This proposal deals with a new strategy to increase the activation of certain anticancer drugs: the use of compounds that stimulate the activity of key regulatory enzymes by antagonizing the effects of feedback inhibitors. 5'-amino-5'-deoxythymidine (5'-AdThd), a non-toxic analog of thymidine, reduces the effects of the feedback inhibition on thymidine kinase (TX) and augments the uptake, cytotoxicity and radiosensitization properties of 5-iododeoxyuridine (IUDR) in various human bladder cancer cell lines. We propose to study this new approach to anticancer drug design in three different levels. First, we will establish the nature of the interaction of 5'-AdThd with TK regarding the possible sites of interactions, primarily through photoaffinity labeling techniques. We will determine whether 5'-AdThd binds the regulatory site or induces changes in the conformation of the enzyme which alters the feedback regulation. In the second level, we will investigate if other human cancer cells from tissues that are traditionally refractory to chemotherapeutic intervention are susceptible to potentiation of the uptake and cytotoxicity of IUDR, fluorodeoxyuridine or bromodeoxyuridine by 5'-AdThd. These studies will expand our limited experience with the human bladder cancer cell lines model in which a selective advantage was reported. We will also test the antitumor effects of IUDR in combination with 5'-AdThd in vivo using the P388 leukemia model in BDF1 mice. In particular, we will study the toxicity of the combination to non-tumor bearing mice and the potential increase in the therapeutic index of IUDR when combined with 5'-AdThd in P388 tumor-bearing mice. Part of the study will entail the examination of the effects of 5'-AdThd on the distribution, metabolism and excretion of IUDR in vivo. Overall, these studies could be an important contribution to the development of more selective and efficacious anticancer drugs, including drugs that are clinical radiosensitizers. They will undoubtedly evidence whether antagonism of feedback regulation of key enzymes that activate important anticancer drugs is a strategy that ought to be developed and extended to other critical drug-enzyme targets.
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