In-depth analysis of Gαs protein activity by probing different fluorescently labeled guanine nucleotides.

G proteins are interacting partners of G protein-coupled receptors (GPCRs) in eukaryotic cells. Upon G protein activation, the ability of the Gα subunit to exchange GDP for GTP determines the intracellular signal transduction. Although various studies have successfully shown that both Gαs and Gαi have an opposite effect on the intracellular cAMP production, with the latter being commonly described as "more active", the functional analysis of Gαs is a comparably more complicated matter. Additionally, the thorough investigation of the ubiquitously expressed variants of Gαs, Gαs(short) and Gαs(long), is still pending. Since the previous experimental evaluation of the activity and function of the Gαs isoforms is not consistent, the focus was laid on structural investigations to understand the GTPase activity. Herein, we examined recombinant human Gαs by applying an established methodological setup developed for Gαi characterization. The ability for GTP binding was evaluated with fluorescence and fluorescence anisotropy assays, whereas the intrinsic hydrolytic activity of the isoforms was determined by a GTPase assay. Among different nucleotide probes, BODIPY FL GTPγS exhibited the highest binding affinity towards the Gαs subunit. This work provides a deeper understanding of the Gαs subunit and provides novel information concerning the differences between the two protein variants.

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

Bicyclic peptides are important chemical tools that can function, for example, as bioactive ligands switching on/off signaling pathways mediated by guanine nucleotide-binding proteins as bicycles are more broadly applicable. Despite their relevance in medicinal chemistry, the synthesis of such peptides is challenging, and the final yield is highly dependent on the chemical composition and physicochemical properties of the scaffold. We recently discovered novel, state-specific peptide modulators targeting the Gαi protein, namely, GPM-2/GPM-3, by screening a one-bead-two-compound combinatorial library. A more detailed analysis, including sequence alignments and computer-assisted conformational studies based on the hit compounds, revealed the new peptide 10 as a potential macrobicyclic Gαi ligand sharing high sequence similarity to the known Gαi modulators. The Gαs protein was included in this study for comparison and to unravel the criteria for the specificity of modulator binding to Gαi versus Gαs. This work provides in-depth computer-assisted experimental studies for the analysis of novel macrobicyclic, library-derived Gαi protein ligands. The sequence and structural comparison of 10 with the lead compounds GPM-2 and GPM-3 reveals the importance of the size and amino acid composition of one ring of the bicyclic system and suggests features enhancing the binding affinity of the peptides to the Gαi protein.

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.

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

Gα proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor- mediated intracellular signaling. The role of the Gα subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and Gα protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein. Herein, we report the development of a reliable fluorescence anisotropy-based method to determine the affinity of ligands at the GTP-binding site and to quantify the fraction of active Gαi1 protein. An advanced bacterial expression protocol was applied to produce active human Gαi1 protein, whose GTP binding capability was determined with novel fluorescently labeled guanine nucleotides acting as high-affinity Gαi1 binders compared to the commonly used BODIPY FL GTPγS. This study thus contributes a new method for future investigations of the characterization of Gαi and other Gα protein subunits, exploring their corresponding signal transduction systems and potential for biomedical applications.

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.

Strategies towards Targeting Gαi/s Proteins: Scanning of Protein-Protein Interaction Sites To Overcome Inaccessibility.

Heterotrimeric G proteins are classified into four subfamilies and play a key role in signal transduction. They transmit extracellular signals to intracellular effectors subsequent to the activation of G protein-coupled receptors (GPCRs), which are targeted by over 30 % of FDA-approved drugs. However, addressing G proteins as drug targets represents a compelling alternative, for example, when G proteins act independently of the corresponding GPCRs, or in cases of complex multifunctional diseases, when a large number of different GPCRs are involved. In contrast to Gαq, efforts to target Gαi/s by suitable chemical compounds has not been successful so far. Here, a comprehensive analysis was conducted examining the most important interface regions of Gαi/s with its upstream and downstream interaction partners. By assigning the existing compounds and the performed approaches to the respective interfaces, the druggability of the individual interfaces was ranked to provide perspectives for selective targeting of Gαi/s in the future.