Inhibition Of Nicotinic Acetylcholine Receptors Research Articles

De novo designed proteins neutralize lethal snake venom toxins.

Snakebite envenoming remains a devastating and neglected tropical disease, claiming over 100,000 lives annually and causing severe complications and long-lasting disabilities for many more1,2. Three-finger toxins (3FTx) are highly toxic components of elapid snake venoms that can cause diverse pathologies, including severe tissue damage3 and inhibition of nicotinic acetylcholine receptors (nAChRs) resulting in life-threatening neurotoxicity4. Currently, the only available treatments for snakebite consist of polyclonal antibodies derived from the plasma of immunized animals, which have high cost and limited efficacy against 3FTxs5,6,7. Here, we use deep learning methods to de novo design proteins to bind short- and long-chain α-neurotoxins and cytotoxins from the 3FTx family. With limited experimental screening, we obtain protein designs with remarkable thermal stability, high binding affinity, and near-atomic level agreement with the computational models. The designed proteins effectively neutralize all three 3FTx sub-families in vitro and protect mice from a lethal neurotoxin challenge. Such potent, stable, and readily manufacturable toxin-neutralizing proteins could provide the basis for safer, cost-effective, and widely accessible next-generation antivenom therapeutics. Beyond snakebite, our computational design methodology should help democratize therapeutic discovery, particularly in resource-limited settings, by substantially reducing costs and resource requirements for development of therapies to neglected tropical diseases.

Microbiota-derived acetylcholine can promote gut motility in Drosophila melanogaster.

The gut microbiota is crucial for intestinal health, including gastrointestinal (GI) motility. How commensal bacterial species influence GI motility has not been fully elucidated. A major factor of GI motility is the gut contraction promoting the propulsive movement of orally ingested materials. Here, we developed a method to monitor and quantify gut contractions in living Drosophila melanogaster larvae. We found that the culture medium of an isolated strain Lactiplantibacillus plantarum Lsi promoted gut contraction in vivo, which was not observed in Leuconostoc sp. Leui nor Acetobacter persici Ai culture medium. To identify bacteria-derived metabolites, we performed metabolome analysis of the culture media by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of the 66 metabolites detected, we found that some metabolites changed in a species-specific manner. Among them, acetylcholine was specifically produced by L. plantarum. Feeding exogenous acetylcholine increased the frequency of gut contractions, which was blocked by D-tubocurarine, an inhibitor of nicotinic acetylcholine receptors. In this study, we propose a mechanism by which the gut microbiota influences Drosophila gut motility. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

Inhibition of nicotinic acetylcholine receptors by oligoarginine peptides and polyamine-related compounds.

Oligoarginine peptides, known mostly for their cell-penetrating properties, are also inhibitors of the nicotinic acetylcholine receptors (nAChRs). Since octa-arginine (R8) inhibits α9α10 nAChR and suppresses neuropathic pain, we checked if other polycationic compounds containing amino and/or guanidino groups could be effective and tested the activity of the disulfide-fixed "cyclo"R8, a series of biogenic polyamines (putrescine, spermidine, and spermine), C-methylated spermine analogs, agmatine and its analogs, as well as acylpolyamine argiotoxin-636 from spider venom. Their inhibitory potency on muscle-type, α7 and α9α10 nAChRs was determined using radioligand analysis, electrophysiology, and calcium imaging. "Cyclo"R8 showed similar activity to that of R8 against α9α10 nAChR (IC50 ≈ 60nM). Biogenic polyamines as well as agmatine and its analogs displayed low activity on muscle-type Torpedo californica, as well as α7 and α9α10 nAChRs, which increased with chain length, the most active being spermine and its C-methylated derivatives having IC50 of about 30μM against muscle-type T. californica nAChR. Argiotoxin-636, which contains a polyamine backbone and terminal guanidino group, also weakly inhibited T. californica nAChR (IC50 ≈ 15μM), but it revealed high potency against rat α9α10 nAChR (IC50 ≈ 200nM). We conclude that oligoarginines and similar polycationic compounds effectively inhibiting α9α10 nAChR may serve as a basis for the development of analgesics to reduce neuropathic pain.

Cholinergic collaterals arising from noradrenergic sympathetic neurons in mice.

The sympathetic nervous system vitally regulates autonomic functions, including cardiac activity. Postganglionic neurons of the sympathetic chain ganglia relay signals from the central nervous system to autonomic peripheral targets. Disrupting this flow of information often dysregulates organ function and leads to poor health outcomes. Despite the importance of these sympathetic neurons, fundamental aspects of the neurocircuitry within peripheral ganglia remain poorly understood. Conventionally, simple monosynaptic cholinergic pathways from preganglionic neurons are thought to activate postganglionic sympathetic neurons. However, early studies suggested more complex neurocircuits may be present within sympathetic ganglia. The present study recorded synaptic responses in sympathetic stellate ganglia neurons following electrical activation of the pre- and postganglionic nerve trunks and used genetic strategies to assess the presence of collateral projections between postganglionic neurons of the stellate ganglia. Orthograde activation of the preganglionic nerve trunk, T-2, uncovered high jitter synaptic latencies consistent with polysynaptic connections. Pharmacological inhibition of nicotinic acetylcholine receptors with hexamethonium blocked all synaptic events. To confirm that high jitter, polysynaptic events were due to the presence of cholinergic collaterals from postganglionic neurons within the stellate ganglion, we knocked out choline acetyltransferase in adult noradrenergic neurons. This genetic knockout eliminated orthograde high jitter synaptic events and EPSCs evoked by retrograde activation. These findings suggest that cholinergic collateral projections arise from noradrenergic neurons within sympathetic ganglia. Identifying the contributions of collateral excitation to normal physiology and pathophysiology is an important area of future study and may offer novel therapeutic targets for the treatment of autonomic imbalance. KEY POINTS: Electrical stimulation of a preganglionic nerve trunk evoked fast synaptic transmission in stellate ganglion neurons with low and high jitter latencies. Retrograde stimulation of a postganglionic nerve trunk evoked direct, all-or-none action currents and delayed nicotinic EPSCs indistinguishable from orthogradely-evoked EPSCs in stellate neurons. Nicotinic acetylcholine receptor blockade prevented all spontaneous and evoked synaptic activity. Knockout of acetylcholine production in noradrenergic neurons eliminated all retrogradely-evoked EPSCs but did not change retrograde action currents, indicating that noradrenergic neurons have cholinergic collaterals connecting neurons within the stellate ganglion.

What We Have Gained from Ibogaine: α3β4 Nicotinic Acetylcholine Receptor Inhibitors as Treatments for Substance Use Disorders.

For decades, ibogaine─the main psychoactive alkaloid found in Tabernanthe iboga─has been investigated as a possible treatment for substance use disorders (SUDs) due to its purported ability to interrupt the addictive properties of multiple drugs of abuse. Of the numerous pharmacological actions of ibogaine and its derivatives, the inhibition of α3β4 nicotinic acetylcholine receptors (nAChRs), represents a probable mechanism of action for their apparent anti-addictive activity. In this Perspective, we examine several classes of compounds that have been discovered and developed to target α3β4 nAChRs. Specifically, by focusing on compounds that have proven efficacious in pre-clinical models of drug abuse and have been evaluated clinically, we highlight the promising potential of the α3β4 nAChRs as viable targets to treat a wide array of SUDs. Additionally, we discuss the challenges faced by the existing classes of α3β4 nAChR ligands that must be overcome to develop them into therapeutic treatments.

Betel quid: New insights into an ancient addiction.

The use of areca nuts (areca) in the form of betel quids constitutes the fourth most common addiction in the world, associated with high risk for oral disease and cancer. Areca is a complex natural product, making it difficult to identify specific components associated with the addictive and carcinogenic properties. It is commonly believed that the muscarinic agonist arecoline is at the core of the addiction. However, muscarinic receptor activation is not generally believed to support drug-taking behaviour. Subjective accounts of areca use include descriptions of both sedative and stimulatory effects, consistent with the presence of multiple psychoactive agents. We have previously reported partial agonism of α4-containing nicotinic acetylcholine receptors by arecoline and subsequent inhibition of those receptors by whole areca broth. In the present study, we report the inhibition of nicotinic acetylcholine receptors and other types of neurotransmitter receptors with compounds of high molecular weight in areca and the ability of low molecular weight areca extract to activate GABA and glutamate receptors. We confirm the presence of a high concentration of GABA and glutamate in areca. Additionally, data also indicate the presence of a dopamine and serotonin transporter blocking activity in areca that could account for the reported stimulant and antidepressant activity. Our data suggest that toxic elements of high molecular weight may contribute to the oral health liability of betel quid use, while two distinct low molecular weight components may provide elements of reward, and the nicotinic activity of arecoline contributes to the physical dependence of addiction.

Oligo-basic amino acids, potential nicotinic acetylcholine receptor inhibitors.

Oligo-basic amino acids have been extensively studied in molecular biology and pharmacology, but the inhibitory activity on nicotinic acetylcholine receptors (nAChRs) was unknown. In this study, the inhibitory activity of 8 oligopeptides, including both basic and acidic amino acids, was evaluated on 9 nAChR subtypes by a two-electrode voltage clamp (TEVC). Among them, the oligo-lysine K9, K12, d-K9, d-K9F, and oligo-arginine R9 showed nanomolar inhibitory activity on various nAChRs, especially for α7 and α9α10 nAChRs. d-K9 containing N-Fmoc protecting group (d-K9F) has an enhanced inhibitory activity on most of the nAChRs, including 47-fold promotion on α1β1δε nAChR. However, H9 and H12 only showed weak inhibitory activity on α9α10 and α1β1δε nAChRs, and the acidic oligopeptide D9 has no inhibitory activity on nAChRs. Flexible docking of K9 in α10(+) α9(-) and α7(+) α7(-) binding pockets showed particularly strong dipole-dipole interactions, which may be responsible for the inhibition of nAChRs. These results demonstrated that oligo-basic amino acids have the potential to be the lead compounds as selective nAChR subtype inhibitors, and oligo-lysines deserved to be modified for further exploitation and utilization. On the other hand, the toxicity and side effects of these nAChR inhibitory peptides should be contemplated in the application.

Cortical nicotinic enhancement of tone-evoked heightened activities and subcortical nicotinic enlargement of activated areas in mouse auditory cortex

Systemic nicotine administration regulates neuronal activities in mouse auditory cortex. How nicotine regulates the spread of the activities across auditory cortical areas is not well known. We investigate this using flavoprotein fluorescence imaging. 20 kHz amplitude-modulated (AM) tones increased the peak intensity of flavoprotein fluorescence in presumptive primary auditory cortex (A1). 5 kHz AM tones activated at least three cortical areas, which are presumably A1, anterior auditory field, and secondary auditory cortex. Nicotine enlarged tone-activated cortical areas and enhanced both 20 kHz and 5 kHz tone-evoked fluorescence intensities at their respective, optimal frequency peak sites and at non-optimal frequency peak sites in A1. The extent of this enhancement was greater at non-optimal frequency sites than at optimal frequency sites. A cortical injection of dihydro-β-erythroidine, an inhibitor of nicotinic acetylcholine receptors composed of α4 and β2-subunits (α4β2*-nAChRs), blocked the enhancement of fluorescence intensity at the peak sites but did not appear to block the enlargement of activated areas. These results suggest that nicotine exposure activates cortical α4β2*-nAChRs to enhance tone-evoked local neuronal activities at an optimal frequency site. The nicotine-induced enlargement of a tone-activated area may depend on the nicotinic enhancement of cortical inputs or other activities.

Structure-Function of Neuronal Nicotinic Acetylcholine Receptor Inhibitors Derived From Natural Toxins.

Neuronal nicotinic acetylcholine receptors (nAChRs) are prototypical cation-selective, ligand-gated ion channels that mediate fast neurotransmission in the central and peripheral nervous systems. nAChRs are involved in a range of physiological and pathological functions and hence are important therapeutic targets. Their subunit homology and diverse pentameric assembly contribute to their challenging pharmacology and limit their drug development potential. Toxins produced by an extensive range of algae, plants and animals target nAChRs, with many proving pivotal in elucidating receptor pharmacology and biochemistry, as well as providing templates for structure-based drug design. The crystal structures of these toxins with diverse chemical profiles in complex with acetylcholine binding protein (AChBP), a soluble homolog of the extracellular ligand-binding domain of the nAChRs and more recently the extracellular domain of human α9 nAChRs, have been reported. These studies have shed light on the diverse molecular mechanisms of ligand-binding at neuronal nAChR subtypes and uncovered critical insights useful for rational drug design. This review provides a comprehensive overview and perspectives obtained from structure and function studies of diverse plant and animal toxins and their associated inhibitory mechanisms at neuronal nAChRs.

Peptide Inhibitors of the α-Cobratoxin-Nicotinic Acetylcholine Receptor Interaction.

Venomous snakebites cause >100 000 deaths every year, in many cases via potent depression of human neuromuscular signaling by snake α-neurotoxins. Emergency therapy still relies on antibody-based antivenom, hampered by poor access, frequent adverse reactions, and cumbersome production/purification. Combining high-throughput discovery and subsequent structure–function characterization, we present simple peptides that bind α-cobratoxin (α-Cbtx) and prevent its inhibition of nicotinic acetylcholine receptors (nAChRs) as a lead for the development of alternative antivenoms. Candidate peptides were identified by phage display and deep sequencing, and hits were characterized by electrophysiological recordings, leading to an 8-mer peptide that prevented α-Cbtx inhibition of nAChRs. We also solved the peptide:α-Cbtx cocrystal structure, revealing that the peptide, although of unique primary sequence, binds to α-Cbtx by mimicking structural features of the nAChR binding pocket. This demonstrates the potential of small peptides to neutralize lethal snake toxins in vitro, establishing a potential route to simple, synthetic, low-cost antivenoms.

Recent Research Progress in and Perspectives of Mesoionic Insecticides: Nicotinic Acetylcholine Receptor Inhibitors.

Triflumezopyrim exemplifies a new class of mesoionic insecticides and has attracted increasing attention as a result of its unique structure, high level of insecticidal activity, new mechanisms of action, low toxicity toward non-target organisms, and environmental friendliness. It inhibits the nicotinic acetylcholine receptor and has high potency against sucking pests, including the brown planthopper (Nilaparvata lugens), which has developed serious resistance to conventional neonicotinoids and low cross-resistance to some newly developed neonicotinoids. This review focuses on the discovery, synthesis, structure-activity relationships, and mechanism of action of mesoionic insecticides. Finally, potential directions for the development of mesoionic insecticides are discussed.

Ribbon α-Conotoxin KTM Exhibits Potent Inhibition of Nicotinic Acetylcholine Receptors.

KTM is a 16 amino acid peptide with the sequence WCCSYPGCYWSSSKWC. Here, we present the nuclear magnetic resonance (NMR) structure and bioactivity of this rationally designed α-conotoxin (α-CTx) that demonstrates potent inhibition of rat α3β2-nicotinic acetylcholine receptors (rα3β2-nAChRs). Two bioassays were used to test the efficacy of KTM. First, a qualitative PC12 cell-based assay confirmed that KTM acts as a nAChR antagonist. Second, bioactivity evaluation by two-electrode voltage clamp electrophysiology was used to measure the inhibition of rα3β2-nAChRs by KTM (IC50 = 0.19 ± 0.02 nM), and inhibition of the same nAChR isoform by α-CTx MII (IC50 = 0.35 ± 0.8 nM). The three-dimensional structure of KTM was determined by NMR spectroscopy, and the final set of 20 structures derived from 32 distance restraints, four dihedral angle constraints, and two disulfide bond constraints overlapped with a mean global backbone root-mean-square deviation (RMSD) of 1.7 ± 0.5 Å. The structure of KTM did not adopt the disulfide fold of α-CTx MII for which it was designed, but instead adopted a flexible ribbon backbone and disulfide connectivity of C2–C16 and C3–C8 with an estimated 12.5% α-helical content. In contrast, α-CTx MII, which has a native fold of C2–C8 and C3–C16, has an estimated 38.1% α-helical secondary structure. KTM is the first reported instance of a Framework I (CC-C-C) α-CTx with ribbon connectivity to display sub-nanomolar inhibitory potency of rα3β2-nAChR subtypes.

Differentiating the Pharmacodynamics and Toxicology of Macrolide and Ketolide Antibiotics.

This is a review of the macrolide and ketolide field focusing on differentiating the pharmacodynamics and especially the toxicology of the macrolides and ketolides. We emphasize the diversity in pharmacodynamics and toxicity of the macrolides and ketolides, resulting from even small structural changes, which makes it important to consider the various different compounds separately, not necessarily as a class. The ketolide, telithromycin, was developed because of rising bacterial macrolide resistance but was withdrawn postapproval after visual disturbances, syncope, myasthenia gravis, and hepatotoxicity were noted. These diverse adverse effects could be attributed to inhibition of nicotinic acetylcholine receptors. Solithromycin, a later generation ketolide, was effective in treating bacterial pneumonia, but it was not approved by the U.S. Food and Drug Administration owing, in part, to its structural similarity to telithromycin. This Miniperspective describes that structurally similar macrolides/ketolides have clearly mechanistically distinct effects. Understanding these effects could help in developing and securing regulatory approval of a new macrolide/ketolide that is active against macrolide-resistant pathogenic bacteria.

Anti-cholinergics mecamylamine and scopolamine alleviate motion sickness-induced gastrointestinal symptoms through both peripheral and central actions

Enhanced cholinergic activity contributes to the production of complex autonomic manifestations of motion sickness (MS). However, whether anti-cholinergics exert their anti-MS effects through central or peripheral actions remained unclarified. In the present study, we investigated the effects of mecamylamine (MEC) and scopolamine (SCOP) on rotation-induced gastrointestinal symptoms (conditioned gaping and defecation), locomotion disturbances (hypoactivity and impaired balance performance), hypothermia as well as Fos expression in vestibulo-autonomic regions in rats. We also observed the effects of hexamethonium (HEX) and methyl scopolamine (MSCP) on those MS behavioral responses. The efficacy of all these drugs on rotation-induced emesis and other MS symptoms in cats was also examined. We found that intragastric administration of MEC and SCOP inhibited rotation-induced gaping and defecation in rats, but only MEC showed a dose-dependent manner. MEC aggravated rotation-induced balance disorder and failed to attenuate rotation-induced hypothermia as the SCOP did. MEC was more effective for inhibiting Fos expression in the caudal vestibular nucleus and nucleus of solitary tract than SCOP. Intraperitoneal injection of HEX and MSCP also significantly alleviated rotation-induced gastrointestinal symptoms, and showed benefit to balance performance in rats. In cats, MEC, SCOP and HEX had prophylactic effects against rotation-induced emesis and salivation, and deceased non-retching/vomiting symptoms, but MSCP only attenuated emesis. It suggested that MEC and SCOP might alleviate gastrointestinal symptoms of MS via inhibiting peripheral autonomic nervous system and central vestibulo-autonomic pathways. The nicotinic acetylcholine receptor inhibitors like MEC might be new candidates against gastrointestinal symptoms induced by MS or other vestibular disorders.

Dual Antidepressant Duloxetine Blocks Nicotinic Receptor Currents, Calcium Signals and Exocytosis in Chromaffin Cells Stimulated with Acetylcholine.

The inhibition of nicotinic acetylcholine receptors (nAChRs) has been proposed as a potential strategy to develop new antidepressant drugs. This is based on the observation that antidepressants that selectively block noradrenaline (NA) or serotonin (5-HT) reuptake also inhibit nAChRs. Dual antidepressants blocking both NA and 5-HT reuptake were proposed to shorten the delay in exerting their clinical effects; whether duloxetine, a prototype of dual antidepressants, also blocks nAChRs is unknown. Here we explored this question in bovine chromaffin cells (BCCs) that express native α3, α5, and α7 nAChRs and in cell lines expressing human α7, α3β4, or α4β2 nAChRs. We have found that duloxetine fully blocked the acetylcholine (ACh)-elicited nicotinic currents in BCCs with an IC50 of 0.86 µM. Such blockade seemed to be noncompetitive, voltage dependent, and partially use dependent. The ACh-elicited membrane depolarization, the elevation of cytosolic calcium ([Ca2+]c), and catecholamine release in BCCs were also blocked by duloxetine. This blockade developed slowly, and the recovery of secretion was also slow and gradual. Duloxetine did not affect Na+ or Ca2+ channel currents neither the high-K+-elicited [Ca2+]c transients and secretion. Of interest was that in cell lines expressing human α7, α3β4, and α4β2 nAChRs, duloxetine blocked nicotinic currents with IC50 values of 0.1, 0.56, and 0.85 µM, respectively. Thus, in blocking α7 receptors, which are abundantly expressed in the brain, duloxetine exhibited approximately 10-fold to 100- fold higher potency with respect to reported IC50 values for various antidepressant drugs. This may contribute to the antidepressant effect of duloxetine.

Cigarette toxin 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces experimental pancreatitis through α7 nicotinic acetylcholine receptors (nAChRs) in mice.

Clinical studies have shown that cigarette smoking is a dose-dependent and independent risk factor for acute pancreatitis. Cigarette smoke contains nicotine which can be converted to the potent receptor ligand and toxin, NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]. Previously, we have shown that NNK induces premature activation of pancreatic zymogens in rats, an initiating event in pancreatitis, and this activation is prevented by pharmacologic inhibition of nicotinic acetylcholine receptors (nAChR). In this study, we determined whether NNK mediates pancreatitis through the α7 isoform of nAChR using α7nAChR knockout mice. PCR analysis confirmed expression of non-neuronal α7nAChR in C57BL/6 (WT) mouse and human acinar cells. NNK treatment stimulated trypsinogen activation in acini from WT but not α7nAChR-/- mice. NNK also stimulated trypsinogen activation in human acini. To further confirm these findings, WT and α7nAChR-/- mice were treated with NNK in vivo and markers of pancreatitis were measured. As observed in acini NNK treatment induced trypsinogen activation in WT but not α7nAChR-/- mice. NNK also induced other markers of pancreatitis including pancreatic edema, vacuolization and pyknotic nuclei in WT but not α7nAChR-/- animals. NNK treatment led to increased neutrophil infiltration, a marker of inflammation, in WT mice and to a significantly lesser extent in α7nAChR-/- mice. We also examined downstream targets of α7nAChR activation and found that calcium and PKC activation are involved down stream of NNK stimulation of α7nAChR. In this study we used genetic deletion of the α7nAChR to confirm our previous inhibitor studies that demonstrated NNK stimulates pancreatitis by activating this receptor. Lastly, we demonstrate that NNK can also stimulate zymogen activation in human acinar cells and thus may play a role in human disease.

Orally administered nicotine effects on rat urinary bladder proliferation and carcinogenesis

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. Cigarette smoke inhalation in mice and orally administered nicotine in rats and mice increased urothelial cell proliferation. Nicotine, a major component of smoke, induced cell proliferation in multiple cell types in vitro. In the present study, the enhancing effects of nicotine on F344 rat bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were examined. Nicotine administered in drinking water for 32 weeks following 4 weeks of BBN treatment significantly increased the incidence and number of urothelial carcinomas dose-dependently. Ki67 and pSTAT3 labeling indices and expression of nicotinic acetylcholine receptor alpha 7 (nAChRα7) in non-tumor bladder urothelial lesions were significantly increased by nicotine, but the TUNEL assay for apoptosis showed no increase. In a 4 week study, inhibitors of nicotinic acetylcholine receptor decreased nicotine-induced urothelial simple hyperplasia and Ki67 labeling index in the bladder and kidney pelvis at a single cytotoxic dose of nicotine (40 ppm). Urothelial cytotoxicity with regenerative proliferation was observed by light and scanning electron microscopy. In vitro, nicotine was not cytotoxic to rat or human immortalized urothelial cells (do not express nicotine receptors) below millimolar concentrations, nor in human RT4, T24 or UMUC3 urothelial carcinoma cells (express nicotine receptors). However, nicotine slightly, but statistically significantly, increased cell proliferation at micromolar concentrations in human urothelial carcinoma cells. These data suggest that nicotine enhances urinary bladder carcinogenesis by inducing cytotoxicity with regenerative proliferation. The possible role of direct mitogenesis, involving nAChR and STAT3 signaling and of nicotine receptors requires further investigation at non-cytotoxic doses of nicotine.

Azemiopsin, a Selective Peptide Antagonist of Muscle Nicotinic Acetylcholine Receptor: Preclinical Evaluation as a Local Muscle Relaxant.

Azemiopsin (Az), a linear peptide from the Azemiops feae viper venom, contains no disulfide bonds, is a high-affinity and selective inhibitor of nicotinic acetylcholine receptor (nAChR) of muscle type and may be considered as potentially applicable nondepolarizing muscle relaxant. In this study, we investigated its preclinical profile in regard to in vitro and in vivo efficacy, acute and chronic toxicity, pharmacokinetics, allergenic capacity, immunotoxicity and mutagenic potency. The peptide effectively inhibited (IC50 ~ 19 nM) calcium response of muscle nAChR evoked by 30 μM (EC100) acetylcholine but was less potent (IC50 ~ 3 μM) at α7 nAChR activated by 10 μM (EC50) acetylcholine and had a low affinity to α4β2 and α3-containing nAChR, as well as to GABAA or 5HT3 receptors. Its muscle relaxant effect was demonstrated at intramuscular injection to mice at doses of 30–300 µg/kg, 30 µg/kg being the initial effective dose and 90 µg/kg—the average effective dose. The maximal muscle relaxant effect of Az was achieved in 10 min after the administration and elimination half-life of Az in mice was calculated as 20–40 min. The longest period of Az action observed at a dose of 300 µg/kg was 55 min. The highest acute toxicity (LD50 510 μg/kg) was observed at intravenous injection of Az, at intramuscular or intraperitoneal administration it was less toxic. The peptide showed practically no immunotoxic, allergenic or mutagenic capacity. Overall, the results demonstrate that Az has good drug-like properties for the application as local muscle relaxant and in its parameters, is not inferior to the relaxants currently used. However, some Az modification might be effective to extend its narrow therapeutic window, a typical characteristic and a weak point of all nondepolarizing myorelaxants.

An Approach to Reveal the Possible Underlying Mechanisms Inherent to Oecophylla smaragdina as a Biocontrol Agent

Oecophylla smaragdina, a biological pest control agent attacks its prey with their mandibles followed by ejection of venom; rendering them paralyzed. Even though Oecophylla smaragdina is a successful pest control agent, no attempts have been made to outline its molecular mechanism of action hitherto. The first part of this study intends characterization of venom constituents using preliminary methods followed by coupled gas chromatography and mass spectroscopy (GC–MS) analysis. The results indicate the anticipated presence of neurotoxin in ant venom (2,5-dipropyl-decahydroquinoline). This alkaloid is a known non-competitive inhibitor of nicotinic acetylcholine receptors (nAChRs), but the proper mechanism of inhibition by decahydroquinoline was obscure and has been addressed in this study. Based on the results and known facts, the second part of this study deals with in silico docking and molecular dynamics simulations to portray the molecular level inhibition of nAChRs by decahydroquinolines. Molecular docking and dynamics studies indicate that the inhibition of nAChRs by decahydroquinoline is attained by occluding the pore lumen of nAChRs in the serine–leucine ring. The GC–MS analysis followed by in silico studies thereby indicates that the potent non-competitive inhibitor of nAChRs found in the venom of Oecophylla must be posing paralyzing effects on its prey.

Recipient treatment with acetylcholinesterase inhibitor donepezil attenuates primary graft failure in rats through inhibiting post-transplantational donor heart ischaemia/reperfusion injury.

Ischaemia/reperfusion injury may have deleterious consequences on heart transplantation. The underlying causes such as inflammation may also contribute to the pathogenesis of primary or chronic graft failure. We hypothesize that donepezil (DO), a reversible acetylcholinesterase inhibitor that increases cholinergic receptor activation, may protect the transplanted heart through increasing the level of acetylcholine, which in turn inhibits systemic inflammation in the recipients. First, Lewis-Lewis heart transplantation model was successfully established, and 75 rats were randomly assigned into 3 groups: the DO group (received single dose of intragastric DO treatment), the donepezil + methyllycaconitine group (α7 nicotinic acetylcholine receptor inhibitor) and the control group. Ten rats per group were sacrificed at 24 h after drug administration, whereas the rest of the groups were observed 1 month after surgery. The status of inflammation, survival of the graft and function of the graft were examined. Serum tumour necrosis factor α level was significantly lower in the donepezil group when compared with the control group 24 h after first drug administration; this trend was maintained for 1 month (P < 0.001). Furthermore, DO inhibited CD11b/18-positive cell infiltration (P < 0.001) and myocardiocyte apoptosis (as shown by the percentage of terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick-end labelling-positive nuclei, P = 0.039) in the recipient rats at 24 h after the first drug administration. The percentage of cardiac grafts that survived for 1 month in rats given DO alone was significantly higher (80.0%, 33.3% and 26.7% in the DO, donepezil + methyllycaconitine and control groups, respectively, P = 0.014); the fractional shortening value of the DO group was significantly higher than that in the other 2 groups (29.25 ± 1.84%, 17.92 ± 3.69% and 17.07 ± 2.99% in the DO, donepezil + methyllycaconitine and control group, respectively, P < 0.001). The collagen volume fraction was lower in the DO group than in the other 2 groups (P < 0.001). Our results reveal that treatment of the recipient with DO protects the donor hearts for 1 month after transplantation through suppressing the signalling pathway of inflammation. These results suggest that DO is a novel and clinically feasible strategy to protect the donor heart in transplantation in the long term.