Discovery of PPI inhibitors for the FAK FAT domain

项目概要/摘要 粘着斑激酶（FAK）或蛋白酪氨酸激酶2（PTK2）是一种125 kDa的非受体酪氨酸激酶 和在许多癌症中过度表达或扩增的支架蛋白，包括卵巢癌，乳腺癌，结肠直肠癌， 黑色素瘤胶质母细胞瘤和胰腺癌FAK参与多种生物学途径， 有助于癌症进展，包括细胞迁移、侵袭、淋巴管生成、抗凋亡， 转移、上皮-间质转化（EMT）以及由PI3K/AKT和β- 连环蛋白。FAK已被证实为通过基因操作和FAK敲低来治疗癌症的靶点， 导致癌细胞中细胞凋亡的强烈激活，而对正常细胞的影响最小。很多ATP- 已经报道了竞争性FAK激酶结构域抑制剂;然而，这些仅是细胞抑制性的和/或 对FAK具有中等选择性，并且在临床试验中显示出有限的功效。FAK的激酶非依赖性作用 不被激酶抑制所阻断;特别是，C-末端粘着斑的支架功能 靶向（FAT）结构域。FAK FAT结构域是FAK依赖性抗凋亡的主要调节剂， 由内源性显性负性FAK亚型调控的结构域称为FAK相关非激酶 （法国）。具体而言，脂肪-桩蛋白-蛋白相互作用（PPI）将FAK定位于粘着斑， FAT结构域界面残基的突变扰乱了粘着斑转换、细胞粘附、迁移和 入侵因此，一个主要的未满足的需求是发现阻断FAK FAT的小分子化学探针 域支架。为了靶向FAK FAT结构域，我们先前已经探索了通过以下方法筛选片段： SPR/NMR和烃钉合肽。这些方法导致了基于FAT片段的命中， 微摩尔亲和力和诱导黑色素瘤细胞凋亡的前导钉合肽探针UA-1907（KD = 1 µ M） 细胞在这个项目中，我们将开发利用化学探针UA-1907的新药筛选方法， 破坏FAK的抗凋亡支架功能的小分子FAT配体。具体而言，我们将：1） 设计TR-FRET生物化学测定以筛选35，000个化合物PPI偏向文库中抑制UA的命中， 2）使用SPR和HSQC-NMR方法确认生物化学命中物为FAT配体;和 3)开发测量FAK-桩蛋白结合的NanoBiT细胞测定以验证命中。总之，我们希望这个项目 为了鉴定第一个描述的脂肪-桩蛋白相互作用的基于小分子的抑制剂， 为今后药物化学优化研究奠定基础。

PROJECT SUMMARY/ABSTRACT Focal adhesion kinase (FAK), or protein tyrosine kinase 2 (PTK2), is a 125 kDa non-receptor tyrosine kinase and scaffolding protein that is overexpressed or amplified in many cancers, including ovarian, breast, colorectal, melanoma, glioblastoma, and pancreatic cancers. FAK is involved in multiple biological pathways that can contribute to cancer progression, including cell migration, invasion, lymphangiogenesis, anti-apoptosis, metastasis, epithelial-mesenchymal transition (EMT), and signaling pathways mediated by PI3K/AKT and β- catenin. FAK has been validated as a target in cancer therapy by genetic manipulation, and FAK knockdown results in robust activation of apoptosis in cancer cells, with minimal effects in normal cells. Many ATP- competitive FAK kinase domain inhibitors have been reported; however, these are only cytostatic and/or moderately selective for FAK, and have shown limited efficacy in clinical trials. Kinase-independent roles of FAK are not blocked by kinase inhibition; in particular, the scaffolding function of the C-terminal focal adhesion targeting (FAT) domain. The FAK FAT domain is the major modulator of FAK-dependent anti-apoptosis and is the domain regulated by the endogenous dominant-negative FAK isoform termed FAK-related non-kinase (FRNK). Specifically, the FAT-paxillin protein-protein interaction (PPI) localizes FAK to the focal adhesion, and mutation of residues at the FAT domain interface perturbs focal adhesion turnover, cell adhesion, migration, and invasion. Thus, a major unmet need is the discovery of small molecule chemical probes that block FAK FAT domain scaffolding. In order to target the FAK FAT domain, we have previously explored fragment screening by SPR/NMR and hydrocarbon-stapled peptides. These approaches led to FAT fragment-based hits with micromolar affinity and the lead stapled peptide probe, UA-1907 (KD = 1 µM) that induces apoptosis in melanoma cells. For this project, we will develop novel drug screening assays leveraging chemical probe UA-1907 to identify small molecule FAT ligands that disrupt the anti-apoptotic scaffolding functions of FAK. Specifically, we will: 1) design a TR-FRET biochemical assay to screen a 35,000 compound PPI-biased library for hits that inhibit UA- 1907 binding to FAT; 2) confirm biochemical hits as FAT ligands using SPR and HSQC-NMR approaches; and 3) develop a NanoBiT cellular assay measuring FAK-paxillin binding to validate hits. In all, we expect this project to identify the first described small molecule-based inhibitors of the FAT-paxillin interaction that will serve as the basis for future medicinal chemistry optimization studies.