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.
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.
|Zhao, Rongbao; Najmi, Mitra; Aluri, Srinivas et al. (2018) Concentrative Transport of Antifolates Mediated by the Proton-Coupled Folate Transporter (SLC46A1); Augmentation by a HEPES Buffer. Mol Pharmacol 93:208-215|
|Zhao, Rongbao; Najmi, Mitra; Aluri, Srinivas et al. (2017) Impact of posttranslational modifications of engineered cysteines on the substituted cysteine accessibility method: evidence for glutathionylation. Am J Physiol Cell Physiol 312:C517-C526|
|Zhao, Rongbao; Aluri, Srinivas; Goldman, I David (2017) The proton-coupled folate transporter (PCFT-SLC46A1) and the syndrome of systemic and cerebral folate deficiency of infancy: Hereditary folate malabsorption. Mol Aspects Med 53:57-72|
|Aluri, Srinivas; Zhao, Rongbao; Fiser, Andras et al. (2017) Residues in the eighth transmembrane domain of the proton-coupled folate transporter (SLC46A1) play an important role in defining the aqueous translocation pathway and in folate substrate binding. Biochim Biophys Acta Biomembr 1859:2193-2202|
|Najmi, Mitra; Zhao, Rongbao; Fiser, Andras et al. (2016) Role of the tryptophan residues in proton-coupled folate transporter (PCFT-SLC46A1) function. Am J Physiol Cell Physiol 311:C150-7|
|Zhao, Rongbao; Najmi, Mitra; Fiser, Andras et al. (2016) Identification of an Extracellular Gate for the Proton-coupled Folate Transporter (PCFT-SLC46A1) by Cysteine Cross-linking. J Biol Chem 291:8162-72|
|Visentin, Michele; Unal, Ersin Selcuk; Najmi, Mitra et al. (2015) Identification of Tyr residues that enhance folate substrate binding and constrain oscillation of the proton-coupled folate transporter (PCFT-SLC46A1). Am J Physiol Cell Physiol 308:C631-41|
|Zhao, Rongbao; Visentin, Michele; Goldman, I David (2015) Determinants of the activities of antifolates delivered into cells by folate-receptor-mediated endocytosis. Cancer Chemother Pharmacol 75:1163-73|
|Zhao, Rongbao; Diop-Bove, Ndeye; Goldman, I David (2014) Enhanced receptor-mediated endocytosis and cytotoxicity of a folic acid-desacetylvinblastine monohydrazide conjugate in a pemetrexed-resistant cell line lacking folate-specific facilitative carriers but with increased folate receptor expression. Mol Pharmacol 85:310-21|
|Visentin, Michele; Unal, Ersin Selcuk; Goldman, I David (2014) The impact of 5-formyltetrahydrofolate on the anti-tumor activity of pralatrexate, as compared to methotrexate, in HeLa cells in vitro. Cancer Chemother Pharmacol 73:1055-62|
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