Targeting Helicobacter pylori urease activity and maturation: In-cell high-throughput approach for drug discovery

• Published in: Biochimica et biophysica acta. General subjects Vol. 1862; no. 10; pp. 2245 - 2253
• Main Authors: Tarsia, Cinzia, Danielli, Alberto, Florini, Francesca, Cinelli, Paolo, Ciurli, Stefano, Zambelli, Barbara
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2018
• Subjects: antibiotics; bacteria; catalytic activity; cellular microenvironment; colorimetry; drug design; Drug screening; drugs; enzyme activity; enzyme inhibition; Enzyme inhibitors; Escherichia coli; Helicobacter pylori; human population; Nickel delivery; operon; peptides; plasmids; Protein-protein interactions; proteins; screening; stomach neoplasms; Urease; urease inhibitors; Urease; Enzyme inhibitors; Helicobacter pylori; Nickel delivery; Drug screening; Protein-protein interactions
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2018.07.020

Helicobacter pylori is a bacterium strongly associated with gastric cancer. It thrives in the acidic environment of the gastric niche of large portions of the human population using a unique adaptive mechanism that involves the catalytic activity of the nickel-dependent enzyme urease. Targeting urease represents a key strategy for drug design and H. pylori eradication. Here, we describe a novel method to screen, directly in the cellular environment, urease inhibitors. A ureolytic Escherichia coli strain was engineered by cloning the entire urease operon in an expression plasmid and used to test in-cell urease inhibition with a high-throughput colorimetric assay. A two-plasmid system was further developed to evaluate the ability of small peptides to block the protein interactions that lead to urease maturation. The developed assay is a robust cellular model to test, directly in the cell environment, urease inhibitors. The efficacy of a co-expressed peptide to affect the interaction between UreF and UreD, two accessory proteins necessary for urease activation, was observed. This event involves a process that occurs through folding upon binding, pointing to the importance of intrinsically disordered hot spots in protein interfaces. The developed system allows the concomitant screening of a large number of drug candidates that interfere with the urease activity both at the level of the enzyme catalysis and maturation. As inhibition of urease has the potential of being a global antibacterial strategy for a large number of infections, this work paves the way for the development of new candidates for antibacterial drugs. [Display omitted] •An in-cell method to screen drug candidates against the activity of Helicobacter pylori urease was developed and tested.•The screen evaluates urease activity in a engineered ureolytic Escherichia coli strain using a colorimetric assay.•The efficacy of inhibitors of the urease catalytic activity was confirmed in cell.•A two-plasmid system was set up to co-express peptide sequences along with the urease operon.•Urease maturation is impaired in cell by a peptide that drugs protein interactions among the urease accessory proteins.