The development of the field of chemical biology has highlighted the utility of chemically synthesized small molecules for the targeting and study of various biological processes. Detailed in this work are a few examples of how this concept has come to fruition. First, the design and discovery of histone deacetylase inhibitors is considered. These inhibitors represent a relatively new class of chemotherapy agents, which target the aberrant hypoacetylation of core histone proteins, a process observed in cancer cells. A click chemistry based approach was employed to rapidly develop a small library of potential inhibitors. The inhibition of two histone deacetylase isoforms was then determined by in vitro enzymatic assays, after which the cytotoxicity was evaluated in the National Cancer Institute’s human cancer cell line screen. A lead compound was discovered that was equipotent with a benchmark compound in enzymatic studies and proved active across all cell lines in the National Cancer Institute screen. This click histone deacetylase inhibitor design has thus provided a new chemical scaffold that has not only revealed a lead compound, but one which is amendable to further structural modifications given the modular nature of this approach.
Secondly, a chemical approach to understanding bacterial polysaccharide biosynthesis is described. Polysaccharides constitute a major component of bacterial cell surfaces and play critical roles in bacteria/host interactions. The biosynthesis of such molecules, however, has mainly been characterized through in vivo genetic studies, thus precluding discernment of the details of this pathway. Accordingly, a chemical approach is described which enabled reconstitution of the Escherichia coli O-polysaccharide biosynthetic pathway in vitro. Starting with chemically prepared N-Acetyl-D-galactosamine-diphospho-undecaprenyl, the E. coli O86 oligosaccharide repeating unit was assembled via sequential enzymatic glycosylation. Successful expression of the putative polymerase Wzy via a chaperone co-expression system then allowed demonstration of polymerization in vitro using this substrate. Analysis of additional substrates revealed a defined mode of recognition for Wzy towards the lipid moiety. Specific polysaccharide chain length modality was furthermore demonstrated to result from the action of Wzz. Collectively, polysaccharide biosynthesis was chemically reconstituted in vitro, providing a well-defined system for further underpinning molecular details of this biosynthetic pathway.
Finally, the pathways through which proteins are glycosylated in prokaryotes and eukaryotes are considered. Glycosylation represents one of the most common forms of co-/posttranslational modification that a protein can undergo. The importance of this process has been highlighted by its association with properties including protein stability and bacterial pathogenicity. Accordingly, to better understand eukaryotic glycosylation, an in vitro assay was established in collaboration with the Aebi group which has enabled the characterization of a protozoan oligosaccharyltransferase Stt3d. For prokaryotes, collaborative efforts with the Feldman group are enabling discernment of the substrate specificity of a surprisingly promiscuous bacterial oligosaccharyltransferase PglL.