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Acrorhagin I (U-AITX-Aeq5a) is a disulfide–rich peptide identified in the aggressive organs (acrorhagi) of the sea anemone Actinia equina. Previous studies (Toxicon 2005, 46:768–74) found that the peptide is toxic in crabs, although the structural and functional properties of acrorhagin I have not been reported. In this work, an Escherichia coli (BL21 strain) expression system was established for the preparation of 13C,15N–labelled acrorhagin I, and the solution structure was determined using NMR spectroscopy. Structurally, acrorhagin I is similar to B–IV toxin from the marine worm Cerebratulus lacteus (PDB id 1VIB), with a well–defined helical hairpin structure stabilised by four intramolecular disulfide bonds. The recombinant peptide was tested in patch–clamp electrophysiology assays against voltage-gated potassium and sodium channels, and in bacterial and fungal growth inhibitory assays and haemolytic assays. Acrorhagin I was not active against any of the ion channels tested and showed no activity in functional assays, indicating that this peptide may possess a different biological function. Metal ion interaction studies using NMR spectroscopy showed that acrorhagin I bound zinc and nickel, suggesting that its function might be modulated by metal ions or that it may be involved in regulating metal ion levels and their transport. The similarity between the structure of acrorhagin I and that of B-IV toxin from a marine worm suggests that this fold may prove to be a recurring motif in disulfide-rich peptides from marine organisms.

[Image: see text] We describe a cysteine-rich, membrane-penetrating, joint-targeting, and remarkably stable peptide, EgK5, that modulates voltage-gated K(V)1.3 potassium channels in T lymphocytes by a distinctive mechanism. EgK5 enters plasma membranes and binds to K(V)1.3, causing current run-down by a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. EgK5 exhibits selectivity for K(V)1.3 over other channels, receptors, transporters, and enzymes. EgK5 suppresses antigen-triggered proliferation of effector memory T cells, a subset enriched among pathogenic autoreactive T cells in autoimmune disease. PET-CT imaging with (18)F-labeled EgK5 shows accumulation of the peptide in large and small joints of rodents. In keeping with its arthrotropism, EgK5 treats disease in a rat model of rheumatoid arthritis. It was also effective in treating disease in a rat model of atopic dermatitis. No signs of toxicity are observed at 10–100 times the in vivo dose. EgK5 shows promise for clinical development as a therapeutic for autoimmune diseases.

ShK is a 35-residue disulfide-linked polypeptide produced by the sea anemone Stichodactyla helianthus, which blocks the potassium channels Kv1.1 and Kv1.3 with pM affinity. An analogue of ShK has been developed that blocks Kv1.3 > 100 times more potently than Kv1.1, and has completed Phase 1b clinical trials for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis. Previous studies have indicated that ShK undergoes a conformational exchange that is critical to its function, but this has proved difficult to characterise. Here, we have used high hydrostatic pressure as a tool to increase the population of the alternative state, which is likely to resemble the active form that binds to the Kv1.3 channel. By following changes in chemical shift with pressure, we have derived the chemical shift values of the low- and high-pressure states, and thus characterised the locations of structural changes. The main difference is in the conformation of the Cys17-Cys32 disulfide, which is likely to affect the positions of the critical Lys22-Tyr23 pair by twisting the 21–24 helix and increasing the solvent exposure of the Lys22 sidechain, as indicated by molecular dynamics simulations.

Consistent with their diverse pharmacology, peptides derived from venomous animals have been developed as drugs to treat disorders as diverse as hypertension, diabetes and chronic pain. Melanoma has a poor prognosis due in part to its metastatic capacity, warranting further development of novel targeted therapies. This prompted us to examine the anti-melanoma activity of the spider peptides gomesin (AgGom) and a gomesin-like homolog (HiGom). AgGom and HiGom dose-dependently reduced the viability and proliferation of melanoma cells whereas it had no deleterious effects on non-transformed neonatal foreskin fibroblasts. Concordantly, gomesin-treated melanoma cells showed a reduced G0/G1 cell population. AgGom and HiGom compromised proliferation of melanoma cells via activation of the p53/p21 cell cycle check-point axis and the Hippo signaling cascade, together with attenuation of the MAP kinase pathway. We show that both gomesin peptides exhibit antitumoral activity in melanoma AVATAR-zebrafish xenograft tumors and that HiGom also reduces tumour progression in a melanoma xenograft mouse model. Taken together, our data highlight the potential of gomesin for development as a novel melanoma-targeted therapy This work was supported by Perpetual IMPACT Grant IDIPAP2015/1585 (to J.J.M.), Discovery Grants DP1095728 (to J.J.M.) and DP130103813 (to G.F.K.) from the Australian Research Council, and by QIMR Berghofer Medical Research Institute crowd funding and salary support (to M.P.I.). M.P.I. is a “Marie Curie” AMAROUT Fellow. M.A.F.R. is a fellow supported by the Talent Program from the Madrid Government of Spain (Grant No. T1-BIO-1854 to M.A.F.R.). J.J.M. is supported by a NHMRC Career Development Fellowship (APP1131732) and G.F.K. by a NHMRC Principal Research Fellowship (APP1136889) SI

The malaria vaccine candidate merozoite surface protein 2 (MSP2) has shown promise in clinical trials and is in part responsible for a reduction in parasite densities. However, strain-specific reductions in parasitaemia suggested that polymorphic regions of MSP2 are immuno-dominant. One strategy to bypass the hurdle of strain-specificity is to bias the immune response towards the conserved regions. Two mouse monoclonal antibodies, 4D11 and 9H4, recognise the conserved C-terminal region of MSP2. Although they bind overlapping epitopes, 4D11 reacts more strongly with native MSP2, suggesting that its epitope is more accessible on the parasite surface. In this study, a structure-based vaccine design approach was applied to the intrinsically disordered antigen, MSP2, using a crystal structure of 4D11 Fv in complex with its minimal binding epitope. Molecular dynamics simulations and surface plasmon resonance informed the design of a series of constrained peptides that mimicked the 4D11-bound epitope structure. These peptides were conjugated to keyhole limpet hemocyanin and used to immunise mice, with high to moderate antibody titres being generated in all groups. The specificities of antibody responses revealed that a single point mutation can focus the antibody response towards a more favourable epitope. This structure-based approach to peptide vaccine design may be useful not only for MSP2-based malaria vaccines, but also for other intrinsically disordered antigens.

Merozoite surface protein 2 (MSP2) is a highly abundant, GPI-anchored antigen on the malaria parasite Plasmodium falciparum. MSP2 induces an immune response in the context of natural infections and vaccine trials, and these responses are associated with protection from parasite infection. Recombinant MSP2 is highly disordered in solution but antigenic analyses suggest that it is more ordered on the merozoite surface. We have shown previously that the interaction of recombinant full-length MSP2 with lipid surfaces induces a conformational change in the conserved N-terminal region of MSP2, which contributes to epitope masking in this region. To explore the impacts of lipid interactions on the conformation and antigenicity of the conserved C-terminal region of MSP2, a construct corresponding to this domain, MSP2172-221, was designed. NMR studies indicate that many residues in MSP2172-221 interact with DPC micelles, including some in epitopes recognised by C-terminal-specific monoclonal antibodies, but, in contrast to the MSP2 N-terminus, there is no indication of stable helical conformation. The binding affinities of a panel of monoclonal antibodies indicate that MSP2172-221 is antigenically similar to full-length MSP2 and show that liposome conjugation alters the antigenicity in a manner that may mimic native MSP2 on the merozoite surface. These findings highlight the impact of lipid interactions on the conformation and antigenicity of MSP2172-221 and will assist in the design of recombinant MSP2 immunogens for use as malaria vaccine candidates. This article is protected by copyright. All rights reserved

Disulfide bonds play a key role in the oxidative folding, conformational stability, and functional activity of many peptides. A few disulfide-rich peptides with privileged architecture such as the inhibitor cystine knot motif have garnered attention as templates in drug design. The single disulfide-directed β-hairpin (SDH), a novel fold identified more recently in contryphan-Vc1, has been shown to possess remarkable thermal, conformational, and chemical stability and can accept a short bioactive epitope without compromising the core structure of the peptide. In this study, we demonstrated that the single disulfide bond is critical in maintaining the native fold by replacing both cysteine residues with serine. We also designed an analogue with an additional, non-native disulfide bridge by replacing Gln1 and Tyr9 with Cys. Contryphan-Vc11–22[Q1C, Y9C] was synthesised utilising orthogonal cysteine protection and its solution structure determined using solution NMR spectroscopy. This analogue maintained the overall fold of native contryphan-Vc1. Previous studies had shown that the β-hairpin core of contryphan-Vc1 was resistant to proteolysis by trypsin and α-chymotrypsin but susceptible to cleavage by pepsin. Contryphan-Vc11–22[Q1C, Y9C] proved to be completely resistant to pepsin, thus confirming our design strategy. These results highlight the role of the disulfide bond in maintaining the SDH fold and provide a basis for the design of more stable analogues for peptide epitope grafting.

Transient interactions in which binding partners retain substantial conformational disorder play an essential role in regulating biological networks, challenging the expectation that specificity demands structurally defined and unambiguous molecular interactions. The monoclonal antibody 6D8 recognises a completely conserved continuous nine-residue epitope within the intrinsically disordered malaria antigen, MSP2, yet it has different affinities for the two allelic forms of this antigen. NMR chemical shift perturbations, relaxation rates and paramagnetic relaxation enhancements reveal the presence of transient interactions involving polymorphic residues immediately C-terminal to the structurally defined epitope. A combination of these experimental data with molecular dynamics simulations shows clearly that the polymorphic C-terminal extension engages in multiple transient interactions distributed across much of the accessible antibody surface. These interactions are determined more by topographical features of the antibody surface than by sequence-specific interactions. Thus, specificity arises as a consequence of subtle differences in what are highly dynamic and essentially non-specific interactions.

Grafting bioactive peptide sequences onto small cysteine-rich scaffolds is a promising strategy for enhancing their stability and value as novel peptide-based therapeutics. However, correctly folded disulfide-rich peptides can be challenging to produce by either recombinant or synthetic means. The single disulfide-directed β-hairpin (SDH) fold, first observed in contryphan-Vc1, provides a potential alternative to complex disulfide-rich scaffolds. We have undertaken recombinant production of full-length contryphan-Vc1 (rCon-Vc1[Z1Q]) and a truncated analogue (rCon-Vc11–22[Z1Q]), analyzed the backbone dynamics of rCon-Vc1[Z1Q], and probed the conformational and proteolytic stability of these peptides to evaluate the potential of contryphan-Vc1 as a molecular scaffold. Backbone 15N relaxation measurements for rCon-Vc1[Z1Q] indicate that the N-terminal domain of the peptide is ordered up to Thr19, whereas the remainder of the C-terminal region is highly flexible. The solution structure of truncated rCon-Vc11–...

Merozoite surface protein 2 (MSP2) is an intrinsically disordered antigen that is abundant on the surface of the malaria parasite Plasmodium falciparum. The two allelic families of MSP2, 3D7 and FC27, differ in their central variable regions, which are flanked by highly conserved C-terminal and N-terminal regions. In a vaccine trial, full-length 3D7 MSP2 induced a strain-specific protective immune response despite the detectable presence of conserved region antibodies. This work focuses on the conserved C-terminal region of MSP2, which includes the only disulphide bond in the protein and encompasses key epitopes recognised by the mouse monoclonal antibodies 4D11 and 9H4. Although the 4D11 and 9H4 epitopes are overlapping, immunofluorescence assays have shown that the mouse monoclonal antibody 4D11 binds to MSP2 on the merozoite surface with a much stronger signal than 9H4. Understanding the structural basis for this antigenic difference between these antibodies will help direct the design of a broad-spectrum and MSP2-based malaria vaccine. 4D11 and 9H4 were reengineered into antibody fragments variable region fragment (Fv) and single-chain Fv (scFv)] and were validated as suitable models for their full-sized IgG counterparts by surface plasmon resonance and isothermal titration calorimetry. An alanine scan of the 13-residue epitope 3D7-MSP2(207-222) identified the minimal binding epitope of 4D11 and the key residues involved in binding. A 2.2-angstrom crystal structure of 4D11 Fv bound to the eight-residue epitope NKENCGAA provided valuable insight into the possible conformation of the C-terminal region of MSP2 on the parasite. This work underpins continued efforts to optimise recombinant MSP2 constructs for evaluation as potential vaccine candidates. (C) 2017 Elsevier Ltd. All rights reserved.

In natural proteins and peptides, amino acids exist almost invariably as l-isomers. There are, however, several examples of naturally-occurring peptides containing d-amino acids. In this study we investigated the role of a naturally-occurring d-amino acid in a small peptide identified in the transcriptome of a marine cone snail. This peptide belongs to a family of peptides known as contryphans, all of which contain a single d-amino acid residue. The solution structure of this peptide was solved by NMR, but further investigations with molecular dynamics simulations suggest that its solution behaviour may be more dynamic than suggested by the NMR ensemble. Functional tests in mice uncovered a novel bioactivity, a depressive phenotype that contrasts with the hyperactive phenotypes typically induced by contryphans. Trp3 is important for bioactivity, but this role is independent of the chirality at this position. The d-chirality of Trp3 in this peptide was found to be protective against enzymatic degradation. Analysis by NMR and molecular dynamics simulations indicated an interaction of Trp3 with lipid membranes, suggesting the possibility of a membrane-mediated mechanism of action for this peptide.

MSP2 is an intrinsically disordered protein that is abundant on the merozoite surface and essential to the parasite Plasmodium falciparum. Naturally-acquired antibody responses to MSP2 are biased towards dimorphic sequences within the central variable region of MSP2 and have been linked to naturally-acquired protection from malaria. In a phase IIb study, an MSP2-containing vaccine induced an immune response that reduced parasitemias in a strain-specific manner. A subsequent phase I study of a vaccine that contained both dimorphic forms of MSP2 induced antibodies that exhibited functional activity in vitro. We have assessed the contribution of the conserved and variable regions of MSP2 to the generation of a strain-transcending antibody response by generating MSP2 chimeras that included conserved and variable regions of the 3D7 and FC27 alleles. Robust anti-MSP2 antibody responses targeting both conserved and variable regions were generated in mice, although the fine specificity and the balance of responses to these regions differed amongst the constructs tested. We observed significant differences in antibody subclass distribution in the responses to these chimeras. Our results suggest that chimeric MSP2 antigens can elicit a broad immune response suitable for protection against different strains of P. falciparum.

Amphibian skin secretions are well known as a rich source of bioactive peptides. However, little is known about the presence or role of peptides in the highly toxic, parotid secretion of the cane toad or giant toad, Bufo marinus (Rhinella marina), though small molecule bufadienolides, which act as potent cardiotoxins, have been described. In the current study we used RP-HPLC, MALDI-TOF mass spectrometry and tandem mass spectrometry to analyze and determine the first sequences of peptides from the parotid secretion of B. marinus. We show that peptides in the range of 900-2500 Da are indeed present, however in extremely low abundance. Despite the low abundance, the sequences of 14 peptides were determined, several of which match fragments of larger cellular proteins, yet none share substantial homology with defensive or anti-microbial peptides reported from frog skin secretions. We conclude that peptides are present in the parotid glands of B. marinus only in very low quantities and that they are likely to be breakdown products of proteins involved in cell maintenance. Given these results, we conclude that peptides are unlikely to contribute directly to the high toxicity of the cane toad.

Centipedes are venomous arthropods responsible for a significant number of non-lethal human envenomations. Despite this, information about the composition and function of their venom contents is scarce. In this study, we have used a,structure to function' proteomic approach combining two-dimensional chromatography (2D-LC), electrospray ionization quadrupole/time-of-flight mass spectrometry (ESI-Q-TOF/MS), N-terminal sequencing and similarity searching to better understand the complexities of the venoms from two Brazilian centipede species: Scolopendra viridicornis nigra and Scolopendra angulata. Comparisons between the LC profiles and the mass compositions of the venoms of the two species are provided. The observed molecular masses ranged from 3019.62 to 20996.94 Da in S. viridicornis nigra (total: 62 molecular masses) and from 1304.73 to 22639.15 Da in S. angulata (total: 65 molecular masses). Also, the N-termini of representatives of 10 protein/peptide families were successfully sequenced where nine of them showed no significant similarity to other protein sequences deposited in the Swiss-Prot database. A screening for insecto-toxic activities in fractions from S. viridicornis venom has also been performed. Six out of the 12 tested fractions were responsible for clear toxic effects in house flies. This work demonstrates that centipede venoms might be a neglected but important source of new bioactive compounds. (c) 2007 Elsevier Ltd. All rights reserved.

HsTX1[R14A] is a potent and selective Kv1.3 channel blocker peptide with the potential to treat autoimmune diseases. Given the typically poor oral bioavailability of peptides, we evaluated pulmonary administration of HsTX1[R14A] in rats as an alternative route for systemic delivery. Plasma concentrations of HsTX1[R14A] were measured by liquid chromatography coupled with tandem mass spectrometry in rats receiving intratracheal administration of HsTX1[R14A] in solution (1-4 mg/kg) or a mannitol-based powder (1 mg/kg) and compared with plasma concentrations after intravenous administration (2 mg/kg). HsTX1[R14A] stability in rat plasma and lung tissue was also determined. HsTX1[R14A] was more stable in plasma than in lung homogenate, with more than 90% of the HsTX1[R14A] remaining intact after 5 h, compared with 40.5% remaining in lung homogenate. The terminal elimination half-life, total clearance, and volume of distribution of HsTX1[R14A] after intravenous administration were 79.6 ± 6.5 min, 8.3 ± 0.6 mL/min/kg, and 949.8 ± 71.0 mL/kg, respectively (mean ± SD). After intratracheal administration, HsTX1[R14A] in solution and dry powder was absorbed to a similar degree, with absolute bioavailability values of 39.2 ± 5.2% and 44.5 ± 12.5%, respectively. This study demonstrated that pulmonary administration is a promising alternative for systemically delivering HsTX1[R14A] for treating autoimmune diseases.

Merozoite surface protein 2 (MSP2) is an intrinsically disordered, membrane-anchored antigen of the malaria parasite Plasmodium falciparum. MSP2 can elicit a protective, albeit strain-specific, antibody response in humans. Antibodies are generated to the conserved N- and C-terminal regions but many of these react poorly with the native antigen on the parasite surface. Here we demonstrate that recognition of a conserved N-terminal epitope by mAb 6D8 is incompatible with the membrane-bound conformation of that region, suggesting a mechanism by which native MSP2 escapes antibody recognition. Furthermore, crystal structures and NMR spectroscopy identify transient, strain-specific interactions between the 6D8 antibody and regions of MSP2 beyond the conserved epitope. These interactions account for the differential affinity of 6D8 for the two allelic families of MSP2, even though 6D8 binds to a fully conserved epitope. These results highlight unappreciated mechanisms that may modulate the specificity and efficacy of immune responses towards disordered antigens.

Tetrodotoxin (TTX) is one of the most potent toxin already isolated, which occurs in a wide range of marine as well as terrestrial animals such as in newts and anurans. In this work, the occurrence of TTX and analogues was examined in three brachycephalid species: Brachycephalus ephippium, B. nodoterga and B. pernix using LC-FLD and LC-MS/MS. In toxicity assay (intra-peritonial injection in mice) B. nodoterga extracts were non-toxic, while B. pernix extract exhibit the highest toxicity among the studied species. Skin showed the highest toxic, followed by the liver. Retention time data in the LC-FLD system indicated the presence of TTX, 4-epiTTX, 4,9-anhydroTTX and TDA, SIM data confirmed the presence of these compounds and revealed other analogs such as 11-norTTX-6(S)-ol, 5-deoxyTTX, 11-deoxyTTX, 11-oxoTTX, 6-epiTTX. Two new components were also identified by mass spectrometry (348 and 330 Da). These unknown compounds have daughter ions similar to TTX, suggesting new putative TTX analogues.

11-oxoTTX is an analogue 4–5 times more toxic than TTX itself, been rare even in marine animals. Two ions at m/z 320 and 336 corresponding to TTX and 11-oxoTTX (M+H+), respectively, were detected in the Brachycephalidae frog Brachycephalus ephippium extracts. The fragment ion pattern of 11-oxoTTX is similar to that TTX, although its possible to verify some specific fragments.

Many insect pests have developed resistance to existing chemical insecticides and consequently there is much interest in the development of new insecticidal compounds with novel modes of action. Although spiders have deployed insecticidal toxins in their venoms for over 250 million years, there is no evolutionary selection pressure on these toxins to possess oral activity since they are injected into prey and predators via a hypodermic needle-like fang. Thus, it has been assumed that spider-venom peptides are not orally active and are therefore unlikely to be useful insecticides. Contrary to this dogma, we show that it is possible to isolate spider-venom peptides with high levels of oral insecticidal activity by directly screening for per os toxicity. Using this approach, we isolated a 34-residue orally active insecticidal peptide (OAIP-1) from venom of the Australian tarantula Selenotypus plumipes. The oral LD50 for OAIP-1 in the agronomically important cotton bollworm Helicoverpa armigera was 104.2±0.6 pmol/g, which is the highest per os activity reported to date for an insecticidal venom peptide. OAIP-1 is equipotent with synthetic pyrethroids and it acts synergistically with neonicotinoid insecticides. The three-dimensional structure of OAIP-1 determined using NMR spectroscopy revealed that the three disulfide bonds form an inhibitor cystine knot motif; this structural motif provides the peptide with a high level of biological stability that probably contributes to its oral activity. OAIP-1 is likely to be synergized by the gut-lytic activity of the Bacillus thuringiensis Cry toxin (Bt) expressed in insect-resistant transgenic crops, and consequently it might be a good candidate for trait stacking with Bt.

Bv8, a 77-residue protein isolated from frogs, is the prototypic member of the prokineticin family of cytokines. Prokineticins (PKs) have only recently been identified in vertebrates (including humans), and they are believed to be involved in a number of key physiological processes, such as angiogenesis, neurogenesis, nociception, and tissue development. We used a combination of Boc solid-phase peptide synthesis, native chemical ligation, and in vitro protein folding to establish robust chemical access to this molecule. Synthetic Bv8 was obtained in good yield and exhibited full activity in a human neuroblastoma cell line and rat dorsal root ganglion (DRG) neurons. The 3D structure of the synthetic protein was determined by using NMR spectroscopy and it was found to be homologous with that of mamba intestinal toxin 1, which is the only other known prokineticin structure. Analysis of a truncated mutant lacking five residues at the N terminus that are critical for receptor binding and activation showed no perturbation to the core protein structure. Together with the functional data, this suggests that receptor binding is likely to be a highly cooperative process possibly involving major allosterically driven structural rearrangements. The facile and efficient synthesis presented here will enable preparation of unique chemical analogues of prokineticins, which should be powerful tools for modulating the structure and function of prokineticins and their receptors, and studying the many physiological processes that have been linked to them.

Bufotenin (5-hydroxy-N,N-dimetyltryptamine) is a tryptamine alkaloid widely spread among anuran families as a component of their chemical defense system, acting as a potent hallucinogenic factor, showing similar activity to LSD upon interaction with the 5HT2 human receptor. This work demonstrates the presence of bufotenin in the skin secretion of three arboreal amphibian species of the Osteocephalus genus (Osteocephalus taurinus, Osteocephalus oophagus and Osteocephalus langsdorffii) from the Amazon and the Atlantic rain forests using RP-HPLC, ESI-MS/MS, UV, IR and multidimensional NMR techniques. To our knowledge, this is the first description of bufotenin in the Osteocephalus genus, so far.

Brachycephalus ephippium is a diurnal frog, that shows aposematic colouration and inhabits Atlantic forest leaf litter in south-eastern Brazil. The presence of tetrodotoxin (TTX) in the skin, liver and ovaries of B. ephippium was demonstrated. The skin (260 M.U./g) exhibited the highest toxicity followed by liver (177 M.U./g). TTX and its analogues, tetrodonic acid, 4-epitetrodotoxin and 4,9 anhydrotetrodotoxin were isolated and identified by HPLC followed by fluorimetric analysis. TTX and 11-nortetrodotoxin-6(S)-ol had their presence confirmed by mass spectrometry (MALDI-TOF). The results confirm Brachycephalidae as a fourth family of anurans containing TTX.