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Identification of β-Strand Mediated Protein-Protein Interaction Inhibitors Using Ligand-Directed Fragment Ligation
preprintsubmitted on 04.06.2020, 19:23 and posted on 05.06.2020, 10:59 by Zsófia Hegedüs Zsófia Hegedüs, Fruzsina Hobor, Deborah K. Shoemark, sergio Celis, Lu-Yun Lian, Chi Trinh, Richard Sessions, Thomas Edwards, Andrew Wilson
β-Strand mediated protein-protein interactions (PPIs) represent underexploited targets for chemical probe development despite representing a significant proportion of known and therapeutically relevant PPI targets. β-strand mimicry is challenging given that both amino acid side-chains and backbone hydrogen-bonds are typically required for molecular recognition, yet these are oriented along perpendicular vectors. This paper describes an alternative approach using GKAP/SHANK1 PDZ as a model and dynamic ligation screening to identify small-molecule replacements for tranches of peptide sequence. A peptide truncation of GKAP functionalized at the N- and C-termini with acylhydrazone groups was used as an anchor. Reversible acylhydrazone bond exchange with a library of aldehyde fragments in the presence of the protein as template and in situ screening using a fluorescence anisotropy (FA) assay identified peptide hybrid hits with comparable affinity to the GKAP peptide binding sequence. Identified hits were validated using FA, ITC, NMR and X-ray crystallography to confirm selective inhibition of the target PDZ-mediated PPI and mode of binding. These analyses together with molecular dynamics simulations demonstrated the ligands make transient interactions with an unoccupied basic patch through electrostatic interactions, establishing proof-of-concept that this unbiased approach to ligand discovery represents a powerful addition to the armory of tools that can be used to identify PPI modulators.