Malaria is caused by the protozoan parasite, Plasmodium. The first obligatory developmental step in Plasmodium's human cycle is the infection of the liver by parasite stages termed sporozoites. Within the hepatocyte, the sporozoites differentiate and divide to form liver stages. Liver stages eventually enter the bloodstream and infect erythrocytes causing disease. Therefore, inhibiting sporozoite infection and liver stage development (together termed pre-erythrocytic stages) would block malaria at an early step. Mechanistic insights into sporozoite infection of hepatocytes and intrahepatic development will contribute significantly to the development of novel drugs for malaria prevention. Importantly, these drugs will inhibit the formation of dormant liver stages by P. vivax for which there are few treatment options. In order to facilitate the discovery of drugs that targe Plasmodium's pre-erythrocytic stages, we aim to identify the target of a tri- substituted pyrrole (Tsp). Tsp prevents both sporozoite infection and parasite development in the liver. Interestingly, Tsp has different targets at the two stages. Our goal is to identify Tsp's target during sporozoite infection. We will use biochemical and genetic approaches in the rodent parasite, P. berghei, to determine if two candidate kinases are the targets of Tsp. We will test the sensitivity of recombinant candidate kinases to Tsp using in vitro kinase assays. To test if Tsp sensitivity is determined by specific amino acids, we will mutate these residues and test the mutant enzyme. We will compare the sensitivity of the mutant and wildtype enzyme to Tsp, to determine if the mutant enzyme becomes insensitive to Tsp in vitro. Then, we will generate 'knockout'sporozoites lacking these kinases and test their ability to infect hepatocytes in tissue culture and in vivo. Finally, we will generate additional mutant parasites, carrying Tsp-resistant alleles of the candidate kinases and test if the mutant sporozoites become refractory to Tsp. Thus, our proposal will identify the sporozoite target of Tsp in vivo. By identifying the target of Tsp, our work will functionally annotate a protein essential for sporozoite infection. This proposa will lay the groundwork for rational screening of derivative compounds of greater potency against P. falciparum orthologs of the target kinases.
Malaria is a deadly disease with few effective treatments. We hope to find better drugs against malaria by identifying the target of a molecule that blocks malaria parasites from infecting mammalian liver cells. Identifying the target of this molecule wil allow us to find drugs that block malaria parasites at an early step, before they cause disease.
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