High Throughput Assay to Identify New Drugs Against Vivax Malaria Liver Stages

描述(申请人提供)：间日疟原虫(PV)是第二大人类疟疾寄生虫，每年导致8000多万例疟疾病例，以及以前未被认识到的重大严重疾病和死亡。最近的数据表明，小儿麻痹症疟疾对发展中国家卫生和经济的影响被大大低估了。在美国旅行者中，PV至少造成了30%的疟疾。努力预防 和控制PV疟疾的成功程度低于预期，原因是出现了耐药菌株，以及被称为催眠药的休眠肝期反复复发。国际疟疾研究界最近强调，如果要成功消除这种寄生虫，迫切需要增加对光伏病毒的研究。迫切需要现代方法来开发包括催眠药在内的治疗PV的新药。Sanaria是世界上唯一一家拥有功能测试的实验室，用于研究抗PV肝期药物的影响和可用于此类测试的PV子孢子库(PvSPZ)。许多药物文库可用于筛选，但由于缺乏中高通量分析，无法针对PV进行筛选。这一阶段SBIR的目标是在我们目前的PvSPZ检测和库存的基础上开发这样一种检测方法，启动针对PV肝脏阶段的药物筛选，包括催眠药。长期目标是使用该系统识别和开发新药，以消除所有PV肝期寄生虫，包括催眠虫。这些药物将是旅行者疟疾化学预防的理想选择，也是大规模管理从特定地理区域消除PV的理想选择，因此发达国家和发展中国家的市场估计每年都超过10亿美元。具体地说，我们将：1)开发一种可重复性、一致性、健壮的培养肝期PV的系统，并用它来筛选针对肝期PV的药物。这将包括评估使用HepG2细胞、原代人类肝细胞(PHH)和诱导多能干细胞来源的人肝细胞(IPSDH)来建立基于细胞的最佳筛选系统。2)与麻省理工学院的Bhatia实验室合作，开发了一种可重复的、一致的、稳健的培养PV肝脏阶段至少3-6周的系统，以识别和丰富催眠菌和 筛选抗PV催眠药活性的药物。使用PHH或IPSDH的微图案化共培养系统正在优化以实现这一目标。3)与NIH化学基因组学中心(NCGC)的Inglese团队合作，使用在特定目标1和2中开发的方法来筛选2,500个化合物库，以确定具有抗肝期寄生虫(包括催眠虫)活性的目标化合物。4)与NIAID/NIH的Wellems小组合作，生产、纯化和冷冻保存至少2x108个完全感染的PvSPZ。2010年，我们与Wellems小组合作，生产了108个完全具有传染性的PvSPZ。这些额外的PvSPZ将用于第二阶段，对大型化合物库进行全面筛选，以识别、表征和开发先导化合物，以开发用于抗肝病阶段的药物。1

DESCRIPTION (provided by applicant): Plasmodium vivax (Pv) is the 2nd most important human malaria parasite, causing more than 80 million cases annually, and significant severe disease and mortality that were previously unrecognized. Recent data indicate that the impact of Pv malaria on health and economies of the developing world has been dramatically underestimated. Pv is responsible for at least 30% of malaria in U.S. travelers. Efforts to prevent and control Pv malaria have had less than expected success due to emergence of drug resistant strains and repeated relapses from dormant liver stage called the hypnozoite. The international malaria research community has recently emphasized the critical need for increased research on Pv, if there is be successful elimination of this parasite. Modern approaches to development of new drugs against Pv, including hypnozoites are urgently needed. Sanaria is the only laboratory in the world, which has a functional assay in place for studying the effects of drugs against Pv liver stages and inventory of Pv sporozoites (PvSPZ) that can be used in such an assay. Many drug libraries are available for screening, but cannot be screened against Pv, because of lack of a medium or high throughput assay. The goal of this Phase I SBIR is to build on our current assay and inventory of PvSPZ to develop such an assay, initiate the screen for drugs against the liver stages of Pv, including hypnozoites. The long-term goal is to use the system to identify and develop new drugs that will eliminate all Pv liver stage parasites, including hypnozoites. Such drugs will be ideal for chemoprophylaxis of malaria in travelers and for mass-administration to eliminate Pv from defined geographic areas, therefore having both developed- and developing-world markets estimated to exceed $1 billion annually. Specifically we will: 1) Develop a reproducible, consistent, robust system for culturing liver stages of Pv and use it to screen for drugs against the liver stages of Pv. This will include assessing the use of HepG2 cells, primary human hepatocytes (PHH), and induced pluripotent stem cell-derived human hepatocytes (iPSDH) to establish an optimal cell- based screening system. 2) In collaboration with the Bhatia lab at MIT develop a reproducible, consistent, robust system of culturing Pv liver stages for at least 3-6 wks to identify and enrich for hypnozoites and screen drugs for activity against Pv hypnozoites. A micro-patterned co-culture system using PHH or iPSDH is being optimized to attain this goal. 3) In collaboration with the Inglese group at the NIH Chemical Genomics Center (NCGC) use the methods developed in Specific Aims 1 and 2 to screen a 2,500 compound library to identify target compounds with activity against liver stage parasites, including hypnozoites. 4) In collaboration with the Wellems group at NIAID/NIH produce, purify, and cryopreserve at least 2x108 fully infectious PvSPZ. In 2010, in collaboration with the Wellems group, we produced 108 fully infectious PvSPZ. These additional PvSPZ will be used in Phase II to conduct full scale screening of large compound libraries to identify and then characterize and develop lead compounds for development as anti-liver stage drugs. 1