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April 1st, 2025
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University of Helsinki Faculty of Biological and Environmental Sciences
biochemistry
biorxiv

Expression, purification and preliminary pharmacological characterization of the Plasmodium falciparum membrane-bound pyrophosphatase type 1

Liu, J.Open in Google Scholar•Vidilaseris, K.Open in Google Scholar•Johansson, N. G.Open in Google Scholar•Ribeiro, O.Open in Google Scholar•Dreano, L.Open in Google Scholar•Yli-Kauhaluoma, J.Open in Google Scholar•Xhaard, H.Open in Google Scholar•Goldman, A.Open in Google Scholar

Membrane-bound pyrophosphatases are integral membrane proteins that catalyze the hydrolysis of pyrophosphate into orthophosphate, while simultaneously facilitating the pumping of protons and/or sodium ions. Since mPPases are absent in humans but play a critical role in the life cycle of protist parasite, they represent promising therapeutic targets. We successfully expressed the Plasmodium falciparum type 1 mPPase in the baculovirus/insect cell expression system and purified the protein, yielding 0.3 mg per liter cell culture. Various detergents were tested for solubilization, with the protein remaining active under all selected detergents. n-dodecyl-{beta}-D-maltoside combined with cholesteryl hemisuccinate provided the highest solubility (88%). Finally, the PfPPase-VP1 was assayed against a set of fourteen antimalarial drugs, along with seven Thermotoga maritima mPPase inhibitors and fourteen compounds of unknown activity against mPPases. Only three compounds, all pyrazolo[1,5-a]pyrimidine-based TmPPase inhibitors, retained micromolar IC50 activity against PfPPase-VP1. The expression and purification of the PfPPase-VP1 will allow to conduct structural studies as well as to develop target-based screens, two steps necessary for the development of inhibitors to combat parasite disease.

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