9 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
- View/download PDF
3. 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
- Full Text
- View/download PDF
4. 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
- View/download PDF
5. 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
6. Pain-causing stinging nettle toxins target TMEM233 to modulate NaV1.7 function.
- Author
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Jami, Sina, Deuis, Jennifer R., Klasfauseweh, Tabea, Cheng, Xiaoyang, Kurdyukov, Sergey, Chung, Felicity, Okorokov, Andrei L., Li, Shengnan, Zhang, Jiangtao, Cristofori-Armstrong, Ben, Israel, Mathilde R., Ju, Robert J., Robinson, Samuel D., Zhao, Peng, Ragnarsson, Lotten, Andersson, Åsa, Tran, Poanna, Schendel, Vanessa, McMahon, Kirsten L., and Tran, Hue N. T.
- Subjects
SODIUM channels ,STINGING nettle ,TOXINS ,MEMBRANE proteins ,PEPTIDES ,SENSORY neurons - 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. Voltage-gated sodium channels function as multiprotein signaling complexes. Here, authors show that the dispanin TMEM233 is essential for activity of stinging nettle toxins and that co-expression of TMEM233 modulates the gating properties of NaV 1.7. [ABSTRACT FROM AUTHOR]- Published
- 2023
- Full Text
- View/download PDF
7. On the Utility of Chemical Strategies to Improve Peptide Gut Stability
- Author
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Kremsmayr, Thomas, Aljnabi, Aws, Blanco-Canosa, Juan B., Tran, Hue N. T., Emidio, Nayara Braga, Muttenthaler, Markus, and European Commission
- Subjects
Scaffolds ,Degradation ,Chemical Strategies ,Cyclization ,Monomers ,Drug Discovery ,Molecular Medicine ,Cyclotides ,Peptides and proteins ,Amino Acid Sequence ,Peptides ,Stability - 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., We thank Marina Kujundzic and Johanna Nemec for their help in collecting some of the stability data and peptide synthesis. We are grateful to the laboratory of Prof. David Craik (The University of Queensland) for providing Vc1.1 and cVc1.1. We thank Prof. Christian F.W. Becker (Institute of Biological Chemistry, University of Vienna) for his support of this work. We also thank Dr. Martin Zehl and the Mass Spectrometry Centre at the University of Vienna (a member of Vienna Life-Science Instruments) for assistance with HR-MS analysis. We are grateful to the NIMH PDSP (National Institute of Mental Health’s Psychoactive Drug Screening Program, Contract # HHSN-271-2018-00023-C), which is directed by Bryan L. Roth at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA, for providing agonist functional activity data of OT variants. This research was supported by the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreements no. 714366), by the Australian Research Council Discovery Project (DP190101667) and by the Vienna Science and Technology Fund (WWTF) through project LS18-053. T. Kremsmayr was supported by the Austrian Academy of Sciences through a DOC Fellowship (25139). J.B.B.-C. thanks the Spanish Ministry of Science and Innovation (RTI2018-096323-B-100).
- Published
- 2022
8. µ-Conotoxins Targeting the Human Voltage-Gated Sodium Channel Subtype Na V 1.7.
- Author
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McMahon, Kirsten L., Tran, Hue N. T., Deuis, Jennifer R., Craik, David J., Vetter, Irina, and Schroeder, Christina I.
- Subjects
- *
SODIUM channels , *CONOTOXINS , *PEPTIDES , *STRUCTURE-activity relationships , *IN vivo studies , *HUMAN beings - Abstract
µ-Conotoxins are small, potent, peptide voltage-gated sodium (NaV) 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 NaV1.7 has so far been limited. We recently identified a novel µ-conotoxin, SxIIIC, which potently inhibits human NaV1.7 (hNaV1.7). 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 hNaV1.7. Analysis of other µ-conotoxins at hNaV1.7 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 hNaV1.7. Through mutagenesis studies, we confirmed that charged residues in this region also affect the selectivity for hNaV1.4. Comparison of µ-conotoxin NMR solution structures identified differences that may contribute to the variance in hNaV1.7 inhibition and validated the role of the loop 1 extension in SxIIIC for improving potency at hNaV1.7, when compared to KIIIA. This work could assist in designing µ-conotoxin derivatives specific for hNaV1.7. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
9. The Allosteric Activation of α7 nAChR by α-Conotoxin MrIC Is Modified by Mutations at the Vestibular Site.
- Author
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Gulsevin, Alican, Papke, Roger L., Stokes, Clare, Tran, Hue N. T., Jin, Aihua H., Vetter, Irina, and Meiler, Jens
- Subjects
CONOTOXINS ,NICOTINIC acetylcholine receptors ,AMINO acids ,PEPTIDES - 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. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
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