Substrates and Inhibitors of Enzymes Involved in Exopolysaccharide Dependent Biofilms

Many biofilm-forming bacteria produce a similar partially de-N-acetylated β-1,6-N-acetyl glucosamine homopolymer (dPNAG) to facilitate bacterial adhesion. In many medically important biofilm forming bacterial strains, including Escherichia coli, Staphylococcus epidermidis and Staphylococcus aureus, de-N-acetylation of the β-1,6-N-acetyl glucosamine homopolymer (PNAG) is catalyzed by a metal dependent de-N-acetylase. Sixty-five percent of all human persistent bacterial infections are considered to be biofilm related. In vivo studies have implicated the production and subsequent de-N-acetylation of PNAG is essential to bacterial virulence. In this work, methods of monitoring and targeting PNAG-dependent biofilm processes are explored. A novel chromogenic glycosidase substrate based on a glycosyl carbamate was developed and used to effectively monitor the activity of Dispersin B (DspB), an enzyme capable of degrading PNAG/dPNAG. Additionally, an array of potential deacetylase inhibitors were synthesized with the goal of targeting the essential de-N-acetylase enzymes PgaB and IcaB, from E. coli and S. epidermidis respectively, for biofilm formation. These inhibitors were based on a carbohydrate scaffold containing either a metal chelating moiety or a transition state mimic. Finally, a novel coumarin based substrate was developed to monitor in vitro de-N-acetylase activity of PgaB and IcaB. Using this substrate, the potency of the synthesized inhibitors was evaluated through a competitive fluorogenic assay. The most effective inhibitor, a chemoenzymatically-synthesized pentasaccharide derivative showed a Ki value of 280 µM.Ph.D