Targeting protein-protein interactions of tyrosine phosphatases with microarrayed fragment libraries displayed on phosphopeptide substrate scaffolds.

Chemical library screening approaches that focus exclusively on catalytic events may overlook unique effects of protein-protein interactions that can be exploited for development of specific inhibitors. Phosphotyrosyl (pTyr) residues embedded in peptide motifs comprise minimal recognition elements that determine substrate specificity of protein tyrosine phosphatases (PTPases). Using solid-phase synthesis, we incorporated aminooxy-containing amino acid residues into a 7-residue epidermal growth factor receptor (EGFR)-derived phosphotyrosine-containing peptide and subjected the peptides to on-resin oxime diversification by reacting with aldehyde-bearing drug-like functionalities. The pTyr residue remained unmodified. The resulting derivatized peptide library was printed in microarrays on nitrocellulose-coated glass surfaces for assessment of PTPase catalytic activity, or on gold monolayers for analysis of kinetic interactions by surface plasmon resonance (SPR). Focusing on amino-acid positions and chemical features, we first analyzed dephosphorylation of the peptide pTyr residues within the microarrayed library by the human dual-specificity phosphatases (DUSP) 14 and DUSP22, as well as by PTPases from poxviruses (VH1), and Yersinia pestis (YopH). In order to identify the highest affinity oxime motifs, the binding interactions of the most active derivatized phosphopeptides were examined by SPR using noncatalytic PTPase mutants. We combined high-affinity oxime fragments identified by the two-step catalytic and SPR-based microarray screens to produce an enhanced PTPase substrate as a lead scaffold for the development of inhibitors. Finally, further substitutions were made on the enhanced substrate chemical fragments to yield inhibitory compounds that serve as a final proof-of-principle.