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a-Conotoxins are multiple disulfide bond containing peptides that are isolated from venomous marine cone snails. They display remarkable selectivity for different subtypes of nicotinic acetylcholine receptors (nAChRs). While a-conotoxins display poor resistance to in vivo degradation by proteases, which limits their use as drug leads, N-to-C cyclization via an oligopeptide spacer unit has been previously shown to improve stability. However, the effect of N-to-C cyclization on the formation of the disulfide bond framework is not fully understood. Four N-to-C cyclic analogs of a-conotoxin ImI; cImI-A, cImI-ßA, cImI-AG, and cImI-AGG were synthesized to evaluate the effect of oligopeptide spacer length on disulfide bond selectivity and stability to proteolysis. Different ratios of disulfide bond isomers were obtained for each analog using a nonselective random disulfide bond forming strategy, which was dependent on the length of the spacer. To identify each isomer obtained using the random strategy, and to gain access to disulfide bond isomers otherwise unattainable using the random strategy, both the native (globular) and ribbon isomers were synthesized in good yield and purity using a selective orthogonal cysteine protecting group strategy. As such, a random oxidation strategy showed a clear preference for the ribbon isomer in cImI-A. The cyclic globular isomers showed a high resistance to enzymatic degradation compared to the ribbon isomers, with the cImI-A and cImI-AG globular isomers demonstrating the highest stability. These results suggest that cyclization can improve the biochemical stability of conotoxins with potential applications in the development of drugs. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 307–313, 2010.

Native chemical ligation methodology developed in the laboratory of Stephen Kent is a versatile approach to the linkage of peptide fragments using a native peptide bond. It is readily adaptable to the task of joining the N- and C-termini of peptides to produce cyclic molecules and we have used it for the cyclization of a range of disulfide-rich peptides. Specifically, it has been valuable for the synthesis of cyclotides, naturally occurring peptides characterized by a head-to-tail cyclized backbone and a knotted arrangement of three conserved disulfide bonds. Cyclotides have a diverse range of biological activities, including anti-HIV, antimicrobial, and insecticidal activities. They are ultrastable owing to their cyclic cystine knot motif, and native chemical ligation methodology has been invaluable in the synthesis of a range of native and modified cyclotides to explore their structure-activity relationships and applications in drug design. Similar studies have also been applied to a smaller cyclic peptide produced in sunflower seeds, sunflower trypsin inhibitor-1, which also shows promise as a template in drug design applications. We have also found native chemical ligation to be a valuable methodology for the cyclization of conotoxins, small disulfide-rich peptides from the venoms of marine cone snails. Conotoxins target a range of ions channels and receptors and are exciting leads in drug design applications. The synthetic cyclization of conotoxins with peptide linkers stabilizes them and improves their biopharmaceutical properties. In summary, this article illustrates the use of native chemical ligation technology in the cyclization of cyclotides, sunflower trypsin inhibitor-1, and conotoxins in our laboratory. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 414–422, 2010.

In recent years, the discovery of a large family of macrocyclic peptides, the cyclotides, has revealed Nature's ingenuity in molecular drug design. The incorporation of a cyclic peptide backbone and a knotted arrangement of disulfide bridges into their structures confers extraordinary chemical, thermal, and enzymatic stability on these biologically active peptides. However, these structural attributes present challenges in the identification of cyclotides. Until now, the sequencing of cyclotides has been slow and inefficient owing to inherent difficulties in the separation of these hydrophobic peptides from plants, the multiple chemical and enzymatic derivatization steps required to make them amenable to mass spectrometric sequencing, and the lack of software tools to efficiently deal with these circular permutants. The current bottleneck slowing the speed of cyclotide sequencing is the requirement for multiple HPLC purification steps before analysis. Here, we have applied proteomic strategies to fast-track the discovery of known, modified and novel sequences. Using four fractions from a previously well-characterized cyclotide-containing plant species, Viola odorata, 11 new sequences, as well as a plethora of known and modified cyclotides, were uncovered. In addition, the methodology was validated through analysis of crude leaf extracts of Oldenlandia affinisand Arabidopsis thaliana. The unambiguous identification of a suite of cyclotides in the Oldenlandia affinisextract provided the ultimate proof-of-concept for this application. Major advances in methodology include the use of optimized LC-MS/MS conditions and design of a custom-built cyclotide database, in which mature cyclotide sequences are excised, replicated and appended, marking a new “era” for cyclotide sequencing. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 592–601, 2010.

Cycloviolacin O2 is a small cyclic cysteine-rich protein belonging to the group of plant proteins called cyclotides. This cyclotide has been previously shown to exert cytotoxic activity against a variety of human tumor cell lines as well as primary cultures of human tumor cells in vitro. This study is the first evaluation of its tolerability and antitumor activity in vivo. Maximal-tolerated doses were estimated to 1.5 mg/kg for single intravenous (i.v.) dosing and 0.5 mg/kg for daily repeated dosing, respectively. Two different in vivo methods were used: the hollow fiber method with single dosing (i.v. 1.0 mg/kg) and traditional xenografts with repeated dosing over 2 weeks (i.v. 0.5 mg/kg daily, 5 days a week). The human tumor cell lines used displayed dose-dependent in vitro sensitivity (including growth in hollow fibers to confirm passage of cycloviolacin O2 through the polyvinylidene fluoride fibers), with IC50values in the micromolar range. Despite this sensitivity in vitro, no significant antitumor effects were detected in vivo, neither with single dosing in the hollow fiber method nor with repeated dosing in xenografts. In summary, the results indicate that antitumor effects are minor or absent at tolerable (sublethal) doses, and cycloviolacin O2 has a very abrupt in vivo toxicity profile, with lethality after single injection at 2 mg/kg, but no signs of discomfort to the animals at 1.5 mg/kg. Repeated dosing of 1 mg/kg gave a local-inflammatory reaction at the site of injection after 2–3 days; lower doses were without complications. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 626–634, 2010.

Cyclotides are a large family of plant-derived proteins typified by their head-to-tail cyclic backbone and knotted arrangement of three disulfide bonds. Although they display a diverse range of biological activities, their native function is thought to be plant defense. Here we characterized the expression, three-dimensional structure, and hemolytic activity of the cyclotide kalata B5 from the African plant Oldenlandia affinis. Kalata B5 shows an interesting seasonal variation in its expression and can only be isolated during certain times of the year, when the plant is flowering. It displays a typical tightly folded cyclic cystine knot structure. A range of pH and temperature titrations reveal that a conserved glutamic acid in loop 1 of the structure forms a key hydrogen bond network, similar to that reported previously for other cyclotides. However, specific line broadening in the NMR spectra of kalata B5 suggests that the hydrogen bonding network in this peptide is less rigid than in other cyclotides. Notably, the pKaof Glu6 of 4.5 is higher than the values for other cyclotides studied so far, which range from 3.0 to 4.0, providing a further indication of a weaker hydrogen bond network. Kalata B5 has only moderate hemolytic activity compared with other highly expressed cyclotides, and this reduced activity probably reflects its more flexible structure. As is the case with other cyclotides, kalata B5 has an exposed hydrophobic region on its surface, supporting suggestions that this hydrophobic patch is a key feature for membrane binding and biological activity of cyclotides. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 647–658, 2010.

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14 amino acid cyclic peptide from sunflower seeds, which possesses exceptionally potent trypsin-inhibitory activity, and has promise as a stable peptide-based drug template. Within its compact structure, SFTI-1 combines a head-to-tail cyclized backbone and a disulfide bond. In this study, we synthesized a range of acyclic and disulfide-deficient analogs of SFTI-1 to investigate enzyme-assisted cyclization of the peptide backbone and proteolytic degradation that occurs as a result of incubation with trypsin. Electrospray and matrix-assisted laser desorption ionization mass spectrometry allowed the characterization of a range of novel degradation products and elucidation of the time-course for cyclization and/or proteolysis. Trypsin displayed the ability to resynthesize the scissile bond(s) and hence cyclize two of the linear permutants, whereas irreversible degradation was observed for another two permutants. An interesting ring contraction mediated by trypsin was observed, supporting a role for protease catalyzed splicing as a way of increasing the combinatorial diversity of cyclic peptides in nature. Disulfide-deficient mutants were degraded within minutes, emphasizing the critical role of the cysteine bridge in maintaining proteolytic stability of SFTI-1. Overall, the study provides additional support for the proposal that naturally occurring cyclic peptides like SFTI-1 are biosynthesized by proteolytic enzymes effectively catalyzing the reverse of their normal reaction to make, rather than breakpeptide bonds. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 665–672, 2010.

Cyclotides are small cysteine-rich plant peptides similar in size and processing to the defensins. Long-term growth of the Rubiaceae family plant Oldenlandia affinisunder different conditions reveals a diverse cyclotide gene and peptide expression profile, including tissue specificity, suggesting that different cyclotides are regulated differently both spatially and in response to the environment. To determine whether cyclotide precursor gene regulation was dynamic we exposed O. affinisto a range of abiotic, biotic, and hormonal stimuli and monitored Oak1-4expression over a 48-h period. Unlike some defensins, the genes for cyclotide precursor proteins Oak1-4did not display dynamic change, indicating that they contribute to the basal defense of O. affinis. Despite this lack of dynamism, the cyclotide profile of plants grown on plates differed markedly from field-grown plants and so prompted attempts to discover novel cyclotides and precursor genes. The two most abundant cyclotides from plate-grown O. affiniswere sequenced and one was found to be an unusual linear cyclotide derivative, kalata B20-lin. Degenerate PCR of plate-grown O. affinisobtained five novel cyclotide genes including Oak9which encodes for kalata B20-lin and appears to have arisen by the presence of a premature stop codon. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 635–646, 2010.

CyBase is a database dedicated to the study of the sequences and three-dimensional structures of ribosomally synthesized, backbone cyclized proteins, and their synthetic variants. This article describes CyBase data and tools that are useful in the analysis of circular proteins. Circular proteins have now been discovered in organisms from all kingdoms of life, and given the current rate of discovery they could soon number in the thousands. Presently CyBase manages 427 protein sequences, 106 nucleic acid sequences, and 49 protein three-dimensional structures from 44 different species. Circular proteins are grouped into distinct classes according to their origin and sequence similarities. These classes include trypsin inhibitors, bacterial proteins, mushroom toxins, cyclotides, and cyclic defensins from primates. Several protein classification types are used in CyBase to designate proteins extracted from natural resources (wild type and precursor) or engineered (modified wild type, grafted, mutant, cyclic permutant, and acyclic permutant). CyBase has tools for the analysis of mass spectrum fingerprints of cyclic peptides, and assists in the discovery of new circular proteins. Some of the developments detailed here have been made specifically for the largest class of circular proteins, thecyclotides, but could be adapted for other classes of cyclic proteins. The cyclotide-specific tools include two-dimensional representations of domains and alternative displays of alignments for precursor sequences. This alignment prompted us to propose a revision of the cyclotide precursor organization, in which the repeated regions now include a small C-terminal region, which appears to have a significant role in the biosynthesis of mature cyclotides. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 584–591, 2010.

We recently isolated a protein disulfide isomerase PDI from the Rubiaceae coffee family plant Oldenlandia affinis OaPDI and demonstrated that it facilitates the production of disulfideknotted defense proteins called cyclotides. PDIs are major folding catalysts in the eukaryotic ER where they are responsible for formation, breakage, or shuffling of disulfide bonds in substrate polypeptides and are important chaperones in the secretory pathway. Here, we report the first detailed analysis of the oligomerization behavior of a plant PDI, based on characterization of OaPDI using various biochemical and biophysical techniques, including sizeexclusion chromatography, NMR spectroscopy, surface plasmon resonance and atomic force microscopy. In solution at low concentration OaPDI comprises mainly monomers, but fractions of dimers andor higherorder oligomers were observed at increased conditions, raising the possibility that dimerization andor oligomerization could be a mechanism to adapt to the varioussized polypeptide substrates of PDI. Unlike mammalian PDIs, oligomerization of the plant PDI is not driven by the formation of intermolecular disulfide bonds, but by noncovalent interactions. The information derived in this study advances our understanding of the oligomerization behavior of OaPDI in particular but is potentially of broader interest for understanding the mechanism and role of oligomerization, and hence the catalytic and physiological mechanism, of the ubiquitous folding catalyst PDI. © 2008 Wiley Periodicals, Inc. Biopolymers Pept Sci 92: 35–43, 2009.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymerswiley.com

Cyclotides are disulfide rich macrocyclic plant peptides that are defined by their unique topology in which a head‐to‐tail cyclized backbone is knotted by the interlocking arrangement of three disulfide bonds. This cyclic cystine knot motif gives the cyclotides exceptional resistance to thermal, chemical, or enzymatic degradation. Over 100 cyclotides have been reported and display a variety of biological activities, including a cytoprotective effect against HIV infected cells. It has been hypothesized that cyclotides from one subfamily, the Möbius subfamily, may be more appropriate than bracelet cyclotides as drug candidates given their lower toxicity to uninfected cells. Here, we report the anti‐HIV and cytotoxic effects of three cyclotides, including two from the Möbius subfamily. We show that Möbius cyclotides have comparable inhibitory activity against HIV infection to bracelet cyclotides and that they are generally less cytotoxic to the target cells. To explore the structure activity relationships (SARs) of the 29 cyclotides tested so far for anti‐HIV activity, we modeled the structures of the 21 cyclotides whose structures have not been previously solved. We show that within cyclotide subfamilies there is a correlation between hydrophobicity of certain loop regions and HIV inhibition. We also show that charged residues in these loops impact on the activity of the cyclotides, presumably by modulating membrane binding. In addition to providing new SAR data, this report is a mini‐review that collates all cyclotide anti‐HIV information reported so far and provides a resource for future studies on the therapeutic potential of cyclotides as natural anti‐HIV agents. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 51–60, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Cyclotides are 28–37 amino acid peptides incorporating three disulfide bonds and a cyclic backbone. Their cyclic and knotted topology renders them immune to denaturation by heat or organic solvents and highly resistant to proteolysis. They have a range of interesting and potentially useful pharmaceutical properties and have been proposed as scaffolds within which peptides with drug activities can be stabilized for delivery. Some members of the family also have agricultural applications deriving from their potent insecticidal activity. Labeling peptides with the NMRactive and stable 15N isotope facilitates a range of studies by NMR, including structural and dynamics studies and their use as tracers. However, owing to their headtotail cyclized peptide backbone labeled cyclotides are not amenable to conventional recombinant labeling strategies. We have developed an approach to overcome this limitation by growing the cyclotidebearing plant Oldenlandia affinison nitrogenfree agar media supplemented with 15N salts and obtaining complete labeling at no detriment to plant biomass. We purified the insecticidal cyclotides kalata B1 and kalata B2 as examples and provide heteronuclear single quantum coherence HSQC NMR spectra for each. This method of labeling cyclotides involves only a fraction of the cost of uniform labeling by solidphase peptide synthesis. © 2008 Wiley Periodicals, Inc. Biopolymers Pept Sci 90: 575–580, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymerswiley.com

The χ‐conopeptides MrIA and MrIB are 13‐residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a β‐hairpin with the two strands connected by an inverse γ‐turn. The residues primarily involved in activity have previously been shown to be located in the turn region (Sharpe, I. A.; Palant, E.; Schroder, C. I.; Kaye, D. M.; Adams, D. J.; Alewood, P. F.; Lewis, R. J. J Biol Chem 2003, 278, 40317–40323), which appears to be more flexible than the β‐strands based on disorder in the ensemble of calculated structures. Analogues of MrIA with N‐terminal truncations indicate that the N‐terminal residues play a role in defining a stable conformation and the native disulfide connectivity. In particular, noncovalent interactions between Val3 and Hyp12 are likely to be involved in maintaining a stable conformation. The N‐terminus also affects activity, as a single N‐terminal deletion introduced additional pharmacology at rat vas deferens, while deleting the first two amino acids reduced χ‐conopeptide potency. © 2005 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 80: 815–823, 2005This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Pyrrhocoricin is a naturally occurring antimicrobial peptide from the European fire bug Pyrrhocoris apterus. It has submicromolar activity against a range of Gram‐negative bacterial strains and has created recent interest as a lead for the development of novel antibiotic compounds. In this study, we have used NMR spectroscopy to determine the solution structures of pyrrhocoricin and a synthetic macrocyclic derivative that has improved in vivo pharmaceutical properties. Native pyrrhocoricin is largely disordered in solution, but there is evidence of a subpopulation with ordered turn regions over residues 2–5, 4–7, and 16–19. The macrocyclic derivative incorporates a nine amino acid linker joining the N‐ and C‐termini, which does not adversely affect the antimicrobial potency but leads to a broader spectrum of activity. The NMR data suggest that the turn conformations in the cyclic derivative are similar to those in the native form, thus implicating them in the biological function. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004