Ligand-Specific Allosteric Coupling Controls G-Protein-Coupled Receptor Signaling.

Allosteric coupling describes a reciprocal process whereby G-protein-coupled receptors (GPCRs) relay ligand-induced conformational changes from the extracellular binding pocket to the intracellular signaling surface. Therefore, GPCR activation is sensitive to both the type of extracellular ligand and intracellular signaling protein. We hypothesized that ligand-specific allosteric coupling may result in preferential (i.

Carbachol dimers with primary carbamate groups as homobivalent modulators of muscarinic receptors.

Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers.

N-Ethylmaleimide differentiates between the M- and M-autoreceptor-mediated inhibition of acetylcholine release in the mouse brain.

Muscarinic M and M receptors resemble each other in brain distribution, function, and G protein signaling. However, there is evidence from human recombinant receptors that the M receptor also couples to G protein whereas such an alternative signaling is of minor importance for its M counterpart. The question arises whether this property is shared by native receptors, e.

Repurposing HAMI3379 to Block GPR17 and Promote Rodent and Human Oligodendrocyte Differentiation.

Identification of additional uses for existing drugs is a hot topic in drug discovery and a viable alternative to de novo drug development. HAMI3379 is known as an antagonist of the cysteinyl-leukotriene CysLT receptor, and was initially developed to treat cardiovascular and inflammatory disorders. In our study we identified HAMI3379 as an antagonist of the orphan G protein-coupled receptor GPR17.

Allosteric modulators targeting CNS muscarinic receptors.

Muscarinic acetylcholine receptors are G protein-coupled receptors (GPCRs) which are broadly expressed in the central nervous system (CNS) and other tissues in the periphery. They emerge as important drug targets for a number of diseases including Alzheimer's disease, Parkinson's disease, and schizophrenia. Muscarinic receptors are divided into five subtypes (M-M) of which M-M have been crystalized.

Ligand-Specific Restriction of Extracellular Conformational Dynamics Constrains Signaling of the M Muscarinic Receptor.

G protein-coupled receptors transmit extracellular signals across cell membranes via different G protein classes and β-arrestins. Some pathways may be therapeutically beneficial, whereas others may be detrimental under certain pathophysiological conditions. For many GPCRs, biased agonists are available, which preferentially signal through one pathway or a subset of pathways, and harnessing biased agonism could be a potential novel therapeutic strategy.

The Orphan Receptor GPR17 Is Unresponsive to Uracil Nucleotides and Cysteinyl Leukotrienes.

Pairing orphan G protein–coupled receptors (GPCRs) with their cognate endogenous ligands is expected to have a major impact on our understanding of GPCR biology. It follows that the reproducibility of orphan receptor ligand pairs should be of fundamental importance to guide meaningful investigations into the pharmacology and function of individual receptors. GPR17 is an orphan receptor characterized by some as a dualistic uracil nucleotide/cysteinyl leukotriene receptor and by others as inactive toward these stimuli altogether.

A New Molecular Mechanism To Engineer Protean Agonism at a G Protein-Coupled Receptor.

Protean agonists are of great pharmacological interest as their behavior may change in magnitude and direction depending on the constitutive activity of a receptor. Yet, this intriguing phenomenon has been poorly described and understood, due to the lack of stable experimental systems and design strategies. In this study, we overcome both limitations: First, we demonstrate that modulation of the ionic strength in a defined experimental set-up allows for analysis of G protein-coupled receptor activation in the absence and presence of a specific amount of spontaneous receptor activity using the muscarinic M acetylcholine receptor as a model.

Engineered Context-Sensitive Agonism: Tissue-Selective Drug Signaling through a G Protein-Coupled Receptor.

Drug discovery strives for selective ligands to achieve targeted modulation of tissue function. Here we introduce engineered context-sensitive agonism as a postreceptor mechanism for tissue-selective drug action through a G protein-coupled receptor. Acetylcholine M-receptor activation is known to mediate, among other actions, potentially dangerous slowing of the heart rate.

Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor.

G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic.

A Molecular Mechanism for Sequential Activation of a G Protein-Coupled Receptor.

Ligands targeting G protein-coupled receptors (GPCRs) are currently classified as either orthosteric, allosteric, or dualsteric/bitopic. Here, we introduce a new pharmacological concept for GPCR functional modulation: sequential receptor activation. A hallmark feature of this is a stepwise ligand binding mode with transient activation of a first receptor site followed by sustained activation of a second topographically distinct site.

The experimental power of FR900359 to study Gq-regulated biological processes.

Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action.

Lack of Gαi2 leads to dilative cardiomyopathy and increased mortality in β1-adrenoceptor overexpressing mice.

Aims: Inhibitory G (Gi) proteins have been proposed to be cardioprotective. We investigated effects of Gαi2 knockout on cardiac function and survival in a murine heart failure model of cardiac β1-adrenoceptor overexpression.

Methods And Results: β1-transgenic mice lacking Gαi2 (β1-tg/Gαi2 (-/-)) were compared with wild-type mice and littermates either overexpressing cardiac β1-adrenoceptors (β1-tg) or lacking Gαi2 (Gαi2 (-/-)).

Rational design of partial agonists for the muscarinic m1 acetylcholine receptor.

Aiming to design partial agonists for a G-protein-coupled receptor based on dynamic ligand binding, we synthesized three different series of bipharmacophoric ligands composed of the orthosteric building blocks iperoxo and 1 linked to allosteric modulators (BQCA-derived compounds, BQCAd; TBPB-derived compound, TBPBd). Their interactions were studied with the human muscarinic acetylcholine M1-receptor (hM1) with respect to receptor binding and Gq-protein signaling. Results demonstrate that iperoxo/BQCAd (2, 3) and 1/BQCAd hybrids (4) act as M1 partial agonists, whereas 1/TBPBd hybrids (5) did not activate M1-receptors.

Pilot the pulse: controlling the multiplicity of receptor dynamics.

G protein-coupled receptors (GPCRs) are involved in almost every (patho)physiological process, which explains their importance as drug targets. GPCRs have long been regarded as on/off-switches, which is reflected by direct activation or blockade of these receptors through the majority of marketed GPCR drugs. In recent years, however, our view of GPCRs has changed dramatically.

Dualsteric muscarinic antagonists--orthosteric binding pose controls allosteric subtype selectivity.

Bivalent ligands of G protein-coupled receptors have been shown to simultaneously either bind to two adjacent receptors or to bridge different parts of one receptor protein. Recently, we found that bivalent agonists of muscarinic receptors can simultaneously occupy both the orthosteric transmitter binding site and the allosteric vestibule of the receptor protein. Such dualsteric agonists display a certain extent of subtype selectivity, generate pathway-specific signaling, and in addition may allow for designed partial agonism.

A cell-permeable inhibitor to trap Gαq proteins in the empty pocket conformation.

In spite of the crucial role of heterotrimeric G proteins as molecular switches transmitting signals from G protein-coupled receptors, their selective manipulation with small molecule, cell-permeable inhibitors still remains an unmet challenge. Here, we report that the small molecule BIM-46187, previously classified as pan-G protein inhibitor, preferentially silences Gαq signaling in a cellular context-dependent manner. Investigations into its mode of action reveal that BIM traps Gαq in the empty pocket conformation by permitting GDP exit but interdicting GTP entry, a molecular mechanism not yet assigned to any other small molecule Gα inhibitor to date.

New insight into active muscarinic receptors with the novel radioagonist [³H]iperoxo.

Activation of G protein-coupled receptors involves major conformational changes of the receptor protein ranging from the extracellular transmitter binding site to the intracellular G protein binding surface. GPCRs such as the muscarinic acetylcholine receptors are commonly probed with radioantagonists rather than radioagonists due to better physicochemical stability, higher affinity, and indifference towards receptor coupling states of the former. Here we introduce tritiated iperoxo, a superagonist at muscarinic M₂ receptors with very high affinity.

Bis(ammonio)alkane-type agonists of muscarinic acetylcholine receptors: synthesis, in vitro functional characterization, and in vivo evaluation of their analgesic activity.

In this study, we synthesized and tested in vitro and in vivo two groups of bis(ammonio)alkane-type compounds, 6a-9a and 6b-9b, which incorporate the orthosteric muscarinic agonist iperoxo into a molecular fragment of the M2-selective allosteric modulators W84 and naphmethonium. The agonist potency and efficacy of these hybrid derivatives at M1, M2 and M3 muscarinic receptor subtypes and their anticholinesterase activity were evaluated on isolated tissue preparations. Their analgesic action was then assayed in vivo in the acetic acid writhing test and the occurrence of peripheral and central cholinergic side effects was also determined.

Dynamic ligand binding dictates partial agonism at a G protein-coupled receptor.

We present a new concept of partial agonism at G protein-coupled receptors. We demonstrate the coexistence of two functionally distinct populations of the muscarinic M2 receptor stabilized by one dynamic ligand, which binds in two opposite orientations. The ratio of orientations determines the cellular response.

Decoding signaling and function of the orphan G protein-coupled receptor GPR17 with a small-molecule agonist.

Replacement of the lost myelin sheath is a therapeutic goal for treating demyelinating diseases of the central nervous system (CNS), such as multiple sclerosis (MS). The G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) GPR17, which is phylogenetically closely related to receptors of the "purinergic cluster," has emerged as a modulator of CNS myelination. However, whether GPR17-mediated signaling positively or negatively regulates this critical process is unresolved.

Dualsteric GPCR targeting and functional selectivity: the paradigmatic M(2) muscarinic acetylcholine receptor.

Muscarinic acetylcholine receptors belong to Class Aseven transmembrane helical receptors and serve as important drug targets in the treatment of various diseases such as chronic obstructive pulmonary disease, overactive bladder, bronchial asthma and glaucoma. Despite intensive research the discovery of experimental ligands which activate or block specific muscarinic receptor subtypes has only been successful for the M1 and M4 subtypes but remains a challenging task at the other subtypes. In recent years, ligands have been introduced which bind simultaneously to the acetylcholine binding site, that is, the orthosteric site, and to an allosteric binding site.

Molecular alliance-from orthosteric and allosteric ligands to dualsteric/bitopic agonists at G protein coupled receptors.

Cell-membrane-spanning G protein coupled receptors (GPCRs) belong to the most important therapeutic target structures. Endogenous transmitters bind from the outer side of the membrane to the "orthosteric" binding site either deep in the binding pocket or at the extracellular N-terminal end of the receptor protein. Exogenous modulators that utilize a different, "allosteric", binding site unveil a pathway to receptor subtype-selectivity.