title: 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.
authors.affiliation: †Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
authors.forename: Brittany L
authors.initials: BL
authors.lastname: Howard
authors.affiliation: ‡Macfarlane Burnet Institute, Melbourne, Victoria, Australia.|∥University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Harvey
authors.affiliation: ⊥Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Stewart
authors.affiliation: ‡Macfarlane Burnet Institute, Melbourne, Victoria, Australia.
authors.forename: Mauro F
authors.initials: MF
authors.lastname: Azevedo
authors.affiliation: †Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.|‡Macfarlane Burnet Institute, Melbourne, Victoria, Australia.|∥University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Brendan S
authors.initials: BS
authors.lastname: Crabb
authors.affiliation: †Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
authors.forename: Ian G
authors.initials: IG
authors.lastname: Jennings
authors.affiliation: ‡Macfarlane Burnet Institute, Melbourne, Victoria, Australia.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Sanders
authors.affiliation: †Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
authors.forename: David T
authors.initials: DT
authors.lastname: Manallack
authors.affiliation: †Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
authors.forename: Philip E
authors.initials: PE
authors.lastname: Thompson
authors.affiliation: ⊥Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Tonkin
authors.affiliation: †Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.|‡Macfarlane Burnet Institute, Melbourne, Victoria, Australia.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Gilson
mesh_terms: D000981:Antiprotozoal Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D060326:Chemistry Techniques, Synthetic; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005260:Female; D006801:Humans; D010726:Phosphodiesterase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000502:physiology; D011688:Purinones / Q000494:pharmacology; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology; D011744:Pyrimidinones / Q000737:chemistry / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D014122:Toxoplasma / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology
chemical_list: C000607182:5-benzyl-3-isopropyl-1H-pyrazolo(4,3-d)pyrimidin-7(6H)-one; D000981:Antiprotozoal Agents; D010726:Phosphodiesterase Inhibitors; D011688:Purinones; D011720:Pyrazoles; D011744:Pyrimidinones; D000242:Cyclic AMP; C011145:zaprinast; D006152:Cyclic GMP