A collaboration between the laboratories of Professor Hiroaki Suga at the University of Tokyo and Professor Toru Sengoku at Yokohama City University has developed a macrocyclic peptide discovery platform that yielded exceptionally potent and selective inhibitors of the kinase GSK3β. Published in the Journal of the American Chemical Society, the work introduces an ATP-mimetic warhead amino acid into RaPID selections, enabling discovery of what the authors term "supra-bivalent" inhibitors that engage multiple binding sites simultaneously through an unexpected knob-into-hole interaction.
Protein kinase inhibitors typically target the highly conserved ATP-binding pocket, which often leads to off-target effects against related kinases. Bivalent inhibitors spanning both the ATP site and a secondary surface offer improved selectivity, but their rational design requires structural knowledge of allosteric sites that remains unavailable for most kinases. The Japanese team addressed this challenge by incorporating p-(pyrimidin-2-ylamino)phenylalanine, designated Z, into thioether-cyclized peptide libraries. This aminopyrimidine-containing residue was designed based on known GSK3β inhibitor scaffolds that form hydrogen bonds with the hinge region connecting the kinase N- and C-lobes.
Five rounds of RaPID selection against GSK3β enriched sequences containing a conserved ZRZ motif. Because library convergence remained modest, the researchers implemented an affinity-based equilibrium selection scheme that partitioned species into binding and non-binding fractions, calculating log2Y enrichment scores from next-generation sequencing data. These scores correlated with binding free energies, enabling identification of the tightest binders from a complex mixture. The representative peptide BiS3 exhibited a dissociation constant of 0.3 nM and an IC50 of 4.8 nM against GSK3β while showing no inhibition of seven other CMGC family kinases at 50 nM concentration.
Crystal structures of six peptide complexes at resolutions between 1.39 and 2.07 Å revealed the mechanism underlying this remarkable selectivity. The RZ dipeptide occupies the ATP pocket, with Z forming bifurcated hydrogen bonds to V135 in the hinge region while arginine mimics magnesium coordination to catalytic residues. Beyond this expected bivalent binding, the ZRZXXS motif creates a central channel that accommodates the guanidinium group of GSK3β R141 in a knob-into-hole arrangement stabilized by hydrogen bonding and aromatic stacking. Because R141 is not conserved among other CMGC kinases, this interaction likely drives the observed selectivity. A third contact point engages a previously unexploited hydrophobic pocket on the C-lobe. Conjugation to a cyclic deca-arginine cell-penetrating peptide enabled the inhibitors to reduce viability of HCT116 colorectal cancer cells, providing preliminary evidence of intracellular target engagement.
