Natural products as antifilarial asparaginyl-tRNA synthetase inhibitors

描述（由申请人提供）：淋巴丝虫病由寄生虫马来丝虫和班氏吴策线虫引起，是世界卫生组织（WHO）的“十大被忽视的热带疾病”之一，感染了全球2亿多人，并使10亿多人处于危险之中。目前可用的药物不足以预防疾病，也不能杀死蠕虫。Kron实验室已经证明，世卫组织批准的一种新的分子靶点--丝虫天冬酰胺酰-tRNA合成酶（AsnRS）在寄生虫生命周期的所有阶段都有表达，并对寄生虫的生存能力起着至关重要的多种作用。目前使用的药物不靶向AsnRS，因此丝虫AsnRS抑制剂将构成一类新的药物，绕过目前已在动物和人中使用超过40年的少数抗丝虫药物所遇到的耐药性问题。我们过去广泛的研究表明，AsnRS在两种丝虫中是相同的，重组AsnRS（rBmAsnRS）的化学稳定性足以用于HTS。我们已经（1）验证了一种使用rBmAsnRS鉴定产生先前未知AsnRS抑制剂的放线菌菌株的创新方法，（2）应用这种新的生物测定指导筛选算法严格检查了约73，420个微发酵液（从36，760株分别在两种培养基中培养的菌株中）并鉴定出20株产生AsnRS抑制剂的先导放线菌菌株，以及（3）从前三株菌株中，分离三类天然产物，代表三种结构上不同的 支架，作为迄今未知的BmAsnRS特异性抑制剂，其在低纳摩尔浓度下杀死成虫，但通常对人细胞没有细胞毒性。在这些寄生虫AsnRS特异性抑制剂中观察到的一般细胞毒性的缺乏支持了我们的新的非放射性AsnRS抑制测定的效用，该测定包括用于监测ATP结合酶的非特异性抑制剂的内部对照。这些发现现在为我们提供了一个突出的阶段，以询问（1）我们可以从高度预选的20种主要菌株中识别出多少结构独特的天然产物支架，以及（2）哪些新的天然产物支架将代表开发新型抗寄生虫药物的最高优先级。我们建议通过以下两个具体目标来解决这些知识差距。目的1：鉴定和结构表征20种主要菌株中的所有AsnRS抑制剂。目的2：收集数据，优先考虑新的AsnRS抑制剂作为抗丝虫药物。我们预计，这项研究的结果将是第一个收集的抗寄生虫AsnRS抑制剂。由于我们的集体设施用于快速药代动力学测试，并且现有的体外和体内模型用于确定抗丝虫功效，因此可以快速进行该集合中的化合物的优先级排序。将向世卫组织提交有前景的先导化合物，供其考虑作为新的人类治疗药物。天然产物铅的微生物来源以及由此通过放大发酵获得的它们的可用性，应极大地促进将最有希望的铅作为抗寄生虫药物推进临床试验所需的后续机制和临床前研究。

DESCRIPTION (provided by applicant): Lymphatic filariasis, caused by the parasites Brugia malayi and Wuchereria bancrofti, is one of the World Health Organization's (WHO) "Top Ten Neglected Tropical Diseases," infects more than 200 million people worldwide and places more than one billion people at risk. Currently available medicines are inadequate for disease prevention and cannot kill adult worms. The Kron lab has shown that a new molecular target approved by WHO, the filarial asparaginyl-tRNA synthetase (AsnRS), is expressed in all stages of the parasite life cycles and plays multiple roles essential to parasite viability. Currently use medicines do not target AsnRS, and thus filarial AsnRS inhibitors would constitute a new class of medicines bypassing drug resistance issues encountered with the few current antifilarial medicines that have been used for more than 40 years in both animals and man. Our extensive past studies indicate that AsnRS is identical in the two filarial species and recombinant AsnRS (rBmAsnRS) is chemically stable enough for use in HTS. We have (1) validated an innovative method using rBmAsnRS to identify actinomycete strains producing previously unknown AsnRS inhibitors, (2) applied this new bioassay-guided screening algorithm to rigorously examine ~73,420 microfermentation broths (from 36,760 strains cultivated in two media each) and identified 20 lead actinomycete strains that produce AsnRS inhibitors, and (3) from the first three strains, isolated three classes of natural products, representing three structurally distinct scaffolds, as heretofore unknown BmAsnRS specific inhibitors that kill adult filaria at low nanomolar concentrations yet are not generally cytotoxic to human cells. The absence of general cytotoxicity observed in these parasitic AsnRS specific inhibitors supports the utility of our new non-radioactive AsnRS inhibition assay which includes an internal control to monitor for nonspecific inhibitors of ATP binding enzymes. These findings have now set an outstanding stage for us to ask (1) how many structurally unique natural product scaffolds we can identify from the highly preselected 20 lead strains and (2) which new natural product scaffolds will represent the highest priority for development as novel anti-parasitic drugs. We propose to address these knowledge gaps with the following two specific aims. Aim 1: Identify and structurally characterize all AsnRS inhibitors in the 20 lead strains. Aim 2: Collect data to prioritize new AsnRS inhibitors as anti-filarial drugs. We anticipate that the outcome of this research will be the first collection of anti-parasite AsnRS inhibitors. Prioritization of compound in this collection can proceed rapidly because of our collective facilities for rapid pharmacokinetic testing, and existing in vitro and in vivo models to determine anti-filarial efficacy. Promising lead compounds will be presented to WHO for consideration as new human therapeutics. The microbial origin of the natural product leads and thereby their availability by scale-up fermentation, should greatly facilitate the follow-up mechanistic and preclinical studies needed to advance the most promising leads into clinical trials as anti-parasite drugs.