Improving SH3 domain ligand selectivity using a non-natural scaffold

Background: Src homology 3 (SH3) domains bind sequences bearing the consens us motif PxxP (where P is proline and x is any amino acid), wherein domain specificity is mediated largely by sequences flanking the PxxP core. This s pecificity is limited, however, as most SH3 domains show high ligand cross- reactivity. We have recently shown that diverse N-substituted residues (pep toids) can replace the prolines in the PxxP motif, yielding a new source of ligand specificity. Results: We have tested the effects of combining multiple peptoid substitut ions with specific flanking sequences on ligand affinity and specificity. W e show that by varying these different elements, a ligand can be selectivel y tuned to target a single SH3 domain in a test set. In addition, we show t hat by making multiple peptoid substitutions, high-affinity ligands can be generated that completely lack the canonical PxxP motif. The resulting liga nds can potently disrupt natural SH3-mediated interactions. Conclusions: Peptide-peptoid hybrid scaffolds yield SH3 ligands with marked ly improved domain selectivity, overcoming one of the principal challenges in designing inhibitors against these domains. These compounds represent im portant leads in the search for orthogonal inhibitors of SH3 domains, and c an serve as tools for the dissection of complex signaling pathways.