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，导致骨骼疾病和无法治疗的后果。骨转移是发病率和死亡率的重要因素。大约75%的晚期乳腺癌患者在骨骼中产生显著的肿瘤负荷，并导致严重的骨溶骨性病变。目前对骨转移的分子机制了解有限，然而，研究表明骨微环境在溶骨性转移中起作用，并且通过肿瘤细胞和骨吸收破骨细胞之间的相互作用，涉及骨溶解和癌细胞生长之间的耦合。富含脯氨酸的酪氨酸激酶（PYK 2）是一种细胞粘附激酶，在破骨细胞中高水平表达，并在破骨细胞活化过程中导致细胞骨架重组和密封区形成的粘附依赖性整合素介导的信号传导中发挥重要作用。PYK 2的自磷酸化以及与Src激酶和CAS（含SH 2和SH 3分子的对接蛋白）的相互作用是信号传导所必需的。然而，关于PYK 2调节破骨细胞功能的分子机制知之甚少。我们的长期研究目标是了解PYK 2在破骨细胞活化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特异性抑制剂，改变PYK 2在骨质溶解中所需的功能。为了实现这一目标，我们将确定PYK 2与底物类似物和相互作用蛋白质结构域的复合物的结构。我们推测，通过抑制PYK 2活性来阻断这一信号通路，可以阻止破骨细胞的活化，并最终破坏骨溶解和癌细胞生长的恶性循环。通过基于结构和活性的方法相结合，我们将确定PYK 2特异性抑制剂的候选先导物，这不仅可以作为探针来验证这一假设，进一步研究PYK 2在破骨细胞活化中的作用机制，而且还可以导致潜在的治疗药物。癌症诱导的骨吸收。本研究的主要目的是：1）表达和纯化PYK 2的全长和不同结构域的活性蛋白，研究PYK 2的激酶活性及其与Src的SH 2结构域和CAS的SH 3结构域的结合活性; 2）结晶PYK 2并确定PYK 2及其结构域的三维结构; 3）使用基于结构和细胞活性的方法的组合来鉴定靶向PYK 2激酶结构域的特异性PYK 2抑制剂的候选先导物，可能是其在破骨细胞活化中的功能所需的蛋白质-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.