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

Muscarinic acetylcholine receptors are membrane receptors involved in many physiological processes. Malfunction of muscarinic signaling is a cause of various internal diseases, as well as psychiatric and neurologic conditions. Cholesterol, neurosteroids, neuroactive steroids, and steroid hormones are molecules of steroid origin that, besides having well-known genomic effects, also modulate membrane proteins including muscarinic acetylcholine receptors. Here, we review current knowledge on the allosteric modulation of muscarinic receptors by these steroids. We give a perspective on the research on the non-genomic effects of steroidal compounds on muscarinic receptors and drug development, with an aim to ultimately exploit such knowledge.

• Keywords
• allosteric modulation, cholesterol, muscarinic receptors, neuroactive steroids, neurosteroids,
• MeSH
• Cholesterol MeSH
• Hormones metabolism   MeSH
• Neurosteroids * pharmacology   MeSH
• Receptors, Muscarinic MeSH
• Steroids pharmacology   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Cholesterol MeSH
• Hormones MeSH
• Neurosteroids * MeSH
• Receptors, Muscarinic MeSH
• Steroids MeSH

Muscarinic receptors (mAChRs) are typical members of the G protein-coupled receptor (GPCR) family and exist in five subtypes from M1 to M5. Muscarinic receptor subtypes do not sufficiently differ in affinity to orthosteric antagonists or agonists; therefore, the analysis of receptor subtypes is complicated, and misinterpretations can occur. Usually, when researchers mainly specialized in CNS and peripheral functions aim to study mAChR involvement in behavior, learning, spinal locomotor networks, biological rhythms, cardiovascular physiology, bronchoconstriction, gastrointestinal tract functions, schizophrenia, and Parkinson's disease, they use orthosteric ligands and they do not use allosteric ligands. Moreover, they usually rely on manufacturers' claims that could be misleading. This review aimed to call the attention of researchers not deeply focused on mAChR pharmacology to this fact. Importantly, limited selective binding is not only a property of mAChRs but is a general attribute of most neurotransmitter receptors. In this review, we want to give an overview of the most common off-targets for established mAChR ligands. In this context, an important point is a mention the tremendous knowledge gap on off-targets for novel compounds compared to very well-established ligands. Therefore, we will summarize reported affinities and give an outline of strategies to investigate the subtype's function, thereby avoiding ambiguous results. Despite that, the multitargeting nature of drugs acting also on mAChR could be an advantage when treating such diseases as schizophrenia. Antipsychotics are a perfect example of a multitargeting advantage in treatment. A promising strategy is the use of allosteric ligands, although some of these ligands have also been shown to exhibit limited selectivity. Another new direction in the development of muscarinic selective ligands is functionally selective and biased agonists. The possible selective ligands, usually allosteric, will also be listed. To overcome the limited selectivity of orthosteric ligands, the recommended process is to carefully examine the presence of respective subtypes in specific tissues via knockout studies, carefully apply "specific" agonists/antagonists at appropriate concentrations and then calculate the probability of a specific subtype involvement in specific functions. This could help interested researchers aiming to study the central nervous system functions mediated by the muscarinic receptor.

• Keywords
• allosteric, multitarget, muscarinic agonist, muscarinic antagonist, muscarinic receptors, orthosteric,
• Publication type
• Journal Article MeSH
• Review 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