INTERACTION OF BG AND RELATED COMPOUNDS WITH AGT

BG 及相关化合物与 AGT 的相互作用

• 批准号: 6237279
• 负责人: ANTHONY E PEGG
• 金额: $ 9.28万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 1998-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6237279
• 关键词: Escherichia coli; N methyl N' nitro N nitrosoguanidine; active sites; adduct; alkyltransferase; antineoplastics; astrocytoma; carmustine; drug design /synthesis /production; drug interactions; drug resistance; drug screening /evaluation; enzyme activity; enzyme inhibitors; enzyme structure; enzyme substrate; gene mutation; guanine analog; mutant; neoplastic cell; oligonucleotides; pharmacokinetics; tissue /cell culture

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.

在上一个支持期间进行的工作已证明 BG充当AGT的底物，导致S-形成 活性位点的苄半胱氨酸，从而使蛋白质失活。 这些 研究还表明，可以使AGT的突变体具有抵抗力 BG灭活。 未来的实验将进行 实验室计划1帮助设计改进的抑制剂。 在 为此，实验将提供对 负责突变AGT蛋白BG耐药性的机制 并研究AGT的相互作用与AGT的作用机理 底物和抑制剂。 详细的具体目的是：（1）研究 详细的AGT突变导致对BG的抗性。 频谱 人类AGT中可能导致对BG的抗性的突变体将被识别 利用蛋白质保护大肠杆菌免受MNNG杀死的能力 BG作为活动的屏幕的存在； （2）设计和测试 使BG抗AGT灭活的抑制剂。 这些研究将是 使用纯化的重组AGT和耐BG的G156A和 P140A AGT突变蛋白和与星形细胞瘤细胞一起表达这些 蛋白质。 对这些抗性AGT的活性化合物的能力 通过BCNU和Temozolomide增强这些细胞的杀死也将是 决定。 AIM引起的其他抗BG的突变体（1）也将 在可用时进行测试； （3）研究BG和 蛋白质的其他已知的灭活剂。 能力 控制和抗BG的人AGT以及对控制和对BG敏感的AGT 与蛋白质。 控制和耐BG的人类AGT的能力 对照和对BG敏感的ADAC烷基转移酶与低结合 分子量底物，包括非常短的寡脱氧核苷酸 将确定含有O6-甲基和O6-苄基加合物。 动力学 烷基转移酶和抑制剂相互作用的测量 将制作以及化合物充当抑制剂的能力 [8-3H] BG的[8 = 3H]鸟嘌呤的形成将被用作测定； （4）至 研究AGT的结构（以及来自E的AD-C烷基转移酶 大肠杆菌）和BG和其他抑制剂反应性改变的突变体。 这 在存在和不存在的情况下，Agt蛋白的晶体结构 将确定抑制剂和DNA底物。 其中一些研究 将使用无效的C145A突变体AGT，该突变体绑定底物但不能 与它们反应，在活性部位形成S-烷基半胱氨酸。 造型 并直接确定突变AGT和ADA-C蛋白的结构 随着与抑制剂反应的能力改变。