Pharmacological manipulation of specific neural circuits to optimize therapeutic index is an unrealized goal in neurology and psychiatry. AMPA receptors are important for excitatory synaptic ...transmission, and their antagonists are antiepileptic. Although efficacious, AMPA-receptor antagonists, including perampanel (Fycompa), the only approved antagonist for epilepsy, induce dizziness and motor impairment. We hypothesized that blockade of forebrain AMPA receptors without blocking cerebellar AMPA receptors would be antiepileptic and devoid of motor impairment. Taking advantage of an AMPA receptor auxiliary protein, TARP γ-8, which is selectively expressed in the forebrain and modulates the pharmacological properties of AMPA receptors, we discovered that LY3130481 selectively antagonized recombinant and native AMPA receptors containing γ-8, but not γ-2 (cerebellum) or other TARP members. Two amino acid residues unique to γ-8 determined this selectivity. We also observed antagonism of AMPA receptors expressed in hippocampal, but not cerebellar, tissue from an patient with epilepsy. Corresponding to this selective activity, LY3130481 prevented multiple seizure types in rats and mice and without motor side effects. These findings demonstrate the first rationally discovered molecule targeting specific neural circuitries for therapeutic advantage.
Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. ...An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon) that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S). Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with 3H-NMS (N-methyl-scopolamine) demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
6-(1S)-1-1-5-(2-hydroxyethoxy)-2-pyridylpyrazol-3-ylethyl- 3H-1,3-benzothiazol-2-one (LY3130481 or CERC-611) is a selective antagonist of AMPA receptors containing transmembrane AMPA receptor ...regulatory protein (TARP) γ-8 that is under development for epilepsy. The present study provided a broad inquiry into its anticonvulsant properties. LY3130481 was anticonvulsant in multiple acute seizure provocation models in mice and rats. In addition, LY3130481 was effective against absence seizures in the GAERS genetic model and in the Frings mouse model. Likewise, LY3130481 attenuated convulsions in mice and rats with long-term induction of seizures (e.g., corneal, pentylenetetrazole, hippocampal, and amygdala kindled seizures). In slices of epileptic human cortex, LY3130481 significantly decreased neuronal firing frequencies. LY3130481 displaced from rat brain a radioligand specific for AMPA receptors associated with TARP γ-8 whereas non-TARP-selective molecules did not. Binding was also observed in hippocampus freshly transected from a patient.
Taken as a whole, the findings reported here establish the broad anticonvulsant efficacy of LY3130481 indicating that blockade of AMPA receptors associated with TARP γ-8 is sufficient for these protective effects.
Conventional antidepressants lack efficacy for many patients (treatmentresistant depression or TRD) and generally take weeks to produce full therapeutic response in others. Emerging data has ...identified certain drugs such as ketamine as rapidly-acting antidepressants for major depressive disorder and TRD. Scopolamine, a drug used to treat motion sickness and nausea, has also been demonstrated to function as a rapidly-acting antidepressant. The mechanisms associated with efficacy in TRD patients and rapid onset of action have been suggested to involve a-Amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid (AMPA) receptor and mammalian target of rapamycin (mTOR) signaling. Since the work on these mechanisms with scopolamine has been limited, the present set of experiments was designed to further explore these mechanisms of action.
Male, NIH Swiss mice demonstrated a robust and immediate antidepressant signature with ketamine or scopolamine when studied under the forced-swim test.
The AMPA receptor antagonist NBQX prevented this antidepressant-like effect of scopolamine and ketamine. An orally-bioavilable mTOR inhibitor (AZD8055) also attenuated the antidepressant- like effects of scopolamine and ketamine. Scopolamine was also shown to augment the antidepressant- like effect of the selective serotonin reuptake inhibitor citalopram. When given in combination, scopolamine and ketamine acted synergistically to produce antidepressant-like effects. Although drug interaction data suggested that additional mechanisms might be at play, metabolomic analysis of frontal cortex and plasma from muscarinic M1+/+ and M1 -/- mice given scopolamine or vehicle did not reveal any hints as to the nature of these additional mechanisms of action.
Overall, the data substantiate and extend the idea that AMPA and mTOR signaling pathways are necessary for the antidepressant-like effects of scopolamine and ketamine, mechanisms that appear to be of general significance for TRD therapeutic agents.
1-Aminobenzotriazole (ABT) has been widely used as a nonspecific mechanism-based inhibitor of cytochrome P450 (P450) enzymes. It is extensively used in preclinical studies to determine the relative ...contribution of oxidative metabolism mediated by P450 in vitro and in vivo. The aim of present study was to understand the translation of fraction metabolized by P450 in dog hepatocytes to in vivo using ABT, for canagliflozin, known to be cleared by P450-mediated oxidation and UDP-glucuronosyltransferases-mediated glucuronidation, and 3 drug discovery project compounds mainly cleared by hepatic metabolism. In a dog hepatocyte, intrinsic clearance assay with and without preincubation of ABT, 3 Lilly compounds exhibited a wide range of fraction metabolized by P450. Subsequent metabolite profiling in dog hepatocytes demonstrated a combination of metabolism by P450 and UDP-glucuronosyltransferases. In vivo, dogs were pretreated with 50 mg/kg ABT or vehicle at 2 h before intravenous administration of canagliflozin and Lilly compounds. The areas under the concentration-time curve (AUC) were compared for the ABT-pretreated and vehicle-pretreated groups. The measured AUC
/AUC
ratios were correlated to fraction of metabolism by P450 in dog hepatocytes, suggesting that in vitro ABT inhibition in hepatocytes is useful to rank order compounds for in vivo fraction of metabolism assessment.
AMPA receptors mediate fast excitatory transmission in the brain. Neuronal AMPA receptors comprise GluA pore-forming principal subunits and can associate with multiple modulatory components, ...including transmembrane AMPA receptor regulatory proteins (TARPs) and CNIHs (cornichons). AMPA receptor potentiators and non-competitive antagonists represent potential targets for a variety of neuropsychiatric disorders. Previous studies showed that the AMPA receptor antagonist GYKI-53655 displaces binding of a potentiator from brain receptors but not from recombinant GluA subunits. Here, we asked whether AMPA receptor modulatory subunits might resolve this discrepancy. We find that the cerebellar TARP, stargazin (γ-2), enhances the binding affinity of the AMPA receptor potentiator 3H-LY450295 and confers sensitivity to displacement by non-competitive antagonists. In cerebellar membranes from stargazer mice, 3H-LY450295 binding is reduced and relatively resistant to displacement by non-competitive antagonists. Coexpression of AMPA receptors with CNIH-2, which is expressed in the hippocampus and at low levels in the cerebellar Purkinje neurons, confers partial sensitivity of 3H-LY450295 potentiator binding to displacement by non-competitive antagonists. Autoradiography of 3H-LY450295 binding to stargazer and γ-8-deficient mouse brain sections, demonstrates that TARPs regulate the pharmacology of allosteric AMPA potentiators and antagonists in the cerebellum and hippocampus, respectively. These studies demonstrate that accessory proteins define AMPA receptor pharmacology by functionally linking allosteric AMPA receptor potentiator and antagonist sites.
LY2812223 (1
,2
,4
,5
,6
)-2-amino-4-(1
-1,2,4-triazol-3-ylsulfanyl)bicyclo3.1.0hexane-2,6-dicarboxylic acid was identified via structure-activity studies arising from the potent metabotropic ...glutamate mGlu2/3 receptor agonist LY354740 (+)-2-aminobicyclo3.1.0 hexane-2,6-dicarboxylic acid as an mGlu2-preferring agonist. This pharmacology was determined using stably transfected cells containing either the human mGlu2 or mGlu3 receptor. We extended the pharmacological evaluation of LY2812223 to native brain tissues derived from relevant species used for preclinical drug development as well as human postmortem brain tissue. This analysis was conducted to ensure pharmacological translation from animals to human subjects in subsequent clinical studies. A guanosine 5'-
-(3-
Sthio)triphosphate (GTP
S) functional binding assay, a method for measuring G
-coupled signaling that is inherent to the group 2 mGlu receptors, was used to evaluate LY2812223 pharmacology of native mGlu receptors in mouse, rat, nonhuman primate, and human cortical brain tissue samples. In native tissue membranes, LY2812223 unexpectedly acted as a partial agonist across all species tested. Activity of LY2812223 was lost in cortical membranes collected from mGlu2 knockout mice, but not those from mGlu3 knockout mice, providing additional support for mGlu2-preferring activity. Other signal transduction assays were used for comparison with the GTP binding assay (cAMP, calcium mobilization, and dynamic mass redistribution). In ectopic cell line-based assays, LY2812223 displayed near maximal agonist responses at the mGlu2 receptor across all assay formats, while it showed no functional agonist activity at the mGlu3 receptor except in the cAMP assay. In native brain slices or membranes that express both mGlu2 and mGlu3 receptors, LY2812223 displayed unexpected partial agonist activity, which may suggest a functional interplay between these receptor subtypes in the brain.
We describe a novel, potent and selective orexin-2 (OX2)/hypocretin-2 receptor antagonist with in vivo activity in an animal model predictive of antidepressant-like efficacy. ...N-biphenyl-2-yl-4-fluoro-N-(1H-imidazol-2-ylmethyl) benzenesulfonamide HCl (LSN2424100) binds with high affinity to recombinant human OX2 receptors (Ki = 4.5 nM), and selectivity over OX1 receptors (Ki = 393 nM). LSN2424100 inhibited OXA-stimulated intracellular calcium release in HEK293 cells expressing human and rat OX2 receptors (Kb = 0.44 and 0.83 nM, respectively) preferentially over cells expressing human and rat OX1 (Kb = 90 and 175 nM, respectively). LSN2424100 exhibits good exposure in Sprague-Dawley rats after IP, but not PO, administration of a 30 mg/kg dose (AUC0-6 h = 1300 and 269 ng(*)h/mL, respectively). After IP administration in rats and mice, LSN2424100 produces dose-dependent antidepressant-like activity in the delayed-reinforcement of low-rate (DRL) assay, a model predictive of antidepressant-like efficacy. Efficacy in the DRL model was lost in mice lacking OX2, but not OX1 receptors, confirming OX2-specific activity. Importantly, antidepressant-like efficacy of the tricyclic antidepressant, imipramine, was maintained in both OX1 and OX2 receptor knock-out mice. In conclusion, the novel OX2 receptor antagonist, LSN2424100, is a valuable tool compound that can be used to explore the role of OX2 receptor-mediated signaling in mood disorders.
Alzheimer's disease and schizophrenia are characterized by expression of psychotic, affective, and cognitive symptoms. Currently, there is a lack of adequate treatment for the cognitive symptoms ...associated with these diseases. Cholinergic signaling and, in particular, M1 muscarinic acetylcholine receptor (m1AChR) signaling have been implicated in the regulation of multiple cognitive domains. Thus, the M1AChR has been identified as a therapeutic drug target for diseases, such as schizophrenia and Alzheimer's disease, that exhibit marked cognitive dysfunction as part of their clinical manifestation. Unfortunately, the development of selective M1 agonist medications has not been successful, mostly because of the highly conserved orthosteric acetylcholine binding site among the five muscarinic receptor subtypes. More recent efforts have focused on the development of allosteric M1AChR modulators that target regions of the receptor distinct from the orthosteric site that are less conserved between family members. However, orthosteric and allosteric ligands may differentially modulate receptor function and ultimately downstream signaling pathways. Thus, the need for highly selective M1AChR orthosteric agonists still exists, not only as a potential therapeutic but also as a pharmacological tool to better understand the physiologic consequences of M1AChR orthosteric activation. Here, we describe the novel, potent and selective M1AChR orthosteric partial agonist LY593093 N-(1R,2R)-6-({(1E)-1-(4-fluorobenzyl)(methyl)aminoethylidene})amino)-2-hydroxy-2,3-dihydro-1H-inden-1-ylbiphenyl-4-carboxamide. This compound demonstrates modest to no activity at the other muscarinic receptor subtypes, stimulates Gα(q)-coupled signaling events as well as β-arrestin recruitment, and displays significant efficacy in in vivo models of cognition.
Centrally administered neuropeptide Y (NPY) produces anxiolytic and orexigenic effects by interacting with Y1 and Y5 receptors that are colocalized in many brain regions. Therefore, we tested the ...hypothesis that co-expression of Y1 and Y5 receptors results in heterodimerization, altered pharmacological properties and altered desensitization. To accomplish this, the carboxyl-termini of Y1 and Y5 receptors were fused with
Renilla luciferase and green flourescent protein and the proximity of the tagged receptors assessed using bioluminescent resonance energy transfer. Under basal conditions, cotransfection of tagged Y1 receptor and Y5 produced a substantial dimerization signal that was unaffected by the endogenous, nonselective agonists, NPY and peptide YY (PYY). Selective Y5 agonists produced an increase in the dimerization signal while Y5 antagonists also produced a slight but significant increase. In the absence of agonists, selective antagonists decreased dimerization. In functional studies, Y5 agonists produced a greater inhibition of adenylyl cyclase activity in Y1/Y5 cells than cells expressing Y5 alone while NPY and PYY exhibited no difference. With PYY stimulation, the Y1 antagonist became inactive and the Y5 antagonist exhibited uncompetitive kinetics in the Y1/Y5 cell line. In confocal microscopy studies, Y1/Y5 co-expression resulted in increased Y5 signaling following PYY stimulation. Addition of both Y1 and Y5 receptor antagonists was required to significantly decrease PYY-induced internalization.
Therefore, Y1/Y5 co-expression results in heterodimerization, altered agonist and antagonist responses and reduced internalization rate. These results may account for the complex pharmacology observed when assessing the responses to NPY and analogs
in vivo.
PDF
