Abstract

Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, the causative agents of severe malaria and toxoplasmosis respectively, undergo several critical developmental transitions during their lifecycle. Most important for human pathogenesis is the asexual cycle, in which parasites undergo rounds of host cell invasion, replication and egress (exit), destroying host cell tissue in the process. Previous work has identified important roles for Protein Kinase G (PKG) and Protein Kinase A (PKA) in parasite egress and invasion, yet little is understood about the regulation of cyclic nucleotides, cGMP and cAMP, that activate these enzymes. To address this we have focused upon the development of inhibitors of 3',5'-cyclic nucleotide phosphodiesterases (PDEs) to block the breakdown of cyclic nucleotides. This was done by repurposing hPDE inhibitors noting various similarities of the human and apicomplexan PDE binding sites. The most potent inhibitors blocked the in vitro proliferation of P. falciparum and T. gondii more potently than the benchmark compound, Zaprinast. 5-Benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (BIPPO) was found to be a potent inhibitor of recombinant PfPDEalpha and activated PKG-dependent egress of T. gondii and P. falciparum likely by promoting the exocytosis of micronemes, an activity that was reversed by a specific Protein Kinase G inhibitor. BIPPO also promotes cAMP-dependent phosphorylation of a P. falciparum ligand critical for host cell invasion suggesting that the compound inhibits single or multiple PDE isoforms that regulate both cGMP and cAMP levels. BIPPO is therefore a useful tool for the dissection of signal transduction pathways in apicomplexan parasites.