Proton-coupled folate/antifolate transport

描述（由申请人提供）：本资助期内的研究重点是表征在人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”转运蛋白。这是通过鉴定“质子偶联叶酸转运蛋白”（PCFT）来完成的。PCFT的药理作用通过其对新一代抗叶酸剂培美曲塞活性的有益影响得到证实。其生理作用通过遗传性叶酸吸收不良（HFM）中PCFT基因的功能缺失突变来证明，HFM是一种常染色体隐性遗传疾病，其特征在于肠道叶酸吸收和叶酸转运到中枢神经系统中的严重缺陷。PCFT似乎还提供了叶酸从酸化内体的输出途径，这与叶酸受体介导的转运中的作用一致。当前提案建立在已发表的发现沿着初步结果的基础上，这些结果支持四个具体目标的可行性：具体目标1是在模拟临床方案中药物药代动力学的条件下，在接近实体瘤中发现的pH值下，表征PCFT介导的转运及其对培美曲塞和甲氨蝶呤体外药理学活性的影响。具体目标2是进一步评估PCFT在FR 1和FR 2介导的内吞作用中的作用，重点是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学分支。具体目标3是使用三种方法表征PCFT的拓扑结构和结构-功能-定点诱变、抗叶酸选择性压力下的化学诱变和取代的半胱氨酸可及性诱变。PCFT残基的功能作用进行了评估模型人类细胞系和电流的诱导和酸化电压钳爪蟾卵母细胞的运输动力学。结果被认为是一个不断发展的背景下PCFT的底物易位途径和潜在的结合和相互作用的残基可以预测和实验评估的结构模型。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。从药理学角度来看，培美曲塞是一种重要的抗叶酸剂，最近获批用于治疗恶性黑色素瘤和非小细胞肺癌，目前正在评估其在其他恶性肿瘤中的疗效。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。从生理学的角度来看，PCFT在叶酸的肠道吸收和维持叶酸充足方面起着关键作用。叶酸缺乏是结直肠癌的一个危险因素，叶酸过量可能会增加已经形成的肿瘤的进展。因此，阐明PCFT的生物学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症发病机制的影响。公共卫生相关性：该建议的重点是质子偶联叶酸转运蛋白（PCFT），最近在这个实验室发现，负责膳食叶酸在肠道中的吸收和叶酸进入大脑的运动。对PCFT的遗传损伤导致叶酸缺乏的遗传性疾病，遗传性叶酸吸收不良。成年人叶酸缺乏与癌症风险增加有关。PCFT还有助于一种新的抗癌药物培美曲塞渗透到癌细胞中。这项研究将拓宽我们对PCFT功能的理解，这可能会改善培美曲塞的癌症治疗，新的癌症预防策略，以及预防叶酸缺乏症。

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.