Foreword for ICCP2012 issue of Biopolymers Peptide Science Circular Proteins: Never ending possibilities

I welcome readers to this special issue of Biopolymers Peptide Science, which includes a selection of papers presented at the 2nd International Conference on Circular Proteins. The conference was held on Heron Island, Queensland, Australia, from the 14–17th October 2012 and was attended by approximately 60 delegates from 10 countries, including Austria, Australia, Canada, China, Finland, Germany, Japan, Singapore, Sweden, and the USA. It included talks from more than 30 speakers, along with 27 poster presentations covering all aspects of the discovery, synthesis, biological activity and applications of circular proteins from a variety of organisms, including bacteria, plants, fungi and animals. The first article in this special issue of Biopolymers Peptide Science is a “Commentary” from a group of independent laboratories in Asia, Australia, Europe and the USA working on the discovery of cyclic peptides from natural products, and in particular on cyclotides. Cyclotides are the largest known family of circular proteins and are characterized by their combined head-to-tail cyclic backbone and knotted arrangement of three conserved disulfide bonds. One theme of the commentary is that cyclotides have now been discovered in a number of diverse plant families, including the Rubiaceae (coffee), Violaceae (violet), Cucurbitaceae (cucurbit), Solanaceae (potato) and Fabaceae (legume) families, but there is a vast difference in the hit rate in discoveries between the different plant families. For example, cyclotides are ubiquitous in every species so far examined in the Violaceae but relatively sparse in the other plant families. Published papers reporting the discovery of cyclotides, by their nature, focus on plants that are found to be ‘cyclotide-positive', which has meant that there is potentially a vast unpublished literature on plant species that have been examined, but found to be negative for cyclotides. One purpose of the Commentary is to encourage researchers working on cyclotides to document any ‘negative' findings, to avoid reinventing the wheel and rescreening plants for which there were no cyclotide hits. Of course, this is a tricky area. It could be that cyclotides were present, but not detected, in a particular plant depending on how and when the sampling was done. For example, seasonal or other variations between expression levels of cyclotides might result in their lack of detection at particular times of the year. Therefore, it is never possible to say that an individual plant species is totally devoid of cyclotides unless this is established at the genomic level. Notwithstanding this caveat, the Commentary aims to simulate discussion as to how to document both positive and negative findings of cyclotides across the plant kingdom. To this end, a role was identified for CyBase, a database documenting discoveries of circular proteins, to include provision for documenting apparently ‘cyclotide-negative' plants. The next series of articles in the issue also is centered on cyclotides, and covers aspects of their discovery and applications. The first of these articles focuses on the characterization of cyclotides and their phylogenetic distribution within plants from the order Gentianales. Specifically, Koehbach et al., report a revised protocol for the screening of plants for cyclotides and provide up-to-date information on the distribution of cyclotides in the plant kingdom. They examined approximately 300 species from more than 40 different plant families, using a refined screening methodology that combines chemical analysis of plant extracts with bioinformatic analysis of transcript databases. Using mass spectrometry and transcriptome mining they identified nine novel cyclotide-containing species and delineated their related precursor genes. Characterization of these novel peptide sequences emphasized the high variability and plasticity of the cyclotide framework, as suggested in earlier studies but now confirmed in a large scale screening program. Overall, this work confirms that cyclotides are probably one of the largest peptide families within the plant kingdom and suggested that their total number may exceed tens of thousands. The next article describes some recent biophysical studies of cyclotides and in particular attempts to unravel the molecular details connected with the self-association of cyclotides. Researchers had earlier postulated that cyclotides can associate into tetramers and octamers, but neither the mechanistic nor functional basis of this self-association had been definitively elucidated. Rosengren et al., report that in aqueous solution the self-association of cyclotides to tetramers appears to occur primarily via clumping of cyclotides around their hydrophobic patches. They proposed that this self-association might have evolved as a protective mechanism for the storage of high concentrations of cyclotides in plant vacuoles prior to their release for defense purposes. Although cyclotides are thought to be host defense agents that kill or debilitate insects and other pests via binding to vital biological membranes, the mechanism by which cyclotides avoid disrupting the membranes of their producing plants has long been a mystery. Rosengren et al., provide one possible explanation for this lack of self-toxicity if it can be assumed that sequestering cyclotides into tetramers/octamers limits their membrane binding in plant vacuoles. The paper points out that this mechanism for self-association at high concentration in solution (and hence presumably in vacuoles) would allow dissociation to monomers upon dilution (e.g. in the gut of a foraging insect), facilitating membrane interactions. The mechanism by which cyclotides disrupt membranes is beginning to be unraveled but remains the subject of further investigation. The effectiveness of cyclotides in killing nematode pests is demonstrated in the next article, by Malagon et al., who reported that the cyclotides kalata B1 and B2 are active in vitro against the digenean flatworm Schistosoma japonicum. Schistosomal disease affects more than 200 million people in sub-Saharan Africa, South America and China, and causes an estimated 200,000 deaths annually. There are concerns that current treatments will succumb to the development of resistance by the parasite, making it important for new agents to be developed. Given the promising in vitro activity seen for cyclotides in initial studies, the most potent molecule, kalata B2, was tested in vivo and was found effective in suppressing schistosomal disease in infected mice. Kalata B2 caused a 15–60% reduction in worm burden in the livers of S. japonicum infected mice when applied in a single dose 28 days post infection. Electron microscopy studies showed that the mechanism of action probably involves disruption of the tegument membranes of the schistosomes. Increased potency would be required to make cyclotides practical as anti-schistosomal agents, but given the hundreds of cyclotides known and the few so far tested, there is promise that improved activity will be discovered. Gerlach et al., describe a novel application of cyclotides, not as direct bioactive molecules themselves, but as adjuvants to improve the anti-viral efficacy of existing anti-HIV drugs. They noted that despite the efficacy of highly active anti-retroviral therapy there is still an urgent need for new treatments for HIV-1 infection. They evaluated the therapeutic effects of the cyclotide cycloviolacin O2 on cell viability, membrane disruption, and proviral integration in a monocytic cell line model of HIV-1 latency and re-activation. They demonstrated that cycloviolacin O2 kills productively infected cells and that sub-toxic concentrations of cycloviolacin O2 disrupted plasma membranes in both latently infected and productively infected cells and enhanced the anti-viral efficacy of nelfinavir, a HIV-1 protease inhibitor. They conclude that cyclotides might be promising agents for inclusion in adjuvant therapy approaches to HIV elimination. Poth et al., provide a review on recently published literature on the use of cyclotides as frameworks for ‘grafting' applications in drug design. The concept here is that biologically active peptide sequences can be stabilized by inserting (grafting) them into a cyclotide framework, typically into one of the exposed loops. The authors highlight a series of successfully grafted cyclotides that have potential uses as drug candidates for cancer, cardiovascular disease, infectious disease and inflammatory pain. These studies on grafted cyclotides have been underpinned by previous fundamental studies that have provided a vast amount of knowledge on the sequence variability, importance of the CCK framework and on the biological activities of cyclotides. The various applications of cyclotides as pharmaceutical agents described above have been greatly facilitated by novel methods for the production of cyclotides and other cyclic peptides, to develop structure-activity relationships and to optimize potency. The next two articles describe recently developed chemical and biological strategies, respectively, to produce cyclotides for a wide range of such applications. Tam and colleagues reported on the biomimetic synthesis of cyclic peptides using novel thioester surrogates. They noted that acyl shifts involving N-S and S-S rearrangements are crucial in the breaking of peptide bonds and the formation of thioester intermediates in intein-catalyzed protein splicing, which ultimately leads to the formation of a new peptide bond by an uncatalysed S-N acyl shift reaction. To mimic these various acyl shift reactions in thioester formation and subsequent peptide ligation processes, the authors describe the development of two Fmoc-compatible thioester surrogates that can undergo uncatalyzed N-S, S-S and S-N acyl shifts used in preparing thioesters and cyclic peptides. They illustrated the applications of these surrogates in the synthesis of cyclic conotoxins and the cyclic sunflower-derived peptide SFTI-1. The former class of molecule has been used in the development of drugs for pain and the latter is a small stable peptide template with applications in drug design. The development of robust methods for the synthesis of these classes of cyclic peptide thus has the potential to facilitate peptide-based drug development. In an article that focuses on alternatives to solid phase synthesis for the production of cyclic peptides, Borra and Camarero provide a review of biological approaches to the synthesis of cyclic polypeptides, including cyclotides. These include thioester-mediated ligation using expressed protein ligation or genetic reprogramming, intein-mediated protein trans-splicing, and protease catalysed protein transpeptidation. They describe the advantages and disadvantages of these various biological approaches to cyclic peptide synthesis and in particular point out the opportunities they provide to generate libraries of bioactive cyclic peptides. The next series of articles are primary research papers that describe applications of specific classes of cyclic peptides. Harris and colleagues focus on sunflower trypsin inhibitor-1 (SFTI-1), a 14 amino acid cyclic peptide containing a single disulfide bond that has proven to be a particularly useful framework in drug design, as noted above. They used this framework to design an inhibitor of the kallikrein-related peptidase 7 (KLK7), a protease implicated in skin disorders. The lead molecule was selected from a versatile library of SFTI-1 variants whose diversity was tailored to exploit distinctive surfaces present in the active site of serine proteases. Overall the authors noted that their approach provides a robust and rapid way of designing novel protease inhibitors. In the next article, Clark and colleagues describe an attempt to use cyclization to improve the biopharmaceutical properties of hepcidin, a peptide hormone that is a key regulator of iron homeostasis. It represents an important lead molecule for the design of agents to treat diseases associated with the mis-regulation of iron. This peptide is amenable to cyclization as its N and C-termini are proximate and indeed the authors demonstrated that cyclization was readily achievable using solid phase peptide chemistry and resulted in improved serum stability over the parent linear peptide. Unfortunately, a series of synthetic cyclic derivatives turned out to be biologically inactive and the study thus provides an important illustration of the sometimes conflicting requirements of stability and bioactivity. In this case, sequestration of the termini via cyclization apparently altered the conformation and accessibility of the N-terminus, which is thought to be crucial to binding to the ferroportin receptor. In the penultimate article, Marahiel and colleagues describe a genome mining approach for the discovery of ‘lasso' peptides from proteobacteria. Although not cyclic in the sense of a head-to-tail cyclic backbone, the lasso peptides contain an embedded ring formed from the covalent linkage of a sidechain carboxyl group at around position 8 in the sequence with the N-terminus. Unusually, this ring is threaded by the C-terminal tail of the molecules, leading to the lasso designation. Their similarity with head-to-tail cyclic peptides is indeed strong, as originally this family of peptides was exemplified by microcin j25, whose structure was initially but wrongly thought to a head-to-tail cyclic peptide, given its exceptional stability. Subsequent studies defined the lasso structure and since the original discovery the field has blossomed and lasso peptides are more widely distributed than was originally thought. In their article Marahiel and colleagues describe the unique three-dimensional structures of selected lasso peptides and confirm their ribosomal origin, involving post-translational modifications by two enzymes that share little homology to enzymes outside the class of lasso peptide biosynthetic gene clusters. Their unique biosynthetic mechanism provides a unique way targeting these peptides in genome mining studies and Marahiel and colleagues were able to identify more than100 putative lasso peptides from 87 different proteobacterial strains. Finally, the series of articles associated with ICCP2012 concludes with a paper by Gunzburg et al., who developed small cyclic peptides to interact with Grb7, an adapter protein involved in the propagation of signals associated with cancer cell migration and proliferation. Their original 11-mer peptide, G7-18NATE is cyclized via a thioether linker and in the new study they describe the addition of a second linkage in an attempt to lock the peptide into a defined binding conformation. In particular, an O-allyl Ser tether enhanced binding affinity. Further studies are underway to develop second generation binders, but it seems an appropriate point to finish the discussion here by noting that adding two cycles to a peptide can be better than one. Perhaps the next ICCP will include more examples of bi-cycles. I hope that readers find this series of articles interesting and I would like to take this opportunity to thank the authors for their valuable participation in ICCP2012 and for the outstanding quality of their articles. Sincerely, David J. Craik Institute for Molecular Bioscience The University of Queensland Brisbane, Qld 4072, Australia

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