In vitro activity: KDU691 is a Plasmodium PI4 kinase (PI4K) inhibitor. It has been tested in vivo as a causal prophylactic and radical-cure agent for Plasmodium cynomolgi sporozoite-infected rhesus macaques, based on its in vitro activity against liver stages. Animals were infected with P. cynomolgi sporozoites, and KDU691 was dosed orally. KDU691 was fully protective when administered prophylactically. In contrast, when tested for radical cure, five daily doses of 20 mg/kg of KDU691 did not prevent relapse, as all animals experienced a secondary infection due to the reactivation of hypnozoites in the liver. These findings indicate that Plasmodium PI4K is a potential drug target for malaria prophylaxis but not radical cure. Longer in vitro culture systems will be required to assess KDU691's activity on established hypnozoites and predict radical cure in vivo.

Kinase Assay: In vitro infections of primary rhesus hepatocytes with P. cynomolgi sporozoites were performed according to methods described previously by Zeeman et al. At day 6 postinfection (p.i.), the assay mixtures were fixed and stained with anti-P. cynomolgi Hsp70 rabbit antiserum and a fluorescein isothiocyanate (FITC)-labeled secondary antibody (goat anti-rabbit). Plates were analyzed with the Operetta high-content imaging system, differentially counting hypnozoites and developing extraerythrocytic forms (EEFs), based on parasite size.

Cell Assay: Sporozoites were harvested from P. cynomolgi-infected mosquitoes, washed with phosphate-buffered saline (PBS), and diluted to 100,000 sporozoites (spz)/ml in PBS. One-milliliter aliquots of sporozoites were prepared and injected into monkeys via intravenous (i.v.) injection.