Furoxan derivatives with antimalarial activity that disrupt P. falciparum endoplasmic reticulum calcium homeostasis

Malaria, caused by Plasmodium spp. parasites, presents treatment challenges due to the emergence of resistant strains to frontline antimalarials. In this study, we explored activities of 24 furoxan derivatives, originally designed for leishmaniasis treatment, against intraerythrocytic Plasmodium falciparum parasites. The studies were carried out with genetically-modified P. falciparum expressing GCaMP3, a cytosolic calcium indicator, and maintained in human red blood cell culture. The most potent compound Furoxan (1,2,5-oxidiazole-2N-oxide) 4m exhibited significant antimalarial activity (EC₅₀ = 2.8 μM). By contrast, the benzofuroxan derivative compounds showed limited inhibition of parasite growth, with the most effective compound, 4r, achieving 40% inhibition at 10 μM. Compound 4m contains the N-acyl hydrazone moiety, known for inhibiting cysteine proteases, and shows low similarity with other antimalarials observed by Tanimoto's score analysis. We assessed the ability of this compound to inhibit falcipains, the main cysteine proteases of P. falciparum, found in the parasite digestive vacuole and responsible for hemoglobin degradation. In contrast to the cysteine protease inhibitor E64, 4m did not inhibit hemoglobin degradation. Additionally, 4m and the other derivatives showed no inhibition of falcipain-2 in vitro. Among the active compounds, we observed that only 4m selectively increased cytosolic calcium concentration ([Ca²⁺]_c) in intra-erythrocytic parasites. This Ca²⁺ release occurred from the cyclopiazonic acid (CPA) sensitive endoplasmic reticulum compartment and not from the nigericin-sensitive acidic compartments, including the food vacuole. 4m can release NO; however, the ability of 4m to increase [Ca²⁺]_c is not dependent on NO, as Ca²⁺ release was not blocked by the NO scavenger CPTIO or mimicked by the NO donor diethylamine NONOate. These findings suggest that 4m warrants further exploration as a new scaffold for developing antimalarial drugs with distinct mechanisms of action compared to available antimalarials.