Interrogating plasmodial endocytosis with mefloquine-based affinity probes

摘要 人类疟疾寄生虫恶性疟原虫输入并分解代谢 大量的宿主红细胞胞浆（主要是血红蛋白）在其 无性繁殖周期血红蛋白摄取的机制和调节， 通过一种称为细胞口的特殊原生动物结构发生， 不太了解。在这里，我们提出了新的数据，连同以前的 发表的工作，强烈表明，抗疟疾行动的甲氟喹， (MQ)与抑制血红蛋白内吞作用密切相关。在这 建议，我们将测试的假设，MQ结合蛋白质的目标，发挥作用， 在寄生虫内吞宿主红细胞血红蛋白中起关键作用。在目标1中， 我们将合成基于MQ的光亲和探针库， 药效团探头设计将侧重于纳入“一体化” 能够引入光活化和亲和基团的部分， 最小的结构扰动将进行表型和寄生虫活力测定， 用于表征探针效力并验证MQ的模式保留， 行动上在目标2中，这些探针将用于不可逆地标记蛋白质靶标 的MQ。将通过与未修饰的竞争来评估标签的特异性 MQ和所选探针的纯化对映体的表征。推定 目标将被亲和纯化，通过质谱鉴定，并验证 使用一套互补的方法。这些研究很有希望 解决长期存在的血红蛋白分子机制问题 内吞作用和MQ的作用方式，从而确定潜在的 高价值抗疟疾靶点。

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.