Proline homeostasis: a novel mediator of drug tolerance in Plasmodium falciparum

项目摘要 有效药物的持续供应对于全世界根除疟疾的努力至关重要。的出现 目前的抗疟疗法的耐药性的传播仍然是一个紧迫的问题， 青蒿素类治疗的失败增加了对新型抗疟化疗的需求。因此 发现新的药物靶点和途径，包括那些对多个生命阶段至关重要的靶点和途径， 下一代治疗方法发展的主要挑战。 使用整合的化学基因组学方法，我们已经确定了细胞质脯氨酰tRNA合成酶， 恶性疟原虫（PfcPRS）作为常山酮长期寻求的生化靶标。而且我们 通过调节脯氨酸的体内平衡，揭示了寄生虫中前所未有的药物耐受机制。 在这个提议中，我们试图了解寄生虫感知和进化能力的分子基础 通过适应性脯氨酸反应（APR）对常山酮的抗性。我们采用综合方法 结合我们在分子寄生虫学、代谢组学、遗传学和合成化学方面的专业知识， 这些方面的氨酰tRNA合成酶生物学和抑制寄生虫。 我们将研究对PfcPRS抑制剂常山酮耐药的非遗传机制。我们确定 常山酮处理引起细胞内脯氨酸增加主要原因及对策 以规避此过程。 我们将确定是否增加脯氨酸水平的寄生虫表现出的APR介导的变化， 在基因组或蛋白质组水平的关键代谢途径，使用高覆盖率的DNA测序， 基于定量质谱的蛋白质组学技术。 我们将探索寄生虫中aaRS抑制的APR非依赖性机制，评估PRS抑制剂， 具有不同的绑定模式。

PROJECT SUMMARY Sustained availability of efficacious drugs is essential for worldwide efforts to eradicate malaria. The emergence and spread of drug resistance to current antimalarial therapies remains a pressing concern with reports of artemisinin-based treatment failures escalating the need for novel antimalarial chemotherapies. Thus the discovery of new druggable targets and pathways, including those that are critical for multiple life stages, is a major challenge for the development of next-generation therapeutics. Using an integrated chemogenomic approach, we have identified the cytoplasmic prolyl tRNA synthetase in Plasmodium falciparum (PfcPRS) as the long-sought biochemical target of halofuginone. Furthermore, we uncovered an unprecedented mechanism of drug-tolerance in the parasite by modulation of proline homeostasis. In this proposal, we seek to understand the molecular basis of the parasite’s ability to sense and evolve resistance to halofuginone via the Adaptive Proline Response (APR). We bring an integrated approach combining our expertise in molecular parasitology, metabolomics, genetics, and synthetic chemistry to probe these aspects of aminoacyl tRNA synthetase biology and inhibition in the parasite. We will investigate a non-genetic mechanism of resistance to the PfcPRS inhibitor, halofuginone. We identify the primary source of increased intracellular proline in response to halofuginone treatment and strategies to circumvent this process. We will determine if increased proline levels in parasites exhibiting the APR are mediated by changes in key metabolic pathways at the genomic or proteomic level, using high-coverage DNA sequencing and quantitative mass spectrometry-based proteomic technologies. We will explore APR-independent mechanisms of aaRS inhibition in the parasite, evaluating PRS inhibitors with differing binding modes.