The broad objective of this renewal application is to develop biochemical rationales for the use of deoxynucleoside analogs in the therapy of cancer. This objective is to be achieved by several related efforts: (a) study of the kinases acting on deoxynucleosides; (b) study in intact cells of the metabolism and metabolic effects of selected deoxynucleoside analogs, such as the 2-halo derivatives of deoxyadenosine and arabinosyladenine; and (c) study of the effects of analog triphosphates on the suspected target enzymes, DNA polymerases and ribonucleotide reductase. Because of current confusion about the number and substrate specificity of the deoxynucleoside kinases we propose to isolate from a single source (L1210 cells) all of the kinases acting on deoxynucleosides. For each of these enzymes we will determine kinetic constants for phosphorylation of both the natural deoxynucleosides and the analog nucleosides that serve as substrates and perform the usual characterizations such as pH dependence and inhibition by substrates and products. We have shown that the phosphorylated enzyme is an intermediate in the adenosine kinase reaction; we will determine if phosphorylated enzymes are intermediates in the reactions catalyzed by the other kinases. Lines of L1210 cells selected for resistance to various nucleotides and deficient in specific nucleoside kinases will be used as adjuncts in the study of the metabolism of new analogs and the substrate specificity of kinases; certain of these cell lines will be used as sources for isolation of kinases. Selected analog triphosphates will be assayed as inhibitors of DNA polymerase alpha and ribonucleotide reductase, and their effectiveness compared with that of known inhibitors, with the objective of identifying structural features required for inhibition of these critical enzymes.
| Bennett Jr, L L; Brockman, R W; Allan, P W et al. (1988) Alterations in nucleotide pools induced by 3-deazaadenosine and related compounds. Role of adenylate deaminase. Biochem Pharmacol 37:1233-44 |
