Giardia drug targets: Structure, function and inhibitors

描述(申请人提供)：蓝氏贾第鞭毛虫是一种在发展中国家流行的食物和水传播寄生虫，也是美国爆发腹泻的主要原因，已被疾病控制中心列为生物恐怖主义B类生物，因为水和食物供应受损可能会影响美国人口和外国军队。抗厌氧原虫的药物通常用于治疗贾第鞭毛虫病。然而，它们会产生不良的副作用，免疫功能低下和免疫功能正常的患者都可能出现临床耐药，复发率很高。该项目的目标是为贾第鞭毛虫病的替代治疗开发新的药物靶点。兰氏革兰氏菌基因组序列是可获得的，因此，已采取基因组学方法与生物学洞察力相结合的方法来鉴定对有机体的生存至关重要的酶，这些酶不存在或与人类酶有足够的差异，以利用抑制剂设计中的这些差异。所选择的酶将被克隆并在大肠杆菌中使用高通量技术表达，那些以可溶性形式表达的酶将通过转录干扰方法在兰氏革兰氏菌中得到验证。将为结构和功能研究准备必要的酶。晶体结构将被确定，并将用于指导针对贾第虫酶的抑制剂的设计。将开发测定酶的动力学常数的分析方法，并将研究催化机理，以便于在体外评价抑制剂。酶/抑制剂复合体的晶体结构将揭示如何进一步改进抑制剂。这些抑制剂对兰氏革兰氏菌生长的影响将被确定。该项目的长期目标是建立一个治疗贾第虫病的潜在药物靶点的数据库，了解它们的作用机制，并准备好准备进行临床前评估的抑制剂。

DESCRIPTION (provided by applicant): Giardia lamblia is a food and waterborne parasite prevalent in developing countries, and a major cause of outbreaks of diarrhea in the United Sates, that has been classified as a bioterrorism category B organism by the Center for Disease Control because compromised water and food supply could affect US populations as well as army units in foreign countries. Drugs commonly used against anaerobic protozoa are used to treat giardiasis. However, they produce undesirable side effects, clinical resistance may occur in both immunocompromised and immunocompetent patients, and the rate of recurrence is high. The goal of this project is to develop new drug targets for alternative treatments of giardiasis. The sequence of the G. lamblia genome is available, thus, a genomic approach combined with biological insight has been taken to identify enzymes essential for the survival of the organism that are absent or are sufficiently divergent from the human enzymes to exploit those differences in the design of inhibitors. The selected enzymes will be cloned and expressed in E. coli using high throughput techniques, and those that are expressed in soluble form will be validated in G. lamblia by transcription interference methods. Essential enzymes will be prepared for structural and functional studies. Crystal structures will be determined, and will serve to guide the design of inhibitors specific to the Giardia enzymes. Assays to determine the kinetic constants of the enzymes will be developed and the catalytic mechanism will be studied to facilitate the in vitro evaluation of the inhibitors. Crystal structures of enzyme/inhibitor complexes will reveal how the inhibitor may be further improved. The effect of the inhibitors on the growth of G. lamblia will be determined. The long range goals of the project is to establish a database of potential drug targets against giardiasis, to gain insight about their mechanism of action, and to prepare inhibitors that are ready for preclinical evaluation.