Molecular Regulation of Folate and Antifolate Transport

描述（由申请人提供）：近年来，人们重新强调基于叶酸的癌症治疗，反映了肿瘤选择性膜转运的能力。本申请探索了人质子偶联叶酸转运蛋白（hPCFT）的治疗潜力。与人还原叶酸载体（hRFC）不同，hPCFT是广泛表达并具有中性pH最适的主要组织叶酸转运蛋白，其显示出较窄的组织分布并以酸性pH最适为特征。hPCFT在酸性pH下显示出大量的转运，表征了肿瘤微环境，在中性pH下显示出有限的转运。我们发现hPCFT在人肿瘤细胞系和原发性肿瘤中广泛表达。在人肿瘤细胞中，hPCFT转录物在蛋白质印迹和转运活性上改变hPCFT蛋白的水平。在人类肿瘤细胞中，hPCFT报告基因活性与hPCFT转录物的不同水平相关。我们启动了hPCFT结构-功能研究，并表征了跨膜结构域（TMD）2和3之间的机械和结构上重要的“折返环”结构域，并定位了TMD 2内的底物结合区。我们证明了hPCFT寡聚化在功能上的重要作用，并定位了一个重要的寡聚体界面TMD 6。在另一项研究中，我们鉴定了新的6-取代吡咯并[2，3-d]嘧啶噻吩甲酰抗叶酸剂，其选择性地通过hPCFT而不是hRFC转运，尽管叶酸受体的摄取也被保留。这些努力促进了一种新的范式，即基于主要叶酸转运蛋白PCFT和FR的肿瘤特异性表达和/或功能的肿瘤靶向治疗的合理发展。我们将继续对主要促进性叶酸转运蛋白进行全面研究，并对基于叶酸的癌症治疗和预防性叶酸补充癌症产生影响。我们的重点是优化我们的新型hPCFT选择性治疗肿瘤靶向，通过探索这种生理上重要的转运蛋白的生物学，从一个令人信服的理由，通过这种机制靶向实体瘤。在具体目标1中，我们将表征hPCFT靶向癌症治疗的分子和细胞决定因素，包括建立hRFC维斯hPCFT的功能化学计量、hPCFT和hRFC以及叶酸受体的伴随肿瘤表达的治疗影响以及人肿瘤细胞中hPCFT转录调控的机制。在目标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蛋白中功能重要的结构域和残基，以及hPCFT同源寡聚体的结构、功能和调控特征。我们的目标是利用hPCFT结构和功能的生物化学研究，以及分子同源性建模，来帮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疗效。我们提出的研究得到了我们初步和已发表结果以及关键试剂和细胞系可用性的有力支持，包括第一代特异性和有效的hPCFT选择性肿瘤靶向抗叶酸剂，这些抗叶酸剂不被hRFC转运。我们的研究是独特的，因为它们的新奇和可能性提供了关键的新见解，这些生理和非生理重要的维生素转运蛋白的机制和调节，可以转化为临床。

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.