Abstract
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.
Highlights
Acute myeloid leukemia (AML) is a hematologic cancer correlated with advancing age and often poor prognosis (Dohner et al, 2015; Khwaja et al, 2016)
We report the development of an automated HTS assay for the discovery of small molecule modulators of the Srcfamily kinase, Hck
We identified a series of ligands for the Hck regulatory region that share a common pyrimidine diamine scaffold based on their ability to inhibit the FP signal resulting from the binding of a peptide probe to a Hck U32L target protein
Summary
Acute myeloid leukemia (AML) is a hematologic cancer correlated with advancing age and often poor prognosis (Dohner et al, 2015; Khwaja et al, 2016). Two classes of mutations are associated with ligand-independent Flt activation in AML. These include internal tandem duplications (ITDs) in the regulatory juxta-membrane domain of the kinase, and activating point mutations within the kinase domain (Gu et al, 2011; Konig and Levis, 2015). More selective inhibitors, including quizartinib, crenolanib, and gilteritinib (Perl et al, 2017), show enhanced specificity for the Flt kinase domain and improved efficacy as single agents (Zarrinkar et al, 2009; Zimmerman et al, 2013). Inhibitor resistance, often involving acquired mutations in the Flt kinase domain, may limit their effectiveness (Pratz and Luger, 2014; Sudhindra and Smith, 2014; Konig and Levis, 2015)
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