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Estimation of agonist activity at g protein-coupled receptors: Analysis of M2 muscarinic receptor signaling through Gi/o, G s, and G15

We developed novel methods for analyzing the concentrationresponse curve of an agonist to estimate the product of observed affinity and intrinsic efficacy, expressed relative to that of a standard agonist. This parameter, termed intrinsic relative activity (RAi), is most applicable for the analysis of responses at G protein-coupled receptors. RAi is equivalent to the potency ratios that agonists would exhibit in a hypothetical, highly sensitive assay in which all agonists behave as full agonists, even those with little intrinsic efficacy. We investigated muscarinic responses at the M2 receptor, including stimulation of phosphoinositide hydrolysis through Galpha15 in HEK 293T cells, inhibition of cAMP accumulation through Gi in Chinese hamster ovary (CHO) cells, and stimulation of cAMP accumulation through Gs in CHO cells treated with pertussis toxin. The RAi values of carbachol, oxotremorine-M, and the enantiomers of aceclidine were approximately the same in the three assay systems. In contrast, the activity of 4-[[N-[3-chlorophenyl]carbamoy] oxy-2-butynyl]trimethylammonium chloride (McN-A-343) was ?10-fold greater at M2 receptors coupled to Galpha15 in HEK 293T cells compared with M2 receptors coupled to Gi in the same cells or in CHO cells. Our results show that the RAi estimate is a useful measure for quantifying agonist activity across different assay systems and for detecting agonist directed signaling. Copyright

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Synthesis, biochemical activity and behavioral effects of a series of 1,4,5,6-tetrahydropyrimidines as novel ligands for M1 receptors

A series of novel tetrahydropyrimidines was synthesized and examined for M1 muscarinic receptor activity. 1,4,5,6-tetrahydro-5-methoxycarbonyl-pyrimidine hydrobromide (1a; CDD-0034-C) displayed a high affinity for muscarinic receptors in rat brain and stimulated PI metabolism in rat hippocampus. Compound 1a ameliorated memory deficits associated with lesions of the septohippocampal cholinergic system in rats.

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Visible-Light-Mediated Metal-Free Difunctionalization of Alkenes with CO2 and Silanes or C(sp3)?H Alkanes

Catalytic alkene difunctionalization via Si?H and C?H activations represents an ideal atom- and step-economic pathway for quick assembly of molecular complexity. We herein developed a visible-light-promoted metal-free difunctionalization of alkenes using abundant CO2 and readily available Si?H and C(sp3)?H bonds as feedstocks. Through the merger of photoredox and hydrogen-atom-transfer catalysis, a variety of value-added compounds, such as beta-silacarboxylic acids and acids bearing a gamma-heteroatom (e.g., N, O, S) could be directly accessed from simple alkenes in a redox-neutral fashion.

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Muscarinic receptor agonists, like dopamine receptor antagonist antipsychotics, inhibit conditioned avoidance response in rats

The purpose of our studies was to determine the effects of muscarinic receptor agonists on conditioned avoidance responding in the rat. Rats were trained to avoid or escape an electric shock delivered to the feet in a discrete trial procedure. The muscarinic receptor agonists pilocarpine and [2-ethyl-8-methyl-2,8-diazaspiro(4.5)decane-1,3-dione] hydrochloride (RS86) and the cholinesterase inhibitor physostigmine all decreased the percentage of avoidance responses at doses that produced less than approximately 30% response failures. Similar results were obtained with the antipsychotic drugs haloperidol, trifluoperazine, chlorpromazine, and clozapine. However, the benzodiazepine anxiolytic diazepam did not decrease avoidance responding up to doses that produced ataxia. On the other hand, oxotremorine and arecoline decreased avoidance responding only by producing response failures, whereas aceclidine produced intermediate changes. The muscarinic receptor antagonists scopolamine, trihexyphenidyl, and benztropine were without effect when administered alone but antagonized the decreases in avoidance responding produced by pilocarpine and RS86. Scopolamine had little effect on the decreases in avoidance responding produced by haloperidol. The newer muscarinic receptor partial agonists or agonist/antagonists [R-(Z)-(+)-alpha- (methoxyimino)-1-azabicyclo[2.2.2]octane-3-acetonitrile] hydrochloride, talsaclidine, milameline, and xanomeline also produced dose-related decreases in avoidance responding. Our results demonstrate that muscarinic receptor agonists can decrease avoidance responding in a manner similar to dopamine- receptor antipsychotic drugs, suggesting that muscarinic receptor agonists may provide an alternative approach to the treatment of psychosis.

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TREATMENT OF SEXUAL DYSFUNCTION AND FOR IMPROVED SEXUAL QUALITY OF LIFE

Compositions and methods for treating sexual dysfunction and enhancing sexual satisfaction using topical application of alpha-1 adrenergic receptor agonists, muscarinic acetylcholine receptor agonists, nicotinic acetylcholine receptor agonists, and cholinesterase inhibitors are disclosed.

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In Silico Appraisal, Synthesis, Antibacterial Screening and DNA Cleavage for 1,2,5-thiadiazole Derivative

Background: Thiadiazole not only acts as ?hydrogen binding domain? and ?two-electron donor system? but also as constrained pharmacophore. Methods: The maleate salt of 2-((2-hydroxy-3-((4-morpholino-1, 2,5-thiadiazol-3-yl) oxy) propyl) amino)- 2-methylpropan-1-ol (TML-Hydroxy)(4) has been synthesized. This methodology involves preparation of 4-morpholino-1, 2,5-thiadiazol-3-ol by hydroxylation of 4-(4-chloro-1, 2,5-thiadiazol-3-yl) morpholine followed by condensation with 2-(chloromethyl) oxirane to afford 4-(4-(oxiran-2-ylmethoxy)-1,2,5-thiadiazol- 3-yl) morpholine. Oxirane ring of this compound was opened by treating with 2-amino-2-methyl propan-1- ol to afford the target compound TML-Hydroxy. Structures of the synthesized compounds have been elucidated by NMR, MASS, FTIR spectroscopy. Results: The DSC study clearly showed that the compound 4-maleate salt is crystalline in nature. In vitro antibacterial inhibition and little potential for DNA cleavage of the compound 4 were explored. We extended our study to explore the inhibition mechanism by conducting molecular docking, ADMET and molecular dynamics analysis by using Schroedinger. The molecular docking for compound 4 showed better interactions with target 3IVX with docking score of -8.508 kcal/mol with respect to standard ciprofloxacin (docking score= -3.879 kcal/mol). TML-Hydroxy was obtained in silico as non-carcinogenic and non-AMES toxic with good percent human oral absorption profile (69.639%). TML-Hydroxy showed the moderate inhibition against Mycobacteria tuberculosis with MIC 25.00 mug/mL as well as moderate inhibition against S. aureus, Bacillus sps, K. Pneumoniae and E. coli species. Conclusion: In view of the importance of the 1,2,5-thiadiazole moiety involved, this study would pave the way for future development of more effective analogs for applications in medicinal field.

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Paneth cells during viral infection and pathogenesis

Paneth cells are major secretory cells located in the crypts of Lieberkuehn in the small intestine. Our understanding of the diverse roles that Paneth cells play in homeostasis and disease has grown substantially since their discovery over a hundred years ago. Classically, Paneth cells have been characterized as a significant source of antimicrobial peptides and proteins important in host defense and shaping the composition of the commensal microbiota. More recently, Paneth cells have been shown to supply key developmental and homeostatic signals to intestinal stem cells in the crypt base. Paneth cell dysfunction leading to dysbiosis and a compromised epithelial barrier have been implicated in the etiology of Crohn?s disease and susceptibility to enteric bacterial infection. Our understanding of the impact of Paneth cells on viral infection is incomplete. Enteric alpha-defensins, produced by Paneth cells, can directly alter viral infection. In addition, alpha-defensins and other antimicrobial Paneth cell products may modulate viral infection indirectly by impacting the microbiome. Here, we discuss recent insights into Paneth cell biology, models to study their function, and the impact, both direct and indirect, of Paneth cells on enteric viral infection.

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COMPOSITIONS AND METHODS FOR THE TREATMENT OF PRESBYOPIA

The invention provides compositions and methods for the treatment of presbyopia. The compositions preferably use aceclidine separate or together with a cycloplegic agent and/or with a nonionic surfactant and viscosity enhancer, and or with low concentrations of a selective a-2 adrenergic receptor agonist.

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The cholinergic system and spatial learning

Acetlylcholine (ACh) in the central nervous system is critical for a multitude of functions. Here, we concentrate on declarative memory in humans, and its equivalent episodic-like memory in rodents and highlight current understanding of cholinergic system in these processes. Spatial memory formation represents a simple form of episodic-like memory in rodents that engages the basal forebrain cholinergic system and its target structures. In these, ACh exerts numerous functions. During spatial acquisition learning, ACh efflux into the extracellular space is immediate in hippocampus and cortex; during consolidation of spatial reference memory, ACh levels are low. These requirements explain why ACh receptor blockade during acquisition blocks memory formation, and it is also consonant with the notion that an unspecific enhancement of cholinergic activity during consolidation is detrimental to memory formation. Working and short-term memory for spatial locations engages the nucleus basalis – prefrontal cortex ACh system. ACh activity is trial related and maintained for some time post-training. Striatal cholinergic activity is increased during stimulus-response learning and behavioural flexibility (reversal learning, extinction) providing a possible switch between different behavioural strategies. At present, there is no clear difference between muscarinic and nicotinergic systems with respect to spatial learning. Antagonists of the respective receptors impair memory formation, agonists can reverse these deficits or may, under specific conditions act more like a general cognitive enhancers by way of improving attention. Data reviewed here do not provide conclusive evidence for muscarinic or nicotinic receptors presenting as novel therapeutic targets, and there is no clear indication for ACh derived novel biomarkers for translational medicine.Unresolved and contradictory results are highlighted and discussed.

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M1 receptor agonist activity is not a requirement for muscarinic antinociception.

The analgesic effects of a series of muscarinic agonists were investigated by use of the mouse acetic acid writhing, grid-shock, hot-plate and tail-flick tests. The compounds tested were oxotremorine, pilocarpine, arecoline, aceclidine, RS86 and four 3-3(substituted-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahy-dro-1 -methyl pyridines (substituted TZTP), these being propoxy-TZTP, 3-Cl-propylthio-TZTP, xanomeline (hexyloxy-TZTP) and hexylthio-TZTP. These agonists were also assayed for their ability to displace [3H]oxotremorine-M and [3H]pirenz-epine binding and for their functional selectivity at pharmaco-logic M1, M2 and M3 receptors. These compounds all produced dose-dependent antinociceptive effects in all of the mouse analgesia tests. The effects of oxotremorine in the writhing test were fully antagonized by the muscarinic antagonist scopolamine (0.1 mg/kg), but only partially antagonized by methsco-polamine (10 mg/kg) and unaffected by the opioid antagonist naltrexone. 3-Cl-propylthio-TZTP and propoxy-TZTP had virtually no effect at the M1 receptor subtype as measured by the human m1 clone expressed in baby hamster kidney cells or the rabbit vas deferens assay. These compounds, however, were more potent in the analgesia tests than the selective M1 agonists xanomeline and hexylthio-TZTP. These data suggest that muscarinic analgesia is mediated by central muscarinic receptors. However, activity at the M1 receptor subtype is not a requirement for antinociceptive activity.

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