Induced ubiquitination of the partially disordered Estrogen Receptor alpha protein via a 14-3-3-directed molecular glue-based PROTAC design

Verhoef, C. J. A.; Crowe, C.; Nakesone, M. A.; de la Cuadra Baste, A.; Harzing, T.; Span, N. A. S.; Sathe, G.; Iso, K.; Ottmann, C.; Brunsveld, L.; Ciulli, A.; Cossar, P. J.

doi:10.1101/2025.11.17.688825

bioRxiv · 10.1101/2025.11.17.688825

Induced ubiquitination of the partially disordered Estrogen Receptor alpha protein via a 14-3-3-directed molecular glue-based PROTAC design

Verhoef, C. J. A.·Crowe, C.·Nakesone, M. A.·de la Cuadra Baste, A.·Harzing, T.·Span, N. A. S.·Sathe, G.·Iso, K.·Ottmann, C.·Brunsveld, L.·Ciulli, A.·Cossar, P. J.

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Abstract

Proteins lacking defined ligandable pockets remain challenging drug targets. Here, we develop a molecular glue-based PROTAC (MGPROTACs) approach that chemically conjugates a molecular glue stabilizer to a VHL-recruiting ligand to capture and ubiquitinate the 14-3-3/Estrogen Receptor (ER) complex. Our designed MGPROTACs engage a composite interface between 14-3-3 and the disordered F-domain of ER, promoting cooperative complex formation and target ubiquitination. Biophysical characterization revealed distinct linker-dependent cooperativities across the MGPROTAC series, which influenced both cellular permeability and ubiquitination efficiency. Cryo-EM of the most cooperative MGPROTAC uncovers de novo VHL-14-3-3{zeta} contacts, while molecular dynamics simulations rationalize the stabilizing interactions underlying cooperativity. Strikingly, fine-tuning linker design enables selective ubiquitination of distinct complex subunits. These findings establish a structural and mechanistic framework for integrating molecular glue and PROTAC principles, expanding the scope of drug discovery to previously intractable protein complexes.

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Verhoef, C. J. A., Crowe, C., Nakesone, M. A., de la Cuadra Baste, A., Harzing, T., Span, N. A. S., Sathe, G., Iso, K., Ottmann, C., Brunsveld, L., Ciulli, A., Cossar, P. J.. 2025-11-17. Induced ubiquitination of the partially disordered Estrogen Receptor alpha protein via a 14-3-3-directed molecular glue-based PROTAC design. https://doi.org/10.1101/2025.11.17.688825

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