Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signalling

J Biol Chem. 2016 Mar 1. pii: jbc.M115.703546. [Epub ahead of print]

Sidik SM¹, Hortua Triana MA², Paul AS³, El Bakkouri M⁴, Hackett CG¹, Tran F⁵, Westwood NJ⁵, Hui R⁴, Zuercher WJ⁶, Duraisingh MT³, Moreno SN², Lourido S⁷.

The lifecycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca²⁺. Such fluxes are necessary for events like motility and egress from host cells. We used genetically-encoded Ca²⁺ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca²⁺ signalling 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 (PKG) to raise levels of cytosolic Ca²⁺. We define the pool of Ca²⁺ 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 Ca²⁺ signalling. Two such compounds constitute novel PKG inhibitors, and prevent zaprinast from increasing cytosolic Ca²⁺. The enhancers identified are capable of releasing intracellular Ca²⁺ 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 Ca²⁺-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca²⁺ 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 signalling pathways regulated by Ca²⁺, underscoring the importance of these pathways and the therapeutic potential of their inhibition.
Copyright © 2016, The American Society for Biochemistry and Molecular Biology.

KEYWORDS:

calcium; calcium intracellular release; drug screening; parasitology; protein kinase G (PKG); signal transduction

PMID:

26933036

[PubMed - as supplied by publisher]