scholarly journals Synthesis, Biological, and Computational Evaluation of Antagonistic, Chiral Hydrobenzoin Esters of Arecaidine Targeting mAChR M1

2020 ◽  
Vol 13 (12) ◽  
pp. 437
Author(s):  
Marius Ozenil ◽  
Jonas Aronow ◽  
Daniela Piljak ◽  
Chrysoula Vraka ◽  
Wolfgang Holzer ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Acetylcholine Receptors ◽  
Radioligand Binding ◽  
Chinese Hamster ◽  
Muscarinic Acetylcholine Receptors ◽  
Ovary Cell ◽  
Computational Evaluation ◽  
Subtype Selectivity ◽  
Pet Tracer ◽  
Positron Emission

Muscarinic acetylcholine receptors (mAChRs) are a pivotal constituent of the central and peripheral nervous system. Yet, therapeutic and diagnostic applications thereof are hampered by the lack of subtype selective ligands. Within this work, we synthesized and chemically characterized three different stereoisomers of hydrobenzoin esters of arecaidine by NMR, HR-MS, chiral chromatography, and HPLC-logP. All compounds are structurally eligible for carbon-11 labeling and show appropriate stability in Dulbecco’s phosphate-buffered saline (DPBS) and F12 cell culture medium. A competitive radioligand binding assay on Chinese hamster ovary cell membranes comprising the human mAChR subtypes M1-M5 showed the highest orthosteric binding affinity for subtype M1 and a strong influence of stereochemistry on binding affinity, which corresponds to in silico molecular docking experiments. Ki values toward M1 were determined as 99 ± 19 nM, 800 ± 200 nM, and 380 ± 90 nM for the (R,R)-, (S,S)-, and racemic (R,S)-stereoisomer, respectively, highlighting the importance of stereochemical variations in mAChR ligand development. All three stereoisomers were shown to act as antagonists toward mAChR M1 using a Fluo-4 calcium efflux assay. With respect to future positron emission tomography (PET) tracer development, the (R,R)-isomer appears especially promising as a lead structure due to its highest subtype selectivity and lowest Ki value.

scholarly journals Preparation of a First 18F-Labeled Agonist for M1 Muscarinic Acetylcholine Receptors

Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2880 ◽  
Author(s):  
Boris D. Zlatopolskiy ◽  
Felix Neumaier ◽  
Till Rüngeler ◽  
Birte Drewes ◽  
Niklas Kolks ◽  
...  
Keyword(s):  
Acetylcholine Receptors ◽  
Radiochemical Yield ◽  
Muscarinic Acetylcholine Receptors ◽  
Preparative Scale ◽  
Muscarinic Acetylcholine ◽  
Pet Tracer ◽  
Positron Emission ◽  
Diagnostic Applications ◽  
M1 Muscarinic ◽  
Subtype Selective

M1 muscarinic acetylcholine receptors (mAChRs) are abundant in postsynaptic nerve terminals of all forebrain regions and have been implicated in the cognitive decline associated with Alzheimer’s disease and other CNS pathologies. Consequently, major efforts have been spent in the development of subtype-selective positron emission tomography (PET) tracers for mAChRs resulting in the development of several 11C-labeled probes. However, protocols for the preparation of 18F-labeled mAChR-ligands have not been published so far. Here, we describe a straightforward procedure for the preparation of an 18F-labeled M1 mAChR agonist and its corresponding pinacol boronate radiolabeling precursor and the non-radioactive reference compound. The target compounds were prepared from commercially available aryl fluorides and Boc protected 4-aminopiperidine using a convergent reaction protocol. The radiolabeling precursor was prepared by a modification of the Miyaura reaction and labeled via the alcohol-enhanced Cu-mediated radiofluorination. The developed procedure afforded the radiotracer in a non-decay-corrected radiochemical yield of 17 ± 3% (n = 3) and in excellent radiochemical purity (>99%) on a preparative scale. Taken together, we developed a straightforward protocol for the preparation of an 18F-labeled M1 mAChR agonist that is amenable for automation and thus provides an important step towards the routine production of a 18F-labeled M1 selective PET tracer for experimental and diagnostic applications.

scholarly journals Update on PET Tracer Development for Muscarinic Acetylcholine Receptors

2021 ◽  
Vol 14 (6) ◽  
pp. 530
Author(s):  
Marius Ozenil ◽  
Jonas Aronow ◽  
Marlon Millard ◽  
Thierry Langer ◽  
Wolfgang Wadsak ◽  
...  
Keyword(s):  
Nervous System ◽  
Acetylcholine Receptors ◽  
Muscarinic Acetylcholine Receptors ◽  
Muscarinic Acetylcholine ◽  
Pet Tracer ◽  
Positron Emission ◽  
Muscarinic Cholinergic ◽  
Imaging Properties ◽  
Tracer Development

The muscarinic cholinergic system regulates peripheral and central nervous system functions, and, thus, their potential as a therapeutic target for several neurodegenerative diseases is undoubted. A clinically applicable positron emission tomography (PET) tracer would facilitate the monitoring of disease progression, elucidate the role of muscarinic acetylcholine receptors (mAChR) in disease development and would aid to clarify the diverse natural functions of mAChR regulation throughout the nervous system, which still are largely unresolved. Still, no mAChR PET tracer has yet found broad clinical application, which demands mAChR tracers with improved imaging properties. This paper reviews strategies of mAChR PET tracer design and summarizes the binding properties and preclinical evaluation of recent mAChR tracer candidates. Furthermore, this work identifies the current major challenges in mAChR PET tracer development and provides a perspective on future developments in this area of research.

Neuromuscular Relaxants as Antagonists for M2and M3Muscarinic Receptors 

1998 ◽  
Vol 88 (3) ◽  
pp. 744-750 ◽  
Author(s):  
Vivian Y. Hou ◽  
Carol A. Hirshman ◽  
Charles W. Emala
Keyword(s):  
Muscarinic Receptor ◽  
Muscarinic Receptors ◽  
Radioligand Binding ◽  
Chinese Hamster ◽  
Neuromuscular Relaxants ◽  
Ovary Cell ◽  
Relative Order ◽  
Parasympathetic Nerves ◽  
M2 Muscarinic Receptor ◽  
M3 Muscarinic Receptor

Background Neuromuscular relaxants such as pancuronium bind to M2 and M3 muscarinic receptors as antagonists. Blockade of muscarinic receptors in atria of the M2 subtype mediates tachycardia. In the lung, blockade of M2 receptors on parasympathetic nerves potentiates vagally induced bronchospasm, whereas blockade of M3 receptors on bronchial smooth muscle inhibits bronchospasm. The current study was designed to quantify the affinity of a series of neuromuscular relaxants for the M2 and M3 muscarinic receptors, which were individually stably transfected in Chinese hamster ovary cell lines. Methods Competitive radioligand binding assays determined the relative binding affinities of the neuromuscular relaxants pancuronium, succinylcholine, mivacurium, doxacurium, atracurium, rocuronium, gallamine, and pipecuronium for the muscarinic receptor in the presence of a muscarinic receptor antagonist (3H-QNB) in membranes prepared from cells individually expressing either the M2 or M3 muscarinic receptor. Results All muscle relaxants evaluated displaced 3H-QNB from muscarinic receptors. The relative order of potency for the M2 muscarinic receptor (highest to lowest) was pancuronium, gallamine, rocuronium, atracurium, pipecuronium, doxacurium, mivacurium, and succinylcholine. The relative order of potency for the M3 muscarinic receptor (highest to lowest) was pancuronium, atracurium, pipecuronium, rocuronium, mivacurium, gallamine, succinylcholine, and doxacurium. Conclusions All neuromuscular relaxants studied had affinities for the M2 and M3 muscarinic receptor, but only pancuronium and gallamine had affinities within the range of concentrations achieved with clinical use. The high affinities of gallamine and pancuronium for the M2 muscarinic receptor are consistent with a mechanism of M2 receptor blockade in relaxant-induced tachycardia.

Development of Flashplate™ Technology to Measure [35S]GTPyS Binding to Chinese Hamster Ovary Cell Membranes Expressing the Cloned Human 5-HTIB Receptor

1998 ◽  
Vol 3 (2) ◽  
pp. 101-105 ◽  
Author(s):  
J. Watson ◽  
J. V. Selkirk ◽  
A. M. Brown
Keyword(s):  
G Protein ◽  
Chinese Hamster Ovary ◽  
Radioligand Binding ◽  
Partial Agonist ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Intrinsic Activity ◽  
Filtration Method ◽  
Gtpγs Binding

With the exponential rate at which proposed drugs are being produced for disease therapy, it is now essential to automate assays used to screen these compounds and increase throughput. This has been rapidly adopted for simple radioligand binding assays but is less amenable for certain functional screens. [35S]GTPγS binding represents a convenient method for screening ligands that bind to G protein-coupled receptors and, ultimately, stimulate G-protein activation. In this study we have investigated the use of 96-well FlashPlates™ (NEN DuPont, Stevenage, England) to measure [35S]GTPγS binding to human 5-HT1B receptors expressed in Chinese hamster ovary cells. The cells were added to the individual wells of the FlashPlate and incubated with [35S]GTPγS in the presence or absence of test drug and bound radioactivity measured in a 96-well spectrometer. 5-HT produced a stimulation of basal [35S]GTPγS binding, which was robust within and between experiments, with pEC50 = 8.1. The 5-HT1B partial agonist GR127935 (2′n-methyl-4′-5-methyl-1,2,4 oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide) caused a partial stimulation (pEC5o = 8.3, intrinsic activity = 0.7), and the selective 5-HTIB receptor antagonist SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2′-methyl-4′-[(5-methyl-1,2,4-oxadiazole-3-yl)biphenyl-4-yl]carbonyl}furo[2,3-flindole-3-spiro-4′-piperidine oxalate) displayed inverse agonism with pEC50 = 7.6. These results are similar to those obtained using the conventional filtration method and indicate that FlashPlate technology can provide a rapid method for measuring [35S]GTPγS binding.

Direct Functional Effects of Parathion and Paraoxon on Neuronal Nicotinic and Muscarinic M3 Acetylcholine Receptors

1996 ◽  
Vol 24 (2) ◽  
pp. 191-199
Author(s):  
Ingeborg van den Beukel ◽  
Regina G.D.M. van Kleef ◽  
Marga Oortgiesen
Keyword(s):  
Cho Cells ◽  
Acetylcholine Receptors ◽  
Ganglion Cells ◽  
Chinese Hamster ◽  
Cell Voltage ◽  
Muscarinic Acetylcholine Receptors ◽  
M3 Receptors ◽  
M3 Receptor ◽  
Inward Currents ◽  
Neuronal Type

The direct effects of two organophosphates, parathion and paraoxon, on nicotinic and muscarinic acetylcholine receptors in cells from different species were investigated, by using the whole-cell voltage clamp technique. The effects of neuronal type nicotinic receptors in mouse N1E-115, human SH-SY5Y and locust thoracic ganglion cells, and on human muscarinic M3 receptors transfected in Chinese hamster ovary (CHO) cells, were analysed. Parathion and paraoxon inhibit acetylcholine-induced nicotinic inward currents at concentrations in the micromblar range. Surprisingly, parathion is a more potent inhibitor than its active acetyl-cholinesterase-inhibiting metabolite, paraoxon. Moderate differences in sensitivity to the blocking action of the organophosphates appear to exist between cells from different species. Both parathion (100nM) and paraoxon (1μM) induce ion currents in M3-transfected CHO cells. They appear to act as agonists on the human muscarinic M3 receptor, affecting the receptors in a different manner to that of acetylcholine.

scholarly journals Regulation of extracellular-signal regulated kinase and c-Jun N-terminal kinase by G-protein-linked muscarinic acetylcholine receptors

1999 ◽  
Vol 338 (3) ◽  
pp. 619-628 ◽  
Author(s):  
Paul G. WYLIE ◽  
R. A. John CHALLISS ◽  
Jonathan L. BLANK
Keyword(s):  
G Protein ◽  
Acetylcholine Receptors ◽  
Chinese Hamster ◽  
Muscarinic Acetylcholine Receptors ◽  
Significant Component ◽  
Inflammatory Reactions ◽  
Erk Activation ◽  
Muscarinic Acetylcholine ◽  
Extracellular Signal ◽  
Jnk Activation

Extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs, or stress-activated protein kinases) are activated by diverse extracellular signals and mediate a variety of cellular responses, including mitogenesis, differentiation, hypertrophy, inflammatory reactions and apoptosis. We have examined the involvement of Ca2+ and protein kinase C (PKC) in ERK and JNK activation by the human G-protein-coupled m2 and m3 muscarinic acetylcholine receptors (mAChR) expressed in Chinese hamster ovary (CHO) cells. We show that the Ca2+-mobilizing m3 AChR is efficiently coupled to JNK and ERK activation, whereas the m2 AChR activates ERK but not JNK. Activation of JNK in CHO-m3 cells by the agonist methacholine (MCh) was delayed in onset and more sustained relative to that of ERK in either CHO-m2 or CHO-m3 cells. The EC50 values for MCh-induced ERK activation in both cell types were essentially identical and similar to that for JNK activation in CHO-m3 cells, suggesting little amplification of the response. Agonist-stimulated Ins(1,4,5)P3 accumulation in CHO-m3 cells was insensitive to pertussis toxin (PTX), consistent with a Gq/phosphoinositide-specific phospholipase C-β mediated pathway, whereas a significant component of ERK and JNK activation in CHO-m3 cells was PTX-sensitive, indicating Gi/o involvement. Using manipulations that prevent receptor-mediated extracellular Ca2+ influx and intracellular Ca2+-store release, we also show that ERK activation by m2 and m3 receptors is Ca2+-independent. In contrast, a significant component (> 50%) of JNK activation mediated by the m3 AChR was dependent on Ca2+, mainly derived from extracellular influx. PKC inhibition and down-regulation studies suggested that JNK activation was negatively regulated by PKC. Conversely, ERK activation by both m2 and m3 AChRs required PKC, suggesting a novel mechanism for PKC activation by PTX-sensitive m2 AChRs. In summary, mAChRs activate JNK and ERK via divergent mechanisms involving either Ca2+ or PKC respectively.

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