Evaluation of Small Molecule Inhibitors of Hemoglobin Transport as Antimalarials

描述(由申请人提供)：我们开发了一种新的模型，用于在红细胞内恶性疟原虫中将血红蛋白从宿主红细胞胞浆内化和运输到消化液泡(DV)。用电子显微镜观察感染疟疾的红细胞(IRBC)的连续切片，发现细胞气孔延伸到寄生虫的消化液泡(DV)并与之融合，从而向寄生虫运送血红蛋白。这一过程依赖于肌动蛋白的动力学，因为稳定肌动蛋白细丝的试剂阻止了血红蛋白向DV的输送。此外，我们证明了将血红蛋白输送到DV是一个专有的过程，因为抑制这一过程会抑制寄生虫的发展并导致寄生虫死亡。最近，使用一种特定的小分子GTP酶抑制剂，我们破坏了细胞孔的形态，阻止了血红蛋白向DV的运输，从而阻止了寄生虫的发展。我们的新发现表明，抑制血红蛋白输送到DV对恶性疟原虫是致命的，结果证实这一过程是抗疟疾治疗的新靶点。我们试图利用我们的观察结果来开发一种体外分析方法来监测血红蛋白向DV的运输(作为读出)，并使用它来筛选小分子库中抑制这一过程和寄生虫发展的化合物。在进入体内测试之前，将进行小分子筛查阳性命中的体外验证实验。干扰血红蛋白运输系统是开发抗疟疾药物的一种独特方法。鉴于迫切需要新的药物靶标和抗疟疾药物，以及全世界数百万人深受疟疾之苦，该项目的潜在影响非常大。 公共卫生相关性：消化性空泡血红蛋白蛋白酶抑制剂正在被评估为抗疟疾药物。有报道指出，这些蛋白水解酶的功能存在很大的冗余性，而且似乎治疗需要抑制许多不同的蛋白水解酶。我们认为上游的细胞造口血红蛋白运输系统可能是一个更好的靶点。针对恶性疟原虫感染红细胞而不是宿主细胞中发生的独特过程的新靶向可能导致全新的抗疟疾化学类型，这些化学类型通过不受现有治疗药物交叉耐药性困扰的新机制发挥作用。如果确定了抑制剂，那么我们将有一个合理的目标，通过一个合理的过程获得，它可以发展成一个针对严重健康问题的药物发现计划。

DESCRIPTION (provided by applicant): We've developed a new model for the internalization and transport of hemoglobin from host erythrocyte cytosol to the digestive vacuole (DV) in intraerythrocytic Plasmodium falciparum malaria parasites. The examination of serial sections by electron microscopy of malaria infected erythrocytes (IRBC) revealed that cytostomes extend to and fuse with the parasite digestive vacuole (DV), thereby delivering hemoglobin to the parasite. This process was dependent on actin dynamics, as agents that stabilize actin filaments prevented the delivery of hemoglobin to the DV. Furthermore, we demonstrated that the delivery of hemoglobin to the DV was an obligate process, as inhibition of this process inhibited parasite development and caused parasite death. More recently, using a specific small molecule GTPase inhibitor, we disrupted the cytostome morphology and prevented hemoglobin transport to the DV, resulting in the arrest of parasite development. Our new discoveries reveal that the inhibition of hemoglobin delivery to the DV is lethal to P. falciparum parasites and as a result validated this process as a new target for antimalarial therapy. We seek to capitalize on our observations to develop an in vitro assay to monitor hemoglobin transport to the DV (as a read out) and use it to screen a small molecule library for compounds that inhibit this process and parasite development. Validation experiments in vitro of positive hits from the small molecule screen will be performed prior to moving into in vivo testing down the road. Perturbing the hemoglobin transport system represents a unique approach for developing antimalarials. Given the urgent need for new drug targets and antimalarials and the millions of people worldwide afflicted by malaria, the potential impact of this project is very high. PUBLIC HEALTH RELEVANCE: Inhibitors of digestive vacuole hemoglobin proteases are being evaluated as antimalarials. Reports indicate great functional redundancy in these proteases and it appears that therapy would need to inhibit many different proteases. We believe the upstream cytostomal hemoglobin transport system may be a better target. Novel targeting of a unique process that occurs in Plasmodium falciparum infected erythrocytes and not in host cells could lead to entirely new antimalarial chemotypes that act by novel mechanisms untroubled by cross resistance to existing therapeutics. If inhibitors are identified then we will have a rational target, obtained by a rational process, which can be developed into a drug discovery program for a serious health problem.