Structure-Function Studies of the Proton-Gated Urea Channel from H. Pylori

幽门螺杆菌质子门控尿素通道的结构功能研究

• 批准号: 8579827
• 负责人: HARTMUT LUECKE
• 金额: $ 46.25万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8579827
• 关键词: Affect; Affinity; Amides; Amino Acids; Antibiotic Therapy; Antibiotics; Architecture; Asthma; Binding; Biological Assay; C-terminal; Carboxylic Acids; Cessation of life; Charge; Chronic; Clarithromycin; Complement; Complement Inactivators; Developed Countries; Development; Discrimination; Drug Targeting; Duodenal Ulcer; Environment; Failure; Funding; Gastric Adenocarcinoma; Gastric mucosa; Gastritis; Gastroesophageal reflux disease; Goals; Helicobacter Infections; Helicobacter pylori; Histidine; Homo; Hydrogen Bonding; Incidence; Individual; Infection; Intervention; Intestinal Diseases; Knock-out; Knowledge; Lead; Measurement; Measures; Meta-Analysis; Metronidazole; Metronidazole resistance; Molecular Conformation; Mutagenesis; Mutate; Mutation; Organism; Outcome; Oxygen; Pathway interactions; Patients; Peptic Ulcer; Pharmaceutical Preparations; Point Mutation; Population; Positioning Attribute; Preventive; Protomer; Proton Pump Inhibitors; Protons; Research; Resistance; Resolution; Role; Side; Site; Specificity; Stomach; Stomach Carcinoma; Stomach Diseases; Structure; Structure-Activity Relationship; Surface; System; Tandem Repeat Sequences; Testing; Thiourea; Thrombocytopenic Purpura; Urea; Urease; Work; Xenopus oocyte; acid stress; base; design; disorder prevention; field study; high throughput screening; improved; infectious disease treatment; inhibitor/antagonist; innovation; malignant stomach neoplasm; microbiome; mimetics; mucosa-associated lymphoid tissue lymphoma; mutant; novel; pathogen; periplasm; prevent; proteoliposomes; public health relevance; small molecule; success; three dimensional structure; urea transporter

DESCRIPTION (provided by applicant): Infection of the gastric mucosa by Helicobacter pylori remains a worldwide problem and contributes to peptic ulcer disease and gastric cancer. Without active intervention, at least 20% of the population of developed countries will continue to be infected by this gastric pathogen. Eradication of the organism would contribute to prevention of disease. Current eradication requires triple therapy, a proton-pump inhibitor and two antibiotics given twice a day for 10 to 14 days. Resistance to either clarithromycin or metronidazole is > 20% and rising. No monotherapy is effective. Gastric infection by H. pylori depends on the expression of a channel unique to this pathogen, HpUreI. HpUreI is a proton-gated urea channel necessary for rapid access of urea to intrabacterial urease, essential for maintaining the periplasm at pH 6.1 in the acidic environment of the stomach, as low as pH 2.5, thus allowing colonization of the stomach. Expression of the channel is increased in the stomach. The channel has 195 residues with six transmembrane segments and three periplasmic regions. Mutagenesis studies have shown that the proton gating of HpUreI is regulated by hydrogen bonding between histidines and carboxylic acid residues in these three periplasmic regions. In the first funding cycle we determined the 3-dimensional structure of this channel, and we now propose to exploit that knowledge to increase our understanding of its proton-gating mechanism and to identify binding modes of inhibitors, some of which have been discovered already. Inhibition of this channel would be expected to result in specific and effective monotherapy for eradication of the organism and usher in an era of test and treat, rather than only treating symptomatic patients. This would provide a preventive approach to serious upper gastro-intestinal diseases, particularly stomach cancer, which causes approximately 750,000 deaths annually. Our specific aims are 1. Generate higher-resolution crystals and analyze the open and closed forms of HpUreI using new N- or C-terminal cleavable 6His- tag constructs of HpUreI. 2. Confirm the urea pathway and selectivity filter suggested by our HpUreI crystal structure by determining the effects of site-directed mutations on transport and on substrate selectivity of HpUreI using Xenopus oocyte and proteoliposome assays for urea transport as well as mutants expressed in H. pylori ureI knock-out strains. 3. Analyze the role of the hexameric arrangement by mutating residues conserved at the protomer interface of the hexamer to disrupt protomer association, followed by activity measurements of the monomeric state using Xenopus oocytes and proteoliposomes. 4. Discover and improve small molecule HpUreI inhibitors based on the structure of channel in the open form; measure their ability to increase the Tm of HpUreI, to inhibit urease activity in intact H. pylori and to affect survival in a novel gastro-mimetic incubation system.

描述（申请人提供）：幽门螺杆菌感染胃粘膜是一个世界性的问题，并导致消化性溃疡疾病和胃癌。如果不积极干预，发达国家至少有20%的人口将继续感染这种胃病原体。消灭这种有机体将有助于预防疾病。目前的根除需要三联疗法，一种质子泵抑制剂和两种抗生素，每天两次，持续10至14天。对克拉霉素或甲硝唑的耐药性为20%，并且还在上升。没有单一疗法是有效的。幽门螺杆菌引起的胃感染取决于这种病原体特有的通道HpUreI的表达。HpUreI是一个质子门控的尿素通道，是尿素快速进入细菌内脲酶所必需的，对于维持胃酸性环境中pH为6.1（低至pH为2.5）的周质至关重要，从而允许胃定植。该通道的表达在胃中增加。该通道有195个残基，6个跨膜段和3个质周区。诱变研究表明，HpUreI的质子门控是由这三个质周区组氨酸和羧酸残基之间的氢键调节的。在第一个资助周期中，我们确定了该通道的三维结构，现在我们建议利用这些知识来增加我们对其质子门控机制的理解，并确定抑制剂的结合模式，其中一些已经被发现。抑制这一通道有望产生特异性和有效的单一疗法，以根除该生物体，并迎来一个检测和治疗的时代，而不仅仅是治疗有症状的患者。这将为预防严重的上消化道疾病，特别是每年造成约75万人死亡的胃癌提供一种方法。我们的具体目标是1。利用HpUreI的N端或c端可切割6His-标签构建更高分辨率的晶体，并分析HpUreI的开放和封闭形式。2. 通过确定位点定向突变对HpUreI转运和底物选择性的影响，以及在幽门螺杆菌ureI敲除菌株中表达的突变体，我们的HpUreI晶体结构建议的尿素途径和选择性过滤器。3. 通过突变原聚体界面上保守的残基来破坏原聚体结合，分析六聚体排列的作用，然后使用爪蟾卵母细胞和蛋白脂质体测量单体状态的活性。4. 基于开放通道结构的HpUreI小分子抑制剂的发现与改进；测量它们增加HpUreI Tm的能力，抑制完整幽门螺杆菌中脲酶活性的能力，以及影响新型模拟胃孵育系统中存活的能力。