Publications by authors named "Raymond S Norton"

216 Publications

Alkyne-Bridged α-Conotoxin Vc1.1 Potently Reverses Mechanical Allodynia in Neuropathic Pain Models.

J Med Chem 2021 Mar 16;64(6):3222-3233. Epub 2021 Mar 16.

School of Chemistry, Monash University, Clayton, Victoria 3800, Australia.

Several -derived venom peptides are promising lead compounds for the management of neuropathic pain, with α-conotoxins being of particular interest. Modification of the interlocked disulfide framework of α-conotoxin Vc1.1 has been achieved using on-resin alkyne metathesis. Although introduction of a metabolically stable alkyne motif significantly disrupts backbone topography, the structural modification generates a potent and selective GABA receptor agonist that inhibits Ca2.2 channels and exhibits dose-dependent reversal of mechanical allodynia in a behavioral rat model of neuropathic pain. The findings herein support the hypothesis that analgesia can be achieved via activation of GABARs expressed in dorsal root ganglion (DRG) sensory neurons.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02151DOI Listing
March 2021

Lipopolysaccharide influences the plasma and brain pharmacokinetics of subcutaneously-administered HsTX1[R14A], a K1.3-blocking peptide.

Toxicon 2021 May 6;195:29-36. Epub 2021 Mar 6.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia. Electronic address:

K1.3 is a voltage-gated potassium channel that is upregulated in neuroinflammatory conditions, such as Alzheimer's disease and Parkinson's disease. HsTX1[R14A] is a potent and selective peptide blocker of K1.3 with the potential to block microglial K1.3, but its brain uptake is expected to be limited owing to the restrictive nature of the blood-brain barrier. To assess its peripheral and brain exposure, a LC-MS/MS assay was developed to quantify HsTX1[R14A] concentrations in mouse plasma and brain homogenate that was reliable and reproducible in the range of 6.7-66.7 nM (r = 0.9765) and 15-150 pmol/g (r = 0.9984), respectively. To assess if neuroinflammation affected HsTX1[R14A] disposition, C57BL/6 mice were administered HsTX1[R14A] subcutaneously (2 mg/kg) 24 h after an intraperitoneal dose of Escherichia coli lipopolysaccharide (LPS), which is commonly used to induce neuroinflammation; brain and plasma concentrations of HsTX1[R14A] were then quantified over 120 min. LPS treatment significantly retarded the decline in HsTX1[R14A] plasma concentrations, presumably as a result of reducing renal clearance, and led to substantial brain uptake of HsTX1[R14A], presumably through disruption of brain inter-endothelial tight junctions. This study suggests that HsTX1[R14A] may reach microglia in sufficient concentrations to block K1.3 in neuroinflammatory conditions, and therefore has the potential to reduce neurodegenerative diseases.
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http://dx.doi.org/10.1016/j.toxicon.2021.03.002DOI Listing
May 2021

Liposome engraftment and antigen combination potentiate the immune response towards conserved epitopes of the malaria vaccine candidate MSP2.

Vaccine 2021 Mar 20;39(12):1746-1757. Epub 2021 Feb 20.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia; ARC Centre for Fragment-Based Design, Monash University, Parkville, Victoria 3052, Australia. Electronic address:

Merozoite surface protein 2 (MSP2) is a highly abundant, GPI-anchored surface antigen on merozoites of the malaria parasite Plasmodium falciparum. It consists of highly conserved N- and C-terminal domains, and a central polymorphic region that allows all MSP2 alleles to be categorized into the 3D7 or FC27 family. Previously it has been shown that epitope accessibility differs between lipid-bound and lipid-free MSP2, suggesting that lipid interactions modulate the conformation and antigenicity in a way that may better mimic native MSP2 on the merozoite surface. Therefore, we have immunised mice with MSP2 engrafted onto liposomes using a C-terminal tether that mimics the native GPI anchor. To improve the immunogenicity of the formulated antigen, liposomes were supplemented with Pathogen Associated Molecular Pattern molecules, specifically agonists of the Toll-like receptor 4 (TLR4) or TLR2. Induced antibodies were directed mostly towards conserved epitopes, predominantly in the conserved C-terminal region of MSP2. We also found that immunisation with a combination of 3D7 and FC27 MSP2 enhanced antibody responses to conserved epitopes, and that the overall responses of mice immunised with MSP2-engrafted liposomes were comparable in magnitude to those of mice immunised with MSP2 formulated in Montanide ISA720. The antibodies elicited in mice by immunising with MSP2-engrafted liposomes recognised the native form of parasite MSP2 on western blots and were found to be cross-reactive with isolated 3D7 and FC27 merozoites when investigated by ELISA. The liposome-tethered MSP2 induced higher titres of complement-fixing antibodies to 3D7 and FC27 MSP2 than did MSP2 formulated in Montanide ISA720. Our results indicate that liposomal formulation represents a viable strategy for eliciting a strong immune response that favours conserved epitopes in MSP2 and thus a strain-transcendent immune response.
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http://dx.doi.org/10.1016/j.vaccine.2021.02.010DOI Listing
March 2021

A disulfide-stabilised helical hairpin fold in acrorhagin I: An emerging structural motif in peptide toxins.

J Struct Biol 2020 Dec 30;213(2):107692. Epub 2020 Dec 30.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia; ARC Centre for Fragment-Based Design, Monash University, Parkville, Victoria 3052, Australia. Electronic address:

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 C,N-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.
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http://dx.doi.org/10.1016/j.jsb.2020.107692DOI Listing
December 2020

Assessing the cellular toxicity of peptide inhibitors of intracellular protein-protein interactions by microinjection.

Bioorg Med Chem 2021 01 3;29:115906. Epub 2020 Dec 3.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic, Australia; ARC Centre for Fragment-Based Design, Monash University, Parkville, Victoria 3052, Australia. Electronic address:

Inhibitors of protein-protein interactions can be developed through a number of technologies to provide leads that include cell-impermeable molecules. Redesign of these impermeable leads to provide cell-permeable derivatives can be challenging and costly. We hypothesised that intracellular toxicity of leads could be assessed by microinjection prior to investing in the redesign process. We demonstrate this approach for our development of inhibitors of the protein-protein interaction between inducible nitric-oxide synthase (iNOS) and SPRY domain-containing SOCS box proteins (SPSBs). We microinjected a lead molecule into AD-293 cells and were able to perform an intracellular toxicity assessment. We also investigated the intracellular distribution and localisation of injected inhibitor using a fluorescently-labelled analogue. Our findings show that a lead peptide inhibitor, CP2, had no toxicity even at intracellular concentrations four orders of magnitude higher than its K for binding to SPSB2. This early toxicity assessment justifies further development of this cell-impermeable lead to confer cell permeability. Our investigation highlights the utility of microinjection as a tool for assessing toxicity during development of drugs targeting protein-protein interactions.
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http://dx.doi.org/10.1016/j.bmc.2020.115906DOI Listing
January 2021

Merozoite surface protein 2 adsorbed onto acetalated dextran microparticles for malaria vaccination.

Int J Pharm 2021 Jan 10;593:120168. Epub 2020 Dec 10.

Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, USA; Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, USA; Department of Microbiology and Immunology, UNC School of Medicine, University of North Carolina, Chapel Hill, NC, USA. Electronic address:

Malaria remains a global health threat, with significant morbidity and mortality worldwide despite current interventions. The human disease is caused by five different parasitic species, with Plasmodium falciparum being the deadliest. As a result, vaccine research against P. falciparum is a global priority. Merozoite surface protein 2 (MSP2) is a promising vaccine antigen as MSP2-specific antibodies have been shown previously to be protective against malaria infection. In this study, the formulation of an MSP2 vaccine was explored to enhance antigen uptake and achieve both an antibody and Th1 immune response by adsorbing MSP2 antigen onto a biomaterial carrier system. Specifically, MSP2 antigen was adsorbed onto acetalated dextran (Ace-DEX) microparticles (MPs). IgG and IgG2a titers elicited by the Ace-DEX MP platform were compared to titer levels elicited by MSP2 adsorbed to an FDA-approved alum adjuvant, MSP2 alone, and PBS alone. Both adsorption of MSP2 to Ace-DEX MPs and to alum elicited antibody responses in vivo, but only the formulation containing Ace-DEX MPs was able to elicit a significant Th1-biased response needed to combat the intracellular pathogen. As such, MSP2 adsorbed to Ace-DEX MPs demonstrates promise as a malaria vaccine.
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http://dx.doi.org/10.1016/j.ijpharm.2020.120168DOI Listing
January 2021

Correction to: NMR in pharmaceutical discovery and development.

J Biomol NMR 2020 Nov;74(10-11):477

Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Virchow-16.3.249, 4002, Basel, Switzerland.

The article "Boeszoermenyi A, Ogórek B, Jain A, Arthanari H, Wagner G (2020) The precious fluorine on the ring: fluorine NMR for biological systems. J Biomol NMR. https ://doi.org/10.1007/s10858-020-00331-z" was written for the "Special Issue: NMR in Pharmaceutical Discovery and Development". However, unfortunately, it was published in an earlier issue of this journal owing to a publisher error. Further, the ORCID ID of author Wolfgang Jahnke is updated in the article. The original article has been corrected.
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http://dx.doi.org/10.1007/s10858-020-00351-9DOI Listing
November 2020

Prolonged Plasma Exposure of the Kv1.3-Inhibitory Peptide HsTX1[R14A] by Subcutaneous Administration of a Poly(Lactic-co-Glycolic Acid) (PLGA) Microsphere Formulation.

J Pharm Sci 2021 03 13;110(3):1182-1188. Epub 2020 Oct 13.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia. Electronic address:

This study evaluated the impact of poly(lactic-co-glycolic acid) (PLGA) microsphere formulations on in vitro release and in vivo plasma exposure of HsTX1[R14A], a potent inhibitor of the voltage-gated potassium channel Kv1.3, with potential to treat autoimmune conditions. Microspheres containing HsTX1[R14A] were prepared using different PLGA materials, including Resomer® RG502H, RG503H and PURASORB® PDLG 5004 (Purac). After assessing encapsulation efficiency and in vitro release, plasma concentrations of HsTX1[R14A] were quantified by LCMS/MS following subcutaneous administration of HsTX1[R14A]-loaded RG503H microspheres (15 mg/kg) or HsTX1[R14A] solution (4 mg/kg) to Sprague-Dawley rats. Microspheres prepared with Purac exhibited the greatest encapsulation efficiency (45.5 ± 2.4% (mean ± SD)) and RG502H the lowest (22.0 ± 6.4%). Release of HsTX1[R14A] was fastest in vitro for RG502H microspheres (maximum release at 31 days) and slowest for Purac (82 days). With a relatively rapid burst release of 20.0 ± 0.4% and a controlled release profile of up to 41 days, HsTX1[R14A]-loaded RG503H microspheres were selected for subcutaneous administration, resulting in detectable plasma concentrations for 11 days relative to 8 h following subcutaneous administration of HsTX1[R14A] solution. Therefore, subcutaneous administration of RG503H PLGA microspheres is a promising approach to be exploited for delivery of this immune modulator.
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http://dx.doi.org/10.1016/j.xphs.2020.10.014DOI Listing
March 2021

NMR in pharmaceutical discovery and development.

J Biomol NMR 2020 Nov 4;74(10-11):473-476. Epub 2020 Sep 4.

Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Virchow-16.3.249, 4002, Basel, Switzerland.

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http://dx.doi.org/10.1007/s10858-020-00345-7DOI Listing
November 2020

Modulation of Lymphocyte Potassium Channel K1.3 by Membrane-Penetrating, Joint-Targeting Immunomodulatory Plant Defensin.

ACS Pharmacol Transl Sci 2020 Aug 14;3(4):720-736. Epub 2020 May 14.

School of Physics, University of Sydney, Sydney, New South Wales 2006, Australia.

We describe a cysteine-rich, membrane-penetrating, joint-targeting, and remarkably stable peptide, EgK5, that modulates voltage-gated K1.3 potassium channels in T lymphocytes by a distinctive mechanism. EgK5 enters plasma membranes and binds to K1.3, causing current run-down by a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. EgK5 exhibits selectivity for K1.3 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 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 dose. EgK5 shows promise for clinical development as a therapeutic for autoimmune diseases.
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http://dx.doi.org/10.1021/acsptsci.0c00035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432667PMC
August 2020

α-Conotoxin Peptidomimetics: Probing the Minimal Binding Motif for Effective Analgesia.

Toxins (Basel) 2020 08 6;12(8). Epub 2020 Aug 6.

School of Chemistry, Monash University, Clayton, Victoria 3800, Australia.

Several analgesic α-conotoxins have been isolated from marine cone snails. Structural modification of native peptides has provided potent and selective analogues for two of its known biological targets-nicotinic acetylcholine and γ-aminobutyric acid (GABA) G protein-coupled (GABA) receptors. Both of these molecular targets are implicated in pain pathways. Despite their small size, an incomplete understanding of the structure-activity relationship of α-conotoxins at each of these targets has hampered the development of therapeutic leads. This review scrutinises the -terminal domain of the α-conotoxin family of peptides, a region defined by an invariant disulfide bridge, a turn-inducing proline residue and multiple polar sidechain residues, and focusses on structural features that provide analgesia through inhibition of high-voltage-activated Ca channels. Elucidating the bioactive conformation of this region of these peptides may hold the key to discovering potent drugs for the unmet management of debilitating chronic pain associated with a wide range of medical conditions.
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http://dx.doi.org/10.3390/toxins12080505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472027PMC
August 2020

The voltage-gated potassium channel K1.3 as a therapeutic target for venom-derived peptides.

Biochem Pharmacol 2020 11 10;181:114146. Epub 2020 Jul 10.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia; ARC Centre for Fragment-Based Design, Monash University, Parkville, VIC 3052, Australia. Electronic address:

The voltage-gated potassium channel K1.3 is a well-established therapeutic target for a range of autoimmune diseases, in addition to being the site of action of many venom-derived peptides. Numerous studies have documented the efficacy of venom peptides that target K1.3, in particular from sea anemones and scorpions, in animal models of autoimmune diseases such as rheumatoid arthritis, psoriasis and multiple sclerosis. Moreover, an analogue of the sea anemone peptide ShK (known as dalazatide) has successfully completed Phase 1 clinical trials in mild-to-moderate plaque psoriasis. In this article we consider other potential therapeutic applications of inhibitors of K1.3, including in inflammatory bowel disease and neuroinflammatory conditions such as Alzheimer's and Parkinson's diseases, as well as fibrotic diseases. We also summarise strategies for facilitating the entry of peptides to the central nervous system, given that this will be a pre-requisite for the treatment of most neuroinflammatory diseases. Venom-derived peptides that have been reported recently to target K1.3 are also described. The increasing number of autoimmune and other conditions in which K1.3 is upregulated and is therefore a potential therapeutic target, combined with the fact that many venom-derived peptides are potent inhibitors of K1.3, suggests that venoms are likely to continue to serve as a rich source of new pharmacological tools and therapeutic leads targeting this channel.
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http://dx.doi.org/10.1016/j.bcp.2020.114146DOI Listing
November 2020

Improving Membrane Permeation in the Beyond Rule-of-Five Space by Using Prodrugs to Mask Hydrogen Bond Donors.

ACS Chem Biol 2020 08 20;15(8):2070-2078. Epub 2020 Jul 20.

Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.

A wide range of drug targets can be effectively modulated by peptides and macrocycles. Unfortunately, the size and polarity of these compounds prevents them from crossing the cell membrane to reach target sites in the cell cytosol. As such, these compounds do not conform to standard measures of drug-likeness and exist in beyond the rule-of-five space. In this work, we investigate whether prodrug moieties that mask hydrogen bond donors can be applied in the beyond rule-of-five domain to improve the permeation of macrocyclic compounds. Using a cyclic peptide model, we show that masking hydrogen bond donors in the natural polar amino acid residues (His, Ser, Gln, Asn, Glu, Asp, Lys, and Arg) imparts membrane permeability to the otherwise impermeable parent molecules, even though the addition of the masking group increases the overall compound molecular weight and the number of hydrogen bond acceptors. We demonstrate this strategy in PAMPA and Caco2 membrane permeability assays and show that masking with groups that reduce the number of hydrogen-bond donors at the cost of additional mass and hydrogen bond acceptors, a donor-acceptor swap, is effective.
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http://dx.doi.org/10.1021/acschembio.0c00218DOI Listing
August 2020

A structurally minimized yet fully active insulin based on cone-snail venom insulin principles.

Nat Struct Mol Biol 2020 07 1;27(7):615-624. Epub 2020 Jun 1.

Department of Biochemistry, University of Utah, Salt Lake City, UT, USA.

Human insulin and its current therapeutic analogs all show propensity, albeit varyingly, to self-associate into dimers and hexamers, which delays their onset of action and makes blood glucose management difficult for people with diabetes. Recently, we described a monomeric, insulin-like peptide in cone-snail venom with moderate human insulin-like bioactivity. Here, with insights from structural biology studies, we report the development of mini-Ins-a human des-octapeptide insulin analog-as a structurally minimal, full-potency insulin. Mini-Ins is monomeric and, despite the lack of the canonical B-chain C-terminal octapeptide, has similar receptor binding affinity to human insulin. Four mutations compensate for the lack of contacts normally made by the octapeptide. Mini-Ins also has similar in vitro insulin signaling and in vivo bioactivities to human insulin. The full bioactivity of mini-Ins demonstrates the dispensability of the PheB24-PheB25-TyrB26 aromatic triplet and opens a new direction for therapeutic insulin development.
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http://dx.doi.org/10.1038/s41594-020-0430-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374640PMC
July 2020

Structure-Activity Relationship Study Reveals the Molecular Basis for Specific Sensing of Hydrophobic Amino Acids by the Chemoreceptor Tlp3.

Biomolecules 2020 05 11;10(5). Epub 2020 May 11.

Infection and Immunity Program, Monash Biomedicine Discovery Institute, Clayton, Victoria 3800, Australia.

Chemotaxis is an important virulence factor of the foodborne pathogen . Inactivation of chemoreceptor Tlp3 reduces the ability of to invade human and chicken cells and to colonise the jejunal mucosa of mice. Knowledge of the structure of the ligand-binding domain (LBD) of Tlp3 in complex with its ligands is essential for a full understanding of the molecular recognition underpinning chemotaxis. To date, the only structure in complex with a signal molecule is Tlp3 LBD bound to isoleucine. Here, we used in vitro and in silico screening to identify eight additional small molecules that signal through Tlp3 as attractants by directly binding to its LBD, and determined the crystal structures of their complexes. All new ligands (leucine, valine, α-amino-N-valeric acid, 4-methylisoleucine, β-methylnorleucine, 3-methylisoleucine, alanine, and phenylalanine) are nonpolar amino acids chemically and structurally similar to isoleucine. X-ray crystallographic analysis revealed the hydrophobic side-chain binding pocket and conserved protein residues that interact with the ammonium and carboxylate groups of the ligands determine the specificity of this chemoreceptor. The uptake of hydrophobic amino acids plays an important role in intestinal colonisation by , and our study suggests that seeks out hydrophobic amino acids using chemotaxis.
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http://dx.doi.org/10.3390/biom10050744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7277094PMC
May 2020

Characterising Functional Venom Profiles of Anthozoans and Medusozoans within Their Ecological Context.

Mar Drugs 2020 Apr 9;18(4). Epub 2020 Apr 9.

School of Biology and Environmental Science, Science and Engineering Faculty, Queensland University of Technology, Brisbane, QLD 4000, Australia.

This review examines the current state of knowledge regarding toxins from anthozoans (sea anemones, coral, zoanthids, corallimorphs, sea pens and tube anemones). We provide an overview of venom from phylum Cnidaria and review the diversity of venom composition between the two major clades (Medusozoa and Anthozoa). We highlight that the functional and ecological context of venom has implications for the temporal and spatial expression of protein and peptide toxins within class Anthozoa. Understanding the nuances in the regulation of venom arsenals has been made possible by recent advances in analytical technologies that allow characterisation of the spatial distributions of toxins. Furthermore, anthozoans are unique in that ecological roles can be assigned using tissue expression data, thereby circumventing some of the challenges related to pharmacological screening.
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http://dx.doi.org/10.3390/md18040202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7230708PMC
April 2020

Tentacle Transcriptomes of the Speckled Anemone (Actiniaria: Actiniidae: Oulactis sp.): Venom-Related Components and Their Domain Structure.

Mar Biotechnol (NY) 2020 Apr 24;22(2):207-219. Epub 2020 Jan 24.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia.

Cnidarians are one of the oldest known animal lineages (ca. 700 million years), with a unique envenomation apparatus to deliver a potent mixture of peptides and proteins. Some peptide toxins from cnidarian venom have proven therapeutic potential. Here, we use a transcriptomic/proteomic strategy to identify sequences with similarity to known venom protein families in the tentacles of the endemic Australian 'speckled anemone' (Oulactis sp.). Illumina RNASeq data were assembled de novo. Annotated sequences in the library were verified by cross-referencing individuals' transcriptomes or protein expression evidence from LC-MS/MS data. Sequences include pore-forming toxins, phospholipases, peptidases, neurotoxins (sodium and potassium channel modulators), cysteine-rich secretory proteins and defensins (antimicrobial peptides). Fewer than 4% of the sequences in the library occurred across the three individuals examined, demonstrating high sequence variability of an individual's arsenal. We searched for actinoporins in Oulactis sp. to assess sequence similarity to the only described toxins (OR-A and -G) for this genus and examined the domain architecture of venom-related peptides and proteins. The novel putative actinoporin of Oulactis sp. has a greater similarity to other species in the Actiniidae family than to O. orientalis. Venom-related sequences have an architecture that occurs in single, repeat or multi-domain combinations of venom-related (e.g. ShK-like) and non-venom (e.g. whey acid protein) domains. This study has produced the first transcriptomes for an endemic Australian sea anemone species and the genus Oulactis, while identifying nearly 400 novel venom-related peptides and proteins for future structural and functional analyses and venom evolution studies.
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http://dx.doi.org/10.1007/s10126-020-09945-8DOI Listing
April 2020

Conformational exchange in the potassium channel blocker ShK.

Sci Rep 2019 12 17;9(1):19307. Epub 2019 Dec 17.

Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK.

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.
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http://dx.doi.org/10.1038/s41598-019-55806-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917819PMC
December 2019

Antimicrobial activity and structure of a consensus human β-defensin and its comparison to a novel putative hBD10.

Proteins 2020 01 30;88(1):175-186. Epub 2019 Jul 30.

Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mexico.

The spread of multidrug resistant bacteria owing to the intensive use of antibiotics is challenging current antibiotic therapies, and making the discovery and evaluation of new antimicrobial agents a high priority. The evaluation of novel peptide sequences of predicted antimicrobial peptides from different sources is valuable approach to identify alternative antibiotic leads. Two strategies were pursued in this study to evaluate novel antimicrobial peptides from the human β-defensin family (hBD). In the first, a 32-residue peptide was designed based on the alignment of all available hBD primary structures, while in the second a putative 35-residue peptide, hBD10, was mined from the gene DEFB110. Both hBDconsensus and hBD10 were chemically synthesized, folded and purified. They showed antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Mycobacterium tuberculosis, but were not hemolytic on human red blood cells. The NMR-based solution structure of hBDconsensus revealed that it adopts a classical β-defensin fold and disulfide connectivities. Even though the mass spectrum of hBD10 confirmed the formation of three disulfide bonds, it showed limited dispersion in H NMR spectra and structural studies were not pursued. The evaluation of different β-defensin structures may identify new antimicrobial agents effective against multidrug-resistant bacterial strains.
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http://dx.doi.org/10.1002/prot.25785DOI Listing
January 2020

Structural and functional characterisation of a novel peptide from the Australian sea anemone Actinia tenebrosa.

Toxicon 2019 Oct 11;168:104-112. Epub 2019 Jul 11.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC, 3052, Australia; ARC Centre for Fragment-Based Design, Monash University, Parkville, VIC, 3052, Australia. Electronic address:

Sea anemone venoms have long been recognised as a rich source of peptides with interesting pharmacological and structural properties. Our recent transcriptomic studies of the Australian sea anemone Actinia tenebrosa have identified a novel 13-residue peptide, U-AITx-Ate1. U-AITx-Ate1 contains a single disulfide bridge and bears no significant homology to previously reported amino acid sequences of peptides from sea anemones or other species. We have produced U-AITx-Ate1 using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The solution structure of U-AITx-Ate1 was determined based on two-dimensional nuclear magnetic resonance spectroscopic data. Diffusion-ordered NMR spectroscopy revealed that U-AITx-Ate1 was monomeric in solution. Perturbations in the 1D H NMR spectrum of U-AITx-Ate1 in the presence of dodecylphosphocholine micelles together with molecular dynamics simulations indicated an interaction of U-AITx-Ate1 with lipid membranes, although no binding was detected to 100% POPC and 80% POPC: 20% POPG lipid nanodiscs by isothermal titration calorimetry. Functional assays were performed to explore the biological activity profile of U-AITx-Ate1. U-AITx-Ate1 showed no activity in voltage-clamp electrophysiology assays and no change in behaviour and mortality rates in crustacea. Moderate cytotoxic activity was observed against two breast cancer cell lines.
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http://dx.doi.org/10.1016/j.toxicon.2019.07.002DOI Listing
October 2019

Mapping the chemical and sequence space of the ShKT superfamily.

Toxicon 2019 Jul 4;165:95-102. Epub 2019 May 4.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia.

The ShKT superfamily is widely distributed throughout nature and encompasses a wide range of documented functions and processes, from modulation of potassium channels to involvement in morphogenesis pathways. Cysteine-rich secretory proteins (CRISPs) contain a cysteine-rich domain (CRD) at the C-terminus that is similar in structure to the ShK fold. Despite the structural similarity of the CRD and ShK-like domains, we know little of the sequence-function relationships in these families. Here, for the first time, we examine the evolution of the biophysical properties of sequences within the ShKT superfamily in relation to function, with a focus on the ShK-like superfamily. ShKT data were sourced from published sequences in the protein family database, in addition to new ShK-like sequences from the Australian speckled anemone (Oulactis sp.). Our analysis clearly delineates the ShK-like family from the CRDs of CRISP proteins. The four CRISP subclusters separate out into the main phyla of Mammalia, Insecta and Reptilia. The ShK-like family is in turn composed of seven subclusters, the largest of which contains members from across the eukaryotes, with a continuum of intermediate properties. Smaller sub-clusters contain specialised members such as nematode ShK-like sequences. Several of these ShKT sub-clusters contain no functionally characterised sequences. This chemical space analysis should be useful as a guide to select sequences for functional studies and to gain insight into the evolution of these highly divergent sequences with an ancient conserved fold.
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http://dx.doi.org/10.1016/j.toxicon.2019.04.008DOI Listing
July 2019

The three-dimensional structure of an H-superfamily conotoxin reveals a granulin fold arising from a common ICK cysteine framework.

J Biol Chem 2019 05 11;294(22):8745-8759. Epub 2019 Apr 11.

From the Department of Biology, Linderstrøm-Lang Centre for Protein Science, University of Copenhagen, 2200 Copenhagen N., Denmark,

Venomous marine cone snails produce peptide toxins (conotoxins) that bind ion channels and receptors with high specificity and therefore are important pharmacological tools. Conotoxins contain conserved cysteine residues that form disulfide bonds that stabilize their structures. To gain structural insight into the large, yet poorly characterized conotoxin H-superfamily, we used NMR and CD spectroscopy along with MS-based analyses to investigate H-Vc7.2 from , a peptide with a VI/VII cysteine framework. This framework has Cys-Cys/Cys-Cys/Cys-Cys connectivities, which have invariably been associated with the inhibitor cystine knot (ICK) fold. However, the solution structure of recombinantly expressed and purified H-Vc7.2 revealed that although it displays the expected cysteine connectivities, H-Vc7.2 adopts a different fold consisting of two stacked β-hairpins with opposing β-strands connected by two parallel disulfide bonds, a structure homologous to the N-terminal region of the human granulin protein. Using structural comparisons, we subsequently identified several toxins and nontoxin proteins with this "mini-granulin" fold. These findings raise fundamental questions concerning sequence-structure relationships within peptides and proteins and the key determinants that specify a given fold.
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http://dx.doi.org/10.1074/jbc.RA119.007491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6552430PMC
May 2019

Evolution of cnidarian trans-defensins: Sequence, structure and exploration of chemical space.

Proteins 2019 07 18;87(7):551-560. Epub 2019 Mar 18.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria, Australia.

Many of the small, cysteine-rich ion-channel modulatory peptides found in Cnidaria are distantly related to vertebrate defensins (of the trans-defensin superfamily). Transcriptomic and proteomic studies of the endemic Australian speckled sea anemone (Oulactis sp.) yielded homologous peptides to known defensin sequences. We extended these data using existing and custom-built hidden Markov models to extract defensin-like families from the transcriptomes of seven endemic Australian cnidarian species. Newly sequenced transcriptomes include three species of Actiniaria (true sea anemones); the speckled anemone (Oulactis sp.), Oulactis muscosa, Dofleinia cf. armata and a species of Corallimorpharia, Rhodactis sp. We analyzed these novel defensin-like sequences along with published homologues to study the evolution of their physico-chemical properties in vertebrate and invertebrate fauna. The cnidarian trans-defensins form a distinct cluster within the chemical space of the superfamily, with a unique set of motifs and biophysical properties. This cluster contains identifiable subgroups, whose distribution in chemical space also correlates with the divergent evolution of their structures. These sequences, currently restricted to cnidarians, form an evolutionarily distinct clade within the trans-defensin superfamily.
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http://dx.doi.org/10.1002/prot.25679DOI Listing
July 2019

Identification of the Binding Site of Apical Membrane Antigen 1 (AMA1) Inhibitors Using a Paramagnetic Probe.

ChemMedChem 2019 03 13;14(5):603-612. Epub 2019 Feb 13.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia.

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small-molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin-labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)-based NMR screening, the H relaxation rates of compounds binding close to the probe were enhanced. Compounds experienced different degrees of PRE as a result of their different orientations relative to the spin label while bound to AMA1. Thus, PRE-derived distance constraints can be used to identify binding sites and guide further hit optimisation.
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http://dx.doi.org/10.1002/cmdc.201800802DOI Listing
March 2019

Interaction of merozoite surface protein 2 with lipid membranes.

FEBS Lett 2019 02 10;593(3):288-295. Epub 2019 Jan 10.

School of Life Sciences, Anhui University, Hefei, China.

Merozoite surface protein 2 (MSP2) is a potential vaccine candidate against malaria, although its functional role is yet to be elucidated. Previous studies showed that MSP2 can interact with membranes, which may facilitate merozoite invasion into the host cell. The N-terminal 25 residues of MSP2 (MSP2 ), which may be aggregated on the merozoite surface, play a key role in the interaction with membranes. Here, we investigated the effects of MSP2 -membrane interactions on the conformation and aggregation of MSP2 and on membrane integrity, using nanodiscs and small unilamellar vesicles as mimetics of cell membranes. MSP2 -membrane interactions induced the peptide to form β-structure and to aggregate, depending on the lipid composition of the membrane. Nonfibrillar aggregates in turn disrupted the membrane.
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http://dx.doi.org/10.1002/1873-3468.13320DOI Listing
February 2019

Modulation of the aggregation of an amyloidogenic sequence by flanking-disordered region in the intrinsically disordered antigen merozoite surface protein 2.

Eur Biophys J 2019 Jan 15;48(1):99-110. Epub 2018 Nov 15.

School of Life Sciences, Anhui University, Hefei, Anhui, 230601, China.

The abundant Plasmodium falciparum merozoite surface protein MSP2, a potential malaria vaccine candidate, is an intrinsically disordered protein with some nascent secondary structure present in its conserved N-terminal region. This relatively ordered region has been implicated in both membrane interactions and amyloid-like aggregation of the protein, while the significance of the flanking-disordered region is unclear. In this study, we show that aggregation of the N-terminal conserved region of MSP2 is influenced in a length- and sequence-dependent fashion by the disordered central variable sequences. Intriguingly, MSP2 peptides containing the conserved region and the first five residues of the variable disordered regions aggregated more rapidly than a peptide corresponding to the conserved region alone. In contrast, MSP2 peptides extending 8 or 12 residues into the disordered region aggregated more slowly, consistent with the expected inhibitory effect of flanking-disordered sequences on the aggregation of amyloidogenic ordered sequences. Computational analyses indicated that the helical propensity of the ordered region of MSP2 was modulated by the adjacent disordered five residues in a sequence-dependent manner. Nuclear magnetic resonance and circular dichroism spectroscopic studies with synthetic peptides confirmed the computational predictions, emphasizing the correlation between aggregation propensity and conformation of the ordered region and the effects thereon of the adjacent disordered region. These results show that the effects of flanking-disordered sequences on a more ordered sequence may include enhancement of aggregation through modulation of the conformational properties of the more ordered sequence.
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http://dx.doi.org/10.1007/s00249-018-1337-8DOI Listing
January 2019

Anti-Infective Peptides to Enhance the Host Innate Response: Design, Development and Delivery.

Authors:
Raymond S Norton

Protein Pept Lett 2018 ;25(12):1101-1107

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia.

Background: Inducible Nitric Oxide Synthase (iNOS or NOS2) produces Nitric Oxide (NO) and related reactive nitrogen species, which are critical effectors of the host innate response and play key roles in the intracellular killing of bacterial and parasitic pathogens. The SPRY domain- containing SOCS box proteins SPSB1 and SPSB2 are key physiological regulators of this important enzyme. Disrupting the endogenous SPSB-iNOS interaction should prolong the intracellular lifetime of iNOS and enhance the production of NO, and therefore be beneficial in treating chronic and persistent infections such as tuberculosis. By using structure-based design, potent peptide inhibitors of this interaction have been developed.

Conclusion: Inhibitors of the SPSB-iNOS interaction have therapeutic potential as a novel class of anti-infective agents. Various strategies are being pursued to target these peptide inhibitors to macrophages and deliver them to the cytoplasm of these cells. It will then be possible to assess the efficacy of such inhibitors in boosting the capacity of macrophages to destroy infectious pathogens.
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http://dx.doi.org/10.2174/0929866525666181101104945DOI Listing
February 2019