Biotechnological applications of spider venom peptides

Er, Sing Yan (2017). Biotechnological applications of spider venom peptides PhD Thesis, Institute for Molecular Bioscience, The University of Queensland. doi:10.14264/uql.2017.457

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Author Er, Sing Yan
Thesis Title Biotechnological applications of spider venom peptides
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
DOI 10.14264/uql.2017.457
Publication date 2017-03-27
Thesis type PhD Thesis
Supervisor Glenn King
Lachlan Rash
Maggie Hardy
Total pages 155
Total colour pages 18
Total black and white pages 127
Language eng
Subjects 030403 Characterisation of Biological Macromolecules
111501 Basic Pharmacology
060110 Receptors and Membrane Biology
Formatted abstract
Spider venoms are a rich source of bioactive compounds with potential biotechnological applications. The overall aim of this project was to isolate and characterise novel spider-venom peptides (SVPs) with potential in two key areas: (1) insect control using orally active insecticidal peptides; (2) improvement of human health with peptide modulators that target acid-sensing ion channels (ASICs).

Insect pests are a worldwide problem as they threaten crop yields and spread a multitude of insect-borne diseases. Unfortunately, the armament of commercially available chemical insecticides is rapidly diminishing due to the emergence of insecticide-resistant insect strains as well as deregistrations and use restrictions by regulatory authorities due to environmental and health concerns. This has created an unmet need for new eco-friendly insecticides. Spiders are highly efficient insect predators that utilise venom to rapidly incapacitate prey. As such, many SVPs have potent insecticidal properties. However, as SVPs are injected by the spider, they are generally perceived to lack sufficient oral efficacy to be of practical agrochemical use. Recently, several orally-active insecticidal peptides (OAIP 1–5) were isolated from the venom of the Australian tarantula, Selenotypus plumipes, and shown to be lethal when fed to the termites. Chapter 2 describes the characterisation and structure determination of one of these peptides, OAIP3. The peptide was produced using an efficient E. coli periplasmic expression system that promotes disulfide bond formation in vivo. The insecticidal activity of the recombinant peptide was tested in mealworms, while the structure of the peptide was solved using heteronuclear nuclear magnetic resonance spectroscopy. Structural studies indicated that OAIP3 contains a canonical inhibitor cystine knot motif commonly found in spider venom peptides. In comparison to its native form, recombinant OAIP3 is markedly less potent in mealworms, with ~53% moribund at a dose of 7.6 nmol/g. Sequence and structural homologies with other spider venom peptides indicated that OAIP3 is likely to target insect voltage-gated sodium channels.

Spider venoms are a rich source of peptides that target therapeutically-relevant ion channels such as ASICs. Chapter 3 describes the isolation and characterisation of a novel ASIC1a modulator, Hm3a, isolated from spider venom. ASICs are proton-gated ion channels that respond to acidosis. Since their initial discovery more than a decade ago, they have been implicated in many neurodegenerative diseases, neuronal damage resulting form ischemia, and pain. Given that spider venoms are a rich source of ion channel modulators, we screened a panel of 30 spider venoms for inhibitory activity on ASIC1a. Through an assay-guided fractionation using reverse-phase high performance liquid chromatography, we isolated a peptide modulator of ASIC1a from the venom of the tarantula Heteroscodra maculata. Recombinant peptide was produced and characterised using two-electrode voltage-clamp electrophysiology studies of ASICs expressed in Xenopus laevis oocytes. The peptide, named π-TRTX-Hm3a (Hm3a), is highly selective for ASIC1a over other ASIC subtypes. Hm3a is a close homolog of PcTx1, sharing similar electrophysiological effects on ASICs, but it proved to be more resistant to high temperatures as well as proteolytic degradation by human serum and cerebrospinal fluid. An engineered variant of Hm3a resulted in improved potency and efficacy on ASIC1a.

Since the population of ASIC1a that is therapeutically relevant is located in the brain, an efficient method of administration is needed to deliver the peptide across the blood-brain barrier. Invasive methods are often required to deliver drugs into the brain. An alternative approach is intranasal administration that bypasses the blood-brain barrier and allows direct delivery to the brain via the olfactory bulb. Thus, we hypothesised that intranasal administration may also be an effective method for delivery of ASIC1a modulators into the CNS. As described in Chapter 4, several engineered variants of the prototypical ASIC1a modulator, PcTx1, were produced recombinantly or chemical synthesised. These peptides were then conjugated to moieties for PET, MRI and optical imaging in vivo. The addition of various imaging moieties at the N-terminal of PcTx1 had minimal effect on its activity on ASICs. A pharmacokinetics and biodistribution study of PcTx1 was performed using in vivo PET/CT imaging and traditional ex vivo tissue sampling after intravenous and intranasal administration. We show that brain delivery of PcTx1 in mice can be achieved through intranasal but not intravenous administration.

In summary, the work presented in this thesis demonstrates that spider venoms provide a diverse library of peptides with potential biotechnological applications. In addition, we show that unconventional intranasal delivery is a plausible route for delivering peptide drugs to target sites in the CNS.
Keyword Spider venom peptide
Orally active insecticidal peptide
Acid sensing ion channel
Intranasal administration
Biodistribution
Pharmacokinetics
Blood-brain barrier
Electrophysiology
Two-microelectrode voltage clamp
PcTx1
Hm3a
Additional Notes 8, 9, 11, 14, 15, 16, 22, 25, 26, 38, 39, 41, 42, 54, 55, 64, 66, 90, 91, 95, 97, 98, 100, 101, 103,105, 110, 111

Document type: Thesis
Collections: UQ Theses (RHD) - Official
UQ Theses (RHD) - Open Access
 
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Created: Fri, 17 Mar 2017, 13:30:12 EST by Sing Yan Er on behalf of Learning and Research Services (UQ Library)