Converting a weaker ATP-binding site inhibitor into a potent hetero-bivalent ligand by tethering to a unique peptide sequence derived from the same kinase.

Attaching an additional binding site directed moiety or a ligand to an ATP-binding site inhibitor has been used as a strategy to increase kinase binding affinity and specificity. The moieties typically used here as the second binding partner are varied from simple organic groups to ligands such as peptides derived from substrate binding site sequences. So far these hetero-bivalent ligands were developed targeting additional binding sites closer to the ATP-binding pocket. Here we report a unique expansion of this hetero-bivalent idea by: (I) targeting a new binding site much farther away from ATP-binding site, (II) using a peptide uniquely derived from a portion of the same kinase sequence that has been reported to turn and bind to the above distance binding pocket (used as the second binding ligand), and (III) optimizing a much longer and flexible linker (to connect ATP-binding site inhibitor and above mentioned second peptide sequence) with multistep, yet complete on-bead synthesis approach. We converted a very weak EphA3-kinase ATP-binding site inhibitor-PP2 into a potent hetero-bivalent ligand by tethering to a unique 5-mer peptide sequence that derived from the linker region of EphA3 that connects kinase and sterile alpha motif (SAM) domains. Our design highlight the use of distance binding pockets to ATP-binding site as the second targeted site, while introducing the idea of extracting natural peptide sequences that already exist within the same kinase sequence, by a careful screening of available crystal structures.