Abstract:
Novel antimalarial drug targets, and drugs and that target the causative agents for malaria in humans, Plasmodium falciparum parasites, are constantly needed due to the emergence of drug-resistant malaria parasite strains. Within unexplored biological avenues of the parasite, unique and essential proteins can be identified and studied as potential drug targets. One of these processes is the unique and atypical cell cycle of the P. falciparum parasite and the largely unexplored regulatory mechanisms and essential role players governing these mechanisms. Three genes (pf3d7_0105800, pf3d7_1112100 and pf3d7_1364400), encoding uncharacterised protein kinases with unknown functions within the parasite, were identified from transcriptome analyses of the switch between states of cell cycle arrest to cell cycle re-entry. This study aimed to functionally validate the essential nature of these putative kinases within the parasite life cycle through genetic manipulation, exposing potential novel targets in the kinase drug target space.
The essentiality of the putative kinases was determined by successfully cloning the 5’-gene-encoding fragments of each gene, namely pf3d7_0105800, pf3d7_1112100 and pf3d7_1364400, into a specialised targeted gene disruption system. This generated truncated versions of each protein, with only ~140–282 N-terminal residues remaining. These gene disruptions resulted in each of the three investigated putative kinase proteins (PF3D7_0105800, PF3D7_1112100 and PF3D7_1364400) indicated as having essential functions during parasite asexual proliferation, as parasites were not able to survive without the full, intact proteins. To gain more in-depth understanding of the function of the selected putative kinase proteins, alternative inducible systems were subsequently evaluated. The 3’-fragment of each gene was successfully cloned to allow displacement of the proteins from their site of action (knock sideways). The data from these inducible systems indicated that two of the kinases (PF3D7_0105800 and PF3D7_1112100) were found to localise to the cytoplasm of asexual parasites, but that insufficient displacement occurred to infer functionality with the knock sideways system. Parasites utilising an alternative system, gene knockdown (glmS ribozyme), were therefore also generated for future exploration.Collectively, novel putative antimalarial drug targets were described that are essential to the cell cycle regulation of P. falciparum parasites. This expands our knowledge of the unique kinome of P. falciparum parasites and reveals new potential role players in the parasite’s atypical cell cycle.
