HTS Assay Development for Discovery of Specific PYK2 Inhibitors

描述（由申请人提供）：富含脯氨酸的酪氨酸激酶（PYK2）是一种与粘着斑激酶（FAK）相关的细胞粘附激酶。虽然FAK在各种细胞类型中广泛表达，但PYK 2主要在神经元细胞和来源于造血细胞谱系的细胞（包括破骨细胞）中表达。大量研究表明，PYK2在粘附依赖性、整合素介导的信号转导中起重要作用，该信号转导导致破骨细胞活化和胶质母细胞瘤侵袭。因此，特异性抑制PYK2活性为癌症和骨吸收相关疾病提供了潜在的治疗干预。我们建议开发一种基于酶的激酶测定法，用于用纯化的PYK2和FAK重组蛋白进行高通量筛选（HTS），以广泛寻找对PYK2具有强效和选择性抑制活性的小分子。特异性和有效的PYK2激酶抑制剂将为理解PYK2在细胞信号传导途径和病理条件中的作用提供分子探针。作为正在进行的PYK2蛋白质晶体结构测定和基于结构的抑制剂设计相关研究项目的一部分，我们已经过表达和纯化了全长和激酶结构域的活性PYK2蛋白。我们的具体目标是：1）开发和优化HTS相容的PYK2激酶测定，2）验证用于化合物文库的高通量筛选的测定。为长远造福公众健康，我们将分享此拟议项目产生的分析方案和数据，以促进了解PYK2参与人类疾病的分子机制的研究，并开发PYK2作为治疗靶点。富含脯氨酸的酪氨酸激酶（PYK2）是一种与粘着斑激酶（FAK）相关的细胞粘附激酶，并参与骨吸收和胶质母细胞瘤侵袭中的破骨细胞活化。靶向PYK2为癌症和骨吸收相关疾病提供了潜在的治疗干预。该拟议项目的具体目标是开发和验证PYK2激酶测定法，用于广泛搜索具有选择性生物活性的分子抑制剂，以促进基础研究和治疗开发。

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.