ALKYLTRANSFERASE INHIBITORS FOR CANCER CHEMOTHERAPY

描述：O 6-苄基鸟嘌呤（b6 G）的临床试验正在进行中。的 这种疗法的成功可能会受到以下因素的影响：（a） 抗b6 G的hAGT突变体;（B）缺乏足够的效力和肿瘤 特异性和（c）当AGT时烷化剂治疗的毒性增强 使用抑制剂。实验提出了解决这些问题的建设 上一个支助期间的预付款，其中包括：㈠ 开发b6 G抗性突变体的选择方法;（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b6 G存在下，（iii）衍生hAGT的晶体结构 蛋白质;和（iv）含有b6 G的短寡脱氧核苷酸 是非常有效的hAGT灭活剂。 该研究项目有三个具体目标。第一个具体目标 是表征hAGT和b6 G与包含hAGT和b6 G的寡核苷酸之间的相互作用。 b6G。这方面的研究将测试和完善目前的模型， 通过比较b6 G和相关化合物与hAGT的失活（和结合）， 的）各种b6 G衍生物与hAGT突变体蛋白质，其中残基被认为 与结合有关的基因发生了改变。还计划使用各种 hAGT的突变体如C145 S和抑制剂，以获得 与hAGT的复合物显示结合如何发生。第二个具体目标将 获得对b6 G敏感性改变的hAGT突变体的全面了解， 其他抑制剂。许多点突变（和至少一种天然存在的 hAGT中的多态性变体）使蛋白质抵抗通过以下途径失活： b6G。hAGT的这种突变形式可能在治疗应用中产生问题， b6 G还提供了保护正常细胞如骨髓的机会， 通过基因治疗方法，其中突变形式被表达。这些研究 将提供hAGT蛋白中位点的详细知识， 耐药突变体可以发生，并将研究这些稳定性和活性 突变体在细胞中表达时。此外，针对这一具体目标的研究 旨在分离对b6 G失活更敏感的突变体。最后， 特定目标3中的研究将研究hAGT的失活和致敏作用 细胞的烷化剂所带来的新的代理人灭活hAGT。 将开展研究，开发第二代制剂， 开发在当前赠款支持期内获得的潜在客户，包括： 含有b6 G的寡核苷酸，产生b6 G的前药，以及更好地了解 靶碱基被hAGT翻转到其中的结合口袋。

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.