A Formylglycine-Peptide for the Site-Directed Identification of Phosphotyrosine-Mimetic Fragments

08 April 2022, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Discovery of protein-binding fragments for precisely defined binding sites is an unmet challenge so far. Here, we investigate formylglycine as a molecular probe for the sensitive detection of fragments binding to a spatially defined protein site. Formylglycine peptide 3 was derived from a phosphotyrosine-containing peptide substrate of protein tyrosine phosphatase PTP1B by replacing the phosphorylated amino acid with the reactive electrophile. Fragment ligation with formylglycine occured in aqueous physiological buffer, structures and kinetics were validated by NMR spectroscopy. Screening and hit validation revealed fluorinated and non-fluorinated hit fragments being able to replace the native phosphotyrosine residue. The formylglycine probe identified low-affinity fragments with high spatial resolution as substantiated by molecular modelling. The best fragment hit, 4-amino-phenyl-acetic acid, was converted into a cellularly active, nanomolar inhibitor of the protein tyrosine phosphatase SHP2.


Fragment-based drug discovery
Fragment ligation
Site-directed fragment screening
Phosphatase inhibitors
Phosphotyrosine Mimetics

Supplementary materials

Supporting Information
Supporting Information to the preprint: A Formylglycine-Peptide for the Site-Directed Identification of Phosphotyrosine-Mimetic Fragments


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