256 results on '"Craik, David J."'
Search Results
2. Sortase‐Catalyzed Protein Domain Inversion.
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Zhou, Yan, Durek, Thomas, Craik, David J., and Rehm, Fabian B. H.
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Topological transformations and permutations of proteins have attracted significant interest as strategies to generate new protein functionalities or stability. These efforts have mainly been inspired by naturally occurring post‐translational modifications, such as head‐to‐tail cyclization, circular permutation, or lasso‐like entanglement. Such approaches can be realized experimentally via genetic encoding, in the case of circular permutation, or via enzymatic processing, in the case of cyclization. Notably, these previously described strategies leave the polypeptide backbone orientation unaltered. Here we describe an unnatural protein permutation, the protein domain inversion, whereby a C‐terminal portion of a protein is enzymatically inverted from the canonical N‐to‐C to a C‐to‐C configuration with respect to the N‐terminal part of the protein. The closest conceptually analogous biological process is perhaps the inversion of DNA segments as catalyzed by recombinases. We achieve these inversions using an engineered sortase A, a widely used transpeptidase. Our reactions proceed efficiently under mild conditions at 4–25 °C and are compatible with entirely heterologously‐produced protein substrates. [ABSTRACT FROM AUTHOR]
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- 2024
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3. Deciphering the structure and mechanism of action of computer-designed mastoparan peptides.
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Oshiro, Karen G. N., Freitas, Carlos D. P., Rezende, Samilla B., Orozco, Raquel M. Q., Chan, Lai Y., Lawrence, Nicole, Lião, Luciano M., Macedo, Maria L. R., Craik, David J., Cardoso, Marlon H., and Franco, Octávio L.
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ARTIFICIAL membranes ,PLASMONICS ,PEPTIDES ,SURFACE plasmon resonance ,BILAYER lipid membranes ,MOLECULAR dynamics ,NUCLEAR magnetic resonance spectroscopy ,CIRCULAR dichroism - Abstract
Mastoparans are cationic peptides with multifunctional pharmacological properties. Mastoparan-R1 and mastoparan-R4 were computationally designed based on native mastoparan-L from wasps and have improved therapeutic potential for the control of bacterial infections. Here, we evaluated whether these peptides maintain their activity against Escherichia coli strains under a range of salt concentrations. We found that mastoparan- R1 and mastoparan-R4 preserved their activity under the conditions tested, including having antibacterial activities at physiological salt concentrations. The overall structure of the peptides was investigated using circular dichroism spectroscopy in a range of solvents. No significant changes in secondary structure were observed (random coil in aqueous solutions and a-helix in hydrophobic and anionic environments). The three-dimensional structures of mastoparan-R1 and mastoparan-R4 were elucidated through nuclear magnetic resonance spectroscopy, revealing amphipathic a-helical segments for Leu3-Ile13 (mastoparan-R1) and Leu3-Ile14 (mastoparan- R4). Possible membrane-association mechanisms for mastoparan-R1 and mastoparan-R4 were investigated through surface plasmon resonance and leakage studies with synthetic POPC and POPC/POPG (4:1) lipid bilayers. Mastoparan-L had the highest affinity for both membrane systems, whereas the two analogs had weaker association, but improved selectivity for lysing anionic membranes. This finding was also supported by molecular dynamics simulations, in which mastoparan-R1 and mastoparan-R4 were found to have greater interactions with bacteria-like membranes compared with model mammalian membranes. Despite having a few differences in their functional and structural profiles, the mastoparan-R1 analog stood out with the highest activity, greater bacteriostatic potential, and selectivity for lysing anionic membranes. This study reinforces the potential of mastoparan-R1 as a drug candidate. [ABSTRACT FROM AUTHOR]
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- 2024
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4. Computational Design of α‐Conotoxins to Target Specific Nicotinic Acetylcholine Receptor Subtypes.
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Wu, Xiaosa, Hone, Arik J., Huang, Yen‐Hua, Clark, Richard J., McIntosh, J. Michael, Kaas, Quentin, and Craik, David J.
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NICOTINIC acetylcholine receptors ,CHOLINERGIC receptors ,CONOTOXINS ,CONUS ,MOLECULAR dynamics ,DRUG target ,MOLECULAR models - Abstract
Nicotinic acetylcholine receptors (nAChRs) are drug targets for neurological diseases and disorders, but selective targeting of the large number of nAChR subtypes is challenging. Marine cone snail α‐conotoxins are potent blockers of nAChRs and some have been engineered to achieve subtype selectivity. This engineering effort would benefit from rapid computational methods able to predict mutational energies, but current approaches typically require high‐resolution experimental structures, which are not widely available for α‐conotoxin complexes. Herein, five mutational energy prediction methods were benchmarked using crystallographic and mutational data on two acetylcholine binding protein/α‐conotoxin systems. Molecular models were developed for six nAChR subtypes in complex with five α‐conotoxins that were studied through 150 substitutions. The best method was a combination of FoldX and molecular dynamics simulations, resulting in a predictive Matthews Correlation Coefficient (MCC) of 0.68 (85 % accuracy). Novel α‐conotoxin mutants designed using this method were successfully validated by experimental assay with improved pharmaceutical properties. This work paves the way for the rapid design of subtype‐specific nAChR ligands and potentially accelerated drug development. [ABSTRACT FROM AUTHOR]
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- 2024
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5. A Chemoenzymatic Approach To Produce a Cyclic Analogue of the Analgesic Drug MVIIA (Ziconotide).
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Zhou, Yan, Harvey, Peta J., Koehbach, Johannes, Chan, Lai Yue, Jones, Alun, Andersson, Åsa, Vetter, Irina, Durek, Thomas, and Craik, David J.
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CHEMICAL synthesis ,CALCIUM channels ,CONOTOXINS ,CHRONIC pain ,RING formation (Chemistry) ,OPIOID analgesics ,PEPTIDE synthesis - Abstract
Ziconotide (ω‐conotoxin MVIIA) is an approved analgesic for the treatment of chronic pain. However, the need for intrathecal administration and adverse effects have limited its widespread application. Backbone cyclization is one way to improve the pharmaceutical properties of conopeptides, but so far chemical synthesis alone has been unable to produce correctly folded and backbone cyclic analogues of MVIIA. In this study, an asparaginyl endopeptidase (AEP)‐mediated cyclization was used to generate backbone cyclic analogues of MVIIA for the first time. Cyclization using six‐ to nine‐residue linkers did not perturb the overall structure of MVIIA, and the cyclic analogues of MVIIA showed inhibition of voltage‐gated calcium channels (CaV2.2) and substantially improved stability in human serum and stimulated intestinal fluid. Our study reveals that AEP transpeptidases are capable of cyclizing structurally complex peptides that chemical synthesis cannot achieve and paves the way for further improving the therapeutic value of conotoxins. [ABSTRACT FROM AUTHOR]
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- 2023
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6. Captivity induces a sweeping and sustained genomic response in a starfish.
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Morin, Marie, Jönsson, Mathias, Wang, Conan K., Craik, David J., Degnan, Sandie M., and Degnan, Bernard M.
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STARFISHES ,CAPTIVITY ,GENE expression ,MARINE animals ,CELL communication ,HEPATITIS C virus - Abstract
Marine animals in the wild are often difficult to access, so they are studied in captivity. However, the implicit assumption that physiological processes of animals in artificial environments are not different from those in the wild has rarely been tested. Here, we investigate the extent to which an animal is impacted by captivity by comparing global gene expression in wild and captive crown‐of‐thorns starfish (COTS). In a preliminary analysis, we compared transcriptomes of three external tissues obtained from multiple wild COTS with a single captive COTS maintained in aquaria for at least 1 week. On average, an astonishingly large 24% of the coding sequences in the genome were differentially expressed. This led us to conduct a replicated experiment to test more comprehensively the impact of captivity on gene expression. Specifically, a comparison of 13 wild with 8 captive COTS coelomocyte transcriptomes revealed significant differences in the expression of 20% of coding sequences. Coelomocyte transcriptomes in captive COTS remain different from those in wild COTS for more than 30 days and show no indication of reverting back to a wild state (i.e. no evidence of acclimation). Genes upregulated in captivity include those involved in oxidative stress and energy metabolism, whereas genes downregulated are involved in cell signalling. These changes in gene expression indicate that being translocated and maintained in captivity has a marked impact on the physiology and health of these echinoderms. This study suggests that caution should be exercised when extrapolating results from captive aquatic invertebrates to their wild counterparts. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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7. Mutagenesis of cyclotide Cter 27 exemplifies a robust folding strategy for bracelet cyclotides.
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Dang, Tien T., Harvey, Peta J., Chan, Lai Yue, Huang, Yen‐Hua, Kaas, Quentin, and Craik, David J.
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BRACELETS ,DRUG design ,CHEMICAL synthesis ,PEPTIDE synthesis ,CHEMICAL inhibitors ,TRYPSIN inhibitors - Abstract
In contrast to Möbius and trypsin inhibitor cyclotides, members of the bracelet subfamily are typically intractable to chemical synthesis and folding. In a significant advance in the field, the bracelet cyclotides ribe 33 and Cter 27 were successfully produced synthetically in moderate yield in a recent study. That synthetic method was a breakthrough as members of the bracelet subfamily of cyclotides had hitherto eluded attempts to be synthetically produced, apart from one report of cyO2 production in which a complicated folding strategy was used. In the current study the successful in vitro folding of three mutants of bracelet cyclotide Cter 27 is reported. This study broadens our understanding of the folding of bracelet cyclotides and elucidates the three dimensional structure of synthetic Cter 27, providing a new class of cyclotide molecular grafting scaffold for drug design applications. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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8. Cystine Knot Peptides with Tuneable Activity and Mechanism.
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Li, Choi Yi, Rehm, Fabian B. H., Yap, Kuok, Zdenek, Christina N., Harding, Maxim D., Fry, Bryan G., Durek, Thomas, Craik, David J., and de Veer, Simon J.
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CYSTINE ,PEPTIDES ,STREPTAVIDIN ,PROTEASE inhibitors ,BIOTIN - Abstract
Knottins are topologically complex peptides that are stabilised by a cystine knot and have exceptionally diverse functions, including protease inhibition. However, approaches for tuning their activity in situ are limited. Here, we demonstrate separate approaches for tuning the activity of knottin protease inhibitors using light or streptavidin. We show that the inhibitory activity and selectivity of an engineered knottin can be controlled with light by activating a second mode of action that switches the inhibitor ON against new targets. Guided by a knottin library screen, we also identify a position in the inhibitor's binding loop that permits insertion of a biotin tag without impairing activity. Using streptavidin, biotinylated knottins with nanomolar affinity can be switched OFF in activity assays, and the anticoagulant activity of a factor XIIa inhibitor can be rapidly switched OFF in human plasma. Our findings expand the scope of engineered knottins for precisely controlling protein function. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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9. Enzymatic C‐to‐C Protein Ligation.
- Author
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Rehm, Fabian B. H., Tyler, Tristan J., de Veer, Simon J., Craik, David J., and Durek, Thomas
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RECOMBINANT proteins ,PEPTIDES ,PEPTIDOMIMETICS ,PROTEINS ,TRANSPEPTIDATION - Abstract
Transpeptidase‐catalyzed protein and peptide modifications have been widely utilized for generating conjugates of interest for biological investigation or therapeutic applications. However, all known transpeptidases are constrained to ligating in the N‐to‐C orientation, limiting the scope of attainable products. Here, we report that an engineered asparaginyl ligase accepts diverse incoming nucleophile substrate mimetics, particularly when a means of selectively quenching the reactivity of byproducts released from the recognition sequence is employed. In addition to directly catalyzing formation of l‐/d‐ or α‐/β‐amino acid junctions, we find C‐terminal Leu‐ethylenediamine (Leu‐Eda) motifs to be bona fide mimetics of native N‐terminal Gly‐Leu sequences. Appending a C‐terminal Leu‐Eda to synthetic peptides or, via an intein‐splicing approach, to recombinant proteins enables direct transpeptidase‐catalyzed C‐to‐C ligations. This work significantly expands the synthetic scope of enzyme‐catalyzed protein transpeptidation reactions. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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10. ɑO‐Conotoxin GeXIVA isomers modulate N‐type calcium (CaV2.2) channels and inwardly‐rectifying potassium (GIRK) channels via GABAB receptor activation.
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Yousuf, Arsalan, Wu, Xiaosa, Bony, Anuja R., Sadeghi, Mahsa, Huang, Yen‐Hua, Craik, David J., and Adams, David J.
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CONOTOXINS ,CALCIUM channels ,POTASSIUM channels ,ISOMERS ,NICOTINIC acetylcholine receptors ,G protein coupled receptors ,DORSAL root ganglia - Abstract
αO‐Conotoxin GeXIVA is a 28 amino acid peptide derived from the venom of the marine snail Conus generalis. The presence of four cysteine residues in the structure of GeXIVA allows it to have three different disulfide isomers, that is, the globular, ribbon or bead isomer. All three isomers are active at α9α10 nicotinic acetylcholine receptors, with the bead isomer, GeXIVA[1,2], being the most potent and exhibiting analgesic activity in animal models of neuropathic pain. The original report of GeXIVA activity failed to observe any effect of the isomers on high voltage‐activated (HVA) calcium channel currents in rat dorsal root ganglion (DRG) neurons. In this study, we report, for the first time, the activity of globular GeXIVA[1,3] at G protein‐coupled GABAB receptors (GABABR) inhibiting HVA N‐type calcium (Cav2.2) channels and reducing membrane excitability in mouse DRG neurons. The inhibition of HVA Ba2+ currents and neuroexcitability by GeXIVA[1,3] was partially reversed by the selective GABABR antagonist CGP 55845. In transfected HEK293T cells co‐expressing human GABABR1 and R2 subunits and Cav2.2 channels, both GeXIVA[1,3] and GeXIVA[1,4] inhibited depolarization‐activated Ba2+ currents mediated by Cav2.2 channels, whereas GeXIVA[1,2] had no effect. The effects of three cyclized GeXIVA[1,4] ribbon isomers were also tested, with cGeXIVA GAG being the most potent at human GABABR‐coupled Cav2.2 channels. Interestingly, globular GeXIVA[1,3] also reversibly potentiated inwardly‐rectifying K+ currents mediated by human GIRK1/2 channels co‐expressed with GABABR in HEK293T cells. This study highlights GABABR as a potentially important receptor target for the activity of αO‐conotoxin GeXIVA to mediate analgesia. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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11. Development of novel frog‐skin peptide scaffolds with selectivity towards melanocortin receptor subtypes.
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Malik, Uru, Chan, Lai Yue, Cai, Minying, Hruby, Victor J., Kaas, Quentin, Daly, Norelle L., and Craik, David J.
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MELANOCORTIN receptors ,PEPTIDES ,SKIN cancer ,IMPOTENCE ,OBESITY ,LIGANDS (Biochemistry) - Abstract
Melanocortin receptors are pharmaceutically important receptors that are involved in complex physiological functions. They have been associated with various diseases including obesity, erectile dysfunction, acne, and skin cancer. It has been challenging to transform nonselective endogenous agonist and antagonist ligands into selective and potent ligands. In this study, we investigated naturally occurring peptides derived from frog skin secretions for selectivity and activity toward melanocortin receptors. Three peptides (ORB, ORB2K and ranacyclin‐T) were found to have selectivity towards the melanocortin receptor 5 (MC5R). ORB and ORB2K had partial binding affinity at nanomolar concentrations, whereas ranacyclin‐T had 57% binding efficiency at 1.6 μM. Backbone cyclization of ORB and ORB2K altered the binding efficiency to melanocortin receptors. Our results suggest that these frog‐skin peptides could be modified for developing melanocortin‐specific ligands and potentially future therapeutics. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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12. Asparaginyl Ligases: New Enzymes for the Protein Engineer's Toolbox.
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Rehm, Fabian B. H., Tyler, Tristan J., Xie, Jing, Yap, Kuok, Durek, Thomas, and Craik, David J.
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- 2021
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13. Increased Valency Improves Inhibitory Activity of Peptides Targeting Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9).
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Tombling, Benjamin J., Lammi, Carmen, Bollati, Carlotta, Anoldi, Anna, Craik, David J., and Wang, Conan K.
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- 2021
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14. Antimicrobial and Anticancer Properties of Synthetic Peptides Derived from the Wasp Parachartergus fraternus.
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Muller, Jessica A. I., Lawrence, Nicole, Chan, Lai Yue, Harvey, Peta J., Elliott, Alysha G., Blaskovich, Mark A. T., Gonçalves, Jacqueline C., Galante, Priscilla, Mortari, Marcia R., Toffoli‐Kadri, Mônica C., Koehbach, Johannes, and Craik, David J.
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- 2021
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15. Sunflower Trypsin Inhibitor‐1 (SFTI‐1): Sowing Seeds in the Fields of Chemistry and Biology.
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Veer, Simon J., White, Andrew M., and Craik, David J.
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CYCLIC peptides ,TRYPSIN ,SUNFLOWERS ,SEEDS - Abstract
Nature‐derived cyclic peptides have proven to be a vast source of inspiration for advancing modern pharmaceutical design and synthetic chemistry. The focus of this Review is sunflower trypsin inhibitor‐1 (SFTI‐1), one of the smallest disulfide‐bridged cyclic peptides found in nature. SFTI‐1 has an unusual biosynthetic pathway that begins with a dual‐purpose albumin precursor and ends with the production of a high‐affinity serine protease inhibitor that rivals other inhibitors much larger in size. Investigations on the molecular basis for SFTI‐1′s rigid structure and adaptable function have planted seeds for thought that have now blossomed in several different fields. Here we survey these applications to highlight the growing potential of SFTI‐1 as a versatile template for engineering inhibitors, a prototypic peptide for studying inhibitory mechanisms, a stable scaffold for grafting bioactive peptides, and a model peptide for evaluating peptidomimetic motifs and platform technologies. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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16. Der Sonnenblumen‐Trypsin‐Inhibitor 1 (SFTI‐1) in der Chemie und Biologie.
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Veer, Simon J., White, Andrew M., and Craik, David J.
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NUNS - Abstract
Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
- Published
- 2021
- Full Text
- View/download PDF
17. Improved Asparaginyl‐Ligase‐Catalyzed Transpeptidation via Selective Nucleophile Quenching.
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Rehm, Fabian B. H., Tyler, Tristan J., Yap, Kuok, Durek, Thomas, and Craik, David J.
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TRANSPEPTIDATION ,SORTASES ,GENERATING functions ,LIGASES ,ENDOPEPTIDASES - Abstract
The use of enzymes for the site‐specific modification of proteins/peptides has become a highly accessible, widespread approach to study protein/peptide functions or to generate therapeutic conjugates. Asparaginyl endopeptidases (AEPs) that preferentially catalyze transpeptidation reactions (AEP ligases) have emerged as enticing alternatives to established approaches, such as bacterial sortases, due to their catalytic efficiency and short tripeptide recognition motifs. However, under standard conditions, a substantial excess of the nucleophile to be conjugated is needed to reach desirable yields. Herein we report a versatile approach to shift the AEP‐catalyzed transpeptidation equilibrium toward product formation via selectively quenching the nucleophilicity of the competing leaving‐group peptide. Our metal‐complexation‐based strategy enables efficient peptide/protein labeling at the N‐ or C‐terminus with near‐equimolar concentrations of nucleophile label. Furthermore, we show that this approach can enhance protein–protein ligation and facilitate the formation of transpeptidation products that are otherwise unattainable. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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18. Cyclotide Structures Revealed by NMR, with a Little Help from X‐ray Crystallography.
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Handley, Thomas N. G., Wang, Conan K., Harvey, Peta J., Lawrence, Nicole, and Craik, David J.
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- 2020
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19. Antimicrobial Peptide Mimetics Based on a Diphenylacetylene Scaffold: Synthesis, Conformational Analysis, and Activity.
- Author
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Peacock, Hayden, Henriques, Sónia Troeira, Benfield, Aurélie H., Elliott, Alysha G., Luo, Jinghui, Luccarelli, James, Nagano, Masanobu, Craik, David J., and Hamilton, Andrew D.
- Published
- 2020
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20. Application and Structural Analysis of Triazole‐Bridged Disulfide Mimetics in Cyclic Peptides.
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White, Andrew M., Veer, Simon J., Wu, Guojie, Harvey, Peta J., Yap, Kuok, King, Gordon J., Swedberg, Joakim E., Wang, Conan K., Law, Ruby H. P., Durek, Thomas, and Craik, David J.
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CYCLIC peptides ,DISULFIDES ,X-ray crystallography ,PEPTIDES ,PROTEASE inhibitors ,PEPTIDOMIMETICS ,TRYPSIN - Abstract
Ruthenium‐catalysed azide–alkyne cycloaddition (RuAAC) provides access to 1,5‐disubstituted 1,2,3‐triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross‐linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor‐1. NMR and X‐ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole‐bridged peptides also displayed superior half‐lives in liver S9 stability assays compared to disulfide‐bridged peptides. This work establishes a foundation for the application of 1,5‐disubstituted 1,2,3‐triazoles as disulfide mimetics. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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21. EGF‐like and Other Disulfide‐rich Microdomains as Therapeutic Scaffolds.
- Author
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Tombling, Benjamin J., Wang, Conan K., and Craik, David J.
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CHEMICAL potential ,ANIMALS ,CONOTOXINS ,DRUG design - Abstract
Disulfide bonds typically introduce conformational constraints into peptides and proteins, conferring improved biopharmaceutical properties and greater therapeutic potential. In our opinion, disulfide‐rich microdomains from proteins are potentially a rich and under‐explored source of drug leads. A survey of the UniProt protein database shows that these domains are widely distributed throughout the plant and animal kingdoms, with the EGF‐like domain being the most abundant of these domains. EGF‐like domains exhibit large diversity in their disulfide bond topologies and calcium binding modes, which we classify in detail here. We found that many EGF‐like domains are associated with disease phenotypes, and the interactions they mediate are potential therapeutic targets. Indeed, EGF‐based therapeutic leads have been identified, and we further propose that these domains can be optimized to expand their therapeutic potential using chemical design strategies. This Review highlights the potential of disulfide‐rich microdomains as future peptide therapeutics. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
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22. EGF‐artige und andere disulfidreiche Mikrodomänen als therapeutische Molekülgerüste.
- Author
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Tombling, Benjamin J., Wang, Conan K., and Craik, David J.
- Abstract
Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
- Published
- 2020
- Full Text
- View/download PDF
23. In Silico Design of MDM2‐Targeting Peptides from a Naturally Occurring Constrained Peptide.
- Author
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Ma, He, Qiu, Peiju, Harvey, Peta J., Li, Xiao, Zhang, Zheyu, Xu, Qingliang, Liang, Jiazhen, Kaas, Quentin, Craik, David J., Yang, Jinbo, Jiang, Tao, Bi, Xiaolin, and Yu, Rilei
- Published
- 2019
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24. Molecular dynamics simulations of dihydro-β-erythroidine bound to the human α4β2 nicotinic acetylcholine receptor.
- Author
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Yu, Rilei, Tae, Han‐Shen, Xu, Qingliang, Craik, David J., Adams, David J., Jiang, Tao, Kaas, Quentin, and Tae, Han-Shen
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NICOTINIC acetylcholine receptors ,MOLECULAR dynamics ,CHOLINERGIC receptors - Abstract
Background and Purpose: The heteromeric α4β2 nicotinic acetylcholine receptor (nAChR) is abundant in the human brain and is associated with a range of CNS disorders. This nAChR subtype has been recently crystallised in a conformation that was proposed to represent a desensitised state. Here, we investigated the conformational transition mechanism of this nAChR from a desensitised to a closed/resting state.Experimental Approach: The competitive antagonist dihydro-β-erythroidine (DHβE) was modelled by replacement of the agonist nicotine in the α4β2 nAChR experimental structure. DHβE is used both in vitro and in vivo for its ability to block α4β2 nAChRs. This system was studied by three molecular dynamics simulations with a combined simulation time of 2.6 μs. Electrophysiological studies of mutated receptors were performed to validate the simulation results.Key Results: The relative positions of the extracellular and transmembrane domains in the models are distinct from those of the desensitised state structure and are compatible with experimental structures of Cys-loop receptors captured in a closed/resting state.Conclusions and Implications: Our model suggests that the side chains of α4 L257 (9') and α4 L264 (16') are the main constrictions in the transmembrane pore. The involvement of position 9' in channel gating is well established, but position 16' was only previously identified as a gate for the bacterial channels, ELIC and GLIC. L257 but not L264 was found to influence the slow component of desensitisation. The structure of the antagonist-bound state proposed here should be valuable for the development of therapeutic or insecticide compounds. [ABSTRACT FROM AUTHOR]- Published
- 2019
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25. NaV1.6 regulates excitability of mechanosensitive sensory neurons.
- Author
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Israel, Mathilde R., Tanaka, Brian S., Castro, Joel, Thongyoo, Panumart, Robinson, Samuel D., Zhao, Peng, Deuis, Jennifer R., Craik, David J., Durek, Thomas, Brierley, Stuart M., Waxman, Stephen G, Dib‐Hajj, Sulayman D., and Vetter, Irina
- Subjects
SENSORY neurons ,PERIPHERAL nervous system ,DORSAL root ganglia ,SODIUM channels ,ACTION potentials - Abstract
Key points: Voltage‐gated sodium channels are critical for peripheral sensory neuron transduction and have been implicated in a number of painful and painless disorders.The β‐scorpion toxin, Cn2, is selective for NaV1.6 in dorsal root ganglion neurons.NaV1.6 plays an essential role in peripheral sensory neurons, specifically at the distal terminals of mechanosensing fibres innervating the skin and colon.NaV1.6 activation also leads to enhanced response to mechanical stimulus in vivo.This works highlights the use of toxins in elucidating pain pathways moreover the importance of non‐peripherally restricted NaV isoforms in pain generation. Peripheral sensory neurons express multiple voltage‐gated sodium channels (NaV) critical for the initiation and propagation of action potentials and transmission of sensory input. Three pore‐forming sodium channel isoforms are primarily expressed in the peripheral nervous system (PNS): NaV1.7, NaV1.8 and NaV1.9. These sodium channels have been implicated in painful and painless channelopathies and there has been intense interest in them as potential therapeutic targets in human pain. Emerging evidence suggests NaV1.6 channels are an important isoform in pain sensing. This study aimed to assess, using pharmacological approaches, the function of NaV1.6 channels in peripheral sensory neurons. The potent and NaV1.6 selective β‐scorpion toxin Cn2 was used to assess the effect of NaV1.6 channel activation in the PNS. The multidisciplinary approach included Ca2+ imaging, whole‐cell patch‐clamp recordings, skin–nerve and gut–nerve preparations and in vivo behavioural assessment of pain. Cn2 facilitates NaV1.6 early channel opening, and increased persistent and resurgent currents in large‐diameter dorsal root ganglion (DRG) neurons. This promotes enhanced excitatory drive and tonic action potential firing in these neurons. In addition, NaV1.6 channel activation in the skin and gut leads to increased response to mechanical stimuli. Finally, intra‐plantar injection of Cn2 causes mechanical but not thermal allodynia. This study confirms selectivity of Cn2 on NaV1.6 channels in sensory neurons. Activation of NaV1.6 channels, in terminals of the skin and viscera, leads to profound changes in neuronal responses to mechanical stimuli. In conclusion, sensory neurons expressing NaV1.6 are important for the transduction of mechanical information in sensory afferents innervating the skin and viscera. Key points: Voltage‐gated sodium channels are critical for peripheral sensory neuron transduction and have been implicated in a number of painful and painless disorders.The β‐scorpion toxin, Cn2, is selective for NaV1.6 in dorsal root ganglion neurons.NaV1.6 plays an essential role in peripheral sensory neurons, specifically at the distal terminals of mechanosensing fibres innervating the skin and colon.NaV1.6 activation also leads to enhanced response to mechanical stimulus in vivo.This works highlights the use of toxins in elucidating pain pathways moreover the importance of non‐peripherally restricted NaV isoforms in pain generation. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
26. Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor.
- Author
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Chambers, Chris, Cutler, Penny, Huang, Yen‐Hua, Goodchild, James A., Blythe, Judith, Wang, Conan K., Bigot, Aurélien, Kaas, Quentin, Craik, David J., Sabbadin, Davide, and Earley, Fergus G.
- Subjects
NICOTINIC acetylcholine receptors ,CHOLINERGIC receptors ,STRUCTURE-activity relationships ,TOXINS ,POTASSIUM channels ,INSECT development - Abstract
The insecticidal effects of ω‐hexatoxin‐Hv1a, κ‐hexatoxin‐Hv1c and ω/κ‐hexatoxin‐Hv1h are currently attributed to action at calcium and potassium channels. By characterizing the binding of these toxins to neuronal membranes, we show that they have more potent effects as positive allosteric modulators (PAMs) of insect nicotinic acetylcholine receptors (nAChRs), consistent with their neuroexcitatory toxicology. Alanine scanning analysis of ω‐hexatoxin‐Hv1a reveals a structure–activity relationship for binding that mirrors that for insecticidal activity. Spinosyn A does not compete with ω‐hexatoxin‐Hv‐1a for binding, and we show that these two PAMs have distinct pharmacology of binding indicating that they act at different receptor populations. These toxins represent valuable tools for the characterization of insect nAChRs and for the development of more selective agrochemicals. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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27. Efficient Enzymatic Cyclization of Disulfide‐Rich Peptides by Using Peptide Ligases.
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Schmidt, Marcel, Huang, Yen‐Hua, Texeira de Oliveira, Eduardo F., Toplak, Ana, Wijma, Hein J., Janssen, Dick B., van Maarseveen, Jan H., Craik, David J., and Nuijens, Timo
- Published
- 2019
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28. Anchor Residues Guide Form and Function in Grafted Peptides.
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Yin, Huawu, Craik, David J., and Wang, Conan K.
- Subjects
- *
SCAFFOLD proteins , *PEPTIDE drugs , *THERMODYNAMICS , *EPITOPES , *PROTEIN conformation - Abstract
Loops at protein–protein interfaces are a rich source of peptide leads that have high specificity and low toxicity. Although such peptides typically need to be constrained to overcome thermodynamic and metabolic limitations, design guidelines to obtain a successfully constrained peptides, and thus facilitate the transition from loop to drug, are relatively poorly formulated. In this work, we surveyed the structures of interface loops and found the position of the terminal residues to be a key determinant of conformation. We used this knowledge to improve the process of molecular grafting, a valuable approach for constraining and stabilising peptides by fusing them to a suitable scaffold. We show that an informed choice of where a loop is "anchored" to a scaffold improves its form and function. This knowledge can help guide the choice of loop and its matching scaffold, and thus increase the success rate for designing stable and potent peptide drug leads. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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29. Where cone snails and spiders meet: design of small cyclic sodium-channel inhibitors.
- Author
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Peigneur, Steve, Cheneval, Olivier, Maiti, Mohitosh, Leipold, Enrico, Heinemann, Stefan H., Lescrinier, Eveline, Herdewijn, Piet, De Lima, Maria Elena, Craik, David J., Schroeder, Christina I., and Tytgat, Jan
- Published
- 2019
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30. Characterising the Subsite Specificity of Urokinase‐Type Plasminogen Activator and Tissue‐Type Plasminogen Activator using a Sequence‐Defined Peptide Aldehyde Library.
- Author
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Li, Choi Yi, de Veer, Simon J., Law, Ruby H. P., Whisstock, James C., Craik, David J., and Swedberg, Joakim E.
- Published
- 2019
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31. Cyclic analogues of α-conotoxin Vc1.1 inhibit colonic nociceptors and provide analgesia in a mouse model of chronic abdominal pain.
- Author
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Castro, Joel, Grundy, Luke, Deiteren, Annemie, Harrington, Andrea M., O'Donnell, Tracey, Maddern, Jessica, Moore, Jessi, Garcia‐Caraballo, Sonia, Rychkov, Grigori Y., Yu, Rilei, Kaas, Quentin, Craik, David J., Adams, David J., Brierley, Stuart M., O'Donnell, Tracey, and Garcia-Caraballo, Sonia
- Subjects
IRRITABLE colon ,VISCERAL pain ,ANALGESICS ,CELLULAR signal transduction ,LABORATORY mice ,PATIENTS ,ABDOMINAL pain ,ANALGESIA ,ANIMAL experimentation ,BIOLOGICAL models ,CELL culture ,CHRONIC diseases ,COLON (Anatomy) ,COMPARATIVE studies ,MARINE toxins ,RESEARCH methodology ,MEDICAL cooperation ,MICE ,NOCICEPTORS ,RESEARCH ,EVALUATION research - Abstract
Background and Purpose: Patients with irritable bowel syndrome suffer from chronic visceral pain (CVP) and limited analgesic therapeutic options are currently available. We have shown that α-conotoxin Vc1.1 induced activation of GABAB receptors on the peripheral endings of colonic afferents and reduced nociceptive signalling from the viscera. However, the analgesic efficacy of more stable, cyclized versions of Vc1.1 on CVP remains to be determined.Experimental Approach: Using ex vivo colonic afferent preparations from mice, we determined the inhibitory actions of cyclized Vc1.1 (cVc1.1) and two cVc1.1 analogues on mouse colonic nociceptors in healthy and chronic visceral hypersensitivity (CVH) states. Using whole-cell patch clamp recordings, we also assessed the inhibitory actions of these peptides on the neuronal excitability of colonic innervating dorsal root ganglion neurons. In vivo, the analgesic efficacy of these analogues was assessed by determining the visceromotor response to colorectal distension in healthy and CVH mice.Key Results: cVc1.1 and the cVc1.1 analogues, [C2H,C8F]cVc1.1 and [N9W]cVc1.1, all caused concentration-dependent inhibition of colonic nociceptors from healthy mice. Inhibition by these peptides was greater than those evoked by linear Vc1.1 and was substantially greater in colonic nociceptors from CVH mice. cVc1.1 also reduced excitability of colonic dorsal root ganglion neurons, with greater effect in CVH neurons. CVH mice treated with cVc1.1 intra-colonically displayed reduced pain responses to noxious colorectal distension compared with vehicle-treated CVH mice.Conclusions and Implications: Cyclic versions of Vc1.1 evoked significant anti-nociceptive actions in CVH states, suggesting that they could be novel candidates for treatment of CVP.Linked Articles: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc. [ABSTRACT FROM AUTHOR]- Published
- 2018
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32. Lysine to arginine mutagenesis of chlorotoxin enhances its cellular uptake.
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Ojeda, Paola G., Henriques, Sónia Troeira, Pan, Yijun, Nicolazzo, Joseph A., Craik, David J., and Wang, Conan K.
- Abstract
Chlorotoxin (CTX), a disulfide-rich peptide from the scorpion Leiurus quinquestriatus, has several promising biopharmaceutical properties, including preferential affinity for certain cancer cells, high serum stability, and cell penetration. These properties underpin its potential for use as a drug design scaffold, especially for the treatment of cancer; indeed, several analogs of CTX have reached clinical trials. Here, we focus on its ability to internalize into cells-a trait associated with a privileged subclass of peptides called cell-penetrating peptides-and whether it can be improved through conservative substitutions. Mutants of CTX were made using solid-phase peptide synthesis and internalization into human cervical carcinoma (HeLa) cells was monitored by fluorescence and confocal microscopy. CTX_M1 (ie, [K15R/K23R]CTX) and CTX_M2 (ie, [K15R/K23R/Y29W]CTX) mutants showed at least a twofold improvement in uptake compared to CTX. We further showed that these mutants internalize into HeLa cells largely via an energy-dependent mechanism. Importantly, the mutants have high stability, remaining intact in serum for over 24 h; thus, retaining the characteristic stability of their parent peptide. Overall, we have shown that simple conservative substitutions can enhance the cellular uptake of CTX, suggesting that such type of mutations might be useful for improving uptake of other peptide toxins. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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33. Structural and functional characterization of chimeric cyclotides from the Möbius and trypsin inhibitor subfamilies.
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Abdul Ghani, Hafiza, Henriques, Sónia Troeira, Huang, Yen‐Hua, Swedberg, Joakim E., Schroeder, Christina I., and Craik, David J.
- Abstract
Cyclotides are plant-derived host defense peptides displaying exceptional stability due to their cyclic cystine knot comprising three intertwined disulfide bonds and a cyclic backbone. Their six conserved cysteine residues are separated by backbone loops with diverse sequences. Prototypical cyclotides from the Möbius (kalata B1) and trypsin inhibitor (MCoTI-II) subfamilies lack sequence homology with one another, but both are able to penetrate cells, apparently via different mechanisms. To delineate the influence of the sequences of the loops on the structure and cell internalization of these two cyclotide subfamilies, a series of Möbius/trypsin inhibitor loop-chimeras of kalata B1 and MCoTI-II were synthesized, and structurally and functionally characterized. NMR analysis showed that the structural fold of the majority of chimeric peptides was minimally affected by the loop substitutions. Substituting loops 3, 5, or 6 of MCoTI-II into the corresponding loops of kalata B1 attenuated its hemolytic and cytotoxic activities, and greatly reduced its cell-penetrating properties. On the other hand, replacing loops of MCoTI-II with the corresponding loops of kalata B1 did not introduce cytotoxicity into the chimeras. Loops 2, 3, and 4 of MCoTI-II were found to contribute little to cell-penetrating properties. Overall, this study provides valuable insights into the structural basis for the hemolytic, cytotoxic, and cell-penetrating properties of kalata B1 and MCoTI-II, which could be useful for future engineering of cyclotides to carry bioactive epitopes to intracellular targets. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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34. Development of cell-penetrating peptide-based drug leads to inhibit MDMX:p53 and MDM2:p53 interactions.
- Author
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Philippe, Grégoire, Huang, Yen‐Hua, Cheneval, Olivier, Lawrence, Nicole, Zhang, Zhen, Fairlie, David P, Craik, David J., de Araujo, Aline Dantas, and Henriques, Sónia Troeira
- Abstract
The transcription factor p53 has a tumor suppressor role in leading damaged cells to apoptosis. Its activity is regulated/inhibited in healthy cells by the proteins MDM2 and MDMX. Overexpression of MDM2 and/or MDMX in cancer cells inactivates p53, facilitating tumor development. A 12-mer dual inhibitor peptide (pDI) was previously reported to be able to target and inhibit MDMX:p53 and MDM2:p53 interactions with nanomolar potency in vitro. With the aim of improving its cellular inhibitory activity, we produced a series of constrained pDI analogs featuring lactam staples that stabilize the bioactive helical conformation and fused them with a cell-penetrating peptide to increase cytosol delivery. We compared pDI and its analogs on their inhibitory potency, toxicity, and ability to enter cancer cells. Overall, the results show that these analogs keep their nanomolar affinity for MDM2 and MDMX and are highly active against cancer cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 853-863, 2016. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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35. Cyclic peptide oral bioavailability: Lessons from the past.
- Author
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Wang, Conan K and Craik, David J.
- Abstract
Achieving high oral bioavailability for drugs is a key design objective in drug development. It is not surprising then that with the growing expectation of peptides as future drugs, there has also been an increasing interest in developing oral peptide therapeutics. Brought to the fore are questions such as what makes peptides orally bioavailable and how this can be achieved; questions which have inspired research into the area for decades. Early research in the area focused on linear peptides with more recent literature focusing on cyclic peptides, motivated in part by cyclic peptides like cyclosporine A that have demonstrated drug-like oral bioavailability. In this review, we take a look at research on the oral bioavailability of peptides, focusing on factors that affect passive permeability. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 901-909, 2016. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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36. Effects of linker sequence modifications on the structure, stability, and biological activity of a cyclic α-conotoxin.
- Author
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Carstens, Bodil B., Swedberg, Joakim, Berecki, Géza, Adams, David J., Craik, David J., and Clark, Richard J.
- Abstract
The cyclic conotoxin analogue cVc1.1 is a promising lead molecule for the development of new treatments for neuropathic and chronic pain. The design of this peptide includes a linker sequence that joins the N and C termini together, improving peptide stability while maintaining the structure and activity of the original linear Vc1.1. The effect of linker length on the structure, activity and stability of cyclised conotoxins has been studied previously but the effect of altering the composition of the linker sequence has not been investigated. In this study, we designed three analogues of cVc1.1 with linker sequences that varied in charge, hydrophobicity and hydrogen bonding capacity and examined the effect on structure, stability, membrane permeability and biological activity. The three designed peptides were successfully synthesized using solid phase peptide synthesis approaches and had similar structures and stability compared with cVc1.1. Despite modifications in charge, hydrophobicity and hydrogen bonding potential, which are all factors that can affect membrane permeability, no changes in the ability of the peptides to pass through membranes in either PAMPA or Caco-2 cell assay were observed. Surprisingly, modification of the linker sequence was deleterious to biological activity. These results suggest the linker sequence might be a useful part of the molecule for optimization of bioactivity and not just the physiochemical properties of cVc1.1. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 864-875, 2016. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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37. Discovery, isolation, and structural characterization of cyclotides from Viola sumatrana Miq.
- Author
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Niyomploy, Ploypat, Chan, Lai Yue, Poth, Aaron G., Colgrave, Michelle L., Sangvanich, Polkit, and Craik, David J.
- Abstract
Cyclotides are cyclic peptides from plants in the Violaceae, Rubiaceae, Fabaceae, Cucurbitaceae, and Solanaceae families. They are sparsely distributed in most of these families, but appear to be ubiquitous in the Violaceae, having been found in every plant so far screened from this family. However, not all geographic regions have been examined and here we report the discovery of cyclotides from a Viola species from South-East Asia. Two novel cyclotides (Visu 1 and Visu 2) and two known cyclotides (kalata S and kalata B1) were identified in V. sumatrana. NMR studies revealed that kalata S and kalata B1 had similar secondary structures. Their biological activities were determined in cytotoxicity assays; both had similar cytotoxic activity and were more toxic to U87 cells compared with other cell lines. Overall, the study strongly supports the ubiquity of cyclotides in the Violaceae and adds to our understanding of their distribution and cytotoxic activity. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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38. Nomenclature of homodetic cyclic peptides produced from ribosomal precursors: An IUPAC task group interim report.
- Author
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Craik, David J., Young Shim, Youn, Göransson, Ulf, Moss, Gerard P., Tan, Ninghua, Jadhav, Pramodkumar D., Shen, Jianheng, and Reaney, Martin J. T.
- Abstract
In 2015, an International Union of Pure and Applied Chemistry (IUPAC) Task Group was formed to develop nomenclature recommendations for homodetic cyclic peptides produced from ribosomal precursors. Delegates of the 2015 International Conference on Circular Proteins (ICCP) were presented with the strengths and weaknesses of four published approaches to homodetic cyclic peptide nomenclature, and a summary of the ensuing discussion is presented here. This interim report presents a potentially novel suggestion-the use of Cahn-Ingold-Prelog rules to specify amino acid priority in homodetic peptides for consistent numbering. Indeed, this might be the first extension of the Cahn-Ingold-Prelog rules in five decades. The authors invite interested parties to contact the corresponding author with suggestions for the improvement of the proposed nomenclature; these ideas will be discussed and considered for inclusion in the final report. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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39. The N-terminal pro-domain of the kalata B1 cyclotide precursor is intrinsically unstructured.
- Author
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Daly, Norelle L., Gunasekera, Sunithi, Clark, Richard J., Lin, Feng, Wade, John D., Anderson, Marilyn A., and Craik, David J.
- Abstract
Cyclotides are plant-derived, gene-encoded, circular peptides with a range of host-defense functions, including insecticidal activity. They also have potential as pharmaceutical scaffolds and understanding their biosynthesis is important to facilitate their large-scale production. Insights into the biosynthesis of cyclotides are emerging but there are still open questions, particularly regarding the influence of the structure of the precursor proteins on processing/biosynthetic pathways. The precursor protein of kalata B1, encoded by the plant Oldenlandia affinis, contains N- and C-terminal propeptides that flank the mature cyclotide domain. The C-terminal region (ctr) is important for the cyclization process, whereas the N-terminal repeat (ntr) has been implicated in vacuolar targeting. In this study we examined the structure and folding of various truncated constructs of the ntr coupled to the mature domain of kalata B1. Despite the ntr having a well-defined helical structure in isolation, once coupled to the natively folded mature domain there is no evidence of an ordered structure. Surprisingly, the ntr appears to be highly disordered and induces self-association of the precursor. This self-association might be associated with the role of the ntr as a vacuolar-targeting signal, as previously shown for unrelated storage proteins. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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40. Efficient enzymatic cyclization of an inhibitory cystine knot-containing peptide.
- Author
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Kwon, Soohyun, Bosmans, Frank, Kaas, Quentin, Cheneval, Olivier, Conibear, Anne C., Rosengren, K. Johan, Wang, Conan K., Schroeder, Christina I., and Craik, David J.
- Abstract
ABSTRACT Disulfide-rich peptides isolated from cone snails are of great interest as drug leads due to their high specificity and potency toward therapeutically relevant ion channels and receptors. They commonly contain the inhibitor cystine knot (ICK) motif comprising three disulfide bonds forming a knotted core. Here we report the successful enzymatic backbone cyclization of an ICK-containing peptide κ-PVIIA, a 27-amino acid conopeptide from Conus purpurascens, using a mutated version of the bacterial transpeptidase, sortase A. Although a slight loss of activity was observed compared to native κ-PVIIA, cyclic κ-PVIIA is a functional peptide that inhibits the Shaker voltage-gated potassium (Kv) channel. Molecular modeling suggests that the decrease in potency may be related to the loss of crucial, but previously unidentified electrostatic interactions between the N-terminus of the peptide and the Shaker channel. This hypothesis was confirmed by testing an N-terminally acetylated κ-PVIIA, which shows a similar decrease in activity. We also investigated the conformational dynamics and hydrogen bond network of cyc-PVIIA, both of which are important factors to be considered for successful cyclization of peptides. We found that cyc-PVIIA has the same conformational dynamics, but different hydrogen bond network compared to those of κ-PVIIA. The ability to efficiently cyclize ICK peptides using sortase A will enable future protein engineering for this class of peptides and may help in the development of novel therapeutic molecules. Biotechnol. Bioeng. 2016;113: 2202-2212. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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41. Cyclisation of Disulfide-Rich Conotoxins in Drug Design Applications.
- Author
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Wu, Xiaosa, Huang, Yen‐Hua, Kaas, Quentin, and Craik, David J.
- Subjects
RING formation (Chemistry) ,DISULFIDES ,CONOTOXINS ,CHEMICAL reactions ,NEUROTOXIC agents ,DRUG design ,ORGANIC chemistry - Abstract
Conotoxins are disulfide-rich peptides found in the venoms of marine snails of the genus Conus. They have attracted great attention from the pharmaceutical industry because of their potential uses as drug leads, but like most peptides, conotoxins are susceptible to proteolysis and typically are not orally bioavailable. Here we discuss approaches that have been used to stabilise conotoxins to improve their potential pharmaceutical use. Specifically, we focus on the use of backbone cyclisation to improve their stability in biological fluids. The Microreview provides an introduction to the various classes of conotoxins, including their frameworks (cysteine patterns) and a background on the receptors that they interact with, as well as an analysis of the binding interactions between conotoxins and their receptors. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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42. Biodistribution of the cyclotide MCoTI-II, a cyclic disulfide-rich peptide drug scaffold.
- Author
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Wang, Conan K., Stalmans, Sofie, De Spiegeleer, Bart, and Craik, David J.
- Abstract
Disulfide-rich macrocyclic peptides are promising templates for drug design because of their unique topology and remarkable stability. However, little is known about their pharmacokinetics. In this study, we characterize the biodistribution in mice of Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a cyclic three-disulfide-containing peptide that has been used in a number of studies as a drug scaffold. The distribution of MCoTI-II was compared with that of chlorotoxin, which is a four-disulfide-containing peptide that has been used to develop brain tumor imaging agents; dermorphin, which is a disulfide-less peptide; and bovine serum albumin, a large protein. Both MCoTI-II and chlorotoxin distributed predominantly to the serum and kidneys, confirming that they are stable in serum and suggesting that they are eliminated from the blood through renal clearance. Although cell-penetrating peptides have been reported to be able to transport across the blood-brain barrier, MCoTI-II, which is a cell-penetrating peptide, showed no uptake into the brain. The uptake of chlorotoxin was higher than that of MCoTI-II but lower than that of dermorphin, which is considered to have low uptake into the brain. This study provides insight into the behavior of disulfide-rich peptides in vivo. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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43. Gene coevolution and regulation lock cyclic plant defence peptides to their targets.
- Author
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Gilding, Edward K., Jackson, Mark A., Poth, Aaron G., Henriques, Sónia Troeira, Prentis, Peter J., Mahatmanto, Tunjung, and Craik, David J.
- Subjects
CYCLIC peptides ,PLANT toxins ,PLANT defenses ,GENETIC polymorphisms in plants ,PLANT genetics ,GENETIC research - Abstract
Plants have evolved many strategies to protect themselves from attack, including peptide toxins that are ribosomally synthesized and thus adaptable directly by genetic polymorphisms. Certain toxins in Clitoria ternatea (butterfly pea) are cyclic cystine-knot peptides of c. 30 residues, called cyclotides, which have co-opted the plant's albumin-1 gene family for their production. How butterfly pea albumin-1 genes were commandeered and how these cyclotides are utilized in defence remain unclear. The role of cyclotides in host plant ecology and biotechnological applications requires exploration., We characterized the sequence diversity and expression dynamics of precursor and processing proteins implicated in butterfly pea cyclotide biosynthesis by expression profiling through RNA-sequencing ( RNA-seq). Peptide-enriched extracts from various organs were tested for activity against insect-like membranes and the model nematode Caenorhabditis elegans., We found that the evolution and deployment of cyclotides involved their diversification to exhibit different chemical properties and expression between organs facing different defensive challenges. Cyclotide-enriched fractions from soil-contacting organs were effective at killing nematodes, whereas similar enriched fractions from aerial organs contained cyclotides that exhibited stronger interactions with insect-like membrane lipids., Cyclotides are employed as versatile and combinatorial mediators of defence in C. ternatea and have specialized to affect different classes of attacking organisms. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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44. Structure-Activity Studies of Cysteine-Rich α-Conotoxins that Inhibit High-Voltage-Activated Calcium Channels via GABAB Receptor Activation Reveal a Minimal Functional Motif.
- Author
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Carstens, Bodil B., Berecki, Géza, Daniel, James T., Lee, Han Siean, Jackson, Kathryn A. V., Tae, Han-Shen, Sadeghi, Mahsa, Castro, Joel, O'Donnell, Tracy, Deiteren, Annemie, Brierley, Stuart M., Craik, David J., Adams, David J., and Clark, Richard J.
- Subjects
CONOTOXIN structure ,DRUG design ,STRUCTURE-activity relationships ,CYSTEINE ,CALCIUM channels ,GABA receptors ,PEPTIDE drugs - Abstract
α-Conotoxins are disulfide-rich peptides that target nicotinic acetylcholine receptors. Recently we identified several α-conotoxins that also modulate voltage-gated calcium channels by acting as G protein-coupled GABA
B receptor (GABAB R) agonists. These α-conotoxins are promising drug leads for the treatment of chronic pain. To elucidate the diversity of α-conotoxins that act through this mechanism, we synthesized and characterized a set of peptides with homology to α-conotoxins known to inhibit high voltage-activated calcium channels via GABAB R activation. Remarkably, all disulfide isomers of the active α-conotoxins Pu1.2 and Pn1.2, and the previously studied Vc1.1 showed similar levels of biological activity. Structure determination by NMR spectroscopy helped us identify a simplified biologically active eight residue peptide motif containing a single disulfide bond that is an excellent lead molecule for developing a new generation of analgesic peptide drugs. [ABSTRACT FROM AUTHOR]- Published
- 2016
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- View/download PDF
45. Chlorotoxin: Structure, activity, and potential uses in cancer therapy.
- Author
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Ojeda, Paola G., Wang, Conan K., and Craik, David J.
- Abstract
ABSTRACT Chlorotoxin is a disulfide-rich stable peptide from the venom of the Israeli scorpion Leiurus quinquestriatus, which has potential therapeutic applications in the treatment of cancer. Its ability to preferentially bind to tumor cells has been harnessed to develop an imaging agent to help visualize tumors during surgical resection. In addition, chlorotoxin has attracted interest as a vehicle to deliver anti-cancer drugs specifically to cancer cells. Given its interesting structural and biological properties, chlorotoxin also has the potential to be used in a variety of other biotechnology and biomedical applications. Here, we review the structure, activity and potential applications of chlorotoxin as a drug design scaffold. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 25-36, 2016. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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- View/download PDF
46. Approaches to the stabilization of bioactive epitopes by grafting and peptide cyclization.
- Author
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Conibear, Anne C., Chaousis, Stephanie, Durek, Thomas, Johan Rosengren, K., Craik, David J., and Schroeder, Christina I.
- Abstract
ABSTRACT Peptides are attracting increasing interest from the pharmaceutical industry because of their specificity and ability to address novel targets, including protein-protein interactions. However, typically they require stabilization for therapeutic applications owing to their susceptibility to degradation by proteases. Advances in the ability to chemically synthesize peptides and the development of new side-chain and backbone ligation strategies provide new tools to stabilize bioactive peptide epitopes. Two such epitopes are LyP1, a nine residue peptide that localizes to tumor cells and has potential as an anticancer therapeutic, and RGDS, a tetrapeptide shown to bind to survivin and induce apoptosis. Here we applied a variety of strategies for the stabilization of LyP1 and RGDS, including side-chain cyclization using 'click' chemistry and 'grafting' the epitopes into two naturally occurring cyclic peptide scaffolds, i.e., θ-defensins and cyclotides. NMR data showed that the three-disulfide θ-defensin and cyclotide scaffolds accommodated the LyP1 and RGDS epitopes but that scaffolds with fewer disulfide bonds were structurally compromised by inclusion of the LyP1 epitope. LyP1, LyP1-, and RGDS-grafted peptides that were largely unstructured also had reduced resistance to degradation in human serum, showing that grafting into a stable cyclic scaffold is an effective strategy for increasing the stability of a bioactive peptide epitope. Overall, the study demonstrates several methods for stabilizing peptide epitopes using side-chain or backbone cyclization and illustrates their potential in peptide drug design. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 89-100, 2016. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
47. Transforming conotoxins into cyclotides: Backbone cyclization of P-superfamily conotoxins.
- Author
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Akcan, Muharrem, Clark, Richard J., Daly, Norelle L., Conibear, Anne C., Faoite, Andrew, Heghinian, Mari D., Sahil, Talwar, Adams, David J., Marí, Frank, and Craik, David J.
- Abstract
ABSTRACT Peptide backbone cyclization is a widely used approach to improve the activity and stability of small peptides but until recently it had not been applied to peptides with multiple disulfide bonds. Conotoxins are disulfide-rich conopeptides derived from the venoms of cone snails that have applications in drug design and development. However, because of their peptidic nature, they can suffer from poor bioavailability and poor stability in vivo. In this study two P-superfamily conotoxins, gm9a and bru9a, were backbone cyclized by joining the N- and C-termini with short peptide linkers using intramolecular native chemical ligation chemistry. The cyclized derivatives had conformations similar to the native peptides showing that backbone cyclization can be applied to three disulfide-bonded peptides with cystine knot motifs. Cyclic gm9a was more potent at high voltage-activated (HVA) calcium channels than its acyclic counterpart, highlighting the value of this approach in developing active and stable conotoxins containing cyclic cystine knot motifs. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 104: 682-692, 2015. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
48. Key challenges for the creation and maintenance of specialist protein resources.
- Author
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Holliday, Gemma L., Bairoch, Amos, Bagos, Pantelis G., Chatonnet, Arnaud, Craik, David J., Finn, Robert D., Henrissat, Bernard, Landsman, David, Manning, Gerard, Nagano, Nozomi, O'Donovan, Claire, Pruitt, Kim D., Rawlings, Neil D., Saier, Milton, Sowdhamini, Ramanathan, Spedding, Michael, Srinivasan, Narayanaswamy, Vriend, Gert, Babbitt, Patricia C., and Bateman, Alex
- Abstract
ABSTRACT As the volume of data relating to proteins increases, researchers rely more and more on the analysis of published data, thus increasing the importance of good access to these data that vary from the supplemental material of individual articles, all the way to major reference databases with professional staff and long-term funding. Specialist protein resources fill an important middle ground, providing interactive web interfaces to their databases for a focused topic or family of proteins, using specialized approaches that are not feasible in the major reference databases. Many are labors of love, run by a single lab with little or no dedicated funding and there are many challenges to building and maintaining them. This perspective arose from a meeting of several specialist protein resources and major reference databases held at the Wellcome Trust Genome Campus (Cambridge, UK) on August 11 and 12, 2014. During this meeting some common key challenges involved in creating and maintaining such resources were discussed, along with various approaches to address them. In laying out these challenges, we aim to inform users about how these issues impact our resources and illustrate ways in which our working together could enhance their accuracy, currency, and overall value. Proteins 2015; 83:1005-1013. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
49. Improving on Nature: Making a Cyclic Heptapeptide Orally Bioavailable.
- Author
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Nielsen, Daniel S., Hoang, Huy N., Lohman, Rink‐Jan, Hill, Timothy A., Lucke, Andrew J., Craik, David J., Edmonds, David J., Griffith, David A., Rotter, Charles J., Ruggeri, Roger B., Price, David A., Liras, Spiros, and Fairlie, David P.
- Subjects
CYCLIC peptides ,HYDROGEN bonding ,DRUG bioavailability ,METHYLATION ,RING formation (Chemistry) - Abstract
The use of peptides in medicine is limited by low membrane permeability, metabolic instability, high clearance, and negligible oral bioavailability. The prediction of oral bioavailability of drugs relies on physicochemical properties that favor passive permeability and oxidative metabolic stability, but these may not be useful for peptides. Here we investigate effects of heterocyclic constraints, intramolecular hydrogen bonds, and side chains on the oral bioavailability of cyclic heptapeptides. NMR-derived structures, amide H-D exchange rates, and temperature-dependent chemical shifts showed that the combination of rigidification, stronger hydrogen bonds, and solvent shielding by branched side chains enhances the oral bioavailability of cyclic heptapeptides in rats without the need for N-methylation. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
50. Racemic and Quasi-Racemic X-ray Structures of Cyclic Disulfide-Rich Peptide Drug Scaffolds.
- Author
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Wang, Conan K., King, Gordon J., Northfield, Susan E., Ojeda, Paola G., and Craik, David J.
- Subjects
X-rays ,DISULFIDES ,PEPTIDE drugs ,CRYSTALLIZATION ,CHEMICAL bonds ,CYCLIC peptides - Abstract
Cyclic disulfide-rich peptides have exceptional stability and are promising frameworks for drug design. We were interested in obtaining X-ray structures of these peptides to assist in drug design applications, but disulfide-rich peptides can be notoriously difficult to crystallize. To overcome this limitation, we chemically synthesized the L- and D-forms of three prototypic cyclic disulfide-rich peptides: SFTI-1 (14-mer with one disulfide bond), cVc1.1 (22-mer with two disulfide bonds), and kB1 (29-mer with three disulfide bonds) for racemic crystallization studies. Facile crystal formation occurred from a racemic mixture of each peptide, giving structures solved at resolutions from 1.25 Å to 1.9 Å. Additionally, we obtained the quasi-racemic structures of two mutants of kB1, [G6A]kB1, and [V25A]kB1, which were solved at a resolution of 1.25 Å and 2.3 Å, respectively. The racemic crystallography approach appears to have broad utility in the structural biology of cyclic peptides. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
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