Bicyclic Peptide Library Screening for the Identification of Gαi Protein Modulators

Abstract

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.

Design, synthesis, and analysis of macrobicyclic peptides for targeting the Gαi protein

“…As a result, peptide 10 was found to bind to the target protein Gαi1 with high affinity, and based on the computational analysis, it showed high sequence and structural similarity to the earlier reported Gαi-modulators GPM-2 and GPM-3. 7 This study thus allows us to suggest physicochemical properties of macrobicyclic peptides that are favorable for the efficient binding to the Gαi protein supporting the future development of Gαi modulators in contrast to Gαs.…”

“…Out of these, two more hits, herein referred to as peptides 9 and 10, exhibited sequence similarities to the previously selected peptides in accordance with the consensus sequence. 7 However, the final yields for the synthesis of these peptides were rather low, thus hampering further in-depth analysis in our first study. 7 In the present work, sequential and structural prerequisites for activity modulation of the Gαi in contrast to Gαs through interaction with peptide tools were studied by employing the Gαi-binding peptides 9 and 10, 7 derived from the aforementioned OBTC combinatorial peptide library, but not yet considered for further investigation.…”

“…7 However, the final yields for the synthesis of these peptides were rather low, thus hampering further in-depth analysis in our first study. 7 In the present work, sequential and structural prerequisites for activity modulation of the Gαi in contrast to Gαs through interaction with peptide tools were studied by employing the Gαi-binding peptides 9 and 10, 7 derived from the aforementioned OBTC combinatorial peptide library, but not yet considered for further investigation. In addition, novel candidates (peptides 11-18), originally identified as Gαs binders, were included in the study for reasons of comparison.…”

“…26 Recently, we introduced two bicyclic peptides containing nonproteinogenic residues that were obtained from the screening of an OBTC combinatorial peptide library, 13,18,22 GPM-2 class-and state-selective to the Gαi protein subunit (Table 1). 7 In addition, cellular assays revealed a guanine nucleotide exchange factorlike activity for GPM-2, whereas GPM-3 was identified as the first GTPase-activating protein modulator for the Gαi1 protein. 7 Apart from the peptides 1-8, 7 the peptide library screening, however, identified further ligands 7 specific for the Gαi subunit.…”

“…7 In addition, cellular assays revealed a guanine nucleotide exchange factorlike activity for GPM-2, whereas GPM-3 was identified as the first GTPase-activating protein modulator for the Gαi1 protein. 7 Apart from the peptides 1-8, 7 the peptide library screening, however, identified further ligands 7 specific for the Gαi subunit. Out of these, two more hits, herein referred to as peptides 9 and 10, exhibited sequence similarities to the previously selected peptides in accordance with the consensus sequence.…”