Alan Robertson - US grants

DESCRIPTION (provided by applicant): The Neglected Tropical Diseases (NTDs) include the soil-transmitted helminthiases (STHs) which are caused by diverse groups of intestinal nematodes. The parasites include Ascaris, Trichuris and hookworms. These infections are common. Ascariasis for example, affects 1.4 billion people worldwide and is most common in children between the ages of 3 and 8. Control of these nematode parasites relies on an effective supply of anthelmintics. Taking veterinary medicine as an example, we know that continued use of anthelmintic compounds for mass chemotherapy will lead to drug resistance. There is a need to identify novel target sites for anthelmintic development. Once such site is the nicotinic acetylcholine receptor on the nematode pharynx. We have discovered this receptor does not respond to currently used anrthelmintic drugs. Approach: The specific aims of this application are: 1. Characterize nAChR responses in A. suum pharynx: using current-, voltage- &patch- clamp techniques. We will test the hypothesis that the A. suum pharynx nAChRs are pharmacologically distinct from the muscle nAChRs activated by cholinergic anthelmintics. 2. Identify &clone pharyngeal specific nicotinic acetylcholine receptor subunits. We will identify the nAChR subunit genes present in the pharynx required to produce functional receptors. 3. Characterize the pharyngeal cholinergic receptors in Xenopus laevis oocytes using voltage- &patch-clamp techniques. We will test the hypothesis that expression of pharyngeal nAChR subunits results in functioning receptors that are pharmacologically similar to those found in vivo. On completion of this project we will have characterized the pharmacology of an important new potential drug target in a parasitic nematode. We will have identified the genes that encode these receptors. Finally, we will have reconstituted the receptors in an accessible platform suitable for screening potential new anthelmintics. The longer term goal of this research is provide ion- channel drug targets from a range of important parasite species in a platform (Xenopus oocytes) suitable for drug discovery screening. PUBLIC HEALTH RELEVANCE: The Neglected Tropical Diseases (NTDs) include soil transmitted nematode parasites like ascariasis, trichuriasis and hookworm. Ascariasis, for example, is one of the most common human parasitic infections. 1.5 billion people worldwide have ascariasis, and the disease is most common in children between the ages of 3 and 8. Treatment of helminthiasis includes use of nicotinic anthelmintics like pyrantel and oxantel which selectively paralyze nematodes by activating cholinergic ion-channels (nAChRs) on their muscle. However, resistance to anthelmintics drugs is a real concern. We have identified a novel potential target site for new anthelmintics, the nAChR on the nematode pharynx, which is not sensitive to currently used drugs. We propose to characterize the pharmacological properties of this receptor, clone the component subunit genes and express the functional receptor in Xenopus oocytes. On completion of the project we will have characterized a potential new drug target (the pharynx nAChR) and reconstituted it in a platform (Xenopus laevis oocytes) suitable for screening compound libraries to identify new anthelmintic compounds.

Objective: The objective of the program is to develop an integrated droplet-based microfluidic system for parallel screening of phenotypic changes in nematode worms within droplet microenvironments of varying chemical compositions. This work aims to provide a radical translation from existing low-throughput worm motility assays to a truly high-throughput, whole-organism assay for testing multi-drug compounds against nematodes.

Intellectual Merit: The intellectual merit is to provide a powerful lab-on-a-chip assay system for biological studies on whole-animal models with unprecedented high throughput. The system will uniquely combine automated generation and modulation of drug-coded pharmacological droplet libraries, guided movement stimulation, locomotion assay, and electrophysiological recording for single organisms inside droplets. This research is transformative because the system can provide unique details of neurophysiological changes in nematodes with drug exposures, facilitating experiments that are impossible by current techniques. The proposed technology is generic because the system can be adapted to test a wide range of important nematodes and drug compounds.

Broader Impacts: The proposed research will help answer fundamental questions in diagnosing, controlling and predicting drug resistance in nematode parasites, thereby unraveling complex mechanisms of host-parasite interactions. This research will generate broad educational opportunities for both undergraduate and graduate students, and benefit curriculum development for a new Undergraduate Bioengineering Minor Program of the Iowa State. Women and minority students, and middle school students will be attracted into science and engineering through open-lab tours, and in-class presentations. High school science teachers will be collaborated to develop K-12 instructional materials in the topics of micro/nanotechnology and bioengineering.

? DESCRIPTION (provided by applicant): The Neglected Tropical Diseases (NTDs) include the soil-transmitted helminthiases (STHs) which are caused by diverse groups of intestinal nematodes. The parasites include Ascaris, Trichuris and hookworms. These infections are common. Ascariasis for example, affects 1.4 billion people worldwide and is most common in children between the ages of 3 and 8. Control of these nematode parasites relies on an effective supply of anthelmintics. Taking veterinary medicine as an example, we know that continued use of anthelmintic compounds for mass chemotherapy will lead to drug resistance. There is a need to identify novel target sites for anthelmintic development. Once such site is the nicotinic acetylcholine receptor on the nematode pharynx. We have discovered this receptor does not respond to currently used anrthelmintic drugs. Approach: The specific aims of this application are: 1. Optimize expression of C. elegans eat-2 and eat 18 and pharmacologically characterize the resulting receptor. We will investigate optimal conditions for receptor expression and characterize the resulting receptor responses using a range of agonists, antagonists and potential allosteric modulators. 2. Clone and express A. suum eat-2 and eat-18. Are other subunits required for the pharyngeal receptor? We will identify orthologs of eat-2 and eat-18 from Ascaris suum, clone, and express them in Xenopus oocytes for characterization. Additionally we will test the hypothesis that other subunit genes are required to form the receptor. 3. Do eat-2 and eat 18 combine to form the mature receptor? We will test the novel hypothesis that eat-2 and eat-18 combine to form a functional nAChR. On completion of this project we will have expressed in a heterologous system an important new potential drug target from a parasitic nematode. We will have characterized an extremely novel nAChR (one that lacks any alpha subunits). We will have identified the genes that encode these receptors in the parasitic nematode Ascaris suum. Finally, we will have determined whether the small (71 a.a.) protein eat-18 is required in the mature ion channel receptor.