TUMOR SENSITIZATION TO PURINE ANALOGS BY E COLI PNP

在肿瘤细胞中选择性表达非人类基因， 从无毒化合物中生产有毒化合物正在考虑 治疗各种实体瘤， 化学治疗剂。 国家癌症研究所的一项临床试验 评估更昔洛韦在不能手术的脑中激活的可能性 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， HSV-TK基因对人肿瘤细胞体内的杀伤作用不充分 杀死未转导HSV-TK的邻近细胞的能力 基因代表了这种基因研究方法中的两个主要实际障碍 治疗癌症。 由于使用HSV-TK的这些问题， 激活化合物，我们已经开发出一种策略， 这些化合物不会被捕获在它们形成的细胞中。 我们利用了人与大肠杆菌之间底物的差异。杆菌 嘌呤核苷磷酸化酶（PNP）以激活无毒前药， 有毒的嘌呤碱。E. coliPNP识别含腺嘌呤核苷 作为底物，而人PNP不作为底物。初步研究表明 E.大肠杆菌PNP在不到1%的人类癌细胞中， 培养导致几乎所有旁观者细胞死亡， 用前体药物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， 转化为MeP，一种有毒的嘌呤碱。coli PNP，但不被人感染 菲律宾国家警察另外，我们的初步研究表明， 以这种方式创造出来的生物， 复制细胞，这进一步区分了我们的方法， 目前使用的大多数抗肿瘤药物 剂. NCDDG的长期目标是开发癌症治疗策略 基于E. coli PNP在肿瘤细胞中的表达， 活化无毒嘌呤类似物前药。 该NCDDG由4个 方案和一个核心。该国家疾病发展指导方针各组成部分的目标是： 1)分子生物学计划，以制定程序，有选择地 将E.大肠杆菌PNP基因导入肿瘤细胞的全过程 2）生物化学程序，以充分表征生物化学 嘌呤核苷类似物及其相应碱基的药理学 帮助合理设计新的前药; 3）化学计划， 设计合成无毒的嘌呤核苷前药， 在表达E.杆菌 PNP基因; 4）X射线晶体学程序，以确定 E.大肠杆菌PNP，并与哺乳动物酶的结构进行比较， 有助于新前药的合理设计;和5）化疗核心， 评价本NCDDG中开发的前药的抗肿瘤活性， 借助分子生物学技术开发的相关动物肿瘤模型 生物学课程。

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.