Neuroprotective Potential of Synthetic Mono-Carbonyl Curcumin Analogs Assessed by Molecular Docking Studies.

Cognitive decline in dementia is associated with deficiency of the cholinergic system. In this study, five mono-carbonyl curcumin analogs were synthesized, and on the basis of their promising in vitro anticholinesterase activities, they were further investigated for in vivo neuroprotective and memory enhancing effects in scopolamine-induced amnesia using elevated plus maze (EPM) and novel object recognition (NOR) behavioral mice models. The effects of the synthesized compounds on the cholinergic system involvement in the brain hippocampus and their binding mode in the active site of cholinesterases were also determined. Compound h2 (p < 0.001) and h3 (p < 0.001) significantly inhibited the cholinesterases and reversed the effects of scopolamine by significantly reducing TLT (p < 0.001) in EPM, while (p < 0.001) increased the time exploring the novel object. The % discrimination index (DI) was significantly increased (p < 0.001) in the novel object recognition test. The mechanism of cholinesterase inhibition was further validated through molecular docking study using MOE software. The results obtained from the in vitro, in vivo and ex vivo studies showed that the synthesized curcumin analogs exhibited significantly higher memory-enhancing potential, and h3 could be an effective neuroprotective agent. However, more study is suggested to explore its exact mechanism of action.

Oxidative testicular injury: effect of L-leucine on redox, cholinergic and purinergic dysfunctions, and dysregulated metabolic pathways.

The antioxidant and anti-proinflammatory activities of L-leucine were investigated on oxidative testicular injury, ex vivo. In vitro analysis revealed L-leucine to be a potent scavenger of free radicals, while inhibiting acetylcholinesterase activity. Oxidative injury was induced in testicular tissues using FeSO4. Treatment with L-leucine led to depletion of oxidative-induced elevated levels of NO, MDA, and myeloperoxidase activity, with concomitant elevation of reduced glutathione and non-protein thiol levels, SOD and catalase activities. L-leucine caused a significant (p < 0.05) alteration of oxidative-elevated acetylcholinesterase and chymotrypsin activities, while concomitantly elevating the activities of ATPase, ENTPDase and 5'-nucleotidase. L-leucine conferred a protective effect against oxidative induced DNA damage. Molecular docking revealed molecular interactions with COX-2, IL-1 beta and iNOS. Treatment with L-leucine led to restoration of oxidative depleted ascorbic acid-2-sulfate, with concomitant depletion of the oxidative induced metabolites: D-4-Hydroxy-2-oxoglutarate, L-cystine, adenosine triphosphate, maleylacetoacetic acid, cholesteryl ester, and 6-Hydroxy flavin adenine dinucleotide. Treatment with L-leucine reactivated glycolysis while concomitantly deactivating oxidative-induced citrate cycle and increasing the impact-fold of purine metabolism pathway. L-leucine was predicted not to be an inhibitor of CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4, with a predicted LD50 value of 5000 mg/Kg and toxicity class of 5. Additionally, L-leucine showed little or no in vitro cytotoxicity in mammalian cells. These results suggest the therapeutic potentials of L-leucine on oxidative testicular injury, as evident by its ability to attenuate oxidative stress and proinflammation, while stalling cholinergic dysfunction and modulating nucleotide hyrolysis; as well as modulate oxidative dysregulated metabolites and their pathways.

Tacrine - Benzothiazoles: Novel class of potential multitarget anti-Alzheimers drugs dealing with cholinergic, amyloid and mitochondrial systems.

A series of tacrine - benzothiazole hybrids incorporate inhibitors of acetylcholinesterase (AChE), amyloid ß (Aß) aggregation and mitochondrial enzyme ABAD, whose interaction with Aß leads to mitochondrial dysfunction, into a single molecule. In vitro, several of 25 final compounds exerted excellent anti-AChE properties and interesting capabilities to block Aß aggregation. The best derivative of the series could be considered 10w that was found to be highly potent and selective towards AChE with the IC50 value in nanomolar range. Moreover, the same drug candidate exerted absolutely the best results of the series against ABAD, decreasing its activity by 23% at 100 µM concentration. Regarding the cytotoxicity profile of highlighted compound, it roughly matched that of its parent compound - 6-chlorotacrine. Finally, 10w was forwarded for in vivo scopolamine-induced amnesia experiment consisting of Morris Water Maze test, where it demonstrated mild procognitive effect. Taking into account all in vitro and in vivo data, highlighted derivative 10w could be considered as the lead structure worthy of further investigation.

Repositioning Dequalinium as Potent Muscarinic Allosteric Ligand by Combining Virtual Screening Campaigns and Experimental Binding Assays.

Structure-based virtual screening is a truly productive repurposing approach provided that reliable target structures are available. Recent progresses in the structural resolution of the G-Protein Coupled Receptors (GPCRs) render these targets amenable for structure-based repurposing studies. Hence, the present study describes structure-based virtual screening campaigns with a view to repurposing known drugs as potential allosteric (and/or orthosteric) ligands for the hM2 muscarinic subtype which was indeed resolved in complex with an allosteric modulator thus allowing a precise identification of this binding cavity. First, a docking protocol was developed and optimized based on binding space concept and enrichment factor optimization algorithm (EFO) consensus approach by using a purposely collected database including known allosteric modulators. The so-developed consensus models were then utilized to virtually screen the DrugBank database. Based on the computational results, six promising molecules were selected and experimentally tested and four of them revealed interesting affinity data; in particular, dequalinium showed a very impressive allosteric modulation for hM2. Based on these results, a second campaign was focused on bis-cationic derivatives and allowed the identification of other two relevant hM2 ligands. Overall, the study enhances the understanding of the factors governing the hM2 allosteric modulation emphasizing the key role of ligand flexibility as well as of arrangement and delocalization of the positively charged moieties.

Kinetics of Chemical Processes in the Human Brain. The Cholinergic Synapse-Mechanisms of Functioning and Control Methods.

In the framework of the kinetic model, the functioning of the cholinergic synapse is considered. The results of mathematical modeling of changes in the level of acetylcholine, induced pH impulse, the influence of the frequency of impulse transmission and inhibition of acetylcholinesterase are presented. Physicochemical explanation for a number of important physiological phenomena, such as neuromuscular paralysis, the molecular mechanism of neurological memory, and actions of nerve poisons and toxins, is given.

Design, synthesis and biological evaluation of novel naturally-inspired multifunctional molecules for the management of Alzheimer's disease.

In our overall goal to overcome the limitations associated with natural products for the management of Alzheimer's disease and to develop in-vivo active multifunctional cholinergic inhibitors, we embarked on the development of ferulic acid analogs. A systematic SAR study to improve upon the cholinesterase inhibition of ferulic acid with analogs that also had lower logP was carried out. Enzyme inhibition and kinetic studies identified compound 7a as a lead molecule with preferential acetylcholinesterase inhibition (AChE IC50 = 5.74 ± 0.13 µM; BChE IC50 = 14.05 ± 0.10 µM) compared to the parent molecule ferulic acid (% inhibition of AChE and BChE at 20 µM, 15.19 ± 0.59 and 19.73 ± 0.91, respectively). Molecular docking and dynamics studies revealed that 7a fits well into the active sites of AChE and BChE, forming stable and strong interactions with key residues Asp74, Trp286, and Tyr337 in AChE and with Tyr128, Trp231, Leu286, Ala328, Phe329, and Tyr341 in BChE. Compound 7a was found to be an efficacious antioxidant in a DPPH assay (IC50 = 57.35 ± 0.27 µM), and it also was able to chelate iron. Data from atomic force microscopy images demonstrated that 7a was able to modulate aggregation of amyloid ß1-42. Upon oral administration, 7a exhibited promising in-vivo activity in the scopolamine-induced AD animal model and was able to improve spatial memory in cognitive deficit mice in the Y-maze model. Analog 7a could effectively reverse the increased levels of AChE and BChE in scopolamine-treated animals and exhibited potent ex-vivo antioxidant properties. These findings suggest that 7a can act as a lead molecule for the development of naturally-inspired multifunctional molecules for the management of Alzheimer's and other neurodegenerative disorders.

Red-Emitting Dibenzodiazepinone Derivatives as Fluorescent Dualsteric Probes for the Muscarinic Acetylcholine M2 Receptor.

Fluorescently labeled dibenzodiazepinone-type muscarinic acetylcholine receptor (MR) antagonists, including dimeric ligands, were prepared using red-emitting cyanine dyes. Probes containing a fluorophore with negative charge showed high M2R affinities (pKi (radioligand competition binding): 9.10-9.59). Binding studies at M1 and M3-M5 receptors indicated a M2R preference. Flow cytometric and high-content imaging saturation and competition binding (M1R, M2R, and M4R) confirmed occupation of the orthosteric site. Confocal microscopy revealed that fluorescence was located mainly at the cell membrane (CHO-hM2R cells). Results from dissociation and saturation binding experiments (M2R) in the presence of allosteric M2R modulators (dissociation: W84, LY2119620, and alcuronium; saturation binding: W84) were consistent with a competitive mode of action between the fluorescent probes and the allosteric ligands. Taken together, these lines of evidence indicate that these ligands are useful fluorescent molecular tools to label the M2R in imaging and binding studies and suggest that they have a dualsteric mode of action.

T-495, a novel low cooperative M1 receptor positive allosteric modulator, improves memory deficits associated with cholinergic dysfunction and is characterized by low gastrointestinal side effect risk.

M1 muscarinic acetylcholine receptor (M1 R) activation can be a new therapeutic approach for the treatment of cognitive deficits associated with cholinergic hypofunction. However, M1 R activation causes gastrointestinal (GI) side effects in animals. We previously found that an M1 R positive allosteric modulator (PAM) with lower cooperativity (α-value) has a limited impact on ileum contraction and can produce a wider margin between cognitive improvement and GI side effects. In fact, TAK-071, a novel M1 R PAM with low cooperativity (α-value of 199), improved scopolamine-induced cognitive deficits with a wider margin against GI side effects than a high cooperative M1 R PAM, T-662 (α-value of 1786), in rats. Here, we describe the pharmacological characteristics of a novel low cooperative M1 R PAM T-495 (α-value of 170), using the clinically tested higher cooperative M1 R PAM MK-7622 (α-value of 511) as a control. In rats, T-495 caused diarrhea at a 100-fold higher dose than that required for the improvement of scopolamine-induced memory deficits. Contrastingly, MK-7622 showed memory improvement and induction of diarrhea at an equal dose. Combination of T-495, but not of MK-7622, and donepezil at each sub-effective dose improved scopolamine-induced memory deficits. Additionally, in mice with reduced acetylcholine levels in the forebrain via overexpression of A53T α-synuclein (ie, a mouse model of dementia with Lewy bodies and Parkinson's disease with dementia), T-495, like donepezil, reversed the memory deficits in the contextual fear conditioning test and Y-maze task. Thus, low cooperative M1 R PAMs are promising agents for the treatment of memory deficits associated with cholinergic dysfunction.

Rapid and efficient synthesis of a novel cholinergic muscarinic M1 receptor positive allosteric modulator using flash chemistry.

Continuous flow-flash synthesis of a 2-bromobenzaldehyde derivative 18 as a key intermediate of a novel cholinergic muscarinic M1 positive allosteric modulator 1 bearing an isoindolin-1-one ring system as a pharmacophore has been achieved using flow microreactors through selective I/Li exchange of 1-bromo-2-iodobenzene derivative 17 with BuLi and subsequent formylation at -40 °C of the highly reactive 2-bromophenyllithium intermediate using DMF, which is difficult to achieve by a conventional batch process due to the conversion of the highly reactive 2-bromophenyllithium intermediate into benzyne even at -78 °C. Late-stage cyclization to give the isoindolin-1-one ring system, through reductive amination of 18 followed by palladium-catalyzed carbonylation with carbon monoxide and intramolecular cyclization, efficiently afforded 1 for its further research and development.

Effects of Three Anti-Seizure Drugs on Cholinergic and Metabolic Activity in Experimental Status Epilepticus.

PURPOSE: Status epilepticus (SE) is characterized by recurrent seizure activity and can be drug- resistant. Knowledge of neuronal and metabolic activity of the brain during SE may be helpful to improve medical care. We here report the effects of three anti-seizure drugs on changes of acetylcholine energy metabolites and oxidative stress during SE. METHODS: We used the lithium-pilocarpine model in rats to induce SE and in vivo- microdialysis to monitor cholinergic and metabolic activity in the hippocampus. We measured extracellular concentrations of acetylcholine, glucose, lactate, pyruvate, glycerol and isoprostanes before and during SE, and after acute treatment with pregabalin, valproic acid, and levetiracteam. RESULTS: Upon onset of  SE, acetylcholine (ACh) release increased six- to eightfold. Glucose was increased only transiently by 30% but lactate levels rose four-fold, and extracellular concentrations of glycerol ten-fold. Isoprostanes are markers of oxidative stress and increased more than 20-fold. Two hours after pilocarpine adminstration, rats were treated with pregabalin (100 mg/kg), levetiracetam (200 mg/kg) or valproic acid (400 mg/kg) by i.p. injection. All three drugs stopped seizure activity in a delayed fashion, but at the doses indicated, only animals that received levetiracetam reached consciousness. All drugs reduced ACh release within 60-120 minutes. Lactate/pyruvate ratios, glycerol and isoprostanne levels were also reduced significantly after drug administration. CONCLUSIONS: Hippocampal ACh release closely follows seizure activity in SE and is attenuated when SE subsides. Pregabalin, valproic acid and levetiracetam all terminate seizures in the rat SE model and attenuate cholinergic and metabolic changes within two hours.

Isoorientin improves scopolamine-induced cognitive impairments by restoring the cholinergic system, antioxidant defense, and p-CREB/BDNF signaling in the hippocampus and frontal cortex.

Isoorientin (ISO) is considered one of the most important flavonoids with various pharmacological effects such as antioxidant, anti-inflammatory, and anti-cancer activities. Despite these beneficial activities, the effects of ISO on learning and memory have not been investigated so far. The current study evaluated the memory-enhancing effects of ISO in a scopolamine-treated mouse model by using the Y-maze and passive avoidance tests. The results showed that ISO (5 and 10 mg/kg, p.o.) treatment significantly improved the cognitive impairments caused by scopolamine. Additionally, ISO significantly decreased scopolamine-induced acetylcholinesterase and thiobarbituric acid reactive substance activities in both the hippocampus and frontal cortex of mice. In addition, ISO significantly increased the levels of total superoxide dismutase induced by scopolamine in the hippocampus and frontal cortex. Moreover, Western blot results indicated that ISO reversed the decreases in expression of phosphorylated cAMP response element binding (CREB) and brain-derived neurotrophic factor (BDNF) in the hippocampus and frontal cortex of scopolamine-treated mice. Thus, our results provide initial evidence that ISO ameliorates scopolamine-induced memory and cognitive impairments partly by restoring the cholinergic system, antioxidant defense, and p-CREB/BDNF signaling pathway, thereby exhibiting memory-enhancing activities.

Hwangryunhaedok-Tang Exerts Neuropreventive Effect on Memory Impairment by Reducing Cholinergic System Dysfunction and Inflammatory Response in a Vascular Dementia Rat Model.

Hwangryunhaedok-tang (HRT) is a traditional oriental herbal formula used in Asian countries for treating inflammatory diseases and controlling fever. Our present study aimed to determine whether HRT has therapeutic effects for patients with vascular dementia (VaD) using a bilateral common carotid artery occlusion (BCCAO) rat model and assessing spatial memory impairment and activation of neuroinflammation. BCCAO was performed in male Sprague Dawley rats to induce VaD, and oral HRT was administered daily for 30 d. Our data showed that HRT ameliorated BCCAO-induced memory and cognitive impairment in behavioral tests. In addition, HRT reversed cholinergic dysfunction and neuronal damage in the hippocampus of BCCAO rats. Furthermore, HRT attenuated microglial activation and reduced the phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase (JNK) induced by BCCAO. Simultaneous high-performance liquid chromatography analysis of HRT using index compounds from the herbal composition revealed that both HRT ethanol extract and commercial HRT granules primarily comprise geniposide, baicalin, and berberine. Our study showed that HRT administration resulted in the prevention of neuronal injury induced by BCCAO through improvement of cholinergic dysfunction and inhibition of neuroinflammatory responses, suggesting that HRT may have potential as a treatment for VaD.

Donepezil-butylated hydroxytoluene (BHT) hybrids as Anti-Alzheimer's disease agents with cholinergic, antioxidant, and neuroprotective properties.

The multifactorial nature of Alzheimer's disease (AD) calls for the development of multitarget agents addressing key pathogenic processes. A novel family of donepezil-butylated hydroxytoluene (BHT) hybrids were designed, synthesized and evaluated as multifunctional ligands against AD. The optimal compound 7d displayed a balanced multifunctional profile covering an intriguing acetylcholinesterase (AChE) inhibition (IC50, 0.075 µM for eeAChE and 0.75 µM for hAChE) and Monoamine oxidase B (MAO-B) inhibition (IC50, 7.4 µM for hMAO-B), excellent antioxidant activity (71.7 µM of IC50 by DPPH method, 0.82 and 1.62 trolox equivalent by ABTS method and ORAC method respectively), and inhibitory effects on self-induced, hAChE-induced Aß aggregation. Moreover, 7d possessed neuroprotective potency against H2O2-induced oxidative damage on PC12 cells and Lipopolysaccharides (LPS)-stimulated inflammation on BV2 cells. Compound 7d was capable of penetrating BBB and presented good liver microsomal metabolic stability. Importantly, compound 7d could dose-dependently reverse scopolamine-induced memory deficit in mice without acute toxicity. Taken together, those outstanding results highlight the donepezil-BHT hybrid 7d as a promising prototype in the research of innovative compound for AD.

New Flavone-Cyanoacetamide Hybrids with a Combination of Cholinergic, Antioxidant, Modulation of ß-Amyloid Aggregation, and Neuroprotection Properties as Innovative Multifunctional Therapeutic Candidates for Alzheimer's Disease and Unraveling Their Mechanism of Action with Acetylcholinesterase.

In line with the modern multi-target-directed ligand paradigm of Alzheimer's disease (AD), a series of 19 compounds composed of flavone and cyanoacetamide groups have been synthesized and evaluated as multifunctional agents against AD. Biological evaluation demonstrated that compounds 7j, 7n, 7o, 7r, and 7s exhibited excellent inhibitory potency (AChE, IC50 of 0.271 ± 0.012 to 1.006 ± 0.075 µM) and good selectivity toward acetylcholinesterase, significant antioxidant activity, good modulation effects on self-induced Aß aggregation, low cytotoxicity, and neuroprotection in human neuroblastoma SK-N-SH cells. Further, an inclusive study on the interaction of 7j, 7n, 7o, 7r, and 7s with AChE at physiological pH 7.2 using fluorescence, circular dichroism, and molecular docking methods suggested that these derivatives bind strongly to the peripheral anionic site of AChE mostly through hydrophobic interactions. Overall, the multifunctional profiles and strong AChE binding affinity highlight these compounds as promising prototypes for further pursuit of innovative multifunctional drugs for AD.

Discovery and Optimization of Potent and CNS Penetrant M5-Preferring Positive Allosteric Modulators Derived from a Novel, Chiral N-(Indanyl)piperidine Amide Scaffold.

The pharmacology of the M5 muscarinic acetylcholine receptor (mAChR) is the least understood of the five mAChR subtypes due to a historic lack of selective small molecule tools. To address this shortcoming, we have continued the optimization effort around the prototypical M5 positive allosteric modulator (PAM) ML380 and have discovered and optimized a new series of M5 PAMs based on a chiral N-(indanyl)piperidine amide core with robust SAR, human and rat M5 PAM EC50 values <100 nM and rat brain/plasma Kp values of ∼0.40. Interestingly, unlike M1 and M4 PAMs with unprecedented mAChR subtype selectivity, this series of M5 PAMs displayed varying degrees of PAM activity at the other two natively Gq-coupled mAChRs, M1 and M3, yet were inactive at M2 and M4.

Structure-Activity Relationships of Pan-Gαq/11 Coupled Muscarinic Acetylcholine Receptor Positive Allosteric Modulators.

Recent years have seen a large increase in the discovery of allosteric ligands targeting muscarinic acetylcholine receptors (mAChRs). One of the challenges in screening such compounds is to understand their mechanisms of action and define appropriate parameter estimates for affinity, cooperativity and efficacy. Herein we describe the mechanisms of action and structure-activity relationships for a series of "pan-Gq-coupled" muscarinic acetylcholine (ACh) receptor (mAChR) positive allosteric modulators (PAMs). Using a combination of radioligand binding, functional inositol phosphate accumulation assays, receptor alkylation and operational data analysis, we show that most compounds in the series derive their variable potency and selectivity from differential cooperativity at the M1, M3 and M5 mAChRs. None of the PAMs showed greater than 10-fold subtype selectivity for the agonist-free receptor, but VU6007705, VU6007678, and VU6008555 displayed markedly increased cooperativity compared to the parent molecule and M5 mAChR-preferring PAM, ML380 (αß > 100), in the presence of ACh. Most of the activity of these PAMs derives from their ability to potentiate ACh binding affinity at mAChRs, though VU6007678 was notable for also potentiating ACh signaling efficacy and robust allosteric agonist activity. These data provide key insights for the future design of more potent and subtype-selective mAChR PAMs.

Novel cinnamamide-dibenzylamine hybrids: Potent neurogenic agents with antioxidant, cholinergic, and neuroprotective properties as innovative drugs for Alzheimer's disease.

By using fragments endowed with interesting and complementary properties for the treatment of Alzheimer's disease (AD), a novel series of cinnamamide-dibenzylamine hybrids have been designed, synthesized, and evaluated biologically. In vitro assay indicated that most of the target compounds exhibited a significant ability to inhibit ChEs, strong potency inhibitory of self-induced ß-amyloid (Aß) aggregation and to act as potential antioxidants and biometal chelators. A Lineweaver-Burk plot and molecular modeling study showed that compound 7f targeted both the CAS and PAS of AChE. In addition, compound 7f could chelate metal ions, reduce PC12 cells death induced by oxidative stress and penetrate the blood-brain barrier (BBB). Overall, all of these outstanding in vitro results in combination with promising in vivo outcomes highlighted derivative 7f as the lead structure worthy of further investigation.

HIPP neurons in the dentate gyrus mediate the cholinergic modulation of background context memory salience.

Cholinergic neuromodulation in the hippocampus controls the salience of background context memory acquired in the presence of elemental stimuli predicting an aversive reinforcement. With pharmacogenetic inhibition we here demonstrate that hilar perforant path-associated (HIPP) cells of the dentate gyrus mediate the devaluation of background context memory during Pavlovian fear conditioning. The salience adjustment is sensitive to reduction of hilar neuropeptide Y (NPY) expression via dominant negative CREB expression in HIPP cells and to acute blockage of NPY-Y1 receptors in the dentate gyrus during conditioning. We show that NPY transmission and HIPP cell activity contribute to inhibitory effects of acetylcholine in the dentate gyrus and that M1 muscarinic receptors mediate the cholinergic activation of HIPP cells as well as their control of background context salience. Our data provide evidence for a peptidergic local circuit in the dentate gyrus that mediates the cholinergic encoding of background context salience during fear memory acquisition.Intra-hippocampal circuits are essential for associating a background context with behaviorally salient stimuli and involve cholinergic modulation at SST+ interneurons. Here the authors show that the salience of the background context memory is modulated through muscarinic activation of NPY+ hilar perforant path associated interneurons and NPY signaling in the dentate gyrus.