title: Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling.
abstract: The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca(2+) Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca(2+) indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca(2+) signaling in the model apicomplexan Toxoplasma gondii In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca(2+) We define the pool of Ca(2+) regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca(2+) signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca(2+) The enhancers identified are capable of releasing intracellular Ca(2+) stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum Inhibition of Ca(2+)-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca(2+) stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca(2+), underscoring the importance of these pathways and the therapeutic potential of their inhibition.
authors.affiliation: From the Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.
authors.forename: Saima M
authors.initials: SM
authors.lastname: Sidik
authors.affiliation: the Center for Tropical and Emerging Global Diseases, Department of Cellular Biology, University of Georgia, Athens, Georgia 30602.
authors.forename: Miryam A
authors.initials: MA
authors.lastname: Hortua Triana
authors.affiliation: the Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts 02115.
authors.forename: Aditya S
authors.initials: AS
authors.lastname: Paul
authors.affiliation: the Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
authors.forename: Majida
authors.initials: M
authors.lastname: El Bakkouri
authors.affiliation: From the Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.
authors.forename: Caroline G
authors.initials: CG
authors.lastname: Hackett
authors.affiliation: the School of Chemistry and Biomedical Sciences Research Complex, University of St. Andrews and EaStCHEM, North Haugh, St. Andrews, Fife KY16 9ST, Scotland, United Kingdom, and.
authors.forename: Fanny
authors.initials: F
authors.lastname: Tran
authors.affiliation: the School of Chemistry and Biomedical Sciences Research Complex, University of St. Andrews and EaStCHEM, North Haugh, St. Andrews, Fife KY16 9ST, Scotland, United Kingdom, and.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Westwood
authors.affiliation: the Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
authors.forename: Raymond
authors.initials: R
authors.lastname: Hui
authors.affiliation: the Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: William J
authors.initials: WJ
authors.lastname: Zuercher
authors.affiliation: the Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts 02115.
authors.forename: Manoj T
authors.initials: MT
authors.lastname: Duraisingh
authors.affiliation: the Center for Tropical and Emerging Global Diseases, Department of Cellular Biology, University of Georgia, Athens, Georgia 30602.
authors.forename: Silvia N J
authors.initials: SN
authors.lastname: Moreno
authors.affiliation: From the Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, lourido@wi.mit.edu.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Lourido
mesh_terms: D020013:Calcium Signaling / Q000187:drug effects*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D010963:Plasmodium falciparum / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011688:Purinones / Q000494:pharmacology*; D014122:Toxoplasma / Q000235:genetics / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins; D011688:Purinones; D017869:Cyclic GMP-Dependent Protein Kinases; C011145:zaprinast
keywords: calcium; calcium intracellular release; drug screening; parasitology; protein kinase G (PKG); signal transduction