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Description
Macromolecular X-ray crystallography is a powerful, sensitive technique that allows the identification of ligand-protein complexes, but it depends on crystals of high resolution and high tolerance to inorganic solvents. In this study, we investigated possible alternatives to the sole treatment of schistosomiasis, Praziquantel, by identifying small molecules that can interact with Schistosoma mansoni Glyceraldehyde-3-phosphate dehydrogenase (SmGAPDH). Identified in all life stages of parasitic worms causing schistosomiasis, a chronic parasitic disease of poverty that causes significant morbidity and mortality, accounting for 70 million disability-adjusted life years lost annually. SmGAPDH is characterised as a potential therapeutic target that plays a pivotal role in the parasite’s evasion of the human host and correlates with drug resistance. For this part of the study, full-length SmGAPDH was produced in Escherichia coli cells, purified to homogeneity using immobilised-nickel affinity chromatography and size-exclusion chromatography, and subjected to crystallisation trials. X-ray diffraction data were collected and used to analyse the electron density map, conduct structural analysis and functional studies, and run fragment screening. The full-length SmGAPDH was successfully purified and formed crystals within 24 hours under Morpheus and ShotGun1 screening, yielding high-resolution X-ray diffraction data at 1.8 Å and reproducible 2.5 Å data in the presence of DMSO. Evaluation of the electron density map shows a density for NAD+, unobserved in the previous studies. A life soak test identified 12 binding pockets and 5 potential ligands. These findings provide a fundamental structural basis for applying crystallographic screening to determine the functional aspects of SmGAPDH exploitable in fragment-based drug discovery and design, while prioritising the NAD-binding site.
