L-Selectin mediates the initial attachment of blood-borne lymphocytes to endothelial cells during lymphocyte homing to secondary lymphoid organs. In addition, L-selectin participates in the similar process of leukocyte adhesion and extravasation at sites of chronic inflammation. The L-selectin ligands are mucin-like glycoproteins adorned with the unusual sulfated carbohydrate epitopes, 6-sulfo sialyl Lewis x and 6'-sulfo sialyl Lewis x. Sulfation of these epitopes on the N-acetylglucosamine (GlcNAc) and galactose (Gal) residues, respectively, converts inactive glycoforms to high-avidity L-selectin ligands. Furthermore, sulfation of these ligands is restricted in the vasculature to sites of sustained lymphocyte recruitment such as peripheral lymph nodes and chronically inflamed tissues. Therefore, the GlcNAc-6- and Gal-6-sulfotransferases that install the sulfate esters may be key modulators of lymphocyte recruitment to lymph nodes and chronically inflamed tissues, and potential targets for anti-inflammatory therapy. Through a collaborative effort with two other laboratories, three human carbohydrate sulfotransferase clones that may be involved in L- selectin ligand biosynthesis have been identified. The broad objectives of this proposal are the biochemical characterization of these enzymes and the design and synthesis of selective inhibitors.
The first Aim of the proposed research is to develop a modular approach to inhibitor design based on the conjugation of two independently-derived compounds, one optimized to bind the carbohydrate binding site and the other optimized to bind the 3'-phosphoadenosine-S'-phosphosulfate (PAPS) binding site. In order to establish a framework for the design of carbohydrate binding site inhibitors, the substrate specificity of each enzyme and the structural features required for recognition will be defined. Preferred carbohydrate substrates will then serve as leads for the design of glycomimetic inhibitors. In parallel, PAPS binding site inhibitors will be identified through the synthesis and screening of aromatic heterocycle libraries. The pharmacophores derived from these parallel efforts will be tethered to produce potent and selective sulfotransferase inhibitors.
The second Aim of the proposal is to define the features of carbohydrate sulfotransferase sequences that relate to function by site-directed and domain swapping mutagenesis. The results will contribute to a model for predicting sulfotransferase specificity based on genomic sequence analysis.
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