Inactivation of human O-6-alkylguanine-DNA alkyltransferase by modified oligodeoxyribonucleotides containing O-6-benzylguanine

Inactivation of the DNA repair protein O-6-alkylguanine-DNA alkyltransferas e (AGT) enhances tumor cell killing by therapeutic alkylating agents. O-6-B enzylguanine (b(6)G) can inactivate AGT and is currently in clinical trials to enhance therapy. Short oligodeoxyribonucleotides containing b(6)G are m uch more effective inactivators, but their use for therapeutic purposes is likely to be compromised by metabolic instability. We have therefore examin ed the ability to inactivate AGT of an 11-mer oligodeoxyribonucleotide cont aining b(6)G (11-mpBG) when modified with terminal methylphosphonate linkag es to protect it from nucleases. This modification did not reduce the abili ty to serve as a substrate/inactivator for AGT, and 11-mpBG had an ED50 val ue of 1.3 nM, more than 300-fold lower than that for b(6)G. A similar oligo deoxyribonucleotide containing O-6-methyl-guanine (m(6)G) was also found to be a good substrate (ED50 value of 10 nM), but the benzylated form was rep aired more rapidly and preferentially. When added to HT29 cell cultures, 5 muM 11-mpBG was able to cause a prolonged inactivation of cellular AGT for at least 72 h and to greatly sensitize the cells to killing by 1,3-bis(2-ch loroethyl)-1-nitrosourea (BCNU). The 11-mpMG was ineffective at up to 20 mu M, suggesting that the benzyl group allows better uptake into the cell. How ever, even with 11-mpBG, the 1000-fold decrease in potency toward AGT in HT 29 cells compared to that toward the protein in vitro suggests that uptake may be a limiting factor. These results suggest that oligodeoxyribonucleoti des such as 11-mpBG may prove to be useful drugs for potentiation of alkyla ting agent chemotherapy if uptake can be improved.