Activation of p16 gene silenced by DNA methylation in cancer cells by phosphoramidate derivatives of 2'-deoxyzebularine

Abstract

We report herein the application of the phosphoramidate ProTide technology to improve the metabolism of the DNA methytransferase inhibitor, zebularine (Z). Zebularine is a riboside that must undergo a complex metabolic transformation before reaching the critical 2'-deoxyzebularine 5'-triphosphate (dZTP). Because 2'-deoxyzebularine (dZ) is not phosphorylated and therefore inactive, the ProTide strategy was employed to bypass the lack of phosphorylation of dZ and the inefficient reduction of zebularine 5'-diphosphate by ribonucleotide-diphosphate reductase required for zebularine. Several compounds were identified as more potent inhibitors of DNA methylation and stronger inducers of p16 tumor suppressor gene than zebularine. However, their activity was dependent on the administration of thymidine to overcome the potent inhibition of thymidylate synthase (TS) and deoxycytidine monophosphate (dCMP) deaminase by dZMP, which deprives cells of essential levels of thymidine. Intriguingly, the activity of the ProTides was cell line-dependent, and activation of p16 was manifest only in Cf-Pac-1 pancreatic ductal adenocarcinoma cells.

Figure 1. Effect of 2’-deoxyzebularine ProTides 7a, 7b, 7b’ and 10 on p16 expression in Cf-Pac-1 pancreatic cancer cells

Cf-Pac-1 cells were treated with ProTides continuously for 8 days. RNA was collected and the levels of p16 expression were measured by real time RT-PCR. GADPH was used as a reference gene. All compounds induce a strong p16 expression in the presence of 100 µM thymidine.

Figure 2. DNA methylation analysis of D4DZ in Cf-Pac-1 pancreatic cancer cells treated with 2’-deoxyzebularine ProTides 7a, 7b, 7b’ and 10

Cf-Pac-1 cells were treated with ProTides continuously for 8 days and DNA was collected. DNA methylation status of the D4DZ repeats expressed in percent methylation was analyzed by quantitative Ms-SNuPE. Treatment with these compounds caused demethylation at the D4DZ locus.

Scheme 1

Synthesis of 2’-deoxyzebularine phosphoramidates