RESEARCH GROUP: Diabetes Research Group

Project Title: Investigation of the insulinotropic peptides from the venom of chalcodes and Grammostola rosea tarnatulas.

Supervisor(s): Dr Yasser Abdel-Wahab, Dr Stephen McClean and Professor Victor Gault Contact Details: [email protected]

Level: PhD

Background to the project : In studying the venom of Theraphosidae we have uncovered a number of peptides that have potent in vitro activity promoting insulin release from the rodent insulin secreting cell line, BRIN-BD11. One of these, a 28-amino acid peptide from the venom of Aphonopelma chalcodes was purified by chromatography and structurally characterised using mass spectrometry and Edman degradation sequencing. The peptide was named Δ-theraphotoxin-Ac1 (Δ-TRTX-Ac1) according to existing nomenclature conventions.

Our studies have shown that a synthetic version of Δ-TRTX-Ac1 is a potent insulinotropic peptide without any cytotoxic effects on rodent beta cells. Mechanistic studies revealed that Δ-TRTX-Ac1 was able to activate PKC-dependent pathway. While initial experiments have provided promising data, this novel peptide must be comprehensively tested in established models to fully determine its possible utility for type 2 diabetes therapy.

In addition to Δ-TRTX-Ac1, a number of other insulinotropic peptides from Aphonopelma chalcodes and Grammostola rosea have been identified and require further investigation through full structural characterisation and functional activity assays.

Objectives of the research project : The objectives of the research will be to investigate the insulinotropic properties of peptides derived from the species Aphonopelma chalcodes and Grammostola rosea. There will be two aspects to the PhD project, the first being an investigation of the Δ-TRTX-Ac1 peptide in animal models of diabetes and obesity. The second aspect will be the structural characterisation of peptides and peptide fractions from Aphonopelma chalcodes and Grammostola rosea venoms.

In vitro and in vivo aims and objectives to include:

In vitro objectives

 Stability studies of the Δ-TRTX-Ac1 peptide in protease-rich environments; e.g. mouse plasma over a 12 hour period  Engineering of the peptide structure to provide greater stability and investigating new analogues in vitro and in vivo.  Purification and full characterisation of peptides from Aphonopelma chalcodes and Grammostola rosea already shown to have demonstrated insulin release.  Molecular characterisation of novel peptides determined by mass spectrometry and automated Edman degradation sequencing tools.  NMR studies on the folding of cysteine-knot structures present in theraphotoxin peptides and related families.  Evaluate the effect of venom peptides on cell viability by MTT assay  Investigate the dose and glucose dependent (in presence of 1.1, 11.1 and 16.7 mmol/L glucose) insulinotropic actions of venom peptides in clonal pancreatic BRIN BD11 cells  Investigate the dose and glucose dependent (in presence of 1.1, 11.1 and 16.7 mmol/L glucose) insulinotropic actions of venom peptides in clonal pancreatic human 1.1B4 cells  Investigate the dose and glucose dependent (in presence of 1.1, 11.1 and 16.7 mmol/L glucose) insulinotropic actions of venom peptides in isolated islets from lean mice  Examine the effect of venom peptides on intracellular Ca++ and membrane potential in clonal pancreatic BRIN BD11 cells and identify the mechanisms of action

In vivo objectives

 Investigate the acute glucose lowering and insulin releasing actions of venom peptides (5-100 nmol/kg bw) in lean and diabetic mice  Identify the persistent glucose lowering and insulin releasing actions of other venom peptides (5-100 nmol/kg bw) in diabetic mice

Methods to be used : Purification of venom peptides will be purified using LC MS technologies. Molecular characterisation will take place using MALDI-ToF mass spectrometry and MS/MS techniques for structural analysis. Sequencing of novel structures will take place using automated Edman degradation sequencing. BLAST searching will be used to determine structural homology with existing sequences and solid phase peptide synthesis used to produce peptide analogues. NMR analysis will be used to determine folding of peptides to form a cysteine knot structure.

Various assays will be used to assess the activity of peptides and will include:  Insulin secretion in BRIN BD11 cells  Effects of venom peptides on membrane potential and intracellular Ca2+  Insulin release studies in BRIN BD11 cells desensitised for PKA and PKC pathways  Determination of cell viability by MTT assay  The actions of native and modified peptides will be assessed in a variety of animal models of diabetes.

Skills required of applicant : The applicant should have a bioscience background and be willing to undertake training in the use of biochemical assays and mass spectrometry techniques. Animal handling will be an integral aspect of the study.

References :

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Furman BL. The development of Byetta (exenatide) from the venom of the Gila monster as an anti-diabetic agent. Toxicon. 2012 Mar 15;59(4):464-71.

Gault VA, Bhat VK, Irwin N, Flatt PR. A novel glucagon-like peptide-1 (GLP-1)/glucagon hybrid peptide with triple-acting agonist activity at glucose-dependent insulinotropic polypeptide, GLP-1, and glucagon receptors and therapeutic potential in high fat-fed mice. J Biol Chem. 2013 Dec 6;288(49):35581-91.

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Moore, SWM., Bhat, VK, Flatt, P, Gault, V and McClean, S (2016) Isolation and Characterisation of Insulin- Releasing Compounds from Pseudechis australis and Pseudechis butleri Venom. International Journal of Peptide Research and Therapeutics, 22 . pp. 211-218.

Moore, SWM, Bhat, VK, Flatt, P, Gault, V and McClean, S (2015) Isolation and characterisation of insulin- releasing compounds from Crotalus adamanteus, Crotalus vegrandis and Bitis nasicornis venom. Toxicon, 101 . 48 - 54.

Moore, S, Smyth, F, Gault, V, O'Kane, E and McClean, S (2009) Mass spectrometric characterisation and quantitation of selected low molecular mass compounds from the venom of Haplopelma lividum (Theraphosidae). Rapid Communications in Mass Spectrometry, 23 (12). pp. 1747-1755.

Marenah L, Flatt PR, Orr DF, Shaw C, Abdel-Wahab YH. Skin secretions of Rana saharica frogs reveal antimicrobial peptides esculentins-1 and -1B and brevinins-1E and -2EC with novel insulin releasing activity. J Endocrinol. 2006 Jan;188(1):1-9.