Jan Jakubik - Academia.edu (original) (raw)
Papers by Jan Jakubik
Scientific Reports
Agonist efficacy denoting the “strength” of agonist action is a cornerstone in the proper assessm... more 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...
Expert Opinion on Drug Discovery, Nov 2, 2022
Pharmaceuticals, Aug 30, 2010
An allosteric modulator is a ligand that binds to an allosteric site on the receptor and changes ... more An allosteric modulator is a ligand that binds to an allosteric site on the receptor and changes receptor conformation to produce increase (positive cooperativity) or decrease (negative cooperativity) in the binding or action of an orthosteric agonist (e.g., acetylcholine). Since the identification of gallamine as the first allosteric modulator of muscarinic receptors in 1976, this unique mode of receptor modulation has been intensively studied by many groups. This review summarizes over 30 years of research on the molecular mechanisms of allosteric interactions of drugs with the receptor and for new allosteric modulators of muscarinic receptors with potential therapeutic use. Identification of positive modulators of acetylcholine binding and function that enhance neurotransmission and the discovery of highly selective allosteric modulators are milestones on the way to novel therapeutic agents for the treatment of schizophrenia, Alzheimer's disease and other disorders involving impaired cognitive function.
Journal of Biological Chemistry, Oct 8, 2004
PubMed, 2004
Muscarinic acetylcholine receptors mediate transmission of an extracellular signal represented by... more Muscarinic acetylcholine receptors mediate transmission of an extracellular signal represented by released acetylcholine to neuronal or effector cells. There are five subtypes of closely homologous muscarinic receptors which are coupled by means of heterotrimeric G-proteins to a variety of signaling pathways resulting in a multitude of target cell effects. Endogenous agonist acetylcholine does not discriminate among individual subtypes and due to the close homology of the orthosteric binding site the same holds true for most of exogenous agonists. In addition to the classical binding site muscarinic receptors have one or more allosteric binding sites at extracellular domains. Binding of allosteric modulators induces conformational changes in the receptor that result in subtype-specific changes in orthosteric binding site affinity for both muscarinic agonists and antagonists. This overview summarizes our recent experimental effort in investigating certain aspects of M2 muscarinic receptor functioning concerning i) the molecular determinants that contribute to the binding of allosteric modulators, ii) G-protein coupling specificity and subsequent cellular responses and iii) possible functional assays that exploit the unique properties of allosteric modulators for characterization of muscarinic receptor subtypes in intact tissue. A detailed knowledge of allosteric properties of muscarinic receptors is required to permit drug design that will modulate signal transmission strength of specific muscarinic receptor subtypes. Furthermore, allosteric modulation of signal transmission strength is determined by cooperativity rather than concentration of allosteric modulator and thus reduces the danger of overdose.
British Journal of Pharmacology, Dec 1, 1994
By measuring the binding of N-[3H-methyl]-scopolamine ([3H]-NMS) and of unlabelled subtypespecifi... more By measuring the binding of N-[3H-methyl]-scopolamine ([3H]-NMS) and of unlabelled subtypespecific muscarinic antagonists, two populations of muscarinic binding sites can be distinguished in the membranes of cardiac ventricles taken from 1-day-old chicks. One of them, corresponding to approximately 80% of [3H]-NMS binding sites, has higher affinities for AF-DXl 16 (pKi = 6.42) and methoctramine (pKi = 7.33); the rate of [3H]NMS dissociation from these sites is fast. The other population, corresponding to approximately 20% of [3H]-NMS binding sites, has lower affinities for AF-DXl 16 (pKi = 5.00) and methoctramine (pKi = 6.19); the rate of [3H]-NMS dissociation from these sites is slow. Both populations have high affinities for pirenzepine, but the affinity of the former (major) population is lower (pKi = 7.99) than that of the latter (minor) population (pKi = 10.14). 2 Since it has been shown earlier that two mRNAs for muscarinic receptors are expressed in the chick heart, one of them close to the genetically defined m2 and the other to the m4 subtype, we propose that the major population of binding sites with high affinities for AF-DX1 16 and methoctramine and the lower affinity for pirenzepine represents the M2-like receptors, while the minor population represents the M4-like receptors. 3 It proved possible to obtain isolated samples of either population by selectively protecting the M2-like sites with AF-DXl 16 and the M4-like sites with pirenzepine, and by inactivating the unprotected sites with benzilylcholine mustard. The properties of the isolated populations corresponded to those derived from the analysis of [3H]-NMS binding to the original mixed population. 4 Alcuronium exerted positive allosteric action on the binding of [3H]-NMS both to the M2-like and the M4-like population and severely slowed down [3H]-NMS dissociation from them; its affinity for the M2-like sites was 3-10 times higher.
Journal of Pharmacology and Experimental Therapeutics, Jun 1, 2002
Chemical Biology & Drug Design, Jul 17, 2017
Muscarinic receptors are known to play important biological roles and are drug targets for severa... more Muscarinic receptors are known to play important biological roles and are drug targets for several human diseases. In a pilot study, novel muscarinic antagonists were synthesized and used as chemical probes to obtain additional information of the muscarinic pharmacophore. The design of these ligands made use of current orthosteric and allosteric models of drug-receptor interactions together with chemical motifs known to achieve muscarinic receptor selectivity. This approach has led to the discovery of several non-competitive muscarinic ligands that strongly bind at a secondary
Journal of Neurochemistry, Nov 23, 2002
We have found earlier that the neuromuscular blocker alcuronium binds to cardiac muscarinic recep... more We have found earlier that the neuromuscular blocker alcuronium binds to cardiac muscarinic receptors simultaneously with their specific antagonist [3 H]methyl-N-scopolamine ([3 H]NMS) and allosterically increases their affinity to this ligand. Nothing is known about the allosteric site with which alcuronium interacts. To gain an insight, we have now investigated how the binding of [3 H]NMS is affected by agents known to modify specific residues in proteins and how their effects are altered by alcuronium. Reagents that covalently modify the tyrosyl residues (p-nitrobenzenesulfonyl fluoride and 4-chloro-7nitrobenzofurazan) and the carboxyl groups of aspartate and glutamate [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, NN'-dicyclohexylcarbodiimide, and N-ethyl-5-phenylisoxazolium-3'-sulfonate] blocked the binding of [3 H]NMS to receptors in rat heart atria. Their action was probably due to the modification of tyrosyl and aspartyl residues directly in the muscarinic binding sites because it was antagonized by atropine and carbamoylcholine. Alcuronium and gallamine, another allosteric ligand, also protected the [ 3H]NMS binding sites against the inactivation by tyrosine-and carboxyl-directed chemical modifiers just as well as by benzilylcholine mustard, known to attach covalently to the muscarinic binding sites. Protection by alcuronium has also been observed on cerebrocortical muscarinic receptors. The effect of alcuronium indicates that the drug interferes with the access of chemical modifiers to the muscarinic sites. In view of the unspecific nature of most of the modifiers used (with regard to muscarinic mechanisms), the protection by alcuronium appears to be best explained on the assumption that the drug binds in close vicinity of the "classical" muscarinic site and sterically blocks the access to this site .
FEBS Journal, Aug 4, 2009
Neurobiology of Aging, Mar 1, 2008
We assessed the integrity of cholinergic neurotransmission in parietal cortex of young adult (7 m... more We assessed the integrity of cholinergic neurotransmission in parietal cortex of young adult (7 months) and aged (17 months) transgenic APPswe/PS1dE9 female mice compared to littermate controls. Choline acetyltransferase and acetylcholinesterase activity declined agedependently in both genotypes, whereas both age-and genotype-dependent decline was found in butyrylcholinesterase activity, vesicular acetylcholine transporter density, muscarinic receptors and carbachol stimulated binding of GTP␥S in membranes as a functional indicator of muscarinic receptor coupling to G-proteins. Notably, vesicular acetylcholine transporter levels and muscarinic receptor-G-protein coupling were impaired in transgenic mice already at the age of 7 months compared to wild type littermates. Thus, brain amyloid accumulation in this mouse model is accompanied by a serious deterioration of muscarinic transmission already before the mice manifest significant cognitive deficits.
Molecular Pharmacology, May 4, 2006
Journal of Pharmacology and Experimental Therapeutics, Apr 19, 2007
Wash-resistantly bound xanomeline inhibits acetylcholine release by persistent activation of pres... more Wash-resistantly bound xanomeline inhibits acetylcholine release by persistent activation of presynaptic M 2 and M 4 muscarinic receptors in rat brain.
Journal of Pharmacology and Experimental Therapeutics, Oct 20, 2003
Journal of Biological Chemistry, Oct 1, 2004
Evidence for a tandem two-site model of ligand binding to muscarinic acetylcholine receptors.
Physiological Research, 2008
There are five subtypes of muscarinic receptors that serve various important physiological functi... more There are five subtypes of muscarinic receptors that serve various important physiological functions in the central nervous system and the periphery. Mental functions like attention, learning, and memory are attributed to the muscarinic M 1 subtype. These functions decline during natural aging and an early deficit is typical for Alzheimer´s disease. In addition, stimulation of the M 1 receptor increases non-amyloidogenic processing of the amyloid precursor protein and thus prevents accumulation of noxious β-amyloid fragments. The selectivity of classical muscarinic agonists among receptor subtypes is very low due to the highly conserved nature of the orthosteric binding site among receptor subtypes. Herein we summarize some recent studies with the functionally-selective M 1 agonist xanomeline that indicate complex pharmacological profile of this drug that includes interactions with and activation of receptor from both orthosteric and ectopic binding sites, and the time-dependent changes of ligand binding and receptor activation. These findings point to potential profitability of exploitation of ectopic ligands in the search for truly selective muscarinic receptor agonists.
British Journal of Pharmacology, Jan 28, 2011
BACKGROUND AND PURPOSE Conventional determination of agonist efficacy at G-protein coupled recept... more BACKGROUND AND PURPOSE Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5′-g-thiotriphosphate (GTPgS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M2 muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. EXPERIMENTAL APPROACH Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [ 35 S]GTPgS and [ 3 H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M2 muscarinic acetylcholine receptor. KEY RESULTS Agonists displayed biphasic competition curves with the antagonist [ 3 H]-N-methylscopolamine. GTPgS (1 mM) changed the competition curves to monophasic with low affinity and 50 mM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [ 3 H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPgS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from Gi/o G-proteins but only its dissociation from Gs/olf G-proteins. CONCLUSIONS AND IMPLICATIONS These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of Gi/o versus Gs/olf G-proteins that are not identified by conventional GTPgS binding.
Molecular Pharmacology, Nov 1, 1998
Scientific Reports
Agonist efficacy denoting the “strength” of agonist action is a cornerstone in the proper assessm... more 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...
Expert Opinion on Drug Discovery, Nov 2, 2022
Pharmaceuticals, Aug 30, 2010
An allosteric modulator is a ligand that binds to an allosteric site on the receptor and changes ... more An allosteric modulator is a ligand that binds to an allosteric site on the receptor and changes receptor conformation to produce increase (positive cooperativity) or decrease (negative cooperativity) in the binding or action of an orthosteric agonist (e.g., acetylcholine). Since the identification of gallamine as the first allosteric modulator of muscarinic receptors in 1976, this unique mode of receptor modulation has been intensively studied by many groups. This review summarizes over 30 years of research on the molecular mechanisms of allosteric interactions of drugs with the receptor and for new allosteric modulators of muscarinic receptors with potential therapeutic use. Identification of positive modulators of acetylcholine binding and function that enhance neurotransmission and the discovery of highly selective allosteric modulators are milestones on the way to novel therapeutic agents for the treatment of schizophrenia, Alzheimer's disease and other disorders involving impaired cognitive function.
Journal of Biological Chemistry, Oct 8, 2004
PubMed, 2004
Muscarinic acetylcholine receptors mediate transmission of an extracellular signal represented by... more Muscarinic acetylcholine receptors mediate transmission of an extracellular signal represented by released acetylcholine to neuronal or effector cells. There are five subtypes of closely homologous muscarinic receptors which are coupled by means of heterotrimeric G-proteins to a variety of signaling pathways resulting in a multitude of target cell effects. Endogenous agonist acetylcholine does not discriminate among individual subtypes and due to the close homology of the orthosteric binding site the same holds true for most of exogenous agonists. In addition to the classical binding site muscarinic receptors have one or more allosteric binding sites at extracellular domains. Binding of allosteric modulators induces conformational changes in the receptor that result in subtype-specific changes in orthosteric binding site affinity for both muscarinic agonists and antagonists. This overview summarizes our recent experimental effort in investigating certain aspects of M2 muscarinic receptor functioning concerning i) the molecular determinants that contribute to the binding of allosteric modulators, ii) G-protein coupling specificity and subsequent cellular responses and iii) possible functional assays that exploit the unique properties of allosteric modulators for characterization of muscarinic receptor subtypes in intact tissue. A detailed knowledge of allosteric properties of muscarinic receptors is required to permit drug design that will modulate signal transmission strength of specific muscarinic receptor subtypes. Furthermore, allosteric modulation of signal transmission strength is determined by cooperativity rather than concentration of allosteric modulator and thus reduces the danger of overdose.
British Journal of Pharmacology, Dec 1, 1994
By measuring the binding of N-[3H-methyl]-scopolamine ([3H]-NMS) and of unlabelled subtypespecifi... more By measuring the binding of N-[3H-methyl]-scopolamine ([3H]-NMS) and of unlabelled subtypespecific muscarinic antagonists, two populations of muscarinic binding sites can be distinguished in the membranes of cardiac ventricles taken from 1-day-old chicks. One of them, corresponding to approximately 80% of [3H]-NMS binding sites, has higher affinities for AF-DXl 16 (pKi = 6.42) and methoctramine (pKi = 7.33); the rate of [3H]NMS dissociation from these sites is fast. The other population, corresponding to approximately 20% of [3H]-NMS binding sites, has lower affinities for AF-DXl 16 (pKi = 5.00) and methoctramine (pKi = 6.19); the rate of [3H]-NMS dissociation from these sites is slow. Both populations have high affinities for pirenzepine, but the affinity of the former (major) population is lower (pKi = 7.99) than that of the latter (minor) population (pKi = 10.14). 2 Since it has been shown earlier that two mRNAs for muscarinic receptors are expressed in the chick heart, one of them close to the genetically defined m2 and the other to the m4 subtype, we propose that the major population of binding sites with high affinities for AF-DX1 16 and methoctramine and the lower affinity for pirenzepine represents the M2-like receptors, while the minor population represents the M4-like receptors. 3 It proved possible to obtain isolated samples of either population by selectively protecting the M2-like sites with AF-DXl 16 and the M4-like sites with pirenzepine, and by inactivating the unprotected sites with benzilylcholine mustard. The properties of the isolated populations corresponded to those derived from the analysis of [3H]-NMS binding to the original mixed population. 4 Alcuronium exerted positive allosteric action on the binding of [3H]-NMS both to the M2-like and the M4-like population and severely slowed down [3H]-NMS dissociation from them; its affinity for the M2-like sites was 3-10 times higher.
Journal of Pharmacology and Experimental Therapeutics, Jun 1, 2002
Chemical Biology & Drug Design, Jul 17, 2017
Muscarinic receptors are known to play important biological roles and are drug targets for severa... more Muscarinic receptors are known to play important biological roles and are drug targets for several human diseases. In a pilot study, novel muscarinic antagonists were synthesized and used as chemical probes to obtain additional information of the muscarinic pharmacophore. The design of these ligands made use of current orthosteric and allosteric models of drug-receptor interactions together with chemical motifs known to achieve muscarinic receptor selectivity. This approach has led to the discovery of several non-competitive muscarinic ligands that strongly bind at a secondary
Journal of Neurochemistry, Nov 23, 2002
We have found earlier that the neuromuscular blocker alcuronium binds to cardiac muscarinic recep... more We have found earlier that the neuromuscular blocker alcuronium binds to cardiac muscarinic receptors simultaneously with their specific antagonist [3 H]methyl-N-scopolamine ([3 H]NMS) and allosterically increases their affinity to this ligand. Nothing is known about the allosteric site with which alcuronium interacts. To gain an insight, we have now investigated how the binding of [3 H]NMS is affected by agents known to modify specific residues in proteins and how their effects are altered by alcuronium. Reagents that covalently modify the tyrosyl residues (p-nitrobenzenesulfonyl fluoride and 4-chloro-7nitrobenzofurazan) and the carboxyl groups of aspartate and glutamate [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, NN'-dicyclohexylcarbodiimide, and N-ethyl-5-phenylisoxazolium-3'-sulfonate] blocked the binding of [3 H]NMS to receptors in rat heart atria. Their action was probably due to the modification of tyrosyl and aspartyl residues directly in the muscarinic binding sites because it was antagonized by atropine and carbamoylcholine. Alcuronium and gallamine, another allosteric ligand, also protected the [ 3H]NMS binding sites against the inactivation by tyrosine-and carboxyl-directed chemical modifiers just as well as by benzilylcholine mustard, known to attach covalently to the muscarinic binding sites. Protection by alcuronium has also been observed on cerebrocortical muscarinic receptors. The effect of alcuronium indicates that the drug interferes with the access of chemical modifiers to the muscarinic sites. In view of the unspecific nature of most of the modifiers used (with regard to muscarinic mechanisms), the protection by alcuronium appears to be best explained on the assumption that the drug binds in close vicinity of the "classical" muscarinic site and sterically blocks the access to this site .
FEBS Journal, Aug 4, 2009
Neurobiology of Aging, Mar 1, 2008
We assessed the integrity of cholinergic neurotransmission in parietal cortex of young adult (7 m... more We assessed the integrity of cholinergic neurotransmission in parietal cortex of young adult (7 months) and aged (17 months) transgenic APPswe/PS1dE9 female mice compared to littermate controls. Choline acetyltransferase and acetylcholinesterase activity declined agedependently in both genotypes, whereas both age-and genotype-dependent decline was found in butyrylcholinesterase activity, vesicular acetylcholine transporter density, muscarinic receptors and carbachol stimulated binding of GTP␥S in membranes as a functional indicator of muscarinic receptor coupling to G-proteins. Notably, vesicular acetylcholine transporter levels and muscarinic receptor-G-protein coupling were impaired in transgenic mice already at the age of 7 months compared to wild type littermates. Thus, brain amyloid accumulation in this mouse model is accompanied by a serious deterioration of muscarinic transmission already before the mice manifest significant cognitive deficits.
Molecular Pharmacology, May 4, 2006
Journal of Pharmacology and Experimental Therapeutics, Apr 19, 2007
Wash-resistantly bound xanomeline inhibits acetylcholine release by persistent activation of pres... more Wash-resistantly bound xanomeline inhibits acetylcholine release by persistent activation of presynaptic M 2 and M 4 muscarinic receptors in rat brain.
Journal of Pharmacology and Experimental Therapeutics, Oct 20, 2003
Journal of Biological Chemistry, Oct 1, 2004
Evidence for a tandem two-site model of ligand binding to muscarinic acetylcholine receptors.
Physiological Research, 2008
There are five subtypes of muscarinic receptors that serve various important physiological functi... more There are five subtypes of muscarinic receptors that serve various important physiological functions in the central nervous system and the periphery. Mental functions like attention, learning, and memory are attributed to the muscarinic M 1 subtype. These functions decline during natural aging and an early deficit is typical for Alzheimer´s disease. In addition, stimulation of the M 1 receptor increases non-amyloidogenic processing of the amyloid precursor protein and thus prevents accumulation of noxious β-amyloid fragments. The selectivity of classical muscarinic agonists among receptor subtypes is very low due to the highly conserved nature of the orthosteric binding site among receptor subtypes. Herein we summarize some recent studies with the functionally-selective M 1 agonist xanomeline that indicate complex pharmacological profile of this drug that includes interactions with and activation of receptor from both orthosteric and ectopic binding sites, and the time-dependent changes of ligand binding and receptor activation. These findings point to potential profitability of exploitation of ectopic ligands in the search for truly selective muscarinic receptor agonists.
British Journal of Pharmacology, Jan 28, 2011
BACKGROUND AND PURPOSE Conventional determination of agonist efficacy at G-protein coupled recept... more BACKGROUND AND PURPOSE Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5′-g-thiotriphosphate (GTPgS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M2 muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. EXPERIMENTAL APPROACH Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [ 35 S]GTPgS and [ 3 H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M2 muscarinic acetylcholine receptor. KEY RESULTS Agonists displayed biphasic competition curves with the antagonist [ 3 H]-N-methylscopolamine. GTPgS (1 mM) changed the competition curves to monophasic with low affinity and 50 mM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [ 3 H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPgS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from Gi/o G-proteins but only its dissociation from Gs/olf G-proteins. CONCLUSIONS AND IMPLICATIONS These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of Gi/o versus Gs/olf G-proteins that are not identified by conventional GTPgS binding.
Molecular Pharmacology, Nov 1, 1998