Arylmethylamino steroid compound 1o interferes with Plasmodium falciparum's hemoglobin metabolism

The anti-parasitic compound arylmethylamino steroid 1o (1o) is a promising drug candidate with low nanomolar activity against the malaria parasite Plasmodium falciparum, but with a so far unknown mode of action. To address this, we applied previously developed live-cell ATP and pH assays to measure effects upon exposure of parasites to 1o. Furthermore, we analyzed the parasites' heme species distribution, the ultrastructural morphology, food vacuole (FV) appearance, and lifecycle development of different parasite stages. We found that 1o increases cytosolic [ATP] level and causes a slight drop in pH, similar to the effects of arylamino alcohols such as mefloquine. The compound also prevents chloroquine (CQ)-mediated proteolysis and limits cytosol acidification within the range of its EC₅₀. Additionally, 1o prevents CQ-mediated heme and hemoglobin accumulation, and preserves ultrastructural FV integrity. Furthermore, we can demonstrate that 1o blocks the development of ring and early trophozoite stages, while late trophozoite stages were unaffected. These findings suggest that the mechanism underlying the killing activity of 1o may be the interference of a pathway within or upstream of hemoglobin digestion, particularly during the highly metabolically active earlier parasite stages. Our data open a new perspective on the compound's mode of action, information critically needed for target identification and further drug development.