High Throughput Screen Development for Modulators of Heme Transporters

描述（由申请人提供）：为了维持其生长和繁殖，寄生虫表现出独特的适应性，使其能够从宿主单向获取营养。一个最好的例子是锥虫和蠕虫寄生虫利用的血红素获取途径-最初在遗传上易处理的蛔虫C中发现。优雅利用C.在线虫中，PI最近鉴定了HRG-1蛋白家族，这是第一个真正的后生动物血红素输入/转运蛋白。目前还没有药理学工具来帮助研究后生动物血红素转运蛋白的细胞和生理作用。在这里，我们建议开发和验证一种基于细胞的，HTS兼容的筛选原型C.使用简单的生长测定在酵母中表达的线虫血红素转运蛋白。初步的低通量测定表明，这种方法是可行的转化为HTS格式。一个强大的组合，在体外，细胞为基础的，和生理分析也到位，以确认和优先级的化合物，以这种方式确定。成功执行这一建议将需要两名研究人员在感兴趣的分子靶点和HTS开发方面的专业知识的合作。预计在该筛选中鉴定的试剂最终将用于确定靶向该途径治疗蠕虫感染、锥虫病、肠线虫病、动体病、淋巴丝虫病、盘尾丝虫病和利什曼病以及血红素和铁代谢的人类遗传性疾病的可行性。探索用于治疗此类疾病的新的选择性靶点特别令人感兴趣，因为目前使用的大多数药物要么过于昂贵，毒性高，要么已经促进了寄生虫抗性菌株的发展。 公共卫生相关性：我们建议开发一种用于高通量筛选血红素转运蛋白小分子拮抗剂的检测方法。血红素转运的研究对于治疗人类铁缺乏症（全球十大死亡因素之一）以及对抗人类和农业寄生虫具有重要意义。这些研究将极大地促进这种干扰配体的鉴定，目前这些目标都不存在。

DESCRIPTION (provided by applicant): To sustain their growth and reproduction, parasites exhibit distinct adaptations that allow them to acquire nutrients unidirectionally from the host. An example par excellence is the heme acquisition pathway exploited by trypanosomatid and helminth parasites - initially discovered in the genetically tractable roundworm C. elegans. Using C. elegans, the PI recently identified the HRG-1 family of proteins, the first bona fide metazoan heme importers/transporters. Currently there are no pharmacological tools to aid in the study of the cellular and physiological roles of metazoan heme transporters. Here, we propose the development and validation of a cell-based, HTS-compatible screen for small molecule antagonists of a prototypical C. elegans heme transporter expressed in yeast using a simple growth assay. Preliminary low-throughput assays suggest that this approach is feasible for conversion to an HTS format. A powerful combination of in vitro, cell-based, and physiological assays are also in place to confirm and prioritize compounds identified in this manner. Successful execution of this proposal will require the collaboration of two researchers having expertise in the molecular target of interest and HTS development. It is anticipated that reagents identified in this screen will ultimately be used to establish the feasibility of targeting this pathway for the treatment of helminth infections, Trypanosomiasis, intestinal nematodes, kinetoplastid diseases, lymphatic filariasis, onchocerciasis, and Leishmaniasis, as well as human genetic disorders of heme and iron metabolism. The exploration of new selective targets for the treatment of such diseases is of particular interest as most drugs currently in use are either prohibitively expensive, have high toxicity, or have already promoted the development of resistant strains of parasites. PUBLIC HEALTH RELEVANCE: We propose to develop an assay for high-throughput screening of small molecule antagonists of a heme transporter. The study of heme trafficking has great importance for the treatment of human iron deficiency, one of the top ten mortality factors worldwide, and for combating human and agricultural parasites. These studies will be greatly facilitated by the identification of such interfering ligands, none of which currently exist for these targets.