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剂重新受到重视。本申请探索了人质子偶联叶酸转运蛋白（hPCFT）的治疗潜力，hPCFT是最近发现的叶酸和抗叶酸剂的膜转运系统，其在功能上和（在某种程度上）解剖学上不同于广泛表达的还原叶酸载体（RFC），主要的组织叶酸转运蛋白。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，不通过RFC运输的手段。这个概念是基于许多人实体瘤中频繁和高水平的hPCFT表达，hPCFT维斯维斯RFC的最佳酸性pH与实体瘤的pH微环境平行，最重要的是，鉴定了第一个由hPCFT而不是RFC选择性转运的小分子细胞毒素。对于类似物AG 17、AG 71和AG 94，体外细胞毒性由hPCFT转运和在甘氨酰胺核糖核苷酸甲酰转移酶处从头嘌呤生物合成的抑制引起。另一种细胞毒性hPCFT底物AG 112是胸苷酸合酶的有效抑制剂。在SCID小鼠中针对皮下人肝癌细胞在体内测试了AG 71，并且显示出有效的hPCFT靶向活性而没有显著毒性。该项目的目标是开发新一代有效的肿瘤靶向化疗药物，其基于hPCFT对RFC的细胞摄取的选择性能力。在目标1中，我们将基于AG 17、AG 71、AG 94和AG 112的构效关系，以及对环系统、连接结构域和末端谷氨酸进行修饰的分子建模，从22个系列的化合物中合成新型双环和三环类似物。在目标2中，我们在具有RFC和hPCFT表达的已建立模式的同基因仓鼠和人肿瘤细胞系模型中测试来自目标1的化合物的细胞毒性，通过核苷保护、原位代谢标记和细胞内代谢物的分析来鉴定分子靶标，并且用分离的酶进行研究。另外的研究将表征具有hPCFT的新型类似物维斯于维斯于RFC的转运性质，以及代谢为聚谷氨酸盐。最后，在目标3中，我们将通过在植入SCID小鼠中的表达hPCFT的人肿瘤中的体内毒性和功效试验来评估最有效的hPCFT靶向类似物的体内功效。我们的研究结果将定义一个全面的结构-活性关系的运输hPCFT维斯一个维斯RFC，并提供优化的类似物与有效的和选择性的抗肿瘤活性对hPCFT表达的肿瘤在体外和体内。本研究将确定新型hPCFT靶向细胞毒性类似物的作用机制，并可能提供临床使用的药物，尽管与目前使用的药物相比，具有不同的抗肿瘤活性谱和降低的毒性。 公共卫生相关性：本申请探索了人质子偶联叶酸转运蛋白（hPCFT）的潜在治疗应用，hPCFT是最近发现的用于许多人组织和实体瘤中的叶酸和抗叶酸剂的膜转运系统，其在功能上和生物学上是可接受的。（在某种程度上）在解剖学上不同于普遍表达的人还原叶酸载体（hRFC），人细胞和组织中的主要组织叶酸转运系统，和高亲和力叶酸受体（FR）。我们假设，hPCFT代表了一种新的和选择性的机制，治疗靶向小分子细胞毒素，不通过其他主要的（抗）叶酸转运系统，最显着的hRFC。这一概念是基于许多人实体瘤中频繁和高水平的hPCFT表达，hPCFT维斯维斯hRFC的最佳酸性pH，其与实体瘤报道的低pH微环境相似，最重要的是，鉴定了第一种由hPCFT而不是hRFC选择性转运的小分子细胞毒素（典型的是化合物AG 17、AG 71、AG 94和AG 112）。本申请的一个重要目标是合理开发hPCFT的特异性转运底物，其将提供对主要使用hRFC进行（抗）叶酸摄取的正常细胞无毒性的抗肿瘤剂。据我们所知，一位专门从事抗癌药物分子药理学的有成就的生物化学家和一位具有抗叶酸药物设计的杰出药物化学家之间的合作代表了唯一能够为所有治疗相关（抗）叶酸转运蛋白（包括hRFC和hRFC）和FR产生独特药效团的奋进。

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.