Proton-coupled folate/antifolate transport

描述（由申请人提供）：当前资助期的研究主要集中在人类实体肿瘤和正常细胞中广泛表达的新型低ph叶酸运输活性的表征。第二个目标是克隆“低ph值”转运蛋白。这是通过“质子偶联叶酸转运体”（PCFT）的鉴定完成的。PCFT对新一代抗叶酸药培美曲塞活性的有益影响证明了其药理作用。在遗传性叶酸吸收不良（HFM）中，PCFT基因的功能缺失突变证明了其生理作用，HFM是一种常染色体隐性遗传病，其特征是肠道叶酸吸收和叶酸转运到中枢神经系统的严重缺陷。PCFT似乎也提供了叶酸从酸化内体出口的途径，这与叶酸受体介导的运输的作用一致。目前的提案建立在已发表的研究结果以及支持四个具体目标可行性的初步结果的基础上：具体目标1是在模拟临床方案中的药物药代动力学和接近实体肿瘤的pH值的条件下，表征pcft介导的转运及其对培美曲塞和甲氨蝶呤的体外药理活性的影响。具体目标2是进一步评估PCFT在FR1和FR2介导的内吞作用中的作用，重点关注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学影响。具体目标3是使用三种方法来表征PCFT的拓扑结构和结构功能-位点定向诱变，抗叶酸选择压力下的化学诱变和替代半胱氨酸可及性诱变。PCFT残基的功能作用通过模型人类细胞系的转运动力学和电压箝位非洲爪蟾卵母细胞的电流诱导和酸化来评估。结果考虑了一个不断发展的PCFT结构模型，其中底物易位途径和潜在的结合和相互作用残基可以通过实验预测和评估。特异性Aim 4关注PCFT表达的调控，包括肿瘤微环境中的另一个因素缺氧上调PCFT表达的机制。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药物，最近被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药物的临床应用。从生理角度来看，PCFT在叶酸的肠道吸收和维持叶酸充足方面起着至关重要的作用。叶酸缺乏是结直肠癌的一个危险因素，叶酸过量可能会加速已经形成的肿瘤的进展。因此，阐明PCFT的生物学特性有助于理解人体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。

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.