HTS Assay Development for Discovery of Specific PYK2 Inhibitors

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DESCRIPTION (provided by applicant): Proline-rich tyrosine kinase (PYK2) is a cellular adhesion kinase related to focal adhesion kinase (FAK). While FAK is wildly expressed in various cell types, PYK2 is primarily expressed in neuron cells and cells derived from hematopoietic cell lineages, including osteoclasts. Numbers of studies suggest that PYK2 plays an important role in the adhesion-dependent, integrin-mediated signaling transduction that leads to osteoclast activation and glioblastoma invasion. Therefore, specific inhibition of PYK2 activity offers a potential therapeutic intervention for cancer and bone resorption-related disorders. We proposed to develop an enzyme-based kinase assay for high throughput screening (HTS) with purified PYK2 and FAK recombinant proteins for a broad search of small molecules with potent and selective inhibition activity to PYK2. Specific and potent PYK2 kinase inhibitors will provide molecular probes for understanding the role of PYK2 in cellular signaling pathways and in pathological conditions. As part of on-going research project related to protein crystal structure determination of PYK2 and structure-based inhibitor design, we have overexpressed and purified the active PYK2 proteins in full-length and kinase domain. Our specific aims are: 1) to develop and optimize a HTS-compatible PYK2 kinase assay, 2) to validate the assay for high throughput screening of compound library. With the long-term goal of benefiting public health, we will share the assay protocols and data generated from this proposed project to facilitate research of understanding molecular mechanisms of PYK2 involved in human disease and development of PYK2 as therapeutic target. Proline-rich tyrosine kinase (PYK2) is a cellular adhesion kinase related to focal adhesion kinase (FAK) and is involved in osteoclast activation in bone resorption and glioblastoma invasion. Targeting PYK2 offers a potential therapeutic intervention for cancer and bone resorption-related disorders. The specific aims of this proposed project is to develop and validate a PYK2 kinase assay for a broad search of molecular inhibitors with selective biological activity to facilitate basic research and therapeutic development.