A series of potential inhibitors of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) were synthesized, characterized in detail by NMR, and tested for their ability to deplete MGMT activity in vitro. The new compounds, omega-[O(6)-R-guan-9-yl]-(CH(2))(n)-beta-d-glucosides with R = benzyl or 4-bromothenyl and omega = n = 2, 4,. 12, were compared with the established inhibitors O(6)-benzylguanine (O(6)-BG), 8-aza-O(6)-benzylguanine (8-aza-BG), and O(6)-(4-bromothenyl)guanine (4-BTG), which exhibit in an in vitro assay IC(50) values of 0.62, 0.038, and 0.009 microM, respectively. Potential advantages of the glucosides are improved water solubility and selective uptake in tumor cells. The 4-BTG glucosides with n = 2, 4, 6 show moderate inhibition with an IC(50) of ca. 0.5 microM, while glucosides derived from BG and 8-aza-BG showed significantly poorer inhibition compared to the parent compounds. The 4-BTG glucosides with n = 8, 10, 12 were effective inhibitors with IC(50) values of ca. 0.03 microM. To understand this behavior, extensive molecular modeling studies were performed using the published crystal structure of MGMT (PDB entry: ). The inhibitor molecules were docked into the BG binding pocket, and molecular dynamics simulations with explicit water molecules were carried out. Stabilization energies for the interactions of specific regions of the inhibitor and individual amino acid residues were calculated. The alkyl spacer is located in a cleft along helix 6 of MGMT. With increasing spacer length there is increasing interaction with several amino acid residues which play an important role in the proposed nucleotide flipping mechanism required for DNA repair.

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

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