The Gastric Acid Pump as a Target for Ulcer Treatment

胃酸泵作为溃疡治疗的目标

• 批准号: 7216934
• 负责人: George Sachs
• 金额: $ 24.85万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-15 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7216934
• 关键词: antiulcer drug; benzimidazole analog; binding sites; chemical models; confocal scanning microscopy; enzyme activity; enzyme inhibitors; enzyme structure; gastric acid; hydrogen potassium exchanging ATPase; hydrogen transport; immunocytochemistry; laboratory mouse; laboratory rat; molecular dynamics; pharmacokinetics; protein protein interaction; site directed mutagenesis; western blottings

DESCRIPTION (provided by applicant): The gastric H+, K+ ATPase catalyzes the final step of gastric acid secretion thereby generating a proton gradient across the canalicular membrane of greater than a million fold. It has been a focus of this laboratory for 30 years. Our interest in recent years has been the structure-function relationships of this pump and the mechanism of inhibition of this pump by covalent inhibitors (the proton pump inhibitors, PPIs) and the K+ -competitive reversible inhibitors (the acid pump antagonists, APAs).In the proposed studies, we plan to continue our site-directed mutagenesis approach coupled with detailed homology analysis and modeling using the 4 available crystal structures of the SERCA Ca ATPase as a template to better define transport by the gastric acid pump. The Ca ATPase although only 29% homologous to the H+-K+-ATPase, has a very similar overall structure and also uses carboxylic acid clusters in the membrane domain as the ion transport- binding and export-import sites as does the Na, K+ ATPase that is 75% homologous to the H+,K+ ATPase. We plan to delineate pathways for transport of hydronium ion from cytoplasm to lumen and K+ from lumen to cytoplasm by analyzing various enzyme activities including phosphorylation and dephosphorylation of selected site mutants. We have developed a hypothesis that the unique Lys791 insertion into one cluster of acids (D814, E820, E795) allows release of proton at the required pH~1.0 and K+ binding to luminal carbonyl groups displaces two of the carboxylic acids bound to lys 791, thereby allowing return ofIys791 and replacement by K+ at this site. New mutants will further define the ion transport pathways of the H+,K+ ATPase by homology modeling. Since acid- pump antagonists are in final clinical trials, we plan to define their site of binding to the enzyme more precisely by synthesizing a new class of compounds and identifying the amino acid residues whose mutation alters the affinity or nature of inhibition by these new compounds as we have done for the now classical imidazo-1,2a prydine class that often show unexpected negative side effects. Since an important step of acid secretory regulation involves a morphological transformation of the parietal cell wherein the ATPase moves from a cytoplasmic membrane location to the microvilli of the secretory canaliculus, we will continue our study of trafficking and sorting of the stably expressed ¿ subunit of the enzyme in polarized gastric cells. In addition, we will study the distribution of a YFP- ¿ subunit knock in- construct in the mouse stomach, living mouse gastric glands and in other tissuess such as the kidney where the enzyme is expressed but function is unknown. The scaffold proteins interacting with the (3 subunit will be elucidated using the the split ubiquitin method which is capable of defining those proteins associated with a particular membrane inserted protein. These studies will aid in clarifying the role of translocation of the pump in regulation of acid secretion. The results of the proposed research will further improve the agents used for the treatment of acid-related diseases and also our knowledge of the ATPase and cellular events involved in regulation of its activity.

描述（由申请人提供）：胃 H+、K+ ATP 酶催化胃酸分泌的最后一步，从而在小管膜上产生大于 100 万倍的质子梯度，30 年来一直是我们实验室的重点。近年来人们感兴趣的是该泵的结构-功能关系以及共价抑制剂（质子泵抑制剂，PPI）和 K+ 抑制该泵的机制。 -竞争性可逆抑制剂（酸泵拮抗剂，APAs）。在拟议的研究中，我们计划继续我们的定点诱变方法，结合详细的同源性分析和建模，使用 SERCA Ca ATPase 的 4 种可用晶体结构作为模板更好地定义胃酸泵的转运，虽然 Ca ATP 酶与 H+-K+-ATP 酶只有 29% 同源，但具有非常相似的整体结构，并且也使用羧酸簇。膜结构域作为离子转运结合和输出-输入位点，Na、K+ ATP 酶与 H+、K+ ATP 酶同源性为 75%。通过分析各种酶活性，包括选定位点突变体的磷酸化和去磷酸化，我们提出了一种假设，即独特的 Lys791 插入到一个酸簇中。 (D814、E820、E795)允许在所需的pH〜1.0下释放质子，并且与管腔羰基结合的K+取代了与lys 791结合的两个羧酸，从而允许Lys791返回并在该位点被K+取代。由于酸泵拮抗剂正在进行最终的临床试验，我们计划通过同源模型进一步定义 H+、K+ ATP 酶的离子转运途径。通过合成一类新的化合物并鉴定其突变改变这些新化合物的亲和力或抑制性质的氨基酸残基，更精确地定义它们与酶的结合位点，就像我们对现在经典的咪唑-1,2a所做的那样由于酸分泌调节的一个重要步骤涉及壁细胞的形态转变，ATP酶从细胞质膜位置移动到分泌小管的微绒毛，因此吡啶类药物经常表现出意想不到的副作用。继续我们对稳定表达的贩运和分类的研究 ¿此外，我们将研究 YFP-¿ 的分布。亚基敲入构建体在小鼠胃、活体小鼠胃腺和其他组织（例如肾脏）中表达，但功能未知，将使用分裂泛素方法阐明与（3亚基）相互作用的支架蛋白。能够确定与特定插入膜蛋白相关的蛋白质。这些研究将有助于阐明泵的易位在酸分泌调节中的作用。所提出的研究结果将进一步改进用于治疗酸的药物。 -有关的疾病以及我们对 ATP 酶和参与其活性调节的细胞事件的了解。