Discovery of Supra-Bivalent GSK3β Inhibitory Peptides Containing an ATP-Mimetic Amino Acid

De novo discovery of highly selective inhibitors of protein
kinases
(PKs) remains a significant challenge in drug discovery. Although
ATP-site-directed bivalent inhibitors targeting a distinct binding
site are an emerging alternative, their rational design is impeded
by limited knowledge about allosteric binding sites unique to the
target kinase and the complexity of the linker design. Here, we report
a strategy that overcomes the above-mentioned issue using a macrocyclic
peptide library containing an ATP-mimetic “warhead”
amino acid, p-(pyrimidin-2-ylamino)phenylalanine
(termed Z), performing the RaPID selection for the discovery of highly
potent and selective inhibitors against the kinase GSK3β. The
experiment led to the enrichment of macrocycles containing a sequence
motif “ZRZ”, whose enrichment score log2Y was calculated for the identification of high-affinity
binders by means of next-generation sequencing analysis. We identified
several highly potent inhibitors, with a representative potent peptide
(BiS3) showing a KD of 0.3 nM and an IC50 of 4.8 nM against GSK3β. The X-ray structural analysis
revealed the unique binding mode of the peptides with “RZ”
mimicking ATP and Mg2+ in the ATP site. Moreover, the rest
of the peptide motifs engaged in multisite interactions via a substrate-competitive site and an unexpected knob-into-hole-like
conformation of the macrocycle, and thus, they act as “supra-bivalent”
kinase inhibitors. This unique binding mode conferred both potency
and selectivity toward GSK3β. This work demonstrated that our
strategy using the warhead amino acid Z is effective in obtaining
specific GSK3β inhibitors, which is likely expandable to other
PK families.