Medical Research Council Cancer Unit, University of Cambridge, Hills Road, Cambridge CB2 0XZ, UK.
Telethon Kids Institute, Centre for Child Health Research, University of Western Australia & PYC Therapeutics Limited, Nedlands, WA 6009, Australia.
Cell Chem Biol. 2021 Nov 18;28(11):1602-1615.e9. doi: 10.1016/j.chembiol.2021.05.009. Epub 2021 Jun 9.
Genetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth. In a crystal structure, the hit peptide occupies the phosphopeptide-binding groove of 14-3-3ε in a conformation distinct from its natural peptide substrates. A biophysical screen identifies drug-like small molecules that displace the hit peptide from 14-3-3ε, providing starting points for structure-guided development. Our findings exemplify "protein interference," an approach using evolutionarily diverse, natural peptides to rapidly identify, validate, and develop chemical probes against MMIs essential for complex cellular phenotypes.
用于鉴定和验证对复杂途径至关重要的大分子相互作用（MMIs）的遗传筛选技术仍然是系统生物学和治疗开发的一个重要未满足的需求。在这里，我们使用来自不同原核基因组的肽文库筛选促进叉头框 O3（FOXO3a）核重定位的 MMIs，FOXO3a 作为一种肿瘤抑制因子，比突变更容易通过翻译后修饰失活。一个命中肽通过磷酸化依赖性相互作用与 14-3-3 家族的信号调节剂结合，调节 FOXO3a 介导的转录，并抑制癌细胞生长。在晶体结构中，命中肽占据 14-3-3ε 的磷酸肽结合槽，构象与天然肽底物明显不同。生物物理筛选可识别出可置换 14-3-3ε 上命中肽的类药小分子，为基于结构的开发提供了起点。我们的研究结果例证了“蛋白质干扰”，这是一种使用进化上多样化的天然肽快速鉴定、验证和开发针对复杂细胞表型至关重要的 MMIs 的化学探针的方法。
