Date of Award

1-1-2009

Degree Name

Doctor of Philosophy

Department

Pharmacology

First Advisor

Copello,Julio

Second Advisor

Evans,M. Steven

Abstract

In this investigation, venom from the giant Neotropical hunting ant Dinoponera australis (Order: Hymenoptera) has been harvested and subjected to chromatographic separations for the purpose of elucidating possible peptides that display neuroactivity by bioassay guided venom fractionation (BGVF). The venom of this arcane solitary predator paralyzes small invertebrate prey and causes highly exaggerated pain in large vertebrates. The hypothesis that the venom has a peptide component highly effective in modulating neuronal conduction by depolarization of cellular membranes has been tested and subsequent biochemical characterization has been performed to elucidate the primary structure. The data suggests that the modulation of neuronal conduction appears to result from the formation of a de novo pore that allows non-selective ion movements in a concentration dependent manner. The venom contains a variety of proteinaceous candidates and one particular peptide from the venom, -Dinoponeratoxin Da-1837, has been observed to cause very fast, large and sustained depolarization in two types of normally quiescent peripheral neurons (primary cultures of trigeminal and dorsal root ganglia) in whole cell patch clamp recordings. The profound depolarization is due to non-selective cationic flux which is irreversible at high concentrations. Preliminary studies suggest that the peptide also has a minor inhibitory effect on voltage-gated sodium channels, which does not contribute to the depolarization. Membrane assays with microsomes, fluorescent probes and lipid bilayers confirmed peptide-induced non-selective and concentration dependent permeabilization of the membrane. The primary structure of the peptide was determined by iterations of product ion scans in multiple configurations utilizing high resolution tandem mass spectrometry, commonly referred to as MS-MS data dependent acquisition. -DpTx Da-1837 is an eighteen residue peptide that is highly hydrophobic, positively charged at physiological pH and has one atypical post translational modification, i.e. C-terminal peptidyl-lysine. The authentication of the toxin was confirmed by the successful solid phase synthesis of an analog that showed neither biochemical nor physiological variation from the properties of the peptide isolated from Dinoponera australis. The conclusion of this study was the creation of derivative analogs that provide the platform for the first fundamental step in drug discovery: establishing the structure-function relationship. Although the purpose of these cytolytic peptides in venom may be to capture prey or discourage predation, the discoveries of new molecules that affect cell viability by interactions with the cellular envelope provide the genesis for studies of targeted cell death. As a novel anti-microbial agent or as a potent tumor suppressor, the development of peptide derivatives could also help direct the development of new therapeutic interventions.

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