The Skaggs Institute
for Chemical Biology

Bioorganic Chemistry and Drug Discovery

C.-H. Wong, C. Behrens, C. Bennett, M. Best, A. Brik, S. Dean, M. Fujio, S. Hanson, Z. Hong, J. Hsu, T.-L. Hsu, C.-Y. Huang, D.-R. Hwang, A. Krebs, J.-C. Lee, F.-S. Liang, H. Liu, L. Liu, M. Numa, T. Polat, M. Sawa, P. Schanen, M. Sugiyama, D. Thayer, S.-K. Wang, L. Whalen, C.-Y. Wu, D. Wu, M. Wuchrer, Y.-Y. Yang

In continuation of our interest in developing new chemical and enzymatic methods for organic synthesis, we developed new aldolases via directed evolution and structure-based directed mutagenesis and used the modified enzymes in synthesis of iminocyclitol derivatives as glycosidase inhibitors. We also developed a method to assemble saccharide and aminoglycoside microarrays in microtiter plates for high-throughput screening. The arrays were then used in a high-throughput assay for discovery of glycosyltransferase inhibitors as potential drug candidates for the treatment of inflammatory diseases, for dissection of carbohydrate specificity, and for identification of selective binding of aminoglycosides to certain RNA sequences.

A current major interest in our laboratory is the development of new synthetic methods and strategies to tackle major problems associated with carbohydrate-mediated biological recognition, such as bacterial and viral infection, cancer metastasis, and immunoresponses. We developed the programmable 1-pot method for oligosaccharide synthesis and covalent and noncovalent methods for arraying oligosaccharides for the high-throughput analysis of sugar-protein interactions.

In our efforts to develop enzyme inhibitors as potential therapeutic agents, we continue to develop new methods to rapidly identify novel inhibitors. We use a well-designed core structure with acceptable binding affinity for an enzyme of interest to mimic the reaction transition state of the enzymatic reaction, and we search for a single group that can be added to the core to further improve the affinity and specificity of the core structure for the enzyme. This additional group is identified from an array of readily available and synthetic building blocks through a reaction with the core in microtiter plates without manipulation to isolate or protect any groups and is followed by high-throughput screening in situ. We used this strategy with the amide bond–forming reaction, the triazole-forming reaction, the epoxide-opening reaction with amines, and the tetrabutylammonium fluoride–mediated N- and O-alkylation, and we rapidly identified several molecules that inhibit, at low nanomolar concentrations, glycosidases, glycosyltransferases, sulfotransferases, HIV protease, and the anthrax lethal factor.

In our work on glycoarrays, we developed a new method to prepare arrays in microtiter plates covalently and showed the usefulness of this method in the high-throughput analysis of the interactions of the polymannose moieties on HIV with the broadly neutralizing antibody 2G12. We used the programmable 1-pot synthesis method to prepare various oligomannoses and spotted them on the surface of microtiter plates containing the succinimide ester groups, through a chemical or photocleavable linker. With this method, we defined the epitope specificity of 2G12, and the optimal epitope identified has been used in the design of vaccines for immunization. Our goal is to develop new carbohydrate-based vaccines for the treatment of AIDS.

Publications

Brik, A., Alexandratos, J., Lin, Y.-C., Elder, J.H., Olson, A.J., Wlodawer, A., Goodsell, D.S., Wong, C.-H. 1,2,3-Triazole as a peptide surrogate in the rapid synthesis of HIV-1 protease inhibitors. Chembiochem 6:1167, 2005.

Brik, A., Wu, C.-Y., Best, M.D., Wong, C.-H. Tetrabutylammonium fluoride-assisted rapid N^9-alkylation on purine ring: application to combinatorial reactions in microtiter plates for the discovery of potent sulfotransferase inhibitors in situ. Bioorg. Med. Chem. 13:4622, 2005.

Calarese, D.A., Lee, H.-K., Huang, C.-Y., Best, M.D., Astronomo, R.D., Stanfield, R.L., Katinger, H., Burton, D.R., Wong, C.-H., Wilson, I.A. Dissection of the carbohydrate specificity of the broadly neutralizing anti-HIV-1 antibody 2G12. Proc. Natl. Acad. Sci. U. S. A. 102:13372, 2005.

Fan, G.-T., Pan, Y.-S., Lu, K.-C., Cheng, Y.-P., Lin, W.-C., Lin, S., Lin, C.-H., Wong, C.-H., Fang, J.-M., Lin, C.-C. Synthesis of α-galactosyl ceramide and the related glycolipids for evaluation of their activities on mouse splenocytes. Tetrahedron 61:1855, 2005.

Fridman, M., Belakhov, V., Lee, L.V., Liang, F.-S., Wong, C.-H., Baasov, T. Dual effect of synthetic aminoglycosides: antibacterial activity against Bacillus anthracis and inhibition of anthrax lethal factor. Angew. Chem. Int. Ed. 44:447, 2005.

Hsu, C.-C., Hong, Z., Wada, M., Franke, D., Wong, C.-H. Directed evolution of D-sialic acid aldolase L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolases. Proc. Natl. Acad. Sci. U. S. A. 102:9122, 2005.

Kinjo, Y., Wu, D., Kim, G., Xing, G.-W., Poles, M.A., Ho, D.D., Tsuji, M., Kawahara, K., Wong, C.-H., Kronenberg, M. Recognition of bacterial glycosphingolipids by natural killer T cells. Nature 434:520, 2005.

Klostermeier, D., Sears, P., Wong, C.-H., Millar, D.P., Williamson, J.R. A three-fluorophore FRET assay for high-throughput screening of small-molecule inhibitors of ribosome assembly. Nucleic Acids Res. 32:2707, 2004.

Liang, F.-S., Wong C.-H. Surface plasmon resonance study of HCV IRES RNA-aminoglycoside interactions. Methods Mol. Biol., in press.

Lin, H., Thayer, D.A., Wong, C.-H., Walsh, C.T. Macrolactamization of glycosylated peptide thioesters by the thioesterase domain of tyrocidine synthetase. Chem. Biol. 11:1635, 2004.

Mocharla, V.P., Colasson, B., Lee, L.V., Romper, S., Sharpless, K.B., Wong, C.-H., Kolb, H.C. In situ click chemistry: enzyme-generated inhibitors of carbonic anhydrase II. Angew. Chem. Int. Ed. 44:116, 2004.

Numa, M.M.D., Lee, L.V., Hsu, C.-C., Bower, K.E., Wong, C.-H. Identification of novel anthrax lethal factor inhibitors generated by combinatorial Pictet-Spengler reaction followed by screening in situ. Chembiochem 6:1002, 2005.

Shie, J.-J., Fang, J.-M., Kuo, C.-J., Kuo, T.-H., Liang, P.-H., Huang, H.-J., Yang, W.-B., Lin, C.-H., Chen, J.-L., Wu, Y.T., Wong, C.-H. Discovery of potent anilide inhibitors against the severe acute respiratory syndrome 3CL protease. J. Med. Chem. 48:4469, 2005.

Shie, J.-J., Fang, J.-M., Kuo, T.-H., Kuo, C.-J., Liang, P.-H., Huang, H.-J., Wu, Y.-T., Jan, J.-T., Cheng, Y.-S.E., Wong, C.-H. Inhibition of the severe acute respiratory syndrome 3CL protease by peptidomimetic α,β-unsaturated esters. Bioorg. Med. Chem. 13:5240, 2005.

Thayer, D.A., Yu, H.N., Galan, M.C., Wong, C.-H. A general strategy toward S-linked glycopeptides. Angew. Chem. Int. Ed. 44:4596, 2005.

Tolbert, T.J., Franke, D., Wong, C.-H. A new strategy for glycoprotein synthesis: ligation of synthetic glycopeptides with truncated proteins expressed in E coli as TEV protease cleavable fusion protein. Bioorg. Med. Chem. 13:909, 2005.

Tolbert, T.J., Wong, C.-H. Carbohydrate chains: enzymatic and chemical synthesis. In: Encyclopedia of Biological Chemistry. Lennarz, W.J., Lane, M.D. (Eds.). Academic Press, San Diego, 2005, Vol. 1, p. 307.

Wei, S.H., Rosen, H., Matheu, M.P., Sanna, M.G., Wang, S.-K., Wong, C.-H., Parker, I., Cahalan, M.D. Sphingosine 1-phosphate type 1 receptor agonism inhibits transendothelial migration of medullary T cells to lymphatic sinuses. Nat. Immunol. 6:1228, 2005.

Wong, C.-H. Protein glycosylation: new challenges and opportunities. J. Org. Chem. 70:4219, 2005.

Wu, C.-Y., Brik, A., Wang, S.-K., Chen, Y.-H., Wong, C.-H. Tetrabutylammonium fluoride-mediated rapid alkylation reaction in microtiter plates for the discovery of enzyme inhibitors in situ. Chembiochem. 6:2176, 2005.

Wu, C.-Y., King, K.-Y., Kuo, C.-J., Liang, P.-H., Wu, Y.-T., Ho, M.-Y., Liao, C.-L., Fang, J.-M., Wong, C.-H. Discovery of stable benzotriazol esters as potent inhibitors of the severe acute respiratory syndrome 3CL protease. Chem. Biol., in press.

Wu, D., Xing, G.-W., Poles, M., Kinjo, Y., Sullivan, B., Bodmer-Narkevitch, V., Plettenburg, O., Kronenberg, M., Tsuji, M., Ho, D.D., Wong, C.-H. Bacterial glycolipids and analogs as antigens for CD1d-restricted NKT cells. Proc. Natl. Acad. Sci. U. S. A. 102:1351, 2005.

Xing, G.-W., Wu, D., Poles, M.A., Horowitz, A., Tsuji, M., Ho, D.D., Wong, C.-H. Synthesis and human NKT cell stimulating properties of 3-O-sulfo-α/β-galactosylceramides. Bioorg. Med. Chem. 13:2907, 2005.