title: High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases.
abstract: The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland College Park , 3114 Bioscience Research Building, College Park, Maryland 20742, United States.
authors.forename: Ori J
authors.initials: OJ
authors.lastname: Lieberman
authors.forename: Mona W
authors.initials: MW
authors.lastname: Orr
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Vincent T
authors.initials: VT
authors.lastname: Lee
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001393:Azoles / Q000737:chemistry / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects / Q000254:growth & development; D002197:Capillary Action; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D054833:Isoindoles; D008024:Ligands; D008958:Models, Molecular; D016566:Organoselenium Compounds / Q000737:chemistry / Q000494:pharmacology*; D019761:Phosphorus-Oxygen Lyases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D011552:Pseudomonas Infections / Q000188:drug therapy / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000201:enzymology* / Q000502:physiology
chemical_list: D000900:Anti-Bacterial Agents; D001393:Azoles; D029968:Escherichia coli Proteins; D054833:Isoindoles; D008024:Ligands; D016566:Organoselenium Compounds; C042986:ebselen; C062025:bis(3',5')-cyclic diguanylic acid; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP