Targeting Gαi/s Proteins with Peptidyl Nucleotide Exchange Modulators

Abstract

Chemical probes that specifically modulate the activity of heterotrimeric G proteins provide excellent tools for investigating G protein-mediated cell signaling. Herein, we report a family of selective peptidyl Gαi/s modulators derived from peptide library screening and optimization. Conjugation to a cellpenetrating peptide rendered the peptides cell-permeable and biologically active in cell-based assays. The peptides exhibit potent guanine-nucleotide exchange modulator-like activity toward Gαi and Gαs. Molecular docking and dynamic simulations revealed the molecular basis of the protein−ligand interactions and their effects on GDP binding. This study demonstrates the feasibility of developing direct Gαi/s modulators and provides a novel chemical probe for investigating cell signaling through GPCRs/G proteins.

Bicyclic Peptide Library Screening for the Identification of Gαi Protein Modulators

“…We previously demonstrated that linear peptidyl modulators of the Gα protein can be obtained by screening an OBOC combinatorial peptide library. 7 However, linear peptides are proteolytically labile and have poor cell permeability. 44 To overcome these limitations, we opted to screen a bicyclic peptide library, as the latter approach has recently led to the discovery of potent, cellpermeable, and metabolically stable bicyclic peptidyl inhibitors against the monomeric G protein K-Ras, namely, cyclorasin B3 and B4 (Figure 1b).…”

“…In c, f, and i, the chemical structure of each bicyclic peptide and their atom interactions (oxygen in red, nitrogen in blue) with surrounding protein residues at the binding sites are depicted in details. PDB IDs: Gαi1•GTPγS (1GIA 54 ) and the homology model of Gαi1•GDP 7 (PDB IDs 1Y3A, 29 5JS8, 60 and 3UMS 61 ). molecular dynamics (MD) simulations, we observed that GPM-3 and GPM-2 form stable complexes with Gαi via distinct stabilizing interactions (Figure 4a−c and d−f, respectively).…”

“…11 In our recent study, we were able to obtain from a one-bead-one-compound (OBOC) combinatorial peptide library screening a promising linear peptide as a lead compound (GPM-1, RWLRYLRYP), which was further optimized by cyclization and conjugation to a cell-penetrating peptide (CPP). 7 Such high-throughput screening (HTS) technologies, including also the one-bead-two-compound (OBTC) libraries and the random nonstandard peptide integrated discovery (RaPID) system, 12−15 have been frequently used for the identification of high-affinity cyclic peptide binders to a variety of proteins. This can be exemplified with the bicyclic cyclorasin B4−27 16 acting as an inhibitor of the monomeric G protein K-Ras.…”

Fluorescence Anisotropy Assay with Guanine Nucleotides Provides Access to Functional Analysis of Gαi1 Proteins

“…The gene of human (hexahistidine) His 6 -tagged Gαi1 (Uniprot ID: P63096) was cloned into the pET28a (+) expression vector and transformed into E. coli BL21 (DE3) bacteria cells as previously described with slight modifications . Bacteria were grown in LB medium (37 °C, OD 600 of 0.4–0.6) and induction was initiated by the addition of 0.25 mM IPTG (4 h, 30 °C).…”

“…The gene of human (hexahistidine) His 6tagged Gαi1 (Uniprot ID: P63096) was cloned into the pET28a (+) expression vector and transformed into E. coli BL21 (DE3) bacteria cells as previously described with slight modifications. 30 Bacteria were grown in LB medium (37 °C, OD 600 of 0.4−0.6) and induction was initiated by the addition of 0.25 mM IPTG (4 h, 30 °C). Bacterial cells were lysed (1 mg mL −1 lysozyme and protease inhibitors), and the expressed protein was purified by Ni-NTA affinity chromatography and subsequently by size exclusion chromatography (SEC) on an A ̈kta Prime Plus device equipped with a HiLoad 16/600 Superdex gel filtration column.…”