Highlights • Lynx1 and its homologs keep the cholinergic system activity at the physiologically appropriate levels. • Lynx1 has three identified targets: nAChRs, muscarinic acetylcholine receptors, ...and potassium channels. • Water-soluble Lynx1 binds competitively at muscle nAChRs, but noncompetitively to neuronal ones. • Lynx1 and α-neurotoxins appear to differ in their affinity and reversibility of binding to nAChRs.
The nicotinic acetylcholine receptor (nAChR) represents the prototype of ligand-gated ion channels. It is vital for neuromuscular transmission and an important regulator of neurotransmission. A ...variety of toxic compounds derived from diverse species target this receptor and have been of elemental importance in basic and applied research. They enabled milestone discoveries in pharmacology and biochemistry ranging from the original formulation of the receptor concept, the first isolation and structural analysis of a receptor protein (the nAChR) to the identification, localization, and differentiation of its diverse subtypes and their validation as a target for therapeutic intervention. Among the venom-derived compounds, α-neurotoxins and α-conotoxins provide the largest families and still represent indispensable pharmacological tools. Application of modified α-neurotoxins provided substantial structural and functional details of the nAChR long before high resolution structures were available. α-bungarotoxin represents not only a standard pharmacological tool and label in nAChR research but also for unrelated proteins tagged with a minimal α-bungarotoxin binding motif. A major advantage of α-conotoxins is their smaller size, as well as superior selectivity for diverse nAChR subtypes that allows their development into ligands with optimized pharmacological and chemical properties and potentially novel drugs. In the following, these two groups of nAChR antagonists will be described focusing on their respective roles in the structural and functional characterization of nAChRs and their development into research tools. In addition, we provide a comparative overview of the diverse α-conotoxin selectivities that can serve as a practical guide for both structure activity studies and subtype classification.
This article is part of the Special Issue entitled ‘Venom-derived Peptides as Pharmacological Tools.’
•α-Neurotoxins have been invaluable in defining nAChR structure and function and still provide important research tools.•α-Conotoxins are superior pharmacological tools for nAChR subtype differentiation and might be developed into novel drugs.•Novel computational approaches are now needed to predict selectivities and affinities and to design optimized nAChR ligands.
The first toxin to give rise to the three‐finger protein (TFP) family was α‐bungarotoxin (α‐Bgt) from Bungarus multicinctus krait venom. α‐Bgt was crucial for research on nicotinic acetylcholine ...receptors (nAChRs), and in this Review article we focus on present data for snake venom TFPs and those of the Ly6/uPAR family from mammalians (membrane‐bound Lynx1 and secreted SLURP‐1) interacting with nAChRs. Recently isolated from Bungarus candidus venom, αδ‐bungarotoxins differ from α‐Bgt: they bind more reversibly and distinguish two binding sites in Torpedo californica nAChR. Naja kaouthia α‐cobratoxin, classical blocker of nAChRs, was shown to inhibit certain GABA‐A receptor subtypes, whereas α‐cobratoxin dimer with 2 intermolecular disulfides has a novel type of 3D structure. Non‐conventional toxin WTX has additional 5th disulfide not in the central loop, as α‐Bgt, but in the N‐terminal loop, like all Ly6/uPAR proteins, and inhibits α7 and Torpedo nAChRs. A water‐soluble form of Lynx1, ws‐Lynx1, was expressed in E. coli, its 1H‐NMR structure and binding to several nAChRs determined. For SLURP‐1, similar information was obtained with its recombinant analogue rSLURP‐1. A common feature of ws‐Lynx1, rSLURP‐1, and WTX is their activity against nAChRs and muscarinic acetylcholine receptors. Synthetic SLURP‐1, identical to the natural protein, demonstrated some differences from rSLURP‐1 in distinguishing nAChR subtypes. The loop II fragment of the Lynx1 was synthesized having the same µM affinity for the Torpedo nAChR as ws‐Lynx1. This review illustrates the productivity of parallel research of nAChR interactions with the two TFP groups.
Nicotinic acetylcholine receptors (nAChRs) located in the tissues of nervous, immune and other systems are a hot topic and much progress has been brought by X‐ray crystallography and cryo‐electron microscopy of these receptors. However, snake venom neurotoxins (“three‐finger proteins”, TFPs), which played a crucial role about 50 years ago in the isolation and characterization of the first nAChR, still are used as invaluable pharmacological tools for distinguishing different nAChR subtypes and for opening new ways in drug design. A much more novel trend is the TFPs of the Ly6 family which are endogenous regulators of the versatile functions of nAChRs.
SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de ...Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,--non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to 'metabotropic' signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.
The COVID-19 pandemic caused by SARS-CoV-2 requires new treatments both to alleviate the symptoms and to prevent the spread of this disease. Previous studies demonstrated good antiviral and virucidal ...activity of phospholipase A
2
s (PLA
2
s) from snake venoms against viruses from different families but there was no data for coronaviruses. Here we show that PLA
2
s from snake venoms protect Vero E6 cells against SARS-CoV-2 cytopathic effects. PLA
2
s showed low cytotoxicity to Vero E6 cells with some activity at micromolar concentrations, but strong antiviral activity at nanomolar concentrations. Dimeric PLA
2
from the viper
Vipera nikolskii
and its subunits manifested especially potent virucidal effects, which were related to their phospholipolytic activity, and inhibited cell–cell fusion mediated by the SARS-CoV-2 spike glycoprotein. Moreover, PLA
2
s interfered with binding both of an antibody against ACE2 and of the receptor-binding domain of the glycoprotein S to 293T/ACE2 cells. This is the first demonstration of a detrimental effect of PLA
2
s on β-coronaviruses. Thus, snake PLA
2
s are promising for the development of antiviral drugs that target the viral envelope, and could also prove to be useful tools to study the interaction of viruses with host cells.
This review covers briefly the work carried out at our institute (IBCh), in many cases in collaboration with other Russian and foreign laboratories, for the last 50 years. It discusses the ...discoveries and studies of various animal toxins, including protein and peptide neurotoxins acting on the nicotinic acetylcholine receptors (nAChRs) and on other ion channels. Among the achievements are the determination of the primary structures of the α-bungarotoxin-like three-finger toxins (TFTs), covalently bound dimeric TFTs, glycosylated cytotoxin, inhibitory cystine knot toxins (ICK), modular ICKs, and such giant molecules as latrotoxins and peptide neurotoxins from the snake, as well as from other animal venoms. For a number of toxins, spatial structures were determined, mostly by 1H-NMR spectroscopy. Using this method in combination with molecular modeling, the molecular mechanisms of the interactions of several toxins with lipid membranes were established. In more detail are presented the results of recent years, among which are the discovery of α-bungarotoxin analogs distinguishing the two binding sites in the muscle-type nAChR, long-chain α-neurotoxins interacting with α9α10 nAChRs and with GABA-A receptors, and the strong antiviral effects of dimeric phospholipases A2. A summary of the toxins obtained from arthropod venoms includes only highly cited works describing the molecules’ success story, which is associated with IBCh. In marine animals, versatile toxins in terms of structure and molecular targets were discovered, and careful work on α-conotoxins differing in specificity for individual nAChR subtypes gave information about their binding sites.
It was a pleasure to receive a proposal to organize and be a guest editor of a Special Issue ofBiomolecules. This is the field in which I am working and personally know some of the leadingscientists. ...My narrow field is the research on the peptide and protein neurotoxins from animalvenoms and their application as sophisticated tools for analysis of nicotinic acetylcholine receptors(nAChRs) ....
Viruses infect all types of organisms, causing viral diseases, which are very common in humans. Since viruses use the metabolic pathways of their host cells to replicate, they are difficult to ...eradicate without affecting the cells. The most effective measures against viral infections are vaccinations and antiviral drugs, which selectively inhibit the viral replication cycle. Both methods have disadvantages, which requires the development of new approaches to the treatment of viral diseases. In the study of animal venoms, it was found that, in addition to toxicity, venoms exhibit other types of biological activity, including an antiviral one, the first mention of which dates back to middle of the last century, but detailed studies of their antiviral activity have been conducted over the past 15 years. The COVID-19 pandemic has reinforced these studies and several compounds with antiviral activity have been identified in venoms. Some of them are very active and can be considered as the basis for antiviral drugs. This review discusses recent antiviral studies, the found compounds with high antiviral activity, and the possible mechanisms of their action. The prospects for using the animal venom components to create antiviral drugs, and the expected problems and possible solutions are also considered.
Phospholipases A2 (PLA2s) are a large family of snake toxins manifesting diverse biological effects, which are not always related to phospholipolytic activity. Snake venom PLA2s (svPLA2s) are ...extracellular proteins with a molecular mass of 13–14 kDa. They are present in venoms in the form of monomers, dimers, and larger oligomers. The cardiovascular system is one of the multiple svPLA2 targets in prey organisms. The results obtained previously on the cardiovascular effects of monomeric svPLA2s were inconsistent, while the data on the dimeric svPLA2 crotoxin from the rattlesnake Crotalus durissus terrificus showed that it significantly reduced the contractile force of guinea pig hearts. Here, we studied the effects of the heterodimeric svPLA2 HDP-1 from the viper Vipera nikolskii on papillary muscle (PM) contractility and the tension of the aortic rings (ARs). HDP-1 is structurally different from crotoxin, and over a wide range of concentrations, it produced a long-term, stable, positive inotropic effect in PMs, which did not turn into contractures at the concentrations studied. This also distinguishes HDP-1 from the monomeric svPLA2s, which at high concentrations inhibited cardiac function. HDP-1, when acting on ARs preconstricted with 10 μM phenylephrine, induced a vasorelaxant effect, similar to some other svPLA2s. These are the first indications of the cardiac and vascular effects of true vipers’ heterodimeric svPLA2s.
Immune response during sepsis is characterized by hyper-inflammation followed by immunosuppression. The crucial role of macrophages is well-known for both septic stages, since they are involved in ...immune homeostasis and inflammation, their dysfunction being implicated in immunosuppression. The cholinergic anti-inflammatory pathway mediated by macrophage α7 nicotinic acetylcholine receptor (nAChR) represents possible drug target. Although α7 nAChR activation on macrophages reduces the production of proinflammatory cytokines, the role of these receptors in immunological changes at the cellular level is not fully understood. Using α7 nAChR selective agonist PNU 282,987, we investigated the influence of α7 nAChR activation on the expression of cytokines and, for the first time, of the macrophage membrane markers: cluster of differentiation 14 (CD14), human leukocyte antigen-DR (HLA-DR), CD11b, and CD54. Application of PNU 282,987 to THP-1Mϕ (THP-1 derived macrophages) cells led to inward ion currents and Ca
increase in cytoplasm showing the presence of functionally active α7 nAChR. Production of cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 was estimated in classically activated macrophages (M1) and treatment with PNU 282,987 diminished IL-10 expression. α7 nAChR activation on THP-1Mϕ, THP-1M1, and monocyte-derived macrophages (MDMs) increased the expression of HLA-DR, CD54, and CD11b molecules, but decreased CD14 receptor expression, these effects being blocked by alpha (α)-bungarotoxin. Thus, PNU 282,987 enhances the macrophage-mediated immunity via α7 nAChR by regulating expression of their membrane receptors and of cytokines, both playing an important role in preventing immunosuppressive states.
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