SUBSTRATE-SPECIFICITY OF HUMAN O-6-METHYLGUANINE-DNA METHYLTRANSFERASE FOR O-6-BENZYLGUANINE DERIVATIVES IN OLIGODEOXYNUCLEOTIDES

To investigate the substrate specificity of human O-6-methylguanine-DN A methyltransferase (MGMT) for O-6-benzylguanine (6BG) derivatives inc orporated in oligodeoxynucleotides, we prepared 25-mer lengths of sequ ences containing various 6BG derivatives and their related compounds a nd then measured the ability of these derivatives to inactivate MGMT i n vitro. Oligodeoxynucleotides containing a 6BG, O-6-(2-fluorobenzyl)g uanine (2F-6BG), O-6-(3-fluorobenzyl)guanine (3F-6BG), O-6-(4-fluorobe nzyl)guanine (4F-6BG), O-6-benzylhypoxanthine (6BH), or O-6-methylguan ine (6MG) were all good substrates for MGMT, and no obvious difference s were observed among them. Oligodeoxynucleotides containing N-2-isobu tyrylated 6BG and 6MG showed only a slightly reduced capacity for inac tivating MGMT compared to N-2-nonmodified forms of these derivatives. No obvious differences were observed in the corresponding double-stran ded and single-stranded oligodeoxynucleotides. MGMT substrate specific ity for the 6BG derivatives in the oligodeoxynucleotide was found to b e quite different from that seen in our previous study [Mineura, K., e t al. (1994) Int. J. Cancer 58, 706-712; (1995) Int. J. Cancer 63, 148 -151. Kohda, K., et al. (1995) Biol. Pharm. Bull. 18, 424-430] and oth ers [Moschel, R. C., et al. (1992) J. Med. Chem. 35, 4486-4491. Chae, M.-Y., et al. (1994) J. Med. Chem. 37, 342-347] using the correspondin g free bases. In brief, (i) 6BG, 3F-6BG, and 4F-6BG greatly inhibited human MGMT, whereas 2F-6BG, 6BH, and 6MG displayed much weaker activit y; (ii) any modifications at the 2-amino group of the 6BG resulted in severe reductions in the ability to inactivate MGMT. These results obt ained by the experiments using oligodeoxynucleotides and free bases su ggest that human MGMT has low substrate specificity for 6BGs in oligod eoxynucleotides. Conformational changes in human MGMT which favor bind ing to oligodeoxynucleotides containing 6BG derivatives and the subseq uent transfer of their benzyl groups may account for the difference in substrate specificity between the incorporated 6BG derivatives and th eir free base form.