration requires dynamical control of the protein complex within focal adhesions (FAs) and this control is regulated by signaling events involving tyrosine phosphorylation. We screened the SH2 domains present in tyrosine-specific kinases and phosphatases found within FAs, including SRC, SHP1 and SHP2, and examined whether these enzymes transiently target FAs via their SH2 domains. We found that the SRC_SH2 domain and the SHP2_N-SH2 domain are associated with FAs, but only the SRC_SH2 domain is able to be regulated by focal adhesion kinase (FAK). The FAK-dependent association of the SRC_SH2 domain is necessary and sufficient for SRC FA targeting. When the targeting of SRC into FAs is inhibited, there is significant suppression of SRC-mediated phosphorylation of paxillin and FAK; this results in an inhibition of FA formation and maturation and a reduction in cell migration. This study reveals an association between FAs and the SRC_SH2 domain as well as between FAs and the SHP2_N-SH2 domains. This supports the hypothesis that the FAK-regulated SRC_SH2 domain plays an important role in directing SRC into FAs and that this SRC-mediated FA signaling drives cell migration.

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“…1) (17). Previous studies have shown that peptide ligands for the SH3 and SH2 domains can displace these regulatory interactions in recombinant Hck and other Src family members, resulting in stimulation of kinase activity in vitro (22,34). We therefore used a similar approach to investigate the roles of SH3 and SH2 in Fgr kinase domain regulation.…”

Section: Fgr Kinase Activity Is Independent Of Sh3 and Sh2 Domain Conmentioning

confidence: 99%

The Src family kinase Fgr is a transforming oncoprotein that functions independently of SH3-SH2 domain regulation

et al. 2018

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Fgr is a member of the Src family of nonreceptor tyrosine kinases, which are overexpressed and constitutively active in many human cancers. Fgr expression is restricted to myeloid hematopoietic cells and is markedly increased in a subset of bone marrow samples from patients with acute myeloid leukemia (AML). Here, we investigated the oncogenic potential of Fgr using Rat-2 fibroblasts that do not express the kinase. Expression of either wild-type or regulatory tail-mutant constructs of Fgr promoted cellular transformation (inferred from colony formation in soft agar), which was accompanied by phosphorylation of the Fgr activation loop, suggesting that the kinase domain of Fgr functions independently of regulation by its noncatalytic SH3-SH2 region. Unlike other family members, recombinant Fgr was not activated by SH3-SH2 domain ligands. However, hydrogen-deuterium exchange mass spectrometry data suggested that the regulatory SH3 and SH2 domains packed against the back of the kinase domain in a Src-like manner. Sequence alignment showed that the activation loop of Fgr was distinct from that of all other Src family members, with proline rather than alanine at the +2 position relative to the activation loop tyrosine. Substitution of the activation loop of Fgr with the sequence from Src partially inhibited kinase activity and suppressed colony formation. Last, Fgr expression enhanced the sensitivity of human myeloid progenitor cells to the cytokine GM-CSF. Because its kinase domain is not sensitive to SH3-SH2–mediated control, simple overexpression of Fgr without mutation may contribute to oncogenic transformation in AML and other blood cancers.

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A Discovery Strategy for Selective Inhibitors of c‐Src in Complex with the Focal Adhesion Kinase SH3/SH2‐binding Region

Cited by 9 publications

References 54 publications

Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck

Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck

Focal adhesion kinase-dependent focal adhesion recruitment of SH2 domains directs SRC into focal adhesions to regulate cell adhesion and migration

The Src family kinase Fgr is a transforming oncoprotein that functions independently of SH3-SH2 domain regulation

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