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Updated: Nov 2, 2025

Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay
Published on: February 28, 2025
Exploration of a 14-3-3 PPI Pocket by Covalent Fragments as Stabilizers
Eline Sijbesma1, Kenneth K Hallenbeck2, Sebastian A Andrei1
1Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands.
Abstract:
The systematic discovery of functional fragments binding to the composite interface of protein complexes is a first critical step for the development of orthosteric stabilizers of protein-protein interactions (PPIs). We have previously shown that disulfide trapping successfully yielded covalent stabilizers for the PPI of 14-3-3 with the estrogen receptor ERα. Here we provide an assessment of the composite PPI target pocket and the molecular characteristics of various fragments binding to a specific subpocket. Evaluating structure-activity relationships highlights the basic principles for PPI stabilization by these covalent fragments that engage a relatively large and exposed binding pocket at the protein/peptide interface with a "molecular glue" mode of action.
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