Chemical Genetics of Plasmodium Kinases

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DESCRIPTION (provided by applicant): Malaria continues to be a global health problem killing 1-2 million people each year. Because of prevalence drug resistance it is urgent to identify new therapeutics for treatment. However, the development of new drugs by traditional screening approaches has lagged behind the required number of molecular entities to combat the disease and the serious problem of drug resistance. Additionally, there are only a few validated drug targets. This underscores the need for novel approaches to identify targets for drug development. Similar to all eukaryotes, cyclin-dependent kinases (CDKs) are likely to be key regulators of the novel intraerythrocytic cell cycle of the malaria parasite, where DNA replicates more than once per cell cycle without cytokinesis. Although several homologues of CDK-like kinases have been identified in P. falciparum, there is a fundamental gap in understanding about their physiological function. The long-term goal is to develop novel malaria therapeutics targeting Plasmodium CDK-like kinases. The objective of this application is to elucidate PfPK5, an essential closest Plasmodium homologue of metazoan CDK1, substrates through loss-of-function studies using chemical genetic approach. The chemical genetic approach will specifically label substrates of a given kinase using ATP analogs. The rationale for these proposed studies is that once PfPK5 physiological substrates are identified, it is expected that PfPK5- specific sites of protein-protein interactions will be exploited to develop mechanism-based route of interfering with PfPK5 function or the pathway where PfPK5 belongs. Thus, the proposed research is relevant to NIH's mission that pertains to developing fundamental knowledge that will potentially help to reduce the burden of human disease. Guided by strong preliminary data two specific aims will be pursued: (1) Identify P. falciparum proteins that are directly phosphorylated by PfPK5 by generating mutant kinases that are specifically able to utilize ATP analogs compared to the wild type kinases. Phosphorylated proteins will be identified by two-dimensional gel electrophoresis followed by mass spectrometric analysis. Candidate PfPK5 substrates will be validated. (2) Analyze phenotypic changes following loss of PfPK5 function by selective in vivo inhibition with ATP analogs. Global changes in gene expression will be analyzed by microarray analysis and phenotypic changes by microscopic and flow cytometric analysis following specific inhibition in PfPK5 activity. The proposed research is significant, because it will fundamentally advance our understanding of the molecular mechanisms of PfPK5 function in regulating Plasmodium falciparum intraerythrocytic life cycle. PUBLIC HEALTH RELEVANCE The proposed studies are of an important and under-investigated area of cell cycle regulation and signaling in Plasmodium falciparum. The proposed research has relevance to public health, because it will increase our understanding of the function of CDK-like kinases in Plasmodium falciparum that will aid in identifying parasite-specific signaling pathways and novel drug targets.