Abstract

The DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) is an important suicide enzyme involved in the defense against O⁶-alkylating mutagens. It also plays a role in the resistance of tumors to anticancer drugs targeting the O⁶-position of guanine, such as temozolomide and fotemustine. Several potent MGMT inhibitors have been developed sensitizing cells to O⁶-alkylating agents. Aimed at targeting MGMT inhibitors to tumor cells, we synthesized MGMT inhibitory compounds conjugated with glucose to improve uptake in tumor cells. Here, we compared O⁶-benzylguanine, O⁶-2-fluoropyridinylmethylguanine (O⁶FPG), O⁶-3-iodobenzylguanine, O⁶-4-bromothenylguanine, and O⁶-5-iodothenylguanine with the corresponding C8-linker β-d-glucose derivatives. All glucose conjugated inhibitors were 3- to 5-fold less effective than the corresponding nonconjugated drugs as to MGMT inhibition that was measured in cell extracts (in vitro) and cultivated HeLaS3 cells (in vivo). Except for O⁶FPG, IC₅₀ values of the guanine derivatives applied in vitro and in vivo were correlated. A similar correlation was not obvious for the corresponding glucosides, indicating differences in cellular uptake. C8-α-d-glucosides were less effective than β-glucosides. From the newly developed glucose-conjugated inhibitors tested, O⁶-4-bromothenylguanine-C8-β-d-glucoside (O⁶BTG-C8-βGlu) was most potent in inhibiting MGMT both in vitro and in vivo. At a concentration of 0.1 μM, it inhibited cellular MGMT to completion. It was not toxic, even when applied chronically to cells at high dose (up to 20 μM). O⁶BTG-C8-βGlu strongly potentiated the killing effect of fotemustine and temozolomide, causing reversal from MGMT+ to MGMT– phenotype. Therefore, O⁶BTG-C8-βGlu seems to be especially suitable for approaching MGMT inhibitor targeting in tumor therapy.