Inactivation ofO6-methylguanine-DNA methyltransferase by glucose-conjugated inhibitors

Abstract

Methylating and chloroethylating agents have been used in the therapy of various kinds of cancer for a decade. 1 Methylating drugs (e.g., procarbazine, dacarbazine, streptozotocin and temozolomide) and chloroethylating agents (e.g., carmustine, lomustine, semustine and fotemustine) alkylate DNA at various sites, among them the O 6 position of guanine. The resulting DNA lesions, i.e., O 6 -methylguanine and O 6 -chloroethylguanine, are highly pro-mutagenic 2 and pro-carcinogenic 3,4 and act as a trigger of cytotoxicity 5,6 and apoptosis. 7,8 Cytotoxicity and apoptosis caused by O 6 -methylguanine are mediated by mismatch repair, 9,10 whereas when caused by O 6 -chloroethylguanine the result is chemical re-arrangement of the chloroethyl group forming cross-links between N1 of guanine and N3 of cytosine. 11 These cross-links are supposed to block DNA replication and trigger the cytotoxic response.The primary cytotoxic lesions O 6 -methylguanine and O 6 -chloroethylguanine are subject to repair by the DNA-repair protein O 6 -methylguanine-DNA methyltransferase (MGMT; also designated alkyltransferase), which transfers the alkyl group from the O 6 position of guanine onto an internal cysteine residue in its active site, leading to an alkylthioether that cannot be re-activated. Thus, guanine in the DNA is restored and the MGMT molecule inactivated. Due to the suicide mechanism of action, the repair reaction is stoichiometric, depending on the initial number of MGMT molecules per cell and the rate of resynthesis upon alkylation. 12,13 The MGMT-repair protein plays a key role in the resistance of tumor cells to methylating and chloroethylating drugs. This has been demonstrated by comparison of naturally occurring tumor cell variants, 14 by transfection experiments in which MGMT was over-expressed, 6 by MGMT knockout strategies 15 and by MGMTdepletion experiments with anti-sense oligonucleotides 16 or specific inhibitors. 17,18 In isogenic cell lines transfected with MGMT cDNA, the level of resistance was a linear function of cellular MGMT activity up to a maximal saturation level, where nonspecific effects of the drugs come into play. A significant protective effect of MGMT can be achieved with quite low expression levels. 6

Correlation between quantified promoter methylation and enzymatic activity of O 6-methylguanine-DNA methyltransferase in glioblastomas

“…5, 100 μM O 6 -BG completely inhibited the activity of 50 ng of MGMT. The halfmaximal (50%) inhibitory concentration (IC 50 ) of O 6 -BG was 0.88 μM, which was consistent with the results of a previous study in which the IC 50 value of O 6 -BG was 0.62 μM [11].…”

Inhibition of O6-Methylguanine-DNA Methyltransferase by Glucose-Conjugated Inhibitors: Comparison with Nonconjugated Inhibitors and Effect on Fotemustine and Temozolomide-Induced Cell Death

“…This indicates that glucose conjugation reduces the inhibitor's ability to inhibit MGMT. This essentially confirms our previous data with a selected set of MGMT inhibitors (Reinhard et al, 2001b). Glucose conjugation (using a C8 spacer) reduced MGMT inhibition in the in vitro experiments shown here by a factor of 3 to 5.…”

Targeting O 6-methylguanine-DNA methyltransferase with specific inhibitors as a strategy in cancer therapy

“…One possible explanation is that D-glucose in the immediate vicinity of the O 6 -BG moiety prevents access of the free base to the active site in MGMT. Extending the linker to more than six carbon atoms restored most of the activity [115,116]. A linker of eight carbons appeared to be optimal, since it retained activity and, serendipitously, significantly increased water solubility.…”