Structure-Function Studies and Inhibitor Discovery Targeting the Proton-Gated Urea Channel and the Cytoplasmic Urease from H. Pylori

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 above 25% and rising and no monotherapy is effective. Gastric infection by H. pylori depends on the expression of a bacterial urea channel (HpUreI) and a cytoplasmic urease (HpUreAB) unique to this pathogen. HpUreI is a proton-gated urea channel necessary for rapid access of urea to the intra-bacterial urease, essential for maintaining the periplasm at pH 6.1 in the acidic environment of the stomach (as low as pH 1), thus enabling colonization of the stomach. The channel is a membrane protein with 195 residues in six transmembrane segments. Previously, we determined the 3-dimensional structure of this channel (3.2 Å) and of the downstream 1.1 MDa urease complex (1.98 Å), and have in this project exploited that knowledge to increase our understanding of the proton-gating mechanism and to identify binding modes of inhibitors, some of which have been discovered already. Inhibition of this channel or of the cytoplasmic urease complex will hopefully result in specific and effective monotherapy in the future 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. We have used CryoEM technology for structural analysis, and have determined the cryo-EM structure of Helicobacter pylori urease with an inhibitor in the active site at 2.0 Å resolution. We have further developed an easy method to test urease using E. coli cells, and identified several possible urease inhibitors able to penetrate the cytoplasm and act at low pH using the E. coli expressing H. pylori urease operon.

- We determined the cryo-EM structure of Helicobacter pylori urease with an inhibitor in the active site at 2.0 Å resolution. - We developed an easy method to test urease using E. coli cells. - We have identified several possible urease inhibitors able to penetrate the cytoplasm and act at low pH using the E. coli expressing H. pylori urease operon.

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 >25% and rising and no monotherapy is effective. Gastric infection by H. pylori depends on the expression of a bacterial urea channel (HpUreI) and a cytoplasmic urease (HpUreAB) unique to this pathogen. HpUreI is a proton-gated urea channel necessary for rapid access of urea to the intra-bacterial urease, essential for maintaining the periplasm at pH 6.1 in the acidic environment of the stomach (as low as pH 1), thus enabling colonization of the stomach. The channel is a membrane protein with 195 residues in six transmembrane segments. Previously, we determined the 3-dimensional structure of this channel (3.2 Å) and of the downstream 1.1 MDa urease complex (1.98 Å), and we now propose to exploit that knowledge to increase our understanding of the proton-gating mechanism and to identify binding modes of inhibitors, some of which have been discovered already. Inhibition of the cytoplasmic urease complex is 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.