TUMOR SENSITIZATION TO PURINE ANALOGS BY E COLI PNP

非人类基因在肿瘤细胞中的选择性表达 正在考虑从无毒化合物中生产有毒化合物 各种难治性实体肿瘤的治疗 化疗药物。NCI的一项临床试验已经开始 评估更昔洛韦在不能手术的脑中的激活潜力 用单纯疱疹病毒胸苷转导的肿瘤 激酶(HSV-TK)基因。然而，我们认为，低效的交付 HSV-TK基因在体内对人肿瘤细胞的作用及不足 杀伤未转导HSV-TK的邻近细胞的能力 基因代表了这种基因研究方法的两个主要实际障碍 癌症的治疗。由于使用HSV-TK的这些问题 激活化合物，我们已经制定了一种策略，将有毒物质 不会被困在形成它们的细胞中的化合物。 我们利用了人类和大肠杆菌之间的底物差异 嘌呤核苷磷酸化酶(PNP)激活无毒前体药物 有毒的嘌呤碱基。大肠杆菌PNP识别含腺嘌呤核苷 作为底物，而人的PNP不是。初步研究表明 大肠杆菌PNP在不到1%的人癌细胞中的表达 培养导致几乎所有旁观者细胞在 使用前药6-甲基嘌呤-2‘-脱氧核苷(MEP-DR)治疗。MEP- DR是一种相对无毒的脱氧腺苷核苷类似物，可以 被大肠杆菌PNP转化为MEP，一种有毒的嘌呤碱基，但不被人类转化 即插即用。此外，我们的初步研究表明，一些药物 可以以这种方式创建同时杀死复制和非复制的 复制细胞，这进一步区分了我们对 实体瘤的治疗从目前使用的大多数抗肿瘤药物 探员们。 NCDDG的长期目标是开发一种癌症治疗策略 基于大肠杆菌PNP在肿瘤细胞中的选择性表达 激活无毒的嘌呤类似物前药。本NCDDG由4个部分组成 程序和一个核心。NCDDG各组成部分的目标是： 1)分子生物学计划开发程序，选择性地 将E.ColiPNP基因导入或转导荷瘤细胞 动物；2)全面描述生物化学特性的生物化学程序 嘌呤核苷类似物及其碱基的药理作用 帮助合理设计新的前药；3)化学程序以 设计和合成无毒的嘌呤核苷前药 在表达大肠杆菌的肿瘤细胞中转化为有毒的嘌呤碱基 PNP基因；4)X射线结晶学程序确定其结构 并将其与哺乳动物酶的结构进行比较，以期 帮助合理设计新的前体药物；以及5)化疗核心 NCDDG前体药的抗肿瘤活性评价 借助分子生物学技术建立相关动物肿瘤模型 生物课程。

The selective expression in tumor cells of non-human genes that can produce toxic compounds from non-toxic compounds is being considered for the treatment of various solid tumors that are refractory to existing chemotherapeutics agents. A clinical trial at the NCI as begun to evaluate the potential for activating ganciclovir in inoperable brain tumors that have been transduced with the herpes simplex virus thymidine kinase (HSV-TK) gene. However, we believe that the inefficient delivery of the HSV-TK gene to human tumor cells in vivo together with inadequate ability to kill neighboring cells that are not transduced with the HSV-TK gene represent two major practical hurdles in this approach to the gene therapy of cancer. Because of these problems with the use of HSV-TK to activate compounds, we have developed a strategy to deliver toxic compounds that will not be trapped in the cell in which they are formed. We have utilized the substrate differences between human and E. coli purine nucleoside phosphorylase (PNP) to activate non-toxic prodrugs to toxic purine bases. E. coli PNP recognizes adenine-containing nucleosides as substrates whereas human PNP does not. Preliminary studies have shown that expression of E. coli PNP in less than 1% of human cancer cells in culture leads to the death of virtually all bystander cells after treatment with the prodrug 6-methylpurine-2'-deoxyriboside (MeP-dR). MeP- dR is a relatively nontoxic deoxyadenosine nucleoside analog that can be converted to MeP, a toxic purine base, by E. coli PNP, but not by human PNP. In addition, our preliminary studies indicate that some of the agents that could be created in this manner kill both replicating and non- replicating cells, which further distinguishes our approach for the treatment of solid tumors from most of the currently used antitumor agents. The longterm goal of this NCDDG is to develop a cancer treatment strategy based on the selective expression of E. coli PNP in tumor cells to activate nontoxic purine analog prodrugs. This NCDDG is composed of 4 programs and one core. The objectives of the components of this NCDDG are: 1) Molecular Biology Program to develop procedures to selectively transfect or transduce the E. coli PNP gene into tumor cells of whole animals; 2) Biochemistry Program to fully characterize the biochemical pharmacology of the purine nucleoside analogs and their respective bases to aid in the rational design of new prodrugs; 3) Chemistry Program to design and synthesize nontoxic purine nucleoside prodrugs that will be converted into toxic purine bases in tumor cells that express the E. coli PNP gene; 4) X-ray Crystallography Program to determine the structure of E. coli PNP and to compare it with the structure of mammalian enzymes to aid in the rational design of new prodrugs; and 5) Chemotherapy Core to evaluate the antitumor activity of the prodrugs developed in this NCDDG in relevant animal tumor models developed with the aid of the Molecular Biology Program.