INTERACTION OF BG AND RELATED COMPOUNDS WITH AGT

在上一次支助期间开展的工作表明 BG作为AGT的底物导致了S的形成- 活性部位的半胱氨酸，从而使蛋白质失活。这些 研究还表明，可以产生抗药性的agt突变体。 被BG灭活。未来的实验将在 实验室计划1，协助设计改进的抑制剂。在……里面 为了做到这一点，实验将提供更多的了解 突变型agt蛋白BG抗性的机制(S) 并探讨了AGT的相互作用及其作用机制。 底物和抑制剂。具体目标是：(1)学习 详细介绍了导致对BG耐药的AGT突变。的光谱 人类AGT中可能导致对BG产生抗药性的突变将被鉴定 利用该蛋白保护大肠杆菌免受MNNG杀伤的能力 BG的存在作为活动的屏幕；(2)设计和测试 使抗BG的AGT失活的抑制剂。这些研究将是 用纯化的重组AGT和抗BG的G156A和 P140A AGT突变蛋白和表达这些蛋白的星形细胞瘤细胞 蛋白质。抗这些抗药性AGT的化合物的能力 增强BCNU和替莫唑胺对这些细胞的杀伤作用 下定决心。由AIM(1)产生的更多抗BG突变也将 在可用时进行测试；(3)研究BG和 其他已知的AGT与该蛋白质的失活剂。美国人的能力 对照和抗BG的人AGT及对照和BG敏感的AGT 带着蛋白质。控制和抵抗BG的人AGT的能力 而对照和BG敏感的ADAC烷基转移酶结合到Low 包括极短寡核苷酸的分子量底物 将测定含有O6-甲基和O6-苄基加合物的化合物。动能 烷基转移酶与抑制剂相互作用的测定 会被制造出来，而这些化合物作为抑制剂的能力 从[8-~3H]BG生成[8=~3H]鸟嘌呤将被用作分析；(4) 研究了大肠杆菌AGT(及相关的AdC烷基转移酶)的结构。 Coli)和对BG和其他抑制剂具有不同反应能力的突变体。这个 AGT蛋白在存在和不存在时的晶体结构 将确定抑制剂和DNA底物。其中一些研究 将使用非活性C145A突变体AGT，它能结合底物，但不能 与它们反应生成活性中心的S-烷基半胱氨酸。建模 直接测定突变的AGT和Ada-C蛋白的结构 与抑制剂发生反应的能力发生改变的情况将会进行。

Work carried out during the previous period of support has demonstrated that BG acts as a substrate for AGT leading to the formation of S- benzylcysteine at the active site and thus inactivating the protein. These studies have also shown that mutants of AGT can be made that are resistant to inactivation by BG. Future experiments will be carried out in Laboratory Program 1 to assist in the design of improved inhibitors. In order to do this, the experiments will provide a greater understanding of the mechanism(s) responsible for the BG resistance of mutant AGT proteins and investigate the interaction a d mechanism of action of AGT with substrates and inhibitors. The detailed specific aims are: (1) To study in detail mutations in AGT that lead to resistance to BG. The spectrum of mutants in human AGT that can lead to resistance to BG will be identified using the ability of the protein to protect E. coli from killing by MNNG in the presence of BG as a screen for activity; (2) To design and test inhibitors that inactivate BG-resistant AGTs. These studies will be carried out with purified recombinant AGT and the BG-resistant G156A and P140A AGT mutant proteins and with astrocytoma cells that express these proteins. The ability of compounds active against these resistant AGTs to enhance the killing of these cells by BCNU and temozolomide will also be determined. Additional BG-resistant mutants arising from aim (1) will also be tested as they become available; (3) To study the interaction of BG and other known inactivators of AGT with the protein. The ability of the control and BG-resistant human AGTs and the control and BG-sensitive AGT with the protein. The ability of the control and BG-resistant human AGTs and the control and BG-sensitive AdaC alkyltransferases to bind to low molecular weight substrates including very short oligodeoxynucleotides containing O6-methyl and O6-benzyl adducts will be determined. Kinetic measurements of the interaction of the alkyltransferases and the inhibitors will be made and the ability of the compounds to act as inhibitors of the formation of [8=3H]guanine from [8-3H]BG will be used as an assay; (4) To study the structure of AGT (and the related Ad-C alkyltransferase from E. coli) and mutants with altered reactivity for BG and other inhibitors. The crystal structure of the AGT protein in the presence and absence of inhibitors and a DNA substrate will be determined. Some of these studies will use an inactive C145A mutant AGT which binds substrates but cannot react with them to form the S-alkylcysteine at the active site. Modeling and direct determination of the structures of mutant AGT and Ada-C proteins with altered ability to react with the inhibitors will be carried out.