Chemical Synthesis and Insecticidal Activity Research Based on α-Conotoxins.

The escalating resistance of agricultural pests to chemical insecticides necessitates the development of novel, efficient, and safe biological insecticides. Conus quercinus, a vermivorous cone snail, yields a crude venom rich in peptides for marine worm predation. This study screened six α-conotoxins with insecticidal potential from a previously constructed transcriptome database of C. quercinus, characterized by two disulfide bonds. These conotoxins were derived via solid-phase peptide synthesis (SPPS) and folded using two-step iodine oxidation for further insecticidal activity validation, such as CCK-8 assay and insect bioassay. The final results confirmed the insecticidal activities of the six α-conotoxins, with Qc1.15 and Qc1.18 exhibiting high insecticidal activity. In addition, structural analysis via homology modeling and functional insights from molecular docking offer a preliminary look into their potential insecticidal mechanisms. In summary, this study provides essential references and foundations for developing novel insecticides.

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.

Fluorescent α-Conotoxin [Q1G, ΔR14]LvIB Identifies the Distribution of α7 Nicotinic Acetylcholine Receptor in the Rat Brain.

α7 nicotinic acetylcholine receptors (nAChRs) are mainly distributed in the central nervous system (CNS), including the hippocampus, striatum, and cortex of the brain. The α7 nAChR has high Ca2+ permeability and can be quickly activated and desensitized, and is closely related to Alzheimer's disease (AD), epilepsy, schizophrenia, lung cancer, Parkinson's disease (PD), inflammation, and other diseases. α-conotoxins from marine cone snail venom are typically short, disulfide-rich neuropeptides targeting nAChRs and can distinguish various subtypes, providing vital pharmacological tools for the functional research of nAChRs. [Q1G, ΔR14]LvΙB is a rat α7 nAChRs selective antagonist, modified from α-conotoxin LvΙB. In this study, we utilized three types of fluorescein after N-Hydroxy succinimide (NHS) activation treatment: 6-TAMRA-SE, Cy3 NHS, and BODIPY-FL NHS, labeling the N-Terminal of [Q1G, ΔR14]LvΙB under weak alkaline conditions, obtaining three fluorescent analogs: LvIB-R, LvIB-C, and LvIB-B, respectively. The potency of [Q1G, ΔR14]LvΙB fluorescent analogs was evaluated at rat α7 nAChRs expressed in Xenopus laevis oocytes. Using a two-electrode voltage clamp (TEVC), the half-maximal inhibitory concentration (IC50) values of LvIB-R, LvIB-C, and LvIB-B were 643.3 nM, 298.0 nM, and 186.9 nM, respectively. The stability of cerebrospinal fluid analysis showed that after incubation for 12 h, the retention rates of the three fluorescent analogs were 52.2%, 22.1%, and 0%, respectively. [Q1G, ΔR14]LvΙB fluorescent analogs were applied to explore the distribution of α7 nAChRs in the hippocampus and striatum of rat brain tissue and it was found that Cy3- and BODIPY FL-labeled [Q1G, ΔR14]LvΙB exhibited better imaging characteristics than 6-TAMARA-. It was also found that α7 nAChRs are widely distributed in the cerebral cortex and cerebellar lobules. Taking into account potency, imaging, and stability, [Q1G, ΔR14]LvΙB -BODIPY FL is an ideal pharmacological tool to investigate the tissue distribution and function of α7 nAChRs. Our findings not only provide a foundation for the development of conotoxins as visual pharmacological probes, but also demonstrate the distribution of α7 nAChRs in the rat brain.

Structural analysis of a U-superfamily conotoxin containing a mini-granulin fold: Insights into key features that distinguish between the ICK and granulin folds.

We are entering an exciting time in structural biology where artificial intelligence can be used to predict protein structures with greater accuracy than ever before. Extending this level of accuracy to the predictions of disulfide-rich peptide structures is likely to be more challenging, at least in the short term, given the tight packing of cysteine residues and the numerous ways that the disulfide bonds can potentially be linked. It has been previously shown in many cases that several disulfide bond connectivities can be accommodated by a single set of NMR-derived structural data without significant violations. Disulfide-rich peptides are prevalent throughout nature, and arguably the most well-known are those present in venoms from organisms such as cone snails. Here, we have determined the first three-dimensional structure and disulfide connectivity of a U-superfamily cone snail venom peptide, TxVIIB. TxVIIB has a VI/VII cysteine framework that is generally associated with an inhibitor cystine knot (ICK) fold; however, AlphaFold predicted that the peptide adopts a mini-granulin fold with a granulin disulfide connectivity. Our experimental studies using NMR spectroscopy and orthogonal protection of cysteine residues indicate that TxVIIB indeed adopts a mini-granulin fold but with the ICK disulfide connectivity. Our findings provide structural insight into the underlying features that govern formation of the mini-granulin fold rather than the ICK fold and will provide fundamental information for prediction algorithms, as the subtle complexity of disulfide isomers may be not adequately addressed by the current prediction algorithms.

Engineering Enhanced Antimicrobial Properties in α-Conotoxin RgIA through D-Type Amino Acid Substitution and Incorporation of Lysine and Leucine Residues.

Antimicrobial peptides (AMPs), acknowledged as host defense peptides, constitute a category of predominant cationic peptides prevalent in diverse life forms. This study explored the antibacterial activity of α-conotoxin RgIA, and to enhance its stability and efficacy, D-amino acid substitution was employed, resulting in the synthesis of nine RgIA mutant analogs. Results revealed that several modified RgIA mutants displayed inhibitory efficacy against various pathogenic bacteria and fungi, including Candida tropicalis and Escherichia coli. Mechanistic investigations elucidated that these polypeptides achieved antibacterial effects through the disruption of bacterial cell membranes. The study further assessed the designed peptides' hemolytic activity, cytotoxicity, and safety. Mutants with antibacterial activity exhibited lower hemolytic activity and cytotoxicity, with Pep 8 demonstrating favorable safety in mice. RgIA mutants incorporating D-amino acids exhibited notable stability and adaptability, sustaining antibacterial properties across diverse environmental conditions. This research underscores the potential of the peptide to advance innovative oral antibiotics, offering a novel approach to address bacterial infections.

Predatory and Defensive Strategies in Cone Snails.

Cone snails are carnivorous marine animals that prey on fish (piscivorous), worms (vermivorous), or other mollusks (molluscivorous). They produce a complex venom mostly made of disulfide-rich conotoxins and conopeptides in a compartmentalized venom gland. The pharmacology of cone snail venom has been increasingly investigated over more than half a century. The rising interest in cone snails was initiated by the surprising high human lethality rate caused by the defensive stings of some species. Although a vast amount of information has been uncovered on their venom composition, pharmacological targets, and mode of action of conotoxins, the venom-ecology relationships are still poorly understood for many lineages. This is especially important given the relatively recent discovery that some species can use different venoms to achieve rapid prey capture and efficient deterrence of aggressors. Indeed, via an unknown mechanism, only a selected subset of conotoxins is injected depending on the intended purpose. Some of these remarkable venom variations have been characterized, often using a combination of mass spectrometry and transcriptomic methods. In this review, we present the current knowledge on such specific predatory and defensive venoms gathered from sixteen different cone snail species that belong to eight subgenera: Pionoconus, Chelyconus, Gastridium, Cylinder, Conus, Stephanoconus, Rhizoconus, and Vituliconus. Further studies are needed to help close the gap in our understanding of the evolved ecological roles of many cone snail venom peptides.

Large-Scale Molecular Dynamics Simulation Based on Heterogeneous Many-Core Architecture.

As the application of molecular dynamics (MD) simulations continues to evolve, the demand for accelerating large-scale simulation systems and handling of enormous simulation tasks is steadily increasing. We propose a parallel acceleration method for large-scale MD simulations based on Sunway heterogeneous many-core processors. This method integrates task scheduling, simulation calculations, and data storage, effectively tackling issues related to large-scale simulations and numerous simulation tasks. The task scheduling strategy flexibly handles tasks on various scales and enables parallel execution of multiple tasks. During the simulation calculations, we ported GROMACS to the Sunway architecture and accelerated the calculation of short-range forces through a heterogeneous processor. Our method achieves approximately 10-fold acceleration and 90% scalability when executing a single simulation task. When handling numerous simulation tasks, our method achieves parallel execution of all of the tasks with 90% scalability. By employing our method, we carried out 50 ns simulations on over 3000 distinct conotoxin structures individually within just 5 h. Additionally, we evaluated more than 200 protein-ligand complexes, and the simulation efficiency significantly exceeded that of midsized to small GPU clusters.

Dual Antagonism of α9α10 nAChR and GABAB Receptor-Coupled CaV2.2 Channels by an Analgesic αO-Conotoxin Analogue.

Pain severely affects the physical and mental health of patients. The need to develop nonopioid analgesic drugs to meet medical demands is urgent. In this study, we designed a truncated analogue of αO-conotoxin, named GeX-2, based on disulfide-bond deletion and sequence truncation. GeX-2 retained the potency of its parent peptide at the human α9α10 nAChR and exhibited potent inhibitory activity at CaV2.2 channels via activation of the GABAB receptor (GABABR). Importantly, GeX-2 significantly alleviated pain in the rat model of chronic constriction injury. The dual inhibition of GeX-2 at both α9α10 nAChRs and CaV2.2 channels is speculated to synergistically mediate the potent analgesic effects. Results from site-directed mutagenesis assay and computational modeling suggest that GeX-2 preferentially interacts with the α10(+)α10(-) binding site of α9α10 nAChR and favorably binds to the top region of the GABABR2 subunit. The study offers vital insights into the molecular action mechanism of GeX-2, demonstrating its potential as a novel nonopioid analgesic.

Cysteine-free cone snail venom peptides: Classification of precursor proteins and identification of mature peptides.

The cysteine-free acyclic peptides present in marine cone snail venom have been much less investigated than their disulfide bonded counterparts. Precursor protein sequences derived from transcriptomic data, together with mass spectrometric fragmentation patterns for peptides present in venom duct tissue extracts, permit the identification of mature peptides. Twelve distinct gene superfamiles have been identified with precursor lengths between 64 and 158 residues. In the case of Conus monile, three distinct mature peptides have been identified, arising from two distinct protein precursors. Mature acyclic peptides are often post-translationally modified, with C-terminus amidation, a feature characteristic of neuropeptides. In the present study, 20 acyclic peptides from Conus monile and Conus betulinus were identified. The common modifications of C-terminus amidation, gamma carboxylation of glutamic acid (E to ϒ), N-terminus conversion of Gln (Q) to a pyroglutamyl residue (Z), and hydroxylation of Pro (P) to Hyp (O) are observed in one or more peptides identified in this study. Proteolytic trimming of sequences by cleavage at the C-terminus of Asn (N) residues is established. The presence of an asparagine endopeptidase is strengthened by the identification of legumain-like sequences in the transcriptome assemblies from diverse Conus species. Such sequences may be expected to have a cleavage specificity at Asn-Xxx peptide bonds.

Computational Design of α-Conotoxins to Target Specific Nicotinic Acetylcholine Receptor Subtypes.

Nicotinic acetylcholine receptors (nAChRs) are drug targets for neurological diseases and disorders, but selective targeting of the large number of nAChR subtypes is challenging. Marine cone snail α-conotoxins are potent blockers of nAChRs and some have been engineered to achieve subtype selectivity. This engineering effort would benefit from rapid computational methods able to predict mutational energies, but current approaches typically require high-resolution experimental structures, which are not widely available for α-conotoxin complexes. Herein, five mutational energy prediction methods were benchmarked using crystallographic and mutational data on two acetylcholine binding protein/α-conotoxin systems. Molecular models were developed for six nAChR subtypes in complex with five α-conotoxins that were studied through 150 substitutions. The best method was a combination of FoldX and molecular dynamics simulations, resulting in a predictive Matthews Correlation Coefficient (MCC) of 0.68 (85 % accuracy). Novel α-conotoxin mutants designed using this method were successfully validated by experimental assay with improved pharmaceutical properties. This work paves the way for the rapid design of subtype-specific nAChR ligands and potentially accelerated drug development.

α-Conotoxin recombinant protein ImI-AFP3 efficiently inhibits the growth and migration of lung cancer cells.

α-Conotoxin ImI is a selective antagonist of alpha7 nicotinic acetylcholine receptor (α7 nAChR) that is involved in cancer development. Human alpha fetoprotein domain 3 (AFP3) is a prototype of anticancer agents. In an effort to design drugs for anticancer treatments, we fused the ImI peptide to AFP3 as a fusion protein for testing. The fusion protein (ImI-AFP3) was highly expressed in the insect Bac-to-Bac system. The purified fusion protein was found to have improved anticancer activity and synergized with the drug gefitinib to inhibit the growth and migration of A549 and NCI-H1299 lung cancer cells. Our data have demonstrated that the recombinant protein ImI-AFP3 is a promising candidate for drug development to suppress lung cancer cell growth, especially to suppress hepatoid adenocarcinoma of the lung (HAL) cell growth.

Enhancing Stability and Albumin Binding Efficiency of α-Conotoxin GI through Fatty Acid Modification.

α-Conotoxin GI is a competitive blocker of muscle-type acetylcholine receptors and holds the potential for being developed as a molecular probe or a lead compound for drug discovery. In this study, four fatty acid-modified α-conotoxin GI analogues of different lengths were synthesized by using a fatty acid modification strategy. Then, we performed a series of in vitro stability assays, albumin binding assays, and pharmacological activity assays to evaluate these modified mutants. The experimental results showed that the presence of fatty acids significantly enhanced the in vitro stability and albumin binding ability of α-conotoxin GI and that this effect was proportional to the length of the fatty acids used. Pharmacological activity tests showed that the modified mutants maintained a good acetylcholine receptor antagonistic activity. The present study shows that fatty acid modification can be an effective strategy to significantly improve conotoxin stability and albumin binding efficiency while maintaining the original targeting ion channel activity.

Conotoxin Prediction: New Features to Increase Prediction Accuracy.

Conotoxins are toxic, disulfide-bond-rich peptides from cone snail venom that target a wide range of receptors and ion channels with multiple pathophysiological effects. Conotoxins have extraordinary potential for medical therapeutics that include cancer, microbial infections, epilepsy, autoimmune diseases, neurological conditions, and cardiovascular disorders. Despite the potential for these compounds in novel therapeutic treatment development, the process of identifying and characterizing the toxicities of conotoxins is difficult, costly, and time-consuming. This challenge requires a series of diverse, complex, and labor-intensive biological, toxicological, and analytical techniques for effective characterization. While recent attempts, using machine learning based solely on primary amino acid sequences to predict biological toxins (e.g., conotoxins and animal venoms), have improved toxin identification, these methods are limited due to peptide conformational flexibility and the high frequency of cysteines present in toxin sequences. This results in an enumerable set of disulfide-bridged foldamers with different conformations of the same primary amino acid sequence that affect function and toxicity levels. Consequently, a given peptide may be toxic when its cysteine residues form a particular disulfide-bond pattern, while alternative bonding patterns (isoforms) or its reduced form (free cysteines with no disulfide bridges) may have little or no toxicological effects. Similarly, the same disulfide-bond pattern may be possible for other peptide sequences and result in different conformations that all exhibit varying toxicities to the same receptor or to different receptors. We present here new features, when combined with primary sequence features to train machine learning algorithms to predict conotoxins, that significantly increase prediction accuracy.

Pharmacological Classes of Conus Peptides Targeted to Calcium, Sodium, and Potassium Channels.

This review describes the specific features of families of Conus venom peptides (conotoxins or conopeptides) that represent twelve pharmacological classes. Members of these conopeptide families are targeted to voltage-gated ion channels, such as calcium, sodium, and potassium channels. The conopeptides covered in this work include omega-conotoxins and contryphans with calcium channels as targets; mu-conotoxins, muO-conotoxins, muP-conotoxins, delta-conotoxins and iota-conotoxin with sodium channels as targets; and kappa-conotoxins, kappaM-conotoxins, kappaO-conotoxin, conkunitzins, and conorfamide with potassium channels as targets. The review covers the peptides that have been characterized over the last two decades with respect to their physiological targets and/or potential pharmacological applications, or those that have been discovered earlier but with noteworthy features elucidated in more recent studies. Some of these peptides have the potential to be developed as therapies for nerve, muscle, and heart conditions associated with dysfunctions in voltage-gated ion channels. The gating process of an ion channel subtype in neurons triggers various biological activities, including regulation of gene expression, contraction, neurotransmitter secretion, and transmission of electrical impulses. Studies on conopeptides and their interactions with calcium, sodium, and potassium channels provide evidence for Conus peptides as neuroscience research probes and therapeutic leads.

Oxidative Folding Catalysts of Conotoxins Derived from the Venom Duct Transcriptome of C. frigidus and C. amadis.

Two novel redox conopeptides with proline residues outside and within the active site disulfide loop were derived from the venom duct transcriptome of the marine cone snails Conus frigidus and Conus amadis. Mature peptides with possible post-translational modification of 4-trans-hydroxylation of proline, namely, Fr874, Fr890[P1O], Fr890[P2O], Fr906, Am1038, and Am1054, have been chemically synthesized and characterized using mass spectrometry. The estimated reduction potential of cysteine disulfides of synthetic peptides varied from -298 to -328 mV, similar to the active site cysteine disulfide motifs of the redox family of proteins. Fr906/Am1054 exhibited pronounced catalytic activity and assisted in improving the yields of natively folded globular form α-conotoxin ImI. Three-dimensional (3D) structures of the redox conopeptides were optimized using computational methods and verified by 2D-ROESY NMR spectroscopy: C. frigidus peptides adopt an N-terminal helical fold and C. amadis peptides adopt distinct structures based on the Phe4-Pro/Hyp5 peptide bond configuration. The shift in the cis-trans configuration of the Phe4-Pro/Hyp5 peptide bond of Am1038/Am1054 was observed between reduced free thiol and oxidized disulfide forms of the optimized peptides. The report confirms the position-specific effect of hydroxyproline on the oxidative folding of conotoxins and sequence diversity of redox conopeptides in the venom duct of cone snails.

Synthesis, Activity, and Application of Fluorescent Analogs of [D1G, Δ14Q]LvIC Targeting α6ß4 Nicotinic Acetylcholine Receptor.

α6ß4* nicotinic acetylcholine receptor (nAChR) (* represents the possible presence of additional subunits) is mainly distributed in the central and peripheral nervous system and is associated with neurological diseases, such as neuropathic pain; however, the ability to explore its function and distribution is limited due to the lack of pharmacological tools. As one of the analogs of α-conotoxin (α-CTx) LvIC from Conus lividus, [D1G, Δ14Q]LvIC (Lv) selectively and potently blocks α6/α3ß4 nAChR (α6/α3 represents a chimera). Here, we synthesized three fluorescent analogs of Lv by connecting fluorescent molecules 6-carboxytetramethylrhodamine succinimidyl ester (6-TAMRA-SE, R), Cy3 NHS ester (Cy3, C) and BODIPY-FL NHS ester (BDP, B) to the N-terminus of the peptide and obtained Lv-R, Lv-C, and Lv-B, respectively. The potency and selectivity of three fluorescent peptides were evaluated using two-electrode voltage-clamp recording on nAChR subtypes expressed in Xenopus laevis oocytes, and the potency and selectivity of Lv-B were almost maintained with the half-maximal inhibition (IC50) of 64 nM. Then, we explored the stability of Lv-B in artificial cerebrospinal fluid and stained rat brain slices with Lv-B. The results indicated that the stability of Lv-B was slightly improved compared to that of native Lv. Additionally, we detected the distribution of the α6ß4* nAChR subtype in the cerebral cortex using green fluorescently labeled peptide and fluorescence microscopy. Our findings not only provide a visualized pharmacological tool for exploring the distribution of the α6ß4* nAChR subtype in various situ tissues and organs but also extend the application of α-CTx [D1G, Δ14Q]LvIC to demonstrate the involvement of α6ß4 nAChR function in pathophysiology and pharmacology.

Synthesis and insecticidal activity of cysteine-free conopeptides from Conus betulinus.

The cone snail Conus betulinus is a vermivorous species that is widely distributed in the South China Sea. Its crude venom contains various peptides used to prey on marine worms. In previous studies, a systematic analysis of the peptide toxin sequences from C. betulinus was carried out using a multiomics technique. In this study, 10 cysteine-free peptides that may possess insecticidal activity were selected from a previously constructed conopeptide library of C. betulinus using the CPY-Fe conopeptide as a template. These conopeptides were prepared by solid-phase peptide synthesis (SPPS), then characterized by the reverse-phase high performance liquid chromatography (HPLC) and mass spectrometry. Insect cytotoxicity and injection experiments revealed that these cysteine-free peptides exerted favorable insecticidal effects, and two of them (Bt010 and Bt016) exhibited high insecticidal efficacy with LD50 of 9.07 nM and 10.93 nM, respectively. In addition, the 3D structures of these peptides were predicted by homology modeling, and a phylogenetic tree was constructed based on the nucleotide data of conopeptides to analyze the relationships among structures, functions, and evolution. A preliminary mechanism for the insecticidal activity of the cysteine-free conopeptides was predicted by molecular docking. To the best of our knowledge, this is the first study to report the insecticidal activity of cysteine-free conopeptides derived from Conus betulinus, signaling that they could potentially be developed into bioinsecticides with desirable properties such as easy preparation, low cost, and high potency.

A previously unrecognized superfamily of macro-conotoxins includes an inhibitor of the sensory neuron calcium channel Cav2.3.

Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, which defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and 2 additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of recombinantly expressed voltage-gated ion channels, Mu8.1 displayed the highest potency against the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study showcases the potential of uncovering novel structures and bioactivities within the largely unexplored group of macro-conotoxins.

Synthesis of Cystine-Stabilised Dicarba Conotoxin EpI: Ring-Closing Metathesis of Sidechain Deprotected, Sulfide-Rich Sequences.

Recombinant peptide synthesis allows for large-scale production of peptides with therapeutic potential. However, access to dicarba peptidomimetics via sidechain-deprotected sequences becomes challenging with exposed Lewis basicity presented by amine and sulfur-containing residues. Presented here is a combination of strategies which can be used to deactivate coordinative residues and achieve high-yielding Ru-catalyzed ring-closing metathesis. The chemistry is exemplified using α-conotoxin EpI, a native bicyclic disulfide-containing sequence isolated from the marine conesnail Conus episcopatus. Replacement of the loop I disulfide with E/Z-dicarba bridges was achieved with high conversion via solution-phase ring-closing metathesis of the unprotected linear peptide after simple chemoselective oxidation and ion-exchange masking of problematic functionality. Metathesis was also attempted in green solvent choices to further improve the sustainability of dicarba peptide synthesis.

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.