Targeting Gαi/s Proteins with Peptidyl Nucleotide Exchange Modulators.

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 cell-penetrating 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.

High-affinity binding and catalytic activity of His/Tyr-based sequences: Extending heme-regulatory motifs beyond CP.

BACKGROUND & MOTIVATION: Peptides and proteins can interact with heme through His, Tyr, or Cys in heme-regulatory motifs (HRMs). The Cys-Pro dipeptide is a well investigated HRM, but for His and Tyr such a distinct motif is currently unknown. In addition, many heme-peptide complexes, such as heme-amyloid ß, can display a peroxidase-like activity, albeit there is little understanding of how the local primary and secondary coordination environment influences catalytic activity. We thus systematically evaluated a series of His- and Tyr-based peptides to identify sequence features for high-affinity heme binding and their impact on the catalytic activity of heme. METHODS: We employed solid-phase peptide synthesis to produce 58 nonapeptides, which were investigated by UV/vis, resonance Raman, and 2D NMR spectroscopy. A chromogenic assay was used to determine the catalytic activity of the heme-peptide complexes. RESULTS: Heme-binding affinity and binding mode were found to be dependent on the coordinating amino acid and spacer length between multiple potential coordination sites in a motif. In particular, HXH and HXXXH motifs showed strong heme binding. Analysis of the peroxidase-like activity revealed that some of these peptides and also HXXXY motifs enhance the catalytic activity of heme significantly. CONCLUSIONS: We identify HXH, HXXXH, and HXXXY as potential new HRMs with functional properties. Several peptides displayed a strikingly high peroxidase-like activity. GENERAL SIGNIFICANCE: The identification of HRMs allows to discover yet unknown heme-regulated proteins, and consequently, enhances our current understanding of pathologies involving labile heme.