Development of small molecule inhibitors of metabolic enzymes as broad spectrum anthelmintic drugs

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Development of small molecule inhibitors of metabolic enzymes as broad spectrum anthelmintic drugs Abstract Parasitic intestinal nematodes including hookworms, roundworm and whipworms, infect over two billion people worldwide, causing significant morbidity, perpetuation of poverty, and loss of life. Characterization of nematode genomes provides fundamental molecular information essential for accelerating basic and translational research, which is a public health priority due to the limited number of currently available effective drugs and increasing drug resistance. In this proposal, we will pursue post-genomic drug discovery studies to develop small molecule drugs as novel therapeutics to treat infections caused by these devastating parasites. We have established an extensive omics/bioinformatics database for human nematode parasites spanning the major taxonomic clades of Nematoda. Using systems biology and evolutionary principles, we reconstructed metabolic networks for 56 diverse nematode parasites and identified chokepoint enzymes, i.e. metabolic enzymes that uniquely consume a specific substrate or generate a unique product. This led to our central hypothesis that compounds that inhibit conserved chokepoint enzymes have a strong potential for broad control of diverse nematodes. To test this, we identified conserved targets and initial inhibitors with potential for broad-spectrum activity, for which phenotypic screening of parasites at the extremes of the phylogeny have validated our predictions. Furthermore, we established a unique database of nematode-specific molecular features among the chokepoint enzyme targets and experimentally established that active-site differences in the nematode enzymes relative to their human orthologs can rationally guide the design of selective inhibitors. The compounds with the best activity in our phenotypic screens are inhibitors predicted to target three known enzyme classes (CPT, mTOR/PI3K, and PDE). To confirm the putative nematode target(s), we will express nematode proteins and implement biochemical enzyme inhibition assays, employ affinity-based labeling techniques, and test for activity against target knockdown worms (Aim 1). By leveraging parasite-specific active- site features of the confirmed protein targets, we will use a X-ray structure-based drug design (SBDD) to optimize lead inhibitors of the three identified target classes (Aim 2). Optimized lead compounds most effective against the human hookworm Ancylostoma ceylanicum and the whipworm Trichuris muris in vitro will be tested in vivo for their pan-intestinal efficacy in hamster and mouse animal models of nematode infection (Aim 3). Our preliminary results, combined with this proposed research, are highly significant since they provide a better understanding of metabolic functions essential for nematode survival, which can be targeted for drug discovery. The rational targeting of metabolic chokepoint enzymes as anthelminthic agents is innovative, as is the concept of utilizing specific pan-phylum conserved targets to develop anthelmintic drug or drugs with broad spectrum efficacy against nematodes. Collectively, this work has high potential to provide one or more new small molecule therapeutics with broad spectrum activity against parasitic nematode infections.