Inhibition of human leukaemic thymidylate kinase and L1210 ribonucleotide reductase by dinucleotides of adenosine and thymidine and their phosphonate analogues

Dinucleotides of adenosine and thymidine in the ApnT series (n = 3,4,5 and 6) and their corresponding phosphonate analogues, where a methylene group replaces the oxygen between the alpha and beta phosphorus atoms adjacent to thymidine, have been evaluated as inhibitors of human leukaemic thymidylate kinase (dTMP kinase, EC 2.7.4.9) and ribonucleotide reductase (EC 1.17.4.1) from L1210 cells. Ap3T, Ap4T, Ap2cpT and Ap3cpT were poor inhibitors of both enzymes. Ap5T, Ap6T and their phosphonate analogues were potent inhibitors of dTMP kinase, possibly acting as bisubstrate analogues (IC50 values: Ap5T, 7.9 microM; Ap4cpT, 5.8 microM; Ap6T, 5.4 microM; Ap4cpT, 4.0 microM). For CDP reductase, where these compounds may bridge activity/effector sites on the M1 subunit of the enzyme, Ap5T and Ap6T were inhibitors with IC50 values of 14.4 microM and 20.3 microM respectively. The phosphonate series of compounds was far less active. The thymidine moiety of the compounds was essential for inhibition since Ap5A was inactive against both enzymes. dTTP, although a poor inhibitor of thymidylate kinase was a potent negative effector of CDP reductase (IC50, 19.3 microM). Significantly, Ap5T was not hydrolysed to release dTTP under the conditions of the assay. These studies show that the activities of both enzymes may be modulated by nucleotide analogues.