ALKYLTRANSFERASE INHIBITORS FOR CANCER CHEMOTHERAPY

用于癌症化疗的烷基转移酶抑制剂

• 批准号: 2429932
• 负责人: ANTHONY E PEGG
• 金额: $ 18.47万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2429932
• 关键词: Escherichia coli; N methyl N' nitro N nitrosoguanidine; active sites; adduct; alkylating agents; alkyltransferase; antineoplastics; carmustine; chemical binding; chemical kinetics; drug interactions; drug resistance; enzyme activity; enzyme inhibitors; enzyme structure; enzyme substrate; guanine analog; human genetic material tag; mutant; neoplasm /cancer pharmacology; neoplastic cell; nucleic acid sequence; oligonucleotides; site directed mutagenesis; tissue /cell culture

There is compelling evidence indicating that the presence in tumor cells of the DNA repair protein, omicron6-alkylguanine-DNA alkyltransferase (AGT), imparts resistance to chemotherapeutic chloroethylating and methylating agents. Previous studies have shown that omicron6- benzylguanine (BG) inactivates human AGT and that treatment of tumor cells with BG sensitizes them to killing by drugs such as BCNU and temozolomide. Depletion of AGT by BG also improves the therapeutic index for the treatment of human tumor xenografts in nude mice by BCNU and clinical trials of this combination have just begun. The aims of the present experiments are: (a) to identify other AGT inhibitors that would be improvements over BG with respect to potency, ease of delivery, specificity towards tumors and the ability to inactivate AGTs resistant to BG and (b) to provide a greater understanding of the mechanism(s) responsible for the BG resistance of AGTs. The detailed specific aims are: (1) to test compounds designed to be improved inhibitors for the ability to inactivate pure human AGT and a BG-resistant mutant and to modulate AGT activity in cultured tumor cells. The effects of such modulation on killing by BCNU will also be examined; (2) to study the interaction of BG and other known inactivators of AGT with the protein. For this purpose, kinetic measurements of the interaction of the AGT 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; (3) to study the structure of AGT (and the related Ada-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; (4) to investigate the spectrum of mutants in human AGT that can lead to resistance to BG. A system for the general identification of such mutants will be used to determine the range of sites at which such alterations can occur by expressing the human AGT in E. coli thus conferring protection from MNNG or BCNU. Such protection will be abolished by BG and after mutagenesis of the plasmid containing the human AGT cDNA, variants which can provide protection to alkylating agents in the presence of BG will be isolated, sequenced and AGT protein prepared for comparison with control AGT for inactivation by BG and the other inactivators.

有令人信服的证据表明肿瘤细胞的存在 DNA修复蛋白，Omicron6-烷基鸟嘌呤-DNA烷基转移酶 （AGT），赋予化学治疗性氯乙基的耐药性和 甲基化剂。 先前的研究表明，Omicron6- 苯甲胺（BG）使人类AGT失活，并治疗肿瘤 具有BG的细胞使它们敏感到被BCNU和BCNU和 替莫唑胺。 BG对AGT的耗竭也改善了治疗指数 用于通过BCNU和 这种组合的临床试验才刚刚开始。 目的 目前的实验是：（a）确定其他AGT抑制剂 在效力，易于交付方面要改进BG， 对肿瘤的特异性和使AGT抗性抗性的能力 向BG和（b）提供对机制的更多了解 负责AGT的BG抗性。 详细的具体目的是：（1）测试设计为 改善了使纯纯人AGT和A灭活能力的抑制剂 耐BG的突变体并调节培养的肿瘤细胞中的AGT活性。 还将检查这种调节对BCNU杀死的影响； （2）研究BG和其他已知的AGT灭活剂的相互作用 与蛋白质。 为此，动力学测量 将建立AGT和抑制剂的相互作用以及能力 作为形成[8-3h]鸟嘌呤的抑制剂的化合物的 从[8-3H]中，BG将用作测定； （3）研究AGT的结构 （以及来自大肠杆菌的相关ADA-C烷基转移酶）和具有 BG和其他抑制剂的反应性改变。 晶体结构 在存在和不存在抑制剂和DNA的情况下Agt蛋白的 将确定底物。 其中一些研究将使用不活动 C145a突变体AGT结合底物但无法与它们反应 在活性部位形成S-烷基半胱氨酸； （4）调查 人类AGT中突变体的频谱可能导致对BG的抗性。 一个 用于一般识别此类突变体的系统将用于 确定通过 在大肠杆菌中表达人类AGT，从而赋予MNNG的保护 或BCNU。 BG和诱变后将废除这种保护 包含人Agt cDNA的质粒，可以提供的变体 在BG存在下对烷基化剂的保护，将分离出来， 测序和AGT蛋白准备与对照AGT进行比较的AGT蛋白 BG和其他灭活因子失活。