Slo-1K channels, TRP-2 channels, emodepside and diethylcarbamazine in Filaria

项目摘要 丝虫病是一组被忽视的热带疾病，由分支III的微丝蚴感染引起 通过叮咬昆虫传播的寄生线虫。淋巴丝虫病就是一个例子 马来丝虫（Brugia malayi）。淋巴丝虫病是一种使人衰弱和毁容的疾病， 全世界1.2亿人。其他丝虫病是由盘尾丝虫引起的河盲症 由Loa loa引起的肠扭转和肠梗阻。这些线虫寄生虫病的防治 由于没有有效的疫苗，因此依赖于驱虫药物的使用。预防使用 大规模药物管理局（MDA）计划受到现有驱虫药疗效的限制。 乙胺嗪是世界大部分地区治疗淋巴丝虫病和丝虫病的主要药物。 在世界范围内，除了存在盘尾丝虫病的地区，因为它有失明的危险。 乙胺嗪可快速清除微丝蚴，并导致约40%的成虫死亡 （macrofilaricide）。一些研究表明乙胺嗪有一个间接宿主- 介导的作用模式，乙胺嗪通过改变宿主花生四烯酸途径发挥作用。 我们观察到乙胺嗪对丝虫有直接作用。我们提出 初步观察表明，乙胺嗪增加TRP-2通道的开放， 马来丝虫和钙激活的SLO-1 K通道的开放。效果是迅速的，短暂的 运动抑制后恢复：反应适应。 苯缩酚酸是一种新出现的重要的环八缩酚肽类驱虫药， 对微丝蚴和成虫的影响。与现有的治疗方法相比， 大规模药物管理（MDA）计划，需要定期治疗以杀死成虫寄生虫。一 的网站的作用emodepside是对线虫SLO-1钾通道，其中开放的渠道 抑制运动性，但对所有丝虫都无效。在这里，我们建议比较对丝虫SLO的影响- 1 K通道，并检查这两个作用和相互作用 研究药物，探索其作用方式。 我们有三个目标： 目的#1：在体外表征 乙胺嗪和依莫地普酯及其组合对：A）微丝蚴; B）布氏丝虫 成年雌性; C）布鲁日成年雄性。 我们将检验乙胺嗪的作用 和依莫得甙是相加的、协同的或拮抗的，并且依赖于生命周期阶段和性别。 目的#2表征SLO-1 K通道对乙胺嗪的电流响应， 膜片钳技术观察依莫地平对马来丝虫肌瓣的保护作用 我们将测试假设： a）依莫地昔和乙胺嗪的作用相互作用; B）依莫地昔和乙胺嗪的相互作用 乙胺嗪和依莫地普苷通过敲低TRP-2依赖于TRP-2的存在 信道;以及c）TRP-2和SLO-1信道消息和信道开放在 长期暴露于乙胺嗪或依莫地昔。 目的#3：表征以下的比较分子药理学：a）不同的SLO-1 K通道 B）马来丝虫的TRP-2通道和 Loa loa在卵母细胞中表达。 我们将测试的假设，药理学和效力的 依莫地甙和乙胺嗪对布氏丝虫、盘尾丝虫SLO-1 K通道和TRP-2通道的影响 和Loa是不同的，也不同于人类通道同源物。我们将检查通道 脱敏。 该提案具有创新性，使用多种技术组合来测试乙胺嗪的效果 和emodepside的推定的靶位点，SLO-1的K通道的丝虫。使用的总体影响 我们的混合技术，将是发现和比较乙胺嗪的影响， emodepside对不同种类的丝虫TRP-2通道和SLO-1 K通道的作用。知识 这些药物的分子作用需要：a）不同丝虫敏感性的分子检测 物种和耐药性; B）设计新药和联合疗法; c）预测和 了解不同线虫寄生虫物种的敏感性;以及d）预测可能的宿主毒性。

Project Summary Filariases are a group of neglected tropical diseases produced by infection with microfilaria of Clade III parasitic nematodes that are transmitted by biting insects. One example is the lymphatic filariasis produced by Brugia malayi. Lymphatic filariasis is a debilitating and disfiguring disease which occurs in 120 million people worldwide. Other filarial diseases are River Blindness produced by Onchocerca volvulus and loiasis produced by Loa loa. Prevention and treatment of these nematode parasite diseases relies on the use of anthelmintic drugs because no effective vaccines are available. Prophylaxis using Mass Drug Administration [MDA] programs are limited by the efficacy of existing anthelmintics. Diethylcarbamazine is a mainstay for the treatment of lymphatic filariasis and loiasis in most parts of the world, except in areas where onchocerciasis is present because it is contra-indicated by risks of blindness. Diethylcarbamazine produces rapid clearance of microfilaria and causes ~40% mortality of adult parasites (macrofilaricide). A number of studies have suggested that diethylcarbamazine has an indirect host- mediated mode of action and that diethylcarbamazine acts by changing host arachidonic acid pathways. We have observed that diethylcarbamazine has direct effects on filarial nematodes. We present preliminary observations that show that diethylcarbamazine increases the opening of TRP-2 channels in Brugia malayi, and opening of calcium-activated SLO-1 K channels. The effect is a rapid, transient inhibition of motility followed by recovery: the response accommodates. Emodepside is an emerging and important cyclooctadepsipeptide class of anthelmintic that also has effects on microfilaria and adult filaria. Emodepside treatments could allow a major advance over existing mass drug administration (MDA) programs which require regular treatments to kill adult parasites. One of the sites of action of emodepside is on nematode SLO-1 K channels where opening of the channels inhibits motility, but it is not effective against all filaria. Here we propose to compare effects on filarial SLO- 1 K channels from Brugia, Onchocerca and Loa and to examine actions and interactions of these two drugs to explore their mode of action. We have 3 aims: Aim #1: Characterize, in vitro, the concentration motility-inhibition-response relationships of diethylcarbamazine and emodepside and their combination on: A) Brugia microfilaria; B) Brugia adult females; C) Brugia adult males. We will test the hypothesis that effects of diethylcarbamazine and emodepside are additive, synergistic or antagonistic and dependent of life-cycle stage and sex. Aim #2 Characterize the SLO-1 K channel current responses to diethylcarbamazine and emodepside in isolated Brugia malayi muscle flaps under patch-clamp We will test the hypotheses: a) that the effects of emodepside and diethylcarbamazine interact; b) that the interactions of diethylcarbamazine and emodepside are dependent on the presence of TRP-2 by knockdown of TRP-2 channels; and c) that TRP-2 and SLO-1 channel message & channel opening accommodates during prolonged exposure to diethylcarbamazine or emodepside. Aim #3: Characterize the comparative molecular pharmacology of: a) different SLO-1 K channels of Brugia malayi, Onchocerca volvulus and Loa loa and; b) TRP-2 channels of Brugia malayi and Loa loa expressed in oocytes. We will test the hypothesis that the pharmacology and potencies of emodepside and diethylcarbamazine on SLO-1 K channels and TRP-2 channels of, Brugia, Onchocerca and Loa are different and also different to a human channel homologue. We will examine channel desensitization. The proposal is innovative, using a combination of techniques to test the effects of diethylcarbamazine and emodepside on their putative target sites, SLO-1 K channels of filarial. The overall impact of using our mixture of techniques, will be discovery and comparison of effects of diethylcarbamazine and emodepside on different species of filarial TRP-2 channels and SLO-1 K channels. Knowledge of the molecular actions of these drugs is required for: a) molecular detection of sensitivity of different filarial species and resistance; b) designing new drugs and combination therapies; c) predicting and understanding sensitivities of different nematode parasite species; and d) predicting possible host toxicity.