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

描述(申请人提供)：趋化因子，或趋化吸引细胞因子，是重要的信号分子，在体内平衡过程中或在对感染或炎症的反应期间调节免疫细胞的运输。趋化因子CCL21正常功能是将表达趋化因子受体CCR7的抗原提呈树突状细胞和幼稚T细胞募集到淋巴结。随后的抗原呈递导致激活的T细胞，这是使适应性免疫系统与疾病抗争的关键步骤，并最终导致激活的巨噬细胞、产生抗体的B细胞和杀伤T细胞。趋化因子也参与了许多疾病状态。CCL21靶向转移癌细胞至淋巴结，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/CCL5、MCP-1/CCL2和SDF-1/CXCL12，导致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，CXCL12的小分子类药物配体可以阻止CXCR4的激活。然而，目前还缺乏对CCL21的类似结构研究。CCL21的结构和寡聚体状态将在目标1中确定。CCL21激活趋化因子受体CCR7。CCR7是一种七螺旋整合膜G蛋白偶联受体。趋化因子通过“两个状态/两个位点”的机制激活它们的受体，首先与受体的N末端(位点1)结合，然后趋化因子N末端与受体上的第二个位点(位点2)结合，导致高亲和力的结合和激活。虽然最近在产生用于结构研究的GPCRs方面的进展是令人兴奋的，但我们将研究CCL21与CCR7的N末端的相互作用，从而模拟位点1的相互作用。趋化因子受体N末端含有翻译后修饰的硫代酪氨酸氨基酸，可增加与趋化因子的亲和力。最近的研究表明，硫代酪氨酸结合位点可以被小分子抑制剂作为靶点。目的鉴定CCR7和CCR7磺基酪氨酸N末端的CCL21结合位点。随后，这些结合位点将作为与R01机构的合作者一起开发CCL21抑制剂的靶点。CCL21还直接与N-末端P-选择素糖基化配体-1(PSGL-1)结合，这种相互作用可以增强白细胞的趋化作用。目的3验证PSGL-1 N末端的硫代酪氨酸参与CCL21结合的假设，如果硫代酪氨酸对结合是重要的，则将确定CCL21上的PSGL-1硫代酪氨酸结合位点。这一建议试图解决CCL21的结构，同时表征CCL21如何识别生理上有意义的结合伙伴。有关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R01机构的研究人员将在他们的硫酪氨酸结合位点定向药物筛选工作中使用这些知识。 与公共健康相关：趋化因子，如CCL21，通过免疫细胞的运输发挥免疫系统的调节作用。然而，CCL21也将转移的癌细胞招募到通常形成转移的淋巴结。转移或扩散的癌症很难治疗。通过结构研究寻求对CCL21的更多了解，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.