Activating autophagy in filarial worms to identify novel macrofilaricides

世界上超过1.2亿最贫穷的人有很高的风险遭受寄生丝虫病的衰弱效应，寄生丝虫病- - - bancrofti乌切里氏菌，马来布鲁吉亚菌和B. timori - - -生活在淋巴系统中，导致人类淋巴丝虫病，通常被称为象皮病，以及其他严重的疾病。盘尾丝虫病（河盲症）就是其中之一，它是由盘尾丝虫病引起的，盘尾丝虫病存在于皮下结节内，并释放导致眼部或皮肤病变（包括失明）的微丝虫病（Mf）。针对15亿有感染危险人群的国际控制规划侧重于控制感染的传播。不幸的是，用于大规模给药计划的治疗方法（伊维菌素）只产生微丝杀灭作用——它们阻断传播，但对成年寄生虫没有作用。它们还需要长期每年给药，在与罗阿罗阿共流行的地区禁用，对儿童和孕妇豁免，并促进丝虫线虫的耐药性。总之，这些限制表明，迫切需要开发新的抗丝虫药，特别是杀死成虫的大丝虫药。最近的数据表明，使用沃尔巴克氏菌（Wolbachia）作为一种新的化疗靶点来对抗人类丝虫病感染具有独特的潜力。临床研究表明，抗生素（强力霉素）的施用杀死内共生菌和引起大丝虫病的作用。为了探索这一潜力，我们引入了一种新的方法：以宿主（寄生虫）为导向的治疗，诱导宿主先天防御并迫使丝状线虫消除其内共生体。我们打算利用自噬这一真核细胞的机制来维持健康的细胞内环境，并保护细胞免受细胞内入侵者（如细菌、病毒）的侵害。这一过程对于细菌-丝状体的共生是必不可少的，它的改变杀死了沃尔巴克氏体，从而杀死了蠕虫本身。我们获得了证据，氯硝柳胺（自噬诱导剂）激活寄生虫的自噬，显著减少细菌数量，并在成虫中引起胚胎抑制作用。我们的项目将确定自噬诱导剂可以作为候选的巨丝杀灭药物。在Aim 1中，为了证明自噬诱导剂在体内对丝虫寄生虫有效，可以作为大丝虫杀灭药物，我们将评价氯硝柳胺对布鲁吉亚感染禽类的疗效。同时，我们将在体外测试其他23种重组药物（已知的自噬诱导剂）对成年布鲁吉亚寄生虫的影响，并比较它们对沃尔巴克氏体负荷和成虫适应性的影响。Aim 1中的体外试验将导致选择最佳靶点（甚至比氯硝柳胺更好）。在Aim 2中，我们将在体内Brugia/jird感染模型中验证最佳的2个候选药物。我们的最终目标是鉴定出干扰共生关系的有效的新型微丝杀菌剂。我们相信，这一举措将产生一类新的活性化合物，并为重新利用药物开辟前景，作为替代或与目前的抗丝虫病治疗联合使用。

More than 120 million of poorest in the world are at high risk of suffering the debilitating effects of the parasitic filarial nematodes⎯ Wuchereria bancrofti, Brugia malayi, and B. timori ⎯ that live in the lymphatic system and cause human lymphatic filariasis, commonly known as elephantiasis, as well as other severe pathologies. Onchocerciasis (river blindness), for one, is caused by Onchocerca volvulus, which resides within subcutaneous nodules and releases the microfilariae (Mf) that are responsible for ocular or dermal pathologies, including blindness. International control programs, which target 1.5 billion people in danger of infection, focus on controlling the transmission of infections. Unfortunately, the treatments used for mass drug administration initiatives (ivermectin) produce only microfilaricidal effects – they block transmission but have no effect on the adult parasites. They also require long-term yearly administration, are contraindicated in areas co-endemic with Loa loa, exempt children and pregnant women, and promote drug resistance in filarial nematodes. Together, these limitations point to a crucial need for the development of new anti-filarial drugs, specifically macrofilaricidal drugs that kill adult worms. Recent data demonstrate the unique potential of using Wolbachia, the mutualistic bacteria of filarial nematodes, as a novel chemotherapeutic target against human filarial infections. Clinical studies showed that the administration of antibiotics (doxycycline) kills endosymbionts and causes macrofilaricidal effects. To explore this potential, we introduce a novel approach: a host(parasite)- oriented treatment that induces a host innate defense and forces filarial nematodes to eliminate their endosymbiont. We intend to take advantage of autophagy, a mechanism of eukaryotic cells to maintain a healthy intracellular environment and protect cells against intracellular invaders (e.g., bacteria, virus es). The process is essential for bacteria-filaria symbiosis and its alteration killed Wolbachia and, consequently, the worm itself. We obtained proof, that niclosamide (autophagy inducer) activates autophagy in parasites, significantly decreases number of bacteria and causes embryostatic effect in adult worms. Our project will determine that the autophagy inducers can be candidate macrofilaricidal drugs. In Aim 1, to prove that autophagy inducers are effective in vivo against filarial parasites and can be used as macrofilaricidal drugs, we will evaluate the efficacy of niclosamide in Brugia infected jirds. In parallel, we will test the other 23 repurposed drugs (known autophagy inducers) on adult Brugia parasites in vitro and compare their effects on the Wolbachia load and adult worm fitness. The in vitro assays in Aim 1 will result in the selection of the best hits (even better than niclosamide). In Aim 2, the best 2 candidates will be validated in our in vivo Brugia/jird model of infection. Our end goal is to identify effective new microfilaricides that interfere with symbiosis. This initiative, we believe, will yield a new class of active compounds and open perspectives for repurposed drugs, for use as alternatives to or in combination with current anti-filarial treatments.