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 淋巴丝虫病是由丝虫线虫引起的蚊子传播疾病，包括 Brugia Malayi，B。Timori和Wuchereria Bancrofti，会影响贫困中数百万的人 撒哈拉以南非洲和东南亚的震惊地区。丝状战争位于 宿主淋巴系统，阻断淋巴引流，导致四肢肿胀 （象症）和皮肤感染。这些感染导致身体残疾和毁容， 精神压力和降低生产力，阻碍了内在的社会经济发展 地区。淋巴丝虫病的控制和治疗取决于大众药物管理 （MDA）的抗寄生虫药物，ivrmectin，二乙基碳氮嗪和阿苯达唑。这 策略在一定程度上取得了成功，但寄生感染持续存在，主要是 归因于当前抗抗物药物的成人成人效应有限。此外，没有 可用于使用的疫苗，以及人们对人类耐药性的担忧。有 公认的需要鉴定新的化学治疗靶标和大写疗法 对丝状感染的更有效控制。 在这里，我们建议研究和表征UNC-49γ-的药物特性 马来语中的氨基丁酸（GABA）通道有助于寻找新型药物靶标。 大多数市售的驱虫药物靶向Cys-Loop配体门控离子 通道（LGIC），包括最近引入的AirDepside（SLO钾通道）， 单臂（NACHRS）和DERQUANTEL（NACHRS）。但是，线虫离子通道是 未驱动的化学治疗目标已经探索了有限的。离子型 GABA通道是神经肌肉连接处的主要抑制性氯化通道 无脊椎动物，对于协调运动至关重要。但是，GABA接收器作为 仅一种抗寄生虫药物哌嗪的靶标。 UNC-49 GABA频道已经 来自秀丽隐杆线虫和Haemonchus contortus的特征。这些渠道是 与亚基组成，体内功能和 药理特性并显示基于物种的多样性。获得见解至关重要 进入线虫离子通道之间物种多样性的分子机制 更好地了解药物靶标。我们建议执行克隆和异源表达 先前未表征的UNC-49 GABA通道来自B. Malayi。我们将使用 在通道上进行药物表征的电生理方法 在异源系统中表达，爪蟾卵母细胞和HEK293细胞系。我们还将使用 Malayi B. Malayi蠕虫上的RNAi技术敲低编码基因的GABA亚基以验证 GABA作为药物靶标。这些实验产生的结果将有助于发展 有关丝状GABA通道的生物学和药理学的初步数据 线虫。完成该项目后，我们将扩大对 丝状UNC-49通道，并确定了UNC-49同工型对 内源性GABA功能。该项目有两个具体的目标： 目的1：研究UNC-49通道在成人B. Malayi中的身体功能的作用。 目标2：从马来语B. Malayi和功能表达的UNC-49亚基克隆到 特征渠道的药理学。