ALKYLTRANSFERASE INHIBITORS FOR CANCER CHEMOTHERAPY

描述：O6-苄基鸟嘌呤(B6G)的临床试验正在进行中。这个 这种疗法的成功可能会受到以下方面的影响：(A)容易地产生 抗b6G的HAGT突变体；(B)缺乏足够的效力和肿瘤 特异性和(C)烷化剂治疗在AGT时的增强毒性 使用了抑制剂。建议进行实验以解决这些问题 关于上一次支助期间的预付款，其中包括：(1) B6G抗性突变体筛选方法的建立；(Ii)研究 表明这些突变体可以保护哺乳动物细胞免受烷化剂的侵害 在b6G存在下，(Iii)HAGT的晶体结构的推导 蛋白质；和(Iv)含有b6G的短寡核苷酸的迹象 是HAGT的强效灭活剂。 拟议的研究项目有三个具体目标。第一个具体目标 是表征hagt和b6G之间的相互作用以及含有 B6G。这一目标的研究将测试和改进当前的结合模型 B6G及相关化合物与HAGT的结合失活比较 的)Hagt突变蛋白的各种b6G衍生物，其中残基认为 参与结合的基因被改变了。它还计划使用各种不同的 Hagt的突变体，如C145S和抑制剂，以获得 与Hagt的络合物显示结合是如何发生的。第二个具体目标是 获得对b6G和b6G敏感性改变的Hagt突变体的充分了解 其他的抑制剂。许多点突变(至少有一个是自然发生的 多态变异体)通过以下方式使蛋白质对失活产生抗性 B6G。Hagt的这种突变形式可能会在治疗应用中产生问题 B6G还提供了一个机会来保护正常细胞，如骨髓 通过表达突变形式的基因治疗方法。这些研究 将提供Hagt蛋白中哪些位置的详细知识 可能会出现抗药性突变，并将研究这些突变的稳定性和活性。 在细胞中表达的突变体。此外，在这一特定目标下的研究 旨在分离对b6G灭活更敏感的突变体。最后， 特定目标3的研究将研究hagt的失活和致敏作用。 由使HAGT失活的新型试剂所带来的细胞对烷化剂的作用。 将进行研究以开发第二代制剂，并将 发展在当前赠款支助期内获得的线索，包括：短期 含有b6G的寡聚糖，产生b6G的前药物，以及更好的知识 目标底座被Hagt翻转到的捆绑口袋。

DESCRIPTION: Clinical trials with O6-benzylguanine (b6G) are in progress. The success of this therapy may be compromised by: (a) the facile production of mutants of hAGT resistant to b6G; (b) the lack of adequate potency and tumor specificity and (c) the enhanced toxicity of alkylating agent therapy when AGT inhibitors are used. Experiments are proposed to address these issues building on advances made during the previous period of support which include: (i) development of a selection method for b6G-resistant mutants; (ii) studies showing that these mutants can protect mammalian cells from alkylating agents in the presence of b6G, (iii) derivation of the crystal structure of the hAGT protein; and (iv) indications that short oligodeoxynucleotides containing b6G are extremely potent inactivators of hAGT. The proposed research project has three specific aims. The first specific aim is to characterize the interaction between hAGT and b6G and oligos containing b6G. Studies in this aim will test and refine the current models for binding of b6G and related compounds to hAGT by comparing the inactivation by (and binding of) various b6G derivatives to hAGT mutant proteins in which residues thought to be involved in binding are altered. It is also planned to use various mutants of hAGT such as C145S and inhibitors to obtain crystal structures of complexes with hAGT showing how binding occurs. The second specific aim will obtain a full understanding of hAGT mutants with altered sensitivity to b6G and other inhibitors. Many point mutations (and at least one naturally occurring polymorphic variant) in hAGT render the protein resistant to inactivation by b6G. Such mutant forms of hAGT may create problems in the therapeutic use of b6G but also provide an opportunity to protect normal cells such as bone marrow by gene therapy approaches in which a mutant form is expressed. These studies will provide a detailed knowledge of the sites in hAGT protein at which resistant mutants can occur, and will study the stability and activity of these mutants when expressed in cells. Furthermore, the studies in this specific aim are aimed at isolating mutants more sensitive to inactivation by b6G. Finally, studies in Specific Aim 3 will study the inactivation of hAGT and sensitization of cells to alkylating agents brought about by novel agents inactivating hAGT. Studies will be carried out to develop second generation agents and will develop leads obtained in the current grant support period which include: short oligos containing b6G, pro-drugs generating b6G, and better knowledge of the binding pocket into which the target base is flipped by the hAGT.