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: Francesca Spyrakis, Barbara Cellini, Stefano Bruno, Paolo Benedetti, Emanuele Carosati, Gabriele Cruciani, Fabrizio Micheli, Antonio Felici, Pietro Cozzini, Glen E Kellogg, Carla Borri Voltattorni, Andrea Mozzarelli
• Journal: ChemMedChem (ChemMedChem) Vol. 9 Issue 7 Pg. 1501-11 (Jul 2014) ISSN: 1860-7187 [Electronic] Germany
• PMID: 24616267 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemical References

• Anti-Bacterial Agents
• Enzyme Inhibitors
• Recombinant Proteins
• Small Molecule Libraries
• cystalysin
• Cystathionine gamma-Lyase

Topics

• Anti-Bacterial Agents (chemistry, pharmacology)
• Binding Sites
• Catalytic Domain
• Cystathionine gamma-Lyase (antagonists & inhibitors, genetics, metabolism)
• Enzyme Inhibitors (chemistry, pharmacology, therapeutic use)
• Gram-Negative Bacteria (drug effects)
• Gram-Positive Bacteria (drug effects)
• Humans
• Microbial Sensitivity Tests
• Molecular Docking Simulation
• Periodontitis (drug therapy)
• Recombinant Proteins (biosynthesis, chemistry, genetics)
• Small Molecule Libraries (chemistry, pharmacology, therapeutic use)
• Treponema denticola (drug effects, enzymology)