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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：
8384110
负责人：
Purnima Bhanot
金额：
$ 19.75万
依托单位：
UNIV OF MED/DENT OF NJ-NJ 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 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8384110
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 Delivery Systems 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 Screening procedure 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 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. 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.

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。疟原虫在人类周期中的第一个强制性发育步骤是被称为子孢子的寄生虫阶段感染肝脏。在肝细胞内，子孢子分化并分裂形成肝脏阶段。肝脏阶段最终进入血液并感染红细胞引起疾病。因此，抑制子孢子感染和肝脏阶段发育（一起称为红细胞前期）将在早期阻断疟疾。子孢子感染肝细胞和肝内发育的机制的见解将大大有助于开发新的药物预防疟疾。重要的是，这些药物将抑制间日疟原虫休眠肝脏阶段的形成，对此几乎没有治疗选择。为了促进疟原虫红细胞前期药物的发现，我们的目标是确定三取代吡咯（Tsp）的靶点。Tsp可以防止子孢子感染和寄生虫在肝脏中的发育。有趣的是，TSP在两个阶段有不同的目标。我们的目标是确定在子孢子感染Tsp的目标。我们将在啮齿类寄生虫伯氏疟原虫中使用生物化学和遗传学方法来确定两种候选激酶是否是Tsp的靶点。我们将使用体外激酶测定来测试重组候选激酶对Tsp的敏感性。为了测试Tsp敏感性是否由特定氨基酸决定，我们将使这些残基突变并测试突变的酶。我们将比较突变体和野生型酶对Tsp的敏感性，以确定突变体酶是否在体外对Tsp不敏感。然后，我们将产生缺乏这些激酶的'敲除'子孢子，并测试它们感染组织中肝细胞的能力培养和体内。最后，我们将产生额外的突变体寄生虫，携带候选激酶的TSP抗性等位基因，并测试突变体子孢子是否变得对TSP难治。因此，我们的建议将确定子孢子靶的TSP在体内。通过识别目标 Tsp，我们的工作将在功能上注释子孢子感染所必需的蛋白质。这一建议将奠定基础，合理筛选衍生化合物的更大的效力对恶性疟原虫直系同源的目标激酶。公共卫生相关性：疟疾是一种致命的疾病，几乎没有有效的治疗方法。我们希望通过确定一种分子的靶点来找到更好的抗疟疾药物，这种分子可以阻止疟原虫感染哺乳动物的肝细胞。确定这种分子的靶点将使我们能够在疟疾寄生虫引起疾病之前，在早期阶段找到阻断疟疾寄生虫的药物。