Targeted Protein Localization by Covalent 14–3–3 Recruitment

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.