The long-term goal of this research is to design, synthesize and evaluate specific inhibitors of enzymes involved in carbohydrate metabolism, as a means of elucidating the roles played by these enzymes in biologically and clinically significant processes. Data from this laboratory has already demonstrated that l-N-iminosugars are highly potent and specific inhibitors of beta-glycosidases and that its conjugate with a UDP analog is a specific and potent inhibitor of alpha-galactosyltransferase. This competing continuation will focus on two biologically important carbohydrate metabolic processes:
Aim 1. To study the biological role of O-GlcNAc. 0-Linked beta-N-acetylglucosamine (0-GlcNAc) is found in many proteins, and its on-and-off glycosylation is thought to regulate the functions of these proteins. Since glycosylation and phosphorylation often (occur at the same sites in a protein, use of inhibitors of the enzyme that regulate the glycosylation offer a viable alternative to site-directed mutagenesis as an approach to analyzing the role of glycosylation. The biological role of 0-GlcNAc will be elucidated by designing and using specific inhibitors to characterize 0-GIcNAc-specific N-acetylglucosaminidase (0-GlcNAc'ase), the enzyme which removes 0-GlcNAc from proteins. The proposed inhibitors include nagastatin analogs and 1-thio-GIcNAc (S-GlcNAc) derivatives and their peptide-conjugates; one potent and specific inhibitor, a S-GlcNAc derivative has already been synthesized. The most promising inhibitors will be used in several biological systems, including cancer cells, in which 0-GlcNAc is thought to play a key role.
Aim 2. To study the reaction mechanism of DNA N glycosylases. DNA base-excision enzymes (N-glycosylases) play a central role in maintaining genetic integrity; however, their reaction mechanisms are still undetermined. This laboratory has recently solved an x-ray crystal structure of the complex of 1-azaribose-containing DNA and one such glycosylase, 3-methyladenine DNA glycosylase (AlkA), and has identified a potential role for the conserved Asp residue in its catalytic process. As a continuation of this work, more specific molecular probes will be developed as a means of gaining additional insight into the mechanisms for the base flipping and N-glycosidic bond-cleaving reactions of other members of this class of enzymes. Given the potential specificity and potency of the proposed inhibitors, these reagents are expected to contribute not only to the study of the biological roles of such important carbohydrate-metabolizing enzymes but also to the development of inhibitor-based drugs with a wide range of clinical applications.
