N-Terminal Capping of the αO-Conotoxin Analogue GeX-2 Improves the Serum Stability and Selectivity toward the Human α9α10 Nicotinic Acetylcholine Receptor.

α9α10 nicotinic acetylcholine receptors (nAChRs) are a promising nonopioid analgesic target, with α9α10 nAChR antagonists showing efficacy against chemotherapy-induced hyperalgesia and allodynia. GeX-2, a potent analgesic conotoxin antagonist of α9α10 nAChRs, has limited serum stability. This study improved GeX-2 stability by capping its N-terminal with fatty acids or polyethylene glycol chains, which enhanced its serum stability but eliminated activity at G protein-coupled γ-aminobutyric acid type B (GABAB) receptor-coupled CaV2.2 channels while preserving activity at α9α10 nAChRs. In vivo, α9α10 nAChRs antagonism alone did not alleviate neuropathic pain, highlighting the importance of GABAB receptor-coupled CaV2.2 channels in GeX-2's antinociceptive effects in the chronic constriction injury rat model. The GeX-2 analogue, with an N-terminal methyl group, showed improved activity and selectivity for α9α10 nAChRs, increased serum half-life, and strong analgesic effects in oxaliplatin-induced cold allodynia models. AlphaFold3 and molecular dynamics simulations provided insights into the binding modes and the effects of N-terminal capping, which informed future peptide therapeutic developments.

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.

Selective Penicillamine Substitution Enables Development of a Potent Analgesic Peptide that Acts through a Non-Opioid-Based Mechanism.

Venom-derived compounds are of broad interest in neuropharmacology and drug development. α-Conotoxins are small disulfide-containing peptides from Conus snails that target nicotinic acetylcholine receptors (nAChRs) and are in clinical development for non-opioid-based treatment of intractable pain. Although refined by evolution for interaction with target prey receptors, enhancements of pharmacological properties are needed for use in mammalian systems. Therefore, we synthesized analogues of α-conotoxin RgIA using a combination of selective penicillamine substitutions together with natural and non-natural amino acid replacements. This approach resulted in a peptide with 9000-fold increased potency on the human α9α10 nAChR and improved resistance to disulfide shuffling compared to the native peptide. The lead analogue, RgIA-5474, potently blocked α9α10 nAChRs, but not opioid- or other pain-related targets. In addition, RgIA-5474 effectively reversed chemotherapy-induced neuropathic pain.

Synthesis and evaluation of disulfide-rich cyclic α-conotoxin [S9A]TxID analogues as novel α3ß4 nAChR antagonists.

Head-to-tail cyclization is an effective strategy to improve the biological stability of peptides. The α-conotoxin [S9A]TxID is a peptide that inhibits α3ß4 nAChR with high activity and selectivity. Herein, we established a method for cyclizing and oxidative folding of [S9A]TxID, and six cyclic analogues of [S9A]TxID were chemically synthesized with various linker lengths. We used the electrophysiology assay to measure activity values of these cyclic analogues, and obtained the most potent analogue c[S9A]TxID-6, which was more stable than native [S9A]TxID against proteinase K.

Engineered Conotoxin Differentially Blocks and Discriminates Rat and Human α7 Nicotinic Acetylcholine Receptors.

The α7 nicotinic acetylcholine receptor (nAChR) is present in the central nervous system and plays an important role in cognitive function and memory. α-Conotoxin LvIB, identified from genomic DNA of Conus lividus, its three isomers and four globular isomer analogues were synthesized and screened at a wide range of nAChR subtypes. One of the analogues, amidated [Q1G,ΔR14]LvIB, was found to be a potent blocker of rat α7 nAChRs. Importantly, it differentiates between α7 nAChRs of human (IC50: 1570 nM) and rat (IC50: 97 nM). Substitutions between rat and human α7 nAChRs at three key mutation sites revealed that no single mutant could completely change the activity profile of amidated [Q1G,ΔR14]LvIB. Rather, we found that the combined influence of Gln141, Asn184, and Lys186 determines the α7 nAChR species specificity of this peptide. This engineered α4/4 conotoxin has potential applications as a template for designing ligands to selectively block human α7 nAChRs.

Cysteine [2,4] Disulfide Bond as a New Modifiable Site of α-Conotoxin TxIB.

α-Conotoxin TxIB, a selective antagonist of α6/α3ß2ß3 nicotinic acetylcholine receptor, could be a potential therapeutic agent for addiction and Parkinson's disease. As a peptide with a complex pharmacophoric conformation, it is important and difficult to find a modifiable site which can be modified effectively and efficiently without activity loss. In this study, three xylene scaffolds were individually reacted with one pair of the cysteine residues ([1,3] or [2,4]), and iodine oxidation was used to form a disulfide bond between the other pair. Overall, six analogs were synthesized with moderate isolated yields from 55% to 65%, which is four times higher than the traditional two-step oxidation with orthogonal protection on cysteines. The cysteine [2,4] modified analogs, with higher stability in human serum than native TxIB, showed obvious inhibitory effect and selectivity on α6/α3ß2ß3 nicotinic acetylcholine receptors (nAChRs), which was 100 times more than the cysteine [1,3] modified ones. This result demonstrated that the cysteine [2,4] disulfide bond is a new modifiable site of TxIB, and further modification can be a simple and feasible strategy for the exploitation and utilization of α-Conotoxin TxIB in drug discovery.

Chemical Synthesis and NMR Solution Structure of Conotoxin GXIA from Conus geographus.

Conotoxins are disulfide-rich peptides found in the venom of cone snails. Due to their exquisite potency and high selectivity for a wide range of voltage and ligand gated ion channels they are attractive drug leads in neuropharmacology. Recently, cone snails were found to have the capability to rapidly switch between venom types with different proteome profiles in response to predatory or defensive stimuli. A novel conotoxin, GXIA (original name G117), belonging to the I3-subfamily was identified as the major component of the predatory venom of piscivorous Conus geographus. Using 2D solution NMR spectroscopy techniques, we resolved the 3D structure for GXIA, the first structure reported for the I3-subfamily and framework XI family. The 32 amino acid peptide is comprised of eight cysteine residues with the resultant disulfide connectivity forming an ICK+1 motif. With a triple stranded ß-sheet, the GXIA backbone shows striking similarity to several tarantula toxins targeting the voltage sensor of voltage gated potassium and sodium channels. Supported by an amphipathic surface, the structural evidence suggests that GXIA is able to embed in the membrane and bind to the voltage sensor domain of a putative ion channel target.

α-Conotoxin TxID and [S9K]TxID, α3ß4 nAChR Antagonists, Attenuate Expression and Reinstatement of Nicotine-Induced Conditioned Place Preference in Mice.

Tobacco smoking has become a prominent health problem faced around the world. The α3ß4 nicotinic acetylcholine receptor (nAChR) is strongly associated with nicotine reward and withdrawal symptom. α-Conotoxin TxID, cloned from Conus textile, is a strong α3ß4 nAChR antagonist, which has weak inhibition activity of α6/α3ß4 nAChR. Meanwhile, its analogue [S9K]TxID only inhibits α3ß4 nAChR (IC50 = 6.9 nM), and has no inhibitory activity to other nAChRs. The present experiment investigates the effect of α3ß4 nAChR antagonists (TxID and [S9K]TxID) on the expression and reinstatement of nicotine-induced conditioned place preference (CPP) and explores the behaviors of acute nicotine in mice. The animal experimental results showed that TxID and [S9K] TxID could inhibit the expression and reinstatement of CPP, respectively. Moreover, both had no effect in acute nicotine experiment and the locomotor activity in mice. Therefore, these findings reveal that the α3ß4 nAChR may be a potential target for anti-nicotine addiction treatment. [S9K]TxID, α3ß4 nAChR antagonist, exhibit a superior effect for anti-nicotine addiction, which is promising to develop a novel smoking cessation drug.

Synthesis, Pharmacological and Structural Characterization of Novel Conopressins from Conus miliaris.

Cone snails produce a fast-acting and often paralyzing venom, largely dominated by disulfide-rich conotoxins targeting ion channels. Although disulfide-poor conopeptides are usually minor components of cone snail venoms, their ability to target key membrane receptors such as GPCRs make them highly valuable as drug lead compounds. From the venom gland transcriptome of Conus miliaris, we report here on the discovery and characterization of two conopressins, which are nonapeptide ligands of the vasopressin/oxytocin receptor family. These novel sequence variants show unusual features, including a charge inversion at the critical position 8, with an aspartate instead of a highly conserved lysine or arginine residue. Both the amidated and acid C-terminal analogues were synthesized, followed by pharmacological characterization on human and zebrafish receptors and structural investigation by NMR. Whereas conopressin-M1 showed weak and only partial agonist activity at hV1bR (amidated form only) and ZFV1a1R (both amidated and acid form), both conopressin-M2 analogues acted as full agonists at the ZFV2 receptor with low micromolar affinity. Together with the NMR structures of amidated conopressins-M1, -M2 and -G, this study provides novel structure-activity relationship information that may help in the design of more selective ligands.

Structure and Activity Studies of Disulfide-Deficient Analogues of αO-Conotoxin GeXIVA.

αO-conotoxin GeXIVA from Conus generalis is a potent antagonist of the α9α10 nAChR and analgesic in animal models of pain. This peptide has two disulfide bond cross-links, and the bead and ribbon isomers have similar inhibitory activity against α9α10 nAChRs. We synthesized 12 disulfide-deficient analogues of bead GeXIVA, and all remained potent inhibitors of α9α10 nAChR. The most potent disulfide-deficient analogue displayed IC50 values of 6 and 33 nM at rat and human α9α10 nAChRs, respectively, representing less than a 2-fold increase compared with bead GeXIVA. The disulfide-deficient analogs and parent peptides also do not have a well-defined structure according to NMR spectroscopy. Molecular simulations suggest that the disulfide bonds and termini of GeXIVA do not establish stable interactions with the receptor. Overall, this study proposes that the structure of the analgesic peptide GeXIVA could be simplified through disulfide bond deletions and potentially termini truncations.

Degradation kinetics of α-conotoxin TxID.

α-Conotoxin (CTx) TxID is a potent α3ß4 nicotinic acetylcholine receptor (nAChR) antagonist that has been suggested as a potential drug candidate to treat addiction and small cell lung cancer. The function and structure of TxID have been well-studied, but analyses of its stability have not previously been reported. The purpose of this study was to analyze the stability and forced degradation of TxID under various conditions: acid, alkali, water hydrolysis, oxidation, light, thiols, temperature, ionic strength and buffer pH. Different degradation products were formed under various conditions, and the degradation patterns of TxID showed pseudo-first-order kinetics. TxID degraded slowest at pH 3 within a pH range of 2-8. The major degradation products were analyzed using liquid chromatography-tandem mass spectrometry and the activity of the main product with α3ß4 nAChR was analyzed using electrophysiological methods. Our analysis of TxID stability may aid the selection of appropriate conditions for peptide production, packaging and storage.

A rink-amide soluble support: high purity conotoxins and other peptides accessed with minimal reagents.

The use of peptides as therapeutics has been growing due to their biocompatibility. Solid phase peptide synthesis typically used to access these peptides requires excess reagents and/or microwave irradiation to drive reactions to completion because the reaction medium is heterogeneous. Reported herein is a soluble polynorbornene support containing rink amide attached sites for synthesizing oligopeptides and conotoxins in high purity using only 1.2 to 2 equivalents of coupling reagents. The support can be isolated as a precipitate from the reaction medium by adding ether. The loading capacity of the support can be easily determined by spectroscopy and can also be tuned by varying the monomer ratio. This support is promising for accessing peptides as the methodology uses minimal reagents and by-products can be easily separated.

NMR Structure of µ-Conotoxin GIIIC: Leucine 18 Induces Local Repacking of the N-Terminus Resulting in Reduced NaV Channel Potency.

µ-Conotoxins are potent and highly specific peptide blockers of voltage-gated sodium channels. In this study, the solution structure of µ-conotoxin GIIIC was determined using 2D NMR spectroscopy and simulated annealing calculations. Despite high sequence similarity, GIIIC adopts a three-dimensional structure that differs from the previously observed conformation of µ-conotoxins GIIIA and GIIIB due to the presence of a bulky, non-polar leucine residue at position 18. The side chain of L18 is oriented towards the core of the molecule and consequently the N-terminus is re-modeled and located closer to L18. The functional characterization of GIIIC defines it as a canonical µ-conotoxin that displays substantial selectivity towards skeletal muscle sodium channels (NaV), albeit with ~2.5-fold lower potency than GIIIA. GIIIC exhibited a lower potency of inhibition of NaV1.4 channels, but the same NaV selectivity profile when compared to GIIIA. These observations suggest that single amino acid differences that significantly affect the structure of the peptide do in fact alter its functional properties. Our work highlights the importance of structural factors, beyond the disulfide pattern and electrostatic interactions, in the understanding of the functional properties of bioactive peptides. The latter thus needs to be considered when designing analogues for further applications.

Synthesis and Structure Determination of µ-Conotoxin PIIIA Isomers with Different Disulfide Connectivities.

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of µ-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

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.

Effects of serum, enzyme, thiol, and forced degradation on the stabilities of αO-Conotoxin GeXIVA[1,2] and GeXIVA [1,4].

αO-conotoxin GeXIVA, which is a potent antagonist of α9α10 nicotinic acetylcholine receptor (nAChR), is of great interest as a potential analgesic for chronic neuropathic pain. It has three isomers, of which both GeXIVA[1,2] and GeXIVA[1,4] showed similar low nanomolar IC50 s in potent blocking rat α9α10 nAChRs. Here, we first reported stabilities of GeXIVA[1,2] and GeXIVA[1,4] in various biochemical circumstances, including human serum, enzymatic degradation, and thiol, which would be the key factors to affect stabilities of the two isomers in vivo. Simultaneously, forced degradation was carried out to evaluate stabilities of the two isomers. GeXIVA[1,2] and GeXIVA[1,4] were unstable when they were incubated in serum and digestive enzymes at 37°C. Their disulfide bond frameworks were easy to be scrambled in GSH and HSA. For different stress conditions, their stabilities were impacted greatly by oxidation, temperature, and alkaline conditions. The results may provide a foundation for storage conditions, structural modification, and pharmaceutical preparation of GeXIVA[1,2] and GeXIVA[1,4].

A novel α-conopeptide Eu1.6 inhibits N-type (CaV2.2) calcium channels and exhibits potent analgesic activity.

We here describe a novel α-conopeptide, Eu1.6 from Conus eburneus, which exhibits strong anti-nociceptive activity by an unexpected mechanism of action. Unlike other α-conopeptides that largely target nicotinic acetylcholine receptors (nAChRs), Eu1.6 displayed only weak inhibitory activity at the α3ß4 and α7 nAChR subtypes and TTX-resistant sodium channels, and no activity at TTX-sensitive sodium channels in rat dorsal root ganglion (DRG) neurons, or opiate receptors, VR1, KCNQ1, L- and T-type calcium channels expressed in HEK293 cells. However, Eu1.6 inhibited high voltage-activated N-type calcium channel currents in isolated mouse DRG neurons which was independent of GABAB receptor activation. In HEK293 cells expressing CaV2.2 channels alone, Eu1.6 reversibly inhibited depolarization-activated Ba2+ currents in a voltage- and state-dependent manner. Inhibition of CaV2.2 by Eu1.6 was concentration-dependent (IC50 ~1 nM). Significantly, systemic administration of Eu1.6 at doses of 2.5-5.0 µg/kg exhibited potent analgesic activities in rat partial sciatic nerve injury and chronic constriction injury pain models. Furthermore, Eu1.6 had no significant side-effect on spontaneous locomotor activity, cardiac and respiratory function, and drug dependence in mice. These findings suggest α-conopeptide Eu1.6 is a potent analgesic for the treatment of neuropathic and chronic pain and opens a novel option for future analgesic drug design.

Macrocyclic Antimicrobial Peptides Engineered from ω-Conotoxin.

The potent calcium channel blocker ω-conotoxin MVIIA is a linear cystine-knot peptide with multiple basic amino acids at both termini. This work shows that macrocyclization of MVIIA linking two positive-charge terminal clusters as a contiguous segment converts a conotoxin into an antimicrobial peptide. In addition, conversion of disulfide bonds to amino butyric acids improved the antimicrobial activity of the cyclic analogs. Ten macrocyclic analogs, with or without disulfide bonds, were prepared by both Boc and Fmoc chemistry using native chemical ligation. All cyclic analogs were active against selected Gram-positive and Gram-negative bacteria with minimal inhibitory concentrations in a low µM range. In contrast, MVIIA and its linear analog were inactive at concentrations up to 0.5 mM. The cyclic analogs also showed 2 to 3-fold improved chemotactic activity against human monocytes THP-1 compared with MVIIA. Reduction of molecular stability against thermal and acid treatment due to the reduced number of disulfide crosslinks can be partly restored by backbone cyclization. Together, these results show that macrocyclization and side chain modification of a linear conopeptide lead to a gain-of-function, which brings a new perspective in designing and engineering of peptidyl therapeutics.

High-Affinity α-Conotoxin PnIA Analogs Designed on the Basis of the Protein Surface Topography Method.

Despite some success for small molecules, elucidating structure-function relationships for biologically active peptides - the ligands for various targets in the organism - remains a great challenge and calls for the development of novel approaches. Some of us recently proposed the Protein Surface Topography (PST) approach, which benefits from a simplified representation of biomolecules' surface as projection maps, which enables the exposure of the structure-function dependencies. Here, we use PST to uncover the "activity pattern" in α-conotoxins - neuroactive peptides that effectively target nicotinic acetylcholine receptors (nAChRs). PST was applied in order to design several variants of the α-conotoxin PnIA, which were synthesized and thoroughly studied. Among the best was PnIA[R9, L10], which exhibits nanomolar affinity for the α7 nAChR, selectivity and a slow wash-out from this target. Importantly, these mutations could hardly be delineated by "standard" structure-based drug design. The proposed combination of PST with a set of experiments proved very efficient for the rational construction of new bioactive molecules.

Im10A, a short conopeptide isolated from Conus imperialis and possesses two highly concentrated disulfide bridges and analgesic activity.

In the present study, we isolated, synthesized and NMR structurally characterized a novel conopeptide Im10A consisting of 11 amino acids (NTICCEGCMCY-NH2) from Conus imperialis. Unlike other conopeptides with four cysteine residues, Im10A had only two residues in loop 1 and one residue in loop 2 (CC-loop1-C-loop2-C), which formed a stable disulfide connectivity "I-IV, II- III" (framework X) with a type I ß-turn. Interestingly, Im10A exhibited 50.7% analgesic activity on rat partial sciatic nerve ligation (PNL) at 2h after Im10A administration. However, 10µM Im10A exhibited no apparent effect on neuronal nicotinic acetylcholine receptor, and it did not target DRG voltage-dependent sodium, potassium and calcium ion channels and opioid receptor. To our knowledge, Im10A had the most concentrated disulfide bridges among conopeptides with four cysteine residues. This finding provided a new motif for the future development of biomimetic compounds.