This application builds upon this laboratory's discovery of the proton-coupled folate transporter (PCFT), a low-pH transporter with high affinity for the new-generation antifolate, pemetrexed, that uniquely sustains the activity of this agent when the major folate/antifolate transporter, the reduced folate carrier, is absent and tumor cells are highly resistance to methotrexate and other antifolates. Studies over the current funding period have begun to elucidate the structure-function of PCFT encompassing its topology, residues required for its function, and elements that discriminate among folates/antifolates. The studies proposed are focused on developing a comprehensive understanding of the structural basis for PCFT function combining laboratory and computational strategies.
The specific aims are: (i) To identify residues that define the aqueous translocation pathway, that define folate binding and its specificity, and that are determinants of proton binding and coupling. These studies utilize the substituted cysteine accessibility method. (ii) To characterize the interaction between PCFT transmembrane helices that are determinants of tertiary structure, and an extracellular gate, by chemical cross-linking of di-cysteine mutants, and to clarify the basis for the role the first intracellular loop plays in PCFT function. (iii) To determine the impact of structural alterations of PCFT on the antitumor activities of pemetrexed and other antifolates in vitro, particularly under acidic conditions. (iv) To utilize the experimental data obtained, in an iterative approach, to inform, refine and validate a homology model of PCFT and to undertake docking analyses in order to identify elements critical to the avidity and selectivity of the folate/antifolate binding pocket. These studies will also inform the development of novel antifolates designed for selectively delivery by PCFT within the acidic microenvironment of solid tumors. Mutant PCFTs developed in these studies are assessed for their stability, trafficking to the cell membrane, folate/antifolate substrate selectivity, and roles in proton binding and proton coupling by transfection into a unique Hela cell line that lacks endogenous folate transporters. This is complemented by electrophysiological measurements in Xenopus oocytes of current and acidification generated by the transport of the folate-proton-PCFT ternary complex, that also make possible analyses of transport properties of antifolates that are not radiolabled.
This research is focused on drugs that kill cancer cells by blocking their ability to utilize folic acid. The studies proposed are designed to better understan the mechanisms by which these drugs are transported into cancer cells within environment of the solid tumors in which they grow, and how this transport can be improved to enhance their effectiveness.
|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 1859:2193-2202|
|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|
|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|
|Visentin, Michele; Diop-Bove, Ndeye; Zhao, Rongbao et al. (2014) The intestinal absorption of folates. Annu Rev Physiol 76:251-74|
|Shin, Daniel Sanghoon; Zhao, Rongbao; Fiser, Andras et al. (2013) Role of the fourth transmembrane domain in proton-coupled folate transporter function as assessed by the substituted cysteine accessibility method. Am J Physiol Cell Physiol 304:C1159-67|
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