Interaction of mammalian O(6)-alkylguanine-DNA alkyltransferases with O(6)-benzylguanine.
Human O(6)-alkylguanine-DNA alkyltransferase (hAGT) activity is a major factor in providing resistance to cancer chemotherapeutic alkylating agents. Inactivation of hAGT by O(6)-benzylguanine (BG) is a promising strategy for overcoming this resistance. Previous studies, which have focused on the region encompassed by residues Pro138 to Gly173, have identified more than 100 individual mutations located at 23 discrete sites at which alterations can render AGT less sensitive to BG. We have now extended the examination of possible sites in hAGT at which alterations might lead to BG resistance to include the residues from Val130 to Asn137, which also make up part of the binding pocket into which BG is postulated to fit. A further 21 mutations located at positions Gly132, Met134, Arg135, and Gly136 were found to lower sensitivity to BG. Mutants R135L, R135Y, and G136P were the most strikingly resistant, with a 50-fold increase in the amount of BG needed to obtain 50% inactivation. These results therefore increase the number of sites at which BG resistance can occur in response to a single amino acid change to 27. Although mammalian AGTs are very similar in amino acid sequence, mouse AGT (mAGT) is significantly less sensitive to BG than rat AGT (rAGT) or hAGT. Construction of chimeric proteins in which portions came from the rAGT and the mAGT indicated that the difference in inactivation resided solely in the amino acids located in the sequence from residues 150 to 188. Individual mutations of the three residues where rAGT and mAGT differ in this region showed that the principal reason for the reduced ability of the mAGT to react with BG was the presence of a histidine residue at position 161, which is occupied by asparagine in rAGT and hAGT. These experiments indicate that many minor changes in amino acids forming all parts of the nucleoside binding pocket of AGT can alter its ability to react with BG and that the possibility that polymorphisms or variants may occur reducing the effectiveness of combination therapy with BG and alkylating agents must be considered.
Loktionova
Natalia A
NA
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, P.O. Box 850, 500 University Drive, Hershey, PA 17033, USA.
Pegg
Anthony E
AE
D000595:Amino Acid Sequence; D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D004791:Enzyme Inhibitors; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase