Interrogating plasmodial endocytosis with mefloquine-based affinity probes

用基于甲氟喹的亲和探针检测疟原虫内吞作用

• 批准号: 10089405
• 负责人: Paul R Carlier
• 金额: $ 20万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10089405
• 关键词: Address; Affinity; Affinity Chromatography; Alkynes; Amino Acids; Antimalarials; Avidin; Binding; Biochemistry; Biological; Biological Assay; Biology; Biotin; Characteristics; Chemicals; Clinical; Crystallization; Cytosol; Data; Development; Diazomethane; Digestion; Endocytosis; Erythrocytes; Event; Future; Generations; Goals; Growth; Heme; Hemoglobin; Human; Individual; Label; Length; Leucine; Libraries; Light; Malaria; Mass Spectrum Analysis; Mefloquine; Membrane; Molecular; Molecular Probes; Molecular Weight; Parasites; Penetrance; Pharmaceutical Chemistry; Phenotype; Plasmodium; Plasmodium falciparum; Play; Process; Proteins; Proteomics; Protozoa; Publishing; Recording of previous events; Regulation; Reporter; Reporting; Research; Ribosomes; Sepharose; Specificity; Structure; Testing; Trypsin; Validation; Variant; Work; asexual; base; combinatorial; cyclopropene; design; drug development; enantiomer; improved; innovation; insight; interest; knowledge of results; novel; pharmacophore; tool; uptake

ABSTRACT The human malaria parasite Plasmodium falciparum imports and catabolizes massive amounts of host erythrocyte cytosol (mainly hemoglobin) during its asexual replication cycle. The mechanism and regulation of hemoglobin uptake, which occurs through a specialized protozoan structure termed the cytostome, is poorly understood. Here, we present new data which, together with prior published work, strongly suggests that the anti-malarial action of mefloquine (MQ) is closely associated with the inhibition of hemoglobin endocytosis. In this proposal, we will test the hypothesis that MQ binds to a protein target that plays a key role in the parasite’s endocytosis of host erythrocyte hemoglobin. In Aim 1, we will synthesize a library of photoaffinity probes that are based on the MQ pharmacophore. Probe design will focus on the incorporation of “all-in-one” moieties that enable the introduction of photoactivatable and affinity groups with minimal structural perturbation. Phenotypic and parasite viability assays will be employed to characterize probe potency and to verify retention of MQ’s mode of action. In Aim 2, these probes will be used to irreversibly label the protein target of MQ. Specificity of labeling will be assessed by competition with unmodified MQ and the characterization of purified enantiomers of selected probes. Putative targets will be affinity purified, identified by mass spectrometry, and validated using a suite of complementary approaches. These studies hold great promise to address the long-standing questions of the molecular mechanisms of hemoglobin endocytosis and of the mode of action of MQ, thereby identifying a potentially high-value anti-malarial target.

抽象的 人类疟疾寄生虫疟原虫进口和分解代谢 大量宿主红细胞细胞质（主要是血红蛋白） 无性复制周期。血红蛋白吸收的机制和调节， 通过称为细胞抑制剂的专门原生动物结构发生的是 理解不佳。在这里，我们提供了新数据，并与先验一起 出版的工作强烈暗示了甲氟喹的反疟疾作用 （MQ）与抑制血红蛋白内吞作用密切相关。在这个 提案，我们将检验以下假设，即MQ与扮演的蛋白质靶标结合 寄生虫宿主血红蛋白的内吞作用中的关键作用。在AIM 1中， 我们将综合基于MQ的光性问题库 药效团。探针设计将集中于“多合一” 能够引入光活化和亲和力群体的部分 最小的结构扰动。表型和寄生虫生存能力评估将是 用于表征探测效力并验证MQ模式的保留 行动。在AIM 2中，这些问题将用于不可逆地标记蛋白质靶标 MQ标签的特异性将通过未修改的竞争来评估 MQ和所选问题的纯化对映异构体的表征。假定 目标将被亲和力纯化，通过质谱识别并经过验证 使用一套完整的方法。这些研究充满希望 解决了血红蛋白分子机制的长期问题 内吞作用和MQ的作用方式，从而识别潜在的 高价值反疟疾目标。