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α3β4, a vital subtype of neuronal nicotinic acetylcholine receptors (nAChRs), is widely distributed in the brain, ganglia, and adrenal glands, associated with addiction and neurological diseases. However, the lack of specific imaging tools for α3β4 nAChRs has hindered the investigation of their tissue distribution and functions. [D11A]LvIA, a peptide derived from marine cone snails, demonstrates high affinity and potency for α3β4 nAChRs, making it a valuable pharmacological tool for studying this receptor subtype. In this study, three fluorescent conjugates of [D11A]LvIA were synthesized using 6-TAMRA-SE (R), Cy3-NHS-ester (Cy3), and BODIPY-FL NHS ester (BDP) dyes. The electrophysiological activities were assessed in Xenopus laevis oocytes by two-electrodes voltage clamp (TEVC). [D11A]LvIA-Cy3 and [D11A]LvIA-BDP show improved selectivity and affinity, with IC 50 values of 512.70 nM and 343.50 nM, respectively, and [D11A]LvIA-Cy3 exhibits better stability in cerebrospinal fluid (CSF). Utilizing [D11A]LvIA-Cy3, we successfully visualized the distribution of α3β4 nAChRs in rat trigeminal ganglia, retina, adrenal glands, and various brain regions. This novel fluorescent peptide provides a significant pharmacological tool for the exploration and visualization in-situ distribution of α3β4 nAChRs in different tissues and also assists in clarifying the function., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

α-Conotoxins are disulfide-rich peptides obtained from the venom of cone snails, which are considered potential molecular probes and drug leads for nAChR-related disorders. However, low specificity towards different nAChR subtypes restricts the further application of many α-conotoxins. In this work, a series of loop1 amino acid-substituted mutants of α-conotoxin RegIIA were synthesized, whose potency and selectivity were evaluated by an electrophysiological approach. The results showed that loop1 alanine scanning mutants [H5A]RegIIA and [P6A]RegIIA blocked rα7 nAChR with IC 50 s of 446 nM and 459 nM, respectively, while their inhibition against rα3β2 and rα3β4 subtypes was negligible, indicating the importance of the fifth and sixth amino acid residues for RegIIA's potency and selectivity. Then, second-generation mutants were designed and synthesized, among which the analogues [H5V]RegIIA and [H5S]RegIIA showed significantly improved selectivity and comparable potency towards rα7 nAChR compared with the native RegIIA. Overall, these findings provide deep insights into the structure-activity relationship of RegIIA, as well as revealing a unique perspective for the further modification and optimization of α-conotoxins and other active peptides.

Voltage-gated sodium (Na V ) channels are intimately involved in the generation and transmission of action potentials, and dysfunction of these channels may contribute to nervous system diseases, such as epilepsy, neuropathic pain, psychosis, autism, and cardiac arrhythmia. Many venom peptides selectively act on Na V channels. These include conotoxins, which are neurotoxins secreted by cone snails for prey capture or self-defense but which are also valuable pharmacological tools for the identification and/or treatment of human diseases. Typically, conotoxins contain two or three disulfide bonds, and these internal crossbraces contribute to conotoxins having compact, well defined structures and high stability. Of the conotoxins containing three disulfide bonds, some selectively target mammalian Na V channels and can block, stimulate, or modulate these channels. Such conotoxins have great potential to serve as pharmacological tools for studying the functions and characteristics of Na V channels or as drug leads for neurologic diseases related to Na V channels. Accordingly, discovering or designing conotoxins targeting Na V channels with high potency and selectivity is important. The amino acid sequences, disulfide bond connectivity, and three-dimensional structures are key factors that affect the biological activity of conotoxins, and targeted synthetic modifications of conotoxins can greatly improve their activity and selectivity. This review examines Na V channel-targeted conotoxins, focusing on their structures, activities, and designed modifications, with a view toward expanding their applications. SIGNIFICANCE STATEMENT: Na V channels are crucial in various neurologic diseases. Some conotoxins selectively target Na V channels, causing either blockade or activation, thus enabling their use as pharmacological tools for studying the channels' characteristics and functions. Conotoxins also have promising potential to be developed as drug leads. The disulfide bonds in these peptides are important for stabilizing their structures, thus leading to enhanced specificity and potency. Together, conotoxins targeting Na V channels have both immediate research value and promising future application prospects., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

α-Conotoxins, as selective nAChR antagonists, can be valuable tools for targeted drug delivery and fluorescent labeling, while conotoxin-drug or conotoxin-fluorescent conjugates through the disulfide bond are rarely reported. Herein, we demonstrate the [2,4] disulfide bond of α-conotoxin as a feasible new chemical modification site. In this study, analogs of the α-conotoxin LsIA cysteine[2,4] were synthesized by stapling with five linkers, and their inhibitory activities against human α7 and rat α3β2 nAChRs were maintained. To further apply this method in targeted delivery, the alkynylbenzyl bromide linker was synthesized and conjugated with Coumarin 120 (AMC) and Camptothecin (CPT) by copper-catalyzed click chemistry, and then stapled between cysteine[2,4] of the LsIA to construct a fluorescent probe and two peptide-drug conjugates. The maximum emission wavelength of the LsIA fluorescent probe was 402.2 nm, which was essentially unchanged compared with AMC. The cytotoxic activity of the LsIA peptide-drug conjugates on human A549 was maintained in vitro. The results demonstrate that the stapling of cysteine[2,4] with alkynylbenzyl bromide is a simple and feasible strategy for the exploitation and utilization of the α-conotoxin LsIA.

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 IC 50 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., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cyclotides are a type of defense peptide most commonly found in the Violaceae family of plants, exhibiting various biological activities. In this study, we focused on the Viola japonica as our research subject and conducted transcriptome sequencing and analysis using high-throughput transcriptomics techniques. During this process, we identified 61 cyclotides, among which 25 were previously documented, while the remaining 36 were designated as vija 1 to vija 36. Mass spectrometry detection showed that 21 putative cyclotides were found in the extract of V. japonica. Through isolation, purification and tandem mass spectrometry, we characterized and investigated the activities of five cyclotides. Our results demonstrated inhibitory effects of these cyclotides on the growth of Acinetobacter baumannii and Bacillus subtilis, with minimum inhibitory concentrations (MICs) of 4.2 μM and 2.1 μM, respectively. Furthermore, time killing kinetic assays revealed that cyclotides at concentration of 4 MICs achieved completely bactericidal effects within 2 h. Additionally, fluorescence staining experiments confirmed that cyclotides disrupt microbial membranes. Moreover, cytotoxicity studies showed that cyclotides possess cytotoxic effects, with IC 50 values ranging from 0.1 to 3.5 μM. In summary, the discovery of new cyclotide sequences enhances our understanding of peptide diversity and the exploration of their activity lays the foundation for a deeper investigation into the mechanisms of action of cyclotides., (© 2024. The Author(s).)

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 Ca 2+ 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 (IC 50 ) 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.

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.

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.

α6β4* 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/α3β4 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 (IC 50 ) 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 α6β4* 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 α6β4* nAChR subtype in various situ tissues and organs but also extend the application of α-CTx [D1G, Δ14Q]LvIC to demonstrate the involvement of α6β4 nAChR function in pathophysiology and pharmacology.

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.

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.

Metformin is a traditional antidiabetic drug that also shows potential antitumor effects in cervical cancer. However, some of its apoptosis-related mechanisms are still unclear. In this study, flow cytometry, western blotting, and RNA sequencing (RNA-seq) were used to evaluate the molecular mechanisms of metformin in HeLa cells. The results showed that metformin inhibited cell viability and promoted apoptosis, the protein expression level of Caspase-3 (CASP3) was increased and that of BCL-2 was decreased in HeLa cells treated with metformin. The RNA-seq results indicated a total of 239 differentially expressed genes between the metformin and control check (CK) groups, with 136 genes upregulated and 103 genes downregulated, and 14 of them were found to be associated with apoptosis signaling pathways. The DDIT3 and HRK genes were robustly upregulated in HeLa cells by the endoplasmic reticulum (ER) stress and the mitochondrial pathway of apoptosis. Metformin also affects the expression of PPP2R5C , PPP2R5A , and RRAGA , which participate in biological processes such as PI3K-AKT, mTOR, and AMPK signaling pathways. Metformin mediates the expression of related genes to induce apoptosis.

Cone snails, as a type of marine organism, have rich species diversity. Traditionally, classifications of cone snails were based mostly on radula, shell, and anatomical characters. Because of these phenotypic features' high population variability and propensity for local adaptation and convergence, identifying species can be difficult and occasionally inaccurate. In addition, mitochondrial genomes contain high phylogenetic information, so complete mitogenomes have been increasingly employed for inferring molecular phylogeny. To enrich the mitogenomic database of cone snails (Caenogastropoda: Conidae), mitogenomes of four Conus species, i.e., C. imperialis (15,505 bp), C. literatus (15,569 bp), C. virgo (15,594 bp), and C. marmoreus (15,579 bp), were characterized and compared. All 4 of these mitogenomes included 13 protein-coding genes, 2 ribosomal RNA genes, 22 tRNA genes, and non-coding regions. All the Protein Codon Genes (PCGs) of both newly sequenced mitogenomes used TAA or TAG as a terminal codon. Most PCGs used conventional start codon ATG, but an alternative initiation codon GTG was detected in a gene (NADH dehydrogenase subunit 4 (nad4)) of C. imperialis . In addition, the phylogenetic relationships were reconstructed among 20 Conus species on the basis of PCGs, COX1, and the complete mitogenome using both Bayesian Inference (BI) and Maximum Likelihood (ML). The phylogenetic results supported that C. litteratus , C. quercinus , and C. virgo were clustered together as a sister group (PP = 1, BS = 99), but they did not support the phylogenetic relation of C. imperialis and C. tribblei (PP = 0.79, BS = 50). In addition, our study established that PCGs and complete mitogenome are the two useful markers for phylogenetic inference of Conus species. These results enriched the data of the cone snail's mitochondrion in the South China Sea and provided a reliable basis for the interpretation of the phylogenetic relationship of the cone snail based on the mitochondrial genome.

Conotoxins are a class of disulfide-rich peptides found in the venom of cone snails, which have attracted considerable attention in recent years due to their potent activity on ion channels and potential for therapeutics. Among them, α-conotoxin RgIA, a 13-residue peptide, has shown great promise as a potent inhibitor of α9α10 nAChRs for pain management. In this study, we investigated the effect of substituting the naturally occurring L-type arginine at position 11 of the RgIA sequence with its D-type amino acid. Our results indicate that this substitution abrogated the ability of RgIA to block α9α10 nAChRs, but instead endowed the peptide with the ability to block α7 nAChR activity. Structural analyses revealed that this substitution induced significant alteration of the secondary structure of RgIA[11r], which consequently affected its activity. Our findings underscore the potential of D-type amino acid substitution as a promising strategy for designing novel conotoxin-based ligands targeting different types of nAChRs., Competing Interests: The authors declare no conflicts of interest.

Conotoxins are widely distributed and important for studying ligand-gated ion channels. TxIB, a conotoxin consisting of 16 amino acids derived from Conus textile , is a unique selective ligand that blocks rat α6/α3β2β3 nAChR (IC 50 = 28 nM) without affecting other rat subtypes. However, when the activity of TxIB against human nAChRs was examined, it was unexpectedly found that TxIB had a significant blocking effect on not only human α6/α3β2β3 nAChR but also human α6/α3β4 nAChR, with an IC 50 of 537 nM. To investigate the molecular mechanism of this species specificity and to establish a theoretical basis for drug development studies of TxIB and its analogs, different amino acid residues between human and rat α6/α3 and β4 nAChR subunits were identified. Each residue of the human species was then substituted with the corresponding residue of the rat species via PCR-directed mutagenesis. The potencies of TxIB towards the native α6/α3β4 nAChRs and their mutants were evaluated through electrophysiological experiments. The results showed that the IC 50 of TxIB against h[α6 V32L, K61R /α3]β4 L107V, V115I was 22.5 μM, a 42-fold decrease in potency compared to the native hα6/α3β4 nAChR. Val-32 and Lys-61 in the human α6/α3 subunit and Leu-107 and Val-115 in the human β4 subunit, together, were found to determine the species differences in the α6/α3β4 nAChR. These results also demonstrate that the effects of species differences between humans and rats should be fully considered when evaluating the efficacy of drug candidates targeting nAChRs in rodent models.

α4/6-conotoxin TxID, which was identified from Conus textile , simultaneously blocks rat (r) α3β4 and rα6/α3β4 nicotinic acetylcholine receptors (nAChRs) with IC 50 values of 3.6 nM and 33.9 nM, respectively. In order to identify the effects of loop2 size on the potency of TxID, alanine (Ala) insertion and truncation mutants were designed and synthesized in this study. An electrophysiological assay was used to evaluate the activity of TxID and its loop2-modified mutants. The results showed that the inhibition of 4/7-subfamily mutants [+9A]TxID, [+10A]TxID, [+14A]TxID, and all the 4/5-subfamily mutants against rα3β4 and rα6/α3β4 nAChRs decreased. Overall, ala-insertion or truncation of the 9th, 10th, and 11th amino acid results in a loss of inhibition and the truncation of loop2 has more obvious impacts on its functions. Our findings have strengthened the understanding of α-conotoxin, provided guidance for further modifications, and offered a perspective for future studies on the molecular mechanism of the interaction between α-conotoxins and nAChRs.

In the chemical synthesis of conotoxins with multiple disulfide bonds, the oxidative folding process can result in diverse disulfide bond connectivities, which presents a challenge for determining the natural disulfide bond connectivities and leads to significant structural differences in the synthesized toxins. Here, we focus on KIIIA, a μ-conotoxin that has high potency in inhibiting Nav1.2 and Nav1.4. The non-natural connectivity pattern (C1-C9, C2-C15, C4-C16) of KIIIA exhibits the highest activity. In this study, we report an optimized Fmoc solid-phase synthesis of KIIIA using various strategies. Our results indicate that free random oxidation is the simplest method for peptides containing triple disulfide bonds, resulting in high yields and a simplified process. Alternatively, the semi-selective strategy utilizing Trt/Acm groups can also produce the ideal isomer, albeit with a lower yield. Furthermore, we performed distributed oxidation using three different protecting groups, optimizing their positions and cleavage order. Our results showed that prioritizing the cleavage of the Mob group over Acm may result in disulfide bond scrambling and the formation of new isomers. We also tested the activity of synthesized isomers on Nav1.4. These findings provide valuable guidance for the synthesis of multi-disulfide-bonded peptides in future studies.

α6β4 nicotinic acetylcholine receptors (nAChRs) are expressed in the central and peripheral nervous systems, but their functions are not fully understood, largely because of a lack of specific ligands. Here, we characterized a novel α-conotoxin, LvIC, and designed a series of analogues to probe structure-activity relationships at the α6β4 nAChR. The potency and selectivity of these conotoxins were tested using two-electrode voltage-clamp recording on nAChR subtypes expressed in Xenopus laevis oocytes. One of the analogues, [D1G,ΔQ14]LvIC, potently blocked α6/α3β4 nAChRs (α6/α3 is a chimera) with an IC 50 of 19 nM, with minimal activity at other nAChR subtypes, including the structurally similar α6/α3β2β3 and α3β4 subtypes. Using NMR, molecular docking, and receptor mutation, structure-activity relationships of [D1G,ΔQ14]LvIC at the α6/α3β4 nAChR were defined. It is a potent and specific antagonist of α6β4 nAChRs that could potentially serve as a novel molecular probe to explore α6β4 nAChR-related neurophysiological and pharmacological functions.

Nicotinic acetylcholine receptors (nAChRs) play an important role in the functioning of the central and peripheral nervous systems, and other organs of living creatures. There are several subtypes of nAChRs, and almost all of them are considered as pharmacological targets in different pathological states. The crude venom of the sea anemone Metridium senile showed the ability to interact with nAChRs. Four novel peptides (Ms11a-1-Ms11a-4) with nAChR binding activity were isolated. These peptides stabilized by three disulfide bridges have no noticeable homology with any known peptides. Ms11a-1-Ms11a-4 showed different binding activity towards the muscle-type nAChR from the Torpedo californica ray. The study of functional activity and selectivity for the most potent peptide (Ms11a-3) revealed the highest antagonism towards the heterologous rat α9α10 nAChR compared to the muscle and α7 receptors. Structural NMR analysis of two toxins (Ms11a-2 and Ms11a-3) showed that they belong to a new variant of the inhibitor cystine knot (ICK) fold but have a prolonged loop between the fifth and sixth cysteine residues. Peptides Ms11a-1-Ms11a-4 could represent new pharmacological tools since they have structures different from other known nAChRs inhibitors.

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 IC 50 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., Competing Interests: The authors declare no conflicts of interest.

Neuronal nicotinic acetylcholine receptors (nAChRs) are important ion channel membrane proteins that are widely distributed in the central nervous system (CNS) and peripheral nervous system (PNS). As an important member, α3β4 nAChRs are related to pain sensation in PNS and nicotine addiction in CNS. However, research related to the α3β4 nAChRs is greatly limited by the lack of subtype-selective pharmacological tools. The α-conotoxin (α-CTx) TxID from the marine cone snail, Conus textile , is a selective α3β4 nAChR antagonist with relatively high potency. In this study, a fluorescent dye (5-TAMRA SE) was used to label TxID on the N-terminus of α-CTx TxID, and pure TxID-F (fluorescent analogue of TxID) was obtained by HPLC. At the same time, the potency and selectivity of TxID-F were detected by high-performance liquid chromatography (HPLC). Additionally, the potency and selectivity of TxID-F were determined by using a two-electrode voltage-clamp technique on various nAChRs expressed in the Xenopus oocyte expression system. The results obtained by electrophysiology showed that TxID-F maintained the same order of potency (IC 50 73 nM) as the native toxin (IC 50 25 nM) for the α3β4 nAChR subtype. In addition, the results of fluorescent spectroscopy and circular dichroism showed TxID-F has the same fluorescence as 5-TAMRA SE, as well as similar profiles as TxID. The results of flow cytometry showed that the histogram shifted significantly to the right for the RAW264.7 cells expressing α3β4-containing nAChRs stained with TxID-F and confirmed by live cell imaging. The study of fluorescent-labeled α-CTx TxID provides a rich pharmacological tool to explore the structure-function relationship, distribution, and ligand-binding domain of α3β4 nAChR subtype in the future.

Oligo-basic amino acids have been extensively studied in molecular biology and pharmacology, but the inhibitory activity on nicotinic acetylcholine receptors (nAChRs) was unknown. In this study, the inhibitory activity of 8 oligopeptides, including both basic and acidic amino acids, was evaluated on 9 nAChR subtypes by a two-electrode voltage clamp (TEVC). Among them, the oligo-lysine K9, K12, d-K9, d-K9F, and oligo-arginine R9 showed nanomolar inhibitory activity on various nAChRs, especially for α7 and α9α10 nAChRs. d-K9 containing N-Fmoc protecting group (d-K9F) has an enhanced inhibitory activity on most of the nAChRs, including 47-fold promotion on α1β1δε nAChR. However, H9 and H12 only showed weak inhibitory activity on α9α10 and α1β1δε nAChRs, and the acidic oligopeptide D9 has no inhibitory activity on nAChRs. Flexible docking of K9 in α10(+) α9(-) and α7(+) α7(-) binding pockets showed particularly strong dipole-dipole interactions, which may be responsible for the inhibition of nAChRs. These results demonstrated that oligo-basic amino acids have the potential to be the lead compounds as selective nAChR subtype inhibitors, and oligo-lysines deserved to be modified for further exploitation and utilization. On the other hand, the toxicity and side effects of these nAChR inhibitory peptides should be contemplated in the application., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Two new indole diketopiperazines ( 1-2 ) obtained from the fermentation culture of a deep-sea-derived fungus Aspergillus chevalieri MCCC M23426, were characterized, together with nine biogenetic related compounds ( 3-11 ). The structures of 1-2 were assigned based on NMR, MS, NMR calculation, DP4+ analysis, and ECD calculation. The bioactive assay showed that compounds 1 , 5 - 7 significantly inhibited the growth of Staphylococcus aureus . Meanwhile, compound 8 potently reduced the cell viability of gastric cancer cell MKN1 with an IC 50 value of 4.6 μM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lv, Xia, Guan, Lai, Zhang, Lin, Shao, Luo, Zhangsun, Qin and Wang.)

Specific interaction between the postsynaptic density protein 95 (PSD95) and synapse-associated protein 90/postsynaptic density 95-associated protein (SAPAP) is crucial for excitatory synaptic development and plasticity. Designing inhibitors that target the guanylate kinase (GK) domain of PSD95, which is responsible for the interaction, is a promising manipulation tool for the investigation of the function of PSD95 GK and the etiology of its related psychiatric disorders. Herein, we designed new peptide inhibitors of PSD95 GK/SAPAP with higher binding affinity by using molecular dynamics simulations. First, the interactions between PSD95 GK and their reported phosphorylated and unphosphorylated peptides were explored by molecular dynamics simulations. Besides the hydrogen bonding interactions mediated by the phospho-serine (p-Ser) or corresponding phosphomimic residue Asp/Glu, the hydrophobic interactions from the other amino acids also contribute to the PSD95 GK/SAPAP interaction. As an unphosphorylated synthetic peptide with moderate binding affinity and relatively lower molecular weight, the QSF inhibitory peptide was selected for further modification. Based on per-residue energy decomposition results of the PSD95 GK/QSF complex, ten peptides were designed to enhance the binding interactions, especially the hydrophobic interactions. The top-ranked five peptides with lower binding energy were eventually synthesized. The binding affinities of the synthesized peptides were determined using fluorescence polarization (FP) assay. As expected, all peptides have higher binding affinity than the QSF peptide ( K i = 5.64 ± 0.51 μM). Among them, F10W was the most potent inhibitor ( K i = 0.75 ± 0.25 μM), suggesting that enhancement of the hydrophobic interactions is an important strategy for the design of new inhibitory peptides targeting PSD95 GK., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tian, Li, Yan, Gu, Zheng, Luo, Zhangsun, Chen and Ouyang.)

The α7 nicotinic acetylcholine receptor (nAChR) is widely distributed in the central and peripheral nervous systems and is closely related to a variety of nervous system diseases and inflammatory responses. The α7 nAChR subtype plays a vital role in the cholinergic anti-inflammatory pathway. In vivo, ACh released from nerve endings stimulates α7 nAChR on macrophages to regulate the NF-κB and JAK2/STAT3 signaling pathways, thereby inhibiting the production and release of downstream proinflammatory cytokines and chemokines. Despite a considerable level of recent research on α7 nAChR-mediated immune responses, much is still unknown. In this study, we used an agonist (PNU282987) and antagonists (MLA and α-conotoxin [A10L]PnIA) of α7 nAChR as pharmacological tools to identify the molecular mechanism of the α7 nAChR-mediated cholinergic anti-inflammatory pathway in RAW264.7 mouse macrophages. The results of quantitative PCR, ELISAs, and transcriptome analysis were combined to clarify the function of α7 nAChR regulation in the inflammatory response. Our findings indicate that the agonist PNU282987 significantly reduced the expression of the IL-6 gene and protein in inflammatory macrophages to attenuate the inflammatory response, but the antagonists MLA and α-conotoxin [A10L]PnIA had the opposite effects. Neither the agonist nor antagonists of α7 nAChR changed the expression level of the α7 nAChR subunit gene; they only regulated receptor function. This study provides a reference and scientific basis for the discovery of novel α7 nAChR agonists and their anti-inflammatory applications in the future.

α6β4 nAChR is expressed in the peripheral and central nervous systems and is associated with pain, addiction, and movement disorders. Natural α-conotoxins (α-CTxs) can effectively block different nAChR subtypes with higher efficacy and selectivity. However, the research on α6β4 nAChR is relatively poor, partly because of the lack of available target-specific α-CTxs. In this study, we synthesized a novel α-4/7 conotoxin QuIA that was found from Conus quercinus . We investigated the efficacy of this peptide to different nAChR subtypes using a two-electrode voltage-clamp technique. Remarkably, we found α-QuIA inhibited the neuronal α3β2 and α6/α3β4 nAChR subtypes with significantly high affinity (IC 50 was 55.7 nM and 90.68 nM, respectively), and did not block other nAChR subtypes even at a high concentration of 10 μM. In contrast, most α-CTxs have been determined so far to effectively block the α6/α3β4 nAChR subtype while also maintaining a similar higher efficacy against the closely related α6β2β3 and/or α3β4 subtypes, which are different from QuIA. In conclusion, α-QuIA is a novel α4/7-CTx, which has the potential to develop as an effective neuropharmacology tool to detect the function of α6β4 nAChR.

Background and Purpose: Alcohol use disorder (AUD) is a serious public health issue and affects the lives of numerous people. Previous studies have shown a link between nicotinic acetylcholine receptors (nAChR) and alcohol addiction. However, the role of α 6 β 2* nAChR in alcohol addiction remains obscure, and whether α 6 β 2* nAChR can be used as a potential drug target for alcohol withdrawal need to be studied. Methods: Zebrafish ( Danio rerio ) were exposed to 0.2% alcohol for 14 days followed by 7 days of repeated withdrawal and then retro-orbitally injected with α -conotoxin TxIB (a selective α 6 β 2* nAChR antagonist). Open Field Test was applied to characterize zebrafish behavior parameters. The monoamine neurotransmitter amounts and their mRNA expression in the zebrafish brain were identified using ELISA and quantitative real-time PCR (RT-PCR). RNA-sequencing (RNA-seq) and subsequent bioinformatics analysis were employed to explore the potential network regulation of TxIB after alcohol withdrawal. Results: The max speed in the center area of the Open Field Test was significantly higher in the withdrawal group whereas TxIB injection corrected this abnormality. The amount and mRNA expression of monoamine neurotransmitters did not change significantly after alcohol withdrawal and TxIB administration. RNA sequencing of zebrafish brain indicated a total of 657 genes showed aberrant expression and among which 225 were reversed after TxIB injection. These reversed genes were significantly enriched in the calcium ion binding pathway and the gene expression profile was further validated by RT-PCR. Conclusion: Our finding suggests α -conotoxin TxIB improved behavioral abnormality induced by alcohol-withdrawal, and changed gene expression mainly in the calcium signaling pathway. Therefore, α -conotoxin TxIB is expected to become a potential therapeutic agent for alcohol withdrawal., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mao, Li, Chen, Dong, Zhangsun, Zhu and Luo.)

Morphine, the main component of opium, is a commonly used analgesic in clinical practice, but its abuse potential limits its clinical application. Nicotinic acetylcholine receptors (nAChRs) in the mesolimbic circuitry play an important role in the rewarding effects of abused drugs. Previous studies have showed that α6β2* (* designated other subunits) nAChRs are mainly distributed in dopaminergic neurons in the midbrain area, which regulates the release of dopamine. So α6β2* nAChRs are regarded as a new target to treat drug abuse. α-Conotoxin TxIB was discovered in our lab, which is the most selective ligand to inhibit α6β2* nAChRs only. Antagonists of α6β2* nAChRs decreased nicotine, cocaine, and ethanol rewarding effects previously. However, their role in morphine addiction has not been reported so far. Thus, it is worth evaluating the effect of α-conotoxin TxIB on the morphine-induced conditioned place preference (CPP) and its behavioral changes in mice. Our results showed that TxIB inhibited expression and acquisition of morphine-induced CPP and did not produce a rewarding effect by itself. Moreover, repeated injections of TxIB have no effect on learning, memory, locomotor activity, and anxiety-like behavior. Therefore, blocking α6/α3β2β3 nAChRs inhibits the development of morphine-induced CPP. α-Conotoxin TxIB may be a potentially useful compound to mitigate the acquisition and/or retention of drug-context associations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Xiong, Zhang, Zhangsun and Luo.)

Nicotinic acetylcholine receptors (nAChRs) are essential pentameric ligand-gated ion channels that are distributed throughout the central and peripheral nervous systems and non-neuronal tissues in mammalian species that play critical roles in a variety of neural and mental activities. The α3β2 nAChR subtype participates in pain, addiction to nicotine, and other neurophysiological and pathological activities. Owing to the lack of highly selective pharmacological tools targeting α3β2, related research on its tissue distribution and function has been hindered. α-Conotoxin (α-CTx) LtIA, discovered from Conus literatus in our lab, potently and selectively blocks α3β2 nAChR, providing an important molecular probe to study the α3β2 nAChR structure and function. We used the fluorescent molecule 5-carboxytetramethylrhodamine succinimidyl ester, which can react with the N-terminus of LtIA, to obtain a novel fluorescent analogue of LtIA (LtIA-F). The potency and selectivity of LtIA-F were tested using a two-electrode voltage clamp recording on various nAChRs expressed in Xenopus laevis oocytes. LtIA-F potently inhibited ACh-evoked currents at the α3β2 nAChR, with an IC 50 value of 90.66 nM, displaying a ∼4-fold decrease in potency compared with native LtIA without a change in selectivity. The serum stability results indicated that LtIA-F exhibited stability similar to that of native LtIA. This study on an α-CTx LtIA fluorescent analogue provides a wealth of pharmacological tools to explore the structure-function relationship, distribution, and ligand binding domain of the α3β2 nAChR subtype.

Nicotinic acetylcholine receptor (nAChR), a member of pentameric ligand-gated ion channel transmembrane protein composed of five subunits, is widely distributed in the central and peripheral nervous system. The nAChRs are associated with various neurological diseases, including schizophrenia, Alzheimer's disease, Parkinson's disease, epilepsy and neuralgia. Receptors containing the α3 subunit are associated with analgesia, generating our interest in their role in pharmacological studies. In this study, α-conotoxin (α-CTx) LvIF was identified as a 16 amino acid peptide using a genomic DNA clone of Conus lividus (C. lividus) . The mature LvIF with natural structure was synthesized by a two-step oxidation method. The blocking potency of α-CTx lvIF on nAChR was detected by a two-electrode voltage clamp. Our results showed that α-CTx LvIF was highly potent against rα3β2 and rα6/α3β2β3 nAChR subtypes, The half-maximal inhibitory concentration (IC 50 ) values of α-CTx LvIF against rα3β2 and rα6/α3β2β3 nAChRs expressed in Xenopus oocytes were 8.9 nM and 14.4 nM, respectively. Furthermore, α-CTx LvIF exhibited no obvious inhibition on other nAChR subtypes. Meanwhile, we also conducted a competitive binding experiment between α-CTxs MII and LvIF, which showed that α-CTxs LvIF and MII bind with rα3β2 nAChR at the partial overlapping domain. These results indicate that the α-CTx LvIF has high potential as a new candidate tool for the studying of rα3β2 nAChR related neurophysiology and pharmacology.

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 α3β4 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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 (IC 50 : 1570 nM) and rat (IC 50 : 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.

α-Conotoxin TxIB, a selective antagonist of α6/α3β2β3 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/α3β2β3 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.

Tobacco smoking has become a prominent health problem faced around the world. The α3β4 nicotinic acetylcholine receptor (nAChR) is strongly associated with nicotine reward and withdrawal symptom. α-Conotoxin TxID, cloned from Conus textile, is a strong α3β4 nAChR antagonist, which has weak inhibition activity of α6/α3β4 nAChR. Meanwhile, its analogue [S9K]TxID only inhibits α3β4 nAChR (IC 50 = 6.9 nM), and has no inhibitory activity to other nAChRs. The present experiment investigates the effect of α3β4 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 α3β4 nAChR may be a potential target for anti-nicotine addiction treatment. [S9K]TxID, α3β4 nAChR antagonist, exhibit a superior effect for anti-nicotine addiction, which is promising to develop a novel smoking cessation drug.

The α3β2 and α3β4 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the central and peripheral nervous systems, playing critical roles in various physiological processes and in such pathologies as addiction to nicotine and other drugs of abuse. α-Conotoxin LvIA, which we previously isolated from Conus lividus , modestly discriminates α3β2 and α3β4 rat nAChRs exhibiting a ∼17-fold tighter binding to the former. Here, alanine scanning resulted in two more selective analogues [N9A]LvIA and [D11A]LvIA, the former having a >2000-fold higher selectivity for α3β2. The determined crystal structures of [N9A]LvIA and [D11A]LvIA bound to the acetylcholine-binding protein (AChBP) were followed by homologous modeling of the complexes with the α3β2 and α3β4 nAChRs and by receptor mutagenesis, which revealed Phe106, Ser108, Ser113, and Ser168 residues in the β2 subunit as essential for LvIA binding. These results may be useful for the design of novel compounds of therapeutic potential targeting α3β2 nAChRs.

The venom of various Conus species is composed of a rich variety of unique bioactive peptides, commonly referred to as conotoxins (conopeptides). Most conopeptides have specific receptors or ion channels as physiologically relevant targets. In this paper, high-throughput transcriptome sequencing was performed to analyze putative conotoxin transcripts from the venom duct of a vermivorous cone snail species, Conus litteratus native to the South China Sea. A total of 128 putative conotoxins were identified, most of them belonging to 22 known superfamilies, with 43 conotoxins being regarded as belonging to new superfamilies. Notably, the M superfamily was the most abundant in conotoxins among the known superfamilies. A total of 15 known cysteine frameworks were also described. The largest proportion of cysteine frameworks were VI/VII (C-C-CC-C-C), IX (C-C-C-C-C-C) and XIV (C-C-C-C). In addition, five novel cysteine patterns were also discovered. Simple sequence repeat detection results showed that di-nucleotide was the major type of repetition, and the codon usage bias results indicated that the codon usage bias of the conotoxin genes was weak, but the M, O1, O2 superfamilies differed in codon preference. Gene cloning indicated that there was no intron in conotoxins of the B1- or J superfamily, one intron with 1273-1339 bp existed in a mature region of the F superfamily, which is different from the previously reported gene structure of conotoxins from other superfamilies. This study will enhance our understanding of conotoxin diversity, and the new conotoxins discovered in this paper will provide more potential candidates for the development of pharmacological probes and marine peptide drugs.

The α9-containing nicotinic acetylcholine receptor (nAChR) is increasingly emerging as a new tumor target owing to its high expression specificity in breast cancer. αO-Conotoxin GeXIVA is a potent antagonist of α9α10 nAChR. Nevertheless, the anti-tumor effect of GeXIVA on breast cancer cells remains unclear. Cell Counting Kit-8 assay was used to study the cell viability of breast cancer MDA-MD-157 cells and human normal breast epithelial cells, which were exposed to different doses of GeXIVA. Flow cytometry was adopted to detect the cell cycle arrest and apoptosis of GeXIVA in breast cancer cells. Migration ability was analyzed by wound healing assay. Western blot (WB), quantitative real-time PCR (QRT-PCR) and flow cytometry were used to determine expression of α9-nAChR. Stable MDA-MB-157 breast cancer cell line, with the α9-nAChR subunit knocked out (KO), was established using the CRISPR/Cas9 technique. GeXIVA was able to significantly inhibit the proliferation and promote apoptosis of breast cancer MDA-MB-157 cells. Furthermore, the proliferation of breast cancer MDA-MB-157 cells was inhibited by GeXIVA, which caused cell cycle arrest through downregulating α9-nAChR. GeXIVA could suppress MDA-MB-157 cell migration as well. This demonstrates that GeXIVA induced a downregulation of α9-nAChR expression, and the growth of MDA-MB-157 α9-nAChR KO cell line was inhibited as well, due to α9-nAChR deletion. GeXIVA inhibits the growth of breast cancer cell MDA-MB-157 cells in vitro and may occur in a mechanism abolishing α9-nAChR.

α-Conotoxin TxIB specifically blocked α6/α3β2β3 acetylcholine receptors (nAChRs), and it could be a potential probe for studying addiction and other diseases related to α6/α3β2β3 nAChRs. However, as a peptide, TxIB may suffer from low stability, short half-life, and poor bioavailability. In this study, cyclization of TxIB was used to improve its stability. Four cyclic mutants of TxIB (cTxIB) were synthesized, and the inhibition of these analogues on α6/α3β2β3 nAChRs as well as their stability in human serum were measured. All cyclized analogues had similar activity compared to wild-type TxIB, which indicated that backbone cyclization of TxIB had no significant effect on its activity. Cyclization of TxIB with a seven-residue linker improved its stability significantly in human serum. Besides this, the results showed that cyclization maintained the activity of α-conotoxin TxIB, which is conducive to its future application.

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 IC 50 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.

Nicotinic acetylcholine receptors (nAChRs) are membrane receptors and play a major role in tumorigenesis and cancer progression. Here, we have investigated the differential expression of nAChR subunits in human breast cancer cell lines and breast epithelial cell lines at mRNA and protein levels and the effects of the αO-conotoxin GeXIVA, antagonist of α9α10 nAChR, on human breast cancer cells. Reverse transcription polymerase chain reaction (PCR) demonstrated that all nAChR subunits, except α6, were expressed in the 20 tested cell lines. Real time quantitative PCR (QRT-PCR) suggested that the mRNA of α5, α7, α9 and β4 nAChR subunits were overexpressed in all the breast cancer cell lines compared with the normal epithelial cell line HS578BST. α9 nAChR was highly expressed in almost all the breast cancer cell lines in comparison to normal cells. The different expression is prominent ( p < 0.001) as determined by flow cytometry and Western blotting, except for MDA-MB-453 and HCC1395 cell lines. αO-conotoxin GeXIVA that targeted α9α10 nAChR were able to significantly inhibit breast cancer cell proliferation in vitro and merits further investigation as potential agents for targeted therapy.

The α9α10 nicotinic acetylcholine receptor (nAChR) is an effective therapeutic target for neuropathic pain. α-Conotoxin RgIA and Vc1.1 are two well-known peptides blocking α9α10 nAChR potently and selectively, which have been extensively investigated as drug candidates. Several key residues were established in previous experimental research. However, the mechanism of the specific interaction still needs to be elucidated in more detail. In this work, we explored the interaction mechanism between RgIA/Vc1.1 and rat α9α10 nAChR using docking and molecular dynamics (MD) simulations. Energy and network analysis programs were used to reveal key residues responsible for their interaction. Our results indicated that the most critical residues were in accord with previous studies. Importantly, several novel residues, including Tyr95, Trp151 in α9 (+)α10 (-) interface as well as Tyr196, Arg59in α10 (+)α9 (-) interface, were found in our models. Furthermore, we analyzed noncovalent interaction energies between RgIA/Vc1.1 and rat α9α10 nAChR. The results showed that three negatively charged residues (Glu197 in α10 subunit, Asp168 in α9 subunit and Asp205 in α10 subunit) were involved in the interaction between RgIA and rat α9α10 nAChR. In contrast, the interaction between Vc1.1 and rat α9α10 nAChR was mediated by the positively charged residues Arg59, Arg81 in α9 (-) subunit. These findings provided further insights into the molecular mechanisms of interaction between RgIA and Vc1.1 and rat α9α10 nAChR., (Copyright © 2019 Elsevier Inc. All rights reserved.)

α-Conotoxins (α-CTxs) are small disulfide-rich peptides from venom of Conus species that target nicotinic acetylcholine receptors (nAChRs). The muscle-type nAChRs have been recognized as a potential target for several diseases, such as myogenic disorders, muscle dystrophies, and myasthenia gravis. EI, an α4/7-CTx, mainly blocks α1β1δε nAChRs and has an extra N-terminal extension of three amino acids. In this study, the alanine scanning (Ala-scan) mutagenesis was applied in order to identify key residues of EI for binding with mouse α1β1δε nAChR. The Ala-substituted analogues were tested for their abilities of modulating muscle and neuronal nAChRs in Xenopus laevis oocytes using two-electrode voltage clamp (TEVC) recordings. Electrophysiological results indicated that the vital residues for functional activity of EI were His-7, Pro-8, Met-12, and Pro-15. These changes exhibited a significant decrease in potency of EI against mouse α1β1δε nAChR. Interestingly, replacing the critical serine (Ser) at position 13 with an alanine (Ala) residue resulted in a 2-fold increase in potency at the α1β1δε nAChR, and showed loss of activity on α3β2 and α3β4 nAChRs. Selectivity and potency of [S13A] EI was improved compared with wild-type EI (WT EI). In addition, the structure-activity relationship (SAR) of EI revealed that the "Arg1-Asn2-Hyp3" residues at the N-terminus conferred potency at the muscle-type nAChRs, and the deletion analogue △1-3 EI caused a total loss of activity at the α1β1δε nAChR. Circular dichroism (CD) spectroscopy studies demonstrated that activity loss of truncated analogue △1-3 EI for α1β1δε nAChR is attributed to disturbance of the secondary structure. In this report, an Ala-scan mutagenesis strategy is presented to identify crucial residues that are significantly affecting potency of E1 for mouse α1β1δε nAChR. It may also be important in remodeling of some novel ligands for inhibiting muscle-type nAChRs.

α-Conotoxin (CTx) TxID is a potent α3β4 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 α3β4 nAChR was analyzed using electrophysiological methods. Our analysis of TxID stability may aid the selection of appropriate conditions for peptide production, packaging and storage., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

A bstract: α-Conotoxin TxIB is a specific antagonist of α6/α3β2β3(α6β2*) nicotinic acetylcholine receptor (nAChR) with an IC 50 of 28 nM. Previous studies have shown that α6β2* 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 α6β2* nAChR-targeted compound may be a promising drug for nicotine addiction treatment., Competing Interests: All the authors declare that they have no conflicts of interest.

In order to improve stability of a peptide marine drug lead, α-conotoxin TxID, we synthesized and modified TxID at the N-terminal with DSPE-PEG-NHS by a nucleophilic substitution reaction to prepare the DSPE-PEG-TxID for the first time. The reaction conditions, including solvent, ratio, pH, and reaction time, were optimized systematically and the optimal one was reacted in dimethyl formamide at pH 8.2 with triethylamine at room temperature for 120 h. The in vitro stabilities in serum, simulated gastric juice, and intestinal fluid were tested, and improved dramatically compared with TxID. The PEG-modified peptide was functionally tested on α3β4 nicotinic acetylcholine receptor (nAChR) heterologously expressed in Xenopus laevis oocytes. The DSPE-PEG-TxID showed an obvious inhibition effect on α3β4 nAChR. All in all, the PEG modification of TxID was improved in stability, resistance to enzymatic degradation, and may prolong the half-life in vivo, which may pave the way for the future application in smoking cessation and drug rehabilitation, as well as small cell lung cancer., Competing Interests: References

Oxaliplatin is a third-generation platinum drug and is widely used as a first-line therapy for the treatment of colorectal cancer (CRC). However, a large number of patients receiving oxaliplatin develop dose-limiting painful neuropathy. Here, we report that αO-conotoxin GeXIVA[1,2], a highly potent and selective antagonist of the α9α10 nicotinic acetylcholine receptor (nAChR) subtype, can relieve and reverse oxaliplatin-induced mechanical and cold allodynia after single and repeated intramuscular (IM) injections in rats. Treatments were started at 4 days post oxaliplatin injection when neuropathic pain emerged and continued for 8 and 16 days. Cold score and mechanical paw withdrawal threshold (PWT) were detected by the acetone test and von Frey test respectively. GeXIVA[1,2] significantly relieved mechanical and cold allodynia in oxaliplatin-treated rats after a single injection. After repeated treatments, GeXIVA[1,2] produced a cumulative analgesic effect without tolerance and promoted recovery from neuropathic pain. Moreover, the long lasting analgesic effect of GeXIVA[1,2] on mechanical allodynia continued until day 10 after the termination of the 16-day repeated treatment procedure. On the contrary, GeXIVA[1,2] did not affect acute mechanical and thermal pain behaviors in normal rats after repeated injections detected by the von Frey test and tail flick test. GeXIVA[1,2] had no influence on rat hind limb grip strength and body weight after repeated treatments. These results indicate that αO-conotoxin GeXIVA[1,2] could provide a novel strategy to treat chemotherapy-induced neuropathic pain.

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 β4 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 α3β4 nAChR were able to significantly inhibit cervical cancer cell proliferation. These findings may provide a basis for new targets for cervical cancer targeted therapy.