Discovery of novel PCFT-targeted agents

描述(由申请人提供)：近年来，基于肿瘤选择性膜转运能力的叶酸治疗癌症再次受到重视。这项应用探索了人类质子偶联叶酸转运体(HPCFT)的治疗潜力，这是一种新近发现的叶酸和抗叶酸的膜转运系统，在功能和(某种程度上)解剖上与普遍表达的还原叶酸载体(RFC)不同，RFC是主要的组织叶酸转运体。我们假设hPCFT代表了一种新的和选择性的治疗靶向小分子细胞毒素的手段，这些小分子细胞毒素不是由RFC转运的。这一概念是基于hPCFT在许多实体肿瘤中频繁和高水平的表达，hPCFT相对于RFC的最适酸性pH与实体瘤的pH微环境平行，以及最重要的是，鉴定了第一批由hPCFT而不是RFC选择性转运的小分子细胞毒素。对于类似物AG17、AG71和AG94，体外细胞毒性是由于hPCFT转运和甘氨酰胺核糖核苷酸甲酰转移酶抑制从头合成嘌呤所致。另一种细胞毒性hPCFT底物AG112是胸苷合成酶的有效抑制物。在SCID小鼠体内测试AG71对皮下人肝癌细胞的作用，显示出强大的hPCFT靶向活性，没有明显的毒性。该项目的目标是开发新一代有效的肿瘤靶向化疗药物，其基础是hPCFT对细胞摄取的选择性能力高于RFC。在目标1中，我们将根据AG17、AG71、AG94和AG112的构效关系和分子模拟，通过对环系、连接域和末端谷氨酸的修饰，从22个系列的化合物中合成新型的双环和三环类似物。在目标2中，我们测试了来自目标1的化合物在同基因仓鼠和人类肿瘤细胞系模型中的细胞毒性，建立了RFC和hPCFT的表达模式，通过核苷保护、细胞内代谢物的原位代谢标记和分析以及分离的酶的研究来确定分子靶点。更多的研究将表征新型类似物hPCFT相对于RFC的转运特性，以及代谢到聚谷氨酸的特性。最后，在目标3中，我们将通过体内毒性和疗效试验来评估最有效的hPCFT靶向类似物的疗效，将hPCFT表达的人类肿瘤植入SCID小鼠体内。我们的结果将确定hPCFT相对于RFC转运的全面构效关系，并提供优化的类似物，在体外和体内对hPCFT表达的肿瘤具有有效和选择性的抗肿瘤活性。这项研究将确定新型hPCFT靶向细胞毒类似物的作用机制，并可能提供用于临床的药物，尽管其抗肿瘤活性和毒性与目前使用的不同。

DESCRIPTION (provided by applicant): In recent years, there has been renewed emphasis on folate-based therapeutics for cancer based on capacities for tumor-selective membrane transport. This application explores the therapeutic potential for the human proton-coupled folate transporter (hPCFT), a recently discovered membrane transport system for folates and antifolates which is functionally and (to some degree) anatomically distinct from the ubiquitously expressed reduced folate carrier (RFC), the major tissue folate transporter. We hypothesize that hPCFT represents a novel and selective means for therapeutic targeting small molecule cytotoxins that are not transported by RFC. This concept is based on frequent and high level hPCFT expression in many human solid tumors, the acidic pH optimum for hPCFT vis ` vis RFC which parallels the pH microenvironments of solid tumors, and, most significantly, identification of the first small molecule cytotoxins that are selectively transported by hPCFT but not RFC. For analogs AG17, AG71, and AG94, in vitro cytotoxicities resulted from hPCFT transport and inhibition of de novo purine biosynthesis at glycinamide ribonucleotide formyltransferase. Another cytotoxic hPCFT substrate, AG112, was a potent inhibitor of thymidylate synthase. AG71 was tested in vivo against subcutaneous human hepatoma cells in SCID mice and showed potent hPCFT-targeted activity without significant toxicity. The goal of this project is to develop a new generation of potent tumor-targeted chemotherapy agents based on their selective capacities for cellular uptake by hPCFT over RFC. In aim 1, we will synthesize novel bicyclic and tricyclic analogs from 22 series of compounds, based on structure-activity profiles for AG17, AG71, AG94 and AG112, and molecular modeling with modifications of the ring systems, the linker domain, and the terminal glutamate. In aim 2, we test compounds from aim 1 for cytotoxicity in isogenic hamster and human tumor cell line models with established patterns of RFC and hPCFT expression, identify molecular targets by nucleoside protection, in situ metabolic labeling and analysis of intracellular metabolites, and studies with isolated enzymes. Additional studies will characterize transport properties of the novel analogs with hPCFT vis a vis RFC, and metabolism to polyglutamates. Finally, in aim 3, we will evaluate in vivo efficacies of the most potent hPCFT-targeted analogs by in vivo toxicity and efficacy trials in hPCFT-expressing human tumors implanted into SCID mice. Our results will define a comprehensive structure-activity relationship for transport by hPCFT vis a vis RFC and afford optimized analogs with potent and selective antitumor activities against hPCFT-expressing tumors in vitro and in vivo. This study will define mechanisms of action of the novel hPCFT-targeted cytotoxic analogs and potentially provide agents to be used clinically, albeit with a different spectrum of antitumor activities and reduced toxicities than those currently in use.