Degree Type

Dissertation

Date of Award

2019

Degree Name

Doctor of Philosophy

Department

Veterinary Microbiology and Preventive Medicine

Major

Biomedical Sciences

First Advisor

Richard J. Martin

Second Advisor

Alan P. Robertson

Abstract

Parasitic nematodes, particularly soil transmitted helminths (STHs), are among the most frequent causes of physical and intellectual growth retardation in humans. Furthermore, parasitic nematodes of livestock and crops result in substantial economic losses, threatening the overall sustenance of the expanding population. Although rarely lethal, infections typically result in chronic, disabling, and disfiguring morbidity adversely affecting the essential components of human development. Chemotherapy continues to serve as the mainstay for treatment and control of nematode infections as there are no vaccines for human use. The global strategy for the control involves regular administration of anthelminthic drugs to the high risk-populations. STHs mainly effect resource limited and poverty-stricken populations. This segment of the population can neither afford conventional treatment nor improve their infrastructure for better sanitation and hygiene facilities. Furthermore, there is limited research investment in the anthelmintics market due to high costs and modest profit levels, which has slowed down the anthelmintic drug discovery over the years. In addition, the widespread resistance to the limited armamentarium of anthelmintic drugs in the veterinary field has raised concerns in human medicine as well. It is prudent to restore the depleted drug-discovery pipeline before the existing antiparasitic drugs are rendered unviable. Thus, research should be directed towards identification and development of novel antinematodal targets and new therapeutics.

We have described a novel nicotinic acetylcholine receptor formed by a non-α subunit, EAT-2, from the pharynx of Caenorhabditis elegans, a model nematode. We hypothesize that activation of this receptor will lead to pharyngeal paralysis similar to the avermectins, which target glutamate-gated chloride (GluCl-) channels in pharyngeal tissue. For the first time we find that a non-alpha nicotinic subunit can form a homomeric ligand gated ion-channel when expressed in vitro. Even though EAT-2 is most closely related to vertebrate α-7 nAChRs, its pharmacological profile is distinct from previously characterized vertebrate channels and nematode somatic muscle nAChRs. The pharyngeal receptor was not activated by many of the cholinergic anthelmintics (levamisole, tribendimidine, pyrantel) and was insensitive to α-bungarotoxin and dihydro-β-erythroidine (DHβE). We also characterized the homologous receptor from Ascaris suum, a model for human parasitic nematode A. lumbricoides. We investigated the A. suum pharynx using electrophysiology and identified a significant cholinergic component that was pharmacologically similar to the A. suum EAT-2 receptor expressed in oocytes. This receptor was not sensitive to existing cholinomimetic anthelmintics and therefore represents a promising drug target. Importantly, the EAT-2 nAChR from both of the nematode species requires a novel auxiliary protein, EAT-18, for functional expression. EAT-18 directly interacts with the non-α subunits and modifies the pharmacological profile of the receptor. To the best of our knowledge this is only the 2nd auxiliary protein described for nAChRs and the first report of an auxiliary protein that is essential for functional expression in any cys-loop ligand-gated ion channel (LGIC). Both EAT-2 and EAT-18 proteins from A. suum were expressed in tissues other than the pharynx suggesting the receptor may serve multiple physiological functions. Thus, we have identified a non-canonical homomeric channel formed by non-alpha nAChR subunits as a potential novel drug target and a new type of obligate auxiliary protein for nAChRs.

Secondly, we have identified an ACR-16 nAChR subunit homologue from Ancylostoma caninum, a model for human hookworm infections. Aca-ACR-16 forms a functional homomeric channel when expressed in Xenopus laevis oocytes. A. suum ACR-16 nAChRs have been studied previously and reported to be a suitable target for the development of therapeutic drugs. In this study we explored the effects of several cholinergic agonists and antagonists on the expressed A. caninum ACR-16 nAChR using two-electrode voltage-clamp. The pharmacological profile of the Acn-ACR-16 receptor showed some similarities to the A. suum ACR-16 nAChRs. Both the homologues were unresponsive to many of the existing cholinomimetic anthelmintics (levamisole, pyrantel, morantel, bephenium and tribendimidine). Like Asu-ACR-16, the Aca-ACR-16 nAChR was also highly sensitive to mecamylamine and d-TC; moderately sensitive to derquantel and hexamethonium. In addition to similarities, the A. caninum ACR-16 nAChR showed marked differences in pharmacological sensitives from A. suum homologue which makes it scientifically interesting. As opposed to Asu-ACR-16, 3-Bromocytisine was the most potent agonist of Aca-ACR-16 while oxantel failed to activate the A. caninum nAChR. The mean time constants of agonists for desensitization rates were longer for Acn-ACR-16 (between 6.2 and 12.6s) in comparison to Asu-ACR-16 (between 1.5 and 4.8s). In contrast to Asu-ACR-16, the A. caninum receptor was completely inhibited by DhβE and moderately inhibited by α-BTX. In conclusion, we have successfully recapitulated a fully functional homomeric ACR-16 nAChR from A. caninum. The pharmacology of the receptor is distinct from other somatic muscle nematode receptors. The ACR-16 homologue from A. caninum also displayed some pharmacological differences from Asu-ACR-16. Therefore, we suggest that A. caninum ACR-16 may be a valid target site that should be further exploited for the development of agents against hookworm infection.

Lastly, we have shown that the monoterpenoid compounds, menthol and carvacrol, can be potentially used for antinematodal therapy in combination with existing cholinergic compounds. We examined the effects of twelve monoterpenoid phytocompounds on a heterologously expressed levamisole sensitive nAChR from Oesophagostomum dentatum and a nicotine sensitive nAChR from A. suum using the two-electrode voltage-clamp technique. The majority of these compounds acted as antagonists when tested at 100 M concentration. Carvacrol produced significant non-competitive inhibition suggesting the compound acts at site different from the ligand binding sites. This monoterpenoid phytocompound has also been shown to target GABA (gamma amino-butyric acid) and tyramine receptors in previous studies. This multifaceted polypharmacological effect of carvacrol might make it efficacious as an anthelmintic, alone or in combination, and help circumvent the development of resistance. Interestingly, menthol potentiated the amplitude of acetylcholine currents in our antagonist experiments. Further investigation revealed that both 0.1 M and 10 M menthol potentiated acetylcholine and levamisole mediated responses in the levamisole sensitive nAChR. We also investigated the effects of 0.1 M menthol on the contractility of A. suum somatic muscle strips. Menthol significantly potentiated the peak contractions at each concentration of acetylcholine tested. We have provided promising evidence of positive allosteric modulation by menthol in both in vivo and in vitro experiments. Thus, menthol could possibly be used in combination therapy with cholinomimetic drugs like pyrantel or levamisole to produce a more potent anthelmintic effect. In summary, menthol and carvacrol can contribute to antinematodal phytotherapy and alleviate the pressure on the limited classes of antiparasitic agents available.

Copyright Owner

Shivani Choudhary

Language

en

File Format

application/pdf

File Size

240 pages

Available for download on Tuesday, July 14, 2020

Included in

Pharmacology Commons

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