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 非受体酪氨酸激酶 以及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、结直肠癌、 黑色素瘤、胶质母细胞瘤和胰腺癌。 FAK 参与多种生物途径，可以 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 PI3K/AKT 和 β- 介导的信号通路 连环蛋白。通过基因操作和 FAK 敲除，FAK 已被验证为癌症治疗的靶点 导致癌细胞凋亡的强烈激活，而对正常细胞的影响最小。许多 ATP- 竞争性 FAK 激酶结构域抑制剂已被报道；然而，这些只是细胞抑制和/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 的激酶独立作用 不被激酶抑制所阻断；特别是C端粘着斑的支架功能 目标（FAT）域。 FAK FAT 结构域是 FAK 依赖性抗凋亡的主要调节剂， 由称为 FAK 相关非激酶的内源性显性失活 FAK 亚型调节的结构域 （FRNK）。具体而言，FAT-桩蛋白-蛋白质相互作用 (PPI) 将 FAK 定位于粘着斑，并且 FAT 结构域界面残基的突变会扰乱粘着斑周转、细胞粘附、迁移和 入侵。因此，一个未满足的主要需求是发现阻止 FAK FAT 的小分子化学探针 域脚手架。为了针对 FAK FAT 结构域，我们之前探索过片段筛选 SPR/NMR 和碳氢化合物肽。这些方法导致基于 FAT 片段的命中 微摩尔亲和力和铅钉合肽探针 UA-1907 (KD = 1 µM) 可诱导黑色素瘤细胞凋亡 细胞。对于这个项目，我们将开发利用化学探针 UA-1907 的新型药物筛选方法来识别 小分子 FAT 配体可破坏 FAK 的抗凋亡支架功能。具体来说，我们将：1） 设计 TR-FRET 生化测定，筛选 35,000 种化合物 PPI 偏向文库，寻找抑制 UA- 的化合物 1907 与 FAT 结合； 2) 使用 SPR 和 HSQC-NMR 方法确认生化命中作为 FAT 配体；和 3) 开发一种 NanoBiT 细胞测定法，测量 FAK-桩蛋白结合以验证命中。总而言之，我们对这个项目充满期待 鉴定第一个描述的基于小分子的 FAT-桩蛋白相互作用抑制剂，该抑制剂将作为 为未来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.