Cholinergic receptors on the nematode pharynx: an unexploited drug target

描述(申请人提供)：被忽视的热带疾病(NTDS)包括土壤传播的蠕虫硫酶(STH)，它是由不同的肠道线虫群引起的。这些寄生虫包括蛔虫、鞭虫和钩虫。这些感染很常见。例如，蛔虫病影响着全世界14亿人，最常见的是3至8岁的儿童。这些线虫寄生虫的控制依赖于有效的驱虫剂供应。以兽医为例，我们知道继续使用驱虫药进行大规模化疗会导致耐药性。有必要确定驱虫药开发的新靶点。这样的位置曾经是线虫咽部的烟碱型乙酰胆碱受体。我们发现，这种受体对目前使用的抗虫药物不起作用。方法：本研究的具体目的是：1.利用电流、电压和膜片钳技术研究猪鼻咽肌细胞的nAChR反应。我们将检验这一假设，即猪咽曲霉的nAChRs与胆碱能驱虫药激活的肌肉nAChRs在药理上是不同的。2.鉴定和克隆咽特异性烟碱型乙酰胆碱受体亚基。我们将确定咽部存在的nAChR亚单位基因，这是产生功能性受体所必需的。3.用电压-膜片钳技术研究非洲爪哇卵母细胞咽胆碱能受体的特性。我们将验证这样一种假设，即咽部nAChR亚单位的表达导致功能受体的功能与体内发现的受体在药理上相似。在这个项目完成后，我们将描述寄生线虫中一种重要的潜在药物靶标的药理学特征。我们将识别出编码这些受体的基因。最后，我们将在一个可接近的平台上重组受体，适合于筛选潜在的新驱虫剂。这项研究的长期目标是在一个适合于药物发现筛选的平台(非洲爪哇卵母细胞)上提供一系列重要寄生虫物种的离子通道药物靶点。 公共卫生相关性：被忽视的热带疾病(NTDS)包括土壤传播的线虫寄生虫，如蛔虫病、鞭虫病和钩虫。例如，蛔虫病是最常见的人类寄生虫感染之一。全球有15亿人患有蛔虫病，这种疾病在3至8岁的儿童中最为常见。蠕虫病的治疗包括使用烟碱型驱虫剂，如吡虫酮和奥沙尼特，这些驱虫剂通过激活线虫肌肉上的胆碱能离子通道(NAChRs)来选择性地麻痹线虫。然而，对驱虫药的耐药性是一个真正令人担忧的问题。我们已经确定了一个新的潜在的新驱虫剂靶点，线虫咽部的nAChR，它对目前使用的药物不敏感。我们建议鉴定该受体的药理特性，克隆其组成亚单位基因，并在非洲爪哇卵母细胞中表达该功能性受体。项目完成后，我们将表征一个潜在的新药靶标(咽nAChR)，并在一个适合筛选化合物文库的平台(非洲爪哇卵母细胞)中重组它，以鉴定新的驱虫化合物。

DESCRIPTION (provided by applicant): The Neglected Tropical Diseases (NTDs) include the soil-transmitted helminthiases (STHs) which are caused by diverse groups of intestinal nematodes. The parasites include Ascaris, Trichuris and hookworms. These infections are common. Ascariasis for example, affects 1.4 billion people worldwide and is most common in children between the ages of 3 and 8. Control of these nematode parasites relies on an effective supply of anthelmintics. Taking veterinary medicine as an example, we know that continued use of anthelmintic compounds for mass chemotherapy will lead to drug resistance. There is a need to identify novel target sites for anthelmintic development. Once such site is the nicotinic acetylcholine receptor on the nematode pharynx. We have discovered this receptor does not respond to currently used anrthelmintic drugs. Approach: The specific aims of this application are: 1. Characterize nAChR responses in A. suum pharynx: using current-, voltage- & patch- clamp techniques. We will test the hypothesis that the A. suum pharynx nAChRs are pharmacologically distinct from the muscle nAChRs activated by cholinergic anthelmintics. 2. Identify & clone pharyngeal specific nicotinic acetylcholine receptor subunits. We will identify the nAChR subunit genes present in the pharynx required to produce functional receptors. 3. Characterize the pharyngeal cholinergic receptors in Xenopus laevis oocytes using voltage- & patch-clamp techniques. We will test the hypothesis that expression of pharyngeal nAChR subunits results in functioning receptors that are pharmacologically similar to those found in vivo. On completion of this project we will have characterized the pharmacology of an important new potential drug target in a parasitic nematode. We will have identified the genes that encode these receptors. Finally, we will have reconstituted the receptors in an accessible platform suitable for screening potential new anthelmintics. The longer term goal of this research is provide ion- channel drug targets from a range of important parasite species in a platform (Xenopus oocytes) suitable for drug discovery screening. PUBLIC HEALTH RELEVANCE: The Neglected Tropical Diseases (NTDs) include soil transmitted nematode parasites like ascariasis, trichuriasis and hookworm. Ascariasis, for example, is one of the most common human parasitic infections. 1.5 billion people worldwide have ascariasis, and the disease is most common in children between the ages of 3 and 8. Treatment of helminthiasis includes use of nicotinic anthelmintics like pyrantel and oxantel which selectively paralyze nematodes by activating cholinergic ion-channels (nAChRs) on their muscle. However, resistance to anthelmintics drugs is a real concern. We have identified a novel potential target site for new anthelmintics, the nAChR on the nematode pharynx, which is not sensitive to currently used drugs. We propose to characterize the pharmacological properties of this receptor, clone the component subunit genes and express the functional receptor in Xenopus oocytes. On completion of the project we will have characterized a potential new drug target (the pharynx nAChR) and reconstituted it in a platform (Xenopus laevis oocytes) suitable for screening compound libraries to identify new anthelmintic compounds.