Targeting Cystalysin, a Virulence Factor of Treponema denticola-Supported Periodontitis

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 pK(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 mu m. One of them exhibited a minimum inhibitory concentration (MIC) value of 64 mu g mL(-1) on Moraxella catarrhalis ATCC 23246, which proves its ability to cross bacterial membranes.