Novel DNA methyltransferase-1 (DNMT1) depleting anticancer nucleosides, 4′-thio-2′-deoxycytidine and 5-aza-4′-thio-2′-deoxycytidine

Abstract

Purpose Currently approved DNA hypomethylating nucleosides elicit their effects in part by depleting DNA methyltransferase I (DNMT1). However, their low response rates and adverse effects continue to drive the discovery of newer DNMT1 depleting agents. Herein, we identified two novel 2′-deoxycytidine (dCyd) analogs, 4′-thio-2′-deoxycytidine (T-dCyd) and 5-aza-4′-thio-2′-deoxycytidine (aza-T-dCyd) that potently deplete DNMT1 in both in vitro and in vivo models of cancer and concomitantly inhibit tumor growth. Methods DNMT1 protein levels in in vitro and in vivo cancer models were determined by Western blotting and antitumor efficacy was evaluated using xenografts. Effects on CpG methylation were evaluated using methylation-specific PCR. T-dCyd metabolism was evaluated using radiolabeled substrate. Results T-dCyd markedly depleted DNMT1 in CCRF-CEM and Kg1a leukemia and nCI-H23 lung carcinoma cell lines, while it was ineffective in the HCt-116 colon or IgrOV-1 ovarian tumor lines. On the other hand, aza-T-dCyd potently depleted DNMT1 in all of these lines indicating that dCyd analogs with minor structural dissimilarities induce different DNMT1 turnover mechanisms. Although T-dCyd was deaminated to 4′-thio-2′-deoxyuridine, very little was converted to 4′-thio-thymidine nucleotides, suggesting that inhibition of thymidylate synthase would be minimal with 4′-thio dCyd analogs. Both T-dCyd and aza-T-dCyd also depleted DNMT1 in human tumor xenografts and markedly reduced in vivo tumor growth. Interestingly, the selectivity index of aza-T-dCyd was at least tenfold greater than that of decitabine. Conclusions Collectively, these data show that 4′-thio modified dCyd analogs, such as T-dCyd or aza-T-dCyd, could be a new source of clinically effective DNMT1 depleting anticancer compounds with less toxicity.

Epigenetic targets and drug discovery Part 2: Histone demethylation and DNA methylation

“…On the basis of AZA, Fazarabine and DHAC were synthesized and tested in clinical trials (Beisler et al, 1977;Beisler et al, 1979); however, both trials were stopped due to the low efficacy in cancer therapies (Holoye et al, 1987;Casper et al, 1992;BenBaruch et al, 1993;Creagan et al, 1993;Williamson et al, 1995;Samuels et al, 1998;Goffin & Eisenhauer, 2002). Some azacytidine analogues, such as 5,6-dihydroazacytidine (5,6-dihydro-AZA), 5-Fluoro-2'-deoxycytidine (FdCyd), and 1-(beta-d-ribofuranosyl)-1,2-dihydropyrimidin-2-one (Zebularine) were also shown to inhibit DNMTs (Zhou et al, 2002;Cheng et al, 2003;Cheng et al, 2004;Thottassery et al, 2014). FdCyd has been utilized to treat solid tumors combined with tetrahydrouridine (THU), an inhibitor of cytidine deaminase, in clinical studies (Fahy et al, 2012).…”

“…RX-3117 has been approved by the FDA for the treatment of pancreatic cancers. In addition, more nucleoside analogues were designed and studied for their ability to inhibit DNMTs, such as 2-(p-nitrophenyl) ethoxycarbonyl-DAC (NPEOC-DAC), CP-4200, 4'-thio-2'-deoxycytidine (TdCyd), and 5-aza-4'-thio-2'-deoxycytidine (5-aza-T-dCyd) (Patent Numbers WO2009042766, WO2009042767, WO2011109012 and US 20110218170 A1) (Byun et al, 2008;Brueckner et al, 2010;Thottassery et al, 2010;Thottassery et al, 2014) (Table 3). Collectively, these findings suggest we are close to finding low toxicity DNMT-depleting anticancer drugs.…”

DNA Methylation Targeting: The DNMT/HMT Crosstalk Challenge

“…This compound incorporates into the DNA sequence recognized by the bacterial C5 DNA methyltransferase M.HhaI, leading to DNA methylation inhibition. Furthermore , it depletes DNMT1 in both in vitro and in vivo cancer models [29,30]. 5-fluoro-2′-deoxycytidine (FdCyd) ( 7 ) has been enrolled in clinical trials for treatment of advanced solid tumors, AML and MS ( NCT00359606, NCT01041443) [31].…”

HPLC method development, validation, and impurity characterization of a potent antitumor nucleoside, T-dCyd (NSC 764276)

“…In general, DNMT1 active site occupancy by the DNA-incorporated nucleotide analog is essential to cause DNMT1 trapping and ensuing degradation of the enzyme. The currently available agents have several unfavorable features, such as inefficient incorporation into DNA, DNA synthesis inhibition at high doses, chemical instability of the triazine ring, metabolic instability due to enzymatic deamination, and inhibition of other enzymes not related to DNA methylation [16]. These undesirable characteristics are responsible for decreased hypomethylation efficiency and preclude high-dose administration for extended periods.…”

“…T-dCyd is cytotoxic to leukemia lines (CCRF-CEM and KG1a), with an IC 50 of ca . 1 µM [16]. However, its activity towards solid tumor lines (NCI-H23, HCT-116, and IGROV-1) is negligible.…”