Activating autophagy in filarial worms to identify novel macrofilaricides

世界上超过1.2亿最贫穷的人面临着遭受寄生丝状线虫⎯Wuchereria bancrofti、Brgia Malayi和B.Timori⎯的极高风险，这些线虫生活在淋巴系统，并导致人类淋巴丝虫病(俗称象皮病)以及其他严重的病理疾病。例如，盘尾丝虫病(河盲症)是由盘尾丝虫引起的，盘尾丝虫驻留在皮下结节内，释放出微丝虫(Mf)，导致眼睛或皮肤的病理，包括失明。国际控制计划的目标是15亿有感染危险的人，重点是控制感染的传播。不幸的是，用于大规模给药活动(伊维菌素)的治疗只产生微丝杀灭作用--它们阻止传播，但对成虫没有效果。它们还需要长期每年给药，在与LOA LOA共同流行的地区是禁忌，免除儿童和孕妇，并促进丝虫线虫的抗药性。总而言之，这些局限性表明了开发新的抗丝虫病药物的迫切需要，特别是杀死成虫的大杀丝虫药物。最近的数据表明，利用丝虫互生细菌Wolbachia作为治疗人类丝虫感染的新的化疗靶点具有独特的潜力。临床研究表明，给予抗生素(多西环素)可杀死内共生菌，并引起大丝状杀菌作用。为了探索这种潜力，我们引入了一种新的方法：以宿主(寄生虫)为导向的治疗，诱导宿主的固有防御，迫使丝状线虫消除它们的内共生体。我们打算利用自噬，这是真核细胞的一种机制，以维持健康的细胞内环境，保护细胞免受细胞内入侵(例如细菌、病毒ES)的侵袭。这一过程对细菌和丝虫的共生至关重要，它的改变杀死了沃尔巴克氏菌，从而杀死了蠕虫本身。我们得到的证据是，氯硝柳胺(自噬诱导剂)激活了寄生虫的自噬，显著减少了细菌数量，并导致成虫的胚胎停滞效应。我们的项目将确定自噬诱导剂可能是候选的大丝状杀菌药物。在目的1中，为了证明自噬诱导剂对体内丝虫具有有效的杀灭作用，我们将评价氯硝柳胺对丝虫感染沙鼠的疗效。同时，我们将在体外测试其他23种改变用途的药物(已知的自噬诱导剂)对成虫的影响，并比较它们对沃尔巴克氏菌载量和成虫适合性的影响。目标1的体外试验将选出最好的药物(甚至比氯硝柳胺更好)。在目标2中，最好的2个候选者将在我们的体内Brugia/Jird感染模型中得到验证。我们的最终目标是找到干扰共生的有效的新型微丝杀菌剂。我们相信，这一倡议将产生一类新的活性化合物和重新调整用途的药物的开放前景，作为当前抗丝虫病治疗的替代品或与之结合使用。

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.