Degree Type

Dissertation

Date of Award

2017

Degree Name

Doctor of Philosophy

Department

Biomedical Sciences

Major

Toxicology

First Advisor

Richard J. Martin

Second Advisor

Alan P. Robertson

Abstract

Parasitic nematode infections remain a serious global public health threat to humans and animals. These infections cause debilitating conditions in humans and significant economic losses through infection of livestock and crop damage. Control of parasitic nematode diseases has continued to rely on use of anthelmintic drugs as there is presently no effective vaccine for the majority of these infections. Over the years, anthelmintic drug discovery has been very slow, and the majority of anthelmintic drugs used in human medicine today were initially developed for animal use as those affected live in the world’s poorest nations that lack the financial means to afford conventional drugs. However, resistance to all the major anthelmintic drug classes has been reported in numerous veterinary parasite species and there are increasing concerns of resistance development in human parasite species. The emergence of widespread resistance therefore underscores the urgent need for the search of new targets, leads and strategies for anthelmintic drug discovery and development.

We have identified the nicotinic acetylcholine receptor (nAChR) subunit ACR-16 from Ascaris suum and showed that ACR-16 forms a functional homopentameric nAChR when expressed in Xenopus oocytes. A. suum is a gastrointestinal (GI) tract parasitic nematode of pigs that is very closely related to the human equivalent A. lumbricoides. ACR-16 is not activated by cholinergic anthelmintics and, although ACR-16 is most closely related to vertebrate 7 receptors based on amino acid sequence alignment, it has some marked pharmacological differences from vertebrate 7 receptors. As opposed to vertebrate 7 receptors, ACR-16 was insensitive to -bungarotoxin and the effects of ivermectin, genistein and PNU120596 on ACR-16 were inhibitory rather than potentiating. The relative calcium permeability ratio for ACR-16 was about 50x lower than that for vertebrate 7 receptors. We have showed using reverse transcription polymerase chain reaction (RT-PCR) that mRNA for ACR-16 is widely distributed throughout Ascaris tissues; suggesting ACR-16 may have functions other than neurotransmission. Our results showed, for the first time, the characterization of the pharmacology of ACR-16 from a parasitic nematode. Based on the pharmacology and expression pattern of ACR-16 in different tissues of the parasite, we suggest ACR-16 is an attractive new anthelmintic drug target with ‘resistance-busting’ properties that should be further exploited for therapeutic drug development.

Secondly, we have showed that the plants Daniellia oliveri and Psorospermum febrifugum have potential as sources of lead compounds for the development of the much-needed filaricidal drugs to treat onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis). We prepared extracts of different polarities from D. oliveri and P. febrifugum and showed these extracts to be active against Onchocerca ochengi microfilariae and adults, and against adult Brugia pahangi based on visual motility scoring, MTT/formazan assay and the Worminator motility measurement system. Importantly, some extracts with O. ochengi microfilariae activity were also active on the adult worm. These extracts also showed activity against adult B. pahangi. We further fractionated the active extracts using Sep-Pak cartridges and High Performance Liquid Chromatography (HPLC) and showed some of these fractions retained activity against adult B. pahangi. We recommend that the active HPLC fractions be further purified to allow for the isolation of the bioactive compounds. This could significantly contribute to chemotherapeutic control of filarial infections currently hampered by the lack of macrofilaricides (adulticides).

Lastly, we have demonstrated that use of combination therapy over single drug therapy is a potentially useful tool for increasing efficacy, spectrum of action and reducing the likelihood of resistance development. We showed the combination of derquantel and abamectin to produce a greater inhibition of acetylcholine and pyrantel responses of expressed pyrantel/tribendimidine nAChRs from Oesophagostomum dentatum than derquantel or abamectin used alone. These nAChRs comprise of UNC-29, UNC-63 and UNC-38 subunits. Our results also showed abamectin acts on nAChRs, in addition to its known effects on glutamate-gated chloride channels (GluCls), implying abamectin has multiple targets. We further showed the action of abamectin on the expressed O. dentatum pyrantel/tribendimidine nAChRs to be bi-phasic, suggesting two allosteric sites of action: a high affinity negative allosteric modulation (NAM) site causing antagonism at lower concentrations (≤0.1 µM) and a lower affinity positive allosteric modulation (PAM) site causing a reduction in the antagonism at higher concentrations (0.3 µM).

Copyright Owner

Melanie Abongwa

Language

en

File Format

application/pdf

File Size

329 pages

Included in

Toxicology Commons

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