Molecular Regulation of Folate and Antifolate Transport

叶酸和抗叶酸转运的分子调节

基本信息

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
项目类别：
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

项目摘要

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，人们对基于叶酸的癌症的治疗剂进行了更新，这反映了肿瘤选择性膜运输的能力。该应用探讨了人类质子偶联叶酸转运蛋白（HPCFT）的治疗潜力。与人类还原叶酸载体（HRFC）不同，它是普遍表达并具有中性pH最佳的主要组织叶酸转运蛋白，HPCFT显示出较窄的组织分布，其特征是酸pH值最佳。 HPCFT在酸性pHS时显示出大量的运输，这些pH值表征了肿瘤微环境和中性pH时的运输有限。我们表明，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转录本在蛋白质印迹和运输活性上平行于HPCFT蛋白水平。在人类肿瘤细胞中，HPCFT报告基因活性与HPCFT转录物的不同水平平行。我们启动了HPCFT结构功能研究，并在跨膜结构域（TMDS）2和3之间进行了机械和结构上重要的“重点环”结构域的表征，并在TMD2中定位了底物结合区域。我们证明了HPCFT寡聚化的功能重要作用，并将重要的低聚物界面定位于TMD6。在另一项研究中，我们鉴定了新型的6叠二取代的吡咯洛（2,3-d]吡啶胺硫烯酰胺抗染料，尽管也保留了叶酸受体的摄取，但通过HPCFT选择性地通过HPCFT进行了选择性地运输。这些努力促进了一种新的范式，即基于肿瘤特异性表达和/或主要叶酸转运蛋白的功能，PCFT和FR的合理发展。我们将继续对主要促进叶酸转运蛋白的全面研究，对基于叶酸的癌症的疗法以及预防性叶酸的疗法的影响。我们的重点是通过探索该生理上重要的转运蛋白的生物学来优化我们的新型HPCFT选择性疗法，以通过这种机制来靶向实体瘤的生物学。 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.在AIM 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蛋白中具有功能重要的结构域和残基，以及HPCFT Homo-Oligomers的结构，功能和调节特征。目的是使用HPCFT结构和功能以及分子同源性建模的生化研究，以协助我们的药物发现工作，并确定新的方法来增强HPCFT-选择性药物的肿瘤靶向和功效。我们提出的研究得到了我们的初步和发表的结果以及关键试剂和细胞系的可用性，包括第一代特定和有效的HPCFT选择性肿瘤靶向的抗染料，而HRFC未运输。我们的研究对于它们的新颖性和可能性，即对这些生理和药理上重要的维生素转运蛋白提供关键的新见解的新颖性和可能性，可以转化为诊所。