Project summary Lymphatic filariasis is a mosquito-transmitted disease caused by filarial nematodes, including Brugia malayi, B. timori, and Wuchereria bancrofti, that affect millions of people in the poverty- stricken regions of Sub-Saharan Africa and Southeast Asia. The filarial worms are located in the host lymphatic system, blocking lymphatic drainage, resulting in gross swelling of the limbs (elephantiasis) and skin infections. These infections result in physical disability and disfigurement, mental stress, and reduced productivity, hampering social-economic development in endemic regions. Control and treatment of lymphatic filariasis rely on the mass drug administration (MDA) of antiparasitic drugs, ivermectin, diethylcarbamazine, and albendazole. This strategy has been successful to a certain extent, but the parasitic infections persist, mainly attributed to the limited adulticidal effect of current anti-filarial agents. Besides, there are no vaccines available for use, and concerns about developing drug resistance in humans. There is an accepted need to identify novel chemotherapeutic targets and macrofilaricidal therapeutics for more efficient control of filarial infections. Here we propose to investigate and characterize the pharmacological properties of UNC-49 γ- aminobutyric acid (GABA) channels in B. malayi to contribute to the search for novel drug targets. The majority of the commercially available anthelmintic drugs target cys-loop ligand-gated ion channels (LGICs), including the recently introduced emodepside (SLO potassium channels), monepantel (nAChRs), and derquantel (nAChRs). However, nematode ion channels are an underexploited chemotherapeutic target with a limited number already explored. Ionotropic GABA channels are the major inhibitory chloride-gated channels at the neuromuscular junction of invertebrates and are essential for coordinated movement. And yet, GABA receptors serve as the target of only one antiparasitic drug, piperazine. UNC-49 GABA channels have been characterized from Caenorhabditis elegans and Haemonchus contortus. These channels were distinct from the vertebrate receptors in subunit composition, in vivo function, and pharmacological properties and displayed species-based diversity. It is essential to gain insights into the molecular mechanisms of species-based diversity among nematode ion channels to understand drug targets better. We propose to perform cloning and heterologous expression of the previously uncharacterized UNC-49 GABA channels from B. malayi. We will use electrophysiological methods to conduct pharmacological characterization on the channels expressed in a heterologous system, Xenopus oocytes, and HEK293 cell lines. We will also use RNAi techniques on B. malayi worms to knock down GABA subunit encoding genes to validate GABA as a drug target. The results generated through these experiments will help develop preliminary data concerning the biology and pharmacology of GABA channels in the filarial nematode. Upon completing this project, we will have broadened our understanding of the filarial UNC-49 channels and identified the contribution of UNC-49 isoforms to the endogenous GABA function. The project has two specific aims: Aim 1: To study the role of UNC-49 channels' physiological function in adult B. malayi. Aim 2: Cloning of UNC-49 subunits from B. malayi and functional expression to characterize the channels' pharmacology.

Project Narrative Filarial nematodes, Brugia malayi, B. timori, and Wuchereria bancrofti, cause lymphatic filariasis that affects millions of people in the tropical regions of the world. The chronic infections result in characteristic painful swelling of the limbs (elephantiasis) and later lymphedema of the genitals. The associated irreversible physical disability and profound disfiguring cause substantial economic, social, and psychological damage. Mass drug treatment with anthelmintic medicines is recommended to interrupt the transmission cycle of filarial infection. Chemotherapy effectively clears microfilariae from the host and suppresses their reappearance, but they have limited adulticidal activity. Inhibitory GABA-gated chloride channels present at the neuromuscular junction of nematodes are a vital mediator of locomotion. They serve as a target for only one nematocidal compound, piperazine, and have not been explored in filarial nematodes. unc-49 encodes for a GABA receptor in nematodes that mediates contralateral body muscle inhibition during locomotion. UNC-49 GABA channel has been characterized from Caenorhabditis elegans and Haemonchus contortus. These receptors are pharmacologically distinct from vertebrate channels and display species-based diversity. We will explore the biological significance and potential of Bma-UNC-49 channels as a novel drug target. This project will characterize the filarial UNC-49 receptors providing a better understanding of its pharmacology and the molecular basis of the activity of selective GABA channels. This proposal will have implications for identifying the molecular basis of the activity of GABA channel activators, exploring selective drugs, and contributing to the development of novel macrofilaricidal compounds in the future.