PYK2 & Therapeutic Strategies-Cancer-induced Osteolysis

描述(由申请人提供)： 常见的癌症，如乳腺癌、前列腺癌和肺癌，经常转移到骨骼，导致骨骼紊乱和无法治愈的后果。骨转移极大地增加了发病率和死亡率。大约75%的晚期乳腺癌患者在骨骼中出现明显的肿瘤负担，并导致严重的骨溶解损害。目前对骨转移的分子机制了解有限，但研究表明，骨微环境在溶骨转移中发挥作用，并通过肿瘤细胞与骨吸收破骨细胞之间的相互作用参与骨溶解与癌细胞生长之间的耦合。富含脯氨酸的酪氨酸激酶(PYK2)是一种细胞黏附蛋白，在破骨细胞中高水平表达，在黏附依赖的整合素介导的信号转导中发挥重要作用，在破骨细胞激活过程中导致细胞骨架重组和封闭区的形成。PYK2的自动磷酸化以及与Src激酶和CAS的相互作用是信号传递所必需的。CAS是SH2和SH3分子的对接蛋白。然而，关于PYK2调节破骨细胞功能的分子机制却知之甚少。我们的长期研究目标是了解PYK2在破骨细胞激活中的结构生物学和功能，探索其结构、功能和动力学之间的关系，并利用这种关系设计改变PYK2的S功能的特异性抑制剂。为了实现这一目标，我们将通过底物类似物和相互作用的蛋白质结构域来确定PYK2的结构。我们假设，通过抑制PYK2活性来阻断这一信号通路可以阻止破骨细胞的激活，并最终破坏骨溶解和癌细胞生长的恶性循环。通过结合基于结构和活性的方法，我们将寻找PYK2特异性抑制剂的候选先导，这些先导不仅作为探针来验证这一假说，并进一步研究PYK2在破骨细胞激活中的作用机制，而且还将导致一种潜在的癌症诱导性骨吸收的治疗剂。我们提出了一项全面的合作努力，最初专注于以下特定目标：1)在全长和单独的结构域中表达和纯化活性PYK2，表征PYK2与Src的SH2结构域和CAS的SH3结构域的结合活性；2)结晶PYK2并确定PYK2及其结构域的三维结构；3)结合基于结构和细胞活性的方法来确定针对PYK2激酶结构域的特定PYK2抑制剂的候选先导，可能是其在破骨细胞激活中发挥作用所需的蛋白质-蛋白质相互作用。

DESCRIPTION (provided by applicant): Common cancers, such as those of the breast, prostate and lung, frequently metastasize to bone and lead to bone disorder and untreatable consequences. Bone metastases contribute heavily to morbidity and mortality. About 75% of patients with advanced breast carcinoma developed significant tumor burden in the skeleton and resulted in severe bone osteolytic lesions. Current understanding of the molecular mechanism of bone metastasis is limited, however, studies indicate that bone microenvironment plays a role in osteolytic metastasis and involves a coupling between osteolysis and cancer cell growth through interactions between the tumor cells and the bone-resorbing osteoclasts. Proline-rich tyrosine kinase (PYK2), a cellular adhesion kinase, is expressed in high levels in osteoclasts and plays an important role in the adhesion-dependent, integrin-mediated signaling that leads to cytoskeletal reorganization and formation of the sealing zone during osteoclast activation. Autophosphorylation of PYK2 and interactions with Src kinase and CAS, a docking protein for SH2 and SH3-containg molecules, is required for the signaling. However, little is known concerning the molecular mechanism by which PYK2 regulates osteoclast function. Our long-term research goal is to understand the structural biology and function of PYK2 in osteoclast activation, explore the relationship among its structure, function, and dynamics, and exploit this relation for the design of specific inhibitors that alter PYK2's function required for osteolysis. Towards this goal, we will determine the structure of PYK2 in complex with substrate analogs and interacting protein domains. We hypothesize that interruption of this signaling pathway by inhibition of PYK2 activity prevents osteoclast activation and eventually disrupts the vicious cycle of osteolysis and cancer cell growth. Through a combination of structure and activity-based approaches, we will identify candidate leads of PYK2 specific inhibitors which not only serve as a probe to test this hypothesis and further study the mechanism by which PYK2 acts in osteoclast activation, but also lead to a potential therapeutic agent for cancer-induced bone resorption. We propose a comprehensive and collaborative effort by focusing initially on the following specific aims: 1) to express and purify active PYK2 in full length and separate domains, to characterize the kinase activity and binding activity of PYK2 with the SH2 domain of Src and the SH3 domain of CAS; 2) to crystallize PYK2 and determine the three-dimensional structure of PYK2 and its domains; 3) to use a combination of structure- and cellular activity-based approaches to identify candidate leads of specific PYK2 inhibitors that target the PYK2 kinase domain, possibly the protein-protein interactions that are required for its function in osteoclast activation.