Natural products as antifilarial asparaginyl-tRNA synthetase inhibitors

描述(申请人提供)：淋巴丝虫病，由寄生虫马来丝虫和班克罗夫氏伍氏原虫引起，是世界卫生组织(WHO)“十大被忽视的热带病”之一，感染全球2亿多人，使10亿多人面临风险。目前可用的药物不足以预防疾病，不能杀死成虫。克伦实验室已经证明，世界卫生组织批准的一个新的分子靶标，丝虫天冬酰胺-tRNA合成酶(AsnRS)，在寄生虫生活史的所有阶段都有表达，并对寄生虫的生存起着至关重要的多种作用。目前使用的药物并不针对AsnRs，因此丝虫AsnRs抑制剂将构成一类新的药物，绕过目前在动物和人类中使用了40多年的少数几种抗丝虫病药物遇到的耐药性问题。我们过去的大量研究表明，AsnRs在这两种丝虫中是相同的，重组AsnRs(RBmAsnRS)具有足够的化学稳定性，可以用于HTS。我们已经(1)验证了一种使用rBmAsnRS来鉴定产生未知AsnRS抑制剂的放线菌的创新方法；(2)应用这种新的生物测定指导的筛选算法严格地检测了~73,420个微发酵液(从两种介质中各培养的36,760个菌株中)，鉴定了20个产生AsnRS抑制剂的主要放线菌；(3)从前三个菌株中分离出三类天然产物，代表三种结构不同的 支架，作为迄今未知的BmAsnRS特异性抑制剂，在低纳摩尔浓度下杀死成虫丝虫，但通常对人类细胞没有细胞毒性。在这些寄生性AsnRS特异性抑制剂中没有观察到普遍的细胞毒性，这支持了我们新的非放射性AsnRS抑制试验的实用性，其中包括监测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舞台，让我们去问(1)我们可以从高度预选的20个先导菌株中识别出多少结构独特的天然产品支架，以及(2)哪些新的天然产品支架将代表作为新型抗寄生虫药物开发的最高优先事项。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：鉴定和鉴定20株先导菌株中的所有AsnRs抑制剂。目标2：收集数据，优先选择新的AsnRs抑制剂作为抗丝虫病药物。我们预计，这项研究的结果将是第一批抗寄生虫AsnRS抑制剂的集合。这个集合中化合物的优先顺序可以快速进行，因为我们有快速药代动力学测试的集体设施，以及现有的体外和体内模型来确定抗丝虫病的效果。有希望的先导化合物将作为新的人类疗法提交世卫组织审议。天然产物铅的微生物来源以及它们在放大发酵中的可用性，应该会极大地促进后续机制和临床前研究，以推动最有希望的先导化合物作为抗寄生虫药物进入临床试验。

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.