Α9α10 Nicotinic Acetylcholine Receptor Research Articles

Aspartic acid mutagenesis of αO-Conotoxin GeXIVA isomers reveals arginine residues crucial for inhibition of the α9α10 nicotinic acetylcholine receptor

The two most active disulfide bond isomers of the analgesic αO-conotoxin GeXIVA, namely GeXIVA[1, 2] and GeXIVA[1, 4], were subjected to Asp-scanning mutagenesis to determine the key amino acid residues for activity at the rat α9α10 nicotinic acetylcholine receptor (nAChR). These studies revealed the key role of arginine residues for the activity of GeXIVA isomers towards the α9α10 nAChR. Based on these results, additional analogues with 2–4 mutations were designed and tested. The analogues [T1A,D14A,V28K]GeXIVA[1, 2] and [D14A,I23A,V28K]GeXIVA[1, 4] were developed and showed sub-nanomolar activity for the α9α10 nAChR with IC50 values of 0.79 and 0.38 nM. The latter analogue had exceptional selectivity for the α9α10 receptor subtype over other nAChR subtypes and can be considered as a drug candidate for further development. Molecular dynamics of receptor-ligand complexes allowed us to make deductions about the possible causes of increases in the affinity of key GeXIVA[1, 4] mutants for the α9α10 nAChR.

Tabernanthalog and ibogainalog inhibit the α7 and α9α10 nicotinic acetylcholine receptors via different mechanisms and with higher potency than the GABAA receptor and CaV2.2 channel

In this study, we have investigated the pharmacological activity and structural interaction of two novel psychoplastogens, tabernanthalog (TBG) and ibogainalog (IBG) at heterologously-expressed rat (r) and human (h) nicotinic acetylcholine receptors (nAChRs), the rα1β2γ2L γ-aminobutyric acid type A receptor (GABAAR), and the human voltage-gated N-type calcium channel (CaV2.2 channel). Both compounds inhibited the nAChRs with the following receptor selectivity: α9α10 > α7 > α3β2 ≅ α3β4, indicating that β2/β4 subunits are relatively less important for their activity. The potencies of TBG and IBG were comparable at hα7 and hα9α10 subtypes, and comparable to their rat counterparts. TBG- and IBG-induced inhibition of rα7 was ACh concentration-independent and voltage-dependent, whereas rα9α10 inhibition was ACh concentration-dependent and voltage-independent, suggesting that they interact with the α7 ion channel pore and α9α10 orthosteric ligand binding site, respectively. These results were supported by molecular docking studies showing that at the α7 model TBG forms stable interactions with luminal rings at 9′, 13′, and 16′, whereas IBG mostly interacts with the extracellular-transmembrane junction. In the α9α10 model, however, these compounds interacted with several residues from the principal (+) and complementary (–) sides in the transmitter binding site. Ibogaminalog (DM506) also interacted with a non-luminal site at α7, and one α9α10 orthosteric site. TBG and IBG inhibited the GABAAR and CaV2.2 channels with 10 to 30-fold lower potencies. In sum, we show that TBG and IBG inhibit the α7 and α9α10 nAChRs by noncompetitive and competitive mechanisms, respectively, and with higher potency than the GABAAR and CaV2.2 channel.

Rational Design of Potent α-Conotoxin PeIA Analogues with Non-Natural Amino Acids for the Inhibition of Human α9α10 Nicotinic Acetylcholine Receptors.

α-Conotoxins (α-CTxs) are structurally related peptides that antagonize nicotinic acetylcholine receptors (nAChRs), which may serve as new alternatives to opioid-based treatment for pain-related conditions. The non-natural amino acid analogues of α-CTxs have been demonstrated with improved potency compared to the native peptide. In this study, we chemically synthesized Dab/Dap-substituted analogues of α-CTx PeIA and evaluated their activity at heterologously expressed human α9α10 nAChRs. PeIA[S4Dap, S9Dap] had the most potent half-maximal inhibitory concentration (IC50) of 0.93 nM. Molecular dynamic simulations suggested that the side chain amino group of Dap4 formed additional hydrogen bonds with S168 and D169 of the receptor and Dap9 formed an extra hydrogen bond interaction with Q34, which is distinctive to PeIA. Overall, our findings provide new insights into further development of more potent analogues of α-CTxs, and PeIA[S4Dap, S9Dap] has potential as a drug candidate for the treatment of chronic neuropathic pain.

αO-Conotoxin GeXIVA[1,2] Reduced Neuropathic Pain and Changed Gene Expression in Chronic Oxaliplatin-Induced Neuropathy Mice Model.

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting painful neuropathy that occurs commonly during cancer management, which often leads to the discontinuation of medication. Previous studies suggest that the α9α10 nicotinic acetylcholine receptor (nAChR)-specific antagonist αO-conotoxin GeXIVA[1,2] is effective in CIPN models; however, the related mechanisms remain unclear. Here, we analyzed the preventive effect of GeXIVA[1,2] on neuropathic pain in the long-term oxaliplatin injection-induced CIPN model. At the end of treatment, lumbar (L4-L6) spinal cord was extracted, and RNA sequencing and bioinformatic analysis were performed to investigate the potential genes and pathways related to CIPN and GeXIVA[1,2]. GeXIVA[1,2] inhibited the development of mechanical allodynia induced by chronic oxaliplatin treatment. Repeated injections of GeXIVA[1,2] for 3 weeks had no effect on the mice's normal pain threshold or locomotor activity and anxiety-like behavior, as evaluated in the open field test (OFT) and elevated plus maze (EPM). Our RNA sequencing results identified 209 differentially expressed genes (DEGs) in the CIPN model, and simultaneously injecting GeXIVA[1,2] with oxaliplatin altered 53 of the identified DEGs. These reverted genes were significantly enriched in immune-related pathways represented by the cytokine-cytokine receptor interaction pathway. Our findings suggest that GeXIVA[1,2] could be a potential therapeutic compound for chronic oxaliplatin-induced CIPN management.

Structure-Activity Relationships of Alanine Scan Mutants αO-Conotoxins GeXIVA[1,2] and GeXIVA[1,4

αO-Conotoxin GeXIVA is a selective α9α10 nicotinic acetylcholine receptor (nAChR) inhibitor displaying two disulfide bonds that can form three isomers. The bead (GeXIVA[1,2]) and ribbon (GeXIVA[1,4]) isomers possess the highest activity on rat and human α9α10 nAChRs. However, the molecular mechanism by which they inhibit the α9α10 nAChR is unknown. Here, an alanine scan of GeXIVA was used to elucidate key interactions between the peptides and the α9α10 nAChR. The majority of GeXIVA[1,2] analogues preserved affinity at α9α10 nAChR, but [R17A]GeXIVA[1,2] enhanced selectivity on the α9α10 nAChR. The I23A replacement of GeXIVA[1,4] increased activity at both rat and human α9α10 nAChRs by 10-fold. Surprisingly, these results do not support the molecular model of an interaction in the orthosteric binding site proposed previously, but rather may involve allosteric coupling with the voltage-sensitive domain of the α9α10 nAChR. These results could help to guide further development of GeXIVA analogues as analgesics.

DM506 (3-Methyl-1,2,3,4,5,6-hexahydroazepino[4,5-b]indole fumarate), a Novel Derivative of Ibogamine, Inhibits α7 and α9α10 Nicotinic Acetylcholine Receptors by Different Allosteric Mechanisms.

The main objective of this study was to determine the pharmacological activity and molecular mechanism of action of DM506 (3-methyl-1,2,3,4,5,6-hexahydroazepino[4,5-b]indole fumarate), a novel ibogamine derivative, at different nicotinic acetylcholine receptor (nAChR) subtypes. The functional results showed that DM506 neither activates nor potentiates but inhibits ACh-evoked currents at each rat nAChR subtype in a non-competitive manner. The receptor selectivity for DM506 inhibition follows the sequence: α9α10 (IC50 = 5.1 ± 0.3 μM) ≅ α7β2 (5.6 ± 0.2 μM) ∼ α7 (6.4 ± 0.5 μM) > α6/α3β2β3 (25 ± 1 μM) > α4β2 (62 ± 4 μM) ≅ α3β4 (70 ± 5 μM). No significance differences in DM506 potency were observed between rat and human α7 and α9α10 nAChRs. These results also indicated that the β2 subunit is not involved or is less relevant in the activity of DM506 at the α7β2 nAChR. DM506 inhibits the α7 and α9α10 nAChRs in a voltage-dependent and voltage-independent manner, respectively. Molecular docking and molecular dynamics studies showed that DM506 forms stable interactions with a putative site located in the α7 cytoplasmic domain and with two intersubunit sites in the extracellular-transmembrane junction of the α9α10 nAChR, one located in the α10(+)/α10(─) interface and another in the α10(+)/α9(─) interface. This study shows for the first time that DM506 inhibits both α9α10 and α7 nAChR subtypes by novel allosteric mechanisms likely involving modulation of the extracellular-transmembrane domain junction and cytoplasmic domain, respectively, but not by direct competitive antagonism or open channel block.

Mechanism of Action and Structure-Activity Relationship of α-Conotoxin Mr1.1 at the Human α9α10 Nicotinic Acetylcholine Receptor.

α-Conotoxins (α-CTxs) can selectively target nicotinic acetylcholine receptors (nAChRs) and are important drug leads for the treatment of cancer, chronic pain, and neuralgia. Here, we chemically synthesized a formerly defined rat α7 nAChR targeting α-CTx Mr1.1 and evaluated its activity at human nAChRs. Mr1.1 was most potent at the human (h) α9α10 nAChR with a half-maximal inhibitory concentration (IC50) of 92.0 nM. Molecular dynamic simulations suggested that Mr1.1 favorably binds at the α10(+)α9(-) and α9(+)α9(-) sites via hydrogen bonds and salt bridges, stabilizing the channel in a closed conformation. Although Mr1.1 and another antagonist, α-CTx Vc1.1 share high sequence similarity and disulfide-bond framework, Mr1.1 has distinct orientations at hα9α10. Based on the Mr1.1-hα9α10 model, analogues were generated, and the more potent Mr1.1[S4Dap], antagonized hα9α10 with an IC50 of 4.0 nM. Furthermore, Mr1.1[S4Dap] displayed analgesic activity in the rat chronic constriction injury (CCI) pain model and therefore presents a promising drug candidate.

Discovery and Structural and Functional Characterization of a Novel A-Superfamily Conotoxin Targeting α9α10 Nicotinic Acetylcholine Receptor.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels widely distributed in the central peripheral nervous system and muscles which participate in rapid synaptic transmission. The α9α10 nAChR is an acetylcholine receptor subtype and is involved in chronic pain. In the present study, a new A-superfamily conotoxin Bt14.12 cloned from Conus betulinus was found to selectively inhibit α9α10 nAChRs with an IC50 of 62.3 nM. Unlike α-conotoxins and other A-superfamily conotoxins, Bt14.12 contains a four Cys (C-C-C-C) framework with a unique disulfide bond connection "C1-C4, C2-C3". The structure-activity studies of Bt14.12 demonstrate that all amino acid residues contribute to its potency. Interestingly, mutation experiments show that the deletion of Asp2 or the addition of three Arg residues at the N-terminus of Bt14.12 significantly enhances its inhibitory activity (IC50 is 21.9 nM or 12.7 nM, respectively) by 2- or 4-fold compared to the wild-type Bt14.12. The NMR structure of Bt14.12 shows that it contains α-helix- and β-turn-like elements, and further computational modelings of the interaction between Bt14.12 and the α9α10 nAChR demonstrate that Bt14.12 possesses a distinctive mode of action and displays a different structure-activity relationship from known α9α10 nAChR targeting α-conotoxins. Our findings provide a novel conotoxin that potently targets α9α10 nAChRs and a new motif for designing potent inhibitors against α9α10 nAChRs.

Novel αO-conotoxin GeXIVA[1,2] Nonaddictive Analgesic with Pharmacokinetic Modelling-Based Mechanistic Assessment.

αO-conotoxin GeXIVA[1,2] was isolated in our laboratory from Conus generalis, a snail native to the South China Sea, and is a novel, nonaddictive, intramuscularly administered analgesic targeting the α9α10 nicotinic acetylcholine receptor (nAChR) with an IC50 of 4.61 nM. However, its pharmacokinetics and related mechanisms underlying the analgesic effect remain unknown. Herein, pharmacokinetics and multiscale pharmacokinetic modelling in animals were subjected systematically to mechanistic assessment for αO-conotoxin GeXIVA[1,2]. The intramuscular bioavailability in rats and dogs was 11.47% and 13.37%, respectively. The plasma exposure of GeXIVA[1,2] increased proportionally with the experimental dose. The plasma protein binding of GeXIVA[1,2] differed between the tested animal species. The one-compartment model with the first-order absorption population pharmacokinetics model predicted doses for humans with bodyweight as the covariant. The pharmacokinetics-pharmacodynamics relationships were characterized using an inhibitory loss indirect response model with an effect compartment. Model simulations have provided potential mechanistic insights into the analgesic effects of GeXIVA[1,2] by inhibiting certain endogenous substances, which may be a key biomarker. This report is the first concerning the pharmacokinetics of GeXIVA[1,2] and its potential analgesic mechanisms based on a top-down modelling approach.

The α9α10 nicotinic acetylcholine receptor: a compelling drug target for hearing loss?

ABSTRACT Introduction Hearing loss is a major health problem, impacting education, communication, interpersonal relationships, and mental health. Drugs that prevent or restore hearing are lacking and hence novel drug targets are sought. There is the possibility of targeting the α9α10 nicotinic acetylcholine receptor (nAChR) in the prevention of noise-induced, hidden hearing loss and presbycusis. This receptor mediates synaptic transmission between medial olivocochlear efferent fibers and cochlear outer hair cells. This target is key since enhanced olivocochlear activity prevents noise-induced hearing loss and delays presbycusis. Areas covered The work examines the α9α10 nicotinic acetylcholine receptor (nAChR), its role in noise-induced, hidden hearing loss and presbycusis and the possibility of targeting. Data has been searched in Pubmed, the World Report on Hearing from the World Health Organization and the Global Burden of Disease Study 2019. Expert opinion The design of positive allosteric modulators of α9α10 nAChRs is proposed because of the advantage of reinforcing the medial olivocochlear (MOC)-hair cell endogenous neurotransmission without directly stimulating the target receptors, therefore avoiding receptor desensitization and reduced efficacy. The time is right for the discovery and development of α9α10 nAChRs targeting agents and high throughput screening assays will support this.

ɑO-Conotoxin GeXIVA isomers modulate N-type calcium (CaV 2.2) channels and inwardly-rectifying potassium (GIRK) channels via GABAB receptor activation.

αO-Conotoxin GeXIVA is a 28 amino acid peptide derived from the venom of the marine snail Conus generalis. The presence of four cysteine residues in the structure of GeXIVA allows it to have three different disulfide isomers, that is, the globular, ribbon or bead isomer. All three isomers are active at α9α10 nicotinic acetylcholine receptors, with the bead isomer, GeXIVA[1,2], being the most potent and exhibiting analgesic activity in animal models of neuropathic pain. The original report of GeXIVA activity failed to observe any effect of the isomers on high voltage-activated (HVA) calcium channel currents in rat dorsal root ganglion (DRG) neurons. In this study, we report, for the first time, the activity of globular GeXIVA[1,3] at G protein-coupled GABAB receptors (GABAB R) inhibiting HVA N-type calcium (Cav2.2) channels and reducing membrane excitability in mouse DRG neurons. The inhibition of HVA Ba2+ currents and neuroexcitability by GeXIVA[1,3] was partially reversed by the selective GABAB R antagonist CGP 55845. In transfected HEK293T cells co-expressing human GABAB R1 and R2subunits and Cav2.2 channels, both GeXIVA[1,3] and GeXIVA[1,4] inhibited depolarization-activated Ba2+ currents mediated by Cav2.2 channels, whereas GeXIVA[1,2] had no effect. The effects of three cyclized GeXIVA[1,4] ribbon isomers were also tested, with cGeXIVA GAG being the most potent at human GABAB R-coupled Cav2.2 channels. Interestingly, globular GeXIVA[1,3] also reversibly potentiated inwardly-rectifying K+ currents mediated by human GIRK1/2 channels co-expressed with GABAB R in HEK293T cells. This study highlights GABAB R as a potentially important receptor target for the activity of αO-conotoxin GeXIVA to mediate analgesia.

Discovery of Methylene Thioacetal-Incorporated α-RgIA Analogues as Potent and Stable Antagonists of the Human α9α10 Nicotinic Acetylcholine Receptor for the Treatment of Neuropathic Pain.

α9-Containing nicotinic acetylcholine receptors (nAChRs) are key targets for the treatment of neuropathic pain. α-Conotoxin RgIA4 is a peptide antagonist of human α9α10 nAChRs with high selectivity. However, structural rearrangement reveals a potential liability for clinical applications. We herein report our designer RgIA analogues stabilized by methylene thioacetal as nonopioid analgesic agents. We demonstrate that replacing disulfide loop I [CysI-CysIII] with methylene thioacetal in the RgIA skeleton results in activity loss, whereas substitution of loop II [CysII-CysIV] can be accommodated. The lead molecule, RgIA-5524, exhibits highly selective inhibition of α9α10 nAChRs with an IC50 of 0.9 nM and much reduced degradation in human serum. In vivo studies showed that RgIA-5524 relieves chemotherapy-induced neuropathic pain in wild type but not α9 knockout mouse models, demonstrating that α9-containing nAChRs are necessary for the therapeutic effects. This work highlights the application of methylene thioacetal as a disulfide surrogate in conotoxin-based, disulfide-rich peptide drugs.

Loss of Choline Agonism in the Inner Ear Hair Cell Nicotinic Acetylcholine Receptor Linked to the α10 Subunit.

The α9α10 nicotinic acetylcholine receptor (nAChR) plays a fundamental role in inner ear physiology. It mediates synaptic transmission between efferent olivocochlear fibers that descend from the brainstem and hair cells of the auditory sensory epithelium. The α9 and α10 subunits have undergone a distinct evolutionary history within the family of nAChRs. Predominantly in mammalian vertebrates, the α9α10 receptor has accumulated changes at the protein level that may ultimately relate to the evolutionary history of the mammalian hearing organ. In the present work, we investigated the responses of α9α10 nAChRs to choline, the metabolite of acetylcholine degradation at the synaptic cleft. Whereas choline is a full agonist of chicken α9α10 receptors it is a partial agonist of the rat receptor. Making use of the expression of α9α10 heterologous receptors, encompassing wild-type, heteromeric, homomeric, mutant, chimeric, and hybrid receptors, and in silico molecular docking, we establish that the mammalian (rat) α10 nAChR subunit underscores the reduced efficacy of choline. Moreover, we show that whereas the complementary face of the α10 subunit does not play an important role in the activation of the receptor by ACh, it is strictly required for choline responses. Thus, we propose that the evolutionary changes acquired in the mammalian α9α10 nAChR resulted in the loss of choline acting as a full agonist at the efferent synapse, without affecting the triggering of ACh responses. This may have accompanied the fine-tuning of hair cell post-synaptic responses to the high-frequency activity of efferent medial olivocochlear fibers that modulate the cochlear amplifier.

Unraveling the Molecular Players at the Cholinergic Efferent Synapse of the Zebrafish Lateral Line.

The lateral line (LL) is a sensory system that allows fish and amphibians to detect water currents. LL responsiveness is modulated by efferent neurons that aid in distinguishing between external and self-generated stimuli, maintaining sensitivity to relevant cues. One component of the efferent system is cholinergic, the activation of which inhibits afferent activity. LL hair cells (HCs) share structural, functional, and molecular similarities with those of the cochlea, making them a popular model for studying human hearing and balance disorders. Because of these commonalities, one could propose that the receptor at the LL efferent synapse is a α9α10 nicotinic acetylcholine receptor (nAChR). However, the identities of the molecular players underlying ACh-mediated inhibition in the LL remain unknown. Surprisingly, through the analysis of single-cell expression studies and in situ hybridization, we describe that α9, but not the α10, subunits are enriched in zebrafish HCs. Moreover, the heterologous expression of zebrafish α9 subunits indicates that homomeric receptors are functional and exhibit robust ACh-gated currents blocked by α-bungarotoxin and strychnine. In addition, in vivo Ca2+ imaging on mechanically stimulated zebrafish LL HCs show that ACh elicits a decrease in evoked Ca2+ signals, regardless of HC polarity. This effect is blocked by both α-bungarotoxin and apamin, indicating coupling of ACh-mediated effects to small-conductance Ca2+-activated potassium (SKs) channels. Our results indicate that an α9-containing (α9*) nAChR operates at the zebrafish LL efferent synapse. Moreover, the activation of α9* nAChRs most likely leads to LL HC hyperpolarization served by SK channels.SIGNIFICANCE STATEMENT The fish lateral line (LL) mechanosensory system shares structural, functional, and molecular similarities with those of the mammalian cochlea. Thus, it has become an accessible model for studying human hearing and balance disorders. However, the molecular players serving efferent control of LL hair cell (HC) activity have not been identified. Here we demonstrate that, different from the hearing organ of vertebrate species, a nicotinic acetylcholine receptor composed only of α9 subunits operates at the LL efferent synapse. Activation of α9-containing receptors leads to LL HC hyperpolarization because of the opening of small-conductance Ca2+-activated potassium channels. These results will further aid in the interpretation of data obtained from LL HCs as a model for cochlear HCs.

(E)-3-Furan-2-yl-N-p-tolyl-acrylamide and its Derivative DM489 Decrease Neuropathic Pain in Mice Predominantly by α7 Nicotinic Acetylcholine Receptor Potentiation.

The main objective of this study was to determine whether (E)-3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2) and its structural derivative DM489 produce anti-neuropathic pain activity using the streptozotocin (STZ)- and oxaliplatin-induced neuropathic pain animal models. To assess possible mechanisms of action, the pharmacological activity of these compounds was determined at α7 and α9α10 nicotinic acetylcholine receptors (nAChRs) and CaV2.2 channels expressed alone or coexpressed with G protein-coupled GABAB receptors. The animal results indicated that a single dose of 3 mg/kg PAM-2 or DM489 decreases STZ-induced neuropathic pain in mice, and chemotherapy-induced neuropathic pain is decreased by PAM-2 (3 mg/kg) and DM489 (10 mg/kg). The observed anti-neuropathic pain activity was inhibited by the α7-selective antagonist methyllycaconitine. The coadministration of oxaliplatin with an inactive dose (1 mg/kg) of PAM-2 decreased the development of neuropathic pain after 14, but not 7, days of cotreatment. The electrophysiological results indicated that PAM-2 potentiates human (h) and rat (r) α7 nAChRs with 2-7 times higher potency than that for hCaV2.2 channel inhibition and an even greater difference compared to that for rα9α10 nAChR inhibition. These results support the notion that α7 nAChR potentiation is likely the predominant molecular mechanism underlying the observed anti-nociceptive pain activity of these compounds.

Hair cell α9α10 nicotinic acetylcholine receptor functional expression regulated by ligand binding and deafness gene products

Auditory hair cells receive olivocochlear efferent innervation, which refines tonotopic mapping, improves sound discrimination, and mitigates acoustic trauma. The olivocochlear synapse involves α9α10 nicotinic acetylcholine receptors (nAChRs), which assemble in hair cells only coincident with cholinergic innervation and do not express in recombinant mammalian cell lines. Here, genome-wide screening determined that assembly and surface expression of α9α10 require ligand binding. Ion channel function additionally demands an auxiliary subunit, which can be transmembrane inner ear (TMIE) or TMEM132e. Both of these single-pass transmembrane proteins are enriched in hair cells and underlie nonsyndromic human deafness. Inner hair cells from TMIE mutant mice show altered postsynaptic α9α10 function and retain α9α10-mediated transmission beyond the second postnatal week associated with abnormally persistent cholinergic innervation. Collectively, this study provides a mechanism to link cholinergic input with α9α10 assembly, identifies unexpected functions for human deafness genes TMIE/TMEM132e, and enables drug discovery for this elusive nAChR implicated in prevalent auditory disorders.

Development of Conformationally Constrained α-RgIA Analogues as Stable Peptide Antagonists of Human α9α10 Nicotinic Acetylcholine Receptors.

Non-opioid therapeutics for the treatment of neuropathic pain are urgently needed to address the ongoing opioid crisis. Peptides from cone snail venoms have served as invaluable molecules to target key pain-related receptors but can suffer from unfavorable physicochemical properties, which limit their therapeutic potential. In this work, we developed conformationally constrained α-RgIA analogues with high potency, receptor selectivity, and enhanced human serum stability to target the human α9α10 nicotinic acetylcholine receptor. The key lactam linkage introduced in α-RgIA fixed the favored globular conformation and suppressed disulfide scrambling. The NMR structure of the macrocyclic peptide overlays well with that of α-RgIA4, demonstrating that the cyclization does not perturb the overall conformation of backbone and key side-chain residues. Finally, a molecular docking model was used to rationalize the selective binding between a macrocyclic analogue and the α9α10 nicotinic acetylcholine receptor. These conformationally constrained antagonists are therefore promising candidates for antinociceptive therapeutic intervention.

Critical residue properties for potency and selectivity of α-Conotoxin RgIA towards α9α10 nicotinic acetylcholine receptors

The α9α10 nicotinic acetylcholine receptor (nAChR) has been characterized as an effective anti-pain target that functions through a non-opioid mechanism. However, as a pentameric ion channel comprised of two different subunits, the specific targeting of α9α10 nAChRs has proven challenging. Previously the 13-amino-acid peptide, RgIA, was shown to block α9α10 nAChRs with high potency and specificity. This peptide, characterized from the venom of the carnivorous marine snail, Conus regius, produced analgesia in several rodent models of chronic pain. Despite promising pre-clinical data in behavioral assays, the number of specific α9α10 nAChR antagonists remains small and the physiological mechanisms of analgesia remain cryptic. In this study, we implement amino-acid substitutions to definitively characterize the chemical properties of RgIA that contribute to its activity against α9α10 nAChRs. Using this mutational approach, we determined the vital role of biochemical side-chain properties and amino acids in the second loop that are amenable to substitutions to further engineer next-generation analogs for the blockade of α9α10 nAChRs.

Coronaridine congeners decrease neuropathic pain in mice and inhibit α9α10 nicotinic acetylcholine receptors and CaV2.2 channels

The primary aim of this study was to determine the anti-neuropathic activity of (±)-18-methoxycoronaridine [(±)-18-MC] and (+)-catharanthine in mice by using the oxaliplatin-induced neuropathic pain paradigm and cold plate test. The results showed that both coronaridine congeners induce anti-neuropathic pain activity at a dose of 72 mg/kg (per os), whereas a lower dose (36 mg/kg) of (+)-catharanthine decreased the progress of oxaliplatin-induced neuropathic pain. To determine the underlying molecular mechanism, electrophysiological recordings were performed on α9α10, α3β4, and α4β2 nAChRs as well as voltage-gated calcium (CaV2.2) channels modulated by G protein-coupled γ-aminobutyric acid type B receptors (GABABRs). The results showed that (±)-18-MC and (+)-catharanthine competitively inhibit α9α10 nAChRs with potencies higher than that at α3β4 and α4β2 nAChRs and directly block CaV2.2 channels without activating GABABRs. Considering the potency of the coronaridine congeners at Cav2.2 channels and α9α10 nAChRs, and the calculated brain concentration of (+)-catharanthine, it is plausible that the observed anti-neuropathic pain effects are mediated by peripheral and central mechanisms involving the inhibition of α9α10 nAChRs and/or CaV2.2 channels.

Dimerization of α-Conotoxins as a Strategy to Enhance the Inhibition of the Human α7 and α9α10 Nicotinic Acetylcholine Receptors.

The affinity of α-conotoxins, a class of nicotinic acetylcholine receptor (nAChR) peptide inhibitors, can be enhanced by dendrimerization. It has been hypothesized that this improvement arose from simultaneous binding of the α-conotoxins to several spatially adjacent sites. We here engineered several α-conotoxin dimers using a linker length compatible between neighboring binding sites on the same receptor. Remarkably, the dimer of α-conotoxin PeIA compared to the monomer displayed an increase in potency by 11-fold (IC50 = 1.9 nM) for the human α9α10 nAChR. The dimerization of α-conotoxin RgIA# resulted in a dual inhibitor that targets both α9α10 and α7 nAChR subtypes with an IC50 = ∼50 nM. The RgIA# dimer is therapeutically interesting because it is the first dual inhibitor that potently and selectively inhibits these two nAChR subtypes, which are both involved in the etiology of several cancers. We propose that the dimerization of α-conotoxins is a simpler and efficient alternative strategy to dendrimers for enhancing the activity of α-conotoxins.