Bicyclic Peptide Library Screening for the Identification of Gαi Protein Modulators

Noncanonical G protein activation and inactivation, particularly
for the Gαi/s protein subfamilies, have long been a focus of
chemical research. Combinatorial libraries were already effectively
applied to identify modulators of the guanine-nucleotide exchange,
as can be exemplified with peptides such as KB-752 and GPM-1c/d, the
so-called guanine-nucleotide exchange modulators. In this study, we
identified novel bicyclic peptides from a combinatorial library screening
that show prominent properties as molecular switch-on/off modulators
of Gαi signaling. Among the series of hits, the exceptional
paradigm of GPM-3, a protein and state-specific bicyclic peptide,
is the first chemically identified GAP (GTPase-activating protein)
modulator with a high binding affinity for Gαi protein. Computational
analyses identified and assessed the structure of the bicyclic peptides,
novel ligand–protein interaction sites, and their subsequent
impact on the nucleotide binding site. This approach can therefore
lead the way for the development of efficient chemical biological
probes targeting Gαi protein modulation within a cellular context.