Chemical Genetics of Plasmodium Kinases

描述(申请人提供)：疟疾仍然是一个全球健康问题，每年导致100-200万人死亡。由于耐药性的普遍存在，迫切需要寻找新的治疗方法。然而，通过传统筛选方法开发的新药已经落后于抗击该病所需的分子实体数量和严重的耐药性问题。此外，只有少数几个有效的药物靶点。这突出表明需要新的方法来确定药物开发的目标。与所有真核生物相似，细胞周期蛋白依赖性蛋白激酶(CDK)可能是疟疾寄生虫新的红细胞内细胞周期的关键调节因子，其中DNA在每个细胞周期复制一次以上，而不发生胞质分裂。虽然已经在恶性疟原虫中鉴定出几种CDK样蛋白的同源物，但对它们的生理功能的了解还存在着很大的差距。长期目标是开发针对疟原虫CDK样激酶的新型疟疾治疗药物。这项应用的目的是通过使用化学遗传学方法通过功能丧失研究来阐明PfPK5，一种与后生动物CDK1最接近的疟原虫同源物。化学遗传学方法将使用ATP类似物专门标记给定的激酶底物。这些研究的基本原理是，一旦确定了PfPK5的生理底物，就有望利用PfPK5特异的蛋白质-蛋白质相互作用位点来开发基于机制的干扰PfPK5功能或PfPK5所属途径的途径。因此，拟议的研究与NIH的使命相关，该使命与发展基础知识有关，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，将追求两个特定的目标：(1)通过产生与野生型激酶相比能够特异性地利用ATP类似物的突变蛋白，鉴定被PfPK5直接磷酸化的恶性疟原虫蛋白。磷酸化的蛋白质将通过双向凝胶电泳和质谱分析进行鉴定。候选PfPK5底物将得到验证。(2)用ATP类似物进行体内选择性抑制，分析PfPK5功能丧失后的表型变化。基因表达的全球变化将通过微阵列分析进行分析，表型变化将通过显微镜和流式细胞仪分析在特定抑制PfPK5活性后进行。这项研究具有重要意义，因为它将从根本上促进我们对PfPK5功能在调节恶性疟原虫红细胞内生命周期中的分子机制的理解。与公共卫生相关的拟议研究是关于恶性疟原虫细胞周期调节和信号传递的一个重要且未被充分研究的领域。这项拟议的研究与公共卫生相关，因为它将增加我们对恶性疟原虫中CDK样激酶功能的理解，这将有助于识别寄生虫特异的信号通路和新的药物靶点。

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.