13 results on '"Tran, Hue N. T."'
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2. Changes in Potency and Subtype Selectivity of Bivalent Na V Toxins are Knot-Specific.
- Author
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Tran P, Tran HNT, McMahon KL, Deuis JR, Ragnarsson L, Norman A, Sharpe SJ, Payne RJ, Vetter I, and Schroeder CI
- Subjects
- Humans, Peptides pharmacology
- Abstract
Disulfide-rich peptide toxins have long been studied for their ability to inhibit voltage-gated sodium channel subtype Na
V 1.7, a validated target for the treatment of pain. In this study, we sought to combine the pore blocking activity of conotoxins with the gating modifier activity of spider toxins to design new bivalent inhibitors of NaV 1.7 with improved potency and selectivity. To do this, we created an array of heterodimeric toxins designed to target human NaV 1.7 by ligating a conotoxin to a spider toxin and assessed the potency and selectivity of the resulting bivalent toxins. A series of spider-derived gating modifier toxins (GpTx-1, ProTx-II, gHwTx-IV, JzTx-V, CcoTx-1, and Pn3a) and two pore-blocker μ-conotoxins, SxIIIC and KIIIA, were used for this study. We employed either enzymatic ligation with sortase A for C- to N-terminal ligation or click chemistry for N- to N-terminal ligation. The bivalent peptide resulting from ligation of ProTx-II and SxIIIC (Pro[LPATG6 ]Sx) was shown to be the best combination as native ProTx-II potency at hNaV 1.7 was conserved following ligation. At hNaV 1.4, a synergistic effect between the pore blocker and gating modifier toxin moieties was observed, resulting in altered sodium channel subtype selectivity compared to the parent peptides. Further studies including mutant bivalent peptides and mutant hNaV 1.7 channels suggested that gating modifier toxins have a greater contribution to the potency of the bivalent peptides than pore blockers. This study delineated potential benefits and drawbacks of designing pharmacological hybrid peptides targeting hNaV 1.7.- Published
- 2023
- Full Text
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3. Pain-causing stinging nettle toxins target TMEM233 to modulate Na V 1.7 function.
- Author
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Jami S, Deuis JR, Klasfauseweh T, Cheng X, Kurdyukov S, Chung F, Okorokov AL, Li S, Zhang J, Cristofori-Armstrong B, Israel MR, Ju RJ, Robinson SD, Zhao P, Ragnarsson L, Andersson Å, Tran P, Schendel V, McMahon KL, Tran HNT, Chin YK, Zhu Y, Liu J, Crawford T, Purushothamvasan S, Habib AM, Andersson DA, Rash LD, Wood JN, Zhao J, Stehbens SJ, Mobli M, Leffler A, Jiang D, Cox JJ, Waxman SG, Dib-Hajj SD, Neely GG, Durek T, and Vetter I
- Subjects
- Australia, Pain, Peptides, NAV1.7 Voltage-Gated Sodium Channel metabolism, Toxins, Biological, Urtica dioica
- Abstract
Voltage-gated sodium (Na
V ) channels are critical regulators of neuronal excitability and are targeted by many toxins that directly interact with the pore-forming α subunit, typically via extracellular loops of the voltage-sensing domains, or residues forming part of the pore domain. Excelsatoxin A (ExTxA), a pain-causing knottin peptide from the Australian stinging tree Dendrocnide excelsa, is the first reported plant-derived NaV channel modulating peptide toxin. Here we show that TMEM233, a member of the dispanin family of transmembrane proteins expressed in sensory neurons, is essential for pharmacological activity of ExTxA at NaV channels, and that co-expression of TMEM233 modulates the gating properties of NaV 1.7. These findings identify TMEM233 as a previously unknown NaV 1.7-interacting protein, position TMEM233 and the dispanins as accessory proteins that are indispensable for toxin-mediated effects on NaV channel gating, and provide important insights into the function of NaV channels in sensory neurons., (© 2023. The Author(s).)- Published
- 2023
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4. µ-Conotoxins Targeting the Human Voltage-Gated Sodium Channel Subtype Na V 1.7.
- Author
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McMahon KL, Tran HNT, Deuis JR, Craik DJ, Vetter I, and Schroeder CI
- Subjects
- Analgesics chemistry, Analgesics pharmacology, Animals, Cysteine, Humans, NAV1.4 Voltage-Gated Sodium Channel, NAV1.7 Voltage-Gated Sodium Channel, Peptides, Conotoxins chemistry, Conotoxins pharmacology, Voltage-Gated Sodium Channel Blockers chemistry, Voltage-Gated Sodium Channel Blockers pharmacology
- Abstract
µ-Conotoxins are small, potent, peptide voltage-gated sodium (Na
V ) channel inhibitors characterised by a conserved cysteine framework. Despite promising in vivo studies indicating analgesic potential of these compounds, selectivity towards the therapeutically relevant subtype NaV 1.7 has so far been limited. We recently identified a novel µ-conotoxin, SxIIIC, which potently inhibits human NaV 1.7 (hNaV 1.7). SxIIIC has high sequence homology with other µ-conotoxins, including SmIIIA and KIIIA, yet shows different NaV channel selectivity for mammalian subtypes. Here, we evaluated and compared the inhibitory potency of µ-conotoxins SxIIIC, SmIIIA and KIIIA at hNaV channels by whole-cell patch-clamp electrophysiology and discovered that these three closely related µ-conotoxins display unique selectivity profiles with significant variations in inhibitory potency at hNaV 1.7. Analysis of other µ-conotoxins at hNaV 1.7 shows that only a limited number are capable of inhibition at this subtype and that differences between the number of residues in loop 3 appear to influence the ability of µ-conotoxins to inhibit hNaV 1.7. Through mutagenesis studies, we confirmed that charged residues in this region also affect the selectivity for hNaV 1.4. Comparison of µ-conotoxin NMR solution structures identified differences that may contribute to the variance in hNaV 1.7 inhibition and validated the role of the loop 1 extension in SxIIIC for improving potency at hNaV 1.7, when compared to KIIIA. This work could assist in designing µ-conotoxin derivatives specific for hNaV 1.7.- Published
- 2022
- Full Text
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5. On the Utility of Chemical Strategies to Improve Peptide Gut Stability.
- Author
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Kremsmayr T, Aljnabi A, Blanco-Canosa JB, Tran HNT, Emidio NB, and Muttenthaler M
- Subjects
- Amino Acid Sequence, Cyclization, Cyclotides chemistry
- Abstract
Inherent susceptibility of peptides to enzymatic degradation in the gastrointestinal tract is a key bottleneck in oral peptide drug development. Here, we present a systematic analysis of (i) the gut stability of disulfide-rich peptide scaffolds, orally administered peptide therapeutics, and well-known neuropeptides and (ii) medicinal chemistry strategies to improve peptide gut stability. Among a broad range of studied peptides, cyclotides were the only scaffold class to resist gastrointestinal degradation, even when grafted with non-native sequences. Backbone cyclization, a frequently applied strategy, failed to improve stability in intestinal fluid, but several site-specific alterations proved efficient. This work furthermore highlights the importance of standardized gut stability test conditions and suggests defined protocols to facilitate cross-study comparison. Together, our results provide a comparative overview and framework for the chemical engineering of gut-stable peptides, which should be valuable for the development of orally administered peptide therapeutics and molecular probes targeting receptors within the gastrointestinal tract.
- Published
- 2022
- Full Text
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6. Structural and functional insights into the inhibition of human voltage-gated sodium channels by μ-conotoxin KIIIA disulfide isomers.
- Author
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Tran HNT, McMahon KL, Deuis JR, Vetter I, and Schroeder CI
- Subjects
- Disulfides chemistry, Disulfides pharmacology, Humans, Molecular Docking Simulation, Structure-Activity Relationship, Conotoxins chemistry, Conotoxins pharmacology, Voltage-Gated Sodium Channel Blockers chemistry, Voltage-Gated Sodium Channel Blockers pharmacology, Voltage-Gated Sodium Channels chemistry, Voltage-Gated Sodium Channels metabolism
- Abstract
μ-Conotoxins are components of cone snail venom, well-known for their analgesic activity through potent inhibition of voltage-gated sodium channel (Na
V ) subtypes, including NaV 1.7. These small, disulfide-rich peptides are typically stabilized by three disulfide bonds arranged in a 'native' CysI-CysIV, CysII-CysV, CysIII-CysVI pattern of disulfide connectivity. However, μ-conotoxin KIIIA, the smallest and most studied μ-conotoxin with inhibitory activity at NaV 1.7, forms two distinct disulfide bond isomers during thermodynamic oxidative folding, including Isomer 1 (CysI-CysV, CysII-CysIV, CysIII-CysVI) and Isomer 2 (CysI-CysVI, CysII-CysIV, CysIII-CysV), but not the native μ-conotoxin arrangement. To date, there has been no study on the structure and activity of KIIIA comprising the native μ-conotoxin disulfide bond arrangement. Here, we evaluated the synthesis, potency, sodium channel subtype selectivity, and 3D structure of the three isomers of KIIIA. Using a regioselective disulfide bond-forming strategy, we synthetically produced the three μ-conotoxin KIIIA isomers displaying distinct bioactivity and NaV subtype selectivity across human NaV channel subtypes 1.2, 1.4, and 1.7. We show that Isomer 1 inhibits NaV subtypes with a rank order of potency of NaV 1.4 > 1.2 > 1.7 and Isomer 2 in the order of NaV 1.4≈1.2 > 1.7, while the native isomer inhibited NaV 1.4 > 1.7≈1.2. The three KIIIA isomers were further evaluated by NMR solution structure analysis and molecular docking with hNaV 1.2. Our study highlights the importance of investigating alternate disulfide isomers, as disulfide connectivity affects not only the overall structure of the peptides but also the potency and subtype selectivity of μ-conotoxins targeting therapeutically relevant NaV subtypes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022. Published by Elsevier Inc.)- Published
- 2022
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7. Evaluation of Efficient Non-reducing Enzymatic and Chemical Ligation Strategies for Complex Disulfide-Rich Peptides.
- Author
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Tran HNT, Tran P, Deuis JR, McMahon KL, Yap K, Craik DJ, Vetter I, and Schroeder CI
- Subjects
- Bacterial Proteins chemistry, Bacterial Proteins metabolism, Click Chemistry, Animals, Humans, Amino Acid Sequence, Disulfides chemistry, Peptides chemistry, Peptides pharmacology, Aminoacyltransferases metabolism, Aminoacyltransferases chemistry, Cysteine Endopeptidases metabolism, Cysteine Endopeptidases chemistry
- Abstract
Double-knotted peptides identified in venoms and synthetic bivalent peptide constructs targeting ion channels are emerging tools for the study of ion channel pharmacology and physiology. These highly complex and disulfide-rich peptides contain two individual cystine knots, each comprising six cysteines and three disulfide bonds. Until now, native double-knotted peptides, such as Hi1a and DkTx, have only been isolated from venom or produced recombinantly, whereas engineered double-knotted peptides have successfully been produced through enzymatic ligation using sortase A to form a seamless amide bond at the ligation site between two knotted toxins, and by alkyne/azide click chemistry, joining two peptide knots via a triazole linkage. To further pursue these double-knotted peptides as pharmacological tools or probes for therapeutically relevant ion channels, we sought to identify a robust methodology resulting in a high yield product that lends itself to rapid production and facile mutational studies. In this study, we evaluated the ligation efficiency of enzymatic (sortase A5°, butelase 1, wild-type OaAEP 1, C247A-OaAEP 1, and peptiligase) and mild chemical approaches (α-ketoacid-hydroxylamine, KAHA) for forming a native amide bond linking the toxins while maintaining the native disulfide connectivity of each pre-folded peptide. We used two Na
V 1.7 inhibitors: PaurTx3, a spider-derived gating modifier peptide, and KIIIA, a small cone snail-derived pore blocker peptide, which have previously been shown to increase affinity and inhibitory potency on hNaV 1.7 when ligated together. Correctly folded peptides were successfully ligated in varying yields, without disulfide bond shuffling or reduction, with sortase A5° being the most efficient, resulting in 60% ligation conversion within 15 min. In addition, electrophysiology studies demonstrated that for these two peptides, the amino acid composition of the linker did not affect the activity of the double-knotted peptides. This study demonstrates the powerful application of enzymes in efficiently ligating complex disulfide-rich peptides, paving the way for facile production of double-knotted peptides.- Published
- 2021
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8. The Allosteric Activation of α7 nAChR by α-Conotoxin MrIC Is Modified by Mutations at the Vestibular Site.
- Author
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Gulsevin A, Papke RL, Stokes C, Tran HNT, Jin AH, Vetter I, and Meiler J
- Subjects
- Allosteric Regulation drug effects, Animals, Binding Sites, Female, Molecular Docking Simulation, Mutation, Oocytes, Xenopus laevis, alpha7 Nicotinic Acetylcholine Receptor chemistry, alpha7 Nicotinic Acetylcholine Receptor genetics, Conotoxins pharmacology, alpha7 Nicotinic Acetylcholine Receptor metabolism
- Abstract
α-conotoxins are 13-19 amino acid toxin peptides that bind various nicotinic acetylcholine receptor (nAChR) subtypes. α-conotoxin Mr1.7c (MrIC) is a 17 amino acid peptide that targets α7 nAChR. Although MrIC has no activating effect on α7 nAChR when applied by itself, it evokes a large response when co-applied with the type II positive allosteric modulator PNU-120596, which potentiates the α7 nAChR response by recovering it from a desensitized state. A lack of standalone activity, despite activation upon co-application with a positive allosteric modulator, was previously observed for molecules that bind to an extracellular domain allosteric activation (AA) site at the vestibule of the receptor. We hypothesized that MrIC may activate α7 nAChR allosterically through this site. We ran voltage-clamp electrophysiology experiments and in silico peptide docking calculations in order to gather evidence in support of α7 nAChR activation by MrIC through the AA site. The experiments with the wild-type α7 nAChR supported an allosteric mode of action, which was confirmed by the significantly increased MrIC + PNU-120596 responses of three α7 nAChR AA site mutants that were designed in silico to improve MrIC binding. Overall, our results shed light on the allosteric activation of α7 nAChR by MrIC and suggest the involvement of the AA site.
- Published
- 2021
- Full Text
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9. Chemical Synthesis of TFF3 Reveals Novel Mechanistic Insights and a Gut-Stable Metabolite.
- Author
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Braga Emidio N, Meli R, Tran HNT, Baik H, Morisset-Lopez S, Elliott AG, Blaskovich MAT, Spiller S, Beck-Sickinger AG, Schroeder CI, and Muttenthaler M
- Subjects
- Biophysical Phenomena, HEK293 Cells, Humans, Molecular Structure, Oxidation-Reduction, Protein Folding, Structure-Activity Relationship, Trefoil Factor-3 chemistry, Trefoil Factor-3 chemical synthesis, Trefoil Factor-3 metabolism
- Abstract
TFF3 regulates essential gastro- and neuroprotective functions, but its molecular mode of action remains poorly understood. Synthetic intractability and lack of reliable bioassays and validated receptors are bottlenecks for mechanistic and structure-activity relationship studies. Here, we report the chemical synthesis of TFF3 and its homodimer via native chemical ligation followed by oxidative folding. Correct folding was confirmed by NMR and circular dichroism, and TFF3 and its homodimer were not cytotoxic or hemolytic. TFF3, its homodimer, and the trefoil domain (TFF3
10-50 ) were susceptible to gastrointestinal degradation, revealing a gut-stable metabolite (TFF37-54 ; t1/2 > 24 h) that retained its trefoil structure and antiapoptotic bioactivity. We tried to validate the putative TFF3 receptors CXCR4 and LINGO2, but neither TFF3 nor its homodimer displayed any activity up to 10 μM. The discovery of a gut-stable bioactive metabolite and reliable synthetic accessibility to TFF3 and its analogues are cornerstones for future molecular probe development and structure-activity relationship studies.- Published
- 2021
- Full Text
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10. Improving the Gastrointestinal Stability of Linaclotide.
- Author
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Braga Emidio N, Tran HNT, Andersson A, Dawson PE, Albericio F, Vetter I, and Muttenthaler M
- Subjects
- Cell Line, Drug Design, Drug Stability, Gastrointestinal Agents chemical synthesis, Guanylyl Cyclase C Agonists chemical synthesis, Humans, Peptides chemical synthesis, Proteolysis, Gastrointestinal Agents metabolism, Guanylyl Cyclase C Agonists metabolism, Peptides metabolism
- Abstract
High susceptibility to proteolytic degradation in the gastrointestinal tract limits the therapeutic application of peptide drugs in gastrointestinal disorders. Linaclotide is an orally administered peptide drug for the treatment of irritable bowel syndrome with constipation (IBS-C) and abdominal pain. Linaclotide is however degraded in the intestinal environment within 1 h, and improvements in gastrointestinal stability might enhance its therapeutic application. We therefore designed and synthesized a series of linaclotide analogues employing a variety of strategic modifications and evaluated their gastrointestinal stability and pharmacological activity at its target receptor guanylate cyclase-C. All analogues had substantial improvements in gastrointestinal half-lives (>8 h vs linaclotide 48 min), and most remained active at low nanomolar concentrations. This work highlights strategic approaches for the development of gut-stable peptides toward the next generation of orally administered peptide drugs for the treatment of gastrointestinal disorders.
- Published
- 2021
- Full Text
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11. Discovery, Pharmacological Characterisation and NMR Structure of the Novel µ-Conotoxin SxIIIC, a Potent and Irreversible Na V Channel Inhibitor.
- Author
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McMahon KL, Tran HNT, Deuis JR, Lewis RJ, Vetter I, and Schroeder CI
- Abstract
Voltage-gated sodium (Na
V ) channel subtypes, including NaV 1.7, are promising targets for the treatment of neurological diseases, such as chronic pain. Cone snail-derived µ-conotoxins are small, potent NaV channel inhibitors which represent potential drug leads. Of the 22 µ-conotoxins characterised so far, only a small number, including KIIIA and CnIIIC, have shown inhibition against human NaV 1.7. We have recently identified a novel µ-conotoxin, SxIIIC, from Conus striolatus . Here we present the isolation of native peptide, chemical synthesis, characterisation of human NaV channel activity by whole-cell patch-clamp electrophysiology and analysis of the NMR solution structure. SxIIIC displays a unique NaV channel selectivity profile (1.4 > 1.3 > 1.1 ≈ 1.6 ≈ 1.7 > 1.2 >> 1.5 ≈ 1.8) when compared to other µ-conotoxins and represents one of the most potent human NaV 1.7 putative pore blockers (IC50 152.2 ± 21.8 nM) to date. NMR analysis reveals the structure of SxIIIC includes the characteristic α-helix seen in other µ-conotoxins. Future investigations into structure-activity relationships of SxIIIC are expected to provide insights into residues important for NaV channel pore blocker selectivity and subsequently important for chronic pain drug development.- Published
- 2020
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12. Manipulation of a spider peptide toxin alters its affinity for lipid bilayers and potency and selectivity for voltage-gated sodium channel subtype 1.7.
- Author
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Agwa AJ, Tran P, Mueller A, Tran HNT, Deuis JR, Israel MR, McMahon KL, Craik DJ, Vetter I, and Schroeder CI
- Subjects
- Animals, Male, Mice, Mice, Inbred C57BL, NAV1.7 Voltage-Gated Sodium Channel chemistry, NAV1.7 Voltage-Gated Sodium Channel drug effects, Scorpion Venoms toxicity, Lipid Bilayers metabolism, NAV1.7 Voltage-Gated Sodium Channel metabolism, Nociception drug effects, Peptide Fragments pharmacology, Spider Venoms pharmacology
- Abstract
Huwentoxin-IV (HwTx-IV) is a gating modifier peptide toxin from spiders that has weak affinity for the lipid bilayer. As some gating modifier toxins have affinity for model lipid bilayers, a tripartite relationship among gating modifier toxins, voltage-gated ion channels, and the lipid membrane surrounding the channels has been proposed. We previously designed an HwTx-IV analogue (gHwTx-IV) with reduced negative charge and increased hydrophobic surface profile, which displays increased lipid bilayer affinity and in vitro activity at the voltage-gated sodium channel subtype 1.7 (Na
V 1.7), a channel targeted in pain management. Here, we show that replacements of the positively-charged residues that contribute to the activity of the peptide can improve gHwTx-IV's potency and selectivity for NaV 1.7. Using HwTx-IV, gHwTx-IV, [R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV variants, we examined their potency and selectivity at human NaV 1.7 and their affinity for the lipid bilayer. [R26A]gHwTx-IV consistently displayed the most improved potency and selectivity for NaV 1.7, examined alongside off-target NaV s, compared with HwTx-IV and gHwTx-IV. The lipid affinity of each of the three novel analogues was weaker than that of gHwTx-IV, but stronger than that of HwTx-IV, suggesting a possible relationship between in vitro potency at NaV 1.7 and affinity for lipid bilayers. In a murine NaV 1.7 engagement model, [R26A]gHwTx-IV exhibited an efficacy comparable with that of native HwTx-IV. In summary, this study reports the development of an HwTx-IV analogue with improved in vitro selectivity for the pain target NaV 1.7 and with an in vivo efficacy similar to that of native HwTx-IV.- Published
- 2020
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13. Enzymatic Ligation of a Pore Blocker Toxin and a Gating Modifier Toxin: Creating Double-Knotted Peptides with Improved Sodium Channel Na V 1.7 Inhibition.
- Author
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Tran HNT, Tran P, Deuis JR, Agwa AJ, Zhang AH, Vetter I, and Schroeder CI
- Subjects
- Amino Acid Sequence, Animals, HEK293 Cells, Humans, Models, Molecular, Mollusk Venoms chemistry, Mollusk Venoms pharmacology, Snails chemistry, Spider Venoms chemistry, Spider Venoms pharmacology, Spiders chemistry, NAV1.7 Voltage-Gated Sodium Channel metabolism, Peptides chemistry, Peptides pharmacology, Voltage-Gated Sodium Channel Blockers chemistry, Voltage-Gated Sodium Channel Blockers pharmacology
- Abstract
Disulfide-rich animal venom peptides targeting either the voltage-sensing domain or the pore domain of voltage-gated sodium channel 1.7 (Na
V 1.7) have been widely studied as drug leads and pharmacological probes for the treatment of chronic pain. However, despite intensive research efforts, the full potential of NaV 1.7 as a therapeutic target is yet to be realized. In this study, using evolved sortase A, we enzymatically ligated two known NaV 1.7 inhibitors-PaurTx3, a spider-derived peptide toxin that modifies the gating mechanism of the channel through interaction with the voltage-sensing domain, and KIIIA, a small cone snail-derived peptide inhibitor of the pore domain-with the aim of creating a bivalent inhibitor which could interact simultaneously with two noncompeting binding sites. Using electrophysiology, we determined the activity at NaV 1.7, and to maximize potency, we systematically evaluated the optimal linker length, which was nine amino acids. Our optimized synthetic bivalent peptide showed improved channel affinity and potency at NaV 1.7 compared to either PaurTx3 or KIIIA individually. This work shows that novel and improved NaV 1.7 inhibitors can be designed by combining a pore blocker toxin and a gating modifier toxin to confer desired pharmacological properties from both the voltage sensing domain and the pore domain.- Published
- 2020
- Full Text
- View/download PDF
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