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

马来丝虫 GABA 门控氯离子通道的功能表征：抗丝虫治疗的未探索靶点

• 批准号: 10742453
• 负责人: Shivani Choudhary
• 金额: $ 21.67万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-21 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10742453
• 关键词: Adult; Affect; Africa South of the Sahara; Agonist; Albendazole; Anthelmintics; Antiparasitic Agents; Appearance; Biological; Biological Process; Biology; Brugia malayi; Caenorhabditis elegans; Cell Line; Characteristics; Child; Childhood; Chloride Channels; Chlorides; Chronic; Citrates; Cloning; Contralateral; Country; Culicidae; Data; Dedications; Development; Diethylcarbamazine; Drug Targeting; Drug resistance; Economic Development; Economics; Electrophysiology (science); Elephantiasis; Exhibits; Filaria bancrofti; Filarial Elephantiases; Filariasis; Future; GABA Receptor; GABA-A Receptor; Genes; Genital; Genitalia; Haemonchus; Human; Infection; Infectious Skin Diseases; Interruption; Invertebrates; Ion Channel; Ion Channel Gating; Ivermectin; Ligands; Limb structure; Locomotion; Lymphatic; Lymphatic System; Lymphedema; Mediating; Mediator; Medicine; Methods; Molecular; Movement; Muscle; Muscle Contraction; Muscle relaxation phase; Nematoda; Neuromuscular Junction; Neurotransmitters; Obstruction; Pain; Paralysed; Parasites; Parasitic infection; Parasitic nematode; Pathologic; Persons; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Phenotype; Physiological; Piperazines; Potassium Channel; Poverty; Productivity; Property; Protein Isoforms; Psyche structure; RNA Interference; RNA Splicing; Recommendation; Research; Role; Side; Southeastern Asia; Stress; Swelling; System; Techniques; Testing; Therapeutic; Variant; Xenopus oocyte; antagonist; behavioral study; chemotherapeutic agent; chemotherapy; chronic infection; disease transmission; drug resistance development; experimental study; gamma-Aminobutyric Acid; in vivo; insight; knock-down; lymphadenopathy; lymphatic drainage; lymphatic dysfunction; mosquito-borne; neglected tropical diseases; nerve supply; new therapeutic target; novel; pharmacologic; physically handicapped; psychologic; receptor; response; social; therapeutic target; transmission process; vaccine access

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.

项目摘要 淋巴丝虫病是一种由丝虫线虫引起的蚊媒传播疾病， 马来丝虫，B.帝汶和班氏丝虫病，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，阻碍了地方病患者的社会经济发展， 地区控制和治疗淋巴丝虫病要靠群众用药 (MDA)抗寄生虫药伊维菌素乙胺嗪和阿苯达唑。这 该策略在一定程度上取得了成功，但寄生虫感染仍然存在，主要是 这归因于目前抗丝虫剂的杀成虫作用有限。此外，没有 可供使用的疫苗，以及对人类产生耐药性的担忧。有 公认需要鉴定新的化疗靶点和杀巨丝虫治疗剂， 更有效地控制丝虫感染。 在这里，我们建议研究和表征UNC-49 γ-的药理学特性 氨基丁酸（GABA）通道在B。马来西亚将为寻找新的药物靶点作出贡献. 大多数市售的抗蠕虫药物靶向cys环配体门控离子 通道（LGIC），包括最近引入的emodepside（SLO钾通道）， 莫奈太尔（nAChRs）和德匡太尔（nAChRs）。然而，线虫离子通道是 然而，这些靶点尚未充分利用，已经探索了有限数量的化疗靶点。离子型 GABA通道是神经肌肉接头的主要抑制性氯门控通道 是无脊椎动物的重要组成部分，也是协调运动的关键。然而，GABA受体作为 只有一种抗寄生虫药哌嗪的作用靶点GABA-49通道已经被 以秀丽隐杆线虫和捻转血矛线虫为特征。这些渠道是 在亚基组成、体内功能和 药理学性质和显示基于物种的多样性。获得洞察力至关重要 研究线虫离子通道中物种多样性的分子机制， 更好地了解药物靶点。我们建议进行克隆和异源表达， 先前未表征的来自B的γ-49 GABA通道。马来语。我们将使用 电生理学方法对通道进行药理学表征 在异源系统、非洲爪蟾卵母细胞和HEK293细胞系中表达。我们还将使用 B上的RNAi技术。马来蠕虫敲低GABA亚基编码基因，以验证 GABA作为药物靶点。通过这些实验产生的结果将有助于开发 关于丝虫中GABA通道生物学和药理学的初步数据 线虫。在完成这个项目后，我们将扩大我们对 丝虫的α-49通道，并确定了α-49亚型的贡献， 内源性GABA功能该项目有两个具体目标： 目的1：研究β-49通道在成人B细胞生理功能中的作用。马来语。 目的2：从B中克隆β-49亚基。马来语和函数表达式 表征经络的药理作用。