Research in our laboratory involves synthetic and mechanistic organic chemistry employed in combination with mechanistic biochemistry. Our ultimate goal is to design potent and specific "mechanism-based inhibitors" of selected target enzymes for use as drugs in the treatment of cancer and parasitic diseases. Three different types of enzymes have been selected for study: folylpolyglutamate synthetase/ gamma-glutamyl hydrolase, glutathionylspermidine synthetase/amidase, and oligosaccharyltransferase.
Folic acid is a key vitamin in human nutrition. Cellular folates contain a reduced pteridine heterocycle and a poly gamma-glutamyl peptide "tail." Two enzymes, folylpolyglutatmate synthetase (FPGS) and gamma-glutamyl hydrolase (GH), being studied by our research group, catalyze the biosynthesis and hydrolytic cleavage, respectively, of the polyglutamyl portion of cellular folates and antifolate drugs. Recent research has involved the synthesis of several fluoro- and phosphoamino acids and their incorporation into folates and antifolate drugs. These efforts have led to the synthesis of fluoropeptides and phosphapeptides for biochemical investigation as inhibitors or stimulators of the reactions catalyzed by FPGS or GH. In collaborative research, our new compounds are being used in intact mammalian cells to assess the role of polyglutamate formation and hydrolysis in normal folate-dependent one-carbon biochemistry and also in the pharmacology of antifolate drugs used in the treatment of cancer.
Trypanothione (TSH), found exclusively in trypanosomatid parasites, is a derivative of the ubiquitous antioxidant, glutathione (GSH). We have extended our phosphapeptide research mentioned above to the development of new inhibitors of TSH biosynthesis as possible anti-trypanosomal drugs. Based on an understanding of the mechanism of a key TSH biosynthetic enzyme, we have designed and synthesized specific phosphapeptide inhibitors of the synthetase domain and, in collaborative research have used these compounds to study the regulation of catalysis by this bifunctional enzyme.
Oligosaccharyltransferase (OST), an enzyme that catalyzes the N-glycosylation of proteins, is a membrane-bound protein comprised of nine subunits that we isolate from yeast. Current research involves the synthesis and biochemical study of peptide-based photoinactivators to identify the active site subunit, fluorosugars as potential OST inhibitors, and isotopically labeled disaccharide donors designed to answer specific mechanistic questions. As in the research with folate polyglutamates and trypanothione, the goal is to understand the mechanism of the OST-catalyzed reaction in sufficient detail to design and synthesize potent and specific inhibitors for use in the study of cellular glycoprotein biosynthesis.
J.W. Tomsho, R.G. Moran, and J.K. Coward. Concentration-dependent Processivity of Multiple Glutamate Ligations Catalyzed by Folylpoly-?-glutamate Synthetase. Biochemistry, 2008, 47, 9040-9050.
D. Majumdar, M.D. Alexander, and J.K. Coward. Design and Synthesis of Epoxide Peptidomimetics as Inhibitors of ?-Glutamyl Hydrolase. J. Org. Chem., 2009, 74, 617-627.
T.L. Hagena and J.K. Coward. Fluoridolysis of 5,6-Epoxy Carbohydrates: Application to the Synthesis of 5-Fluoro Lactosamine and Isolactosamine Glycosides. Tetrahedron. Asymm., 2009, 20, 781-794.
J.J. McGuire, D.M. Bartley, J.W. Tomsho, W.H. Haile, and J.K. Coward. Inhibition of human folylpolyglutamate synthetase by diastereomeric phosphinic acid mimics of the tetrahedral intermediate. Arch. Biochem. Biophys., 2009, 488, 140-145.
P. Wang, Q. Wang, Y. Yang, J.K. Coward, A. Nzila, P.F.G. Sims, and J.E. Hyde. Characterization of the bifunctional dihydrofolate synthase-folylpolyglutamate synthase from Plasmodium falciparum; a potential novel target for antimalarial antifolate inhibition. Mol. Biochem. Parasitol. 2010, 172, 41-51.
P.A. Frantom, J.K. Coward, and J.S. Blanchard. UDP-(5F)-GlcNAc acts as a slow-binding inhibitor of MshA, a retaining glycosyltransferase. J. Am. Chem. Soc. 2010, 132, 6626-6627.