059 Daridorexant: A dual, equipotent, and insurmountable antagonist of both orexin-1 and orexin-2 receptors

SLEEP ◽  
2021 ◽  
Vol 44 (Supplement_2) ◽  
pp. A25-A25
Author(s):  
Celia Mueller Grandjean ◽  
Manon Kiry ◽  
Catherine Vaillant ◽  
Oliver Nayler ◽  
John Gatfield
Keyword(s):  
Calcium Release ◽  
Brain Activity ◽  
Specific Treatment ◽  
Receptor Occupancy ◽  
Kinetic Properties ◽  
Receptor Subtypes ◽  
Residence Times ◽  
Binding Assays ◽  
Receptor System ◽  
Highly Effective

Abstract Introduction The orexin neuropeptide–receptor system is a central sleep and wake regulator in the brain. The two orexin receptor subtypes, OX1R and OX2R, are expressed either alone or together in all major wake-promoting brain areas. OX1R and OX2R activation by orexins causes elevation of intracellular calcium, which enhances synaptic transmission in secondary, monoaminergic wake- and arousal-promoting neurotransmitter circuits. Orexin receptor antagonists represent a novel and specific treatment of insomnia, which is different from classical therapy that more broadly inhibits brain activity via GABAA activation. Here we describe the molecular pharmacology of daridorexant, an orexin receptor antagonist which has proven highly effective in improving sleep and daytime functioning in insomnia patients. Methods Orexin-A(OxA)-induced calcium release assays in OX1R- and OX2R-expressing recombinant cell lines were applied to measure the antagonistic potency and kinetic properties of daridorexant in functional assays. Whole-cell competitive binding assays, using an orthosteric tracer were employed to determine the Ki of daridorexant. Comparisons were made with suvorexant and lemborexant. Results In OxA-induced calcium release assays with 2-h pre-incubation time, daridorexant displayed apparent Kb values of 0.5 nM (OX1R) and 0.8 nM (OX2R) with insurmountable antagonism on both receptors, demonstrating equipotent and highly effective functional inhibition of both receptor subtypes. On-target residence times of daridorexant (37oC) expressed as receptor occupancy half-lives (ROt1/2) were 4 min (OX1R) and 8 min (OX2R). In binding assays, daridorexant behaved as highly potent orthosteric antagonist. Also suvorexant behaved as dual insurmountable antagonist at OX1R/OX2R (appKb=0.7nM/1.0nM; ROt1/2=9 min/6 min) and as potent orthosteric antagonist in binding assays. Interestingly, lemborexant displayed a different interaction profile at OX1R/OX2R (appKb=13nM/0.4nM, ROt1/2<2min/<2min), i.e. it behaved as preferential OX2R antagonist with a very short on-target residence time and little insurmountability. Conclusion Daridorexant displays the desired target interaction profile of a dual, equipotent, and insurmountable antagonist of both OX1R and OX2R, which ensures equally efficient inhibition of both arousal-/wake-promoting receptor subtypes. Daridorexant′s on-target residence times are long enough to cause insurmountable inhibition, but short enough to avoid pharmacodynamic effects after drug elimination. Support (if any) Funded by Idorsia Pharmaceuticals Ltd.

scholarly journals Mefloquine-Induced Disruption of Calcium Homeostasis in Mammalian Cells Is Similar to That Induced by Ionomycin

2007 ◽  
Vol 52 (2) ◽  
pp. 684-693 ◽  
Author(s):  
D. Caridha ◽  
D. Yourick ◽  
M. Cabezas ◽  
L. Wolf ◽  
T. H. Hudson ◽  
...  
Keyword(s):  
Glutamate Receptor ◽  
Calcium Homeostasis ◽  
Mammalian Cells ◽  
Calcium Release ◽  
Radioligand Binding ◽  
Inositol Phosphate ◽  
Receptor Subtypes ◽  
Binding Assays ◽  
External Calcium ◽  
Er Calcium

ABSTRACT In previous studies, we have shown that mefloquine disrupts calcium homeostasis in neurons by depletion of endoplasmic reticulum (ER) stores, followed by an influx of external calcium across the plasma membrane. In this study, we explore two hypotheses concerning the mechanism(s) of action of mefloquine. First, we investigated the possibility that mefloquine activates non-N-methyl-d-aspartic acid receptors and the inositol phosphate 3 (IP3) signaling cascade leading to ER calcium release. Second, we compared the disruptive effects of mefloquine on calcium homeostasis to those of ionomycin in neuronal and nonneuronal cells. Ionomycin is known to discharge the ER calcium store (through an undefined mechanism), which induces capacitative calcium entry (CCE). In radioligand binding assays, mefloquine showed no affinity for the known binding sites of several glutamate receptor subtypes. The pattern of neuroprotection induced by a panel of glutamate receptor antagonists was dissimilar to that of mefloquine. Both mefloquine and ionomycin exhibited dose-related and qualitatively similar disruptions of calcium homeostasis in both neurons and macrophages. The influx of external calcium was blocked by the inhibitors of CCE in a dose-related fashion. Both mefloquine and ionomycin upregulated the IP3 pathway in a manner that we interpret to be secondary to CCE. Collectively, these data suggest that mefloquine does not activate glutamate receptors and that it disrupts calcium homeostasis in mammalian cells in a manner similar to that of ionomycin.

Muscarinic receptor subtypes in feline tracheal submucosal gland secretion

1992 ◽  
Vol 262 (2) ◽  
pp. L223-L228 ◽  
Author(s):  
H. Ishihara ◽  
S. Shimura ◽  
M. Satoh ◽  
T. Masuda ◽  
H. Nonaka ◽  
...  
Keyword(s):  
Muscarinic Receptor ◽  
Gland Secretion ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Submucosal Gland ◽  
Muscarinic Receptor Subtypes ◽  
Binding Assays ◽  
Submucosal Glands ◽  
Electrolyte Secretion ◽  
Quinuclidinyl Benzilate

To determine what muscarinic receptor subtype regulates [Ca2+]i mediating airway submucosal gland secretion, we examined the effects of atropine (Atr), pirenzepine (PZ), 11([2-(diethylamino)methyl-1-piperidinyl] acetyl)-5,11-dihydro-6H-pyrido (2,3-b)(1,4)-benzo-diazepin-6-one (AF-DX116) and 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) on methacholine (MCh)-evoked [Ca2+]i rise in acinar cells, and compared this with mucus glycoprotein (MGP) and electrolyte secretion evoked by MCh from submucosal glands isolated from feline trachea. [Ca2+]i was measured with the Ca(2+)-sensitive fluorescent dye, fura 2. We determined MGP secretion by measuring TCA-precipitable 3H-labeled glycoconjugates and electrolyte secretion by the change in the rate constant of 22Na-efflux from isolated glands. Half-maximal inhibitory concentrations (IC50) of PZ, AF-DX116, 4-DAMP, and Atr against MCh-evoked [Ca2+]i rise were 10(-7) M, 6 x 10(-6) M, 8 x 10(-9) M, and 6 x 10(-9) M, respectively. IC50 of PZ, AF-DX116, 4-DAMP, and Atr against MCh-evoked MGP secretion were 10(-6) M, 2 x 10(-5) M, 8 x 10(-9) M, and 6 x 10(-9) M, respectively. MCh (10(-5) M)-evoked 22Na efflux was significantly inhibited by 10(-7) M 4-DAMP and 10(-7) M Atr (P less than 0.01, each) but not by 10(-7) M PZ. Receptor binding assays with [3H]quinuclidinyl benzilate showed that the Ki values for PZ, AF-D x 116, 4-DAMP and Atr were 2.2 x 10(-8) M, 6.6 x 10(-7) M, 6.2 x 10(-10) M, and 2.9 x 10(-10) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

GabaA Receptors: Pharmacology, Behavioral Roles, and Motor Disorders

1996 ◽  
Vol 2 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Hartmut Lüddens ◽  
Esa R. Korpi
Keyword(s):  
Gabaa Receptors ◽  
Motor Coordination ◽  
Expression Patterns ◽  
Anion Channel ◽  
Developmental Expression ◽  
Mammalian Brain ◽  
Receptor Subtypes ◽  
Receptor System ◽  
Inhibitory Neurotransmitter ◽  
Gabaa Receptor Subtypes

τ-Aminobutyric acid (GABA), the most prevalent inhibitory neurotransmitter in the mammalian brain, exerts its main action through GABAA receptors. They belong to the superfamily of ligand-gated ion channels and respond to GABA by the opening of an intrinsic anion channel. Multiple GABAA receptor subtypes in the brain show differential regional and developmental expression patterns. The receptors have a pentameric structure and are formed from members of at least three different subunit families (α1–6, β1–3, and τ1–3). The regulation of functional properties by GABA and its analogs and by benzodiazepine (BZ) receptor ligands differs dramatically with the type of α variant in the receptor complex. Additional variations of GABAA receptors result from substitution of γ subunits. The role of the β subunits, which are essential for receptor assembly, is less well defined on a functional basis. Besides their involvement in anxiolysis and sedation, GABAA receptors clearly have an impact on motor coordination. However, with the possible exception of the alcohol-and BZ-sensitive alcohol non-tolerant (ANT) rat line, it is not well documented whether a genetic alteration in this receptor system is directly involved in the impairment of animal or human motor activity.

Autoradiographic analysis and regulation of angiotensin receptor subtypes AT4, AT1, and AT(1—7) in the kidney

2001 ◽  
Vol 281 (5) ◽  
pp. F936-F947 ◽  
Author(s):  
Rajash K. Handa ◽  
Shelly E. Handa ◽  
Monica K. S. Elgemark
Keyword(s):  
Cell Types ◽  
Receptor Subtypes ◽  
Receptor Density ◽  
Angiotensin Receptor ◽  
Puromycin Aminonucleoside ◽  
Water Diuresis ◽  
Receptor System ◽  
Numerous Cell ◽  
Autoradiographic Analysis

Receptor autoradiography revealed that angiotensin AT4 receptors were abundantly expressed in normal mammalian (mouse, rat, gerbil, guinea pig, rabbit) and avian (sparrow, chicken, turkey) kidneys and were more extensively distributed than previously reported (including proximal and distal segments of the nephron, interstitium, renal artery, vein, and ureter). Angiotensin AT4 receptors were generally found to be more abundant than angiotensin AT1 receptors in mammalian kidneys, whereas angiotensin AT(1—7) receptors were not detected in either mammalian or avian kidneys. Rats subjected to various chronic treatments were found to preferentially decrease kidney AT4 receptor density (furosemide, puromycin aminonucleoside, nitro-l-arginine methyl ester), decrease kidney AT1 receptor density (bilateral ureteral obstruction), or increase kidney AT1 receptor distribution in the inner medulla (water diuresis). These results indicate that the AT4 receptor can be expressed in numerous cell types within the normal kidney of several species. Furthermore, several models of renal dysfunction and injury have been identified that selectively alter kidney AT4 density and may potentially aid in elucidating the role of this novel angiotensin receptor system in renal function.

Increased GABAA receptor binding in amygdala after prenatal administration of valproic acid to rats

2016 ◽  
Vol 29 (5) ◽  
pp. 309-314 ◽  
Author(s):  
Freja Bertelsen ◽  
Arne Møller ◽  
Davide Folloni ◽  
Kim Ryun Drasbek ◽  
Jørgen Scheel-Krüger ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Receptor Binding ◽  
Partial Agonist ◽  
Autism Spectrum ◽  
Receptor Subtypes ◽  
Gamma Aminobutyric Acid ◽  
Receptor System ◽  
Gaba System ◽  
Key Factor ◽  
Muscimol Binding

ObjectivePrenatal exposure to valproic acid (VPA) enhances the risk for later development of autism spectrum disorders (ASD). An altered gamma-aminobutyric acid (GABA) system may be a key factor in ASD. Here we investigated possible changes in the GABA system in rats exposed to a low dose of prenatal VPA.MethodWe performed autoradiography with [3H]muscimol, (a GABAA receptor agonist), and [11C]Ro15-4513 (a partial agonist of the GABAA α1+5 receptor subtypes), in brain sections containing amygdala, thalamus and hippocampus of rats treated prenatally with 20 mg/kg VPA or saline from the 12th day of gestation.ResultPrenatal VPA significantly increased [11C]Ro15-4513 binding in the left amygdala compared with controls (p<0.05). This difference was not observed in the hippocampus, thalamus or right amygdala. No differences were observed in [3H]muscimol binding.ConclusionWe observed an asymmetric increase in GABAA receptor binding. Disturbances in the GABAA receptor system have also been detected in human autism with [11C]Ro15-4513.

scholarly journals On transport phenomena and equilibration time scales in thermodenuders

2011 ◽  
Vol 4 (3) ◽  
pp. 571-581 ◽  
Author(s):  
R. Saleh ◽  
A. Shihadeh ◽  
A. Khlystov
Keyword(s):  
Size Distribution ◽  
Time Scales ◽  
Transport Phenomena ◽  
Kinetic Properties ◽  
Equilibration Time ◽  
Residence Times ◽  
Size Distributions ◽  
Laboratory Measurements ◽  
Model Simulations ◽  
Cooling Section

Abstract. This paper presents a theoretical and experimental investigation of thermodenuders that addresses two controversial issues: (1) equilibration time scales and (2) the need for an activated carbon (AC) denuder in the cooling section. We describe a plug flow model for transport phenomena in a TD, which can be used to simulate the rate of vapor build-up in the gas phase and the corresponding change in particle size distribution. Model simulations were found to have excellent agreement with experiments performed with pure and mixed dicarboxylic acid aerosols. Both simulations and experiments showed that the aerosols approached equilibrium within reasonable residence times (15 s–30 s) for aerosol concentrations and size distributions typical for laboratory measurements, and that volatility studies at sufficiently high aerosol loadings, therefore, need not resort to kinetic models for inference of thermodynamic properties. However, for size distributions relevant for ambient aerosols, equilibration time scales were much larger than residence times available with current TD designs. We have also performed dimensional analysis on the problem of equilibration in TDs, and derived a dimensionless equilibration parameter which can be used to determine the residence time needed for an aerosol of given size distribution and kinetic properties to approach equilibrium. It is also shown theoretically and empirically that aerosol volatility has no effect on the equilibration time scales. Model simulations and experiments showed that with aerosol size distributions relevant to both ambient and laboratory measurements re-condensation in the cooling section, with and without an AC denuder, was negligible. Thus, there is no significant benefit in using an AC denuder in the cooling section. Due to the risk of stripping volatile material from the aerosol, the use of AC denuders in the cooling section should be avoided. Finally, we present a rationale for why ΔC is the proper measure of volatility, while using mass fraction remaining (MFR) can be misleading.

Class I antiarrhythmic drug effects on ouabain binding to guinea pig cardiac Na+-K+ATPase

1999 ◽  
Vol 77 (11) ◽  
pp. 866-870 ◽  
Author(s):  
Abdulrahman A Almotrefi ◽  
Chona Basco ◽  
Azadali Moorji ◽  
Nduna Dzimiri
Keyword(s):  
Guinea Pig ◽  
Drug Effects ◽  
Receptor Occupancy ◽  
Class I ◽  
Antiarrhythmic Agents ◽  
Binding Assays ◽  
Ouabain Binding ◽  
Guinea Pig Heart ◽  
Receptor Sites ◽  
Displacement Assays

The notion that the inhibition of the Mg2+-dependent ATP-hydrolytic function of the myocardial Na+-K+ATPase by class I antiarrhythmic agents occurs as a result of their binding to the same receptor sites as the digitalis glycosides was tested by performing competitive binding assays of [3H]ouabain (OUA) with eight drugs: disopyramide, encainide, lidocaine, lorcainide, phenytoin, procainamide, quinidine, and tocainide in guinea pig heart microsomal preparations. In the first set of experiments, 10-200 µM concentrations of the drugs were preincubated with the enzyme and displacement assays performed with 250 nM OUA. The drugs showed receptor occupancy of 19-32% at 50 µM, 25-44% at 100 µM, and 37-56% at 200 µM. Then, 10-500 nM concentrations of OUA were preincubated with the enzyme, and competitive assays were performed using 200 µM concentrations of the drugs. OUA occupied 39-51% of the receptor sites at 100 nM, 44-67% at 250 nM, and 62-82% at 500 nM, displacing the drugs in a concentration-dependent fashion. The results show that antiarrhythmic drugs interact with the same or similar receptor sites as ouabain on the Na+-K+ATPase, pointing to a possible contribution of these interactions to the mechanism for their inhibitory actions on the enzyme, and perhaps their arrhythmogenic effects.Key words: class I antiarrhythmic agents, proarrhythmias, Na+-K+ATPase, ouabain binding.

scholarly journals Contact Map Fingerprints of Protein-Ligand Unbinding Trajectories Reveal Mechanisms Determining Residence Times Computed from Scaled Molecular Dynamics

2021 ◽  
Author(s):  
Marc Bianciotto ◽  
Paraskevi Gkeka ◽  
Daria B. Kokh ◽  
Rebecca Wade ◽  
Minoux Hervé
Keyword(s):  
Molecular Dynamics ◽  
Residence Time ◽  
Bound State ◽  
Kinetic Properties ◽  
Residence Times ◽  
Contact Map ◽  
New Techniques ◽  
Relative Residence Time ◽  
Binding Kinetic

<div>The binding kinetic properties of potential drugs may significantly influence their subsequent clinical efficacy. Predictions of these properties based on computer simulations provide a useful alternative to their expensive and time-demanding experimental counterparts, even at an early drug discovery stage.</div><div>Herein, we perform Scaled Molecular Dynamics (ScaledMD) simulations on a set of 27 ligands of HSP90 belonging to more than 7 chemical series in order to estimate their relative residence time. We introduce two new techniques for the analysis and the classification of the simulated unbinding trajectories. The first technique, which helps in estimating the limits of the free energy well around the bound state and the second one, based on a new contact map fingerprint, allows the description and the comparison of the paths that lead to unbinding.</div><div>Using these analyses, we find that ScaledMD’s relative residence time generally enables the identification of the slowest unbinders. We propose an explanation for the underestimation of the residence times of a subset of compounds and we investigate how the biasing in ScaledMD can affect the mechanistic insights that can be gained from the simulations.</div>

scholarly journals Tonic GABAergic activity facilitates dendritic calcium signaling and short-term plasticity

2020 ◽  
Author(s):  
Chiayu Q. Chiu ◽  
Thomas M. Morse ◽  
Francesca Nani ◽  
Frederic Knoflach ◽  
Maria-Clemencia Hernandez ◽  
...  
Keyword(s):  
Calcium Signaling ◽  
Neuronal Excitability ◽  
Pyramidal Neurons ◽  
Calcium Influx ◽  
Brain Activity ◽  
Receptor Subtypes ◽  
Short Term ◽  
Dendritic Membrane ◽  
Cortical Pyramidal Neurons ◽  
Gabaergic Activity

SummaryBrain activity is highly regulated by GABAergic activity, which acts via GABAARs to suppress somatic spike generation as well as dendritic synaptic integration and calcium signaling. Tonic GABAergic conductances mediated by distinct receptor subtypes can also inhibit neuronal excitability and spike output, though the consequences for dendritic calcium signaling are unclear. Here, we use 2-photon calcium imaging in cortical pyramidal neurons and computational modeling to show that low affinity GABAARs containing an α5 subunit mediate a tonic hyperpolarization of the dendritic membrane potential, resulting in deinactivation of voltage-gated calcium channels and a paradoxical boosting of action potential-evoked calcium influx. We also find that GABAergic enhancement of calcium signaling modulates short-term synaptic plasticity, augmenting depolarization-induced suppression of inhibition. These results demonstrate a novel role for GABA in the control of dendritic activity and suggest a mechanism for differential modulation of electrical and biochemical signaling.

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