26 results on '"Ching-I. A. Wang"'
Search Results
2. Stabilisierung eines cysteinreichen Kegelschneckentoxins, MrIA, in Form eines 1,2,3-Triazol-Disulfidbrückenmimetikums
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Ching-I Anderson Wang, Richard J. Lewis, Alessandro Gori, Renato Longhi, Maria Luisa Gelmi, Rebecca F. Bhola, Paul F. Alewood, K. Johan Rosengren, MacDonald J. Christie, Peta J. Harvey, and Andreas Brust
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General Medicine - Abstract
Die Synthese von Disulfidbruckenmimetika ist eine wichtige Strategie zur Optimierung disulfidreicher Peptide beim Wirkstoff-Design. Wir beschreiben Mimetika des Conotoxins MrIA, die durch selektiven Austausch einzelner Disulfidbindungen gegen Brucken aus einem 1,4-disubstituierten 1,2,3-Triazol erhalten wurden. Eine sequenzielle, Kupfer-katalysierte Azid-Alkin-Klick-Reaktion (CuAAC) mit nachfolgender Disulfidbildung fuhrte regioselektiv zu hybriden Triazol-Disulfid-Analoga von MrIA. Mimetika, in denen Triazol die Cys4-Cys13-Disulfidbindung ersetzte, behielten ihre Tertiarstruktur sowie die volle In-vitro- und In-vivo-Aktivitat als Inhibitoren der Noradrenalin-Wiederaufnahme bei. Diese Mimetika sind resistent gegen Reduktion mit Glutathion, was zu einer verbesserten Plasmastabilitat fuhrt und dadurch interessant fur die Wirkstoff-Entwicklung ist.
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- 2014
3. Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A
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Lai Yue Chan, Yen-Hua Huang, K. Johan Rosengren, David J. Craik, Jason Mulvenna, Chia Chia Tan, Ching-I Anderson Wang, Christina I. Schroeder, Soohyun Kwon, and Xinying Jia
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Protein Conformation ,education ,Molecular Sequence Data ,Cyclotides ,Peptide ,Peptides, Cyclic ,Biochemistry ,Bacterial Proteins ,Sortase ,Peptide bond ,Amino Acid Sequence ,Cysteine ,Molecular Biology ,chemistry.chemical_classification ,Peptide chemical synthesis ,Cell Biology ,Aminoacyltransferases ,Native chemical ligation ,Combinatorial chemistry ,Cyclic peptide ,Cyclotide ,Cysteine Endopeptidases ,chemistry ,Cyclization ,Sortase A ,Protein Structure and Folding ,Conotoxins ,Peptides - Abstract
Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, α-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential.
- Published
- 2014
4. Emerging opportunities for allosteric modulation of G-protein coupled receptors
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Richard J. Lewis and Ching-I Anderson Wang
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Pharmacology ,biology ,Protein Conformation ,Protein Stability ,Allosteric regulation ,Computational biology ,Ligands ,Biochemistry ,Receptors, G-Protein-Coupled ,Molecular recognition ,Allosteric Regulation ,Membrane protein ,Allosteric enzyme ,Rhodopsin ,biology.protein ,Animals ,Humans ,Protein Interaction Domains and Motifs ,Salt bridge ,Receptor ,Signal Transduction ,G protein-coupled receptor - Abstract
Their ubiquitous nature, wide cellular distribution and versatile molecular recognition and signalling help make G-protein binding receptors (GPCRs) the most important class of membrane proteins in clinical medicine, accounting for ∼40% of all current therapeutics. A large percentage of current drugs target the endogenous ligand binding (orthosteric) site, which are structurally and evolutionarily conserved, particularly among members of the same GPCR subfamily. With the recent advances in GPCR X-ray crystallography, new opportunities for developing novel subtype selective drugs have emerged. Given the increasing recognition that the extracellular surface conformation changes in response to ligand binding, it is likely that all GPCRs possess an allosteric site(s) capable of regulating GPCR signalling. Allosteric sites are less structurally conserved than their corresponding orthosteric site and thus provide new opportunities for the development of more selective drugs. Constitutive oligomerisation (dimerisation) identified in many of the GPCRs investigated, adds another dimension to the structural and functional complexity of GPCRs. In this review, we compare 60 crystal structures of nine GPCR subtypes (rhodopsin, ß₂-AR, ß₁-AR, A(2a)-AR, CXCR4, D₃R, H₁R, M₂R, M₃R) across four subfamilies of Class A GPCRs, and discuss mechanisms involved in receptor activation and potential allosteric binding sites across the highly variable extracellular surface of these GPCRs. This analysis has identified a new extracellular salt bridge (ESB-2) that might be exploited in the design of allosteric modulators.
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- 2013
5. Conopeptide ρ-TIA Defines a New Allosteric Site on the Extracellular Surface of the α1B-Adrenoceptor
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Dewi Fajarningsih, Richard J. Lewis, Lotten Ragnarsson, K. Johan Rosengren, Åsa Andersson, Andreas Brust, Thea A. Monks, and Ching-I Anderson Wang
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Models, Molecular ,Agonist ,Turkeys ,medicine.drug_class ,Molecular Sequence Data ,Static Electricity ,Allosteric regulation ,Peptide binding ,Biochemistry ,Protein Structure, Secondary ,Structure-Activity Relationship ,Cricetinae ,Receptors, Adrenergic, alpha-1 ,Membrane Biology ,medicine ,Animals ,Humans ,Inverse agonist ,Computer Simulation ,Amino Acid Sequence ,Amino Acids ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,G protein-coupled receptor ,biology ,fungi ,food and beverages ,Cell Biology ,Recombinant Proteins ,Protein Structure, Tertiary ,Rats ,Allosteric enzyme ,Docking (molecular) ,Mutation ,Adrenergic alpha-1 Receptor Antagonists ,biology.protein ,Biophysics ,Pharmacophore ,Peptides ,Sequence Alignment ,hormones, hormone substitutes, and hormone antagonists ,Allosteric Site - Abstract
The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β1-adrenergic receptor crystal structure, we modeled the α1B-adrenoceptor (α1B-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α1B-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α1-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs. Background: Mechanistic insight into allosteric modulation of GPCRs can facilitate antagonist design. Results: Extracellular surface residues (ECS) of the α1B-adrenoceptor at the base of extracellular loop 3 interact with the allosteric antagonist TIA. Conclusion: The identified ECS pharmacophore provides the first structural constraints for allosteric antagonist design at α1-adrenoceptors. Significance: Binding to the ECS of a GPCR can allosterically inhibit agonist signaling.
- Published
- 2013
6. Solving the [alpha]-conotoxin folding problem: efficient selenium-directed on-resin generation of more potent and stable nicotinic acetylcholine receptor antagonists
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Muttenthaler, Markus, Nevin, Simon T., Grishin, Anton A., Ngo, Shyuan. T., Peng T. Choy, Daly, Norelle L., Shu-Hong Hu, Armishaw, Christopher J., Ching-I. A. Wang, Lewis, Richard J., Martin, Jennifer L., Noakes, Peter G., Craik, David J., Adams, David J., and Alewood, Paul F.
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Cysteine -- Chemical properties ,Nicotinic receptors -- Structure ,Nicotinic receptors -- Chemical properties ,Protein folding -- Analysis ,Xenopus -- Physiological aspects ,Chemistry - Abstract
The application of selenocysteine in a supported phase method to direct native folding and produce [alpha]-conotoxins efficiently with improved biophysical properties is reported. The results obtained provide insight into the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.
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- 2010
7. A Second Extracellular Site Is Required for Norepinephrine Transport by the Human Norepinephrine Transporter
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Soumya Ramu, Ching-I Anderson Wang, Richard J. Lewis, and Nausad Shaikh
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Neurotransmitter transporter ,Stereochemistry ,Norepinephrine ,Leucine ,Fluoxetine ,Chlorocebus aethiops ,Norepinephrine transport ,Extracellular ,medicine ,Animals ,Humans ,Computer Simulation ,Binding site ,Pharmacology ,Binding Sites ,Norepinephrine Plasma Membrane Transport Proteins ,Adrenergic Uptake Inhibitors ,biology ,Chemistry ,fungi ,Biological Transport ,Nisoxetine ,Ligand (biochemistry) ,Molecular Docking Simulation ,Norepinephrine transporter ,COS Cells ,Mutagenesis, Site-Directed ,biology.protein ,Biophysics ,Molecular Medicine ,medicine.drug - Abstract
The human norepinephrine transporter (NET) is implicated in many neurological disorders and is a target of tricyclic antidepressants and nisoxetine (NX). We used molecular docking simulations to guide the identification of residues likely to affect substrate transport and ligand interactions at NET. Mutations to alanine identified a hydrophobic pocket in the extracellular cavity of NET, comprising residues Thr80, Phe317, and Tyr317, which was critical for efficient norepinephrine (NE) transport. This secondary NE substrate site (NESS-2) overlapped the NX binding site, comprising Tyr84, Phe317, and Tyr317, and was positioned ∼11 Å extracellular to the primary site for NE (NESS-1). Thr80 in NESS-2 appeared to be critical in positioning NE for efficient translocation to NESS-1. Three residues identified as being involved in gating the reverse transport of NE (Arg81, Gln314, and Asp473) did not affect NE affinity for NESS-1. Mutating residues adjacent to NESS-2 abolished NET expression (D75A and L76A) or appeared to affect NET folding (S419A), suggesting important roles in stabilizing NET structure, whereas W308A and F388A at the top of NESS-2 abolished both NE transport and NX binding. Our findings are consistent with a multistep model of substrate transport by NET, for which a second, shallow extracellular NE substrate site (NESS-2) is required for efficient NE transport by NET.
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- 2012
8. Berichtigung: Vicinal Disulfide Constrained Cyclic Peptidomimetics: a Turn Mimetic Scaffold Targeting the Norepinephrine Transporter
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Andreas Brust, Ching-I. A. Wang, Norelle L. Daly, Joe Kennerly, Mahsa Sadeghi, Macdonald J. Christie, Richard J. Lewis, Mehdi Mobli, and Paul F. Alewood
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General Medicine - Published
- 2018
9. Emerging structure–function relationships defining monoamine NSS transporter substrate and ligand affinity
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Ching-I Anderson Wang and Richard J. Lewis
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Models, Molecular ,Protein Folding ,Molecular Sequence Data ,Antidepressive Agents, Tricyclic ,Ligands ,Biochemistry ,Substrate Specificity ,Structure-Activity Relationship ,Leucine ,Dopamine ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Homology modeling ,Binding site ,Neurotransmitter sodium symporter ,Serotonin Plasma Membrane Transport Proteins ,Pharmacology ,Dopamine Plasma Membrane Transport Proteins ,Norepinephrine Plasma Membrane Transport Proteins ,Monoamine transporter ,biology ,Chemistry ,Transporter ,Monoamine neurotransmitter ,biology.protein ,Serotonin ,Crystallization ,Selective Serotonin Reuptake Inhibitors ,medicine.drug - Abstract
Monoamine transporters are a group of transmembrane neurotransmitter sodium symporter (NSS) transporters that play a crucial role in regulating biogenic monoamine concentrations at peripheral and central synapses. Given the key role played by serotonin, dopamine and noradrenaline in addictive and disease states, structure-function studies have been conducted to help guide the development of improved central nervous system therapeutics. Extensive pharmacological, immunological and biochemical studies, in conjunction with three-dimensional homology modeling, have been performed to structurally and functionally characterise the monoamine transporter substrate permeation pathway, substrate selectivity, and binding sites for ions, substrates and inhibitors at the molecular level. However, only recently has it been possible to start to construct an accurate molecular interaction network for the monoamine transporters and their corresponding substrates and inhibitors. Crystal structures of Aquifex aeolicus leucine transporter (LeuT(Aa)), a homologous protein to monoamine transporters that has been experimentally demonstrated to share similar structural folds with monoamine transporters, have been determined in complex with amino acids and inhibitors. The molecular interactions of leucine and tricyclic antidepressants (TCA) has supported many of the predictions based on the mutational studies. Models constructed from LeuT(Aa) are now allowing a rational approach to further clarify the molecular determinants of NSS transporter-ligand complexes, and potentially the ability to better manipulate drug specificity and affinity. In this review, we compare the structure-function relationships of other SLC6 NSS family transporters with monoamine transporters, and discuss possible mechanisms involved in substrate binding and transport, and modes of inhibition by TCAs.
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- 2010
10. Purification of the M flax-rust resistance protein expressed in Pichia pastoris
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Bostjan Kobe, Peter N. Dodds, Peter A. Anderson, Simon A. Schmidt, Ching-I Anderson Wang, Pradeep Sornaraj, Simon J. Williams, Ben James, and Jeffrey G. Ellis
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Lysis ,food and beverages ,Cell Biology ,Plant Science ,Biology ,Plant disease resistance ,biology.organism_classification ,Yeast ,Pichia pastoris ,Biochemistry ,Affinity chromatography ,Protein purification ,Protein A/G ,Genetics ,biology.protein ,Pichia - Abstract
The M flax-rust resistance (R) gene is predicted to encode a 150-kDa protein of the Toll-interleukin-like receptor-nucleotide binding site-leucine rich repeat (TIR-NBS-LRR) class of plant disease resistance proteins and provides resistance against the Melampsora lini (flax rust) fungus carrying the AvrM avirulence gene. The extremely low level of this class of R proteins found in plant tissue has precluded their biochemical and structural analysis, and the study of these proteins has been largely restricted to genetic analyses and in vivo investigations. Here we report the production and purification of the M protein in the methalotrophic yeast, Pichia pastoris. Expression trials with five different constructs reveals optimum levels of soluble native M protein can be obtained as an N-terminally 9x His-tagged protein, in which the first 21 amino acids of the predicted wild-type protein are deleted. Expression was achieved using a high cell density fed-batch bioreactor culture at low temperature. M protein was purified to near homogeneity from whole-cell lysates using cation exchange, immobilised metal ion affinity chromatography and gel filtration with a final yield of approximately 3 mg of protein/1000 g wet weight of yeast cells lysed. The successful expression and purification of soluble M protein opens the way for biochemical and structural analysis of this class of important plant proteins.
- Published
- 2007
11. Stabilization of the cysteine-rich conotoxin MrIA by using a 1,2,3-triazole as a disulfide bond mimetic
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MacDonald J. Christie, Maria Luisa Gelmi, Renato Longhi, Paul F. Alewood, Richard J. Lewis, Alessandro Gori, Ching-I Anderson Wang, K. Johan Rosengren, Rebecca F. Bhola, Andreas Brust, and Peta J. Harvey
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1,2,3-Triazole ,Peptidomimetic ,drug design ,Triazole ,Structure-activity relationships ,Catalysis ,Disulfide mimetics ,chemistry.chemical_compound ,Structure-Activity Relationship ,Conotoxin ,Amino Acid Sequence ,Cysteine ,Disulfides ,Norepinephrine Plasma Membrane Transport Proteins ,structure-activity relationships ,General Chemistry ,Triazoles ,Combinatorial chemistry ,Protein tertiary structure ,Cycloaddition ,chemistry ,peptidomimetics ,Drug Design ,click chemistry ,Click chemistry ,Click Chemistry ,Peptidomimetics ,disulfide mimetics ,Conotoxins - Abstract
The design of disulfide bond mimetics is an important strategy for optimising cysteine-rich peptides in drug development. Mimetics of the drug lead conotoxin MrIA, in which one disulfide bond is selectively replaced of by a 1,4-disubstituted-1,2,3-triazole bridge, are described. Sequential copper-catalyzed azide–alkyne cycloaddition (CuAAC; click reaction) followed by disulfide formation resulted in the regioselective syntheses of triazole–disulfide hybrid MrIA analogues. Mimetics with a triazole replacing the Cys4–Cys13 disulfide bond retained tertiary structure and full in vitro and in vivo activity as norepinephrine reuptake inhibitors. Importantly, these mimetics are resistant to reduction in the presence of glutathione, thus resulting in improved plasma stability and increased suitability for drug development. NHMRC 1045964 & 1072113
- Published
- 2014
12. Isolation and structural and pharmacological characterization of α-elapitoxin-Dpp2d, an amidated three finger toxin from black mamba venom
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Irina Vetter, Irene Vergara, Richard J. Lewis, Timothy Reeks, Thomas Durek, Paul F. Alewood, Oleksiy Kovtun, and Ching-I Anderson Wang
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alpha7 Nicotinic Acetylcholine Receptor ,Protein Conformation ,medicine.medical_treatment ,Molecular Sequence Data ,Neurotoxins ,Venom ,Nerve Tissue Proteins ,Nicotinic Antagonists ,Reptilian Proteins ,Receptors, Nicotinic ,medicine.disease_cause ,Biochemistry ,Binding, Competitive ,Cell Line ,Protein structure ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Calcium Signaling ,Elapidae ,Nicotinic Agonists ,Elapid Venoms ,Neurons ,Protease ,Toxin ,Chemistry ,Protein Stability ,Proteolytic enzymes ,Trypsin ,Recombinant Proteins ,Nicotinic agonist ,Epibatidine ,Carrier Proteins ,Protein Processing, Post-Translational ,Sequence Alignment ,medicine.drug - Abstract
We isolated a novel, atypical long-chain three-finger toxin (TFT), α-elapitoxin-Dpp2d (α-EPTX-Dpp2d), from black mamba (Dendroaspis polylepis polylepis) venom. Proteolytic digestion with trypsin and V8 protease, together with MS/MS de novo sequencing, indicated that the mature toxin has an amidated C-terminal arginine, a posttranslational modification rarely observed for snake TFTs. α-EPTX-Dpp2d was found to potently inhibit α7 neuronal nicotinic acetylcholine receptors (nAChR; IC₅₀, 58 ± 24 nM) and muscle-type nAChR (IC₅₀, 114 ± 37 nM) but did not affect α3β2 and α3β4 nAChR isoforms at 1 μM concentrations. Competitive radioligand binding assays demonstrated that α-EPTX-Dpp2d competes with epibatidine binding to the Lymnea stagnalis acetylcholine-binding protein (Ls-AChBP; IC₅₀, 4.9 ± 2.3 nM). The activity profile and binding data are reminiscent of classical long-chain TFTs with a free carboxyl termini, suggesting that amidation does not significantly affect toxin selectivity. The crystal structure of α-EPTX-Dpp2d was determined at 1.7 A resolution and displayed a dimeric toxin assembly with each monomer positioned in an antiparallel orientation. The dimeric structure is stabilized by extensive intermolecular hydrogen bonds and electrostatic interactions, which raised the possibility that the toxin may exist as a noncovalent homodimer in solution. However, chemical cross-linking and size-exclusion chromatography coupled with multiangle laser light scattering (MALLS) data indicated that the toxin is predominantly monomeric under physiological conditions. Because of its high potency and selectivity, we expect this toxin to be a valuable pharmacological tool for studying the structure and function of nAChRs.
- Published
- 2014
13. Vicinal Disulfide Constrained Cyclic Peptidomimetics: a Turn Mimetic Scaffold Targeting the Norepinephrine Transporter
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Richard J. Lewis, Paul F. Alewood, Ching-I Anderson Wang, Mahsa Sadeghi, Norelle L. Daly, MacDonald J. Christie, Andreas Brust, Mehdi Mobli, and Joe Kennerly
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Magnetic Resonance Spectroscopy ,Stereochemistry ,Peptidomimetic ,Drug Evaluation, Preclinical ,Peptide ,biological activity ,Peptides, Cyclic ,Catalysis ,drug discovery ,Turn (biochemistry) ,Norepinephrine reuptake inhibitor ,Animals ,Humans ,Amino Acid Sequence ,Disulfides ,chemistry.chemical_classification ,Norepinephrine Plasma Membrane Transport Proteins ,Adrenergic Uptake Inhibitors ,biology ,cyclic peptides ,General Chemistry ,General Medicine ,Native chemical ligation ,Combinatorial chemistry ,Cyclic peptide ,Protein Structure, Tertiary ,Rats ,chemistry ,Norepinephrine transporter ,peptidomimetics ,biology.protein ,Peptidomimetics ,combinatorial chemistry ,Half-Life - Abstract
Loopy peptides: Peptide turn mimetics of a clinically relevant norepinephrine reuptake inhibitor were developed employing a high-throughput synthesis approach to generate peptide thioesters, with subsequent cyclization through native chemical ligation. The vicinal disulfide constrained cyclic peptidomimetics (see scheme) show high structural and functional similarity to the parent peptide, though with superior metabolic stability. NHMRC Program Grant No. 351446
- Published
- 2013
14. Chemical engineering and structural and pharmacological characterization of the α-scorpion toxin OD1
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David J. Adams, Oliver Knapp, Thomas Durek, Paul F. Alewood, Ching-I Anderson Wang, Irina Vetter, Leonid Motin, and Richard J. Lewis
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Models, Molecular ,Mutant ,Molecular Sequence Data ,Scorpion Venoms ,Peptide ,Voltage-Gated Sodium Channels ,Crystallography, X-Ray ,Biochemistry ,Chemical synthesis ,Scorpions ,Animals ,Humans ,Amino Acid Sequence ,chemistry.chemical_classification ,Scorpion toxin ,Sodium channel ,General Medicine ,Voltage-Gated Sodium Channel Agonists ,Native chemical ligation ,HEK293 Cells ,chemistry ,Chemical engineering ,Molecular Medicine ,Chemical ligation ,Selectivity ,Peptides - Abstract
Scorpion alpha-toxins are invaluable pharmacological tools for studying voltage-gated sodium channels, but few structure-function studies have been undertaken due to their challenging synthesis. To address this deficiency, we report a chemical engineering strategy based upon native chemical ligation. The chemical synthesis of alpha-toxin OD1 was achieved by chemical ligation of three unprotected peptide segments. A high resolution X-ray structure (1.8 angstrom) of synthetic OD1 showed the typical beta alpha beta beta alpha-toxin fold and revealed important conformational differences in the pharrnacophore region when compared with other alpha-toxin structures. Pharmacological analysis of synthetic OD1 revealed potent alpha-toxin activity (inhibition of fast inactivation) at Na(v)1.7, as well as Na(v)1.4 and Na(v)1.6. In addition, OD1 also produced potent beta-toxin activity at Na(v)1.4 and Na(v)1.6 (shift of channel activation in the hyperpolarizing direction), indicating that ODI might interact at more than one site with Na(v)1.4 A and Na(v)1.6. Investigation of nine OD1 mutants revealed that three residues in the reverse turn contributed significantly to selectivity, with the triple ODI mutant (D9K, D10P, K11H) being 40 fold more selective for Na(v)1.7 over Na(v)1.6, while OD1 K11V was 5-fold more selective for Na1.6(v) than Na(v)1.7. This switch in selectivity highlights the importance of the reverse turn for engineering alpha-toxins with altered selectivity at Na-v subtypes.
- Published
- 2013
15. Atypical alpha-conotoxin LtIA from Conus litteratus targets a novel microsite of the alpha3beta2 nicotinic receptor
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Sulan Luo, Paul F. Alewood, Cheryl Dowell, Ching-I Anderson Wang, Norelle L. Daly, Dongting Zhangsun, J. Michael McIntosh, Kalyana B. Akondi, Xiaopeng Zhu, Sean Christensen, Yuanyan Hu, Richard J. Lewis, Yong Wu, and David J. Craik
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Models, Molecular ,Protein Folding ,Xenopus ,Mutant ,Molecular Sequence Data ,Nicotinic Antagonists ,Biology ,In Vitro Techniques ,Receptors, Nicotinic ,Biochemistry ,complex mixtures ,Mice ,Animals ,Amino Acid Sequence ,Binding site ,Receptor ,Molecular Biology ,Nuclear Magnetic Resonance, Biomolecular ,α conotoxin ,Binding Sites ,Wild type ,Conus Snail ,Cell Biology ,biology.organism_classification ,Molecular biology ,Recombinant Proteins ,Cell biology ,Rats ,Conus litteratus ,Nicotinic acetylcholine receptor ,Nicotinic agonist ,nervous system ,Structural Homology, Protein ,Protein Structure and Folding ,Mutagenesis, Site-Directed ,Oocytes ,Female ,Conotoxins ,Oxidation-Reduction - Abstract
Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. alpha-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of alpha-conotoxins may serve as a platform for development of new therapies. Previously characterized alpha-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel alpha-conotoxin LtIA, which lacks this highly conserved motif but potently blocked alpha3beta2 nAChRs with a 9.8 nm IC(50) value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing alpha-conotoxin MII. Nevertheless, pre-block of alpha3beta2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR beta subunit ligand-binding interface mutations were used to examine the1000-fold selectivity difference of LtIA for alpha3beta2 versus alpha3beta4 nAChRs. Unlike MII, LtIA had a900-fold increased IC(50) value on alpha3beta2(F119Q) versus wild type nAChRs, whereas T59K and V111I beta2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the alpha3beta2 nAChR that includes beta2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.
- Published
- 2010
16. Molecular Engineering of Conotoxins: The Importance of Loop Size to α-Conotoxin Structure and Function
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Sébastien Dutertre, Simon T. Nevin, Richard J. Lewis, Ching-I Anderson Wang, David J. Craik, David J. Adams, Norelle L. Daly, Paul F. Alewood, Ai-Hua Jin, Institute for Molecular Bioscience, University of Queensland [Brisbane], Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), and Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)
- Subjects
alpha7 Nicotinic Acetylcholine Receptor ,Molecular model ,Stereochemistry ,Xenopus ,Molecular Sequence Data ,Snails ,Receptors, Nicotinic ,Protein Engineering ,complex mixtures ,Structure-Activity Relationship ,03 medical and health sciences ,0302 clinical medicine ,Drug Discovery ,Animals ,Amino Acid Sequence ,Disulfides ,Conotoxin ,Nuclear Magnetic Resonance, Biomolecular ,Protein secondary structure ,Adrenergic alpha-Antagonists ,030304 developmental biology ,Acetylcholine receptor ,0303 health sciences ,Sequence Homology, Amino Acid ,Chemistry ,Circular Dichroism ,Nicotinic acetylcholine receptor ,Nicotinic agonist ,nervous system ,Docking (molecular) ,[SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology ,Molecular Medicine ,Conotoxins ,030217 neurology & neurosurgery ,Cysteine - Abstract
International audience; Alpha-conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). The majority of currently characterized alpha-conotoxins have a 4/7 loop size, and the major features of neuronal alpha-conotoxins include a globular disulfide connectivity and a helical structure centered around the third of their four cysteine residues. In this study, a novel "molecular pruning" approach was undertaken to define the relationship between loop size, structure, and function of alpha-conotoxins. This involved the systematic truncation of the second loop in the alpha-conotoxin [A10L]PnIA [4/7], a potent antagonist of the alpha7 nAChR. The penalty for truncation was found to be decreased conformational stability and increased susceptibility to disulfide bond scrambling. Truncation down to 4/4[A10L]PnIA maintained helicity and did not significantly reduce electrophysiological activity at alpha7 nAChRs, whereas 4/3[A10L]PnIA lost both alpha7 nAChR activity and helicity. In contrast, all truncated analogues lost approximately 100-fold affinity at the AChBP, a model protein for the extracellular domain of the nAChR. Docking simulations identified several hydrogen bonds lost upon truncation that provide an explanation for the reduced affinities observed at the alpha7 nAChR and AChBP.
- Published
- 2008
17. Crystal structures of flax rust avirulence proteins AvrL567-A and -D reveal details of the structural basis for flax disease resistance specificity
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Jade K. Forwood, Fionna E. Loughlin, Jeffrey G. Ellis, Gregory J. Lawrence, Gregor Gunčar, Joel P. Mackay, Bostjan Kobe, Trazel Teh, Horst Joachim Schirra, Ching-I Anderson Wang, Peter A. Anderson, Ann-Maree Catanzariti, and Peter N. Dodds
- Subjects
Hypersensitive response ,Models, Molecular ,Linum ,Virulence Factors ,DNA Mutational Analysis ,Molecular Sequence Data ,Plant Science ,Plasma protein binding ,Plant disease resistance ,Leucine-rich repeat ,Crystallography, X-Ray ,Structure-Activity Relationship ,Flax ,Amino Acid Sequence ,Peptide sequence ,Research Articles ,Plant Diseases ,Plant Proteins ,chemistry.chemical_classification ,Genetics ,biology ,Basidiomycota ,Cell Biology ,biology.organism_classification ,Immunity, Innate ,Amino acid ,chemistry ,Biochemistry ,Nucleic acid ,Protein Binding - Abstract
The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-Å resolution, respectively. The structures of both proteins are very similar and reveal a β-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.
- Published
- 2007
18. Purification of the M flax-rust resistance protein expressed in Pichia pastoris
- Author
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Simon A, Schmidt, Simon J, Williams, Ching-I A, Wang, Pradeep, Sornaraj, Ben, James, Bostjan, Kobe, Peter N, Dodds, Jeffrey G, Ellis, and Peter A, Anderson
- Subjects
Basidiomycota ,Flax ,Genetic Vectors ,Gene Expression ,Genes, Plant ,Pichia ,Recombinant Proteins ,Plant Diseases ,Plant Proteins ,Plasmids - Abstract
The M flax-rust resistance (R) gene is predicted to encode a 150-kDa protein of the Toll-interleukin-like receptor-nucleotide binding site-leucine rich repeat (TIR-NBS-LRR) class of plant disease resistance proteins and provides resistance against the Melampsora lini (flax rust) fungus carrying the AvrM avirulence gene. The extremely low level of this class of R proteins found in plant tissue has precluded their biochemical and structural analysis, and the study of these proteins has been largely restricted to genetic analyses and in vivo investigations. Here we report the production and purification of the M protein in the methalotrophic yeast, Pichia pastoris. Expression trials with five different constructs reveals optimum levels of soluble native M protein can be obtained as an N-terminally 9x His-tagged protein, in which the first 21 amino acids of the predicted wild-type protein are deleted. Expression was achieved using a high cell density fed-batch bioreactor culture at low temperature. M protein was purified to near homogeneity from whole-cell lysates using cation exchange, immobilised metal ion affinity chromatography and gel filtration with a final yield of approximately 3 mg of protein/1000 g wet weight of yeast cells lysed. The successful expression and purification of soluble M protein opens the way for biochemical and structural analysis of this class of important plant proteins.
- Published
- 2007
19. The use of Co2+ for crystallization and structure determination, using a conventional monochromatic X-ray source, of flax rust avirulence protein
- Author
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Trazel Teh, Peter N. Dodds, Ann-Maree Catanzariti, Bostjan Kobe, Jeffrey G. Ellis, Ching-I Anderson Wang, Gregor Gunčar, and Jade K. Forwood
- Subjects
Biophysics ,chemistry.chemical_element ,Synchrotron radiation ,Crystal structure ,Biology ,Crystallography, X-Ray ,Biochemistry ,law.invention ,Fungal Proteins ,Structural Biology ,law ,Flax ,Genetics ,Crystallization ,Plant Diseases ,Fungal protein ,Virulence ,Basidiomycota ,X-ray ,food and beverages ,Cobalt ,Condensed Matter Physics ,Crystallography ,chemistry ,Absorption edge ,Crystallization Communications ,Monochromatic color - Abstract
Metal-binding sites are ubiquitous in proteins and can be readily utilized for phasing. It is shown that a protein crystal structure can be solved using single-wavelength anomalous diffraction based on the anomalous signal of a cobalt ion measured on a conventional monochromatic X-ray source. The unique absorption edge of cobalt (1.61 A) is compatible with the Cu K alpha wavelength (1.54 A) commonly available in macromolecular crystallography laboratories. This approach was applied to the determination of the structure of Melampsora lini avirulence protein AvrL567-A, a protein with a novel fold from the fungal pathogen flax rust that induces plant disease resistance in flax plants. This approach using cobalt ions may be applicable to all cobalt-binding proteins and may be advantageous when synchrotron radiation is not readily available.
- Published
- 2007
20. Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes
- Author
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Trazel Teh, Michael Ayliffe, Gregory J. Lawrence, Jeffrey G. Ellis, Ann-Maree Catanzariti, Bostjan Kobe, Ching-I Anderson Wang, and Peter N. Dodds
- Subjects
Molecular Sequence Data ,Biology ,Plant disease resistance ,Genes, Plant ,medicine.disease_cause ,Leucine-Rich Repeat Proteins ,Host-Parasite Interactions ,Evolution, Molecular ,Species Specificity ,Flax ,medicine ,Arabidopsis thaliana ,Amino Acid Sequence ,Amino Acids ,Selection, Genetic ,Gene ,Peptide sequence ,Alleles ,Plant Proteins ,Plant Diseases ,Genetics ,Mutation ,Binding Sites ,Multidisciplinary ,Virulence ,Effector ,Fungi ,Proteins ,Biological Sciences ,Plants ,biology.organism_classification ,Commentary ,Effector-triggered immunity ,Function (biology) ,Protein Binding - Abstract
Plant resistance proteins (R proteins) recognize corresponding pathogen avirulence (Avr) proteins either indirectly through detection of changes in their host protein targets or through direct R–Avr protein interaction. Although indirect recognition imposes selection against Avr effector function, pathogen effector molecules recognized through direct interaction may overcome resistance through sequence diversification rather than loss of function. Here we show that the flax rust fungus AvrL567 genes, whose products are recognized by the L5, L6, and L7 R proteins of flax, are highly diverse, with 12 sequence variants identified from six rust strains. Seven AvrL567 variants derived from Avr alleles induce necrotic responses when expressed in flax plants containing corresponding resistance genes ( R genes), whereas five variants from avr alleles do not. Differences in recognition specificity between AvrL567 variants and evidence for diversifying selection acting on these genes suggest they have been involved in a gene-specific arms race with the corresponding flax R genes. Yeast two-hybrid assays indicate that recognition is based on direct R–Avr protein interaction and recapitulate the interaction specificity observed in planta . Biochemical analysis of Escherichia coli -produced AvrL567 proteins shows that variants that escape recognition nevertheless maintain a conserved structure and stability, suggesting that the amino acid sequence differences directly affect the R–Avr protein interaction. We suggest that direct recognition associated with high genetic diversity at corresponding R and Avr gene loci represents an alternative outcome of plant–pathogen coevolution to indirect recognition associated with simple balanced polymorphisms for functional and nonfunctional R and Avr genes.
- Published
- 2006
21. 136. The Allosteric Binding Site for ρ-TIA on the Extracellular Surface of the α1B- Adrenoceptor
- Author
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Andreas Brust, Thea A. Monks, Lotten Ragnarsson, Dewi Fajarningsih, K. Johan Rosengren, Åsa Andersson, Ching-I Anderson Wang, and Richard J. Lewis
- Subjects
Adrenergic receptor ,Chemistry ,α1b adrenoceptor ,Allosteric regulation ,Extracellular ,Biophysics ,Conotoxin ,Toxicology - Published
- 2012
22. Conotoxin engineering: dual pharmacophoric noradrenaline transport inhibitor/integrin binding peptide with improved stability
- Author
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Ching-I Anderson Wang, Zoltan Dekan, Richard J. Lewis, Paul F. Alewood, and Robert K. Andrews
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Models, Molecular ,Integrins ,Integrin ,Peptide ,Protein Engineering ,Peptides, Cyclic ,Biochemistry ,Norepinephrine ,Animals ,Humans ,Conotoxin ,Physical and Theoretical Chemistry ,Receptor ,RGD motif ,Integrin binding ,chemistry.chemical_classification ,Norepinephrine Plasma Membrane Transport Proteins ,biology ,Protein Stability ,Organic Chemistry ,Biological Transport ,Rats ,chemistry ,Norepinephrine transporter ,biology.protein ,Pharmacophore ,Conotoxins ,Oligopeptides - Abstract
A dual-pharmacophoric peptide was engineered by grafting the integrin binding RGD motif between the C- and N-termini of a disulfide-rich noradrenaline transporter inhibiting χ-conotoxin resulting in a stable backbone cyclized peptide. The construct maintained two independent biological activities and showed increased plasma stability with no adverse effects observed following administration to rats, highlighting the potential value of pharmacophore grafting into constrained peptide scaffolds.
- Published
- 2012
23. Correction to 'Analgesic ω-Conotoxins CVIE and CVIF Selectively and Voltage-Dependently Block Recombinant and Native N-Type Calcium Channels': TABLE 1
- Author
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Ching-I Anderson Wang, Leonid Motin, Paul F. Alewood, MacDonald J. Christie, David J. Adams, Roger Drinkwater, Géza Berecki, J. Lewis, S. Vink, Alison Haythornthwaite, Paramjit S. Bansal, and Melissa Moretta
- Subjects
Pharmacology ,Voltage-dependent calcium channel ,Chemistry ,Stereochemistry ,law ,Block (telecommunications) ,Analgesic ,Recombinant DNA ,Molecular Medicine ,Omega-Conotoxins ,law.invention - Published
- 2011
24. Conopeptide ρ-TIA Defines a New Allosteric Site on the Extracellular Surface of the α1B-Adrenoceptor.
- Author
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Ragnarsson, Lotten, Ching-I Anderson Wang, Andersson, Åsa, Fajarningsih, Dewi, Monks, Thea, Brust, Andreas, Rosengren, K. Johan, and Lewis, Richard J.
- Subjects
- *
G protein coupled receptors , *ALLOSTERIC regulation , *ADRENERGIC receptors , *RADIOLIGAND assay , *HYDROGEN bonding , *MEMBRANE proteins - Abstract
The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β1-adrenergic receptor crystal structure, we modeled the α1B-adrenoceptor (α1B-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α1B-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α1-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
25. Molecular Engineering of Conotoxins: The Importance of Loop Size to α-Conotoxin Structure and Function.
- Author
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Ai-Hua Jin, Norelle L. Daly, Simon T. Nevin, Ching-I A. Wang, Sebastien Dutertre, Richard J. Lewis, David J. Adams, David J. Craik, and Paul F. Alewood
- Published
- 2008
- Full Text
- View/download PDF
26. Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A.
- Author
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Xinying Jia, Soohyun Kwon, Ching-I Anderson Wang, Yen-Hua Huang, Lai Y. Chan, Chia Chia Tan, Rosengren, K. Johan, Mulvenna, Jason P., Schroeder, Christina I., and Craik, David J.
- Subjects
- *
DISULFIDES synthesis , *PEPTIDES , *THERAPEUTICS research , *ENZYMES , *BIOACTIVE compounds - Abstract
Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, α-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
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