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.

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.

NMR-Based Structural Characterization of a Two-Disulfide-Bonded Analogue of the FXIIIa Inhibitor Tridegin: New Insights into Structure-Activity Relationships.

The saliva of blood-sucking leeches contains a plethora of anticoagulant substances. One of these compounds derived from Haementeria ghilianii, the 66mer three-disulfide-bonded peptide tridegin, specifically inhibits the blood coagulation factor FXIIIa. Tridegin represents a potential tool for antithrombotic and thrombolytic therapy. We recently synthesized two-disulfide-bonded tridegin variants, which retained their inhibitory potential. For further lead optimization, however, structure information is required. We thus analyzed the structure of a two-disulfide-bonded tridegin isomer by solution 2D NMR spectroscopy in a combinatory approach with subsequent MD simulations. The isomer was studied using two fragments, i.e., the disulfide-bonded N-terminal (Lys1-Cys37) and the flexible C-terminal part (Arg38-Glu66), which allowed for a simplified, label-free NMR-structure elucidation of the 66mer peptide. The structural information was subsequently used in molecular modeling and docking studies to provide insights into the structure-activity relationships. The present study will prospectively support the development of anticoagulant-therapy-relevant compounds targeting FXIIIa.

Deciphering Specificity Determinants for FR900359-Derived Gq α Inhibitors Based on Computational and Structure-Activity Studies.

Direct targeting of intracellular Gα subunits of G protein-coupled receptors by chemical tools is a challenging task in current pharmacological studies and in the development of novel therapeutic approaches. In this study we analyzed novel FR900359-based analogs from natural sources, synthetic cyclic peptides, as well as all so-far known Gq α inhibitors in a comprehensive study to devise a strategy for the elucidation of characteristics that determine interactions with and inhibition of Gq in the specific FR/YM-binding pocket. Using 2D NMR spectroscopy and molecular docking we identified unique features in the macrocyclic structures responsible for binding to the target protein correlating with inhibitory activity. While all novel compounds were devoid of effects on Gi and Gs proteins, no inhibitor surpassed the biological activity of FR. This raises the question of whether depsipeptides such as FR already represent valuable chemical tools for specific inhibition of Gq and, at the same time, are suitable natural lead structures for the development of novel compounds to target Gα subunits other than Gq .

Mechanistic Investigations of Two Bacterial Diterpene Cyclases: Spiroviolene Synthase and Tsukubadiene Synthase.

The mechanisms of two diterpene cyclases from streptomycetes-one with an unknown product that was identified as the spirocyclic hydrocarbon spiroviolene and one with the known product tsukubadiene-were investigated in detail by isotope labeling experiments. Although the structures of the products were very different, the cyclization mechanisms of both enzymes proceed through the same initial cyclization reactions, before they diverge towards the individual products, which is reflected in the close phylogenetic relationship of the enzymes.

Terpene Cyclases from Social Amoebae.

Genome sequences of social amoebae reveal the presence of terpene cyclases (TCs) in these organisms. Two TCs from Dictyostelium discoideum converted farnesyl diphosphate into (2S,3R,6S,9S)-(-)-protoillud-7-ene and (3S)-(+)-asterisca-2(9),6-diene. The enzyme mechanisms and EI-MS fragmentations of the products were studied by labeling experiments.