Selective activation of individual subtypes of muscarinic receptors is a promising way to safely alleviate a wide range of pathological conditions in the central nervous system and the periphery as well. The flexible G-protein interface of muscarinic receptors allows them to interact with several G-proteins with various efficacy, potency, and kinetics. Agonists biased to the particular G-protein mediated pathway may result in selectivity among muscarinic subtypes and, due to the non-uniform expression of individual G-protein alpha subunits, possibly achieve tissue specificity. Here, we demonstrate that novel tetrahydropyridine-based agonists exert specific signalling profiles in coupling with individual G-protein α subunits. These signalling profiles profoundly differ from the reference agonist carbachol. Moreover, coupling with individual Gα induced by these novel agonists varies among subtypes of muscarinic receptors which may lead to subtype selectivity. Thus, the novel tetrahydropyridine-based agonist can contribute to the elucidation of the mechanism of pathway-specific activation of muscarinic receptors and serve as a starting point for the development of desired selective muscarinic agonists.

• MeSH
• Muscarinic Agonists * pharmacology   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Carbachol pharmacology   MeSH
• Humans MeSH
• GTP-Binding Protein alpha Subunits metabolism   genetics   MeSH
• GTP-Binding Proteins metabolism   MeSH
• Pyridines pharmacology   MeSH
• Receptors, Muscarinic * metabolism   MeSH
• Signal Transduction drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Muscarinic Agonists * MeSH
• Carbachol MeSH
• GTP-Binding Protein alpha Subunits MeSH
• GTP-Binding Proteins MeSH
• Pyridines MeSH
• Receptors, Muscarinic * MeSH

Agonist efficacy denoting the "strength" of agonist action is a cornerstone in the proper assessment of agonist selectivity and signalling bias. The simulation models are very accurate but complex and hard to fit experimental data. The parsimonious operational model of agonism (OMA) has become successful in the determination of agonist efficacies and ranking them. In 1983, Black and Leff introduced the slope factor to the OMA to make it more flexible and allow for fitting steep as well as flat concentration-response curves. First, we performed a functional analysis to indicate the potential pitfalls of the OMA. Namely, exponentiation of operational efficacy may break relationships among the OMA parameters. The fitting of the Black & Leff equation to the theoretical curves of several models of functional responses and the experimental data confirmed the fickleness of the exponentiation of operational efficacy affecting estimates of operational efficacy as well as other OMA parameters. In contrast, fitting The OMA based on the Hill equation to the same data led to better estimates of model parameters. In conclusion, Hill equation-based OMA should be preferred over the Black & Leff equation when functional-response curves differ in the slope factor. Otherwise, the Black & Leff equation should be used with extreme caution acknowledging potential pitfalls.

• MeSH
• Models, Biological * MeSH
• Computer Simulation MeSH
• Signal Transduction * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

A complex evaluation of agonist bias at G-protein coupled receptors at the level of G-protein classes and isoforms including non-preferential ones is essential for advanced agonist screening and drug development. Molecular crosstalk in downstream signaling and a lack of sufficiently sensitive and selective methods to study direct coupling with G-protein of interest complicates this analysis. We performed binding and functional analysis of 11 structurally different agonists on prepared fusion proteins of individual subtypes of muscarinic receptors and non-canonical promiscuous α-subunit of G16 protein to study agonist bias. We have demonstrated that fusion of muscarinic receptors with Gα16 limits access of other competitive Gα subunits to the receptor, and thus enables us to study activation of Gα16 mediated pathway more specifically. Our data demonstrated agonist-specific activation of G16 pathway among individual subtypes of muscarinic receptors and revealed signaling bias of oxotremorine towards Gα16 pathway at the M2 receptor and at the same time impaired Gα16 signaling of iperoxo at M5 receptors. Our data have shown that fusion proteins of muscarinic receptors with α-subunit of G-proteins can serve as a suitable tool for studying agonist bias, especially at non-preferential pathways.

• Keywords
• fusion proteins, muscarinic receptors, non-canonical signaling, signaling bias,
• MeSH
• Cyclic AMP metabolism   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Inhibitory Concentration 50 MeSH
• Isoxazoles chemistry   MeSH
• Cricetinae MeSH
• Quaternary Ammonium Compounds chemistry   MeSH
• Humans MeSH
• Molecular Conformation MeSH
• Oxotremorine chemistry   MeSH
• GTP-Binding Protein alpha Subunits, Gq-G11 metabolism   MeSH
• Receptors, Muscarinic metabolism   MeSH
• Recombinant Fusion Proteins chemistry   MeSH
• Signal Transduction * MeSH
• Molecular Dynamics Simulation MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cyclic AMP MeSH
• G protein alpha 16 MeSH Browser
• iperoxo MeSH Browser
• Isoxazoles MeSH
• Quaternary Ammonium Compounds MeSH
• Oxotremorine MeSH
• GTP-Binding Protein alpha Subunits, Gq-G11 MeSH
• Receptors, Muscarinic MeSH
• Recombinant Fusion Proteins MeSH

Proper determination of agonist efficacy is indispensable in the evaluation of agonist selectivity and bias to activation of specific signalling pathways. The operational model (OM) of pharmacological agonism is a useful means for achieving this goal. Allosteric ligands bind to receptors at sites that are distinct from those of endogenous agonists that interact with the orthosteric domain on the receptor. An allosteric modulator and an orthosteric agonist bind simultaneously to the receptor to form a ternary complex, where the allosteric modulator affects the binding affinity and operational efficacy of the agonist. Allosteric modulators are an intensively studied group of receptor ligands because of their selectivity and preservation of physiological space-time pattern of the signals they modulate. We analysed the operational model of allosterically-modulated agonism (OMAM) including modulation by allosteric agonists. Similar to OM, several parameters of OMAM are inter-dependent. We derived equations describing mutual relationships among parameters of the functional response and OMAM. We present a workflow for the robust fitting of OMAM to experimental data using derived equations.

• MeSH
• Allosteric Regulation MeSH
• Kinetics MeSH
• Humans MeSH
• Ligands MeSH
• Receptors, G-Protein-Coupled agonists   metabolism   MeSH
• Drug Synergism * MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ligands MeSH
• Receptors, G-Protein-Coupled MeSH

BACKGROUND AND PURPOSE: More than 30% of currently marketed medications act via GPCRs. Thus, GPCRs represent one of the most important pharmacotherapeutic targets. In contrast to traditional agonists activating multiple signalling pathways, agonists activating a single signalling pathway represent a new generation of drugs with increased specificity and fewer adverse effects. EXPERIMENTAL APPROACH: We have synthesized novel agonists of muscarinic ACh receptors and tested their binding and function (on levels of cAMP and inositol phosphates) in CHO cells expressing individual subtypes of muscarinic receptors, primary cultures of rat aortic smooth muscle cells and suspensions of digested native tissues from rats. Binding of the novel compounds to M2 receptors was modelled in silico. KEY RESULTS: Two of the tested new compounds (1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium and 1-methyl-1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium) only inhibited cAMP synthesis in CHO cells, primary cultures, and native tissues, with selectivity for M2 muscarinic receptors and displaying bias towards the Gi signalling pathway at all subtypes of muscarinic receptors. Molecular modelling revealed interactions with the orthosteric binding site in a way specific for a given agonist followed by agonist-specific changes in the conformation of the receptor. CONCLUSIONS AND IMPLICATIONS: The identified compounds may serve as lead structures in the search for novel non-steroidal and non-opioid analgesics acting via M2 and M4 muscarinic receptors with reduced side effects associated with activation of the phospholipase C signalling pathway.

• MeSH
• Muscarinic Agonists * pharmacology   MeSH
• Muscarinic Antagonists pharmacology   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Cricetinae MeSH
• Rats MeSH
• Receptor, Muscarinic M2 MeSH
• Receptors, Muscarinic * MeSH
• Signal Transduction MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Muscarinic Agonists * MeSH
• Muscarinic Antagonists MeSH
• Receptor, Muscarinic M2 MeSH
• Receptors, Muscarinic * MeSH