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

描述（由申请人提供）：趋化因子或趋化因子是重要的信号分子，在稳态过程或对感染或炎症的反应中调节免疫细胞的运输。趋化因子ccr21的正常功能是将表达趋化因子受体CCR7的抗原呈递树突状细胞和幼稚t细胞招募到淋巴结。随后的抗原呈递导致活化的t细胞，这是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。CCL21靶向转移癌细胞到淋巴结，这是形成转移的常见部位。前哨淋巴结转移的存在被用来判断许多癌症的严重程度。对其他趋化因子的结构研究，包括RANTES/CCL5、MCP-1/CCL2和SDF- 1/CXCL12，导致变异作为拮抗剂或部分激动剂，可以减少或消除细胞向野生型趋化因子的迁移。此外，小的药物样CXCL12配体可以阻止CXCR4的激活。然而，对CCL21的类似结构研究缺乏。CCL21的结构和寡聚物状态将在Aim 1中确定。CCL21激活趋化因子受体CCR7。CCR7是一种七螺旋整体膜g蛋白偶联受体（GPCR）。趋化因子通过“双态/双位点”机制激活其受体，首先结合受体n端（位点1），随后趋化因子n端结合受体的第二个位点（位点2），引起高亲和力结合和激活。尽管最近生产用于结构研究的gpcr的进展令人兴奋，但我们将研究CCL21与CCR7的n端相互作用，从而模拟Site 1相互作用。趋化因子受体n端含有翻译后修饰的巯基酪氨酸氨基酸，增加对趋化因子的亲和力。最近的研究表明，硫代酪氨酸结合位点可以用小分子抑制剂靶向。Aim 2旨在确定CCR7 n端和CCR7磺基酪氨酸的CCL21结合位点。随后，这些结合位点将与R01机构的合作者一起作为开发CCL21抑制剂的靶点。CCL21还直接结合n端p选择素糖配体-1 (PSGL-1)，这种相互作用增强了白细胞的趋化性。Aim 3将验证PSGL-1 n端磺胺基酪氨酸参与CCL21结合的假设，如果磺胺基酪氨酸对CCL21的结合很重要，将确定CCL21上PSGL-1磺胺基酪氨酸的结合位点。本提案旨在解决CCL21的结构，同时表征CCL21如何识别生理上重要的结合伴侣。这些结合事件的信息将直接影响针对趋化因子的药物发现研究，因为这些知识将被R01机构的研究人员用于其磺基酪氨酸结合位点的药物筛选工作。

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.