To investigate the substrate specificity of human O6-methylguanine-DNA methyltransferase (MGMT) for O6-benzylguanine (6BG) derivatives incorporated in oligodeoxynucleotides, we prepared 25-mer lengths of sequences containing various 6BG derivatives and their related compounds and then measured the ability of these derivatives to inactivate MGMT in vitro. Oligodeoxynucleotides containing a 6BG, O6-(2-fluorobenzyl)guanine (2F- 6BG), O6-(3-fluorobenzyl)guanine (3F-6BG), O6-(4-fluorobenzyl)guanine (4F- 6BG), O6-benzylhypoxanthine (6BH), or O6-methylguanine (6MG) were all good substrates for MGMT, and no obvious differences were observed among them. Oligodeoxynucleotides containing N2-isobutyrylated 6BG and 6MG showed only a slightly reduced capacity for inactivating MGMT compared to N2-nonmodified forms of these derivatives. No obvious differences were observed in the corresponding double-stranded and single-stranded oligodeoxynucleotides. MGMT substrate specificity for the 6BG derivatives in the oligodeoxynucleotide was found to be quite different from that seen in our previous study [Mineura, K., et 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 others [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 corresponding free bases. In brief, (i) 6BG, 3F-6BG, and 4F-6BG greatly inhibited human MGMT, whereas 2F- 6BG, 6BH, and 6MG displayed much weaker activity; (ii) any modifications at the 2-amino group of the 6BG resulted in severe reductions in the ability to inactivate MGMT. These results obtained by the experiments using oligodeoxynucleotides and free bases suggest that human MGMT has low substrate specificity for 6BGs in oligodeoxynucleotides. Conformational changes in human MGMT which favor binding to oligodeoxynucleotides containing 6BG derivatives and the subsequent transfer of their benzyl groups may account for the difference in substrate specificity between the incorporated 6BG derivatives and their free base form.
All Science Journal Classification (ASJC) codes
- Drug Discovery
- Organic Chemistry
- Health, Toxicology and Mutagenesis