Human O6-alkylguanine-DNA alkyltransferase was rapidly inactivated by low concentrations of O6-benzylguanine, but the alkyltransferase from the Escherichia coli ogt gene was much less sensitive and alkyltransferases from the E. coli ada gene or from yeast were not affected. O6-Benzyl-2'-deoxyguanosine was less potent than the base, but was still an effective inactivator of the human alkyltransferase and had no effect on the microbial proteins. O6-Allylguanine was somewhat less active, but still gave complete inactivation of both the human and Ogt alkyltransferases at 200 microM in 30 min, slightly affected the Ada protein, and had no effect on the yeast alkyltransferase. O4-Benzylthymidine did not inactivate any of the alkyltransferase proteins tested. Inactivation of the human alkyltransferase by O6-benzylguanine led to the formation of S-benzylcysteine in the protein and to the stoichiometric production of guanine. The rate of guanine formation followed second-order kinetics (k = 600 M-1 s-1). Prior inactivation of the alkyltransferase by reaction with a methylated DNA substrate abolished its ability to convert O6-benzylguanine into guanine. These results indicate that O6-benzylguanine inactivates the protein by acting as a substrate for alkyl transfer and by forming S-benzylcysteine at the acceptor site of the protein. The inability of O6-benzylguanine to inactivate the microbial alkyltransferases may be explained by steric constraints at this site.(ABSTRACT TRUNCATED AT 250 WORDS)

Several new O6-benzylguanine analogs bearing increasingly bulky substituent groups on the benzene ring or at position 9 were tested for their ability to inactivate the human DNA repair protein, O6-alkylguanine-DNA alkyltransferase. Substitution on the benzene ring was well tolerated although activity varied considerably with structural changes in groups attached to position 9. For this site, activity was preserved with large or small lipophilic groups while introduction of non-carbohydrate polar groups generally reduced activity regardless of their size.

Several 8-substituted O6-benzylguanines, 2- and/or 8-substituted 6-(benzyloxy)purines, substituted 6(4)-(benzyloxy)pyrimidines, and a 6-(benzyloxy)-s-triazine were tested for their ability to inactivate the human DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT, alkyltransferase). Two types of compounds were identified as being significantly more effective than O6-benzylguanine (the prototype low molecular weight inactivator) at inactivating AGT in human HT29 colon tumor cell extracts. These were 8-substituted O6-benzylguanines bearing electron-withdrawing groups at the 8-position (e.g. 8-aza-O6-benzylguanine and O6-benzyl-8-bromoguanine) and 5-substituted 2,4-diamino-6-(benzyloxy)pyrimidines bearing electron-withdrawing groups at the 5-position (e.g. 2,4-diamino-6-(benzyloxy)-5-nitroso- and 2,4-diamino-6-(benzyloxy)-5-nitropyrimidine). The latter derivatives were also more effective than O6-benzylguanine at inactivating AGT in intact HT29 colon tumor cells. Provided these types of purines and pyrimidines do not exhibit undesirable toxicity, they may be superior to O6-benzylguanine as chemotherapeutic adjuvants for enhancing the effectiveness of antitumor drugs for which the mechanism of action involves modification of the O6-position of DNA guanine residues.