Molecular Regulation of Folate and Antifolate Transport

叶酸和抗叶酸转运的分子调节

• 批准号: 9025683
• 负责人: Zhanjun Hou
• 金额: $ 26.85万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-02-12 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9025683
• 关键词: Acids; Affinity; Alimta; Anions; Area; Binding; Biochemical; Biological; Biology; Blood; Carrier Proteins; Cell Line; Cells; Clinic; Clinical; Clinical Trials; Cloning; Coupled; Cytotoxin; Deoxycytidine; Development; Diet; Diffusion; Disease; Drug or chemical Tissue Distribution; Drug resistance; Exhibits; Eye; Figs - dietary; Folic Acid; Folic Acid Antagonists; Fostering; Gene Expression Regulation; Gene Structure; Generations; Genetic Polymorphism; Genetic Transcription; Goals; Health; HepG2; Histone Deacetylase Inhibitor; Homo; Homology Modeling; Human; Hydroxymethyltransferases; Immunodeficient Mouse; Immunohistochemistry; Inherited; Intestinal Absorption; Lometrexol; Malabsorption Syndromes; Malignant Neoplasms; Mammalian Cell; Mediating; Membrane; Mesothelioma; Methotrexate; Molecular; Mutate; Neuraxis; Non-Small-Cell Lung Carcinoma; Nucleotide Biosynthesis; Patients; Pemetrexed; Pharmaceutical Preparations; Physiological; Primary Neoplasm; Primary carcinoma of the liver cells; Protons; Publishing; Purine Nucleotides; Pyrimidine; Raltitrexed; Reagent; Regulation; Reporter Genes; Reporting; Research; Resistance; Ribonucleotides; Role; Route; S Phase Arrest; SLC19A1 gene; Solid Neoplasm; Structure; Structure-Activity Relationship; System; Tetrahydrofolates; Therapeutic; Tissues; Transcript; Transcriptional Regulation; Translating; Transmembrane Domain; Transmembrane Transport; Tumor Cell Line; Valproic Acid; Vitamins; Western Blotting; Xenograft procedure; absorption; analog; base; cancer cell; cancer therapy; clinically relevant; cofactor; cytotoxic; design; drug discovery; experience; folate-binding protein; folic acid supplementation; gastrointestinal; glycine amide; human tissue; improved; insight; neoplastic cell; novel; novel strategies; novel therapeutics; polyglutamates; prophylactic; protein structure function; small molecule; stoichiometry; structural biology; targeted cancer therapy; targeted treatment; therapeutic target; tumor; tumor microenvironment; uptake

DESCRIPTION (provided by applicant): In recent years, there has been renewed emphasis on folate-based therapeutics for cancer, reflecting on capacities for tumor-selective membrane transport. This application explores the therapeutic potential for the human proton-coupled folate transporter (hPCFT). Unlike the human reduced folate carrier (hRFC), the major tissue folate transporter which is ubiquitously expressed and has a neutral pH optimum, hPCFT shows a narrower tissue distribution and is characterized by an acid pH optimum. hPCFT shows substantial transport at acid pHs characterizing the tumor microenvironment and limited transport at neutral pH. We showed that hPCFT is widely expressed in human tumor cell lines and primary tumors. In human tumor cells, hPCFT transcripts paralleled levels of hPCFT proteins on Western blots and transport activity. In human tumor cells, hPCFT reporter gene activity paralleled disparate levels of hPCFT transcripts. We initiated hPCFT structure-function studies and characterized a mechanistically and structurally important "re-entrant loop" domain between transmembrane domains (TMDs) 2 and 3 and localized a substrate binding region within TMD2. We demonstrated a functionally important role for hPCFT oligomerization and localized an important oligomer interface to TMD6. In a separate line of study, we identified novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates that are selectively transported by hPCFT but not hRFC, although uptake by folate receptor was also preserved. These efforts have fostered a new paradigm, the rational development of tumor-targeted therapies based on tumor-specific expression and/or function of the major folate transporters, PCFT and FR. We will continue our comprehensive study of the major facilitative folate transporters, with implications to folate-based therapies for cancer, and to prophylactic folate supple- mentation for cancer. Our emphasis is on optimizing our novel hPCFT-selective therapeutics for tumor targeting, by exploring the biology of this physiologically important transporter, drawing from a compelling rationale for targeting solid tumors by this mechanism. In Specific Aim 1, we will characterize molecular and cellular determinants of hPCFT- targeted cancer therapy, including establishing functional stoichiometries for hRFC vis a vis hPCFT, the therapeutic impact of concomitant tumor expression of hPCFT and hRFC, as well as folate receptor , and mechanisms of hPCFT transcriptional regulation in human tumor cells. In Aim 2, we will characterize structural and functional determinants of hPCFT-targeted therapy, including identification of functionally important domains and residues in the hPCFT protein, and the structural, functional, and regulatory features of hPCFT homo-oligomers. The goal is to use biochemical studies of hPCFT structure and function, and molecular homology modeling, to assist our drug discovery efforts, and to identify new approaches to enhance tumor targeting and efficacy of hPCFT-selective drugs. Our proposed studies are strongly supported by our preliminary and published results and the availability of critical reagents and cell lines, includig the first generation of specific and potent hPCFT-selective tumor-targeted antifolates that are not transported by hRFC. Our studies are distinctive for their novelty and likelihood of providing critical new insights into mechanisms and regulation of these physiologically and pharmacologically important vitamin transporters that can be translated to the clinic.

描述（由申请人提供）：近年来，人们重新重视基于叶酸的癌症疗法，反映了肿瘤选择性膜运输的能力。该应用探索了人类质子耦合叶酸转运蛋白 (hPCFT) 的治疗潜力。与人还原叶酸载体 (hRFC)（广泛表达且具有中性 pH 最佳值的主要组织叶酸转运蛋白）不同，hPCFT 显示出较窄的组织分布，并以酸性 pH 为特征。 hPCFT 在表征肿瘤微环境的酸性 pH 下表现出大量转运，而在中性 pH 下则表现出有限的转运。我们发现 hPCFT 在人类肿瘤细胞系和原发性肿瘤中广泛表达。在人类肿瘤细胞中，hPCFT 转录的 hPCFT 蛋白水平与蛋白质印迹和转运活性平行。在人类肿瘤细胞中，hPCFT 报告基因活性与不同水平的 hPCFT 转录本平行。我们启动了 hPCFT 结构功能研究，并表征了跨膜结构域 (TMD) 2 和 3 之间机械和结构上重要的“重入环”结构域，并定位了 TMD2 内的底物结合区域。我们证明了 hPCFT 寡聚化在功能上的重要作用，并将重要的寡聚物界面定位到 TMD6。在另一项研究中，我们发现了新型 6-取代吡咯并[2,3-d]嘧啶噻吩甲酰抗叶酸剂，尽管叶酸受体的摄取也被保留，但它们可以被 hPCFT 而不是 hRFC 选择性转运。这些努力培育了一种新的范例，即基于主要叶酸转运蛋白、PCFT 和 FR 的肿瘤特异性表达和/或功能的肿瘤靶向治疗的合理开发。我们将继续对主要的促进叶酸转运蛋白进行全面研究，这对基于叶酸的癌症疗法以及预防性补充癌症叶酸具有重要意义。我们的重点是通过探索这种生理上重要的转运蛋白的生物学，并从通过这种机制靶向实体瘤的令人信服的原理中得出结论，优化我们用于肿瘤靶向的新型 hPCFT 选择性疗法。在具体目标 1 中，我们将描述 hPCFT 靶向癌症治疗的分子和细胞决定因素，包括建立 hRFC 与 hPCFT 的功能化学计量、hPCFT 和 hRFC 以及叶酸受体同时肿瘤表达的治疗影响，以及人类肿瘤细胞中 hPCFT 转录调节的机制。在目标 2 中，我们将表征 hPCFT 靶向治疗的结构和功能决定因素，包括鉴定 hPCFT 蛋白中功能重要的结构域和残基，以及 hPCFT 同源寡聚物的结构、功能和调控特征。目标是利用 hPCFT 结构和功能的生化研究以及分子同源建模来协助我们的药物发现工作，并确定增强 hPCFT 选择性药物的肿瘤靶向性和功效的新方法。我们提出的研究得到了我们的初步和已发表的结果以及关键试剂和细胞系的可用性的大力支持，包括第一代不由 hRFC 转运的特异性且有效的 hPCFT 选择性肿瘤靶向抗叶酸剂。我们的研究因其新颖性而与众不同，并且有可能为这些生理和药理学重要的维生素转运蛋白的机制和调节提供重要的新见解，并可以转化为临床。