Insertion of Nanoluc into the Extracellular Loops as a Complementary Method To Establish BRET-Based Binding Assays for GPCRs.

Luminescence-based techniques play an increasingly important role in all areas of biochemical research, including investigations on G protein-coupled receptors (GPCRs). One quite recent and popular addition has been made by introducing bioluminescence resonance energy transfer (BRET)-based binding assays for GPCRs, which are based on the fusion of nanoluciferase (Nluc) to the N-terminus of the receptor and the occurring energy transfer via BRET to a bound fluorescent ligand. However, being based on BRET, the technique is strongly dependent on the distance/orientation between the luciferase and the fluorescent ligand.

Shining light on the histamine H receptor: Synthesis of carbamoylguanidine-type agonists as a pharmacological tool to study internalization.

So far, only little is known about the internalization process of the histamine H receptor (HR). One promising approach to study such dynamic processes is the use of agonistic fluorescent ligands. Therefore, a series of carbamoylguanidine-type HR agonists containing various fluorophores, heterocycles, and linkers (28-40) was synthesized.

Quantitative Determination and Imaging of Gα Signaling in Live Cells via Split-Luciferase Complementation.

G Protein-coupled receptors (GPCRs) transduce signals elicited by bioactive chemical agents (ligands), such as hormones, neurotransmitters, or cytokines, across the cellular membrane. Upon ligand binding, the receptor undergoes structural rearrangements, which cause the activation of G proteins. This triggers the activation of signaling cascades involving amplification, which takes place after every stage of the cascade.

Fluorescent H Receptor Squaramide-Type Antagonists: Synthesis, Characterization, and Applications.

Fluorescence labeled ligands have been gaining importance as molecular tools, enabling receptor-ligand-binding studies by various fluorescence-based techniques. Aiming at red-emitting fluorescent ligands for the hHR, a series of squaramides labeled with pyridinium or cyanine fluorophores (-) was synthesized and characterized. The highest hHR affinities in radioligand competition binding assays were obtained in the case of pyridinium labeled antagonists - (p : 7.

UR-DEBa242: A Py-5-Labeled Fluorescent Multipurpose Probe for Investigations on the Histamine H and H Receptors.

Comprehensively characterized fluorescent probes for the histamine H receptor (HR) and especially for the HR orthologs [e.g., human (h) and mouse (m)] are highly needed as versatile complementary tools to radioligands.

Fluorescence Labeling of Neurotensin(8-13) via Arginine Residues Gives Molecular Tools with High Receptor Affinity.

Fluorescence-labeled receptor ligands have emerged as valuable molecular tools, being indispensable for studying receptor-ligand interactions by fluorescence-based techniques such as high-content imaging, fluorescence microscopy, and fluorescence polarization. Through application of a new labeling strategy for peptides, a series of fluorescent neurotensin(8-13) derivatives was synthesized by attaching red-emitting fluorophores (indolinium- and pyridinium-type cyanine dyes) to carbamoylated arginine residues in neurotensin(8-13) analogues, yielding fluorescent probes with high NTSR affinity (p values: 8.15-9.

[H]UR-DEBa176: A 2,4-Diaminopyrimidine-Type Radioligand Enabling Binding Studies at the Human, Mouse, and Rat Histamine H Receptors.

Differences in sequence homology between human (h), mouse (m), and rat (r) histamine H receptors (HR) cause discrepancies regarding affinities, potencies, and/or efficacies of ligands and therefore compromise translational animal models and the applicability of radioligands. Aiming at a radioligand enabling robust and comparative binding studies at the h/m/rHRs, 2,4-diaminopyrimidines were synthesized and pharmacologically investigated. The most notable compounds identified were two (partial) agonists with comparable potencies at the h/m/rHRs: UR-DEBa148 (-neopentyl-4-(1,4,6,7-tetrahydro-5-imidazo[4,5-]pyridin-5-yl)pyrimidin-2-amine bis(2,2,2-trifluoroacetate), ), the most potent [pEC (reporter gene assay) = 9.

Novel BQCA- and TBPB-Derived M Receptor Hybrid Ligands: Orthosteric Carbachol Differentially Regulates Partial Agonism.

Recently, investigations of the complex mechanisms of allostery have led to a deeper understanding of G protein-coupled receptor (GPCR) activation and signaling processes. In this context, muscarinic acetylcholine receptors (mAChRs) are highly relevant due to their exemplary role in the study of allosteric modulation. In this work, we compare and discuss two sets of putatively dualsteric ligands, which were designed to connect carbachol to different types of allosteric ligands.

Split luciferase-based assay for simultaneous analyses of the ligand concentration- and time-dependent recruitment of β-arrestin2.

Functional selectivity of agonists has gained increasing interest in G protein-coupled receptor (GPCR) research, e.g. due to expectations of drugs with reduced adverse effects.

Fluorination of Photoswitchable Muscarinic Agonists Tunes Receptor Pharmacology and Photochromic Properties.

Red-shifted azobenzene scaffolds have emerged as useful molecular photoswitches to expand potential applications of photopharmacological tool compounds. As one of them, tetra- ortho-fluoro azobenzene is well compatible for the design of visible-light-responsive systems, providing stable and bidirectional photoconversions and tissue-compatible characteristics. Using the unsubstituted azobenzene core and its tetra- ortho-fluorinated analogue, we have developed a set of uni- and bivalent photoswitchable toolbox derivatives of the highly potent muscarinic acetylcholine receptor agonist iperoxo.

A split luciferase-based probe for quantitative proximal determination of Gα signalling in live cells.

The earlier an activation of a G protein-dependent signalling cascade at a G protein-coupled receptor (GPCR) is probed, the less amplificatory effects contribute to the measured signal. This is especially useful in case of a precise quantification of agonist efficacies, and is of paramount importance, when determining agonist bias in relation to the β-arrestin pathway. As most canonical assays with medium to high throughput rely on the quantification of second messengers, and assays affording more proximal readouts are often limited in throughput, we developed a technique with a proximal readout and sufficiently high throughput that can be used in live cells.

In Search of NPY YR Antagonists: Incorporation of Carbamoylated Arginine, Aza-Amino Acids, or d-Amino Acids into Oligopeptides Derived from the C-Termini of the Endogenous Agonists.

The cross-linked pentapeptides (2,7)-diaminooctanedioyl-bis(Tyr-Arg-Leu-Arg-Tyr-amide) ((2,7)-BVD-74D, (2,7)-) and octanedioyl-bis(Tyr-Arg-Leu-Arg-Tyr-amide) () as well as the pentapeptide Ac-Tyr-Arg-Leu-Arg-Tyr-amide () were previously described as neuropeptide Y Y receptor (YR) partial agonists. Here, we report on a series of analogues of (2,7)- and in which Arg, Leu, or Arg were replaced by the respective aza-amino acids. The replacement of Arg in with a carbamoylated arginine building block and the extension of the N-terminus by an additional arginine led to the high-affinity hexapeptide Ac-Arg-Tyr--[(4-aminobutyl)aminocarbonyl]Arg-Leu-Arg-Tyr-amide (), which was used as a precursor for a d-amino acid scan.

Structural basis of ligand binding modes at the neuropeptide Y Y receptor.

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology . The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y, Y, Y and Y receptors, with different affinity and selectivity . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y receptor (YR) .

Establishing a Split Luciferase Assay for Proteinkinase G (PKG) Interaction Studies.

Nitric oxide (NO/cyclic guanosine monophosphate (cGMP)-regulated cellular mechanisms are involved in a variety of (patho-) physiological processes. One of the main effector molecules in this system, proteinkinase G (PKG), serves as a molecular switch by phosphorylating different target proteins and thereby turning them on or off. To date, only a few interaction partners of PKG have been described although the identification of protein-protein interactions (PPI) is indispensable for the understanding of cellular processes and diseases.

Fluorescence- and Radiolabeling of [Lys,Nle]hPP Yields Molecular Tools for the NPY Y Receptor.

The neuropeptide Y (NPY) Y receptor (YR) is involved in energy homeostasis and considered a potential drug target for the treatment of obesity. Only a few molecular tools, i.e.

Recruitment of β-arrestin 1 and 2 to the β2-adrenoceptor: analysis of 65 ligands.

Unlabelled: Beyond canonical signaling via Gαs and cAMP, the concept of functional selectivity at β2-adrenoceptors (β2ARs) describes the ability of adrenergic drugs to stabilize ligand-specific receptor conformations to initiate further signaling cascades comprising additional G-protein classes or β-arrestins (βarr). A set of 65 adrenergic ligands including 40 agonists and 25 antagonists in either racemic or enantiopure forms was used for βarr recruitment experiments based on a split-luciferase assay in a cellular system expressing β2AR. Many agonists showed only (weak) partial agonism regarding βarr recruitment.

Structure-bias relationships for fenoterol stereoisomers in six molecular and cellular assays at the β2-adrenoceptor.

Functional selectivity is well established as an underlying concept of ligand-specific signaling via G protein-coupled receptors (GPCRs). Functionally, selective drugs could show greater therapeutic efficacy and fewer adverse effects. Dual coupling of the β2-adrenoceptor (β2AR) triggers a signal transduction via Gsα and Giα proteins.