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Targeting Cystalysin, a Virulence Factor of Treponema denticola‐ Supported Periodontitis
Author(s) -
Spyrakis Francesca,
Cellini Barbara,
Bruno Stefano,
Benedetti Paolo,
Carosati Emanuele,
Cruciani Gabriele,
Micheli Fabrizio,
Felici Antonio,
Cozzini Pietro,
Kellogg Glen E.,
Voltattorni Carla Borri,
Mozzarelli Andrea
Publication year - 2014
Publication title -
chemmedchem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.817
H-Index - 100
eISSN - 1860-7187
pISSN - 1860-7179
DOI - 10.1002/cmdc.201300527
Subject(s) - treponema denticola , chemistry , docking (animal) , biochemistry , virulence , lyase , active site , virulence factor , enzyme , stereochemistry , bacteria , biology , porphyromonas gingivalis , medicine , genetics , nursing , gene
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 p K a 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 ( K i ) values of 25 and 37 μ M . 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.