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Larry H. Matherly - One of the best experts on this subject based on the ideXlab platform.
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Regulation of differential Proton-Coupled Folate Transporter gene expression in human tumors: transactivation by KLF15 with NRF-1 and the role of Sp1
Biochemical Journal, 2019Co-Authors: Carrie O'connor, Josephine Frühauf, Aleem Gangjee, Larry H. MatherlyAbstract:: Tumors can be therapeutically targeted with novel antiFolates (e.g. AGF94) that are selectively transported by the human Proton-Coupled Folate Transporter (hPCFT). Studies were performed to determine the transcription regulation of hPCFT in tumors and identify possible mechanisms that contribute to the highly disparate levels of hPCFT in HepG2 versus HT1080 tumor cells. Transfection of hPCFT-null HT1080 cells with hPCFT restored transport and sensitivity to AGF94 Progressive deletions of the hPCFT promoter construct (-2005 to +96) and reporter gene assays in HepG2 and HT1080 cells confirmed differences in hPCFT transactivation and localized a minimal promoter to between positions -50 and +96. The minimal promoter included KLF15, GC-Box and NRF-1 cis-binding elements whose functional importance was confirmed by promoter deletions and mutations of core consensus sequences and reporter gene assays. In HepG2 cells, NRF-1, KLF15 and Sp1 transcripts were increased over HT1080 cells by ∼5.1-, ∼44-, and ∼2.4-fold, respectively. In Drosophila SL2 cells, transfection with KLF15 and NRF-1 synergistically activated the hPCFT promoter; Sp1 was modestly activating or inhibitory. Chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA) and supershifts confirmed differential binding of KLF15, Sp1, and NRF-1 to the hPCFT promoter in HepG2 and HT1080 cells that paralleled hPCFT levels. Treatment of HT1080 nuclear extracts (NE) with protein kinase A increased Sp1 binding to its consensus sequence by EMSA, suggesting a role for Sp1 phosphorylation in regulating hPCFT transcription. A better understanding of determinants of hPCFT transcriptional control may identify new therapeutic strategies for cancer by modulating hPCFT levels in combination with hPCFT-targeted antiFolates.
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Development and validation of chemical features-based Proton-Coupled Folate Transporter/activity and reduced Folate carrier/activity models (pharmacophores).
Journal of Molecular Graphics & Modelling, 2018Co-Authors: Khushbu Shah, Larry H. Matherly, Sudhir Raghavan, Aleem GangjeeAbstract:Abstract All clinically used antiFolates lack transport selectivity for tumors over normal cells resulting in dose-limiting toxicities. There is growing interest in developing novel tumor-targeted cytotoxic antiFolates with selective transport into tumors over normal cells via the Proton-Coupled Folate Transporter (PCFT) over the ubiquitously expressed reduced Folate carrier (RFC). A lack of X-ray crystal structures or predictive models for PCFT or RFC has hindered structure-aided drug design for PCFT-selective therapeutics. Four-point validated models (pharmacophores) were generated for PCFT/Activity (HBA, NI, RA, RA) and RFC/Activity (HBD, NI, HBA, HBA) based on inhibition (IC50) of proliferation of isogenic Chinese hamster ovary (CHO) cells engineered to express only human PCFT or only RFC. Our results revealed substantial differences in structural features required for transport of novel molecules by these Transporters which can be utilized for developing Transporter-selective antiFolates.
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The promise and challenges of exploiting the Proton-Coupled Folate Transporter for selective therapeutic targeting of cancer
Cancer Chemotherapy and Pharmacology, 2018Co-Authors: Larry H. Matherly, Aleem GangjeeAbstract:This review considers the “promise” of exploiting the Proton-Coupled Folate Transporter (PCFT) for selective therapeutic targeting of cancer. PCFT was discovered in 2006 and was identified as the principal Folate Transporter involved in the intestinal absorption of dietary Folates. The recognition that PCFT was highly expressed in many tumors stimulated substantial interest in using PCFT for cytotoxic drug targeting, taking advantage of its high level transport activity under the acidic pH conditions that characterize many tumors. For pemetrexed, among the best PCFT substrates, transport by PCFT establishes its importance as a clinically important Transporter in malignant pleural mesothelioma and non-small cell lung cancer. In recent years, the notion of PCFT-targeting has been extended to a new generation of tumor-targeted 6-substituted pyrrolo[2,3- d ]pyrimidine compounds that are structurally and functionally distinct from pemetrexed, and that exhibit near exclusive transport by PCFT and potent inhibition of de novo purine nucleotide biosynthesis. Based on compelling preclinical evidence in a wide range of human tumor models, it is now time to advance the most optimized PCFT-targeted agents with the best balance of PCFT transport specificity and potent antitumor efficacy to the clinic to validate this novel paradigm of highly selective tumor targeting.
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Functional and mechanistic roles of the human Proton-Coupled Folate Transporter transmembrane domain 6-7 linker
Biochemical Journal, 2016Co-Authors: M. Roy Wilson, Lucas J. Wilson, Jun Ye, Larry H. MatherlyAbstract:The Proton-Coupled Folate Transporter (PCFT; SLC46A1) is a Folate-proton symporter expressed in solid tumors and is used for tumor-targeted delivery of cytotoxic antiFolates. Topology modeling suggests that PCFT secondary structure includes twelve transmembrane domains (TMDs) with TMDs 6 and 7 linked by an intracellular loop (positions 236 to 265) including His247, implicated as functionally important. Single cysteine mutants were inserted from positions 241 to 251 in cysteine-less PCFT and mutant proteins were expressed in PCFT-null (R1-11) HeLa cells; none were reactive with 2-aminoethyl methanethiosulfonate biotin, suggesting that the TMD6-7 loop is intracellular. Twenty-nine single alanine mutants spanning the entire TMD6-7 loop were expressed in R1-11 cells; activity was generally preserved, with the exception of the 247, 250 and 251 mutants, partly due to decreased surface expression. Co-expression of PCFT TMD1-6 and TMD7-12 half-molecules in R1-11 cells partially restored transport activity, although removal of residues 252-265 from TMD7-12 abolished transport. Chimeric proteins, including non-homologous sequence from a thiamine Transporter (ThTr1) inserted into the PCFT TMD6-7 loop (positions 236 to 250, or 251 to 265), were active, although replacement of the entire loop with ThTr1 sequence resulted in substantial loss of activity. Amino acid replacements (Ala, Arg, His, Gln, Glu) or deletions at position 247 in wild-type and PCFT-ThTr1 chimeras resulted in differential effects on transport. Collectively, our findings suggest that the PCFT TMD6-7 connecting loop confers protein stability and may serve a unique functional role that depends on secondary structure rather than particular sequence elements.
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Abstract 1362: Novel proton coupled Folate Transporter (PCFT) and reduced Folate carrier (RFC) pharmacophore models for development of Transporter-selective antiFolates
Cancer Research, 2016Co-Authors: Sudhir Raghavan, Aleem Gangjee, Larry H. MatherlyAbstract:RFC is a ubiquitously expressed Folate Transporter that is present in normal tissues and tumors. RFC is utilized by reduced Folates and by all the clinically used classical antiFolates. In contrast, PCFT shows more limited expression in normal tissues and is highly effective in Folate transport under low pH conditions associated with hypoxic solid tumors. AntiFolates that are selectively transported by PCFT over RFC offer significant promise for development of targeted therapies of tumors (over)expressing PCFT. We previously reported 6-substituted pyrrolo[2,3-d]pyrimidines and thieno[2,3-d]pyrimidines as part of a continued effort to elucidate the SAR for substrate binding and cellular uptake for RFC, PCFT as well as Folate receptors (FRs). While the X-ray crystal structure of FRs bound to antiFolates has been resolved, the absence of X-ray crystal structures of PCFT and RFC has hindered structure-based medicinal chemistry efforts for development of antiFolates selectively transported by PCFT. To address this, pharmacophore models were developed for the first time using Discovery Studio for PCFT and RFC using a series of thirty six 6-substituted bicyclic pyrrolo[2,3-d]pyrimidines and/or thieno[2,3-d]pyrimidines that included highly potent and selective compounds such as AGF94 (PCFT = 0.3 nM; RFC = 101 nM) previously reported by us. Compounds used for model development displayed a 3-log range inhibition of cancer cells overexpressing PCFT or RFC. The best models display a 4-point pharmacophore with excellent regression values (r2 PCFT = 0.96; RFC = 0.92). The models were internally validated by leave-one-out analysis (q2 PCFT = 0.752; RFC = 0.59) and externally validated using a test set of 6-substituted bicyclic analogs not used in model development. Comparison of the pharmacophore models indicates different conformational and structural requirements for compounds for binding to PCFT or RFC. We have used these models to explain the potent PCFT inhibitory activity and selectivity of compounds such as AGF94. These models are being used in the development of selectively transported targeted antiFolates that would result in decreased toxicity compared to standard antiFolates such as methotrexate (MTX) or pemetrexed (PMX) which utilize all three Folate transport systems. Citation Format: Sudhir Raghavan, Aleem Gangjee, Larry H. Matherly. Novel proton coupled Folate Transporter (PCFT) and reduced Folate carrier (RFC) pharmacophore models for development of Transporter-selective antiFolates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1362.
I. David Goldman - One of the best experts on this subject based on the ideXlab platform.
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Substitutions that lock and unlock the Proton-Coupled Folate Transporter (PCFT-SLC46A1) in an inward-open conformation.
Journal of Biological Chemistry, 2019Co-Authors: Srinivas Aluri, Rongbao Zhao, Daniel Sanghoon Shin, Andras Fiser, I. David GoldmanAbstract:: The Proton-Coupled Folate Transporter (PCFT) mediates intestinal absorption of Folates and their transport from blood to cerebrospinal fluid across the choroid plexus. Substitutions at Asp-109 in the first intracellular loop between the first and second transmembrane domains (TMDs) abolish PCFT function, but protein expression and trafficking to the cell membrane are retained. Here, we used site-directed mutagenesis, the substituted-cysteine accessibility method, functional analyses, and homology modeling to determine whether the D109A substitution locks PCFT in one of its conformational states. Cys-substituted residues lining the PCFT aqueous translocation pathway and accessible in WT PCFT to the membrane-impermeable cysteine-biotinylation reagent, MTSEA-biotin, lost accessibility when introduced into the D109A scaffold. Substitutions at Gly-305 located exofacially within the eighth TMD, particularly with bulky residues, when introduced into the D109A scaffold largely restored function and MTSEA-biotin accessibility to Cys-substituted residues within the pathway. Likewise, Ser-196 substitution in the fifth TMD, predicted by homology modeling to be in proximity to Gly-305, also partially restored function found in solute Transporters, is critical to oscillation of the carrier among its conformational states. Substitutions at Asp-109 and Gly-112 lock PCFT in an inward-open conformation, resulting in the loss of function. However, the integrity of the locked protein is preserved, indicated by the restoration of function after insertion of a second "unlocking" mutation. and accessibility. Similarly, the inactivating G112K substitution within the first intracellular loop was partially reactivated by introducing the G305L substitution. These data indicate that the first intracellular loop, with a sequence identical to "motif A" (GXXXDXXGR(R/K)).
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Concentrative Transport of AntiFolates Mediated by the Proton-Coupled Folate Transporter (SLC46A1); Augmentation by a HEPES Buffer.
Molecular Pharmacology, 2018Co-Authors: Rongbao Zhao, Srinivas Aluri, Mitra Najmi, David C. Spray, I. David GoldmanAbstract:The Proton-Coupled Folate Transporter (PCFT) is ubiquitously expressed in solid tumors to which it delivers antiFolates, particularly pemetrexed, into cancer cells. Studies of PCFT-mediated transport, to date, have focused exclusively on the influx of Folates and antiFolates. This article addresses the impact of PCFT on concentrative transport, critical to the formation of the active polyglutamate congeners, and at pH levels relevant to the tumor microenvironment. An HeLa-derived cell line was employed, in which Folate-specific transport was mediated exclusively by PCFT. At pH 7.0, there was a substantial chemical gradient for methotrexate that decreased as the extracellular pH was increased. A chemical gradient was still detected at pH 7.4 in the usual HEPES-based transport buffer in contrast to what was observed in a bicarbonate/CO2-buffered medium. This antiFolate gradient correlated with an alkaline intracellular pH in the former (pH 7.85), but not the latter (pH 7.39), buffer and was abolished by the protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. The gradient in HEPES buffer at pH 7.4 was the result of the activity of Na+/H+ exchanger(s); it was eliminated by inhibitors of Na+/H+ exchanger (s) or Na+/K+ ATPase. An antiFolate chemical gradient was also detected in bicarbonate buffer at pH 6.9 versus 7.4, also suppressed by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. When the membrane potential is considered, PCFT generates substantial transmembrane electrochemical-potential gradients at extracellular pH levels relevant to the tumor microenvironment. The augmentation of intracellular pH, when cells are in a HEPES buffer, should be taken into consideration in studies that encompass all Proton-Coupled Transporter families.
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Hereditary Folate malabsorption due to a mutation in the external gate of the Proton-Coupled Folate Transporter SLC46A1.
Blood Advances, 2018Co-Authors: Srinivas Aluri, Rongbao Zhao, Andras Fiser, Charlotte M.a. Lubout, Susanna M. I. Goorden, I. David GoldmanAbstract:Hereditary Folate malabsorption (HFM) is an autosomal recessive disorder characterized by impaired intestinal Folate absorption and impaired Folate transport across the choroid plexus due to loss of function of the Proton-Coupled Folate Transporter (PCFT-SLC46A1). We report a novel mutation, causing HFM, affecting a residue located in the 11th transmembrane helix within the external gate. The mutant N411K-PCFT was stable, trafficked to the cell membrane, and had sufficient residual activity to characterize the transport defect and the structural requirements at this site for gate function. The influx V max of the N411K mutant was markedly decreased, as was the affinity for most, but not all, Folate/antiFolate substrates. The greatest loss of activity was for 5-methyltetrahydroFolate. Substitutions with positive charged residues resulted in a loss of activity (arginine > lysine > histidine). Function was retained for the negative charged aspartate, but not the larger glutamate substitutions, whereas the bulky hydrophobic (leucine), or polar (glutamine) substitutions, were tolerated. Homology models of PCFT, in the inward and outward open conformations, based upon the mammalian Glut5 fructose Transporter structures, localize Asn411 protruding into the aqueous pathway. This is most prominent when the carrier is in the inward open conformation when the external gate is closed. Mutations at this site likely result in highly specific steric and electrostatic interactions between the Asn411-substituted, and other, residues in the gate region that impede carrier function. The substrate specificity of the N411K mutant may be due to alterations of substrate flows through the external gate, downstream allosteric alterations in the Folate-binding pocket, or both.
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Substituted-cysteine accessibility and cross-linking identify an exofacial cleft in the 7th and 8th helices of the Proton-Coupled Folate Transporter (SLC46A1)
American Journal of Physiology-cell Physiology, 2017Co-Authors: Srinivas Aluri, Rongbao Zhao, Andras Fiser, I. David GoldmanAbstract:The Proton-Coupled Folate Transporter (PCFT-SLC46A1) is required for Folate transport across the apical membrane of the small intestine and across the choroid plexus. This study focuses on the stru...
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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.
Biochimica et Biophysica Acta, 2017Co-Authors: Srinivas Aluri, Rongbao Zhao, Andras Fiser, I. David GoldmanAbstract:Abstract The Proton-Coupled Folate Transporter (PCFT-SLC46A1) is required for intestinal Folate absorption and Folate transport across the choroid plexus. This report addresses the structure/function of the 8th transmembrane helix. Based upon biotinylation of cysteine-substituted residues by MTSEA-biotin, 14 contiguous exofacial residues to Leu316 were accessible to the extracellular compartment of the 23 residues in this helix (Leu303-Leu325). Pemetrexed blocked biotinylation of six Cys-substituted residues deep within the helix implicating an important role for this region in Folate binding. Accessibility decreased at 4 °C vs RT. The influx K t , K i and V max were markedly increased for the P314C mutant, similar to what was observed for Y315A and Y315P mutants. However, the K t , alone, was increased for the P314Y mutant. To correlate these observations with PCFT structural changes during the transport cycle, homology models were built for PCFT based upon the recently reported structures of bovine and rodent GLUT5 fructose Transporters in the inward-open and outward- open conformations, respectively. The models predict substantial structural alterations in the exofacial region of the eighth transmembrane helix as it cycles between its conformational states that can account for the extended and contiguous aqueous accessibility of this region of the helix. Further, a helix break in one of the two conformations can account for the critical roles Pro314 and Tyr315, located in this region, play in PCFT function. The data indicates that the 8th transmembrane helix of PCFT plays an important role in defining the aqueous channel and the Folate binding pocket.
Aleem Gangjee - One of the best experts on this subject based on the ideXlab platform.
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Regulation of differential Proton-Coupled Folate Transporter gene expression in human tumors: transactivation by KLF15 with NRF-1 and the role of Sp1
Biochemical Journal, 2019Co-Authors: Carrie O'connor, Josephine Frühauf, Aleem Gangjee, Larry H. MatherlyAbstract:: Tumors can be therapeutically targeted with novel antiFolates (e.g. AGF94) that are selectively transported by the human Proton-Coupled Folate Transporter (hPCFT). Studies were performed to determine the transcription regulation of hPCFT in tumors and identify possible mechanisms that contribute to the highly disparate levels of hPCFT in HepG2 versus HT1080 tumor cells. Transfection of hPCFT-null HT1080 cells with hPCFT restored transport and sensitivity to AGF94 Progressive deletions of the hPCFT promoter construct (-2005 to +96) and reporter gene assays in HepG2 and HT1080 cells confirmed differences in hPCFT transactivation and localized a minimal promoter to between positions -50 and +96. The minimal promoter included KLF15, GC-Box and NRF-1 cis-binding elements whose functional importance was confirmed by promoter deletions and mutations of core consensus sequences and reporter gene assays. In HepG2 cells, NRF-1, KLF15 and Sp1 transcripts were increased over HT1080 cells by ∼5.1-, ∼44-, and ∼2.4-fold, respectively. In Drosophila SL2 cells, transfection with KLF15 and NRF-1 synergistically activated the hPCFT promoter; Sp1 was modestly activating or inhibitory. Chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA) and supershifts confirmed differential binding of KLF15, Sp1, and NRF-1 to the hPCFT promoter in HepG2 and HT1080 cells that paralleled hPCFT levels. Treatment of HT1080 nuclear extracts (NE) with protein kinase A increased Sp1 binding to its consensus sequence by EMSA, suggesting a role for Sp1 phosphorylation in regulating hPCFT transcription. A better understanding of determinants of hPCFT transcriptional control may identify new therapeutic strategies for cancer by modulating hPCFT levels in combination with hPCFT-targeted antiFolates.
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Abstract 3980: Dual targeting mitochondrial and cytosolic one-carbon metabolism via the Proton-Coupled Folate Transporter with novel 5-substituted pyrrolo[3,2-d]pyrimidine antiFolates
Cancer Research, 2018Co-Authors: Aamod Dekhne, Khushbu Shah, Lisa Polin, Gregory S. Ducker, Josephine Frühauf, Junayed Nayeen, Adrianne Wallace-povirk, Carrie O'connor, Aleem GangjeeAbstract:Cellular one-carbon (1C) metabolism generates a host of metabolites especially critical to cancer cells. Classical 1C inhibitors often primarily target a single enzyme, commonly leading to drug resistance and necessitating the development of novel compounds targeting multiple enzymes. We synthesized several novel 5-substituted pyrrolo[3,2-d]pyrimidine compounds (AGF291, AGF320, and AGF347) with transport by the Proton-Coupled Folate Transporter (PCFT), a Folate Transporter with a narrow physiological niche, but commonly expressed in many solid tumors. These analogs showed substantial inhibition in proliferation assays with Chinese hamster ovary sublines engineered to individually express PCFT or the ubiquitously-expressed reduced Folate carrier (the major tissue Folate Transporter), as well as in the PCFT-expressing human cancer cell lines H460 (large cell lung carcinoma), HCT-116 (colorectal carcinoma), and MIA PaCa-2 (pancreatic ductal adenocarcinoma). Full abrogation of inhibitory effects for all compounds required co-treatment with both adenosine and glycine, suggesting dual-targeting of cytosolic de novo purine biosynthesis and mitochondrial 1C/glycine metabolism. For cytosolic de novo purine biosynthesis, lack of protection by 5-aminoimidazole-4-carboxamide (AICA) suggested AICA ribonucleotide formyltransferase (AICARFTase), the second Folate-dependent enzyme, as the likely enzyme target. AICARFTase inhibition was confirmed in intact H460 and HCT-116 cells by measuring accumulation of AICAR by targeted metabolomics. Targeting of mitochondrial enzymes in H460 and HCT-116 cells was suggested by selective inhibition of incorporation of [3-14C]serine over [14C]formate into the purine intermediate [14C]formyl glycinamide ribonucleotide. [2,3,3-2H]Serine isotope label scrambling analysis confirmed the mitochondrial target to be serine hydroxymethyltransferase2 (SHMT2). SHMT2 is a potentially promising anticancer drug target as its expression is highly correlated with the malignant phenotype across a broad spectrum of cancers beyond lung and colon, including breast, glioma, and liver. Indeed, our initial in-vivo studies with AGF291 suggest potent efficacy toward both MIA PaCa-2 and H460 xenograft mouse models. Our studies demonstrate the potential for tumor-selective dual targeting of both mitochondrial and cytosolic 1C metabolism to overcome resistance to classical 1C inhibitors. Citation Format: Aamod S. Dekhne, Gregory S. Ducker, Josephine Fruhauf, Khushbu Shah, Md. Junayed Nayeen, Adrianne Wallace-Povirk, Carrie O9Connor, Zhanjun Hou, Lisa Polin, Aleem Gangjee, Joshua D. Rabinowitz, Larry H. Matherly. Dual targeting mitochondrial and cytosolic one-carbon metabolism via the Proton-Coupled Folate Transporter with novel 5-substituted pyrrolo[3,2-d]pyrimidine antiFolates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3980.
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Development and validation of chemical features-based Proton-Coupled Folate Transporter/activity and reduced Folate carrier/activity models (pharmacophores).
Journal of Molecular Graphics & Modelling, 2018Co-Authors: Khushbu Shah, Larry H. Matherly, Sudhir Raghavan, Aleem GangjeeAbstract:Abstract All clinically used antiFolates lack transport selectivity for tumors over normal cells resulting in dose-limiting toxicities. There is growing interest in developing novel tumor-targeted cytotoxic antiFolates with selective transport into tumors over normal cells via the Proton-Coupled Folate Transporter (PCFT) over the ubiquitously expressed reduced Folate carrier (RFC). A lack of X-ray crystal structures or predictive models for PCFT or RFC has hindered structure-aided drug design for PCFT-selective therapeutics. Four-point validated models (pharmacophores) were generated for PCFT/Activity (HBA, NI, RA, RA) and RFC/Activity (HBD, NI, HBA, HBA) based on inhibition (IC50) of proliferation of isogenic Chinese hamster ovary (CHO) cells engineered to express only human PCFT or only RFC. Our results revealed substantial differences in structural features required for transport of novel molecules by these Transporters which can be utilized for developing Transporter-selective antiFolates.
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The promise and challenges of exploiting the Proton-Coupled Folate Transporter for selective therapeutic targeting of cancer
Cancer Chemotherapy and Pharmacology, 2018Co-Authors: Larry H. Matherly, Aleem GangjeeAbstract:This review considers the “promise” of exploiting the Proton-Coupled Folate Transporter (PCFT) for selective therapeutic targeting of cancer. PCFT was discovered in 2006 and was identified as the principal Folate Transporter involved in the intestinal absorption of dietary Folates. The recognition that PCFT was highly expressed in many tumors stimulated substantial interest in using PCFT for cytotoxic drug targeting, taking advantage of its high level transport activity under the acidic pH conditions that characterize many tumors. For pemetrexed, among the best PCFT substrates, transport by PCFT establishes its importance as a clinically important Transporter in malignant pleural mesothelioma and non-small cell lung cancer. In recent years, the notion of PCFT-targeting has been extended to a new generation of tumor-targeted 6-substituted pyrrolo[2,3- d ]pyrimidine compounds that are structurally and functionally distinct from pemetrexed, and that exhibit near exclusive transport by PCFT and potent inhibition of de novo purine nucleotide biosynthesis. Based on compelling preclinical evidence in a wide range of human tumor models, it is now time to advance the most optimized PCFT-targeted agents with the best balance of PCFT transport specificity and potent antitumor efficacy to the clinic to validate this novel paradigm of highly selective tumor targeting.
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Abstract 1362: Novel proton coupled Folate Transporter (PCFT) and reduced Folate carrier (RFC) pharmacophore models for development of Transporter-selective antiFolates
Cancer Research, 2016Co-Authors: Sudhir Raghavan, Aleem Gangjee, Larry H. MatherlyAbstract:RFC is a ubiquitously expressed Folate Transporter that is present in normal tissues and tumors. RFC is utilized by reduced Folates and by all the clinically used classical antiFolates. In contrast, PCFT shows more limited expression in normal tissues and is highly effective in Folate transport under low pH conditions associated with hypoxic solid tumors. AntiFolates that are selectively transported by PCFT over RFC offer significant promise for development of targeted therapies of tumors (over)expressing PCFT. We previously reported 6-substituted pyrrolo[2,3-d]pyrimidines and thieno[2,3-d]pyrimidines as part of a continued effort to elucidate the SAR for substrate binding and cellular uptake for RFC, PCFT as well as Folate receptors (FRs). While the X-ray crystal structure of FRs bound to antiFolates has been resolved, the absence of X-ray crystal structures of PCFT and RFC has hindered structure-based medicinal chemistry efforts for development of antiFolates selectively transported by PCFT. To address this, pharmacophore models were developed for the first time using Discovery Studio for PCFT and RFC using a series of thirty six 6-substituted bicyclic pyrrolo[2,3-d]pyrimidines and/or thieno[2,3-d]pyrimidines that included highly potent and selective compounds such as AGF94 (PCFT = 0.3 nM; RFC = 101 nM) previously reported by us. Compounds used for model development displayed a 3-log range inhibition of cancer cells overexpressing PCFT or RFC. The best models display a 4-point pharmacophore with excellent regression values (r2 PCFT = 0.96; RFC = 0.92). The models were internally validated by leave-one-out analysis (q2 PCFT = 0.752; RFC = 0.59) and externally validated using a test set of 6-substituted bicyclic analogs not used in model development. Comparison of the pharmacophore models indicates different conformational and structural requirements for compounds for binding to PCFT or RFC. We have used these models to explain the potent PCFT inhibitory activity and selectivity of compounds such as AGF94. These models are being used in the development of selectively transported targeted antiFolates that would result in decreased toxicity compared to standard antiFolates such as methotrexate (MTX) or pemetrexed (PMX) which utilize all three Folate transport systems. Citation Format: Sudhir Raghavan, Aleem Gangjee, Larry H. Matherly. Novel proton coupled Folate Transporter (PCFT) and reduced Folate carrier (RFC) pharmacophore models for development of Transporter-selective antiFolates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1362.
Rongbao Zhao - One of the best experts on this subject based on the ideXlab platform.
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Substitutions that lock and unlock the Proton-Coupled Folate Transporter (PCFT-SLC46A1) in an inward-open conformation.
Journal of Biological Chemistry, 2019Co-Authors: Srinivas Aluri, Rongbao Zhao, Daniel Sanghoon Shin, Andras Fiser, I. David GoldmanAbstract:: The Proton-Coupled Folate Transporter (PCFT) mediates intestinal absorption of Folates and their transport from blood to cerebrospinal fluid across the choroid plexus. Substitutions at Asp-109 in the first intracellular loop between the first and second transmembrane domains (TMDs) abolish PCFT function, but protein expression and trafficking to the cell membrane are retained. Here, we used site-directed mutagenesis, the substituted-cysteine accessibility method, functional analyses, and homology modeling to determine whether the D109A substitution locks PCFT in one of its conformational states. Cys-substituted residues lining the PCFT aqueous translocation pathway and accessible in WT PCFT to the membrane-impermeable cysteine-biotinylation reagent, MTSEA-biotin, lost accessibility when introduced into the D109A scaffold. Substitutions at Gly-305 located exofacially within the eighth TMD, particularly with bulky residues, when introduced into the D109A scaffold largely restored function and MTSEA-biotin accessibility to Cys-substituted residues within the pathway. Likewise, Ser-196 substitution in the fifth TMD, predicted by homology modeling to be in proximity to Gly-305, also partially restored function found in solute Transporters, is critical to oscillation of the carrier among its conformational states. Substitutions at Asp-109 and Gly-112 lock PCFT in an inward-open conformation, resulting in the loss of function. However, the integrity of the locked protein is preserved, indicated by the restoration of function after insertion of a second "unlocking" mutation. and accessibility. Similarly, the inactivating G112K substitution within the first intracellular loop was partially reactivated by introducing the G305L substitution. These data indicate that the first intracellular loop, with a sequence identical to "motif A" (GXXXDXXGR(R/K)).
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Concentrative Transport of AntiFolates Mediated by the Proton-Coupled Folate Transporter (SLC46A1); Augmentation by a HEPES Buffer.
Molecular Pharmacology, 2018Co-Authors: Rongbao Zhao, Srinivas Aluri, Mitra Najmi, David C. Spray, I. David GoldmanAbstract:The Proton-Coupled Folate Transporter (PCFT) is ubiquitously expressed in solid tumors to which it delivers antiFolates, particularly pemetrexed, into cancer cells. Studies of PCFT-mediated transport, to date, have focused exclusively on the influx of Folates and antiFolates. This article addresses the impact of PCFT on concentrative transport, critical to the formation of the active polyglutamate congeners, and at pH levels relevant to the tumor microenvironment. An HeLa-derived cell line was employed, in which Folate-specific transport was mediated exclusively by PCFT. At pH 7.0, there was a substantial chemical gradient for methotrexate that decreased as the extracellular pH was increased. A chemical gradient was still detected at pH 7.4 in the usual HEPES-based transport buffer in contrast to what was observed in a bicarbonate/CO2-buffered medium. This antiFolate gradient correlated with an alkaline intracellular pH in the former (pH 7.85), but not the latter (pH 7.39), buffer and was abolished by the protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. The gradient in HEPES buffer at pH 7.4 was the result of the activity of Na+/H+ exchanger(s); it was eliminated by inhibitors of Na+/H+ exchanger (s) or Na+/K+ ATPase. An antiFolate chemical gradient was also detected in bicarbonate buffer at pH 6.9 versus 7.4, also suppressed by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. When the membrane potential is considered, PCFT generates substantial transmembrane electrochemical-potential gradients at extracellular pH levels relevant to the tumor microenvironment. The augmentation of intracellular pH, when cells are in a HEPES buffer, should be taken into consideration in studies that encompass all Proton-Coupled Transporter families.
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Hereditary Folate malabsorption due to a mutation in the external gate of the Proton-Coupled Folate Transporter SLC46A1.
Blood Advances, 2018Co-Authors: Srinivas Aluri, Rongbao Zhao, Andras Fiser, Charlotte M.a. Lubout, Susanna M. I. Goorden, I. David GoldmanAbstract:Hereditary Folate malabsorption (HFM) is an autosomal recessive disorder characterized by impaired intestinal Folate absorption and impaired Folate transport across the choroid plexus due to loss of function of the Proton-Coupled Folate Transporter (PCFT-SLC46A1). We report a novel mutation, causing HFM, affecting a residue located in the 11th transmembrane helix within the external gate. The mutant N411K-PCFT was stable, trafficked to the cell membrane, and had sufficient residual activity to characterize the transport defect and the structural requirements at this site for gate function. The influx V max of the N411K mutant was markedly decreased, as was the affinity for most, but not all, Folate/antiFolate substrates. The greatest loss of activity was for 5-methyltetrahydroFolate. Substitutions with positive charged residues resulted in a loss of activity (arginine > lysine > histidine). Function was retained for the negative charged aspartate, but not the larger glutamate substitutions, whereas the bulky hydrophobic (leucine), or polar (glutamine) substitutions, were tolerated. Homology models of PCFT, in the inward and outward open conformations, based upon the mammalian Glut5 fructose Transporter structures, localize Asn411 protruding into the aqueous pathway. This is most prominent when the carrier is in the inward open conformation when the external gate is closed. Mutations at this site likely result in highly specific steric and electrostatic interactions between the Asn411-substituted, and other, residues in the gate region that impede carrier function. The substrate specificity of the N411K mutant may be due to alterations of substrate flows through the external gate, downstream allosteric alterations in the Folate-binding pocket, or both.
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Substituted-cysteine accessibility and cross-linking identify an exofacial cleft in the 7th and 8th helices of the Proton-Coupled Folate Transporter (SLC46A1)
American Journal of Physiology-cell Physiology, 2017Co-Authors: Srinivas Aluri, Rongbao Zhao, Andras Fiser, I. David GoldmanAbstract:The Proton-Coupled Folate Transporter (PCFT-SLC46A1) is required for Folate transport across the apical membrane of the small intestine and across the choroid plexus. This study focuses on the stru...
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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.
Biochimica et Biophysica Acta, 2017Co-Authors: Srinivas Aluri, Rongbao Zhao, Andras Fiser, I. David GoldmanAbstract:Abstract The Proton-Coupled Folate Transporter (PCFT-SLC46A1) is required for intestinal Folate absorption and Folate transport across the choroid plexus. This report addresses the structure/function of the 8th transmembrane helix. Based upon biotinylation of cysteine-substituted residues by MTSEA-biotin, 14 contiguous exofacial residues to Leu316 were accessible to the extracellular compartment of the 23 residues in this helix (Leu303-Leu325). Pemetrexed blocked biotinylation of six Cys-substituted residues deep within the helix implicating an important role for this region in Folate binding. Accessibility decreased at 4 °C vs RT. The influx K t , K i and V max were markedly increased for the P314C mutant, similar to what was observed for Y315A and Y315P mutants. However, the K t , alone, was increased for the P314Y mutant. To correlate these observations with PCFT structural changes during the transport cycle, homology models were built for PCFT based upon the recently reported structures of bovine and rodent GLUT5 fructose Transporters in the inward-open and outward- open conformations, respectively. The models predict substantial structural alterations in the exofacial region of the eighth transmembrane helix as it cycles between its conformational states that can account for the extended and contiguous aqueous accessibility of this region of the helix. Further, a helix break in one of the two conformations can account for the critical roles Pro314 and Tyr315, located in this region, play in PCFT function. The data indicates that the 8th transmembrane helix of PCFT plays an important role in defining the aqueous channel and the Folate binding pocket.
Sita Kugel Desmoulin - One of the best experts on this subject based on the ideXlab platform.
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Abstract 783: Identification of structural determinants of human Proton-Coupled Folate Transporter oligomerization
Cancer Research, 2014Co-Authors: M. Roy Wilson, Lucas J. Wilson, Sita Kugel Desmoulin, Jenny Huang, Larry H. MatherlyAbstract:Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The human Proton-Coupled Folate Transporter (hPCFT) is expressed in human solid tumors and is active at pHs characterizing the human microenvironment. hPCFT has 459 amino acids with 12 transmembrane domains and cytosolic N- and C-termini. Recent attention has focused on exploiting hPCFT for targeting solid tumors with novel cytotoxic antiFolates (Kugel Desmoulin et al., Cancer Biol Ther 13: 1355-1373, 2012). hPCFT forms homo-oligomers with functional significance (Hou et al., J Biol Chem 287: 4982-4995, 2012). The hPCFT primary sequence includes GXXXG motifs in transmembrane domain (TMD) 2 (G93XXXG97) and 4 (G155XXXG159) that are analogous to dimerization motifs in other amphipathic proteins. To investigate the role of these GXXXG motifs in hPCFT oligomerization, we mutated Gly to Leu to obtain single (G93L, G97L, G155L, G159L) or multiple (G93L/G97L, G155L/G159L, G93L/G97L/G155L/G159L) mutants. Only with the G159L and G93L mutants, was [3H]methotrexate transport at pH 5.5 substantially preserved. Transport activity correlated closely with surface hPCFT levels by sulfo-NHS-SS-biotin labeling and Western blots. When hemagglutin (HA)-tagged G-to-L hPCFT mutants were co-expressed with FLAG-His10-tagged wild-type (WT) hPCFT in hPCFT-null HeLa cells, both mutant and WT proteins were retained on nickel affinity columns, suggesting that the hPCFT GXXXG motifs are not involved in protein oligomerization. This was substantiated by fluorescent resonance energy transfer (FRET) with N-terminal YFP-tagged and C-terminal CFP-tagged hPCFT constructs ectopically expressed in hPCFT-null HeLa cells. The GXXXG motifs in hPCFT are important for proper hPCFT folding and intracellular trafficking. A functional Cys-less (CL) hPCFT construct was generated in which the 7 Cys residues were replaced by Ser. Individual Cys insertion mutants were generated in which Cys was inserted from positions 115-137 in TMD3, and from positions 213-236 in TMD6. Constructs were transfected into hPCFT-null HeLa cells, followed by cross-linking with MTS-6-MTS (TMD3) or MTS-1-MTS (TMD6). Cross-links were detected for V118, A128, V132, Q136 and L137 in TMD 3, and for W213C, A227C, F228C, L234C, K235C, and E236C in TMD6, establishing proximities between vicinal TMDs in separate hPCFT monomers. These results strongly imply that TMD3 and TMD6 provide critical polypeptide interfaces for the formation of hPCFT oligomers. Identification of structural motifs or domains involved in hPCFT oligomerization may lead to novel approaches for therapeutically “rescuing” functionally impaired hPCFT mutants, or enhancing surface expression of hPCFT in tumors treated with hPCFT-selective antiFolates. Key words: Proton-Coupled Folate Transporter, Folate, oligomerization, cross-linking, Transporter. Citation Format: Zhanjun Hou, M. Roy Wilson, Lucas Wilson, Sita Kugel Desmoulin, Jenny Huang, Larry H. Matherly. Identification of structural determinants of human Proton-Coupled Folate Transporter oligomerization. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 783. doi:10.1158/1538-7445.AM2014-783
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The human Proton-Coupled Folate Transporter: Biology and therapeutic applications to cancer
Cancer Biology & Therapy, 2012Co-Authors: Sita Kugel Desmoulin, Aleem Gangjee, Larry H. MatherlyAbstract:This review summarizes the biology of the Proton-Coupled Folate Transporter (PCFT). PCFT was identified in 2006 as the primary Transporter for intestinal absorption of dietary Folates, as mutations in PCFT are causal in hereditary Folate malabsorption (HFM) syndrome. Since 2006, there have been major advances in understanding the mechanistic roles of critical amino acids and/or domains in the PCFT protein, many of which were identified as mutated in HFM patients, and in characterizing transcriptional control of the human PCFT gene. With the recognition that PCFT is abundantly expressed in human tumors and is active at pHs characterizing the tumor microenvironment, attention turned to exploiting PCFT for delivering novel cytotoxic antiFolates for solid tumors. The finding that pemetrexed is an excellent PCFT substrate explains its demonstrated clinical efficacy for mesothelioma and non-small cell lung cancer, and prompted development of more PCFT-selective tumor-targeted 6-substituted pyrrolo[2,3-d]pyrimid...
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Functional loss of the reduced Folate carrier enhances the antitumor activities of novel antiFolates with selective uptake by the Proton-Coupled Folate Transporter.
Molecular Pharmacology, 2012Co-Authors: Sita Kugel Desmoulin, Aleem Gangjee, Christina Cherian, Lei Wang, Lisa Polin, Kathryn White, Juiwanna Kushner, Mark Stout, Larry H. MatherlyAbstract:Uptake of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antiFolates with four or three bridge carbons [compound 1 (C1) and compound 2 (C2), respectively] into solid tumors by the Proton-Coupled Folate Transporter (PCFT) represents a novel therapeutic strategy that harnesses the acidic tumor microenvironment. Although these compounds are not substrates for the reduced Folate carrier (RFC), the major facilitative Folate Transporter, RFC expression may alter drug efficacies by affecting cellular tetrahydroFolate (THF) cofactor pools that can compete for polyglutamylation and/or binding to intracellular enzyme targets. Human tumor cells including wild-type (WT) and R5 (RFC-null) HeLa cells express high levels of PCFT protein. C1 and C2 inhibited proliferation of R5 cells 3 to 4 times more potently than WT cells or R5 cells transfected with RFC. Transport of C1 and C2 was virtually identical between WT and R5 cells, establishing that differences in drug sensitivities between sublines were independent of PCFT transport. Steady-state intracellular [3H]THF cofactors derived from [3H]5-formyl-THF were depleted in R5 cells compared with those in WT cells, an effect exacerbated by C1 and C2. Whereas C1 and C2 polyglutamates accumulated to similar levels in WT and R5 cells, there were differences in polyglutamyl distributions in favor of the longest chain length forms. In severe combined immunodeficient mice, the antitumor efficacies of C1 and C2 were greater toward subcutaneous R5 tumors than toward WT tumors, confirming the collateral drug sensitivities observed in vitro. Thus, solid tumor-targeted antiFolates with PCFT-selective cellular uptake should have enhanced activities toward tumors lacking RFC function, reflecting contraction of THF cofactor pools.
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Abstract 3826: Therapeutic targeting malignant mesothelioma with a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antiFolate via its selective uptake by the Proton-Coupled Folate Transporter
Cancer Research, 2012Co-Authors: Christina Cherian, Sita Kugel Desmoulin, Aleem Gangjee, Lei Wang, Lisa Polin, Kathryn White, Juiwanna Kushner, Mark Stout, Larry H. MatherlyAbstract:Pemetrexed (Pmx; Alimta) is among the most active agents for treating malignant pleural mesothelioma (MPM). Pmx is transported into cells by 3 systems, the reduced Folate carrier (RFC), Folate receptor (FR) β, and Proton-Coupled Folate Transporter (PCFT). RFC is present in all tissues and tumors, whereas PCFT and FRα are more narrowly expressed. By real-time RT-PCR, clinically-relevant MPM cell lines express both RFC and PCFT but not FRα. The reported clinical efficacy of Pmx toward MPM may partly reflect high levels of PCFT in this tumor. Novel 6-substituted pyrrolo[2,3-d]pyrimidine antiFolates with significant PCFT substrate activity but with greater specificities toward PCFT over RFC than Pmx, might offer significant advantages for selective therapeutic targeting MPM with reduced toxicity. We directly tested this notion for AG94, a 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antiFolate with a 3 carbon bridge. In HeLa sublines expressing moderate PCFT (R1-11-PCFT4) or RFC (R1-11-RFC6) levels, AG94 selectively inhibited (>35-fold) proliferation of PCFT-expressing R1-11-PCFT4 cells, in contrast to the nearly identical inhibitions with Pmx toward these sublines. H2452 human MPM cells showed similar sensitivities to the anti-proliferative effects of AG94 and Pmx (IC 50 s of 72 nM and 64 nM, respectively). Analogous results were obtained by colony-forming assays. Growth inhibition by AG94 was reversed with excess adenosine and 5-amino-4-imidazolecarboxamide but not thymidine. Potent inhibition of glycinamide ribonucleotide formyltransferase (GARFTase), the first Folate-dependent step in de novo purine biosynthesis, was confirmed by an in situ GARFTase assay in both R-1-11-PCFT and H2452 cells. Assays with [ 3 H]AG94 with engineered Chinese hamster ovary cells confirmed selective PCFT transport, characterized by an acidic pH optimum and transport superiority over Pmx at both pH 5.5 and pH 6.8. Analogous results were seen with R1-11-PCFT4 and H2452 cells. In both R1-11-PCFT4 and H2452 cells, [ 3 H]Pmx and [ 3 H]AG94 were metabolized to polyglutamates, including di- to heptaglutamyl forms. Efficacy trials with subcutaneous early and upstage H2452 tumors were performed in SCID mice administered AG94, compared to gemcitabine and cisplatin. AG94 showed antitumor efficacies superior to gemcitabine and cisplatin. All mice tolerated the treatment regimens well. Our results demonstrate potent antitumor efficacies of AG94 toward H2452 MPM cells in vitro and in vivo, reflecting its efficient membrane transport by PCFT over RFC, synthesis of polyglutamate conjugates, and inhibition of GARFTase. Selectivity for non-RFC cellular uptake processes by potent antiFolate drugs such as AG94 presents an exciting new opportunity for treating solid tumors including MPM over chemotherapy agents currently available. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3826. doi:1538-7445.AM2012-3826
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Abstract 3825: The impact of the reduced Folate carrier on the collateral sensitivity of novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antiFolates with selective uptake by the Proton-Coupled Folate Transporter
Cancer Research, 2012Co-Authors: Sita Kugel Desmoulin, Aleem Gangjee, Christina Cherian, Lei Wang, Lisa Polin, Kathryn White, Juiwanna Kushner, Mark Stout, Larry H. MatherlyAbstract:The 6-substituted pyrrolo[2,3-d]pyrimidine thienonyl antiFolates with 3 (AG94) or 4 (AG71) carbon bridge lengths represent a new class of agents that inhibit α-glycinamide ribonucleotide (GAR) formyltransferase (GARFTase). AG71 and AG94 are selectively transported by the Proton-Coupled Folate Transporter (PCFT) over the reduced Folate carrier (RFC), the major facilitative Folate Transporter in tissues and tumors. PCFT functions optimally at acidic pHs. Transport of AG71 and AG94 by PCFT and the acidic tumor microenvironment may further increase tumor selectivity. Although AG71 and AG94 are not RFC substrates, levels of RFC could nonetheless impact their anti-proliferative effects, via expansion or contraction of intracellular tetrahydroFolate (THF) pools. Studies were performed with wild-type (WT) and methotrexate resistant R5 HeLa cells in which RFC is deleted. In proliferation assays with AG71 and AG94, R5 cells were more sensitive than were WT cells (3.9- and 2.4-fold, respectively). RFC-transfected R5 cells had IC50s for growth inhibition similar to WT cells. To determine the mechanisms of the collateral sensitivities to AG71 and AG94, we measured steady-state intracellular [3H]THF accumulations at a physiologic concentration of [3H]5-formyl THF in WT and R5 cells. R5 cells had contracted intracellular [3H]THF pools compared to WT cells. The collateral antiFolate sensitivities in R5 cells were independent of PCFT transport, since there were no differences in transport for AG71 and AG94 at any pH between the sublines. Intracellular THF cofactors may compete at the level of polyglutamylation by folylpolyglutamate synthetase and/or GARFTase binding. When incubated with radiolabeled AG71 and AG94, WT cells accumulated slightly lesser amounts of total drug forms (84 and 76% for AG71 and AG94, respectively) than did R5 cells, most of these as polyglutamates. This difference was greatest for the longest chain-length (Glu4-Glu6) polyglutamates (increased ∼2-fold for both AG71 and AG94). GARFTase activity was measured in WT and R5 cells by an in situ metabolic assay which measures incorporation of [14C]glycine into [14C]formyl GAR. R5 and WT cells were differentially sensitive to GARFTase inhibition by AG71 and AG94 and these differences were lost when RFC expression was restored to R5 cells. In an in vivo efficacy trial with subcutaneous WT and R5 cells in SCID mice, antitumor efficacies of both AG71 and AG94 were increased in R5 cells relative to WT cells and this effect was greater for AG71. These findings suggest that novel antiFolates with selective membrane transport by PCFT over RFC would have enhanced activities toward tumors that have lost RFC function, and that levels of RFC may be an important biomarker of chemotherapy efficacy with this new class of solid tumor-selective drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3825. doi:1538-7445.AM2012-3825
