Discovery of novel PCFT-targeted agents

新型 PCFT 靶向药物的发现

• 批准号: 8231365
• 负责人: ALEEM GANGJEE
• 金额: $ 45.88万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2016-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8231365
• 关键词: 4-Aminobenzoic Acid; Affinity; Anabolism; Anions; Antineoplastic Agents; Binding; Biological; Blood; Bone Marrow; Cell Line; Cell Proliferation; Cells; Chinese Hamster; Collaborations; Coupled; Cytotoxic agent; Cytotoxin; Diffusion; Drug Design; Duodenum; Engineering; Enzymes; Evaluation; Exhibits; Folate; Folic Acid; Folic Acid Antagonists; Formates; Generations; Glutamates; Glycine; Goals; Growth; Hamsters; Homeostasis; Human; Hydroxymethyltransferases; Implant; In Situ; In Vitro; Institution; Intestines; Kidney; Label; Lead; Leucovorin; Ligase; Liver; Malignant Neoplasms; Mammalian Cell; Mediating; Membrane; Membrane Transport Proteins; Metabolic; Metabolism; Methotrexate; Modeling; Modification; Molecular; Molecular Models; Molecular Profiling; Molecular Target; Monitor; Non-Small-Cell Lung Carcinoma; Normal Cell; Nucleosides; Nucleotide Biosynthesis; Ovarian Carcinoma; Ovary; Pattern; Pemetrexed; Pharmaceutical Preparations; Pharmacology; Physiological; Primary carcinoma of the liver cells; Property; Protons; Pteridines; Purine Nucleotides; Purines; Radiolabeled; Raltitrexed; Reporting; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleotides; Role; Route; SCID Mice; SLC19A1 gene; Series; Solid Neoplasm; Specificity; Structure; Structure of choroid plexus; Structure-Activity Relationship; System; Testing; Tetrahydrofolates; Therapeutic; Thymidylate Synthase; Thymidylate Synthase Inhibitor; Time; Tissues; Toxic effect; Transmembrane Transport; Tumor Cell Line; absorption; analog; antitumor agent; base; brush border membrane; cancer therapy; chemotherapy; cytotoxic; cytotoxicity; efficacy trial; experience; folate-binding protein; glycine amide; hepatoma cell; human tissue; in vivo; inhibitor/antagonist; interstitial; leukemia; lung Carcinoma; molecular modeling; neoplastic cell; novel; pharmacophore; polyglutamates; public health relevance; purine; radiotracer; small molecule; subcutaneous; therapeutic target; tumor; uptake

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.

描述（由申请人提供）：近年来，基于肿瘤选择性膜运输的能力，人们对基于叶酸的癌症进行了重点。该应用探讨了人类质子偶联叶酸转运蛋白（HPCFT）的治疗潜力，叶酸转运蛋白（HPCFT）是一种最近发现的针对叶酸和抗染料的膜传输系统，该系统在功能上是功能性的，并且在解剖学上与无处不在表达的降低的叶酸载体（RFC），主要组织型牛酸酯转运蛋白携带者。我们假设HPCFT代表了一种新颖的选择性手段，用于靶向未由RFC转运的小分子细胞毒素的治疗方法。该概念基于许多人类实体瘤中的频繁和高水平的HPCFT表达，酸性的pH值是HPCFT VIS`vis RFC的最佳pH，与实体瘤的pH微环境相似，最重要的是，最重要的是，第一个小分子细胞毒素可以通过HPCFT选择为HPCFT选择性地运输，但未RFC。对于类似物的AG17，AG71和AG94，由HPCFT转运和抑制从甘霉素核糖核苷酸甲基转移酶的NovoNe军生物合成产生的体外细胞毒性。另一种细胞毒性HPCFT底物AG112是胸苷酸合酶的有效抑制剂。 AG71在体内对SCID小鼠中皮下人肝癌细胞进行了测试，并显示出有效的HPCFT靶向活性而没有明显的毒性。该项目的目的是基于HPCFT超过RFC的细胞摄取，开发新一代有效的肿瘤化疗剂。在AIM 1中，我们将基于AG17，AG71，AG94和AG112的结构活性谱，以及与环系统，接头域，链接域和末端谷氨酸剂的修饰，基于22系列化合物的新型双环和三环类似物。在AIM 2中，我们测试了AIM 1的化合物在等源性仓鼠和人类肿瘤细胞系模型中具有既定的RFC和HPCFT表达模式，通过核苷保护，原位代谢标记和分析细胞内代谢物的分析以及对分离的enzymes的研究。其他研究将表征新型类似物与HPCFT的转运特性，以及对聚谷酸酯的代谢。最后，在AIM 3中，我们将通过体内毒性和疗效试验在植入SCID小鼠中表达HPCFT的人类肿瘤的体内毒性和疗效试验来评估最有效的HPCFT靶向类似物的体内效率。我们的结果将定义通过HPCFT进行的综合结构活性关系，并提供具有有效和选择性的抗肿瘤活性的优化类似物，以针对表达HPCFT的表达肿瘤的体外和体内。这项研究将定义新型HPCFT靶向细胞毒性类似物的作用机制，并有可能提供临床上使用的药物，尽管具有与当前使用的抗肿瘤活性不同的抗肿瘤活性和降低的毒性。 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亲和叶酸受体（FRS）。我们假设HPCFT代表了一种用于靶向小分子细胞毒素的新型且选择性的机制，该机制不是由其他主要（抗）叶酸转运系统（尤其是HRFC）转运的小分子细胞毒素。这个概念基于许多人类实体瘤中的频繁和高水平的HPCFT表达，酸性pH值最佳的HPCFT相对于HRFC的HRFC与固体瘤报道的低pH微环境相似，最重要的是，最重要的是，最重要的是，最重要的是通过HPCFT选择了第一小分子细胞毒素，但AG9（ag9）是ag9 and hrfc的17，而不是Hrfc（ag9）（ag9）（agn）。 AG112）。该应用的一个重要目标是合理地开发特定的HPCFT运输底物，该底物将为抗肿瘤剂提供对主要使用HRFC进行（抗）叶酸摄取的正常细胞而没有毒性的抗肿瘤剂。据我们所知，这种专门从事抗癌药物分子药理学的成熟的生物化学主义者与一位出色的药物化学家之间的这种合作，并展示了抗生物学药物设计的记录，代表了唯一能够为所有治疗性相关（抗）叶酸转运者（包括HRFC和HRFC和HRFC和HRFC和hrfc和frs和frs and frs和frs and frs and frs and frs and Hrfc and frs and frs and frs and frs and frs and frs and frs and frs and frs and frs and frs and frs and frs and frs and frs and frs and frs and frs。