Targeting the CXCL12/CXCR4 Axis towards 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-蛋白结合的新型化学探针。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）。许多趋化因子已被证实为药物开发的靶标，但几乎所有的发现工作都集中在结合gpcr的药物上。然而，由于传统药物发现方法难以控制的毒性，CXCR4受体拮抗剂破坏广泛的趋化因子-受体界面已被证明是困难的，这强调了关注结合CXCL12趋化因子的药物的替代策略的必要性。最近，我们开发了一种新的基于结构的硅/核磁共振杂交筛选策略，并确定了一个CXCL12配体2，它可以阻断受体识别位点。我们通过设计和合成一个只包含9个四唑衍生物的小片段库来优化初始hit 2。从这个文库中，我们发现了两个配体效率（LE）高于典型PPI干扰物的片段（LE d = 0.24）。其中一个片段5在sY12位点的Kd值为13¿M (LE=0.33)，一个片段9在sY21位点的Kd值为41¿M （LE=0.28）。我们还在sY21位点用1.8¿分辨率的四唑片段9解析了CXCL12的x射线共晶结构。基于这些发现，我们设计并合成了包含12个化合物的第二个文库。我们已经证明，CXCL12结合位点的性质有助于获得在功能性趋化分析中表现出合理活性的高亲和力配体。两轮优化共合成了约20个化合物，其中最佳化合物9c在cxcl12诱导的趋化性中显示出51¿M的IC50，比最初的IC50提高了16倍。我们建议将这些经过验证的CXCL12趋化因子拮抗剂开发成体内化学探针，用于转移性疾病的诊断和治疗。本次修订申请的目的是：1)设计、合成和优化针对CXCL12上sY21结合位点的CXCL12趋化因子拮抗剂。2)设计、合成并优化sY12和sY7结合位点的CXCL12拮抗剂。3)利用片段生长和连接策略设计、合成高亲和力抑制剂。4)利用钙通量、趋化性、体外迁移和结肠癌和黑色素瘤小鼠模型转移的生物测定，以及利用x射线和核磁共振技术测定共络合物的结构，开发有效的选择性抑制剂作为体内化学探针。由于趋化因子信号在癌细胞迁移中起着关键作用，CXCL12趋化因子拮抗剂很可能作为转移性疾病的新型治疗药物而发挥作用。最后，我们将利用动物模型评估优化后的化合物对cxcr4导向的转移性癌症的体内疗效，这在我们最近发表在PNAS上的文章中有描述。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 chemical probes that are capable of potently and selectively disrupting the CXCL12/CXCR4 protein-protein binding. 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). A number of chemokines have been validated as targets for drug development, but virtually all discovery efforts focus on the agents that bind GPCRs. However, disrupting the extensive chemokine- receptor interface by CXCR4 receptor antagonists has proven difficult due to unmanageable toxicities for traditional drug discovery approaches, emphasizing the need for alternative strategies focusing on agents that bind the CXCL12 chemokine. Recently, we developed a novel structure-based hybrid in silico/NMR screening strategy and identified a CXCL12 ligand 2 that occludes the receptor recognition site. We optimized the initial hit 2 by designing and synthesizing a small fragment library containing only nine tetrazole derivatives. From this library, we discovered two fragments with higher ligand efficiency (LE) than the typical PPI disruptors (LE d 0.24). One fragment 5 exhibited a Kd of 13 ¿M (LE=0.33) for CXCL12 binding in sY12 site and one fragment 9 with a Kd of 41 ¿M (LE=0.28) in sY21 site measured by 2D NMR. We also solved an X-ray co-crystal structure of CXCL12 with tetrazole fragment 9 at 1.8 ¿ resolution in sY21 site. Based on these findings, we designed and synthesized a second library containing a dozen compounds. We have demonstrated that the nature of the CXCL12 binding site is conductive to obtaining higher affinity ligands that exhibit reasonable activity in functional chemotaxis assays. The best compound 9c, from a total of ~20 compounds synthesized in two round of optimization, showed an IC50 of 51 ¿M in CXCL12-induced chemotaxis, a 16 fold improvement from the original hit 2. We propose to develop these validated CXCL12 chemokine antagonists into in vivo chemical probes for use in diagnosis and treatment of metastatic disease. The aims of this revised grant application are: 1) Design, synthesize and optimize CXCL12 chemokine antagonists targeting the sY21 binding site on CXCL12. 2) Design, synthesize and optimize CXCL12 antagonists in sY12 and sY7 binding sites. 3) Design, synthesize high-affinity inhibitors using fragment-growing and linking strategy. 4) Develop potent and selective inhibitors as in vivo chemical probes using robust bioassays of calcium flux, chemotaxis and migration in vitro and metastasis in colon and melanoma mouse models as well as structural determination of co-complexes using X-ray and NMR. Because chemokine signaling plays a key role in cancer cell migration, CXCL12 chemokine antagonists are likely to find utility as novel therapeutic agents for metastatic disease. Finally, we will assess the 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.