Plant derived cyclic peptides

Cyclic peptides are widespread throughout the plant kingdom, and display diverse sequences, structures and bioactivities. The potential applications attributed to these peptides and their unusual biosynthesis has captivated the attention of researchers for many years. Several gene sequences for plant cyclic peptides have been discovered over the last two decades but it is only recently that we are beginning to understand the intricacies associated with their biosynthesis. Recent studies have focussed on three main classes of plant derived cyclic peptides, namely orbitides, SFTI related peptides and cyclotides. In this mini-review, we discuss the expansion of the known sequence and structural diversity in these families, insights into the enzymes involved in the biosynthesis, the exciting applications which includes a cyclotide currently in clinical trials for the treatment of multiple sclerosis, and new production methods that are being developed to realise the potential of plant cyclic peptides as pharmaceutical or agricultural agents

Coral venom toxins

The phylum Cnidaria contains a wide variety of unique organisms that possess interesting adaptations evolved over many years to help them survive in a competitive environment. One of these adaptations is the presence of venom, which has been of particular interest for studies aimed at identifying novel drug leads and for understanding the mechanisms involved in envenomation. The potency of the venom varies significantly amongst cnidarians, and although corals are often overshadowed by the jellyfish and sea anemone toxins, they also possess a range of interesting bioactive compounds. In this mini-review, we provide an overview of the toxins present in corals, highlighting the diverse structures and bioactivities

Dual-targeting anti-angiogenic cyclic peptides as potential drug leads for cancer therapy

Peptide analogues derived from bioactive hormones such as somatostatin or certain growth factors have great potential as angiogenesis inhibitors for cancer applications. In an attempt to combat emerging drug resistance many FDA-approved anti-angiogenesis therapies are co-administered with cytotoxic drugs as a combination therapy to target multiple signaling pathways of cancers. However, cancer therapies often encounter limiting factors such as high toxicities and side effects. Here, we combined two anti-angiogenic epitopes that act on different pathways of angiogenesis into a single non-toxic cyclic peptide framework, namely MCoTI-II (Momordica cochinchinensis trypsin inhibitor-II), and subsequently assessed the anti-angiogenic activity of the novel compound. We hypothesized that the combination of these two epitopes would elicit a synergistic effect by targeting different angiogenesis pathways and result in improved potency, compared to that of a single epitope. This novel approach has resulted in the development of a potent, non-toxic, stable and cyclic analogue with nanomolar potency inhibition in in vitro endothelial cell migration and in vivo chorioallantoic membrane angiogenesis assays. This is the first report to use the MCoTI-II framework to develop a 2-in-1 anti-angiogenic peptide, which has the potential to be used as a form of combination therapy for targeting a wide range of cancers

Venom costs and optimization in scorpions

Scorpions use venoms as weapons to improve prey capture and predator defense, and these benefits must be balanced against costs associated with its use. Venom costs involve direct energetic costs associated with the production and storage of toxins, and indirect fitness costs arising from reduced venom availability. In order to reduce these costs, scorpions optimize their venom use via evolutionary responses, phenotypic plasticity, and behavioral mechanisms. Over long timescales, evolutionary adaptation to environments with different selection pressures appears to have contributed to interspecific variation in venomcomposition and stingermorphology. Furthermore, plastic responses may allow scorpions to modify and optimize their venom composition as pressures change. Optimal venomuse can vary when facing each prey itemand potential predator encountered, and therefore scorpions display a range of behaviors to optimize their venom use to the particular situation. These behaviors include varying sting rates, employing dry stings, and further altering the volume and composition of venom injected. Whilst these cost-reducing mechanisms are recognized in scorpions, relatively little is understood about the factors that influence them. Here, we review evidence of the costs associated with venom use in scorpions and discuss the mechanisms that have evolved to minimize them

Characterizing circular peptides in mixtures: sequence fragment assembly of cyclotides from a violet plant by MALDI-TOF/TOF mass spectrometry

Cyclotides are a very abundant class of plant peptides that display significant sequence variability around a conserved cystine-knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability. Their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals. However, laborious isolation and purification prior to de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development. Here we extend the knowledge about their sequence diversity by analysing the cyclotide content of a violet species native to Western Asia and the Caucasus region. Using an experimental approach, which was named sequence fragment assembly by MALDI-TOF/TOF, it was possible to characterize 13 cyclotides from Viola ignobilis, whereof ten (vigno 1-10) display previously unknown sequences. Amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analysing mixtures containing multiple peptides. As a model to further dissect the combinatorial nature of the cyclotide scaffold, we employed in vitro oxidative refolding of representative vigno cyclotides and confirmed the high dependency of folding yield on the inter-cysteine loop sequences. Overall this work highlights the immense structural diversity and plasticity of the unique cyclotide framework. The presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides

Small molecules in the venom of the scorpion Hormurus waigiensis

Despite scorpion stings posing a significant public health issue in particular regions of the world, certain aspects of scorpion venom chemistry remain poorly described. Although there has been extensive research into the identity and activity of scorpion venom peptides, non-peptide small molecules present in the venom have received comparatively little attention. Small molecules can have important functions within venoms; for example, in some spider species the main toxic components of the venom are acylpolyamines. Other molecules can have auxiliary effects that facilitate envenomation, such as purines with hypotensive properties utilised by snakes. In this study, we investigated some non-peptide small molecule constituents of Hormurus waigiensis venom using LC/MS, reversed-phase HPLC, and NMR spectroscopy. We identified adenosine, adenosine monophosphate (AMP), and citric acid within the venom, with low quantities of the amino acids glutamic acid and aspartic acid also being present. Purine nucleosides such as adenosine play important auxiliary functions in snake venoms when injected alongside other venom toxins, and they may have a similar role within H. waigiensis venom. Further research on these and other small molecules in scorpion venoms may elucidate their roles in prey capture and predator defence, and gaining a greater understanding of how scorpion venom components act in combination could allow for the development of improved first aid

Folding of truncated granulin peptides

Granulins are a family of unique protein growth factors which are found in a range of species and have several bioactivities that include cell proliferation and wound healing. They typically contain six disulfide bonds, but the sequences, structures and bioactivities vary significantly. We have previously shown that an N-terminally truncated version of a granulin from the human liver fluke, Opisthorchis viverrini, can fold independently into a “mini-granulin” structure and has potent wound healing properties in vivo. The incorporation of a non-native third disulfide bond, with respect to the full-length granulin module, was critical for the formation of regular secondary structure in the liver fluke derived peptide. By contrast, this third disulfide bond is not required for a carp granulin-1 truncated peptide to fold independently. This distinction led us to explore granulins from the zebrafish model organism. Here we show that the mini-granulin fold occurs in a naturally occurring paragranulin (half-domain) from zebrafish, and is also present in a truncated form of a full-length zebrafish granulin, suggesting this structure might be a common property in either naturally occurring or engineered N-terminally truncated granulins and the carp granulin-1 folding is an anomaly. The in vitro folding yield is significantly higher in the naturally occurring paragranulin, but only the truncated zebrafish granulin peptide promoted the proliferation of fibroblasts consistent with a growth factor function, and therefore the function of the paragranulin remains unknown. These findings provide insight into the folding and evolution of granulin domains and might be useful in the elucidation of the structural features important for bioactivity to aid the design of more potent and stable analogues for the development of novel wound healing agents

Structural characterisation of predicted helical regions in the Chironex fleckeri CfTX-1 toxin

The Australian jellyfish Chironex fleckeri, belongs to a family of cubozoan jellyfish known for their potent venoms. CfTX-1 and -2 are two highly abundant toxins in the venom, but there is no structural data available for these proteins. Structural information on toxins is integral to the understanding of the mechanism of these toxins and the development of an effective treatment. Two regions of CfTX-1 have been predicted to have helical structures that are involved with the mechanism of action. Here we have synthesized peptides corresponding to these regions and analyzed their structures using NMR spectroscopy. The peptide corresponding to the predicted N-terminal amphiphilic helix appears unstructured in aqueous solution. This lack of structure concurs with structural disorder predicted for this region of the protein using the Protein DisOrder prediction System PrDOS. Conversely, a peptide corresponding to a predicted transmembrane region is very hydrophobic, insoluble in aqueous solution and predicted to be structured by PrDOS. In the presence of SDS-micelles both peptides have well-defined helical structures showing that a membrane mimicking environment stabilizes the structures of both peptides and supports the prediction of the transmembrane region in CfTX-1. This is the first study to experimentally analyze the structure of regions of a C. fleckeri protein

Dissecting the oxidative folding of circular cystine knot miniproteins

Cyclotides are plant proteins with exceptional stability owing to the presence of a cyclic backbone and three disulfide bonds arranged in a cystine knot motif. Accordingly, they have been proposed as templates to stabilize bioactive epitopes in drug-design applications. The two main subfamilies, referred to as the Möbius and bracelet cyclotides, require dramatically different in vitro folding conditions to achieve the native fold. To determine the underlying elements that influence cyclotide folding, we examined the in vitro folding of a suite of hybrid cyclotides based on combination of the Möbius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1. The folding pathways of the two cyclotide subfamilies were found to be different and influenced by specific residues within intercysteine loops 2 and 6. Two changes in these loops, a substitution in loop 2 and an addition in loop 6, enabled the folding of a cycloviolacin O1 analogue under conditions in which folding does not occur in vitro for the native peptide. A key intermediate contains a native-like hairpin structure that appears to be a nucleation locus early in the folding process. Overall, these mechanistic findings on the folding of cyclotides are potentially valuable for the design of new drug leads. Copyright Mary Ann Liebert, Inc

Voltage-Gated Sodium Channel Modulation by a New Spider Toxin Ssp1a Isolated From an Australian Theraphosid

Given the important role of voltage-gated sodium (NaV) channel-modulating spider toxins in elucidating the function, pharmacology, and mechanism of action of therapeutically relevant NaV channels, we screened the venom from Australian theraphosid species against the human pain target hNaV1.7. Using assay-guided fractionation, we isolated a 33-residue inhibitor cystine knot (ICK) peptide (Ssp1a) belonging to the NaSpTx1 family. Recombinant Ssp1a (rSsp1a) inhibited neuronal hNaV subtypes with a rank order of potency hNaV1.7 > 1.6 > 1.2 > 1.3 > 1.1. rSsp1a inhibited hNaV1.7, hNaV1.2 and hNaV1.3 without significantly altering the voltage-dependence of activation, inactivation, or delay in recovery from inactivation. However, rSsp1a demonstrated voltage-dependent inhibition at hNaV1.7 and rSsp1a-bound hNaV1.7 opened at extreme depolarizations, suggesting rSsp1a likely interacted with voltage-sensing domain II (VSD II) of hNaV1.7 to trap the channel in its resting state. Nuclear magnetic resonance spectroscopy revealed key structural features of Ssp1a, including an amphipathic surface with hydrophobic and charged patches shown by docking studies to comprise the interacting surface. This study provides the basis for future structure-function studies to guide the development of subtype selective inhibitors