Targeting the Mitochondrion of P. falciparum

描述(申请人提供)：恶性疟原虫的线粒体被剥夺了许多典型的线粒体功能，但保留了对寄生虫生存至关重要的途径。剩下的线粒体功能被证明是抗疟疾药物发现的一组非常有成效的靶点。克服耐药性的一种方法是设计新药，使耐药性的适宜成本限制突变寄生虫在联合疗法中生存的能力，或在缺乏选择的情况下坚持下去。通过整体高通量筛选，我们发现了许多新的化学类型，它们似乎作用于线粒体靶标。我们建议对线粒体采取化学生物学的方法，并将其开发成一套试剂，通过精确定位作用于线粒体的一系列化学类型的作用机制和耐药性，来表征线粒体的功能。我们将研究耐药突变对靶标结构和功能的影响，以及这些突变和随之而来的生化变化对体外寄生虫生长和适应的影响。我们将选择对一系列似乎针对线粒体等的化学抑制剂的抗性，重点是DHODH。使用一种已被证明高效的方法，我们将对抗药性突变体进行表征，以确定化学抑制剂的目标。我们建议进行足够深入的选择，并使用足够多样化的化学方法来采样影响线粒体等的可能靶点和机制的范围，并深入探索DHODH抑制剂的耐药性突变范围，重点是进入临床测试的化合物。我们将筛选以前发现的DHODH抑制剂，筛选那些对耐药寄生虫有活性的药物。我们将绘制新分离的突变株的抗性突变图，并通过测量突变对酶、线粒体功能和有机体生长的影响来评估生物学后果。

DESCRIPTION (provided by applicant): The mitochondrion of P. falciparum is stripped of many of the typical functions of mitochondria, yet retains pathways essential to the parasite survival. The remaining mitochondrial functions are proving to be an exceptionally productive set of targets for antimalarial drug discovery. One approach to overcoming drug resistance is to design new drugs such that the fitness cost of resistance restricts the ability of mutant parasites to survive combination therapy or to persist in the absence of selection. Through whole organism high-throughput screening we have discovered many new chemotypes that appear to act on mitochondrial targets. We propose to take a chemical biology approach to the mitochondrion and develop these into a suite of reagents useful in characterizing mitochondrial function by pinpointing mechanisms of action and resistance for a range of chemotypes that act against the mitochondrion. We will study the effects of the resistance mutations on the structure and function of the targets and the impact of those mutations and consequent biochemical changes on parasite growth and fitness in vitro. We will select resistance to a range of chemical inhibitors that appear to target the mitochondrial ETC, focusing on DHODH. Using an approach that has proven highly productive, we will characterize the resistant mutants to identify the target of the chemical inhibitor. We propose to conduct selections in sufficient depth and using a sufficiently diverse range of chemistry to sample the range of possible targets and mechanisms affecting the mitochondrial ETC, and to explore the range of resistance mutations to DHODH inhibitors intensively, focusing on the compound advancing to clinical testing. We will screen previously identified DHODH inhibitor screening hits for those active against the resistant parasites. We will map the resistance mutations of mutants newly isolated and assess the biological consequences by measuring the effects of the mutations on the enzyme, on mitochondrial function and on the growth of the organism.