Identification and characterization novel of cystine-loop ligand- gated chloride channels from Dirofilaria immitis: pharmacological analysis and novel compound screening

Abstract

Dirofilaria immitis, otherwise known as heartworm, is a parasite that infects the hearts of dogs and causes serious health consequences. While preventative treatments are available drug resistance is developing at an alarming rate. Cystine-loop ligand-gated ion channels are important receptors in nematode neurobiology, and as such are promising drug targets. The UNC-49 (GABA-gated chloride channel) and ACC (acetylcholine-gated chloride channel) family of receptors have been characterized as potential drug targets in other nematodes. However, these receptors have yet to be identified or characterized in D. immitis. This thesis investigates the cloning and pharmacological characterization of 5 novel receptor subunits from D. immitis: UNC-49B and UNC-49C, ACC-1, LGC-46 and LGC-47. Additionally, novel derivatives of the antiparasitic drug levamisole were tested on ACC receptors to determine if any modifications enhanced levamisole action. D. immitis UNC-49B assembled as a homomeric channel and exhibited an EC50 of 5mM for GABA. UNC-49B also formed a functional channel with UNC-49C, which exhibited a decrease in GABA sensitivity. Additionally, the D. immitis UNC-49 receptors were significantly more sensitive to the open channel blocker picrotoxin compared to the same receptors from the sheep parasite Haemonchus contortus. Moreover, D. immitis UNC-49C, unlike other UNC-49C subunits, did not cause a decrease in picrotoxin sensitivity when assembled with UNC-49B. D. immitis ACC-1, LGC-46, and LGC-47 were unable to form functional channels through heterologous expression in Xenopus laevis oocytes. Levamisole derivatives were therefore tested on H. contortus ACC-2, which identified at least one showing a higher sensitivity compared to levamisole. Overall, this study characterized 2 novel UNC-49 receptors, identified 3 potential members of the ACC family in D. immitis and tested 8 novel derivatives of levamisole. This study lays foundation for the identification of more ligand-gated ion channels and will serve as a starting point for future researchers looking at new drug potential targets in D. immitis.