INTERACTION OF BG AND RELATED COMPOUNDS WITH AGT

上一个支助期间开展的工作表明， BG作为AGT的底物，导致S- 苄基半胱氨酸在活性位点，从而使蛋白质失活。 这些 研究还表明，AGT的突变体可以产生抗性， 到BG灭活。 未来的实验将在 实验室计划1，以协助设计改进的抑制剂。 在 为了做到这一点，实验将提供更好的理解， 负责突变AGT蛋白BG抗性的机制 并研究AGT的相互作用和作用机制， 底物和抑制剂。 具体目标是：（1）研究 详细介绍了AGT突变导致对BG的耐药性。 的光谱 将鉴定出可导致BG抗性的人AGT突变体 利用蛋白质保护E. MNNG对大肠杆菌的杀灭作用 BG的存在作为活性的筛选;（2）设计和测试 抑制抗BG AGTs的药物。 这些研究报告将 用纯化的重组AGT和BG抗性G156 A进行， P140 A AGT突变蛋白和表达这些蛋白的星形细胞瘤细胞 proteins. 对这些耐药AGT有活性的化合物， 增强BCNU和替莫唑胺对这些细胞的杀伤作用也将是 测定 由目的（1）产生的其他BG抗性突变体也将 （3）研究BG和 其他已知的AGT灭活剂与蛋白质。 的能力 对照和BG抗性人AGT以及对照和BG敏感性AGT 和蛋白质。 对照和BG抗性人AGT的能力 并且对照和BG敏感性AdaC烷基转移酶结合到低水平。 包括非常短的寡脱氧核苷酸的分子量底物 将测定含有O 6-甲基和O 6-苄基加合物。 动力学 烷基转移酶和抑制剂相互作用的测量 将被制备，并且化合物作为抗肿瘤药物的抑制剂的能力将被提高。 将使用[8- 3 H]BG形成[8= 3 H]鸟嘌呤作为测定;（4） 研究AGT的结构（以及相关的来自E. coli）和对BG和其它抑制剂具有改变的反应性的突变体。 的 AGT蛋白在存在和不存在下的晶体结构 将确定抑制剂和DNA底物。 其中一些研究 将使用无活性的C145 A突变AGT，其结合底物但不能 与它们反应在活性位点形成S-烷基半胱氨酸。 建模 并直接测定突变AGT和Ada-C蛋白的结构 与抑制剂反应的能力改变。

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.