PDE-I2 inhibits the formation of nuclei and daughter merozoites. (C) The morphology of rhoptries was analyzed by fluorescence microscopy of ARO-mCherry at 48 hpi. Box plot represents number of mCherry-foci per parasite at 48 hpi. n(DMSO) = 53; n(PDE-I2) = 50. Bar graph represents percentage of parasites with normal versus abnormal morphology of rhoptries. In total, at least 180 parasites were analyzed per condition. (D) Formation of the IMC was analyzed by fluorescence microscopy of GAPM2-GFP at 48 hpi. Bar graph represents percentage of parasites with normal versus abnormal morphology of IMC. In total, at least 150 parasites were analyzed per condition. (E) Invagination of the PPM was analyzed by fluorescence microscopy of SMS1-mCherry at 48 hpi. Bar graph represents percentage of parasites with normal versus abnormal morphology of PPM. In total, at least 250 parasites were analyzed per condition. All assays were performed with highly synchronous parasites (2-hour synchronicity window) incubated with IC90 of PDE-I2 or vehicle (DMSO) at 2 hpi. The final DMSO concentration did not exceed 0.5%. Scale bars, 8 mM; To visualize these structures, we used three transgenic parasite lines expressing the rhoptry protein PfARO (armadillo repeats only) the IMC protein GAPM2 (glideosome-associated protein with multiple membrane spans or the PPM-associated protein PfSMS1 (Sphingomyelin synthase 1) as mCherry or GFP fusion proteins. We show that while PDE-I2-treated parasites can form rhoptries visible as punctate foci in mature schizonts, their number was significantly reduced (C); however, the most significant defects conferred by PDE-I2 treatment were observed in IMC and PPM formation and organization. While IMC biogenesis is initiated, no subsequent growth along the nascent daughter cells’ polar axis could be observed (D). PDE-I2 treatment also severely impaired PPM formation (E).

Alder A, Struck NS, Xu M, Johnson JW, Wang W, Pallant D, Cook MA, Rambow J, Lemcke S, Gilberger TW, Wright GD. A non-reactive natural product precursor of the duocarmycin family has potent and selective antimalarial activity. Cell Chem Biol. 2021 23:S2451-9456(21)00439-6.