ALKYLTRANSFERASE INHIBITORS FOR CANCER CHEMOTHERAPY

有令人信服的证据表明，肿瘤细胞中的存在 在DNA修复蛋白中，奥米克隆6-烷基鸟嘌呤-DNA烷基转移酶 (AGT)，使人对化疗氯乙基化产生抵抗力 甲基化试剂。先前的研究表明，奥米克隆6- 苯基鸟嘌呤灭活人AGT及肿瘤治疗 含有BG的细胞使它们对BCNU和BCNU等药物的杀伤敏感 替莫唑胺。BG耗竭AGT也可提高治疗指数 BCNU联合BCNU治疗人肿瘤裸鼠移植瘤的实验研究 这种组合的临床试验才刚刚开始。该计划的目标是 目前的实验是：(A)确定其他AGT抑制剂， 与BG相比，BG在效力、传输便利性、 肿瘤的特异性和灭活AGT耐药的能力 向BG和(B)提供对该机制的更多了解(S) 对AGT的BG抗性负责。 具体的具体目标是：(1)测试设计为 用于灭活纯人AGT和a的能力的改进的抑制剂 BG抗性突变体和调节培养的肿瘤细胞的AGT活性。 还将研究这种调制对BCNU杀戮的影响； (2)研究BG与其他已知的AGT灭活剂的相互作用 带着蛋白质。为此目的，动力学测量 AGT和抑制剂之间的相互作用将会发生，并能够 作为[8-~3H]鸟嘌呤形成抑制剂的化合物 从[8-~3H]BG将作为一种分析；(3)研究AGT的结构 (和来自大肠杆菌的相关Ada-C烷基转移酶)和突变体 改变了BG和其他抑制剂的反应性。晶体结构 在有无抑制剂和DNA的情况下对AGT蛋白的影响 底物将被确定。其中一些研究将使用非活动的 C145A突变体AGT与底物结合，但不能与底物反应 在活性中心形成S-烷基半胱氨酸；(4)考察 可导致对BG耐药的人AGT突变谱。一个 这类突变体的一般鉴定系统将用于 通过以下方式确定可能发生此类更改的地点范围 人AGT在大肠杆菌中的表达及其对MNNG的保护作用 或北大。这种保护将被BG废除，并在突变后 含有人AGT cDNA的质粒，其变体可以提供 在BG存在的情况下对烷化剂的保护将被隔离， 对AGT蛋白进行测序，并与对照AGT进行比较 被BG和其他灭活剂灭活。

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.