Isolation, structural characterisation and mode of action of bioactive agents from arachnid and reptile venoms

Abstract

Selected snake and spider venom fractions were screened for in-vitro insulinotropic activity in glucose responsive BRIN-BD11 cells, with insulin secretion measured using radioimmunoassay. Significant insulin secretion was noted for 27 snake venom fractions, containing a diverse range of snake toxin families including phospholipases A2, α-neurotoxins, disintegrins, serine proteinases, CRISP (cysteine-rich secretory proteins), metalloproteinases and nucleotidases. The partial N-terminal sequences are reported for 15 snake venom components. Elevated levels of insulin secretion were recorded for 16 fractions from the Grammostola rosea venom and 31 fractions from the Aphonopelma chalcodes venom. The synthetic version of a novel 28 amino acid residue peptide isolated from the Aphonopelma chalcodes produced a significant concentration dependent increase in insulin secretion. A number of theraphotoxins are proposed as constituents of the active Aphonopelma chalcodes fractions. Partial sequences are presented for 3 unknown Grammostola rosea peptides with insulinotropic activity reported as a novel function of a number of known Grammostola peptides.

A microtitre assay was used to assess antimicrobial activity of snake and spider venom against both Gram-positive and Gram-negative bacteria. Snake venom fractions containing an L-amino acid oxidase or metalloproteinase component showed preferential activity against Staphylococcus aureus, while phospholipases A2 were most active against Bacillus subtilis. Activity against Salmonella typhimurium was greatest for fractions containing L- amino acid oxidase. Escherichia coli was least susceptible to the test fractions. Antimicrobial activity for the Aphonopelma chalcodes crude venom was confined to fractions 26 to 35, containing low mass compounds of m/z 730-830 alongside a peptide component of m/z 2919, for which the novel sequence is reported.

A quantitative study was performed on the selected low molecular mass components isolated in the Haplopelma lividum spider venom using liquid chromatography / electrospray ionisation mass spectrometry. This study of the bioactive constituents of snake and spider venom serves to enhance the existing body of evidence supporting the study of venomics in pursuit of novel leads for pharmaceutical research and development.

Thesis is embargoed until October 2016

Date of Award	Oct 2013
Original language	English
Sponsors	Nicobrand Ltd, Coleraine funded a PhD studentship for Sara W. M. Moore
Supervisor	Stephen McClean (Supervisor), Victor Gault (Supervisor) & Franklin Smyth (Supervisor)