Identification of Potent Phosphodiesterase Inhibitors that Demonstrate Cyclic Nucleotide-Dependent Functions in Apicomplexan Parasites

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 human PDE 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 P. falciparum PfPDEα 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.

Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii

“…Because PKG is activated by the second messenger cGMP, compounds that elevate cGMP levels should stimulate microneme secretion. Consistent with studies showing that zaprinast, an inhibitor of cGMPPDEs, stimulates egress (9), it was later shown that zaprinast stimulates microneme secretion in Toxoplasma, presumably by elevating cGMP (16 min stimulates microneme secretion by activating PKG via increasing cGMP levels. To determine whether serum albumin affects cGMP levels, RH-MIC2-GLuc-C-myc parasites were mock-treated or treated with serum albumin.…”

“…Consistent with this finding, cyclic GMP (cGMP) has emerged as a second signaling molecule that stimulates microneme secretion. Indirect evidence for this pathway is provided by inhibitors of cGMP-specific phosphodiesterases (PDE), such as zaprinast and BIPPO, which stimulate microneme secretion and egress in Toxoplasma (9,16), and Plasmodium falciparum merozoites (17). More directly, chemical-genetic studies showed that inhibition of PKG blocks microneme secretion in Eimeria tenella sporozoites (15), Toxoplasma tachyzoites (15), and P. falciparum merozoites (17).…”

Plasma Membrane Association by N-Acylation Governs PKG Function in Toxoplasma gondii

“…Microneme secretion is controlled by two key signaling pathways, calcium (Ca 2+ ) and cyclic GMP (cGMP), which direct calcium-dependent protein kinases (CDPKs) (6) and protein kinase G (PKG) (7, 8), respectively, to transduce their respective signals through substrate phosphorylation. Phosphoproteomic studies have identified substrates for CDPK1 in T. gondii (9) and PKG in Plasmodium spp.…”

Phenotypic, chemical and functional characterization of cyclic nucleotide phosphodiesterase 4 (PDE4) as a potential anthelmintic drug target

“…In Trypanosoma cruzi , an ortholog of huPDE4, TcPDE4 (TcPDEB1) displayed an inhibition profile characteristic of the PDE4 subfamily, including a specificity for cAMP over cGMP [62]. Lastly, PDE inhibitors block the in vitro proliferation of Plasmodium falciparum and Toxoplasma gondii [63, 64]. …”