The molecular basis of subtype selectivity of human kinin G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) are the most important signal transducers in higher eukaryotes. Despite considerable progress, the molecular basis of subtype-specific ligand selectivity, especially for peptide receptors, remains unknown. Here, by integrating DNP-enhanced solid-state NMR spectroscopy with advanced molecular modeling and docking, the mechanism of the subtype selectivity of human bradykinin receptors for their peptide agonists has been resolved. The conserved middle segments of the bound peptides show distinct conformations that result in different presentations of their N and C termini toward their receptors. Analysis of the peptide-receptor interfaces reveals that the charged N-terminal residues of the peptides are mainly selected through electrostatic interactions, whereas the C-terminal segments are recognized via both conformations and interactions. The detailed molecular picture obtained by this approach opens a new gateway for exploring the complex conformational and chemical space of peptides and peptide analogs for designing GPCR subtype-selective biochemical tools and drugs.

The Aminotriazole Antagonist Cmpd-1 Stabilises a Distinct Inactive State of the Adenosine 2A Receptor.

The widely expressed G-protein coupled receptors (GPCRs) are versatile signal transducer proteins that are attractive drug targets but structurally challenging to study. GPCRs undergo a number of conformational rearrangements when transitioning from the inactive to the active state but have so far been believed to adopt a fairly conserved inactive conformation. Using 19 F NMR spectroscopy and advanced molecular dynamics simulations we describe a novel inactive state of the adenosine 2A receptor which is stabilised by the aminotriazole antagonist Cmpd-1. We demonstrate that the ligand stabilises a unique conformation of helix V and present data on the putative binding mode of the compound involving contacts to the transmembrane bundle as well as the extracellular loop 2.

Discovery of Orally Available and Brain Penetrant AEP Inhibitors.

Alzheimer's Disease (AD) is the most widespread form of dementia, with one of the pathological hallmarks being the formation of neurofibrillary tangles (NFTs). These tangles consist of phosphorylated Tau fragments. Asparagine endopeptidase (AEP) is a key Tau cleaving enzyme that generates aggregation-prone Tau fragments. Inhibition of AEP to reduce the level of toxic Tau fragment formation could represent a promising therapeutic strategy. Here, we report the first orthosteric, selective, orally bioavailable, and brain penetrant inhibitors with an irreversible binding mode. We outline the development of the series starting from reversible molecules and demonstrate the link between inhibition of AEP and reduction of Tau N368 fragment both in vitro and in vivo.

Modulation of PTH1R signaling by an extracellular binding antibody.

Parathyroid hormone receptor 1 (PTH1R) is a class B G-protein coupled receptor with key roles in bone development. The receptor signals through both the Gs and Gq G-proteins as well as through ß-arrestin in a G-protein independent manner. Current treatments for bone disorders, such as osteoporosis, target the PTH1R but are suboptimal in their efficacy. Monoclonal antibodies represent a major growth area in therapeutics as a result of their superior specificity and long serum half-life. Here, we discovered antibodies against the extracellular domain (ECD) of PTH1R from a phage display library. One of these antibodies, ECD-ScFvhFc, binds PTH1R with high affinity and although it has little or no effect on G-protein dependent receptor signaling, it does reduce PTH1R mediated ß-arrestin signaling. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) demonstrated that the ECD-ScFvhFc binding site overlapped partially with that of the cognate ligand, PTH. The results of this study demonstrate the suitability of PTH1R as a target for therapeutic antibody development.

Modulation of PTH1R signaling by an ECD binding antibody results in inhibition of ß-arrestin 2 coupling.

Parathyroid hormone receptor 1 (PTH1R) belongs to the secretin class of G protein coupled receptors (GPCRs) and natively binds parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP). Ligand binding to PTH1R involves binding to the large extracellular domain (ECD) and the orthosteric pocket, inducing conformational changes in the transmembrane domain and receptor activation. PTH1R regulates bone metabolism, signaling mainly through Gs and Gq/11 G-proteins. Here, we used phage display to generate PTH1R ECD-specific antibodies with the aim of modulating receptor functionality. We identified ECD-scFvhFc, which exhibited high affinity binding to both the isolated ECD and to the full-length receptor in styrene-maleic acid (SMA) lipid particles. Epitope mapping using hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicates that the α1 helix of the ECD is ECD-scFvhFc's epitope which may partially overlap with the known PTH (1-34) binding site. However, PTH (1-34)-mediated Gs activation is Undisturbed by ECD-scFvhFc binding. In contrast, ECD-scFvhFc potently inhibits ß-arrestin-2 recruitment after PTH (1-34)-driven receptor activation and thus represents the first monoclonal antibody to selectively inhibit distinct PTH1R signaling pathways. Given the complexity of PTH1R signaling and the emerging importance of biased GPCR activation in drug development, ECD-scFvhFc could be a valuable tool to study PTH1R signaling bias.

Cell-free synthesis of isotopically labelled peptide ligands for the functional characterization of G protein-coupled receptors.

Cell-free systems exploit the transcription and translation machinery of cells from different origins to produce proteins in a defined chemical environment. Due to its open nature, cell-free protein production is a versatile tool to introduce specific labels such as heavy isotopes, non-natural amino acids and tags into the protein while avoiding cell toxicity. In particular, radiolabelled peptides and proteins are valuable tools for the functional characterization of protein-protein interactions and for studying binding kinetics. In this study we evaluated cell-free protein production for the generation of radiolabelled ligands for G protein-coupled receptors (GPCRs). These receptors are seven-transmembrane-domain receptors activated by a plethora of extracellular stimuli including peptide ligands. Many GPCR peptide ligands contain disulphide bonds and are thus inherently difficult to produce in bacterial expression hosts or in Escherichia coli-based cell-free systems. Here, we established an adapted E. coli-based cell-free translation system for the production of disulphide bond-containing GPCR peptide ligands and specifically introduce tritium labels for detection. The bacterial oxidoreductase DsbA is used as a chaperone to favour the formation of disulphide bonds and to enhance the yield of correctly folded proteins and peptides. We demonstrate the correct folding and formation of disulphide bonds and show high-affinity ligand binding of the produced radio peptide ligands to the respective receptors. Thus, our system allows the fast, cost-effective and reliable synthesis of custom GPCR peptide ligands for functional and structural studies.

Expression of membrane proteins in the eyes of transgenic Drosophila melanogaster.

In recent years, improved protein expression and crystallization strategies, as well as advanced synchrotron radiation sources and crystallographic tools considerably increased the number of crystal structures of integral membrane proteins from higher eukaryotes. However, seen as a proportion of the total number of candidate proteins, these achievements still appear meager, reflecting the huge effort that is often required to obtain high-level and functional expression of eukaryotic membrane proteins. Besides bacteria, yeast, insect, or mammalian cells are frequently used for heterologous expression, but despite considerable investments in time, labor, and money, there are numerous drawbacks to these systems. Are there other strategies that allow for an effective, large-scale production of functional membrane proteins? This chapter describes the expression of proteins in photoreceptor cells of transgenic Drosophila as an easily accessible, versatile alternative. We present step-by-step protocols starting from the cloning of the target gene into a suitable vector for fly eye expression and ending with the harvest of transgenic Drosophila and subsequent protein purification from the eye. Our examples span a number of eukaryotic membrane proteins from different classes-including receptors, transporters, channels, and enzymes-that were successfully expressed without further optimization. The protocols provided here are robust and straightforward to follow even without prior experience in Drosophila work.