INSTITUTE OF PHYSIOLOGY CAS

1. Neurochemistry Group

We study physiology, biochemistry and pharmacology of cholinergic neurons at the molecular level and molecular pharmacology of other GPCRs. In our study, we mainly employ cell lines but we also use animal models. Our research is focused mainly on these topics:

biochemical physiology and pharmacology of cholinergic neurons
development and differentiation of cholinergic neurons
synthesis, storage, and release of acetylcholine
presynaptic regulation of acetylcholine release
cholinergic mechanisms in the pathogenesis of Alzheimer´s disease
influence of beta-amyloid on acetylcholine metabolism and muscarinic transmission
molecular pharmacology of muscarinic receptors
allosteric modulation of receptor activation.
interaction of receptors with G-proteins
modelling of muscarinic receptor signal transduction.

2. Group of Biochemistry of Membrane Receptors

The research focuses on the cellular and molecular mechanisms of desensitization of hormone response mediated by the activation of G protein-coupled receptors (GPCR). G protein-coupled receptors are plasma membrane integral proteins that serve as transducers of extracellular signals across the plasma membrane bilayer to the cell interior. GPCR play a key role in the regulation of many physiological processes and functions. Moreover, GPCRs represent one of the most important groups of targets for therapeutics. The main areas of the research are:

role of the cell membrane and membrane domains
opioid receptors and drug addiction
effect of monovalent ions on δ-opioid receptors – analysis of lithium effect in living cells and isolated cell membranes.

Publication 2015 - 2021

Projects

Structure-activity guided design of novel long-acting antagonists of muscarinic receptors

Given the broad range of functions that muscarinic receptors subserve, it is of fundamental importance to find subtype-selective ligands for therapeutic use in specific disorders. More

Molecular mechanisms of functional selectivity of atypical agonists at muscarinic receptors

Ectopic agonists represent a new class of drugs that bind out of the orthosteric site and display unique functional selectivity through mechanisms yet to be defined. We perform a detailed analysis of receptor activation induced by ectopic and classical agonists with the aim to reveal molecular mechanisms underlying functional selectivity. More

Molecular mechanisms of signalling bias at muscarinic receptors

A biased agonist is a ligand which stabilizes a particular active conformation of a receptor, thus stimulating some responses but not others. Biased agonists might represent a novel and uniquely effective type of therapeutic agent with reduced side-effects. More

Cholinergic modulation of striatum-based behaviour

The aim of the project is to determine how disruption of cholinergic activation of striatal GABAergic interneurons alters striatal signalling and striatum-based behaviour by using a mouse model with deletion of the β2 nicotinic acetylcholine receptor subunit in striatal GABAergic interneurons. More

Molecular mechanisms of membrane cholesterol effects on function of muscarinic receptors

Cholesterol has been found to co-crystallize with a number of GPCRs. Our current experiments show that membrane cholesterol specifically binds to a muscarinic receptor and slows down their activation. More

Opioid receptors and drug addiction

Addiction to opioid drugs (opioids) represents one of the most severe forms of drug abuse. Opium is one of the oldest known drugs and is used for its medicinal and euphoric effects for thousands of years. Opioid dependence develops as a consequence of changes in the central nervous system (CNS) induced by prolonged exposure to opioid drugs, e.g., morphine, codein or heroin. More

The effect of monovalent ions on delta-opioid receptors – analysis of lithium effect in living cells and isolated cell membranes

Activity of opioid receptor (OR) is among others modulates by monovalent cations especially by sodium (Na+) and lithium (Li+) which atomic radius is very similar to those of sodium. Lithium is used in psychiatry as a mood stabilizer (see review article Vošahlíková and Svoboda, 2016). Monovalent ions modulate GPCR signalling in not fully understood mechanism. More

The role of cell membrane and membrane domains

Cell (plasma) membrane (PM) serves as a protective barrier, but also plays a role in the cellular communication. The integrity of hydrophobic lipid environment of PM is an important factor affecting function of membrane proteins. More

Achievements

Insights into the operational model of agonism of receptor dimers

Jakubík, J.; Randáková, A. Insights into the Operational Model of Agonism of Receptor Dimers. Expert Opin. Drug Discov. 2022, 17, 1181–1191, doi:10.1080/17460441.2023.2147502. Invited Review. The exact ranking of efficacies and potencies of agonists is indispensable in the discovery of new selective agonists. The operational model of agonism (OMA) is the current standard. Many receptors function as oligomers, requiring an extension of the classical OMA. Extension of OMA by slope factors gives simple equations of functional response that are easy to fit experimental data but results may be inaccurate. Extension of OMA by cooperativity factors gives accurate but complex equations of functional response. Extension of OMA by cooperativity factors should be preferred. More

Functionally selective and biased agonists of muscarinic receptors.

Randáková, A.; Jakubík, J. Functionally Selective and Biased Agonists of Muscarinic Receptors. Pharmacol. Res. 2021, 169, 105641, doi:10.1016/j.phrs.2021.105641. Invited review. More

Nicotinic Acetylcholine Receptors Expressed by Striatal Interneurons Inhibit Striatal Activity and Control Striatal-Dependent Behaviors.

Abbondanza, A.; Ribeiro Bas, I.; Modrak, M.; Capek, M.; Minich, J.; Tyshkevich, A.; Naser, S.; Rangotis, R.; Houdek, P.; Sumova, A.; et al. Nicotinic Acetylcholine Receptors Expressed by Striatal Interneurons Inhibit Striatal Activity and Control Striatal-Dependent Behaviors. J. Neurosci. 2022, 42, 2786–2803, doi:10.1523/JNEUROSCI.1627-21.2022. A large variety of nicotinic acetylcholine receptors (nAChRs) are expressed in the striatum, a brain region that is crucial in the control of behaviour. The complexity of receptors with different functions is hindering our understanding of mechanisms through which striatal acetylcholine modulates behaviour. We focused on the role of a small population of beta2-containing nAChRs. We identified neuronal types expressing these receptors and determined their impact on the control of explorative behaviour, anxiety-like behaviour, learning, and sensitivity to stimulants. Additional experiments showed that these alterations were associated with an overall increased activity of striatal neurons. Thus, the small population of nicotinic receptors represents an interesting target for modulation of response to stimulant drugs and other striatal-based behaviour. More

Neurosteroids and steroid hormones are allosteric modulators of muscarinic receptors

Dolejší, E.; Szánti-Pintér, E.; Chetverikov, N.; Nelic, D.; Randáková, A.; Doležal, V.; Kudová, E.; Jakubík, J. Neurosteroids and Steroid Hormones Are Allosteric Modulators of Muscarinic Receptors. Neuropharmacology 2021, 199, 108798, doi:10.1016/j.neuropharm.2021.108798. Some neurosteroids and steroid hormones bind to muscarinic acetylcholine receptors with the affinity of 100 nM or greater Steroids acting at nanomolar concentrations represent the novel pharmacophore of allosteric modulators of muscarinic receptors Corticosterone and progesterone allosterically modulate muscarinic receptors at physiologically relevant concentrations More

The operational model of allosteric modulation of pharmacological agonism

Proper determination of agonist efficacy is indispensable in the evaluation of agonist selectivity and bias to activation of specific signalling pathways. The operational model of pharmacological agonism is a useful means for achieving this goal. More

Novel M<sub>2</sub>-Selective, Gi-Biased Agonists of Muscarinic Acetylcholine Receptors

We have developed new muscarinic receptor agonists as a pharmacophore for the development of new non-addictive analgesics such as opiates or weakening of immunity as steroid analgesics. These muscarinic analgesics would not cause side effects such as incontinence, excessive salivation and sweating, and others. More

Agonist-specific conformations of the M<sub>2</sub> muscarinic acetylcholine receptor assessed by molecular dynamics

Structurally diverse agonists for a given receptor induce receptor conformations specific to each structural family. These agonist-specific conformations can lead to non-uniform modulation of signalling pathways. This preferential orientation of signalling of a given receptor towards a subset of its signal transducers is termed signalling bias. This property may be employed to develop drugs that selectively produce desired effects while avoiding side effects associated with activation of unwanted signalling pathways. We modelled conformations of the M2 receptor specific to individual agonists, including the newly developed Gi-biased agonists. More

Applications and limitations of fitting of the operational model to determine relative efficacies of agonists

Proper determination of agonist efficacy is essential in the assessment of agonist selectivity and signalling bias. Agonist efficacy is a relative term that is dependent on the system in which it is measured, especially being dependent on receptor expression level. In this work, we analyse limits and pitfalls of fitting OM to experimental data. More

Role of membrane cholesterol in differential sensitivity of muscarinic receptor subtypes to persistently bound xanomeline

Our new publication in journal Neuropharmacology in which we demonstrate that membrane cholesterol plays an important and subtype-specific role in activation of muscarinic acetylcholine receptors. To our knowledge, this is the first demonstration of pharmacological selectivity due to differences in receptor-membrane interactions at any GPCR. The possibility to achieve pharmacological selectivity based on receptor-membrane interactions changes our view on the molecular basis of pharmacological selectivity and opens new ways for the development of a novel pharmaceutics. More

Novel long-acting antagonists of muscarinic ACh receptors

In this study, we have developed new potent and long-acting antagonists of muscarinic acetylcholine receptors with a potential therapeutic use. More

Synthesis of novel and functionally selective non-competitive muscarinic antagonists as chemical probes

In collaboration with Barry University novel muscarinic antagonists were developed. More

Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

This article analyzes in atomistic detail binding of orthosteric ligands to the muscarinic receptors. More

Book: Muscarinic Receptor: From Structure to Animal Models

Dr. Jan Jakubik Institute of Physiology Academy of Sciences and professor. Jaromir Mysliveček Institute of Physiology 1st Faculty of Medicine were approached to write the current list of methods that are used in research of muscarinic receptor as part of a Neuromethods series published by Springer. More

Towards predictive docking at aminergic G-protein coupled receptors.

Procedure described in this paper represents a possible way to predict interactions of antagonists with aminergic GPCRs. More

Lipid-based Diets Improve Muscarinic Neurotransmission in the Hippocampus of Transgenic APPswe/PS1dE9 Mice

Even short-term feeding of transgenic mice with chow containing specific lipid-based dietary supplements can influence markers of cholinergic synapses and rectify impaired muscarinic signal transduction that develops in transgenic mice. More

Publications

Szczurowska; Ewa - Szánti-Pintér; Eszter - Chetverikov; Nikolai - Randáková; Alena - Kudová; Eva - Jakubík; Jan . Modulation of Muscarinic Signalling in the Central Nervous System by Steroid Hormones and Neurosteroids . International Journal of Molecular Sciences. 2023; 24(1)); 507 . IF = 5.6 [ASEP] [ doi ]

Ujčíková; Hana - Roubalová; Lenka - Lee; Y. S. - Slaninová; Jiřina - Brejchová; Jana - Svoboda; Petr . The Dose-Dependent Effects of Multifunctional Enkephalin Analogs on the Protein Composition of Rat Spleen Lymphocytes; Cortex; and Hippocampus; Comparison with Changes Induced by Morphine . Biomedicines. 2022; 10(8)); 1969 . IF = 4.7 [ASEP] [ doi ]

Ujčíková; Hana - Robles; D. - Yue; X. - Svoboda; Petr - Lee; Y. S. - Navratilova; E. Time-Dependent Changes in Protein Composition of Medial Prefrontal Cortex in Rats with Neuropathic Pain . International Journal of Molecular Sciences. 2022; 23(2)); 955 . IF = 5.6 [ASEP] [ doi ]

Ujčíková; Hana - Eckhardt; Adam - Hejnová; L. - Novotný; J. - Svoboda; Petr . Alterations in the Proteome and Phosphoproteome Profiles of Rat Hippocampus after Six Months of Morphine Withdrawal: Comparison with the Forebrain Cortex . Biomedicines. 2022; 10(1)); 80 . IF = 4.7 [ASEP] [ doi ]

Szczurowska; Ewa - Szánti-Pintér; Eszter - Randáková; Alena - Jakubík; Jan - Kudová; Eva . Allosteric Modulation of Muscarinic Receptors by Cholesterol; Neurosteroids and Neuroactive Steroids . International Journal of Molecular Sciences. 2022; 23(21)); 13075 . IF = 5.6 [ASEP] [ doi ]

	Mgr. Jan Jakubík, PhD Head of the Laboratory
	MUDr. Vladimír Doležal, DrSc. Deputy Head of the Laboratory
	MUDr. et Mgr. Helena Janíčková, PhD Scientist
	Mgr. Eva Dolejší, PhD Junior Scientist
	Mgr. Alena Janoušková (née Randáková), PhD Scientist
	Alice Abbondanza, MSc Postgraduate Student
	Nikolai Chetverikov, MSc Postgraduate student
	Mgr. Dominik Nelic Postgraduate student
	Trisha Dhabalia Student
	Bc. Mutale Jane Mulenga Student
	Dana Ungerová Technician

	Biochemistry of Membrane Receptors
	Doc. RNDr. Petr Svoboda, DrSc. Scientist
	RNDr. Lenka Roubalová, PhD. Scientist
	RNDr. Hana Ujčíková, PhD. Junior Scientist

Laboratory of Neurochemistry