We study the physiology, biochemistry and pharmacology of cholinergic neurons at molecular level. 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 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 muscarinic receptors with G-proteins. Modeling of muscarinic receptor signal transduction.
Projects
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.
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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.
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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.
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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.
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Achievements
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.
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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.
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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.
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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.
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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.
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Publications
Randáková; Alena - Nelic; Dominik - Ungerová; Dana - Nwokoye; P. - Su; Q. - Doležal; Vladimír - El-Fakahany; E. E. - Boulos; J. - Jakubík; Jan
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Novel M-2-selective; G(i)-biased agonists of muscarinic acetylcholine receptors
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British Journal of Pharmacology. 2020; 177(9); 2073-2089
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IF = 7.730
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Randáková; Alena - Nelic; Dominik - Doležal; Vladimír - El-Fakahany; E. E. - Boulos; J. - Jakubík; Jan
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Agonist-Specific Conformations of the M-2 Muscarinic Acetylcholine Receptor Assessed by Molecular Dynamics
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Journal of Chemical Information and Modeling. 2020; 60(4); 2325-2338
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IF = 4.549
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Jakubík; Jan - El-Fakahany; E. E.
Current Advances in Allosteric Modulation of Muscarinic Receptors
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Biomolecules. 2020; 10(2)); 325
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IF = 4.082
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Jakubík, Jan - Randáková, Alena - Rudajev, Vladimír - Zimčík, Pavel - El-Fakahany, E. E. - Doležal, Vladimír
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Applications and limitations of fitting of the operational model to determine relative efficacies of agonists
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Scientific Reports 2019, 9(Mar 15)), 4637
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IF = 3.998
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Jakubík, Jan - Randáková, Alena - El-Fakahany, E. E. - Doležal, Vladimír
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Analysis of equilibrium binding of an orthosteric tracer and two allosteric modulators
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PLoS ONE 2019, 14(3)), e0214255
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IF = 2.740
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NPY+ interneurons (green) in mouse striatum injected by AVV5-mCherry (red)
Neurons in mouse gzrus dentatus expressing GFP (green).
Department of Neurochemistry, July 2019
Department of Neurochemistry
Department of Neurochemistry
Department of Neurochemistry, July 2016
Model of postsynaptic membrane. βA fibril, ACh - acetylcholine, APP - amyloid precursor protein, Go G-protein, Kir - inward rectifying potassium channel, M2 muscarinic receptor
Acetylcholine (ACh) is synthesized from choline and acetylcoenzyme A (AcCoA) by cholineacetyl transferase (ChAT) in cytoplasm of synaptic ending of neuron. ACh is transported into vesicles by vesicular acetylcholin transporter (VAChT) where it is stored untill release. Upon arrival of the signal in form of action potential to the synaptic ending vesicles fuse with plasma membrane and ACh is released to synaptic cleft. On post-synaptic membrane ACh either opens non selective cation channels - nicotinic receptors (NAChR) - or activates G protein coupled muscarinic receptors (MAChR). ACh may regulates its release via interaction with presynaptic MAChR. Action of ACh is terminated by acetylcholine esterase that cleaves it to inactive choline and acetate. Choline is transported back by to synaptic ending by choline transporter (ChT).
