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.
Aminoacyl-tRNA synthetases (AARSs) are validated but underutilized molecular targets for drug discovery against parasites. Moreover, the extent to which natural products have been screened for anti-AARS activity is limited. Accordingly, this project utilizes several important new discoveries and technological advances to overcome scientific barriers and thus encompasses a novel strategy to identify lead AARS inhibitors as antiparasitic (antifilarial) agents through the systematic structural and functional characterizatin of selected actinomycete-derived natural products.