Investigation of the relationship between CGMP signalling and calcium mobilisation in Plasmodium falciparum with a focus on the role of phosphodiesterases in sexual development

Malaria kills almost half a million people every year and is a huge public health burden particularly in Sub-Saharan Africa. The current first line treatment is very effective but is threatened by the drug resistance that has been seen in the many effective compounds that have come before it. Consequently, there is a real need to develop new and novel compounds that target multiple stages of the parasite life cycle, as failure of our first line treatment could be devastating. Cell signalling pathways regulate essential events in the parasite lifecycle and as such, components of these pathways are potential targets for parasite disruption. The cyclic nucleotide and Ca2+ signalling cascades have essential functions at multiple stages in the parasite life cycle and often show overlap in their roles. This means that inhibition of one such pathway may disrupt the other. The relationship between 3’-5’-cyclic guanosine monophosphate and Ca2+ signalling is investigated here using Ca2+ assays on both schizont and gametocyte stages using a combination of genetic and pharmacological approaches. Results indicate that zaprinast-induced Ca2+ release through PDE inhibition is dependent upon PKG and in schizonts occurs via inositol triphosphate. Gametogenesis is an essential lifecycle event leading to transmission. The PDE PfPDEδ has been implicated in this event, however little is known about this enzyme. Using a PfPDEδ-ko line and the generation of a HA-tagged PfPDEδ line using the CRISPRCas9 gene editing system, this project seeks to confirm whether PfPDEδ is essential for gametogenesis and to understand more about the role that this protein plays in this event. This includes its cellular localisation, cyclic nucleotide specificity and its role in Ca2+ mobilisation, an event essential to the later stages of gametogenesis. The role of PfPDEδ in erythrocyte-infected gametocyte deformability is also investigated and the phenotype of a PfPDEδ-ko line is dissected. This information will eventually aim to determine whether PfPDEδ would be a good transmission-blocking target. Of particular interest to this thesis are compounds that would prevent transmission because currently the only licenced antimalarial to target transmission stages is Primaquine. A combination of a transmission-blocking agent with one that will inhibit asexual replicating stages would both alleviate clinical symptoms while preventing transmission. PDEs have already been identified and used as effective drug targets in humans. A small panel of Pfizer PDE inhibiters that have shown good activity in asexual blood stages has been tested against the gametocyte stages and this has led to the identification of a human PDE inhibitor that not only kills asexual blood stages in the low nM range, but also one that can kill gametocyte stages at similar concentrations. In addition to this, the mode of action of this compound has been determined.