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
• agonisté muskarinových receptorů * farmakologie   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• karbachol farmakologie   MeSH
• lidé MeSH
• proteiny vázající GTP - alfa-podjednotky metabolismus   genetika   MeSH
• proteiny vázající GTP metabolismus   MeSH
• pyridiny farmakologie   MeSH
• receptory muskarinové * metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• agonisté muskarinových receptorů * MeSH
• karbachol MeSH
• proteiny vázající GTP - alfa-podjednotky MeSH
• proteiny vázající GTP MeSH
• pyridiny MeSH
• receptory muskarinové * 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
• biologické modely * MeSH
• počítačová simulace MeSH
• signální transdukce * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• Klíčová slova
• fusion proteins, muscarinic receptors, non-canonical signaling, signaling bias,
• MeSH
• AMP cyklický metabolismus   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• inhibiční koncentrace 50 MeSH
• isoxazoly chemie   MeSH
• křečci praví MeSH
• kvartérní amoniové sloučeniny chemie   MeSH
• lidé MeSH
• molekulární konformace MeSH
• oxotremorin chemie   MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 metabolismus   MeSH
• receptory muskarinové metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   MeSH
• signální transdukce * MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• AMP cyklický MeSH
• G protein alpha 16 MeSH Prohlížeč
• iperoxo MeSH Prohlížeč
• isoxazoly MeSH
• kvartérní amoniové sloučeniny MeSH
• oxotremorin MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 MeSH
• receptory muskarinové MeSH
• rekombinantní fúzní proteiny MeSH