Peta J Harvey

University of Queensland , Brisbane, Queensland, Australia

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Publications (8)41.01 Total impact

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    ABSTRACT: Cyclotides are plant peptides comprising a circular backbone and three conserved disulfide bonds that confer them with exceptional stability. They were originally discovered in Oldenlandia affinis based on their use in traditional African medicine to accelerate labor. Recently, cyclotides have been identified in numerous plant species of the coffee, violet, cucurbit, pea, potato, and grass families. Their unique structural topology, high stability, and tolerance to sequence variation make them promising templates for the development of peptide-based pharmaceuticals. However, the mechanisms underlying their biological activities remain largely unknown; specifically, a receptor for a native cyclotide has not been reported hitherto. Using bioactivity-guided fractionation of an herbal peptide extract known to indigenous healers as "kalata-kalata," the cyclotide kalata B7 was found to induce strong contractility on human uterine smooth muscle cells. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V1a receptors, members of the G protein-coupled receptor family, as molecular targets for this cyclotide. Furthermore, we show that cyclotides can serve as templates for the design of selective G protein-coupled receptor ligands by generating an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. These observations provide a proof of concept for the development of cyclotide-based peptide ligands.
    Proceedings of the National Academy of Sciences 11/2013; · 9.74 Impact Factor
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    ABSTRACT: The α3β4 nAChRs are implicated in pain sensation in the PNS and addiction to nicotine in the CNS. We identified an α-4/6-conotoxin (CTx) TxIC from Conus textile. The new toxin consists of 15 amino acid residues with two disulfide bonds. TxIC was synthesized using solid phase methods and the synthetic peptide was functionally tested on nAChRs heterologously expressed in Xenopus laevis oocytes. TxIC blocked rat α3β4 nAChRs with a 12.5 nM IC50, which places it amongst the most potent α3β4 nAChR antagonists. TxIC also blocked the closely related α6/α3β4 with a 94 nM IC50 but showed little activity on other nAChR subtypes. NMR analysis showed that two major structural isomers exist in solution, one of which adopts a regular α-CTx fold but with different surface charge distribution to other 4/6 family members. α-CTx TxIC is a novel tool with which to probe the structure and function of α3β4 nAChRs.
    Journal of Medicinal Chemistry 11/2013; · 5.61 Impact Factor
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    ABSTRACT: The cyclotides are a family of small head-to-tail cyclic plant defense proteins. In addition to their cyclic backbone, cyclotides comprise three disulfide bonds in a knotted arrangement, resulting in a highly cross-braced structure that provides exceptional chemical and proteolytic stability. A number of bioactivities have been associated with cyclotides, including insecticidal, antimicrobial, anti-viral and cytotoxic, and these activities are related to an ability to target and disrupt biological membranes. Kalata B2 and to a lesser extent kalata B1, isolated from Oldenlandia affinis, self-associate to tetramers and octamers in aqueous buffers, and this oligomerisation has been suggested to be relevant for their ability to form pores in membranes. Here we demonstrate by solution NMR spectroscopy analysis that the oligomerization of kalata B2 is concentration dependent and that it involves the packing of hydrophobic residues normally exposed on the surface of kalata B2 into a multimeric hydrophobic core. Interestingly, the hydrophobic surface that is "quenched" has previously been shown to be responsible for the ability of kalata B2 to insert into membranes. Thus it seems unlikely that the oligomers observed in aqueous solution are related to any multimeric state present in a membrane environment, and responsible for the formation of pores. The ability to self-associate might alternatively provide a mechanism for preventing self-toxicity when stored at high concentrations in intracellular compartments. © 2013 Wiley Periodicals, Inc. Biopolymers, 2013.
    Biopolymers 07/2013; · 2.88 Impact Factor
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    ABSTRACT: The inhibitor cystine knot (ICK) defines a large superfamily of polypeptides with high structural stability and functional diversity. Here, we describe a new scorpion venom-derived K+ channel toxin (named λ-MeuKTx-1) with an ICK fold through gene cloning, chemical synthesis, nuclear magnetic resonance spectroscopy, Ca2+ release measurements, and electrophysiological recordings. λ-MeuKTx-1 was found to adopt an ICK fold that contains a three-strand anti-parallel β-sheet and a 310-helix. Functionally, this peptide selectively inhibits the Drosophila Shaker K+ channel but is not capable of activating skeletal type Ca2+-release channels/ryanodine receptors, which is remarkably different from the previously known scorpion venom ICK peptides. The removal of two C-terminal residues of λ-MeuKTx-1 led to the loss of the inhibitory activity on the channel whereas the C-terminal amidation resulted in the emergence of activity on four mammalian K+ channels accompanied by the loss of activity on the Shaker channel. A combination of structural and pharmacological data allows recognition of three putative functional sites involved in channel blockade of λ-MeuKTx-1. The presence of a functional dyad in λ-MeuKTx-1 supports functional convergence among scorpion venom peptides with different folds. Furthermore, similarities in precursor organization, exon-intron structure, three-dimensional fold and function suggest that scorpion venom ICK-type K+ channel inhibitors and Ca2+-release channel activators share a common ancestor and their divergence occurs after speciation between buthidae and non-buthids. The structural and functional characterization of the first scorpion venom ICK toxin with K+ channel blocking activity sheds light on functionally divergent and convergent evolution of this conserved scaffold of ancient origin.
    Bioscience Reports 05/2013; · 1.88 Impact Factor
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    ABSTRACT: The θ-defensins are, to date, the only known ribosomally-synthesised cyclic peptides in mammals and they have promising antimicrobial bioactivities. The characteristic structural motif of the θ-defensins is the cyclic cystine ladder, comprising a cyclic peptide backbone and three parallel disulfide bonds. In contrast to the cyclic cystine knot, which characterises the plant cyclotides, the cyclic cystine ladder has not been as well described as a structural motif. Here we report the solution structures and NMR relaxation properties in aqueous solution of three representative θ-defensins from different species. Our data suggest that the θ-defensins are more rigid and structurally-defined than has been previously thought. In addition, all three θ-defensins were found to self-associate in aqueous solution in a concentration-dependent and reversible manner, a property that might have a role in their mechanism of action. The structural definition of the θ-defensins and the cyclic cystine ladder will help to guide exploitation of these molecules as structural frameworks for the design of peptide drugs.
    Biochemistry 11/2012; · 3.38 Impact Factor
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    Shunyi Zhu, Bin Gao, Peta J Harvey, David J Craik
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    ABSTRACT: Fungi are a newly emerging source of peptide antibiotics with therapeutic potential. Here, we report 17 new fungal defensin-like peptide (fDLP) genes and the detailed characterization of a corresponding synthetic fDLP (micasin) from a dermatophyte in terms of its structure, activity and therapeutic potential. NMR analysis showed that synthetic micasin adopts a "hallmark" cysteine-stabilized α-helical and β-sheet fold. It was active on both gram-positive and gram-negative bacteria, and importantly it killed two clinical isolates of methicillin-resistant Staphylococcus aureus and the opportunistic pathogen Pseudomonas aeruginosa at low micromolar concentrations. Micasin killed approximately 100% of treated bacteria within 3 h through a membrane nondisruptive mechanism of action, and showed extremely low hemolysis and high serum stability. Consistent with these functional properties, micasin increases survival in mice infected by the pathogenic bacteria in a peritonitis model. Our work represents a valuable approach to explore novel peptide antibiotics from a large resource of fungal genomes.
    Proceedings of the National Academy of Sciences 05/2012; 109(22):8495-500. · 9.74 Impact Factor
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    ABSTRACT: The peptides present in the venoms of marine snails are used by the snails to capture prey, but they have also attracted the interest of drug designers because of their potent activity against therapeutically important targets. These peptides are typically disulfiderich and target a wide range of ion channels, transporters and receptors with exquisite selectivity. In this article, we discuss structural and biological studies on several classes of conotoxins that have potential as drug leads for the treatment of pain. The chemical re-engineering of conotoxins via cyclization has been particularly valuable in improving their biopharmaceutical properties. An excellent example is the α-conotoxin Vc1.1, for which several cyclized analogs have been made. One of them was shown to be orally active in a rat pain model and this analog is currently undergoing pre-clinical development for the treatment of neuropathic pain. Several other α-conotoxins, including ImI, AuIB and MII, have proved amenable to cyclization and in all cases improvements in stability are obtained upon cyclization, suggesting that cyclization is a generally applicable approach to conotoxin stabilization. A variety of other chemical re-engineering approaches have also been used. Minor re-engineering of χ-conotoxin MrIa to convert its N-terminal residue to pyroglutamic acid proved particularly successful and the modified derivative, Xen2174, is currently in clinical trials for neuropathic pain.
    Current pharmaceutical design 12/2011; 17(38):4242-53. · 4.41 Impact Factor
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    ABSTRACT: Cyclotides are a family of plant defense proteins with a unique cyclic backbone and cystine knot. Their remarkable stability under harsh thermal, enzymatic, and chemical conditions, combined with their range of bioactivities, including anti-HIV activity, underpins their potential as protein drug scaffolds. The vast majority of cyclotides possess a conserved glutamate residue in loop 1 of the sequence that is involved in a structurally important network of hydrogen bonds to an adjacent loop (loop 3). A single native cyclotide sequence, kalata B12, has been discovered that has an aspartic acid in this otherwise conserved position. Previous studies have determined that methylation of the glutamate or substitution with alanine abolishes the membrane disrupting activity that is characteristic of the family. To further understand the role of this conserved structural feature, we studied the folding, structure, stability, and activity of the natural aspartic acid variant kalata B12 and compared it to the prototypical cyclotide kalata B1, along with its glutamate to alanine or aspartate mutants. We show that the overall fold of kalata B12 is similar to the structure of other cyclotides, confirming that the cyclotide framework is robust and tolerant to substitution, although the structure appears to be more flexible than other cyclotides. Modification of the glutamate in kalata B1 or replacing the aspartate in kalata B12 with a glutamate reduces the efficiency of oxidative folding relative to the native peptides. The bioactivity of all modified glutamate cyclotides is abolished, suggesting an important functional role of this conserved residue. Overall, this study shows that the presence of a glutamic acid in loop 1 of the cyclotides improves stability and is essential for the membrane disrupting activity of cyclotides.
    Biochemistry 04/2011; 50(19):4077-86. · 3.38 Impact Factor