Pharmacological Diversity of nAChRs in Clade III Nematodes: Levamisole receptors

描述(申请人提供)：1.被忽视的热带疾病包括蛔虫病和淋巴丝虫病。这些疾病是由寄生线虫引起的，这些线虫由于分子上的相似性而被归入Clade III。蛔虫病是由大肠蛔虫引起的。在世界范围内，它发生在14亿人中。淋巴丝虫病(象皮病)威胁着83个国家的10多亿人；它是由马来丝虫等丝虫引起的。这些线虫寄生虫(成虫或幼虫)的控制依赖于有限数量的驱虫药。有人担心，大规模化疗将导致耐药性的发展。迫切需要进行研究，以便更好地利用现有药物，包括联合用药，以保持其价值，并开发新药。2.近年来，几种新型的线虫选择性胆碱能驱虫药相继问世，如三苯二胺和地奎酮(现与阿维菌素复配)，增加了胆碱能驱虫药的意义。三苯二胺具有单剂量大剂量给药的潜力。Dequantel对对其他胆碱能驱虫药具有抗药性的蠕虫具有‘破坏’作用。线虫尼古丁受体(NAChRs)的药理比人们所了解的要复杂得多。在猪蛔虫第三支线虫中，肌肉nAChRs分为N亚型、L亚型和B亚型。Clade III肌肉受体(左旋咪唑受体)的药理作用受受体亚单位组成的调节。在寄生线虫中，亚型和调控的治疗重要性需要进一步研究。我们有三个目标。3.我们的方法将使用肌肉收缩试验、电流钳、电压钳、膜片钳、克隆和非洲爪哇卵母细胞表达和药物制剂来表征胆碱能驱虫药的作用及其受体的亚型。目的1研究三苯二胺在猪链霉菌中的作用方式和亚型选择性。它将检验这样的假设，即三苯二胺对L亚型的nAChR没有选择性，而对猪链霉菌的另一种类型具有选择性，并确定其作为开放通道阻滞剂的作用是否限制了疗效。目的#2将研究亚基排列和化学计量学对蛔虫nAChRs药理的影响。它将以不同的组合表达来自猪链霉菌的四个特定的尼古丁受体亚基，以检验不同的亚基组成复制天然肌肉受体(左旋咪唑受体)的药理多样性的假设。目的#3描述马来丝虫的nAChR反应和受体，以验证其亚型在功能上与猪分支线虫相似的假设。4.该提案具有创新性，探索了未知的药物效应以及亚基排列对Clade III nAChRs药理特性的影响。我们还将这组研究人员开发的方法扩展到马来丝虫寄生虫的制备。5.总体影响将对以下方面产生强大的影响：扩大和知情使用三苯二胺，这是一种潜在的单一剂量丙二醛；扩大和知情地使用第三级线虫的尼古丁驱虫靶点的药理多样性；使用一种新型的马来杆菌制剂来研究未来药物开发的离子通道靶点；开发或知情地使用组合(例如，德喹与阿维菌素；或亚型选择性胆碱能驱虫药的组合)。

DESCRIPTION (provided by applicant): 1. The Neglected Tropical Diseases include ascariasis and lymphatic filariasis. These diseases are caused by parasitic nematodes that are grouped in Clade III because of their molecular similarities. Ascariasis is caused by the large intestinal roundworm, Ascaris lumbricoides. Worldwide, it occurs in 1.4 billion people. Lymphatic filariasis (elephantiasis) threatens over a billion people in 83 countries; it is caused by filaria nematodes like Brugia malayi. Control of these nematode parasites (adults or larvae) relies on a limited number of anthelmintic drugs. There are concerns that mass chemotherapy will lead to the development of resistance. There is a significant and urgent need for research that will allow better use of existing drugs, including combinations, to preserve their value, and to develop new drugs. 2. Recently, several novel nematode selective cholinergic anthelmintics, like tribendimidine and derquantel (now synergized with abamectin), have been introduced and increased the significance of cholinergic anthelmintics. Tribendimidine has potential for single-dose Mass Drug Administration. Derquantel has 'resistance busting' actions against worms that are resistant to other cholinergic anthelmintics. The pharmacology of nematode nicotinic receptors (nAChRs) is more complex than has been appreciated. The muscle nAChRs are divided into N-, L- and B-subtypes in the Clade III nematode Ascaris suum. The pharmacology of Clade III muscle receptors (levamisole receptors) are modulated by receptor subunit composition. The therapeutic importance of subtypes and modulation demands further investigation in parasitic nematodes. We have three aims. 3. Our approach will use muscle contraction assays, current-clamp, voltage-clamp, patch- clamp, cloning & Xenopus oocyte expression and pharmacological agents, to characterize actions of cholinergic anthelmintics and the subtypes of their receptors. Aim #1 will characterize the mode of action and subtype selectivity of tribendimidine in A. suum. It will test the hypothesis that tribendimidine is not selective for the L-subtype of nAChR but another type in A. suum and determine if its action as an open-channel blocker limits efficacy. Aim #2 will examine the effects of subunit arrangements and stoichiometry on pharmacology of Ascaris nAChRs. It will express four specific nicotinic receptor subunits from A. suum in different combinations to test the hypothesis that different subunit compositions reproduce the pharmacological diversity of native muscle receptors (levamisole receptors). Aim #3 will characterize nAChR responses and receptors in B. malayi, to test the hypothesis that the subtypes are functionally similar to the Clade III nematode, A. suum. 4. The proposal is innovative, exploring unknown drug effects and effects of subunit arrangements on the pharmacological properties of Clade III nAChRs. We also extend, to the Brugia malayi parasite preparation, the methods that this group of investigators has developed. 5. The overall impact will be a powerful influence on: expansion & informed use of tribendimidine, a potential single-dose MDA; the expansion and informed use of the pharmacological diversity of nicotinic anthelmintic target sites in Clade III nematodes; use of a novel B. malayi preparation for studying ion-channel targets for future drug development; development or informed use of combinations (e.g. derquantel with avermectins; or combinations of subtype selective cholinergic anthelmintics).