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Identification of the target of a compound that inhibits plasmodium sporozoites

抑制疟原虫子孢子的化合物靶标的鉴定

基本信息

批准号：
8716838
负责人：
Purnima Bhanot
金额：
$ 23.85万
依托单位：
RBHS-NEW JERSEY MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-25 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8716838
关键词：
Affect Alleles Amino Acids Artemisinins Binding Biochemical Genetics Biological Assay Blood Circulation Cells Combined Modality Therapy Culicidae Cyclic GMP-Dependent Protein Kinases Development Disease Dose Drug Targeting Enzymes Erythrocytes Goals Hepatocyte Human In Vitro Infection Inhibitory Concentration 50 Injection of therapeutic agent Knock-out Life Liver Malaria Malaria prevention Mediating Molecular Models Morbidity - disease rate Mus Mutate Orthologous Gene Parasites Pharmaceutical Preparations Phosphotransferases Plasmodium Plasmodium falciparum Positioning Attribute Predisposition Primaquine Process Prophylactic treatment Proteins Pyrroles Recombinants Recurrent disease Refractory Resistance Rodent Sporozoites Staging Study models Symptoms Testing Toxic effect Toxoplasma gondii Transgenic Organisms Vaccines Work artemisinine base calcium-dependent protein kinase cell motility chemotherapy effective therapy efficacy testing genetic analysis in vivo inhibitor/antagonist insight intrahepatic knockout gene liver infection molecular modeling mutant novel novel strategies prevent response screening tissue culture tissue/cell culture

项目摘要

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.

描述(申请人提供)：疟疾是由原生动物寄生虫疟原虫引起的。疟原虫在人类周期中的第一个强制性发育步骤是由被称为子孢子的寄生虫期感染肝脏。在肝细胞内，子孢子分化和分裂形成肝脏阶段。肝脏阶段最终进入血流，感染红细胞导致疾病。因此，抑制子孢子感染和肝期发育(一起称为红细胞前期)将在早期阻止疟疾。对子孢子感染肝细胞和肝内发育的机械性洞察将对疟疾预防新药的开发有重要贡献。重要的是，这些药物将抑制间日疟原虫形成休眠的肝脏阶段，而对此几乎没有治疗选择。为了便于发现针对疟原虫红细胞前期阶段的药物，我们的目标是确定三取代吡咯(TSP)的靶标。TSP既可防止子孢子感染，又可防止肝脏内寄生虫的发展。有趣的是，TSP在这两个阶段有不同的目标。我们的目标是确定TSP在子孢子感染过程中的靶标。我们将在啮齿动物寄生虫P.berghei中使用生化和遗传学方法来确定是否有两个候选激酶是TSP的靶标。我们将使用体外激酶法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的酶。我们将比较突变型和野生型酶对TSP的敏感性，以确定突变酶在体外是否对TSP不敏感。然后，我们将产生缺乏这些激酶的“敲除”子孢子，并测试它们感染组织中肝细胞的能力培养的和体内的。最后，我们将产生额外的突变寄生虫，携带候选激酶的TSP抗性等位基因，并测试突变的子孢子是否对TSP产生抵抗力。因此，我们的建议将在体内确定TSP的子孢子靶点。通过识别的目标 TSP，我们的工作将从功能上注释一种对子孢子感染至关重要的蛋白质。本研究为合理筛选抗恶性疟原虫靶蛋白同源蛋白的高效衍生物奠定了基础。