The long range goals of the project are to develop potent and specific multisubstrate inhibitors of thymidylate synthase, to use these as probes to study the active site of the enzyme and to develop from this information useful new antitumor agents. Sufficient data on inhibitors of thymidylate synthase (TS) have been gathered during the current grant period to permit the construction of one molecule (or a small set) of the currently studied classes which should have optimal binding and cell transport properties. Such molecules will be formed from the """"""""flexible"""""""" or pyrimidine-based folate analogs, substituted at N10 with propargyl and with or without glutamate esterification. The nucleotide component will bear methylphosphonate rather than phosphate at the thymidylate 5'-position, based upon observations that one such derivative seems to be cytotoxic; i.e., able to penetrate into cells A novel approach to the internalization of multisubstrate analogs will involve replacement of the 5-phosphate with bromoacetamido. This type of substitution on thymidine has been shown by others to give rise to potent, specific, irreversible inhibitors of thymidylate synthetase. Such a substitution on multisubstrate analogs may facilitate cell uptake and provide an even higher degree of specificity because of the multisubstrate nature of the inhibitors. Data currently available suggest that the current generation of active site probes really represent product-substrate rather than bisubstrate analogs. It is expected that introduction of an electron-withdrawing C=0 or CF2 group at nucleotide C5 in place of the electron releasing CH2 will permit true mechanism-based (covalent binding within the TS active site. Several such molecules are proposed for synthesis. Biological data will continue to be gathered by Dr. Y.-C. Cheng, University of North Carolina. Inhibitor studies and detailed kinetic analysis will be performed using purified TS and dihydrofolate reductase from human sources, and evaluation of appropriate compounds against human cells in culture will be conducted.
| Broom, A D (1989) Rational design of enzyme inhibitors: multisubstrate analogue inhibitors. J Med Chem 32:2-7 |
| Brixner, D I; Ueda, T; Cheng, Y C et al. (1987) Folate analogues as inhibitors of thymidylate synthase. J Med Chem 30:675-8 |
| Broom, A D (1986) Design and synthesis of multisubstrate analog inhibitors of one-carbon transfer reactions. Fed Proc 45:2779-83 |
