SEARCH FOR ANTI-AMOEBIC DRUGS W/ LESS TOXICITY IN HUMANS: ENTAMOEBA HISTOLYTICA

寻找对人类毒性较小的抗阿米巴药物：溶组织阿米巴

• 批准号: 7960133
• 负责人: AVELINA ESPINOSA
• 金额: $ 8.02万
• 依托单位: UNIVERSITY OF RHODE ISLAND
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-04 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7960133
• 关键词: Acetates; Affect; Amebiasis; Animal Model; Behavioral Research; Carbon Dioxide; Cell Culture Techniques; Chemicals; Computer Retrieval of Information on Scientific Projects Database; Computer Simulation; Crystallography; Cyst; Disease; Drug toxicity; EhADH2 enzyme; Entamoeba histolytica; Enzymes; Escherichia coli; Ethanol; Fermentation; Food; Funding; Glucose; Goals; Grant; Growth; Human; In Vitro; Ingestion; Institution; Kinetics; Large Intestine; Lethal Dose 50; Life Cycle Stages; Mitochondria; Pharmaceutical Preparations; Procedures; Research; Research Personnel; Resources; SCID Mice; Source; Staging; Study models; Techniques; Testing; Toxic effect; United States National Institutes of Health; Water; antimicrobial; design; in vivo; inhibitor/antagonist; killings; mutant; pathogen; trait

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Anaerobic pathogens have acquired traits to survive and thrive in host guts. As a model, we study the causative agent of human amebiasis, Entamoeba histolytica. This disease affects 50 million people yearly with 100 thousand fatalities worldwide. Entamoeba histolytica's life cycle consists of the disease-causing trophozoite stage and the infectious cyst stage. Cysts are acquired by ingestion of contaminated food or water, and multiplication and differentiation of the emergent trophozoites happen within the human's large intestine. Entamoeba histolytica lacks mitochondria and obtains its energy from the fermentation of glucose, producing carbon dioxide, acetate, and ethanol. The bifunctional Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2) is essential for the growth and survival of E. histolytica trophozoites. That their evolutionary origin apparently differs from that of vertebrate enzymes designates them as appealing antimicrobial targets. The goal of this study is to search for alternative non-toxic drugs that inhibit the growth of E. histolytica trophozoites, by specifically affecting the EhADH2 enzyme. Animal models will be used to determine the toxicity of these drugs in humans. Three specific aims will help achieve this goal: (1) Identify candidate chemicals that inhibit EhADH2. First, test potential inhibitors in vitro using an Escherichia coli AdhE deficient strain, and later in vivo using Entamoeba histolytica trophozoites. Useful candidates should inhibit the enzymatic activity of EhADH2, killing trophozoites with little or no effect to mammalian enzymes. (2) The selected chemicals will be further analyzed in cell cultures. To evaluate the toxicity of these drugs, various concentrations of drugs will be tested in animal models (SCID mice). LD50 standard procedures will be used. (3) Further analysis of EhADH2 will aid in designing anti-amoebic agents. EhADH2 (wild-type, mutants) will be analyzed with identified and designed inhibitors, using classical kinetics, spectrophotometer, computer modeling, and x-ray crystallography techniques.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 中心，不一定是研究者的机构。 厌氧病原体已获得在宿主肠道中生存和繁衍的特征。作为模型，我们研究人类阿米巴病的病原体溶组织内阿米巴。这种疾病每年影响 5000 万人，全世界有 10 万人死亡。溶组织内阿米巴的生命周期由致病滋养体阶段和传染性包囊阶段组成。囊肿是通过摄入受污染的食物或水而获得的，新生滋养体的增殖和分化发生在人类的大肠内。溶组织内阿米巴缺乏线粒体，通过葡萄糖发酵获取能量，产生二氧化碳、乙酸和乙醇。双功能溶组织阿米巴乙醇脱氢酶 2 (EhADH2) 对于溶组织阿米巴滋养体的生长和存活至关重要。它们的进化起源明显不同于脊椎动物酶，这使得它们成为有吸引力的抗菌靶点。本研究的目的是寻找替代的无毒药物，通过特异性影响 EhADH2 酶来抑制溶组织内阿米巴滋养体的生长。动物模型将用于确定这些药物对人类的毒性。三个具体目标将有助于实现这一目标：(1) 确定抑制 EhADH2 的候选化学物质。首先，使用大肠杆菌 AdhE 缺陷菌株在体外测试潜在的抑制剂，然后使用溶组织内阿米巴滋养体在体内测试潜在的抑制剂。有用的候选者应该抑制 EhADH2 的酶活性，杀死滋养体，而对哺乳动物酶影响很小或没有影响。 (2) 所选化学物质将在细胞培养物中进一步分析。为了评估这些药物的毒性，将在动物模型（SCID 小鼠）中测试不同浓度的药物。将使用 LD50 标准程序。 (3)对EhADH2的进一步分析将有助于设计抗阿米巴药物。将使用经典动力学、分光光度计、计算机建模和 X 射线晶体学技术，使用已识别和设计的抑制剂对 EhADH2（野生型、突变体）进行分析。