Targeting the CXCL12/CXCR4 Axis toward the Therapy of Metastatic Cancers

描述(由申请人提供)：该项目的总体目标是发现能够有效和选择性地破坏CXCL12/CXCR4蛋白质-蛋白质界面的新型体内化学探针。趋化因子CXCL12及其G蛋白偶联受体CXCR4是临床上优先考虑的靶点，因为它们与转移性癌症(也与自身免疫性疾病和心血管疾病有关)有关。由于趋化因子同时与两个不同的位点相互作用，以结合和激活它们的受体，GPCR及其配体都是小分子抑制的潜在靶点。最近，我们开发了一种新的基于结构的杂化分子筛查策略，并发现了一种封闭受体识别位点的CXCL12配体。我们使用基于片段的方法合成了一组四唑类化合物，从而优化了最初的HIT。这一系列中最好的分子通过2D核磁共振波谱显示其Kd值为13 mm(LE=0.33)，并且完全抑制了CXCL12介导的CXCR4表达细胞的趋化作用。值得注意的是，我们在1.8A的分辨率下，解决了趋化因子CXCL12与我们的四唑片段之一(2D核磁共振波谱的Kd为41 mm，LE为0.28)的第一个X射线共晶结构。在加州大学旧金山分校的一项平行筛选工作中，通过将300多万个分子对接到CXCR4的晶体结构中，鉴定出一种新的CXCR4拮抗剂，并通过钙通量分析确认其IC50为57 mM。放射配基置换50 PM[I125]-CXCL12的IC50值为310 nM，LE为0.36，并能有效阻断细胞的趋化作用。我们建议将这些经过验证的CXCL12和CXCR4抑制剂开发成体内化学探针，用于诊断和治疗转移性疾病。本次资助申请的目的是：1)设计、合成和优化CXCL12趋化因子抑制剂。2)CXCR4受体拮抗剂的设计、合成和优化。3)开发有效和选择性的缓蚀剂作为体内化学探针，使用稳健的生物测定和使用X射线结晶学和2D核磁共振光谱确定共配合物的结构。由于趋化因子信号在癌细胞迁移中起关键作用，CXCL12抑制剂和CXCR4拮抗剂都有可能成为治疗转移性疾病的新型药物。最后，我们将使用我们最近发表在PNAS上的CXCR4导向转移癌动物模型来评估优化化合物的体内疗效。这是PAR-12-060的关键先决条件。

DESCRIPTION (provided by applicant): The overall goal of this project is to discover novel in vivo chemical probes that are capable of potently and selectively disrupting the CXCL12/CXCR4 protein-protein interface. The chemokine CXCL12 and its G protein-coupled receptor CXCR4 are high-priority clinical targets because of their involvement in metastatic cancers (also implicated in autoimmune disease and cardiovascular disease). Because chemokines simultaneously interact with two distinct sites to bind and activate their receptors, both the GPCR and its ligand are potential targets for inhibition by small molecules. Recently, we developed a novel structure-based hybrid in silico/NMR screening strategy and identified a CXCL12 ligand that occludes the receptor recognition site. We optimized the initial hit by synthesizing a panel of tetrazole derivatives using a fragment-based approach. The best molecules in this series demonstrated a Kd of 13 mM (LE of 0.33) by 2D NMR spectroscopy and completely inhibited CXCL12-mediated chemotaxis of CXCR4-expressing cells. Significantly, we solved the first X-ray co-crystal structure of chemokine CXCL12 with one of our tetrazole fragments (a Kd of 41 mM by 2D NMR spectroscopy, LE of 0.28) at 1.8 A resolution. In a parallel screening effort by our collaborators at UCSF, a new CXCR4 antagonist was identified by docking over three million molecules to the crystal structure of CXCR4 and confirmed with an IC50 of 57 mM by calcium flux assay. The hit exhibited an IC50 value of 310 nM by radioligand displacement of 50 pM [I125]-CXCL12, LE of 0.36, and efficacy in blocking cellular chemotaxis. We propose to develop these validated CXCL12 and CXCR4 inhibitors into in vivo chemical probes for use in diagnosis and treatment of metastatic disease. The aims of this grant application are: 1) design, synthesize and optimize CXCL12 chemokine inhibitors. 2) Design, synthesize and optimize CXCR4 receptor antagonists. 3) Develop potent and selective inhibitors as in vivo chemical probes using robust bioassays and structural determination of co-complexes using both X-ray crystallography and 2D NMR spectroscopy. Because chemokine signaling plays a key role in cancer cell migration, both CXCL12 inhibitors and CXCR4 antagonists are likely to find utility as novel therapeutic agents for metastatic disease. Finally, we will assess he in vivo efficacy of optimized compounds using animal models for CXCR4-directed metastatic cancer as described in our recent publication in PNAS. This is a key prerequisite for PAR-12-060.