Biofilm Lysine Decarboxylase, a New Therapeutic Target for Periodontal Inflammation.

BACKGROUND: Lysine, a nutritionally essential amino acid, enters the oral cavity in gingival crevicular fluid (GCF). During oral hygiene restriction (OHR), lysine decarboxylase (LDC) in dento-gingival biofilms converts lysine to cadaverine. Lysine depletion impairs the dental epithelial barrier to bacterial proinflammatory products. Antibodies to LDC from Eikenella corrodens (Ecor-LDC) inhibit LDC activity and retard gingival inflammation in beagle dogs. Whether E. corrodens is the major source of LDC in dental biofilms and whether the lysine analog tranexamic acid (TA) inhibits LDC activity, biofilm accumulation, and GCF exudation in a human gingivitis model were examined. METHODS: Antibodies raised in goats to LDC-rich extracts from E. corrodens cell surfaces were used to inhibit Ecor-LDC and detect it in biofilm extracts using Western blots. Ecor-LDC activity was measured at pH 4.0 to 11.0 and its TA dissociation constant (Ki) at pH 7.0. Young adults used a 5% or 10% TA mouthwash three times daily during OHR for 1 week. RESULTS: Ecor-LDC antibodies and TA inhibited biofilm LDC. Ki of TA for Ecor-LDC was 940 µM. TA reduced plaque index (PI) by downshifting the PI correlation with biofilm lysine content after OHR without TA. GCF was correspondingly suppressed. However, greater TA retention in saliva partially relieved GCF suppression but not biofilm lysine depletion. CONCLUSIONS: TA slightly inhibits LDC but strongly reduces biofilm by inhibiting bacterial lysine uptake. Unfortunately, TA may impair dental epithelial attachments by also inhibiting lysine transporter uptake. Ecor-LDC inhibitors other than lysine analogs may maintain sufficient lysine levels and attachment integrity to prevent periodontal inflammation.

Luciferase inhibition by a novel naphthoquinone.

The novel naphthoquinone 12,13-dihydro-N-methyl-6,11,13-trioxo-5H-benzo[4,5]cyclohepta[1,2-b]naphthalen-5,12-imine (hereafter called TU100) was created as a potential chemotherapeutic agent. Previous work showed it is an irreversible inhibitor of type I and II topoisomerases that alkylates specific enzyme thiols. While analyzing the effect of TU100 on cancer cells, we discovered it is a potent inhibitor of luciferase derived from both Photinus pyralis (fireflies) and Renilla reniformis (sea pansy). Pre-incubation experiments showed that TU100 does not irreversibly inactivate luciferase, indicating its mechanism is different from that observed with topoisomerases. Firefly luciferase generates light using ATP and luciferin as substrates (bioluminescence). An examination of TU100 inhibition at varying substrate concentrations revealed the drug is uncompetitive with respect to ATP and competitive with respect to luciferin. The TU100 binding constant (K(I)) is 2.5±0.7 µM as determined by Dixon plot analysis. These data suggest TU100 specifically binds the luciferase-ATP complex and prevents its interaction with luciferin. Given the novel structure of TU100, unique mechanism of action, and ability to target luciferase from different species, these results identify TU100 as an important new reagent for investigating and regulating bioluminescent enzymes.

Induction of cell death by a novel naphthoquinone containing a modified anthracycline ring system.

The novel naphthoquinone adduct 12,13-Dihydro-N-methyl-6,11,13-trioxo-5H-benzo[4,5]cyclohepta[1,2-b]naphthalen-5,12-imine (hereafter called TU100) was synthesized as a potential chemotherapeutic agent. TU100 arrests tissue culture cells in S and G2/M phases of the cell cycle, followed by rapid induction of apoptosis. Evaluation by the Developmental Therapeutics Program at the National Cancer Institute revealed TU100 differentially inhibits growth of tissue-specific human cancer cell lines and has in vivo efficacy in a hollow fiber assay. These data were evaluated against previously analyzed compounds using the COMPARE algorithm and predicted that TU100 has a unique mechanism of action. Further analysis revealed TU100 does not intercalate into DNA despite structural similarity to anthracyclines. Cells treated with the drug do exhibit DNA damage, however, as indicated by phosphorylation of histone H2A.X. This damage and effects on cell viability are likely mediated in part by TU100-induced reactive oxygen species. Based on these results, TU100 shows promise as a chemotherapeutic drug owing to its unique structure, cellular targets, and efficacy against selected panels of tissue-specific cancer cell lines.

Topoisomerase I inactivation by a novel thiol reactive naphthoquinone.

The naphthoquinone adduct 12,13-dihydro-N-methyl-6,11,13-trioxo-5H-benzo[4,5]cyclohepta[1,2-b]naphthalen-5,12-imine (hereafter called TU100) contains structural features of both the anthracycline and isoquinone chemotherapeutics. An initial characterization showed TU100 is cytotoxic to mammalian cells and can inhibit topoisomerase I and II. Analysis using topoisomerase I now reveals TU100 is a slow acting inhibitor targeting the enzyme in the absence of DNA. Diluting pre-incubated TU100 and topoisomerase I failed to alleviate inhibition, suggesting the enzyme is being covalently modified. Critical cysteine thiols were identified as the possible target based on the ability of reducing agents to reverse TU100 inhibition. Consistent with this idea, TU100 protected topoisomerase I from inactivation by the sulfhydryl modifying agent N-ethylmaleimide (NEM). Unlike agents nonspecifically reacting with thiols, however, TU100 is specific for topoisomerase because it failed to inhibit a cysteine dependent protease. These results indicate TU100 is a novel naphthoquinone that inactivates free topoisomerase I via alkylation of cysteine residues.

Topoisomerase I/II inhibition by a novel naphthoquinone containing a modified anthracycline ring system.

In an attempt to create more effective chemotherapeutic compounds, the naphthoquinone adduct 12,13-dihydro-N-methyl-6,11,13-trioxo-5H-benzo[4,5]cyclohepta[1,2-b]naphthalen-5,12-imine (hereafter called TU100) was synthesized. Cell viability studies revealed TU100 is specific for eukaryotes and induces cell death. Based on its structural similarities to the anthracyclines and isoquinolines, the ability of TU100 to inhibit topoisomerase I and II was examined. TU100 was an effective inhibitor of both enzymes, as indicated by its ability to prevent topoisomerase-mediated relaxation of supercoiled plasmid DNA. The mechanism of action does not involve TU100 intercalation into DNA, unlike anthracyclines. Pre-incubation of topoisomerase with TU100 dramatically decreased the IC(50), suggesting the drug is a novel slow acting topoisomerase inhibitor that works in the absence of DNA. Taken together these results indicate the novel naphthoquinone adduct TU100 is a dual topoisomerase I/II inhibitor with a unique mechanism of action and chemotherapeutic potential.

Parallel synthesis of an oligomeric imidazole-4,5-dicarboxamide library.

A library of oligomeric compounds was synthesized based on the imidazole-4,5-dicarboxylic acid scaffold along with amino acid esters and chiral diamines derived from amino acids. The final compounds incorporate nonpolar amino acids (Leu, Phe, Trp), polar amino acids (Ser, Asp, Arg), and neutral amino acids (Gly, Ala), and were designed to be useful in screening for inhibitors of protein-protein interactions. Many of the protected and deprotected oligomers show evidence of conformational isomers persistent at room temperature in aqueous solution. A total of 317 final oligomers, out of 441 targeted compounds, were obtained in high analytical purity and of sufficient quantity to submit them for high-throughput screening as part of the NIH Roadmap.

Non-enzymatic hydrolysis of creatine ethyl ester.

The rate of the non-enzymatic hydrolysis of creatine ethyl ester (CEE) was studied at 37 degrees C over the pH range of 1.6-7.0 using (1)H NMR. The ester can be present in solution in three forms: the unprotonated form (CEE), the monoprotonated form (HCEE(+)), and the diprotonated form (H(2)CEE(2+)). The values of pK(a1) and pK(a2) of H(2)CEE(2+) were found to be 2.30 and 5.25, respectively. The rate law is found to be Rate=-dCCEE/dt=k++[H2CEE2+][OH-]+k+[HCEE+][OH-]+k0[CEE][OH-] where the rate constants k(++), k(+), and k(0) are (3.9+/-0.2)x10(6)L mol(-1)s(-1), (3.3+/-0.5)x10(4)L mol(-1)s(-1), and (4.9+/-0.3)x10(4)L mol(-1)s(-1), respectively. Calculations performed at the density functional theory level support the hypothesis that the similarity in the values of k(+) and k(0) results from intramolecular hydrogen bonding that plays a crucial role. This study indicates that the half-life of CEE in blood is on the order of one minute, suggesting that CEE may hydrolyze too quickly to reach muscle cells in its ester form.

Parallel synthesis of a library of symmetrically- and dissymmetrically-disubstituted imidazole-4,5-dicarboxamides bearing amino acid esters.

The imidazole-4,5-dicarboxylic acid scaffold is readily derivatized with amino acid esters to afford symmetrically- and dissymmetrically-disubstituted imidazole-4,5-dicarboxamides with intramolecularly hydrogen bonded conformations that predispose the presentation of amino acid pharmacophores. In this work, a total of 45 imidazole-4,5-dicarboxamides bearing amino acid esters were prepared by parallel synthesis. The library members were purified by column chromatography on silica gel and the purified compounds characterized by LC-MS with LC detection at 214 nm. A selection of the final compounds was also analyzed by (1)H-NMR spectroscopy. The analytically pure final products have been submitted to the Molecular Library Small Molecule Repository (MLSMR) for screening in the Molecular Library Screening Center Network (MLSCN) as part of the NIH Roadmap.

Parallel synthesis of an imidazole-4,5-dicarboxamide library bearing amino acid esters and alkanamines.

The imidazole-4,5-dicarboxylic acid scaffold is readily derivatized with amino acid esters and alkanamines to afford compounds with intramolecularly hydrogen bonded conformations that mimic substituted purines and therefore are hypothesized to be potential inhibitors of kinases through competitive binding to the ATP site. In this work, a total of 126 dissymmetrically disubstituted imidazole-4,5-dicarboxamides with amino acid ester and alkanamide substituents were prepared by parallel synthesis. The library members were purified by column chromatography on silica gel and the purified compounds characterized by LC-MS with LC detection at 214 nm. A selection of the final compounds was also analyzed by (1)H-NMR spectroscopy. The analytically pure final products have been submitted to the Molecular Library Small Molecule Repository (MLSMR) for screening in the Molecular Library Screening Center Network (MLSCN) as part of the NIH Roadmap.

The synthesis of 1-(4-triethoxysilyl)phenyl)-4,4,4-trifluoro-1,3-butanedione, a novel trialkoxysilane monomer for the preparation of functionalized sol-gel matrix materials.

The title compound, 1-(4-triethoxysilyl)phenyl)-4,4,4-trifluoro-1,3-butanedione, was synthesized in a three-step sequence starting from 2-(4-bromophenyl)propene. Containing both a trialkoxysilyl and a substituted 1,3-butanedione functional grouping within its structure, this new silane is a viable starting material for the preparation of functionalized sol-gel materials.