Shao Qian, Duong Tuong Nghi, Park Inji, Nomura Daniel K
Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720 USA.
Innovative Genomics Institute, Berkeley, CA 94720 USA.
bioRxiv. 2023 Nov 6:2023.11.06.565850. doi: 10.1101/2023.11.06.565850.
14-3-3 proteins have the unique ability to bind and sequester a multitude of diverse phosphorylated signaling proteins and transcription factors. Many previous studies have shown that 14-3-3 interactions with specific phosphorylated substrate proteins can be enhanced through small-molecule natural product or fully synthetic molecular glue interactions. However, enhancing 14-3-3 interactions with both therapeutically intractable transcription factor substrates as well as potential neo-substrates to sequester and inhibit their function has remained elusive. One of the 14-3-3 proteins, 14-3-3σ or SFN, has a cysteine C38 at the substrate binding interface near sites where previous 14-3-3 molecular glues have been found to bind. In this study, we screened a fully synthetic cysteine-reactive covalent ligand library to identify molecular glues that enhance 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 that are part of the Hippo transducer pathway. We identified a hit EN171 that covalently targets 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 also could be used as a covalent 14-3-3 recruiter in heterobifunctional molecules to sequester nuclear neo-substrates such as BRD4 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 also 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蛋白具有独特的能力，能够结合并隔离多种不同的磷酸化信号蛋白和转录因子。许多先前的研究表明，通过小分子天然产物或全合成分子胶相互作用，可以增强14-3-3与特定磷酸化底物蛋白的相互作用。然而，增强14-3-3与治疗上难以处理的转录因子底物以及潜在的新底物的相互作用，以隔离并抑制它们的功能，仍然难以实现。14-3-3蛋白之一，即14-3-3σ或SFN，在底物结合界面处靠近先前发现14-3-3分子胶结合的位点，有一个半胱氨酸C38。在本研究中，我们筛选了一个全合成的半胱氨酸反应性共价配体库，以鉴定分子胶，这些分子胶不仅能增强14-3-3σ与可成药转录因子如雌激素受体（ERα）的相互作用，还能增强与具有挑战性的致癌转录因子如YAP和TAZ（它们是Hippo转导途径的一部分）的相互作用。我们鉴定出一个命中物EN171，它以共价方式靶向14-3-3，增强14-3-3与ERα、YAP和TAZ的相互作用，导致雌激素受体和Hippo途径转录活性受损。我们进一步证明，EN171不仅可以用作增强天然蛋白相互作用的分子胶，还可以用作异双功能分子中的共价14-3-3招募剂，将核新底物如BRD4隔离到细胞质中。总体而言，我们的研究揭示了一种共价配体，它作为一种新型的14-3-3分子胶，作用于YAP和TAZ等具有挑战性的转录因子，并且还证明这些分子胶可以潜在地用于异双功能分子中，将核新底物从细胞核中隔离出来并转运到细胞质中，以实现靶向蛋白定位。
