Systematic screening identified broad-spectrum inhibitors of human-pathogenic Encephalitozoon species and revealed no in vitro activity of nitazoxanide

BACKGROUND: Microsporidia are obligate intracellular eukaryotic pathogens infecting a wide range of vertebrate and invertebrate hosts. Therapeutic options for microsporidiosis remain limited in both human and veterinary medicine. OBJECTIVES: The goal was to screen the 400 compounds of the open-access Pandemic Response Box, provided by the Medicines for Malaria Venture foundation, for their anti-microsporidia activity. METHODS: The compounds were screened at 1 µM against Encephalitozoon cuniculi, E. intestinalis and E. hellem using an ELISA-based assay. Selected compounds were further evaluated by determining their in vitro cytotoxicity and their IC50 values. RESULTS: Preliminary screening revealed promising efficacy of eight compounds against the three species. Trimetrexate was the most active compound, with IC50 values < 1 µM and selectivity index > 100 for all three species. Triapine exhibited intermediate activity, with IC50 values ranging from 1 to 2 µM. Nitazoxanide failed to achieve 50% growth inhibition at the highest concentration tested (2 µM) against any of the three species. CONCLUSIONS: Trimetrexate emerged as the most promising candidate for further anti-microsporidia exploration, whereas nitazoxanide showed no detectable activity under the conditions tested, questioning its role in the treatment of human microsporidiosis.