Structural studies of chemokines that target metastatic cancer cells to the lymph

描述（由申请人提供）：趋化因子或化学引诱细胞因子是重要的信号分子，其在稳态过程期间或在对感染或炎症的应答期间调节免疫细胞的运输。趋化因子CCL 21通常用于将抗原呈递树突细胞和表达趋化因子受体CCR 7的初始T细胞募集到淋巴结。随后的抗原呈递导致活化的T细胞，这是允许适应性免疫系统对抗疾病的关键步骤，并最终导致活化的巨噬细胞、产生抗体的B细胞和杀伤T细胞。趋化因子也参与许多疾病状态。CCL 21将转移性癌细胞靶向至淋巴结，淋巴结是形成转移的常见部位。前哨淋巴结中转移的存在被用于对许多癌症的严重程度进行分期。对其他趋化因子的结构研究，包括RANTES/CCL 5、MCP-1/CCL 2和SDF- 1/CXCL 12，产生了作为拮抗剂或部分激动剂的变体，可以减少或消除细胞向野生型趋化因子的迁移。此外，CXCL 12的小的药物样配体可以防止CXCR 4的活化。然而，对CCL 21的类似结构研究缺乏。CCL 21的结构沿着低聚物状态将在目的1中确定。CCL 21激活趋化因子受体CCR 7。CCR 7是七螺旋整合膜G蛋白偶联受体（GPCR）。趋化因子通过“两态/两位点”机制激活其受体，首先结合受体N-末端（位点1），随后趋化因子N-末端结合受体上的第二位点（位点2），引起高亲和力结合和激活。虽然最近的进展，在生产GPCR的结构研究是令人兴奋的，我们将研究CCL 21与CCR 7的N-末端的相互作用，从而模仿网站1的相互作用。趋化因子受体N-末端含有后修饰的磺基酪氨酸氨基酸，其增加对趋化因子的亲和力。最近的研究表明磺基酪氨酸结合位点可以用小分子抑制剂靶向。目的2：鉴定CCL 21在CCR 7 N端的结合位点和CCR 7磺基酪氨酸。随后，这些结合位点将作为与R 01机构合作者开发CCL 21抑制剂的靶标。CCL 21还直接结合N-末端P-选择素糖配体-I（PSGL-1），这是一种增强白细胞趋化性的相互作用。目的3将检验PSGL-1 N-末端中的磺基酪氨酸有助于CCL 21结合的假设，并且如果磺基酪氨酸对于结合是重要的，则将鉴定CCL 21上的PSGL-1磺基酪氨酸结合位点。该提案旨在解决CCL 21的结构，同时表征CCL 21如何识别生理上重要的结合伴侣。关于这些结合事件的信息将直接影响靶向趋化因子的药物发现研究，因为R 01机构的研究人员将在其磺基酪氨酸结合位点定向药物筛选工作中使用这些知识。 公共卫生相关性：趋化因子，如CCL 21，通过运输免疫细胞作为免疫系统的调节剂。然而，CCL 21也将转移性癌细胞募集到通常形成转移的淋巴结。已经转移或扩散的癌症很难治疗。通过结构研究来增加对CCL 21的理解，这可能有助于确定减少癌症转移的方法。

DESCRIPTION (provided by applicant): Chemokines, or chemoattractant cytokines, are important signaling molecules that regulate trafficking of immune cells during homeostatic processes or during a response to infection or inflammation. The chemokine CCL21 normally functions to recruit antigen presenting dendritic cells and naive T-cells expressing the chemokine receptor CCR7 to the lymph nodes. Subsequent antigen presentation results in activated T-cells, a key step in allowing the adaptive immune system to fight disease, and ultimately leads to activated macrophages, antibody producing B-cells and killer T-cells. Chemokines are also involved in numerous disease states. CCL21 targets metastatic cancer cells to the lymph nodes, a common site for formation of metastases. The presence of metastases in sentinel lymph nodes is used to stage the severity of numerous cancers. Structural studies on other chemokines, including RANTES/CCL5, MCP-1/CCL2, and SDF- 1/CXCL12, led to variants that function as antagonists or partial agonists that can reduce or eliminate cellular migration towards the wild-type chemokine. Also, small, drug-like ligands for CXCL12 can prevent activation of CXCR4. However, similar structural studies on CCL21 are lacking. The structure of CCL21 along with the oligmer state, will be determined in Aim 1. CCL21 activates the chemokine receptor CCR7. CCR7 is a heptahelical integral membrane G-protein coupled receptor (GPCR). Chemokines activate their receptors via a "two state/ two site" mechanism by first binding to the receptor N-terminus (Site 1) and subsequently the chemokine N-terminus binds to a second site on the receptor (Site 2) causing high affinity binding and activation. Although recent progress in producing GPCRs for structural studies is exciting, we will investigate the interaction of CCL21 with the N-terminus of CCR7, thus mimicking the Site 1 interaction. Chemokine receptor N-termini contain post-translationally modified sulfotyrosine amino acids that increase affinity for chemokine. Recent studies suggest sulfotyrosine binding sites can be targeted with small molecule inhibitors. Aim 2 seeks to identify the CCL21 binding site for N-terminus of CCR7 and the CCR7 sulfotyrosines. Subsequently, these binding sites will serve as targets for the development of CCL21 inhibitors with collaborators at R01 institutions. CCL21 also binds directly the N-terminus P-selectin glycoligand-1 (PSGL-1), an interaction that enhances chemotaxis of leukocytes. Aim 3 will test the hypothesis that sulfotyrosines in the PSGL-1 N-terminus contribute to CCL21 binding, and, if sulfotyrosines are important for binding, will identify the PSGL-1 sulfotyrosine binding sites on CCL21. This proposal seeks to solve the structure of CCL21 while characterizing how CCL21 recognizes physiological significant binding partners. Information on these binding events will directly influence drug discovery studies that target chemokines, as this knowledge will be used by researchers at R01 institutions in their sulfotyrosine binding site directed drug screening efforts. PUBLIC HEALTH RELEVANCE: Chemokines, like CCL21, function as regulators of the immune system though trafficking of immune cells. However, CCL21 also recruits metastatic cancer cells to the lymph nodes where metastases commonly form. Cancers that have metastasized or spread are difficult to treat. An increased understanding of CCL21 is sought through structural studies that may help identify ways to reduce cancer metastasis.