title: Targeting cystalysin, a virulence factor of treponema denticola-supported periodontitis.
abstract: Cystalysin from Treponema denticola is a pyridoxal 5'-phosphate dependent lyase that catalyzes the formation of pyruvate, ammonia, and sulfide from cysteine. It is a virulence factor in adult periodontitis because its reaction contributes to hemolysis, which sustains the pathogen. Therefore, it was proposed as a potential antimicrobial target. To identify specific inhibitors by structure-based in silico methods, we first validated the crystal structure of cystalysin as a reliable starting point for the design of ligands. By using single-crystal absorption microspectrophotometry, we found that the enzyme in the crystalline state, with respect to that in solution, exhibits: 1) the same absorption spectra for the catalytic intermediates, 2) a close pKa value for the residue controlling the keto enamine ionization, and 3) similar reactivity with glycine, L-serine, L-methionine, and the nonspecific irreversible inhibitor aminoethoxyvinylglycine. Next, we screened in silico a library of 9357 compounds with the Fingerprints for Ligands and Proteins (FLAP) software, by using the three-dimensional structure of cystalysin as a template. From the library, 17 compounds were selected and experimentally evaluated by enzyme assays and spectroscopic methods. Two compounds were found to competitively inhibit recombinant T. denticola cystalysin, with inhibition constant (Ki ) values of 25 and 37 μM. One of them exhibited a minimum inhibitory concentration (MIC) value of 64 μg mL(-1) on Moraxella catarrhalis ATCC 23246, which proves its ability to cross bacterial membranes.
authors.affiliation: Department of Food Sciences, University of Parma, Parma (Italy); Current address: Department of Life Sciences, University of Modena and Reggio Emilia, Modena (Italy).
authors.forename: Francesca
authors.initials: F
authors.lastname: Spyrakis
authors.forename: Barbara
authors.initials: B
authors.lastname: Cellini
authors.forename: Stefano
authors.initials: S
authors.lastname: Bruno
authors.forename: Paolo
authors.initials: P
authors.lastname: Benedetti
authors.forename: Emanuele
authors.initials: E
authors.lastname: Carosati
authors.forename: Gabriele
authors.initials: G
authors.lastname: Cruciani
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Micheli
authors.forename: Antonio
authors.initials: A
authors.lastname: Felici
authors.forename: Pietro
authors.initials: P
authors.lastname: Cozzini
authors.forename: Glen E
authors.initials: GE
authors.lastname: Kellogg
authors.forename: Carla Borri
authors.initials: CB
authors.lastname: Voltattorni
authors.forename: Andrea
authors.initials: A
authors.lastname: Mozzarelli
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D020134:Catalytic Domain; D003542:Cystathionine gamma-Lyase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology / Q000627:therapeutic use; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006801:Humans; D008826:Microbial Sensitivity Tests; D062105:Molecular Docking Simulation; D010518:Periodontitis / Q000188:drug therapy; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use; D048373:Treponema denticola / Q000187:drug effects / Q000201:enzymology*
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; C400145:cystalysin; D003542:Cystathionine gamma-Lyase
keywords: antibiotic agents; crystal microspectrophotometry; cystalysin; enzyme inhibitors; in silico screening