α7- and α9-Containing Nicotinic Acetylcholine Receptors in the Functioning of Immune System and in Pain.

Nicotinic acetylcholine receptors (nAChRs) present as many different subtypes in the nervous and immune systems, muscles and on the cells of other organs. In the immune system, inflammation is regulated via the vagus nerve through the activation of the non-neuronal α7 nAChR subtype, affecting the production of cytokines. The analgesic properties of α7 nAChR-selective compounds are mostly based on the activation of the cholinergic anti-inflammatory pathway. The molecular mechanism of neuropathic pain relief mediated by the inhibition of α9-containing nAChRs is not fully understood yet, but the role of immune factors in this process is becoming evident. To obtain appropriate drugs, a search of selective agonists, antagonists and modulators of α7- and α9-containing nAChRs is underway. The naturally occurring three-finger snake α-neurotoxins and mammalian Ly6/uPAR proteins, as well as neurotoxic peptides α-conotoxins, are not only sophisticated tools in research on nAChRs but are also considered as potential medicines. In particular, the inhibition of the α9-containing nAChRs by α-conotoxins may be a pathway to alleviate neuropathic pain. nAChRs are involved in the inflammation processes during AIDS and other viral infections; thus they can also be means used in drug design. In this review, we discuss the role of α7- and α9-containing nAChRs in the immune processes and in pain.

Human health and snails.

Snails can provide a considerable variety of bioactive compounds for cosmetic and pharmaceutical industries, useful for the development of new formulations with less toxicity and post effects compared to regular compounds used for the purpose. Compounds from crude extract, mucus, slime consist of glycans, polypeptides, proteins, etc., and can be used for curing diseases like viral lesions, warts, and different dermal problems. Some particular uses of snails involve treating post-traumatic stress. Micro RNA of Lymnaea stagnalis, was known to be responsible for the development of long-term memory and treatment of Alzheimer's and Dementia like diseases. This review explores the application of various bioactive compounds from snails with its potential as new translational medicinal and cosmetic applications. Snail bioactive compounds like ω-MVIIA, µ-SIIIA, µO-MrVIB, Xen2174, δ-EVIA, α-Vc1.1, σ-GVIIA, Conantokin-G, and Contulakin-G, conopeptides can be used for the development of anti-cancer drugs. These compounds target the innate immunity and improve the defense system of humans and provide protection against these life-threatening health concerns.AbbreviationsFDA: Food and Drug Administration; UTI: urinal tract infection; nAChRs: nicotinic acetylcholine receptors; NMDA: N-methyl-D-aspartate; CNS: central nervous system; CAR T: chimeric antigen receptors therapy; Micro RNA: micro ribonucleic acid.

Cervical Cancer Correlates with the Differential Expression of Nicotinic Acetylcholine Receptors and Reveals Therapeutic Targets.

Nicotinic acetylcholine receptors (nAChRs) are associated with various cancers, but the relation between nAChRs and cervical cancer remains unclear. Therefore, this study investigated the differential expression of nAChR subunits in human cervical cancer cell lines (SiHa, HeLa, and CaSki) and in normal ectocervical cell lines (Ect1/E6E7) at mRNA and protein levels. Two specific nAChR subtype blockers, αO-conotoxin GeXIVA and α-conotoxin TxID, were then selected to treat different human cervical cancer cell lines with specific nAChR subtype overexpression. The results showed that α3, α9, α10, and ß4 nAChR subunits were overexpressed in SiHa cells compared with that in normal cells. α9 and α10 nAChR subunits were overexpressed in CaSki cells. α*-conotoxins that targeted either α9α10 or α3ß4 nAChR were able to significantly inhibit cervical cancer cell proliferation. These findings may provide a basis for new targets for cervical cancer targeted therapy.

α-Conotoxin TxIB: A Uniquely Selective Ligand for α6/α3ß2ß3 Nicotinic Acetylcholine Receptor Attenuates Nicotine-Induced Conditioned Place Preference in Mice.

Abstract: α-Conotoxin TxIB is a specific antagonist of α6/α3ß2ß3(α6ß2*) nicotinic acetylcholine receptor (nAChR) with an IC50 of 28 nM. Previous studies have shown that α6ß2* nAChRs are abundantly expressed in midbrain dopaminergic neurons and play an important role in mediating the mechanism of nicotine and other drugs reward effect. It provided important targets for the development of anti-addiction drugs. The present study evaluated the pharmacological activity of TxIB in vivo with conditioned place preference (CPP) model, which were induced by subcutaneous injection (s.c.) of nicotine (NIC, 0.5 mg/kg). α-Conotoxin TxIB inhibited the expression and reinstatement of CPP in mice dose-dependently, but had no significant effect on locomotor activity. The concentrations of dopamine (DA), γ-aminobutyric acid (GABA) and noradrenaline (NE) in different brain regions were measured by enzyme-linked immunosorbent assay (ELISA). We found that TxIB could inhibit the concentrations of DA, GABA and NE in different brain regions (such as nucleus accumbens (NAc), hippocampus (HIP) and prefrontal cortex (PFC)) in NIC-induced mice. The concentrations of DA and NE were decreased in ventral tegmental area (VTA), while GABA had little change. The current work described the inhibition activity of TxIB in NIC-induced CPP, suggesting that α6ß2* nAChR-targeted compound may be a promising drug for nicotine addiction treatment.

Alanine-Scanning Mutagenesis of α-Conotoxin GI Reveals the Residues Crucial for Activity at the Muscle Acetylcholine Receptor.

Recently, the muscle-type nicotinic acetylcholine receptors (nAChRs) have been pursued as a potential target of several diseases, including myogenic disorders, muscle dystrophies and myasthenia gravis, etc. α-conotoxin GI isolated from Conus geographus selectively and potently inhibited the muscle-type nAChRs which can be developed as a tool to study them. Herein, alanine scanning mutagenesis was used to reveal the structure⁻activity relationship (SAR) between GI and mouse α1ß1δε nAChRs. The Pro5, Gly8, Arg8, and Tyr11 were proved to be the critical residues for receptor inhibiting as the alanine (Ala) replacement led to a significant potency loss on mouse α1ß1δε nAChR. On the contrary, substituting Asn4, His10 and Ser12 with Ala respectively did not affect its activity. Interestingly, the [E1A] GI analogue exhibited a three-fold potency for mouse α1ß1δε nAChR, whereas it obviously decreased potency at rat α9α10 nAChR compared to wildtype GI. Molecular dynamic simulations also suggest that loop2 of GI significantly affects the interaction with α1ß1δε nAChR, and Tyr11 of GI is a critical residue binding with three hydrophobic amino acids of the δ subunit, including Leu93, Tyr95 and Leu103. Our research elucidates the interaction of GI and mouse α1ß1δε nAChR in detail that will help to develop the novel analogues of GI.

Conotoxins and their regulatory considerations.

Venom derived peptides from marine cone snails, conotoxins, have demonstrated unique pharmacological targeting properties that have been pivotal in advancing medical research. The awareness of their true toxic origins and potent pharmacological nature is emphasized by their 'select agent' classification by the US Centers for Disease Control and Prevention. We briefly introduce the biochemical and pharmacological aspects of conotoxins, highlighting current advancements into their biological engineering, and provide details to the present regulations that govern their use in research.

Alkyne-Bridged α-Conotoxin Vc1.1 Potently Reverses Mechanical Allodynia in Neuropathic Pain Models.

Several Conus-derived venom peptides are promising lead compounds for the management of neuropathic pain, with α-conotoxins being of particular interest. Modification of the interlocked disulfide framework of α-conotoxin Vc1.1 has been achieved using on-resin alkyne metathesis. Although introduction of a metabolically stable alkyne motif significantly disrupts backbone topography, the structural modification generates a potent and selective GABAB receptor agonist that inhibits Cav2.2 channels and exhibits dose-dependent reversal of mechanical allodynia in a behavioral rat model of neuropathic pain. The findings herein support the hypothesis that analgesia can be achieved via activation of GABABRs expressed in dorsal root ganglion (DRG) sensory neurons.

Antinociceptive effects of the marine snail peptides conantokin-G and conotoxin MVIIA alone and in combination in rat models of pain.

There are a number of neurologically active ion channel blocking peptides derived from cone snail venom, such as conantokin-G and omega-conotoxin MVIIA. Conantokin-G inhibits NMDA receptors containing the NR2B subunit whereas omega-conotoxin MVIIA blocks N-type Ca(2+) channels. Separately, these peptides induce antinociceptive effects in pre-clinical pain models following intrathecal injection. In the current study, the efficacies of these peptides were determined separately and in combination by intrathecal injection into rats with a spinal nerve ligation, in rats with a spinal cord compression injury and in the formalin test. Separately, both conantokin-G and omega-conotoxin MVIIA dose-dependently attenuated nociceptive responses in all of these models. However, at high antinociceptive doses for both formalin and nerve injury models, omega-conotoxin MVIIA evoked untoward side effects. Using isobolographic analysis, the combination of sub-antinociceptive doses of peptides demonstrated additive antinociception in rats with a nerve ligation and in the formalin test, without apparent adverse side effects. In a model of neuropathic spinal cord injury pain, which is clinically difficult to treat, the combination of conantokin-G and omega-conotoxin MVIIA resulted in robust synergistic antinociception. These data suggest that a combination of these peptides may be analgesic across diverse clinical pains with limited untoward side effects, and particularly potent for reducing spinal cord injury pain.

Conotoxin venom peptide therapeutics.

Venom peptides offer enormous opportunity for the discovery of peptide drug leads. This review focusses on the potential of cone snails that have developed arrays of small peptides as part of highly evolved venoms used for prey capture and defence. Many of these peptides selectively modulate ion channels and transporters, making them a valuable source of new ligands for studying the role these targets play in normal and disease physiology. A number of these conopeptides reduce pain in animals models and several are now in preclinical and clinical development for the treatment of severe pain often associated with diseases such as cancer.

Structure-Activity Studies Reveal the Molecular Basis for GABAB-Receptor Mediated Inhibition of High Voltage-Activated Calcium Channels by α-Conotoxin Vc1.1.

α-Conotoxins are disulfide-bonded peptides from cone snail venoms and are characterized by their affinity for nicotinic acetylcholine receptors (nAChR). Several α-conotoxins with distinct selectivity for nAChR subtypes have been identified as potent analgesics in animal models of chronic pain. However, a number of α-conotoxins have been shown to inhibit N-type calcium channel currents in rodent dissociated dorsal root ganglion (DRG) neurons via activation of G protein-coupled GABAB receptors (GABABR). Therefore, it is unclear whether activation of GABABR or inhibition of α9α10 nAChRs is the analgesic mechanism. To investigate the mechanisms by which α-conotoxins provide analgesia, we synthesized a suite of Vc1.1 analogues where all residues, except the conserved cysteines, in Vc1.1 were individually replaced by alanine (A), lysine (K), and aspartic acid (D). Our results show that the amino acids in the first loop play an important role in binding of the peptide to the receptor, whereas those in the second loop play an important role for the selectivity of the peptide for the GABABR over α9α10 nAChRs. We designed a cVc1.1 analogue that is >8000-fold selective for GABABR-mediated inhibition of high voltage-activated (HVA) calcium channels over α9α10 nAChRs and show that it is analgesic in a mouse model of chronic visceral hypersensitivity (CVH). cVc1.1[D11A,E14A] caused dose-dependent inhibition of colonic nociceptors with greater efficacy in ex vivo CVH colonic nociceptors relative to healthy colonic nociceptors. These findings suggest that selectively targeting GABABR-mediated HVA calcium channel inhibition by α-conotoxins could be effective for the treatment of chronic visceral pain.

Conotoxin MVIIA improves cell viability and antioxidant system after spinal cord injury in rats.

This study evaluates whether intrathecal MVIIA injection after spinal cord injury (SCI) elicits neuroprotective effects. The test rats were randomly distributed into six groups- sham, placebo, MVIIA 2.5 µM, MVIIA 5 µM, MVIIA 10 µM, and MVIIA 20 µM-and were administered the treatment four hours after SCI. After the optimal MVIIA dose (MVIIA 10 µM) was defined, the best time for application, one or four hours, was analyzed. Locomotor hind limb function and side effects were assessed. Forty-eight hours after the injury and immediately after euthanasia, spinal cord segments were removed from the test rats. Cell viability, reactive oxygen species, lipid peroxidation, and glutamate release were investigated. To examine the MVIIA mechanism of action, the gene expressions of pro-apoptotic (Bax, nNOS, and caspase-3, -8, -9, -12) and anti-apoptotic (Bcl-xl) factors in the spinal cord tissue samples were determined by real-time PCR, and the activities of antioxidant enzymes were also investigated. Application of intrathecal MVIIA 10 µM four hours after SCI prompted a neuroprotective effect: neuronal death decreased (22.46%), oxidative stress diminished, pro-apoptotic factors (Bax, nNOS, and caspase-3, -8) were expressed to a lesser extent, and mitochondrial viability as well as anti-apoptotic factor (Bcl-xl) expression increased. These results suggested that MVIIA provided neuroprotection through antioxidant effects. Indeed, superoxide dismutase (188.41%), and glutathione peroxidase (199.96%), reductase (193.86%), and transferase (175.93%) expressions increased. Therefore, intrathecal MVIIA (MVIIA 10 µM, 4 h) application has neuroprotective potential, and the possible mechanisms are related to antioxidant agent modulation and to intrinsic and extrinsic apoptotic pathways.

Are alpha9alpha10 nicotinic acetylcholine receptors a pain target for alpha-conotoxins?

The synthetic alpha-conotoxin Vc1.1 is a small disulfide bonded peptide currently in development as a treatment for neuropathic pain. Unlike Vc1.1, the native post-translationally modified peptide vc1a does not act as an analgesic in vivo in rat models of neuropathic pain. It has recently been proposed that the primary target of Vc1.1 is the alpha9alpha10 nicotinic acetylcholine receptor (nAChR). We show that Vc1.1 and its post-translationally modified analogs vc1a, [P6O]Vc1.1, and [E14gamma]Vc1.1 are equally potent at inhibiting ACh-evoked currents mediated by alpha9alpha10 nAChRs. This suggests that alpha9alpha10 nAChRs are unlikely to be the molecular mechanism or therapeutic target of Vc1.1 for the treatment of neuropathic pain.

Therapeutic applications of conotoxins that target the neuronal nicotinic acetylcholine receptor.

Pain therapeutics discovered by molecular mining of the expressed genome of Australian predatory cone snails are providing lead compounds for the treatment of neurological diseases such as multiple sclerosis, shingles, diabetic neuropathy and other painful neurological conditions. The high specificity exhibited by these novel compounds for neuronal receptors and ion channels in the brain and nervous system indicates the high degree of selectivity that this class of neuropeptides can be expected to show when used therapeutically in humans. A lead compound, ACV1 (conotoxin Vc1.1 from Conus victoriae), has entered Phase II clinical trials and is being developed for the treatment for neuropathic pain. ACV1 will be targeted initially for the treatment of sciatica, shingles and diabetic neuropathy. The compound is a 16 amino acid peptide [Sandall et al., 2003. A novel alpha-conotoxin identified by gene sequencing is active in suppressing the vascular response to selective stimulation of sensory nerves in vivo. Biochemistry 42, 6904-6911], an antagonist of neuronal nicotinic acetylcholine receptors. It has potent analgesic activity following subcutaneous or intramuscular administration in several preclinical animal models of human neuropathic pain [Satkunanathan et al., 2005. Alpha conotoxin Vc1.1 alleviates neuropathic pain and accelerates functional recovery of injured neurons. Brain. Res. 1059, 149-158]. ACV1 may act as an analgesic by decreasing ectopic excitation in sensory nerves. In addition ACV1 appears to accelerate the recovery of injured nerves and tissues.

Anti-hypersensitive effect of intramuscular administration of αO-conotoxin GeXIVA[1,2] and GeXIVA[1,4] in rats of neuropathic pain.

αO-conotoxin GeXIVA (GeXIVA) is a potent antagonist of α9α10 nicotinic acetylcholine receptors (nAChRs), which has four Cys residues and three disulfide isomers. Among the 3 isomers, both GeXIVA[1,2] (bead isomer) and GeXIVA[1,4] (ribbon isomer) showed potent block on α9α10 nAChRs with close low nanomolar IC50s. Here we report that anti-hypersensitive effects of the bead and ribbon isomers in the chronic constriction injury (CCI) model of neuropathic pain and acute pain model of tail flick test. Treatment was started and continued for 7 or 14days after the development of hyperalgesia which was induced by CCI surgery. GeXIVA[1,2] and GeXIVA[1,4] significantly reduced mechanical allodynia in CCI rats without tolerance, in which GeXIVA[1,2] remained up to two weeks after intramuscular administration of the toxins was ceased. The pain reliever effect of GeXIVA[1,2] on neuropathic rats was slightly better than GeXIVA[1,4]. The two isomers did not suppress the acute thermal pain behaviors significantly when they were tested in the tail flick model by intramuscular bolus injection. Both GeXIVA[1,2] and GeXIVA[1,4] had no significant effect on performance of rats in the accelerating rotarod test after intramuscular injections. This suggests that αO-conotoxin GeXIVA[1,2] and GeXIVA[1,4] may offer new strategies to the treatment of neuropathic pain.

Conotoxins: Structure, Therapeutic Potential and Pharmacological Applications.

Cone snails, also known as marine gastropods, from Conus genus produce in their venom a diverse range of small pharmacologically active structured peptides called conotoxins. The cone snail venoms are widely unexplored arsenal of toxins with therapeutic and pharmacological potential, making them a treasure trove of ligands and peptidic drug leads. Conotoxins are small disulfide bonded peptides, which act as remarkable selective inhibitors and modulators of ion channels (calcium, sodium, potassium), nicotinic acetylcholine receptors, noradrenaline transporters, N-methyl-D-aspartate receptors, and neurotensin receptors. They are highly potent and specific against several neuronal targets making them valuable as research tools, drug leads and even therapeutics. In this review, we discuss their gene superfamily classification, nomenclature, post-translational modification, structural framework, pharmacology and medical applications of the active conopeptides. We aim to give an overview of their structure and therapeutic potential. Understanding these aspects of conopeptides will help in designing more specific peptidic analogues.

The α9α10 nicotinic receptor antagonist α-conotoxin RgIA prevents neuropathic pain induced by oxaliplatin treatment.

Oxaliplatin, a third-generation diaminocyclohexane platinum drug, is widely used alone or in combination with 5-fluorouracil and leucovorin to treat metastatic colorectal, ovarian, and pancreatic cancers. Oxaliplatin long-term treatment is associated with the development of a dose-limiting painful neuropathy that dramatically impairs the patient's quality of life and therapy possibility. To study novel strategies to treat oxaliplatin-induced neuropathy, we evaluated α-conotoxin RgIA, a peptide that potently blocks the α9α10 nicotinic acetylcholine receptor (nAChR) subtype in a rat model of oxaliplatin-dependent neurotoxicity (2.4mgkg(-1) oxaliplatin intraperitoneally daily for 21days). The administration of RgIA (2 and 10nmol injected intramuscularly once a day concomitantly with oxaliplatin treatment), reduced the oxaliplatin-dependent hypersensitivity to mechanical and thermal noxious and non-noxious stimuli. Moreover, morphological modifications of L4-L5 dorsal root ganglia were significantly prevented. In the spinal cord the numerical increase of astrocyte cell density present in oxaliplatin-treated rats is partially prevented by RgIA treatment. Nevertheless, the administration of the α-conotoxin is able per se to elicit a numerical increase and a morphological activation of microglia and astrocytes in specific brain areas.

NMDA/NR2B selective antagonists in the treatment of ischemic brain injury.

Glutamate is the main excitatory neurotransmitter in the central nervous system and it plays a significant role not only in synaptic transmission but also in acute and chronic neuropathologies including stroke. Presently, four receptors for glutamate have been identified and the NMDA receptor family is the most intensively studied. A number of NMDA receptor antagonists have been developed and used for treatment of neurological diseases in patients. However, all of these drugs have been failed in clinical trials either because of intolerable side effects or lack of medical efficacy. Recently, the understanding of molecular structure of NMDA receptors has been advanced and this finding thus provides information for designing subtype-selective antagonists. Using NR2B subunit selective antagonists, ifenprodil and eliprodil, as basic structure models, second and third generation congeners have been developed. Several NR2B-selective compounds showed neuroprotective actions at doses that did not produce measurable side effects in preclinical studies. Some of NR2B subunit selective antagonists have also been tested for the treatment of ischemic brain injury. The present review describes the role of glutamate in ischemic brain injury with an emphasis on the NR2B containing NMDA receptors.

Anti-allodynic efficacy of the chi-conopeptide, Xen2174, in rats with neuropathic pain.

Xen2174 is a structural analogue of Mr1A, a chi-conopeptide recently isolated from the venom of the marine cone snail, Conus marmoreus. Although both chi-conopeptides are highly selective inhibitors of the norepinephrine transporter (NET), Xen2174 has superior chemical stability relative to Mr1A. It is well-known that tricyclic antidepressants (TCAs) are also potent NET inhibitors, but their poor selectivity relative to other monoamine transporters and various G-protein-coupled receptors, results in dose-limiting side-effects in vivo. As TCAs and the alpha(2)-adrenoceptor agonist, clonidine, have established efficacy for the relief of neuropathic pain, this study examined whether intrathecal (i.t.) Xen2174 alleviated mechanical allodynia in rats with either a chronic constriction injury of the sciatic nerve (CCI-rats) or an L5/L6 spinal-nerve injury. The anti-allodynic responses of i.t. Mr1A and i.t. morphine were also investigated in CCI-rats. Paw withdrawal thresholds were assessed using calibrated von Frey filaments. Bolus doses of i.t. Xen2174 produced dose-dependent relief of mechanical allodynia in CCI-rats and in spinal nerve-ligated rats. Dose-dependent anti-allodynic effects were also produced by i.t. bolus doses of Mr1A and morphine in CCI-rats, but a pronounced 'ceiling' effect was observed for i.t. morphine. The side-effect profiles were mild for both chi-conopeptides with an absence of sedation. Confirming the noradrenergic mechanism of action, i.t. co-administration of yohimbine (100 nmol) with Xen2174 (10 nmol) abolished Xen2174s anti-allodynic actions. Xen2174 appears to be a promising candidate for development as a novel therapeutic for i.t. administration to patients with persistent neuropathic pain.

Conotoxin Interactions with α9α10-nAChRs: Is the α9α10-Nicotinic Acetylcholine Receptor an Important Therapeutic Target for Pain Management?

The α9α10-nicotinic acetylcholine receptor (nAChR) has been implicated in pain and has been proposed to be a novel target for analgesics. However, the evidence to support the involvement of the α9α10-nAChR in pain is conflicted. This receptor was first implicated in pain with the characterisation of conotoxin Vc1.1, which is highly selective for α9α10-nAChRs and is an efficacious analgesic in chronic pain models with restorative capacities and no reported side effects. Numerous other analgesic conotoxin and non-conotoxin molecules have been subsequently characterised that also inhibit α9α10-nAChRs. However, there is evidence that α9α10-nAChR inhibition is neither necessary nor sufficient for analgesia. α9α10-nAChR-inhibiting analogues of Vc1.1 have no analgesic effects. Genetically-modified α9-nAChR knockout mice have a phenotype that is markedly different from the analgesic profile of Vc1.1 and similar conotoxins, suggesting that the conotoxin effects are largely independent of α9α10-nAChRs. Furthermore, an alternative mechanism of analgesia by Vc1.1 and other similar conotoxins involving non-canonical coupling of GABAB receptors to voltage-gated calcium channels is known. Additional incongruities regarding α9α10-nAChRs in analgesia are discussed. A more comprehensive characterisation of the role of α9α10-nAChRs in pain is crucial for understanding the analgesic action of conotoxins and for improved drug design.