Proton-coupled folate/antifolate transport

质子耦合叶酸/抗叶酸转运

• 批准号: 7737545
• 负责人: Israel DAVID GOLDMAN
• 金额: $ 52.67万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7737545
• 关键词: Adult; Antineoplastic Agents; Binding; Biology; Brain; Caco-2 Cells; Charge; Chemicals; Clinical; Colorectal Cancer; Coupling; Cysteine; DNA Methylation; Defect; Disease; Drug Kinetics; Endocytosis; Endosomes; Folate; Folic Acid Antagonists; Folic Acid Deficiency; Funding; Generations; Genes; Genetic; Goals; Growth; Homeostasis; Human; Human Cell Line; Hypoxia; In Vitro; Indium; Inherited; Intestinal Absorption; Intestines; Kinetics; Laboratories; Laboratory Study; Maintenance; Malabsorption Syndromes; Malignant Neoplasms; Mediating; Membrane Transport Proteins; Methotrexate; Modeling; Movement; Mutagenesis; Neuraxis; Non-Small-Cell Lung Carcinoma; Normal Cell; Pathogenesis; Pathway interactions; Pemetrexed; Penetration; Pharmaceutical Preparations; Physiological; Play; Prevention; Prevention strategy; Property; Protons; Publishing; Regulation; Research; Response Elements; Risk Factors; Role; Route; Simulate; Site-Directed Mutagenesis; Solid Neoplasm; Structural Models; Structure; Structure-Activity Relationship; System; Treatment Protocols; Tumor Cell Line; Up-Regulation; Xenopus oocyte; absorption; cancer cell; cancer prevention; cancer risk; cancer therapy; chemical function; clinically relevant; folate-binding protein; improved; loss of function mutation; man; meetings; melanoma; neoplastic cell; novel; pressure; promoter; public health relevance; tumor; tumor progression; voltage clamp

DESCRIPTION (provided by applicant): Studies over the current funding period focused on characterization of a novel low-pH folate transport activity widely expressed in human solid tumor and normal cells. The penultimate goal was to clone the "Low-pH" transporter. This was accomplished with the identification of the "Proton-Coupled Folate Transporter" (PCFT). The pharmacological role of PCFT was demonstrated by its salutary impact on the activity of the new-generation antifolate, pemetrexed. Its physiological role was demonstrated by loss-of-function mutations in the PCFT gene in hereditary folate malabsorption (HFM), an autosomal recessive disorder characterized by severe defects in intestinal folate absorption and transport of folates into the central nervous system. PCFT also appears to provide an export route for folates from acidified endosomes consistent with a role in folate receptor - mediated transport. The current proposal builds on published findings along with preliminary results that support the feasibility of the four specific aims: Specific Aim 1 is to characterize PCFT-mediated transport and its impact on the pharmacological activities of pemetrexed and methotrexate in vitro under conditions that simulate drug pharmacokinetics in clinical regimens and at pH's that approximate what is found in solid tumors. Specific Aim 2 is to further assess the role PCFT plays in FR1- and FR2- mediated endocytosis focusing on the physiological and pharmacological ramifications in terms of meeting cellular folate growth requirements and antifolate growth inhibition. Specific Aim 3 is to characterize the topology and structure-function of PCFT using three approaches - site-directed mutagenesis, chemical mutagenesis under antifolate selective pressure, and substituted cysteine accessibility mutagenesis. The functional role of PCFT residues is evaluated by transport kinetics in model human cell lines and current induction and acidification in voltage-clamped Xenopus oocytes. Results are considered with the context of an evolving structural model for PCFT in which the substrate translocation pathway and potential binding and interacting residues can be predicted and evaluated experimentally. Specific Aim 4 is focused on regulation of PCFT expression, including the mechanism of up-regulation with hypoxia, another element in the tumor microenvironment. These studies have clinical relevance. From the pharmacological perspective, pemetrexed is an important antifolate recently approved for the treatment of malignant melanoma and non-small cell lung cancer and is being evaluated for efficacy in other malignancies. Observations from these laboratory studies may contribute to enhancing the clinical utility of this agent. From a physiological perspective, PCFT plays a critical role in intestinal absorption of folates and the maintenance of folate sufficiency. Folate deficiency is a risk-factor for colorectal cancer and folate excess may augment progression of tumors already formed. Hence, elucidation of the biology of PCFT contributes to the understanding of factors that are determinants of folate homeostasis in man and impact on the pathogenesis of cancer and other folate-deficiency disorders. PUBLIC HEALTH RELEVANCE: This proposal is focused on the proton-coupled folate transporter (PCFT), recently discovered in this laboratory, responsible for the absorption of dietary folates in the intestine and movement of folates into the brain. Genetic damage to PCFT results in an inherited disease of folate deficiency, hereditary folate malabsorption. Folate deficiency in adults is associated with an increased risk of cancer. PCFT also contributes to the penetration of a new anticancer drug, pemetrexed, into cancer cells. This research will broaden our understanding of how PCFT functions and this may result in improved cancer treatment with pemetrexed, new cancer prevention strategies, and the prevention of folate-deficiency states.

描述（由申请人提供）：在当前资金期间的研究集中于表征在人类实体瘤和正常细胞中广泛表达的新型低ph叶酸转运活性。倒数第二个目标是克隆“低ph”运输蛋白。这是通过鉴定“质子耦合叶酸转运蛋白”（PCFT）来完成的。 PCFT的药理学作用通过对新一代抗叶酸的活性（Pemetrexed）的活性证明。它的生理作用是通过遗传性叶酸不良吸收（HFM）中PCFT基因的功能丧失突变证明的，这是一种常染色体隐性遗传疾病，其特征是肠道叶酸吸收和叶酸的转运和中枢神经系统中的严重缺陷。 PCFT似乎还为与酸化内体的叶状体提供了与叶酸受体介导的转运作用一致的出口途径。 The current proposal builds on published findings along with preliminary results that support the feasibility of the four specific aims: Specific Aim 1 is to characterize PCFT-mediated transport and its impact on the pharmacological activities of pemetrexed and methotrexate in vitro under conditions that simulate drug pharmacokinetics in clinical regimens and at pH's that approximate what is found in solid tumors.具体目的2是进一步评估PCFT在FR1和FR2介导的内吞作用中的作用，该吞噬作用着重于满足细胞叶酸生长需求和抗叶酸生长抑制的生理和药理分析。具体目的3是使用三种方法 - 定向诱变，抗叶酸选择性压力下的化学诱变以及取代的半胱氨酸可及性诱变的三种方法来表征PCFT的拓扑和结构功能。 PCFT残基的功能作用是通过模型人类细胞系中的转运动力学评估的，电压斜率Xenopus卵母细胞中的电流诱导和酸化。在PCFT的不断发展的结构模型的背景下考虑了结果，在该模型中，可以通过实验预测和评估底物易位途径以及潜在的结合和相互作用残基。具体目标4的重点是调节PCFT表达，包括用缺氧的上调机制，这是肿瘤微环境中的另一个元素。这些研究具有临床意义。从药理学的角度来看，Pemetrexed是一种重要的抗牙糖，最近批准用于治疗恶性黑色素瘤和非小细胞肺癌，并正在评估其他恶性肿瘤的功效。这些实验室研究的观察结果可能有助于增强该药物的临床实用性。从生理的角度来看，PCFT在叶子的肠道吸收和维持叶酸足够的情况下起着至关重要的作用。叶酸缺乏是结直肠癌的风险因素，而叶酸过量可能会增加已经形成的肿瘤。因此，阐明PCFT的生物学有助于理解人类叶酸稳态决定因素的因素，并对癌症和其他叶酸缺陷障碍的发病机理产生影响。公共卫生相关性：该提案的重点是最近在该实验室发现的质子耦合叶酸转运蛋白（PCFT），负责吸收饮食中叶酸酯在肠道和叶酸进入大脑的肠道和运动中。对PCFT的遗传损害导致遗传性叶酸缺乏疾病，遗传叶酸吸收不良。成人的叶酸缺乏与癌症风险增加有关。 PCFT还有助于将新的抗癌药物刺穿到癌细胞中。这项研究将扩大我们对PCFT功能的理解，这可能会导致通过Pemetrexed，新的癌症预防策略和预防叶酸缺陷状态改善癌症治疗。