Role of Receptors in Relation to Plaques and Tangles in Alzheimer's Disease Pathology.

Despite the identification of Aß plaques and NFTs as biomarkers for Alzheimer's disease (AD) pathology, therapeutic interventions remain elusive, with neither an absolute prophylactic nor a curative medication available to impede the progression of AD presently available. Current approaches focus on symptomatic treatments to maintain AD patients' mental stability and behavioral symptoms by decreasing neuronal degeneration; however, the complexity of AD pathology requires a wide range of therapeutic approaches for both preventive and curative treatments. In this regard, this review summarizes the role of receptors as a potential target for treating AD and focuses on the path of major receptors which are responsible for AD progression. This review gives an overall idea centering on major receptors, their agonist and antagonist and future prospects of viral mimicry in AD pathology. This article aims to provide researchers and developers a comprehensive idea about the different receptors involved in AD pathogenesis that may lead to finding a new therapeutic strategy to treat AD.

N1H- and N1-Substituted Phenylguanidines as α7 Nicotinic Acetylcholine (nACh) Receptor Antagonists: Structure-Activity Relationship Studies.

We previously reported that N-(3-chlorophenyl)guanidine (1) represents a novel α7 nicotinic ACh (nACh) receptor antagonist chemotype. In the present study, a small series of compounds was synthesized with the intent to investigate the structure-activity relationship (SAR). Preliminary data suggested that the N-methyl analog of 1, 2, was several times more potent. Therefore, the chloro group at the aryl 3-position of 1 and its N1-methyl counterpart 2 were replaced with a number of substituents considering the electronic, lipophilic, and steric nature of the substituents. The potencies of the compounds to inhibit acetylcholine (ACh)-induced responses were obtained in Xenopus laevis oocytes expressing human α7 nicotinic ACh receptors (nAChRs) using a two-electrode voltage-clamp assay. We found that the nature of the 3-position substituents had relatively little (i.e., <10-fold) effect on potency, and the presence of an N1-isopropyl substituent was tolerated. Here, we report the first SAR investigation of this novel α7 nAChR antagonist chemotype.

Desformylflustrabromine (dFBr), a positive allosteric modulator of the α4ß2 nicotinic receptor modulates the hypnotic response to ethanol.

BACKGROUND: Binge drinking can increase an individual's risk of developing alcohol use disorder (AUD). Ethanol targets multiple neurotransmitter systems; however, not much is known about its effects on the cholinergic system. Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels, the heteromeric α4ß2 nAChR being a commonly expressed subtype. Desformylflustrabromine (dFBr), a positive allosteric modulator (PAM), increases the efficacy of α4ß2 nAChR in vitro and has previously been shown to have translational potential. In this study, we investigated whether dFBr modulates the hypnotic response to ethanol. METHODS: Ethanol-induced loss of righting reflex (LORR) duration was measured in the presence and absence of dFBr. The ß2 nAChR selective antagonist dihydro-ß-erythroidine (DHßE) was used to study the involvement of the ß2 subunit. Additionally, we used a crosslinking-based western blot assay to estimate changes in total versus intracellular α4 nAChR protein in thalamic tissue of rats treated with vehicle, dFBr, ethanol, or ethanol and dFBr. Lastly, using Xenopus oocyte two-electrode voltage clamp (TEVC) studies, we determined the effects of ethanol and dFBr on α4ß2 nAChR. RESULTS: Pretreatment with 6 mg/kg dFBr reduced ethanol-induced LORR duration as compared to rats treated with ethanol alone. LORR studies with DHßE suggest that dFBr reduced ethanol-induced LORR duration via the ß2 nAChR subunit. Crosslinking-based western analyses revealed that ethanol caused early increases in total and presumably surface thalamic α4 nAChR subunit protein levels. This ethanol-induced α4 nAChR upregulation was significantly reduced in rats pretreated with 6 mg/kg dFBr. In TEVC studies, ethanol potentiated ACh-induced currents in α4ß2 nAChR, while it slightly reduced dFBr potentiation of maximal ACh currents. CONCLUSIONS: Our results suggest that thalamic nAChRs containing the α4 subunit are rapidly upregulated by a single intoxicating dose of ethanol. Furthermore, dFBr, an α4ß2 nAChR-selective PAM, significantly attenuates the hypnotic response to ethanol via actions on ß2 nAChR. Overall, these results indicate that dFBr represents an option to reverse ethanol intoxication.

Multi-modal antidepressant-like action of 6- and 7-chloro-2-aminodihydroquinazolines in the mouse tail suspension test.

RATIONALE: 2-Amino-6-chloro-3,4-dihydroquinazoline (e.g., A6CDQ) represents a novel putative antidepressant originally thought to act through a 5-HT3 serotonin receptor antagonist mechanism. Here, we investigated this further by examining a positional isomer of A6CDQ (i.e., A7CDQ). MATERIALS AND METHODS: 5-HT3 receptor and transporter activity (uptake-1 and uptake-2) were investigated using a variety of in vitro assays and the in vivo mouse tail suspension test (TST). RESULTS: Although A7CDQ binds at 5-HT3 receptors with low affinity (Ki = 1975 nM) compared to A6CDQ (Ki = 80 nM), it retained 5-HT3 receptor antagonist action (IC50 = 5.77 and 0.26 µM, respectively). In the mouse TST A7CDQ produced antidepressant-like actions (ED50 = 0.09 mg/kg) comparable to that of A6CDQ. In addition, A6CDQ was found to be a 5-HT releasing agent (Km = 2.8 µM) at hSERT and a reuptake inhibitor (IC50 = 1.8 µM) at hNET, whereas A7CDQ was a weak reuptake inhibitor (Km = 43.6 µM) at SERT but a releasing agent (EC50 = 3.3 µM) at hNET. Moreover, A6CDQ and A7CDQ were potent inhibitors of uptake-2 (e.g.; OCT3 IC50 = 3.9 and 5.9 µM, respectively). CONCLUSIONS: A simple shift of a substituent in a common quinazoline scaffold from one position to another (i.e., a chloro group from the 6- to the 7-position) resulted in a common action in the TST but via a somewhat different mechanism. A6CDQ and A7CDQ might represent the first members of a new class of potential antidepressants with a unique multi-modal mechanism of action.

"Methylene Bridge" to 5-HT3 Receptor Antagonists: Conformationally Constrained Phenylguanidines.

Arylguanidines, depending upon their aromatic substitution pattern, display varying actions at 5-HT3 receptors (e.g., partial agonist, agonist, superagonist). Here, we demonstrate that conformational constraint of these agents as dihydroquinazolines (such as A6CDQ; 1) results in their conversion to 5-HT3 receptor antagonists. We examined the structure-activity relationships of 1. Replacement/removal of any of the guanidinium nitrogen atoms of 1 resulted in decreased affinity. All three nitrogen atoms of 1 are necessary for optimal binding affinity at 5-HT3 receptors. Introduction of substituents as small as an N2-methyl group abolishes affinity. The results are consistent with homology modeling/docking studies and binding data from site-directed mutagenesis studies. Introducing a "methylene bridge" to the arylguanidine structure, regardless of its functional activity, results in a 5-HT3 receptor antagonist.

des-Formylflustrabromine (dFBr): A Structure-Activity Study on Its Ability To Potentiate the Action of Acetylcholine at α4ß2 Nicotinic Acetylcholine Receptors.

The naturally occurring indole alkaloid des-formylflustrabromine (dFBr; 1) is one of the first agents shown to act as a selective positive allosteric modulator (PAM) at α4ß2 nicotinic acetylcholine receptors (nAChRs). We previously deconstructed this agent to determine which of its structural features contribute to its actions and have identified an agent that might serve as the basis for a " working pharmacophore". Here, we elaborate the dFBr (1; EC50 = 0.2 µM) structure to identify how various structural modifications impact its actions. Electrophysiological studies with Xenopus laevis oocytes identified several compounds with dFBr-like potency and one, the 5-bromo analogue of 1 (i.e., 5-bromo dFBr; 25; EC50 = 0.4 µM), with more than twice the efficacy of 1 as a PAM at α4ß2 nAChRs.

Rabies virus modifies host behaviour through a snake-toxin like region of its glycoprotein that inhibits neurotransmitter receptors in the CNS.

Rabies virus induces drastic behaviour modifications in infected hosts. The mechanisms used to achieve these changes in the host are not known. The main finding of this study is that a region in the rabies virus glycoprotein, with homologies to snake toxins, has the ability to alter behaviour in animals through inhibition of nicotinic acetylcholine receptors present in the central nervous system. This finding provides a novel aspect to virus receptor interaction and host manipulation by pathogens in general. The neurotoxin-like region of the rabies virus glycoprotein inhibited acetylcholine responses of α4ß2 nicotinic receptors in vitro, as did full length ectodomain of the rabies virus glycoprotein. The same peptides significantly altered a nicotinic receptor induced behaviour in C. elegans and increased locomotor activity levels when injected into the central nervous system of mice. These results provide a mechanistic explanation for the behavioural changes in hosts infected by rabies virus.

Superagonist, Full Agonist, Partial Agonist, and Antagonist Actions of Arylguanidines at 5-Hydroxytryptamine-3 (5-HT3) Subunit A Receptors.

Introduction of minor variations to the substitution pattern of arylguanidine 5-hydroxytryptamine-3 (5-HT3) receptor ligands resulted in a broad spectrum of functionally-active ligands from antagonist to superagonist. For example, (i) introduction of an additional Cl-substituent(s) to our lead full agonist N-(3-chlorophenyl)guanidine (mCPG, 2; efficacy % = 106) yielded superagonists 7-9 (efficacy % = 186, 139, and 129, respectively), (ii) a positional isomer of 2, p-Cl analog 11, displayed partial agonist actions (efficacy % = 12), and (iii) replacing the halogen atom at the meta or para position with an electron donating OCH3 group or a stronger electron withdrawing (i.e., CF3) group resulted in antagonists 13-16. We posit based on combined mutagenesis, crystallographic, and computational analyses that for the 5-HT3 receptor, the arylguanidines that are better able to simultaneously engage the primary and complementary subunits, thus keeping them in close proximity, have greater agonist character while those that are deficient in this ability are antagonists.

Attenuation of Compulsive-Like Behavior Through Positive Allosteric Modulation of α4ß2 Nicotinic Acetylcholine Receptors in Non-Induced Compulsive-Like Mice.

Nicotinic α4ß2 receptors are the most abundant subtypes of nicotinic acetylcholine receptors (nAChRs) expressed in brain regions implicated in obsessive compulsive disorder (OCD). These receptors are known to modify normal and addictive behaviors by modulating neuronal excitability. Desformylflustrabromine (dFBr) is a novel, positive allosteric modulator (PAM) of high acetylcholine sensitivity (HS) and low acetylcholine sensitivity (LS) α4ß2 nAChRs. The present study tested the hypothesis that positive allosteric modulation of α4ß2 receptors by dFBr will attenuate compulsive-like behavior in a non-induced compulsive-like mouse model. Male mice (Mus musculus) selected for compulsive-like nesting behavior (NB; 48 animals; 12 per group) received acute (once) and chronic (every day for 32 days) subcutaneous injection of dFBr at 2, 4 and 6 mg/kg doses. Saline was used as a control (0 mg/kg). Compulsive-like NB was assessed after 1, 2, 3, 4, 5 and 24 h, while compulsive-like marble burying (MB) and anxiety-like open field (OF) behaviors were performed 2 h after dFBr administration. In the acute administration protocol, dFBr dose dependently attenuated NB and MB. Rapid effects (1-2 h after drug administration) of dFBr on MB and NB were observed for the chronic administration which was in congruence with the acute study. Chronic administration also revealed sustained suppression of NB by dFBr following 5 weeks of treatment. In both the acute and chronic regimen dFBr did not modulate OF behaviors. This research demonstrates the novel role of positive allosteric modulation of α4ß2 nicotinic receptors by dFBr as a translational potential for OCD.

Desformylflustrabromine Modulates α4ß2 Neuronal Nicotinic Acetylcholine Receptor High- and Low-Sensitivity Isoforms at Allosteric Clefts Containing the ß2 Subunit.

Alterations in expression patterns of α4ß2 nicotinic acetylcholine receptors have been demonstrated to alter cholinergic neurotransmission and are implicated in neurologic disorders, including autism, nicotine addiction, Alzheimer's disease, and Parkinson's disease. Positive allosteric modulators (PAMs) represent promising new leads in the development of therapeutic agents for the treatment of these disorders. This study investigates the involvement of the ß2-containing subunit interfaces of α4ß2 receptors in the modulation of acetylcholine (ACh)-induced responses by the PAM desformylflustrabromine (dFBr). Eight amino acids on the principal face of the ß2 subunit were mutated to alanine to explore the involvement of this region in the potentiation of ACh-induced currents by dFBr. ACh-induced responses obtained from wild-type and mutant α4ß2 receptors expressed in Xenopus laevis oocytes were recorded in the presence and absence of dFBr using two-electrode voltage clamp electrophysiology. Wild-type and mutant receptors were expressed in both high and low ACh sensitivity isoforms by using biased injection ratios of 1:5 or 5:1 α4 to ß2 complementary RNA. Mutations were made in the B, C, and A loops of the principal face of the ß2 subunit, which are regions not involved in the binding of ACh. Mutant ß2(Y120A) significantly eliminated dFBr potency in both isoform preparations. Several other mutations altered dFBr potentiation levels in both preparations. Our findings support the involvement of the principal face of the ß2 subunit in dFBr modulation of ACh-induced responses. Findings from this study will aid in the improved design of dFBr-like PAMs for potential therapeutic use.

Allosteric modulation of alpha4beta2 nicotinic acetylcholine receptors by HEPES.

A number of new positive allosteric modulators (PAMs) have been reported that enhance responses of neuronal alpha7 and alpha4beta2 nicotinic acetylcholine receptor subtypes to orthosteric ligands. PAMs represent promising new leads for the development of therapeutic agents for disorders involving alterations in nicotinic neurotransmission including Autism, Alzheimer's and Parkinson's disease. During our recent studies of alpha4beta2 PAMs, we identified a novel effect of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The effects of HEPES were evaluated in a phosphate buffered recording solution using two-electrode voltage clamp techniques and alpha4beta2 and alpha7 nicotinic acetylcholine receptor subtypes expressed in Xenopus laevis oocytes. Acetylcholine induced responses of high-sensitivity alpha4beta2 receptors were potentiated 190% by co-exposure to HEPES. Responses were inhibited at higher concentrations (bell-shaped concentration/response curve). Coincidentally, at concentrations of HEPES typically used in oocyte recording (5-10mM), the potentiating effects of HEPES are matched by its inhibitory effects, thus producing no net effect. Mutagenesis results suggest HEPES potentiates the high-sensitivity stoichiometry of the alpha4beta2 receptors through action at the beta2+/beta2- interface and is dependent on residue beta2D218. HEPES did not potentiate low-sensitivity alpha4beta2 receptors and did not produce any observable effect on acetylcholine induced responses on alpha7 nicotinic acetylcholine receptors.

2-Amino-6-chloro-3,4-dihydroquinazoline: A novel 5-HT3 receptor antagonist with antidepressant character.

2-Amino-6-chloro-3,4-dihydroquinazoline HCl (A6CDQ, 4) binds at 5-HT3 serotonin receptors and displays antidepressant-like action in the mouse tail suspension test (TST). Empirically, 4 was demonstrated to be a 5-HT3 receptor antagonist (two-electrode voltage clamp recordings using frog oocytes; IC50=0.26µM), and one that should readily penetrate the blood-brain barrier (logP=1.86). 5-HT3 receptor antagonists represent a potential approach to the development of new antidepressants, and 4 is an example of a structurally novel 5-HT3 receptor antagonist that is active in a preclinical antidepressant model (i.e., the mouse TST).

Deconstruction of the α4ß2 nicotinic acetylcholine receptor positive allosteric modulator desformylflustrabromine.

Desformylflustrabromine (dFBr; 1), perhaps the first selective positive allosteric modulator of α4ß2 neuronal nicotinic acetylcholine (nACh) receptors, was deconstructed to determine which structural features contribute to its actions on receptors expressed in Xenopus ooycytes using two-electrode voltage clamp techniques. Although the intact structure of 1 was found to be optimal, several deconstructed analogs retained activity. Neither the 6-bromo substituent nor the entire 2-position chain is required for activity. In particular, reduction of the olefinic side chain of 1, as seen with 6, not only resulted in retention of activity/potency but in enhanced selectivity for α4ß2 versus α7 nACh receptors. Pharmacophoric features for the allosteric modulation of α4ß2 nACh receptors by 1 were identified.

Pharmacological characterization of the allosteric modulator desformylflustrabromine and its interaction with alpha4beta2 neuronal nicotinic acetylcholine receptor orthosteric ligands.

Neuronal nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of ligand-gated ion channels. nAChRs are involved in modulating nicotinic-based signal transmission in the central nervous system and are implicated in a range of disorders. Desformylflustrabromine (dFBr) is a positive allosteric modulator that potentiates alpha4beta2 nAChRs. It has been reported that dFBr is selective for the alpha4beta2 receptor relative to other common nAChR subtypes (Neurosci Lett 373:144-149, 2005). Coapplication of dFBr with acetylcholine (ACh) produces a bell-shaped dose-response curve with a peak potentiation of more than 265% (Bioorg Med Chem Lett 17:4855-4860, 2007) at dFBr concentrations <10 microM and inhibition of responses at concentrations >10 microM. The potentiation and inhibition components of dFBr-modulated responses were examined by using two-electrode voltage clamp and human alpha4beta2 nAChRs expressed in Xenopus laevis oocytes. Currents to both partial and full agonists were potentiated by dFBr. Responses to low-efficacy agonists were potentiated significantly more than responses to high-efficacy agonists. Antagonist pIC(50) values were unaffected by coapplication of dFBr. In addition to its potentiating effects, dFBr was able to induce current spikes when applied to desensitized receptors, suggestive of a shift in equilibrium from the desensitized to open conformation. In contrast to potentiation, inhibition of ACh responses by dFBr depends on membrane potential and is probably the result of open-channel block by dFBr and ACh. Our data indicate distinct mechanisms for the potentiation and inhibition components of dFBr action. dFBr could prove useful for therapeutic enhancement of responses at alpha4beta2-containing synapses.

A comprehensive study on the 5-hydroxytryptamine(3A) receptor binding of agonists serotonin and m-chlorophenylbiguanidine.

Serotonin type 3 receptors (5-HT(3)R) are members of the ligand gated ion channel receptor family. In this study, the interactions of the agonists serotonin (5-HT) and m-chlorophenylbiguanidine (mCPBG) at the binding site of the 5-HT(3A)R were investigated at an atomic level. Site-directed mutagenesis studies in Loop B and E along with our earlier published results from mutations within Loops A, C, and D provide comprehensive data on the interaction of 5-HT and mCPBG with 5-HT(3A)Rs. Using this data we have constructed a refined homology model of the 5-HT(3A)R that considers all of the available experimental data. 5-HT and mCPBG were docked into the newly constructed homology model and the amino acid residues critical in binding of these agonists were compared and analyzed. Our docking results reveal many similar binding interactions for 5-HT and mCPBG. Namely, residues THR181, TRP183, PHE226, ILE228, TYR234 and GLU129 were all found to play key roles in binding of both 5-HT and mCPBG. However, the results also revealed two important differences that exist between the interactions of the two agonists. In our model, a hydrogen bond is formed between the indole hydrogen of 5-HT and the residue TYR153. This interaction is not present in the case of mCPBG. Conversely, a hydrogen bond exists between SER182 and a protonated nitrogen of mCPBG, which does not exist in 5-HT. Our modeling results were found to be in accordance with experimental data.

Microcantilever biosensors based on conformational change of proteins.

Microcantilevers (MCLs) hold a position as a cost-effective and highly sensitive sensor platform for medical diagnostics, environmental analysis and fast throughput analysis. MCLs are unique in that adsorption of analytes on the microcantilever (MCL) surface changes the surface characteristics of the MCL and results in bending of the MCL. Surface stress due to conformation change of proteins and other polymers has been a recent focus of MCL research. Since conformational changes in proteins can be produced through binding of anylates at specific receptor sites, MCLs that respond to conformational change induced surface stress are promising as transducers of chemical information and are ideal for developing microcantilever-based biosensors. The MCL can also potentially be used to investigate conformational change of proteins induced by non-binding events such as post-translational modification and changes in temperature or pH. This review will provide an overview of MCL biosensors based on conformational change of proteins bound to the MCL surface. The models include conformational change of proteins, proteins on membranes, enzymes, DNA and other polymers.

Improved surface modification approach for micromechanical biosensors.

We have investigated the sensing performance of protein-based microcantilever biosensors prepared from multiple surface conjugation chemistries. The 11-mercaptoundecanoic acid monolayers were prepared according to both traditional and modified processes. In three protein-based biosensors, the modified process improved microcantilever sensing performance by increasing the bending amplitude, a critical step toward developing a cost-effective microcantilever-based sensor platform for medical diagnostics and environmental and drug screening applications. Scanning electron microscopy (SEM) images demonstrated that proteins immobilized on the microcantilever surface using the modified chemistry approach formed a compact layer.

Synthesis of desformylflustrabromine and its evaluation as an alpha4beta2 and alpha7 nACh receptor modulator.

Desformylflustrabromine (dFBr; 1) and desformylflustrabromine-B (dFBr-B; 2) have been previously isolated from natural sources, and the former has been demonstrated to be a novel and selective positive allosteric modulator of alpha4beta2 nicotinic acetylcholine (nACh) receptors. The present study describes the synthesis of water-soluble salts of 1 and 2, and confirms and further investigates the actions of 1 and 2 using two-electrode voltage clamp recordings.

Interactions of granisetron with an agonist-free 5-HT3A receptor model.

A new homology model of type-3A serotonin receptors (5-HT(3A)Rs) was built on the basis of the electron microscopic structure of the nicotinic acetylcholine receptor and with an agonist-free binding cavity. The new model was used to re-evaluate the interactions of granisetron, a 5-HT(3A)R antagonist. Docking of granisetron identified two possible binding modes, including a newly identified region for antagonists formed by loop B, C, and E residues. Amino acid residues L184-D189 in loop B were mutated to alanine, while Y143 and Y153 in loop E were mutated to phenylalanine. Mutation H185A resulted in no detectable granisetron binding, while D189A resulted in a 22-fold reduction in affinity. Y143F and Y153F decreased granisetron affinity to the same extent as Y143A and Y153A mutations, supporting the role of the OH groups of these tyrosines in loop E. Modeling and mutation studies suggest that granisetron plays its antagonist role by hindering the closure of the back wall of the binding cavity.

The loop C region of the murine 5-HT3A receptor contributes to the differential actions of 5-hydroxytryptamine and m-chlorophenylbiguanide.

Sequence and predicted structural similarities between members of the Cys loop superfamily of ligand-gated ion channel receptors and the acetylcholine binding protein (AChBP) suggest that the ligand-binding site is formed by six loops that intersect at subunit interfaces. We employed site-directed mutagenesis to investigate the role of amino acids from the loop C region of the murine 5-HT(3AS)R in interacting with two structurally different agonists, serotonin (5-HT) and m-chlorophenylbiguanide (mCPBG). Mutant receptors were evaluated using radioligand binding, two-electrode voltage clamp, and immunofluorescence studies. Electrophysiological assays were employed to identify changes in response characteristics and relative efficacies of mCPBG and the partial agonist, 2-methyl 5-HT (2-Me5-HT). We have also constructed novel 5-HT and mCPBG docked models of the receptor binding site based on homology models of the AChBP. Both ligand-docked models correlate well with results from mutagenesis and electrophysiological assays. Four key amino acids were identified as being important to ligand binding and/or gating of the receptor. Among these, I228 and D229 are specific for effects mediated by 5-HT compared to mCPBG, indicating a differential interaction of these ligands with loop C. Residues F226 and Y234 are important for both 5-HT and mCPBG interactions. Mutations at F226, I228, and Y234 also altered the relative efficacies of agonists, suggesting a role in the gating mechanism.