The α3ß4 nAChR tissue distribution identified by fluorescent α-conotoxin [D11A]LvIA.

α3ß4, 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 α3ß4 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 α3ß4 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 IC50 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 α3ß4 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 α3ß4 nAChRs in different tissues and also assists in clarifying the function.

Single Amino Acid Substitution in Loop1 Switches the Selectivity of α-Conotoxin RegIIA towards the α7 Nicotinic Acetylcholine Receptor.

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 IC50s of 446 nM and 459 nM, respectively, while their inhibition against rα3ß2 and rα3ß4 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.

Stapling Cysteine[2,4] Disulfide Bond of α-Conotoxin LsIA and Its Potential in Target Delivery.

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 α3ß2 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.

Conotoxins Targeting Voltage-Gated Sodium Ion Channels.

Voltage-gated sodium (NaV) 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 NaV 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 NaV 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 NaV channels or as drug leads for neurologic diseases related to NaV channels. Accordingly, discovering or designing conotoxins targeting NaV 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 NaV channel-targeted conotoxins, focusing on their structures, activities, and designed modifications, with a view toward expanding their applications. SIGNIFICANCE STATEMENT: NaV channels are crucial in various neurologic diseases. Some conotoxins selectively target NaV 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 NaV channels have both immediate research value and promising future application prospects.

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

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

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

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

Characterization and evaluation of cytotoxic and antimicrobial activities of cyclotides from Viola japonica.

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 IC50 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.

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

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

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

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

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

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

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

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

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

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

Substitution of D-Arginine at Position 11 of α-RgIA Potently Inhibits α7 Nicotinic Acetylcholine Receptor.

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.

Potential Mechanisms of Metformin-Induced Apoptosis in HeLa Cells.

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.

Mitogenome Characterization of Four Conus Species and Comparative Analysis.

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.

Loop2 Size Modification Reveals Significant Impacts on the Potency of α-Conotoxin TxID.

α4/6-conotoxin TxID, which was identified from Conus textile, simultaneously blocks rat (r) α3ß4 and rα6/α3ß4 nicotinic acetylcholine receptors (nAChRs) with IC50 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 rα3ß4 and rα6/α3ß4 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.

Molecular Determinants of Species Specificity of α-Conotoxin TxIB towards Rat and Human α6/α3ß4 Nicotinic Acetylcholine Receptors.

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/α3ß2ß3 nAChR (IC50 = 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/α3ß2ß3 nAChR but also human α6/α3ß4 nAChR, with an IC50 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 ß4 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/α3ß4 nAChRs and their mutants were evaluated through electrophysiological experiments. The results showed that the IC50 of TxIB against h[α6V32L, K61R/α3]ß4L107V, V115I was 22.5 µM, a 42-fold decrease in potency compared to the native hα6/α3ß4 nAChR. Val-32 and Lys-61 in the human α6/α3 subunit and Leu-107 and Val-115 in the human ß4 subunit, together, were found to determine the species differences in the α6/α3ß4 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.

Synthesis of the Most Potent Isomer of µ-Conotoxin KIIIA Using Different Strategies.

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.

Development of sertraline analogues as potential anti-ischemic stroke agents.

Ischemic stroke (IS) is harmful to human health and social development, and there is no medicine available at present. To find the hit compound for treating ischemic stroke, we screened 28 FDA approved nervous system drugs by using an in vitro OGD-induced stroke model. Notably, our in vitro and in vivo studies demonstrated that low-dose sertraline had good neuroprotective activities, while high-dose sertraline showed significant toxicity. Interestingly, the same high-dose sertraline in the control group did not exhibit any obvious toxic effect. Therefore, it is important to modify the structure of sertraline to improve the activity and reduce the toxicity. Stereoisomers of sertraline were first investigated to analyze the influence of stereochemistry on the neuroprotective activities, which showed no obvious difference. Then we evaluated the activity of our previously reported sertraline analogues and found that introducing amide or alkane groups to the amino moiety might be beneficial to enhance the activity and reduce the toxicity. Thus, 10 new analogues were designed, synthesized, and evaluated. Among them, compound OY-201 showed the best safety and neuroprotective activity in both in vitro and in vivo models. Moreover, it exhibited good blood-brain barrier (BBB) permeability, indicating its potential for the development of anti-ischemic stroke drugs.

Discovery, Characterization, and Engineering of LvIC, an α4/4-Conotoxin That Selectively Blocks Rat α6/α3ß4 Nicotinic Acetylcholine Receptors.

α6ß4 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 α6ß4 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/α3ß4 nAChRs (α6/α3 is a chimera) with an IC50 of 19 nM, with minimal activity at other nAChR subtypes, including the structurally similar α6/α3ß2ß3 and α3ß4 subtypes. Using NMR, molecular docking, and receptor mutation, structure-activity relationships of [D1G,ΔQ14]LvIC at the α6/α3ß4 nAChR were defined. It is a potent and specific antagonist of α6ß4 nAChRs that could potentially serve as a novel molecular probe to explore α6ß4 nAChR-related neurophysiological and pharmacological functions.