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

描述（由申请人提供）：本项目的总体目标是发现能够有效和选择性破坏CXCL 12/CXCR 4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是高度优先的临床靶点，因为它们参与转移性癌症（也涉及自身免疫性疾病和心血管疾病）。由于趋化因子同时与两个不同的位点相互作用以结合并激活其受体，因此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混合物在硅/NMR筛选策略，并确定了CXCL12配体，封闭受体识别位点。我们通过使用基于片段的方法合成一组四唑衍生物来优化初始命中。该系列中最好的分子通过2D NMR光谱显示Kd为13 mM（LE为0.33），并完全抑制CXCR 4表达细胞的CXCL 12介导的趋化性。值得注意的是，我们以1.8 A分辨率解析了趋化因子CXCL 12与我们的四唑片段之一（通过2D NMR光谱法测定的Kd为41 mM，LE为0.28）的第一个X射线共晶结构。在我们的合作者在UCSF进行的平行筛选工作中，通过将超过300万个分子与CXCR 4的晶体结构对接，鉴定出一种新的CXCR 4拮抗剂，并通过钙通量测定确认其IC50为57 mM。通过50 pM [I125]-CXCL 12的放射性配体置换，命中表现出310 nM的IC50值，LE为0.36，并且在阻断细胞趋化性方面具有效力。我们建议将这些经验证的CXCL12和CXCR4抑制剂开发成用于诊断和治疗转移性疾病的体内化学探针。本研究的主要目的是：1）设计、合成和优化CXCL 12趋化因子抑制剂。2)设计、合成和优化CXCR4受体拮抗剂。3)开发有效的和选择性的抑制剂作为体内化学探针，使用强大的生物测定和使用X射线晶体学和2D NMR光谱学的共复合物的结构测定。由于趋化因子信号传导在癌细胞迁移中起关键作用，CXCL12抑制剂和CXCR4拮抗剂都可能作为转移性疾病的新型治疗剂而被发现。最后，我们将使用CXCR4导向的转移性癌症的动物模型评估优化化合物的体内功效，如我们最近在PNAS上的出版物中所述。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.