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基于叶酸的癌症治疗再次受到重视。该应用程序探索了人类质子偶联叶酸转运体（hPCFT）的治疗潜力，hPCFT是最近发现的叶酸和反叶酸的膜转运系统，在功能上和（某种程度上）解剖学上不同于普遍表达的减少叶酸载体（RFC），主要的组织叶酸转运体。我们假设hPCFT代表了一种新的、选择性的治疗方法，用于靶向不通过RFC运输的小分子细胞毒素。这个概念是基于hPCFT在许多人类实体肿瘤中频繁和高水平的表达，hPCFT相对于RFC的最佳酸性pH值与实体肿瘤的pH微环境相似，最重要的是，确定了第一个由hPCFT而不是RFC选择性运输的小分子细胞毒素。对于类似物AG17、AG71和AG94，体外细胞毒性是由hPCFT转运和抑制甘氨酸酰胺核糖核苷酸甲酰转移酶的嘌呤生物新合成引起的。另一种具有细胞毒性的hPCFT底物AG112是胸苷酸合成酶的有效抑制剂。AG71在SCID小鼠体内对皮下人肝癌细胞进行了体内试验，显示出强大的hpcft靶向活性，且无明显毒性。该项目的目标是开发新一代有效的肿瘤靶向化疗药物，基于它们对hPCFT在RFC上的细胞摄取的选择性能力。在目标1中，我们将基于AG17、AG71、AG94和AG112的结构-活性谱，以及对环系统、连接域和末端谷氨酸进行修饰的分子模型，从22个系列的化合物中合成新的双环和三环类似物。在目标2中，我们通过建立RFC和hPCFT表达模式，测试目标1中的化合物在等基因仓鼠和人类肿瘤细胞系模型中的细胞毒性，通过核苷保护，原位代谢标记和细胞内代谢物分析确定分子靶点，并使用分离酶进行研究。进一步的研究将表征新型类似物与hPCFT相对于RFC的运输特性，以及对多谷氨酸的代谢。最后，在目标3中，我们将通过在植入SCID小鼠的表达hpcft的人类肿瘤中进行体内毒性和功效试验，评估最有效的hpcft靶向类似物的体内疗效。我们的研究结果将定义hPCFT与RFC之间的全面结构-活性关系，并提供优化的类似物，在体外和体内对表达hPCFT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靶向hpcft的细胞毒性类似物的作用机制，并有可能提供临床使用的药物，尽管与目前使用的药物具有不同的抗肿瘤活性和降低的毒性。

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. PUBLIC HEALTH RELEVANCE: This application explores potential therapeutic applications of human proton-coupled folate transporter (hPCFT), a recently discovered membrane transport system for folates and antifolates in many human tissues and solid tumors which is functionally and (to some degree) anatomically distinct from the ubiquitously expressed human reduced folate carrier (hRFC), the major tissue folate transport system in human cells and tissues, and the high affinity folate receptors (FRs). We hypothesize that hPCFT represents a novel and selective mechanism for therapeutic targeting small molecule cytotoxins which are not transported by other major (anti)folate transport systems, most notably hRFC. This concept is based on the frequent and high level hPCFT expression in many human solid tumors, the acidic pH optimum for hPCFT vis a vis hRFC which parallels the low pH microenvironments reported for solid tumors, and, most significantly, identification of first small molecule cytotoxins that are selectively transported by hPCFT but not hRFC (typified by compounds AG17, AG71, AG94, and AG112). An important goal of this application is to rationally develop specific transport substrates for hPCFT that will afford antitumor agents without toxicity to normal cells that primarily use hRFC for (anti)folate uptake. To our knowledge, this collaboration between an accomplished biochemist specializing in molecular pharmacology of anticancer drugs and an outstanding medicinal chemist with a demonstrated track record of antifolate drug design represents the only such endeavor capable of generating unique pharmacophores for all the therapeutically relevant (anti)folate transporters, including hRFC and hRFC, and FRs.