Natural products as antifilarial asparaginyl-tRNA synthetase inhibitors

描述（由申请人提供）：淋巴丝虫病是由马来西亚布鲁氏菌和班氏乌切里亚寄生虫引起的，是世界卫生组织（WHO）“十大被忽视的热带病”之一，全世界有超过2亿人感染，超过10亿人处于危险之中。目前可用的药物不足以预防疾病，也不能杀死成虫。Kron实验室已经证明，世卫组织批准的一种新的分子靶标——丝虫天冬酰胺- 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重作用。目前使用的药物不针对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多年的少数抗丝虫药物所遇到的耐药性问题。我们过去的大量研究表明，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来鉴定产生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微发酵液（来自36,760株在两种培养基中培养的菌株），并鉴定出20种产生AsnRS抑制剂的放线菌菌株，并且(3)从前三种菌株中分离出三种天然产物，代表三种结构不同

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.