Proton-coupled folate/antifolate transport

描述(由申请人提供)：当前资助期的研究集中于一种新的低pH叶酸转运活性的特征，该活性在人类实体肿瘤和正常细胞中广泛表达。倒数第二个目标是克隆“低pH值”转运蛋白。这是通过鉴定“质子偶联叶酸转运蛋白”(PCFT)来实现的。PCFT对新一代抗叶酸培美曲塞活性的有益影响证明了其药理作用。遗传性叶酸吸收不良(HFM)是一种常染色体隐性遗传性疾病，其特征是肠道对叶酸的吸收和叶酸向中枢神经系统的运输严重缺陷。PCFT似乎还提供了一条从酸化的内体中输出叶酸的途径，这与叶酸受体介导的转运过程中的作用一致。目前的建议建立在已发表的研究结果和支持四个特定目标可行性的初步结果的基础上：特定目标1表征PCFT介导的转运及其对培美曲塞和甲氨蝶呤体外药理活性的影响，条件是模拟临床方案中的药物药代动力学，并在与实体瘤相似的pH条件下进行。具体目的2是进一步评估PCFT在FR1和FR2介导的内吞作用中所起的作用，重点是在满足细胞叶酸生长需求和抗叶酸生长抑制方面的生理和药理影响。具体目的3是用三种方法--定点突变、抗叶酸选择压力下的化学突变和取代半胱氨酸可及性突变来表征PCFT的拓扑结构和结构功能。PCFT残基的功能作用通过在模型人类细胞系中的转运动力学和在电压钳制的非洲爪哇卵母细胞中的电流诱导和酸化来评估。结果是在PCFT的演变结构模型的背景下考虑的，在该模型中，底物转位途径以及潜在的结合和相互作用残基可以通过实验进行预测和评估。具体目标4集中于PCFT表达的调节，包括缺氧上调的机制，缺氧是肿瘤微环境中的另一个因素。这些研究具有临床意义。从药理学的角度来看，培美曲塞是一种重要的抗叶酸药物，最近被批准用于治疗恶性黑色素瘤和非小细胞肺癌，目前正在评估其对其他恶性肿瘤的疗效。这些实验室研究的观察结果可能有助于提高该制剂的临床实用价值。从生理角度来看，PCFT在叶酸的肠道吸收和维持叶酸充足中起着关键作用。叶酸缺乏是结直肠癌的危险因素，而叶酸过量可能会加剧已经形成的肿瘤的进展。因此，阐明PCFT的生物学特性有助于了解人类叶酸稳态的决定因素，以及对癌症和其他叶酸缺乏性疾病发病机制的影响。与公众健康相关：这项建议的重点是质子偶联叶酸转运体(PCFT)，它是本实验室最近发现的，负责饮食中的叶酸在肠道中的吸收和叶酸进入大脑的运动。PCFT的遗传损伤会导致一种遗传性叶酸缺乏疾病，即遗传性叶酸吸收不良。成年人缺乏叶酸会增加患癌症的风险。PCFT还有助于一种新的抗癌药物培美曲塞渗透到癌细胞中。这项研究将扩大我们对PCFT功能的理解，这可能会导致培美曲塞改善癌症治疗，新的癌症预防策略，以及预防叶酸缺乏状态。

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.