Sulfotyrosine-guided discovery of small molecule chemokine inhibitors

描述(申请人提供)：该项目的目标是开发一个发现小分子趋化因子抑制剂的创新平台。趋化因子主要通过结合和激活一系列G蛋白偶联受体来指导细胞迁移，这些受体在翻译后被酪氨酸硫化修饰。趋化因子的功能需要硫酸盐化，这表明受体磺基酪氨酸直接参与趋化因子配体的特异性结合。最近，我们通过解析趋化因子SDF1/CXCL12与其受体CXCR4的含硫酪氨酸片段之间的复合体的核磁共振结构，揭示了趋化因子家族识别磺基酪氨酸的分子基础。CXCR4的N-端胞外区含有三个潜在的硫化位点，每个硫代酪氨酸在CXC12表面占据一个独特的裂隙。我们假设，由于硫代酪氨酸识别位点对于高亲和力受体结合是必不可少的，所以每个识别位点都代表了针对趋化因子配体的小分子的抑制靶点。在特定目标1中，我们建议在CXCL12表面定义额外的硫代酪氨酸识别位点，并结合高通量对接计算、2D核磁共振验证位点特异性配体结合和功能分析来测试趋化因子抑制。我们将在目标2中改进我们的电子筛选方法，使用CXCL12/CXCR4复合体的结构作为测试案例，以评估柔性侧链对接和针对核磁共振构象集合的片段筛选。将利用合成孔径雷达分析，通过设计和合成与相邻硫基酪氨酸识别位点的配体相结合的新化合物，对特定目标3的亲和力和选择性进行命中到铅和铅的优化。与CXCL12结合并在基于细胞的趋化因子分析中作为有效和选择性抑制剂发挥作用的化合物将提供给现有的合作者用于动物临床前研究。为了评估硫代酪氨酸诱导的抑制的更广泛的潜力，我们将开发一种简化的方法来在特定目标4中识别位点，并将我们的筛选方法应用于一组具有代表性的趋化因子靶标。硫代酪氨酸识别指导的基于结构的药物发现是一项概念性的进步，可以应用于趋化因子家族的所有50个成员和其他需要这种蛋白质修饰才能在细胞外空间发挥作用的复合体。应用这一策略出现的炎症性和自身免疫性疾病或癌症的新治疗方法将对人类健康产生重大影响。

DESCRIPTION (provided by applicant): The goal of this project is the development of an innovative platform for discovery of small molecule chemokine inhibitors. Chemokines direct cell migration primarily by binding and activating a family of G protein-coupled receptors that are post-translationally modified by tyrosine sulfation. Sulfation is required for chemokine function, suggesting that receptor sulfotyrosines participate directly in specific binding of the chemokine ligand. We recently revealed the molecular basis for sulfotyrosine recognition in the chemokine family by solving the NMR structure of a complex between the chemokine SDF1/CXCL12 and a sulfotyrosine-containing fragment of its receptor CXCR4. The N-terminal extracellular domain of CXCR4 contains three potential sulfation sites, and each sulfotyrosine occupies a unique cleft on the CXC12 surface. We hypothesize that because sulfotyrosine recognition sites are essential for high affinity receptor binding, each one represents a target for inhibition using small molecules directed at the chemokine ligand. In specific aim 1 we propose to define additional sulfotyrosine recognition sites on the CXCL12 surface and use a combination of high-throughput docking calculations, validation of site-specific ligand binding by 2D NMR, and functional assays to test for chemokine inhibition. We will refine our in silico screening methods in aim 2 using the structure of the CXCL12/CXCR4 complex as a test case for evaluation of flexible side chain docking and fragment screening against the ensemble of NMR conformers. Hit-to-lead and lead optimization will be performed for affinity and selectivity in specific aim 3 using SAR analysis and by designing and synthesizing novel compounds that combine ligands from adjacent sulfotyrosine recognition sites. Compounds that bind CXCL12 and function as potent and selective inhibitors in cell-based chemokine assays will be made available to existing collaborators for preclinical studies in animals. To assess the broader potential of sulfotyrosine- directed inhibition, we will develop a streamlined approach to site identification in specific aim 4 and apply our screening method to a representative set of chemokine targets. Structure-based drug discovery guided by sulfotyrosine recognition is a conceptual advance that can be applied to all 50 members of the chemokine family and other complexes that require this protein modification to function in the extracellular space. New treatments for inflammatory and autoimmune disease or cancer that emerge from application of this strategy will have a major impact on human health.