Trehalose-6-phosphate phosphatase inhibitors as anti-helminthics

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PROJECT SUMMARY/ABSTRACT Parasitic nematodes are responsible for numerous chronically incapacitating and deforming diseases in Africa, Asia, and the Americas. Among these diseases is lymphatic filariasis, which is a mosquito-transmitted disease, endemic to 81 countries. It is estimated that 120 million people are infected with this disease. Enzymes that are essential to the parasitic nematodes but that do not have a human homologue are potential drug targets for therapeutic intervention. The availability of the genome from B. malayi, the representative organism for filarial nematodes, has enabled the ranking of potential drug targets from this parasitic organism. One such enzyme is trehalose-6-phosphate phosphatase (T6PP), which is required for the biosynthesis of trehalose. The oblation of T6PP activity in the C. elegans model commonly used for parasitic nematodes ultimately leads to organism death, probably due to the accumulation of trehalose 6-phosphate (T6P). In our collaborative research project we have solved the structure of B. malayi T6PP, determined those residues critical to T6P binding, developed substrate analogs, established a high-throughput assay for inhibitor screening and utilized that screen to find two nanomolar-affinity lead inhibitors. One of the lead compounds discovered by screening, Closantel, shows in vivo activity against B. malayi, and notably against adult worms which current treatments fail to achieve. The proposed objective is to develop advanced leads based on the compounds already identified via screening to enhance potency and availability and to determine the mechanism of T6P toxicity. The research plan is focused on four Aims. Aim 1 (R21) will deliver 1) structure-activity relationship analysis on principle hits Closantel and Cephalosporin C via synthesis and testing in vitro on B. malayi T6PP and in vivo on C. elegans 2) in vitro stability tests in rat plasma and liver S9 fractions 3) X-ray crystal structures of B. malayi T6PP complexed with inhibitors, 4) tests of principle hits and variants in a SAXS-based screen as additional insight into SAR and mechanism of action. Aim 2 (R21) will 1) provide counter-screening against phosphatases from the same and other phosphatase families to ensure selectivity, 2) develop a panel of orthologous T6PP enzymes from other parasitic nematodes for inhibition kinetics (and possibly crystallography) to provide the basis for broad- spectrum anti-helminthics. Aim 3 (R33) will 1) test for inhibitory activity in vivo in B. malayi 2) perform metabolism, preliminary pharmacokinetic and cytotoxicity studies 3) determine the mechanism of the anti- metabolite activity of T6P by quantifying levels of T6P accompanying toxicity and assessing the effect of T6P on primary metabolic enzymes. Aim 4 (R33) will test the advanced leads in a B. malayi gerbil infection model. The proposed research will deliver advanced leads that show selective killing action for the treatment of disease(s) inflicting the large segment of the world’s population suffering from infection by parasitic nematodes.