Chemical Genetics of Plasmodium Kinases

疟原虫激酶的化学遗传学

基本信息

批准号：
7645600
负责人：
DEBOPAM CHAKRABARTI
金额：
$ 35.62万
依托单位：
UNIVERSITY OF CENTRAL FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-01 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7645600
关键词：
Address Affect Alleles Area Binding Binding Sites CDC2 Protein Kinase Catalytic Domain Cell Cycle Cell Cycle Regulation Cell Extracts Cells Characteristics Cyclin-Dependent Kinases Cytokinesis DNA DNA biosynthesis Data Development Disease Drug Delivery Systems Drug Design Drug resistance Electrostatics Engineering Environment Enzymes Erythrocytes Eukaryota Eukaryotic Cell Exhibits Family Gene Expression Gene Expression Microarray Analysis Gene Targeting Generations Global Change Goals Growth Haploidy Health Homologous Gene Hot Spot Hydrogen Bonding Knock-out Knowledge Label Life Cycle Stages Malaria Microarray Analysis Microscopic Mission Molecular Outcome Parasites Pathway interactions Pharmaceutical Preparations Phenotype Phosphotransferases Physiological Plasmodium Plasmodium falciparum Preparation Prevalence Property Proteins Public Health Research Research Personnel Role Route Screening procedure Shapes Signal Pathway Signal Transduction Site Solutions Specificity Substrate Interaction Techniques Therapeutic Two-Dimensional Gel Electrophoresis Work analog asexual base cell growth cellular targeting chemical genetics combat drug development experience human disease in vivo inhibitor/antagonist innovation killings loss of function mutant novel novel strategies novel therapeutics protein protein interaction public health relevance resistant strain therapeutic target

项目摘要

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.

描述（由申请人提供）：疟疾仍然是全球健康问题，每年杀死1-200万人。由于耐药性耐药性，迫切需要识别新的治疗疗法。但是，通过传统筛查方法开发新药物已经落后于所需的分子实体数量，以应对疾病和耐药性的严重问题。此外，只有少数经过验证的药物靶标。这强调了对识别药物开发目标的新方法的需求。与所有真核生物相似，细胞周期蛋白依赖性激酶（CDK）可能是疟原虫寄生虫的新型肠内细胞周期的关键调节剂，其中DNA在没有细胞因子的情况下每个细胞周期复制了一次以上。尽管在恶性疟原虫中已经鉴定出了几种CDK样激酶的同源物，但了解其生理功能存在根本差距。长期目标是开发针对疟原虫CDK样激酶的新型疟疾疗法。该应用的目的是阐明PFPK5，这是一种基于化学遗传方法的功能丧失研究，这是Metazoan CDK1的最接近的疟原虫同源物。化学遗传方法将使用ATP类似物特异性标记给定激酶的底物。这些提出的研究的基本原理是，一旦确定了PFPK5生理底物，就可以预计将利用PFPK5-蛋白质 - 蛋白质相互作用的特定位点来开发基于机制的基于机制的干扰PFPK5功能或PFPK5属性的途径。因此，拟议的研究与NIH的使命相关，该使命与发展基本知识有关，这可能有助于减轻人类疾病的负担。在强大的初步数据的指导下，将追求两个具体目标：（1）通过产生与野生型动力酶相比，通过产生特定能够利用ATP类似物的突变激酶来确定PFPK5直接磷酸化的恶性疟原虫蛋白。磷酸化蛋白将通过二维凝胶电泳鉴定，然后进行质谱分析。候选PFPK5基材将得到验证。（2）通过选择性在体内抑制ATP类似物来分析PFPK5功能丧失后的表型变化。在PFPK5活性中特定的抑制后，将通过微阵列分析和表型变化来分析基因表达的全球变化。拟议的研究很重要，因为它从根本上可以提高我们对PFPK5功能的分子机制在调节恶性疟原虫内肠内生命周期中的理解。公共卫生相关性拟议的研究是恶性疟原虫中细胞周期调节和信号传导的重要且不足的区域。拟议的研究与公共卫生有关，因为它将增加我们对恶性疟原虫中CDK样激酶功能的理解，这将有助于鉴定寄生虫特异性的信号通路和新型药物靶标。