Discovery of PPI inhibitors for the FAK FAT domain

项目摘要/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 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参与了多条生物通路，这些通路可以 促进癌症进展，包括细胞迁移、侵袭、淋巴管生成、抗细胞凋亡、 PI3K/AKT和β介导的肿瘤转移、上皮-间充质转化及其信号转导途径 连锁素。通过基因操作和FAK基因敲除，FAK已被确认为癌症治疗的靶点 结果强烈地激活了癌细胞的凋亡，而对正常细胞的影响最小。许多ATP- 已经报道了竞争性的FAK激酶结构域抑制剂；然而，这些仅仅是细胞抑制和/或 对FAK有中等选择性，临床试验显示疗效有限。FAK的不依赖于激酶的作用 不被激酶抑制所阻断；特别是C-末端局部黏附的支架功能 瞄准(FAT)域。FAK脂肪域是FAK依赖的抗细胞凋亡的主要调节器，是 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)。具体地说，脂肪-巴西林蛋白-蛋白相互作用(PPI)将FAK定位于局部粘连，并且 脂肪结构域界面残基的突变扰乱了焦点黏附转换、细胞黏附、迁移和 入侵。因此，一个尚未得到满足的主要需求是发现能阻断FAK脂肪的小分子化学探针。 域脚手架。为了针对FAK脂肪结构域，我们之前已经探索了通过以下方式筛选片段 SPR/核磁共振和碳氢化合物结合的多肽。这些方法导致了基于脂肪片段的命中 微摩尔亲和力和铅修饰的多肽探针UA-1907(Kd=1µM)诱导黑色素瘤细胞凋亡 细胞。对于这个项目，我们将利用化学探针UA-1907来开发新的药物筛选分析方法来识别 破坏FAK抗细胞凋亡支架功能的小分子脂肪配体。具体来说，我们将：1) 设计一种TRRET生化分析方法来筛选35,000个基于PPI的化合物文库，以寻找抑制UA-2的HITS 1907与脂肪结合；2)使用SPR和HSQC-核磁共振方法确认生化打击为脂肪配体；以及 3)建立检测FAK-帕西林结合的纳米比特细胞测定法，以验证HITS。总而言之，我们期待这个项目 为了鉴定第一个被描述的基于小分子的脂肪-帕西林相互作用的抑制剂，它将作为 为今后的药物化学优化研究奠定了基础。

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.