Investigations into Phosphodieterases as Targets for Antimalarial Drug Discovery

Phosphodiesterases are key enzymes in cyclic-nucleotide signalling pathways, regulating the levels of cAMP and cGMP in the cell. Cyclic nucleotides play an important role in regulating progression of the complex parasite life cycle. There are four Plasmodium PDEs, PDEα-δ. PDEβ has proved refractory to deletion and is predicted to be essential in asexual blood stages. PDEs α, γ and δ have been successfully disrupted in previous studies, which revealed stage-specific roles for PDEγ and PDEδ in the mosquito. PDEβ is a promising drug target due to the proven ability to create specific inhibitors for individual human PDEs (e.g. Viagra inhibits human PDE5), the established safety record of current inhibitors and the predicted essential nature of PDEβ in P. falciparum blood stages. The lack of useful reagents; knockout strains, tagged lines and recombinant proteins, has severely limited progress in this field. Conditional genetic disruption (required to study essential genes) is notoriously difficult in P. falciparum. This project has attempted to generate a conditional knock-out using the destabilisation domain system and the novel DiCre system, which involves recombinase-mediated genomic excision of the target DNA upon introduction of a drug. A PDEβ haemagglutanin (HA) tagged line has been successfully generated and used to investigate the cellular biology of PDEβ, including, the subcellular localisation and cyclic nucleotide specificity of PDEβ, which until now has remained speculative. A small library of PDE inhibitors generated by Pfizer has been evaluated using a parasite growth inhibition assay and a PDE assay, with compounds active at sub-micromolar concentrations against the parasite and the protein. These assays have used wild type parasites and also a PDEα KO line which has no obvious phenotype in blood stage parasites.