title: In Silico Discovery and In Vitro Validation of Catechol-Containing Sulfonohydrazide Compounds as Potent Inhibitors of the Diguanylate Cyclase PleD.
abstract: Biofilm formation is responsible for increased antibiotic tolerance in pathogenic bacteria. Cyclic di-GMP (c-di-GMP) is a widely used second-messenger signal that plays a key role in bacterial biofilm formation. c-di-GMP is synthesized by diguanylate cyclases (DGCs), a conserved class of enzymes absent in mammals and hence considered attractive molecular targets for the development of antibiofilm agents. Here, the results of a virtual screening approach aimed at identifying small-molecule inhibitors of the DGC PleD from Caulobacter crescentus are described. A three-dimensional (3D) pharmacophore model, derived from the mode of binding of GTP to the active site of PleD, was exploited to screen the ZINC database of compounds. Seven virtual hits were tested in vitro for their ability to inhibit the activity of purified PleD by using circular dichroism spectroscopy. Two drug-like molecules with a catechol moiety and a sulfonohydrazide scaffold were shown to competitively inhibit PleD at the low-micromolar range (50% inhibitory concentration [IC50] of ∼11 μM). Their predicted binding mode highlighted key structural features presumably responsible for the efficient inhibition of PleD by both hits. These molecules represent the most potent in vitro inhibitors of PleD identified so far and could therefore result in useful leads for the development of novel classes of antimicrobials able to hamper biofilm formation.
  
  
  OBJECTIVE
  Biofilm-mediated infections are difficult to eradicate, posing a threatening health issue worldwide. The capability of bacteria to form biofilms is almost universally stimulated by the second messenger c-di-GMP. This evidence has boosted research in the last decade for the development of new antibiofilm strategies interfering with c-di-GMP metabolism. Here, two potent inhibitors of c-di-GMP synthesis have been identified in silico and characterized in vitro by using the well-characterized DGC enzyme PleD from C. crescentus as a structural template and molecular target. Given that the protein residues implied as crucial for enzyme inhibition are found to be highly conserved among DGCs, the outcome of this study could pave the way for the future development of broad-spectrum antibiofilm compounds.
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Fernicola
authors.affiliation: Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Paiardini
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Giardina
authors.affiliation: Department of Science, University Roma Tre, Rome, Italy.
authors.forename: Giordano
authors.initials: G
authors.lastname: Rampioni
authors.affiliation: Department of Science, University Roma Tre, Rome, Italy.
authors.forename: Livia
authors.initials: L
authors.lastname: Leoni
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Cutruzzolà
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy serena.rinaldo@uniroma1.it.
authors.forename: Serena
authors.identifier: http://orcid.org/0000-0003-0682-023X
authors.initials: S
authors.lastname: Rinaldo
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018441:Biofilms / Q000254:growth & development; D002396:Catechols / Q000737:chemistry*; D016935:Caulobacter crescentus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D055808:Drug Discovery / Q000379:methods*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008954:Models, Biological; D008958:Models, Molecular; D019761:Phosphorus-Oxygen Lyases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D001426:Bacterial Proteins; D002396:Catechols; D029968:Escherichia coli Proteins; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase