ALKYLTRANSFERASE INHIBITORS FOR CANCER CHEMOTHERAPY

有令人信服的证据表明，在肿瘤细胞的存在， DNA修复蛋白，奥米克隆6-烷基鸟嘌呤-DNA烷基转移酶 (AGT)赋予对化学治疗的氯乙基化的抗性， 甲基化剂 先前的研究表明，omicron 6- 苄基鸟嘌呤（BG）灭活人AGT和肿瘤治疗 具有BG的细胞使它们对药物如BCNU的杀伤敏感， 替莫唑胺 BG消耗AGT也可提高治疗指数 用于通过BCNU治疗裸鼠中的人肿瘤异种移植物， 这种组合的临床试验才刚刚开始。 的目标 目前的实验是：（a）鉴定其他AGT抑制剂， 与BG相比，在效力、易于输送、 对肿瘤的特异性和抗AGTs的能力 和（B）提供对机制的更好理解 负责AGT的BG抗性。 具体目标是：（1）测试设计为 用于抑制纯人AGT的能力的改进的抑制剂， BG抗性突变体和调节AGT活性在培养的肿瘤细胞。 还将检查这种调节对BCNU杀伤的影响; (2)研究BG与其他已知AGT灭活剂的相互作用 和蛋白质。 为此，动力学测量的 AGT和抑制剂的相互作用将产生， 作为[8- 3 H]鸟嘌呤形成抑制剂的化合物 （3）研究AGT的结构 (and从E.大肠杆菌）和突变体， 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， 将确定衬底。 其中一些研究将使用非活性的 C145 A突变AGT，结合底物但不能与底物反应， 在活性位点形成S-烷基半胱氨酸;（4）研究 人AGT中可导致BG抗性的突变体谱。 一 用于一般鉴定这种突变体的系统将用于 确定可能发生这种改变的位点范围， 在E.大肠杆菌，从而赋予对MNNG的保护 或者北加大这种保护作用将被BG和诱变后消除 含有人AGT cDNA的质粒的变体，其可以提供 在BG存在下对烷基化剂的保护将被分离， 测序并制备AGT蛋白，用于与对照AGT进行比较， BG和其他灭活剂的灭活。

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.