Identification of the target of a compound that inhibits plasmodium sporozoites

描述（由申请人提供）：疟疾是由原生动物寄生虫疟原虫引起的。在疟原虫的人类周期中，第一个必要的发育步骤是被称为孢子虫的寄生虫阶段感染肝脏。在肝细胞内，孢子子分化并分裂形成肝期。肝脏阶段最终进入血液并感染红细胞导致疾病。因此，抑制孢子虫感染和肝期发育（统称为红细胞前阶段）将在早期阶段阻断疟疾。对肝细胞孢子感染和肝内发育机制的深入了解将对疟疾预防新药的开发具有重要意义。重要的是，这些药物将抑制间日疟原虫的休眠肝脏阶段的形成，而这一阶段的治疗方法很少。为了促进发现针对疟原虫红细胞前阶段的药物，我们的目标是确定三取代吡咯（Tsp）的靶标。茶氨酸可防止肝内孢子虫感染和寄生虫的发育。有趣的是，Tsp在两个阶段有不同的目标。我们的目标是在孢子虫感染期间确定Tsp的靶标。我们将在啮齿动物寄生虫伯氏疟原虫中使用生化和遗传方法来确定两种候选激酶是否为Tsp的靶标。我们将使用体外激酶试验来测试重组候选激酶对Tsp的敏感性。为了测试Tsp的敏感性是否由特定的氨基酸决定，我们将突变这些残基并测试突变酶。我们将比较突变型酶和野生型酶对Tsp的敏感性，以确定突变型酶是否在体外对Tsp不敏感。然后，我们将产生缺乏这些激酶的“敲除”孢子子，并测试它们在组织中感染肝细胞的能力

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.