Giardia drug targets: Structure, function and inhibitors

描述（由申请人提供）：兰氏贾第虫是一种在发展中国家流行的食物和水传播寄生虫，也是美国腹泻爆发的主要原因，已被疾病控制中心列为生物恐怖主义B类生物，因为受损的水和食物供应可能影响美国人口以及外国军队。通常用于抗厌氧原生动物的药物被用于治疗贾第虫病。然而，它们会产生不良的副作用，在免疫功能低下和免疫功能正常的患者中都可能出现临床耐药性，并且复发率很高。该项目的目标是为贾第虫病的替代治疗开发新的药物靶点。兰氏螺旋体基因组的序列是可用的，因此，结合生物学见解的基因组方法已被用于识别生物体生存所必需的酶，这些酶缺失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。所选择的酶将通过高通量技术在大肠杆菌中克隆和表达，而那些以可溶性形式表达的酶将通过转录干扰方法在兰氏杆菌中进行验证。必要的酶将准备用于结构和功能研究。晶体结构将被确定，并将用于指导贾第鞭毛虫酶特异性抑制剂的设计。将建立测定酶的动力学常数的方法，并研究其催化机制，以促进抑制剂的体外评价。酶/抑制剂复合物的晶体结构将揭示如何进一步改进抑制剂。测定抑制剂对兰氏杆菌生长的影响。该项目的长期目标是建立一个针对贾第虫病的潜在药物靶点数据库，深入了解其作用机制，并准备用于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.