14-3-3 proteins have a unique ability to bind and sequester a multitude of diverse phosphorylated signaling proteins and transcription factors. Many previous studies have shown that interactions of 14-3-3 with specific phosphorylated substrate proteins can be enhanced through small-molecule natural products or fully synthetic molecular glue interactions. However, enhancing 14-3-3 interactions with both therapeutically intractable transcription factor substrates and potential neo-substrates to sequester and inhibit their function remains elusive. One of the 14-3-3 proteins, 14-3-3σ or SFN, has cysteine C38 at the substrate-binding interface, near the sites where previous 14-3-3 molecular glues have been found to bind. In this study, we screen a fully synthetic cysteine-reactive covalent ligand library to identify molecular glues that enhance the interaction of 14-3-3σ with not only druggable transcription factors such as estrogen receptor (ERα) but also challenging oncogenic transcription factors such as YAP and TAZ, which are part of the Hippo transducer pathway. We identify a hit EN171 that covalently targets both C38 and C96 on 14-3-3 to enhance 14-3-3 interactions with ERα, YAP, and TAZ, leading to impaired estrogen receptor and Hippo pathway transcriptional activity. We further demonstrate that EN171 could not only be used as a molecular glue to enhance native protein interactions but could also be used as a covalent 14-3-3 recruiter in heterobifunctional molecules to sequester nuclear neo-substrates such as BRD4 and BLC6 into the cytosol. Overall, our study reveals a covalent ligand that acts as a novel 14-3-3 molecular glue for challenging transcription factors such as YAP and TAZ and demonstrates that these glues can be potentially utilized in heterobifunctional molecules to sequester nuclear neo-substrates out of the nucleus and into the cytosol to enable targeted protein localization.

14-3-3蛋白具有独特的结合和隔离多种不同磷酸化信号蛋白和转录因子的能力。许多先前的研究表明，通过小分子天然产物或完全合成的分子胶作用可以增强14-3-3与特定磷酸化底物蛋白质之间的相互作用。然而，增强14-3-3与治疗困难的转录因子底物及潜在新底物之间的作用，以隔离并抑制其功能仍然难以实现。其中一种14-3-3蛋白，即14-3-3σ或SFN，在底物结合界面上有一个半胱氨酸C38，该位置靠近先前发现的14-3-3分子胶的结合位点附近。在这项研究中，我们筛选了一个完全合成的半胱氨酸反应共价配体库，以识别能够增强14-3-3σ与包括雌激素受体（ERα）在内的可药物转录因子以及具有挑战性的致癌转录因子如YAP和TAZ之间相互作用的分子胶。这些转录因子是Hippo信号通路的一部分。我们发现了一个名为EN171的化合物，它共价靶向14-3-3上的C38和C96位点，从而增强了其与ERα、YAP和TAZ的相互作用，并导致雌激素受体及Hippo信号途径转录活性受损。此外，我们还证明了EN171不仅可以作为增强天然蛋白间相互作用的分子胶使用，还可以用于异双功能分子中作为共价14-3-3招募剂，将核内新底物如BRD4和BLC6隔离到细胞质中。总的来说，我们的研究揭示了一种能够作为新型14-3-3分子胶用于挑战性的转录因子（如YAP和TAZ）的共价配体，并表明这些胶可以潜在地在异双功能分子中使用以将核内新底物从核隔离到细胞质中，从而实现蛋白质定位的靶向调控。