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

描述（由申请人提供）：幽门螺杆菌感染胃粘膜仍然是一个世界性问题，并导致消化性溃疡和胃癌。如果不积极干预，发达国家至少20%的人口将继续受到这种胃病原体的感染。消灭这种生物将有助于预防疾病。目前的根除需要三联疗法，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率> 20%，且呈上升趋势。没有单一疗法是有效的。胃感染H. pylori依赖于该病原体特有的通道HpUreI的表达。HpUreI是尿素快速进入细菌内尿素酶所必需的质子门控尿素通道，对于在胃的酸性环境中维持周质在pH 6.1（低至pH 2.5）是必需的，从而允许胃的定殖。通道的表达在胃中增加。该通道具有195个残基，具有六个跨膜区段和三个周质区。突变研究表明，HpUreI的质子门控是由这三个周质区的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，我们确定了这个通道的三维结构，我们现在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。抑制这一通道将有望产生特异性和有效的单一疗法来根除微生物，并迎来一个测试和治疗的时代，而不仅仅是治疗有症状的患者。这将为严重的上消化道疾病，特别是每年造成约750 000人死亡的胃癌提供一种预防办法。我们的具体目标是1。产生更高分辨率的晶体，并使用新的N-或C-末端可切割的HpUreI 6 His-标签构建体分析HpUreI的开放和闭合形式。2.通过使用爪蟾卵母细胞和蛋白脂质体尿素转运试验以及在H. pylori ureI敲除菌株。3.通过突变六聚体的原聚体界面处保守的残基来分析六聚体排列的作用，以破坏原聚体缔合，然后使用爪蟾卵母细胞和脂蛋白体对单体状态进行活性测量。4.发现并改进基于开放式通道结构的小分子HpUreI抑制剂，测定其提高HpUreI Tm的能力，抑制完整H. pylori，并影响新型胃模拟孵育系统中的存活率。

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.