A functional characterization of Brugia malayi GABA-gated chloride channels: an unexplored target for antifilarial therapeutics

项目总结 淋巴丝虫病是一种由丝虫引起的蚊子传播疾病，包括 马来丝虫、铁莫里丝虫和班克罗夫提丝虫影响着数百万贫困人口-- 撒哈拉以南非洲和东南亚的受灾地区。丝虫位于 宿主淋巴系统，阻塞淋巴引流，导致四肢严重肿胀 (象皮病)和皮肤感染。这些感染会导致身体残疾和毁容， 精神压力和生产力下降，阻碍了地方病的社会经济发展 地区。防治淋巴丝虫病有赖于群众性用药 抗寄生虫药伊维菌素、乙胺嘧啶和阿苯达唑的丙二醛。这 策略在一定程度上取得了成功，但寄生虫感染仍在持续，主要是 归因于目前的抗丝虫剂的成虫杀灭作用有限。再说了，没有 可供使用的疫苗，以及对人类产生抗药性的担忧。的确有 公认的需要确定新的化疗靶点和大丝虫杀灭疗法 更有效地控制丝虫病感染。 在此，我们建议对unc-49γ的药理性质进行研究和表征。 马来丝虫体内的氨基丁酸(GABA)通道有助于寻找新的药物靶点。 大多数市售的驱虫药都是以环配体门控离子为靶标的。 通道(LGIC)，包括最近推出的表情苷(SLO钾通道)， Monepantel(NAChRs)和derquantel(NAChRs)。然而，线虫离子通道是一种 未充分开发的化疗靶点，已探索的数量有限。电离性 GABA通道是神经肌肉接头的主要抑制性氯离子门控通道 属于无脊椎动物，是协调运动所必需的。然而，GABA受体作为 只有一种抗寄生虫药的目标，哌嗪。UNC-49 GABA通道已被 其特征是秀丽线虫和缠绕血圆线虫。这些频道是 与脊椎动物受体在亚基组成、体内功能和 药理特性和基于物种的多样性。获得洞察力是至关重要的。 探讨线虫离子通道间物种多样性的分子机制 更好地了解药物靶标。我们建议进行克隆和异源表达 马来芽胞杆菌的UNC-49 GABA通道是以前未知的。我们将使用 对经络进行药理学表征的电生理学方法 在异种系统、非洲爪哇卵母细胞和HEK293细胞系中表达。我们还将使用 用RNAi技术敲除马来丝虫GABA亚单位编码基因验证 GABA作为药物靶点。通过这些实验产生的结果将有助于开发 丝虫体内GABA通道的生物学和药理学研究初报 线虫。在完成这个项目后，我们将扩大对 并确定了UNC-49亚型对丝虫的贡献 内源性GABA功能。该项目有两个具体目标： 目的1：研究UNC-49通道在马来丝虫成虫体内的生理功能。 目的2：马来丝虫UNC-49亚基的克隆及功能表达 描述这些通道的药理作用。

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.