Rodrigo A.V. Morales

Isolation of an Orally Active Insecticidal Toxin from the Venom of an Australian Tarantula

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.

Chemical synthesis and structure of the prokineticin Bv8.

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.

Pulmonary Delivery of the Kv1.3-Blocking Peptide HsTX1[R14A] for the Treatment of Autoimmune Diseases

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.

Structure and activity of contryphan-Vc2: Importance of the d-amino acid residue

Abstract 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. Graphical abstract Figure. No caption available. HighlightsContryphan‐Vc2 contains a natural d‐amino acid residue.Both d‐ and l‐analogues exhibit the same bioactivity.Both analogues interact with lipid membranes.Possible membrane‐mediated mechanism of action.

The Use of Imaging Mass Spectrometry to Study Peptide Toxin Distribution in Australian Sea Anemones

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic. 3052, Australia. Centre for Advanced Imaging, University of Queensland, St Lucia, Qld 4072, Australia. Centre for Microscopy and Microanalysis, University of Queensland, St Lucia, Qld 4072, Australia. Institute for Molecular Bioscience, University of Queensland, St Lucia, Qld 4072, Australia. Bioinformatics Division, Walter & Eliza Hall Institute of Research, Parkville, Vic. 3052, Australia. Department of Biochemistry and Molecular Biology and Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, Vic. 3800, Australia. Corresponding author. Email: michela.mitchell@monash.edu

Structure, folding and stability of a minimal homologue from Anemonia sulcata of the sea anemone potassium channel blocker ShK

Graphical abstract AsK132958 is a 29‐residue peptide identified in a transcriptomic study of Anemonia sulcata. It has the same disulfide framework and a similar structure to ShK. AsK132958 is not active against KV1.3 channels, owing to the lack of a Lys‐Tyr dyad and other functionally important amino acid residues. AsK132958 is more resistant to proteolysis than ShK. Introducing a Lys‐Tyr functional dyad to the AsK132958 structural scaffold may be a useful way of developing a proteolytically stable KV1.3 blocker. Figure. No caption available. HighlightsAsK132958 is one of the shortest peptides with a ShK/BgK‐like cysteine framework.AsK132958 is a structural homologue of ShK.Despite having an ShK‐like scaffold, AsK132958 is not active against KV1.3 or related potassium channels.AsK132958 is more resistant to proteases than ShK and is a promising scaffold for engineering other activities.AsK132958 could be an evolutionary precursor of peptides with ShK‐like scaffold and activity. ABSTRACT Peptide toxins elaborated by sea anemones target various ion‐channel sub‐types. Recent transcriptomic studies of sea anemones have identified several novel candidate peptides, some of which have cysteine frameworks identical to those of previously reported sequences. One such peptide is AsK132958, which was identified in a transcriptomic study of Anemonia sulcata and has a cysteine framework similar to that of ShK from Stichodactyla helianthus, but is six amino acid residues shorter. We have determined the solution structure of this novel peptide using NMR spectroscopy. The disulfide connectivities and structural scaffold of AsK132958 are very similar to those of ShK but the structure is more constrained. Toxicity assays were performed using grass shrimp (Palaemonetes sp) and Artemia nauplii, and patch‐clamp electrophysiology assays were performed to assess the activity of AsK132958 against a range of voltage‐gated potassium (KV) channels. AsK132958 showed no activity against grass shrimp, Artemia nauplii, or any of the KV channels tested, owing partly to the absence of a functional Lys‐Tyr dyad. Three AsK132958 analogues, each containing a Tyr in the vicinity of Lys19, were therefore generated in an effort to restore binding, but none showed activity against any of KV channels tested. However, AsK132958 and its analogues are less susceptible to proteolysis than that of ShK. Our structure suggests that Lys19, which might be expected to occupy the pore of the channel, is not sufficiently accessible for binding, and therefore that AsK132958 must have a distinct functional role that does not involve KV channels.

Structure and Characterisation of a Key Epitope in the Conserved C-Terminal Domain of the Malaria Vaccine Candidate MSP2

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-MSP2207-222 identified the minimal binding epitope of 4D11 and the key residues involved in binding. A 2.2-Å 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.

Lipid interactions modulate the structural and antigenic properties of the C‐terminal domain of the malaria antigen merozoite surface protein 2

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.

Gomesin inhibits melanoma growth by manipulating key signaling cascades that control cell death and proliferation

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.

Transient antibody-antigen interactions mediate the strain-specific recognition of a conserved malaria epitope

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.Krishnarjuna et al. show that multiple transient interactions mediate monoclonal antibody recognition of an epitope within a disordered malaria antigen, MSP2. These results explain the antibody’s differential affinities for two allelic forms of the antigen.