The Development of Substrates and Inhibitors of the PNAG Biosynthetic Machinery

Biofilms are surface-attached colonies of bacteria that are encapsulated in an extracellular matrix composed of a milieu of proteins, nucleic acids and polysaccharides. In a wide variety of clinically relevant bacteria including Staphylococcus epidermidis and Escherichia coli the composition of the biofilm matrix is dominated by a partially deacetylated polymer of β-(1,6)-linked N-acetylglucosamine (PNAG). The protein components that are responsible for the synthesis, modification and export of the PNAG polymer are encoded by the ica and pga operons in S. epidermidis and E. coli respectively. It has been shown that deletions of any of the gene products comprising the Pga or Ica biosynthetic machinery result in the absence of a biofilm-forming phenotype and severely attenuated virulence. This work explores new ways of understanding the activity of enzymes involved in the biosynthesis of the PNAG polysaccharide. An NMR-based assay was developed to more directly observe the rates of PNAG biosynthesis in vitro. Analogues of UDP-GlcNAc incorporating NMR-active nuclei were synthesized using a chemoenzymatic approach and validated with the processive glycosyltransferase PgaCD. Additionally, substrate-based inhibitors of the deacetylase enzymes PgaB and SpPgda were designed and synthesized. A 2-C gluco scaffold was accessed through glycosidation of a cylcopropyl donor and functionalized with either a methylphosphonate or sulfonamide moiety, meant to mimic the anionic tetrahedral deacetylase transition state. In a separate effort, using structure- and fragment-based inhibitor design strategies in combination with in silico docking, inhibitors of PgaB were developed and synthesized. Finally, new fluorogenic substrates of deacetylase enzymes were synthesized and tested against PgaB and IcaB. The most promising new substrate, AMMU, facilitated high throughput screening of IcaB using a library of over 130 000 compounds.

Department dc:contributor.department

Chemistry

Year dc:date.issued

2018

Author dc:creator

• DiFrancesco, Benjamin Robert

Advisor dc:contributor.advisor

• Nitz, Mark

• Assay Development
• Biofilms
• Deacetylase
• Drug Design
• Enzymology
• PNAG

Handle dc:identifier.uri

http://hdl.handle.net/1807/91837

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/91837