PROJECT SUMMARY/ABSTRACT Schistosome parasites infect 200 million people, resulting in significant morbidity and more than 200,000 deaths annually. Schistosomiasis control strategies rely almost exclusively on chemotherapy, and tens of millions of people are treated with the only available drug, praziquantel (PZQ). There are no new drugs in the clinical pipeline. With projected levels of PZQ use, it is inevitable that PZQ-resistant parasites will evolve. Therefore, it is imperative to find new drug targets and drugs for schistosomiasis treatment, our long-term objective. We identified a highly promising drug target: the worm selenocysteine-containing enzyme thioredoxin glutathione reductase (TGR). We established that TGR is a central and essential mediator of antioxidant defenses in the worm. The antioxidant defenses of vertebrates are diversified to three enzymes, glutathione reductase, thioredoxin reductase, and glutaredoxin, whereas schistosomes rely solely on TGR. TGR is a chokepoint and its inhibition leads to rapid worm death in all developmental stages. In contrast, PZQ has poor activity against juvenile worms, often resulting in partial cures. TGR is a defined molecular target, active as a recombinant protein, and we have established biochemical assays amenable to rapid compound screening, SAR, and optimization. We initiated several HTS of large compound libraries, which identified TGR inhibitors that have been used to obtain both liganded and ligand-free crystal structures of TGR, allowing a structure-based approach to hit optimization. These studies have elucidated an inhibitory mechanism that is completely novel for this family of proteins, allowing the development of non-covalent inhibitors. We hypothesize that it will be possible to optimize our novel TGR inhibitors for potency for TGR inhibition and selectivity against human glutathione reductase and thioredoxin reductase enzymes in vitro and schistosomicidal efficacy in vivo, our short-term objectives. Our aims are to optimize novel TGR inhibitors using cutting-edge structure- and ligand-based computer-aided design and medicinal chemistry to improve potency, selectivity, solubility, toxicity, and bioavailability. This will be complemented by X-ray crystallography and cryo-electron microscopy. Medicinal chemistry will be informed by enzymatic analysis of TGR and orthologous human enzymes, metabolic stability, in vitro cell toxicity, and activity against ex vivo worms. Finally, select compounds will be assessed for PK/PD properties and efficacy against schistosome infections in mice. To accomplish these aims, we assembled a team of experts in schistosome biochemistry and drug discovery, medicinal chemistry, computer-aided molecular design, chemical and structural biology of TGR. The varied and synergistic expertise of the team will facilitate overcoming critical barriers to development of schistosomicidal therapeutics. While additional preclinical studies would be needed, discovery o...