Naledi Pilusa

South Africa

Fragment screening on S.mansoni glyderaldehyde-3-phosphate dehydrogenase by x-ray crystallography towards the discovery of anti-schistosomal compounds

Naledi Pilusa1, Lizbé Koekemoer2, Julia Griese3; Abidemni Paul Kappo1

1Molecular Biophysics and Structural Biology Group, Department of Biochemistry, University of Johannesburg, Johannesburg, South Africa
2Centre for Medicines Discovery, University of Oxford, NDM Research Building, Oxford, Oxfordshire, UK
3 Department of Cell and Molecular Biology, Uppsala University, 751 24 Uppsala, Sweden

Abstract

Background

Schistosomiasis is a chronic parasitic disease of poverty that causes significant morbidity and mortality, accounting for 70 million disability-adjusted life years lost annually. Treatments solely rely on Praziquantel, but it’s limitations including to drug pressure and ineffectiveness against early-stage infections, signify the need for alternative treatment. Schistosoma mansoni Glyceraldehyde-3-phosphate Dehydrogenase (SmGAPDH) is characterized as a potential therapeutic target, shown to play a role in human host evasion and to be present in all stages of the infection, correlating with drug resistance. This study explored the structure of SmGAPDH using X-ray crystallography to identify novel binding matter and binding surfaces for targeted drug design and development.

Methods

Computational modelling was used to predict SmGAPDH’s NAD-binding domain structure and evaluated its antigenicity. For experimental validation, both the NAD-binding domain and the full length SmGAPDH were produced in Escherichia coli BL21(DE3) cells, purified using nickel-affinity and size-exclusion chromatography, and subjected to crystallization trials. X-ray diffraction data was collected, and fragment screening campaign was conducted.

Results

In silico modelling predicted SmGAPDH’s NAD-binding domain structure to have two α-helices and six β-sheets, and identified antigenic sites. Both the NAD-binding domain and full length SmGAPDH showed successful purification. Formed crystals under Morpheus and ShotGun1 screening conditions, that grow in 24 hours yielding high resolution X-ray diffraction data, 1.8 Å. A life soak test identified 12 binding pockets and 5 potential ligands.

Conclusions

This research project validates the characterization of SmGAPDH as a potential therapeutic target. Crystallographic structure confirmed computational predictions, offering enhanced resolution and clearly depicting density for NAD+, unobserved in the published structure. These findings form fundamental basis for developing inhibitors against SmGAPDH using fragment-based drug design.