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1.  Role of Lysine 411 in Substrate Carboxyl Group Binding to the Human Reduced Folate Carrier, as Determined by Site-directed Mutagenesis and Affinity Inhibition 
Molecular pharmacology  2008;73(4):10.1124/mol.107.043190.
Reduced folate carrier (RFC) is the major membrane transporter for folates and antifolates in mammalian tissues. Recent studies used radioaffinity labeling with N-hydroxysuccinimide (NHS)-3H-methotrexate (MTX) to localize substrate binding to residues in transmembrane domain (TMD) 11 of human RFC. To identify the modified residue(s), seven nucleophilic residues in TMD11 were mutated to Val or Ala and mutant constructs expressed in RFC-null HeLa cells. Only Lys411Ala RFC was not inhibited by NHS-MTX. By radioaffinity labeling with NHS-3H-MTX, wild type (wt) RFC was labeled; for Lys411Ala RFC, radiolabeling was abolished. When Lys411 was replaced with Ala, Arg, Gln, Glu, Leu, and Met, only Lys411Glu RFC showed substantially decreased transport. Nine classical diamino furo[2,3-d]pyrimidine antifolates with unsubstituted α- and γ-carboxylates (1), hydrogen- or methyl-substituted α- (2, 3) or γ- (4, 5) carboxylates, or substitutions of both α- and γ-carboxylates (6, 7, 8, 9) were used to inhibit 3H-MTX transport with RFC-null K562 cells expressing wt and Lys411Ala RFCs. For wt and Lys411Ala RFCs, inhibitory potencies were in the order 4>5>1>3>2; 6-9 were poor inhibitors. Inhibitions decreased in the presence of physiologic anions. When NHS esters of 1, 2, and 4 were used to covalently modify wt RFC, inhibitory potencies were in the order 2>1>4; inhibition was abolished for Lys411Ala RFC. These results suggest that Lys411 participates in substrate binding via an ionic association with the substrate γ-carboxylate, however, this is not essential for transport. An unmodified α-carboxylate is required for high affinity substrate binding to RFC, whereas the γ-carboxyl is not essential.
doi:10.1124/mol.107.043190
PMCID: PMC3806200  PMID: 18182479
2.  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 
The 5-substituted pyrrolo[2,3-d]pyrimidine antifolate pemetrexed (Pmx) is an active agent for malignant pleural mesothelioma (MPM). Pmx is transported into MPM cells by the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). We tested the notion that a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate (compound 2) might be an effective treatment for MPM, reflecting its highly selective membrane transport by PCFT over RFC. Compound 2 selectively inhibited proliferation of a HeLa subline expressing exclusively PCFT (R1-11-PCFT4) over an isogenic subline expressing only RFC (R1-11-RFC6). By outgrowth, H2452 human MPM cells were highly sensitive to the inhibitory effects of compound 2. By colony-forming assays, following an intermittent (24 h) drug exposure, 2 was cytotoxic. Cytotoxic activity by 2 was due to potent inhibition of glycinamide ribonucleotide formyltransferase (GARFTase) in de novo purine biosynthesis, as confirmed by nucleoside protection and in situ GARFTase assays with [14C]glycine. Assays with [3H]compound 2 and R1-11-PCFT4 or R1-11-RFC6 cells directly confirmed selective membrane transport by PCFT over RFC. PCFT transport was also confirmed for H2452 cells. In R1-11-PCFT4 and H2452 cells, [3H]compound 2 was metabolized to polyglutamates. Potent in vivo efficacy was confirmed toward early- and upstage H2452 xenografts in severe combined immunodeficient mice administered intravenous compound 2. Our results demonstrate potent antitumor efficacy of compound 2 toward H2452 MPM in vitro and in vivo, reflecting its efficient membrane transport by PCFT over RFC, synthesis of polyglutamates, and inhibition of GARFTase. Selectivity for non-RFC cellular uptake processes by novel tumor-targeted antifolates such as compound 2 presents an exciting new opportunity for treating solid tumors.
doi:10.1007/s00280-013-2094-0
PMCID: PMC3769948  PMID: 23412628
proton-coupled folate transporter; mesothelioma; folate; antifolate; pemetrexed
3.  Synthesis and biological activity of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers as inhibitors of de novo purine biosynthesis with selectivity for cellular uptake by high affinity folate receptors and the proton-coupled folate transporter over the reduced folate carrier 
Journal of Medicinal Chemistry  2012;55(4):1758-1770.
We reported the selective transport of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl-for-benzoyl-substituted side chain and a 3- (3a) and 4-carbon (3b) bridge. Compound 3a was more potent than 3b against tumor cells; While 3b was completely selective for transport by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) over reduced folate carrier (RFC), 3a was not. To determine if decreasing the distance between the bicyclic scaffold and L-glutamate in 3b would preserve transport selectivity and potency against human tumor cells, 3b regioisomers with [1,3] (7 and 8) and [1,2] (4, 5 and 6) substitutions on the thienoyl ring, and with acetylenic insertions in the 4-atom bridge, were synthesized and evaluated. Compounds 7 and 8 were potent nanomolar inhibitors of KB and IGROV1 human tumor cells with complete selectivity for FRα and PCFT over RFC.
doi:10.1021/jm201688n
PMCID: PMC3288238  PMID: 22243528
4.  Synthesis, biological and antitumor activity of a highly potent 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitor with proton-coupled folate transporter and folate receptor selectivity over the reduced folate carrier that inhibits β-glycinamide ribonucleotide formyltransferase 
Journal of medicinal chemistry  2011;54(20):7150-7164.
2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoyl side chain (compounds 1–3, respectively) were synthesized for comparison with compound 4, the previous lead compound of this series. Conversion of hydroxyl acetylen-thiophene carboxylic esters to thiophenyl-α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidine compounds of type 18 and 19. Coupling with L-glutamate diethyl ester, followed by saponification, afforded 1–3. Compound 3 selectively inhibited proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including human tumor cells KB and IGROV1 much more potently than 4. Compound 3 was more inhibitory than 4 toward β-glycinamide ribonucleotide formyltransferase (GARFTase). Both 3 and 4 depleted cellular ATP pools. In SCID mice with IGROV1 tumors, 3 was more efficacious than 4. Collectively, our results show potent antitumor activity for 3 in vitro and in vivo, associated with its selective membrane transport by FRs and PCFT over RFC and inhibition of GARFTase, clearly establishing the 3-atom bridge as superior to the 1, 2 and 4-atom bridge lengths for the activity of this series.
doi:10.1021/jm200739e
PMCID: PMC3209708  PMID: 21879757
5.  Synthesis and biological activity of a novel series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitors of purine biosynthesis with selectivity for high affinity folate receptors and the proton-coupled folate transporter over the reduced folate carrier for cellular entry† 
Journal of medicinal chemistry  2010;53(3):1306-1318.
2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl side chain and 4-6 carbon bridge lengths (compounds 1-3) were synthesized as substrates for folate receptors (FRs) and the proton-coupled folate transporter (PCFT). Conversion of acetylene carboxylic acids to α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidines. Sonogashira coupling with (S)-2-[(5-bromo-thiophene-2-carbonyl)-amino]-pentanedioic acid diethyl ester, followed by hydrogenation and saponification, afforded 1-3. Compounds 1 and 2 potently inhibited KB and IGROV1 human tumor cells that express FRα, reduced folate carrier (RFC), and PCFT. The analogs were selective for FR- and PCFT over RFC. Glycinamide ribonucleotide formyltransferase was the principal cellular target. In SCID mice with KB tumors, 1 was highly active against both early (3.5 log kill, 1/5 cures) and advanced (3.7 log kill, 4/5 complete remissions) stage tumors. Our results demonstrate potent in vitro and in vivo antitumor activity for 1 due to selective transport by FRs and PCFT over RFC.
doi:10.1021/jm9015729
PMCID: PMC2836843  PMID: 20085328
6.  Substrate-specific binding and conformational changes involving Ser313 and transmembrane domain 8 of the human reduced folate carrier, as determined by site-directed mutagenesis and protein cross-linking 
Biochemical Journal  2010;430(Pt 2):265-274.
RFC (reduced folate carrier) is the major transporter for reduced folates and antifolates [e.g. MTX (methotrexate)]. RFC is characterized by two halves, each with six TMD (transmembrane domain) α helices connected by a hydrophilic loop, and cytoplasmic N- and C-termini. We previously identified TMDs 4, 5, 7, 8, 10 and 11 as forming the hydrophilic cavity for translocation of (anti)folates. The proximal end of TMD8 (positions 311–314) was implicated in substrate binding from scanning-cysteine accessibility methods; cysteine replacement of Ser313 resulted in loss of transport. In the present study, Ser313 was mutated to alanine, cysteine, phenylalanine and threonine. Mutant RFCs were expressed in RFC-null R5 HeLa cells. Replacement of Ser313 with cysteine or phenylalanine abolished MTX transport, whereas residual activity was preserved for the alanine and threonine mutants. In stable K562 transfectants, S313A and S313T RFCs showed substantially decreased Vmax values without changes in Kt values for MTX compared with wild-type RFC. S313A and S313T RFCs differentially impacted binding of ten diverse (anti)folate substrates. Cross-linking between TMD8 and TMD5 was studied by expressing cysteine-less TMD1–6 (N6) and TMD7–12 (C6) half-molecules with cysteine insertions spanning these helices in R5 cells, followed by treatment with thiol-reactive homobifunctional cross-linkers. C6–C6 and N6–N6 cross-links were seen for all cysteine pairs. From the N6 and C6 cysteine pairs, Cys175/Cys311 was cross-linked; cross-linking increased in the presence of transport substrates. The results of the present study indicate that the proximal end of TMD8 is juxtaposed to TMD5 and is conformationally active in the presence of transport substrates, and TMD8, including Ser313, probably contributes to the RFC substrate-binding domain.
doi:10.1042/BJ20100181
PMCID: PMC2947195  PMID: 20557288
antifolate; cross-linking; folate; major facilitator superfamily; mutagenesis; oligomer; reduced folate carrier; transporter; BMH, 1,6-bis(maleimido)hexane; C6, transmembrane domains 7–12; cl, cysteine-less; HA, haemagglutinin; MFS, major facilitator superfamily; MTSES, 2-sulfonatoethyl methanethiosulfonate; MTX, methotrexate; N6, transmembrane domains 1–6; p-PDM, p-phenylenedimaleimide; RFC, reduced folate carrier; hRFC, human RFC; TMD, transmembrane domain; wt, wild-type
7.  The impact of NOTCH1, FBW7 and PTEN mutations on prognosis and downstream signaling in pediatric T- cell acute lymphoblastic leukemia: A report from the Children's Oncology Group 
We explored the impact of mutations in the NOTCH1, FBW7 and PTEN genes on prognosis and downstream signaling in a well-defined cohort of 47 pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. In T-ALL lymphoblasts, we identified high frequency mutations in NOTCH1 (n=16), FBW7 (n=5) and PTEN (n=26). NOTCH1 mutations resulted in 1.3-3.3-fold increased transactivation of a HES1 reporter construct over wild-type NOTCH1; mutant FBW7 resulted in further augmentation of reporter gene activity. NOTCH1 and FBW7 mutations were accompanied by increased median transcripts for NOTCH1 target genes (HES1, DELTEX1, cMYC). However, none of these mutations were associated with treatment outcome. Elevated HES1, DELTEX1 and cMYC transcripts were associated with significant increases in transcript levels of several chemotherapy relevant genes, including MDR1, ABCC5, reduced folate carrier, asparagine synthetase, thiopurine methyltranserase, Bcl-2 and dihydrofolate reductase. PTEN transcripts positively correlated with HES1 and cMYC transcript levels. Our results suggest that (1) multiple factors should be considered with attempting to identify molecular-based prognostic factors for pediatric T-ALL, and (2) depending on the NOTCH1 signaling status, modifications in the types or dosing of standard chemotherapy drugs for T-ALL, or combinations of agents capable of targeting NOTCH1, AKT and/or mTOR with standard chemotherapy agents may be warranted.
doi:10.1038/leu.2009.64
PMCID: PMC2726275  PMID: 19340001
acute lymphoblastic leukemia; NOTCH1; FBW7; PTEN; HES1; DELTEX1; cMYC; chemotherapy; T-cell
8.  Synthesis and Discovery of High Affinity Folate Receptor-Specific Glycinamide Ribonucleotide Formyltransferase Inhibitors With Antitumor Activity 
Journal of medicinal chemistry  2008;51(16):5052-5063.
A series of 6-substituted classical pyrrolo[2,3-d]pyrimidine antifolates with a 3- to 6-carbon bridge between the heterocycle and the benzoyl-L-glutamate (compounds 2, 3, 4 and 5, respectively) was synthesized starting from methyl 4-formylbenzoate and a Wittig reaction with the appropriate triphenylphosphonium bromide, followed by reduction and conversion to the α-bromomethylketones. Cyclocondensation of 2,4-diamino-4-oxopyrimidine with the α-bromoketones, coupling with diethyl-L-glutamate and saponification afforded 2–5. Compounds 2–5 had negligible substrate activity for RFC but showed variably potent (nanomolar) and selective inhibitory activities toward Chinese hamster ovary cells that expressed FRα or FRβ, and toward FRα-expressing KB and IGROV1 human tumor cells. Inhibition of KB cell colony formation was also observed. Glycinamide ribonucleotide formyl transferase (GARFTase) was identified as the primary intracellular target of the pyrrolo[2,3-d]pyrimidines. The combined properties of selective FR targeting, lack of RFC transport, and GARFTase inhibition resulting in potent antitumor activity are unprecedented and warrant development of these analogs as antitumor agents.
doi:10.1021/jm8003366
PMCID: PMC2748117  PMID: 18680275
9.  Synthesis and biological activity of a novel series of 6-substituted thieno[2,3-d]pyrimidine antifolate inhibitors of purine biosynthesis with selectivity for high affinity folate receptors over the reduced folate carrier and proton-coupled folate transporter for cellular entry 
Journal of medicinal chemistry  2009;52(9):2940-2951.
A series of seven 2-amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines, with bridge length variations (from 2-8 carbon atoms) were synthesized as selective folate receptor (FR) α and β substrates and as antitumor agents. The syntheses were accomplished from appropriate allylalcohols and 4-iodobenzoate to afford the aldehydes which were converted to the appropriate 2-amino-4-carbethoxy-5-substituted thiophenes 23-29. Cyclization with chlorformamidine afforded the thieno[2,3-d]pyrimidines 30-36 which were hydrolyzed and coupled with diethyl-L-glutamate, followed by saponification to give the target compounds 2-8. Compounds 3-6 were potent growth inhibitors (IC50 4.7 to 334 nM) of human tumor cells (KB and IGROV1) that express FRs. In addition, compounds 3-6 inhibited the growth of Chinese hamster ovary (CHO) cells that expressed FRs but not the reduced folate carrier (RFC) or proton-coupled folate transporter (PCFT). However, the compounds were inactive toward CHO cells that lacked FRs but contained either the RFC or PCFT. By nucleoside and 5-amino-4-imidazole carboxamide (AICA) protection studies, along with in vitro and in situ enzyme activity assays, the mechanism of antitumor activity was identified as the dual inhibition of glycinamide ribonucleotide formyltransferase and, likely, AICA ribonucleotide formyltransferase. The dual inhibitory activity of the active thieno[2,3-d]pyrimidine antifolates and the FR specificity represent unique mechanistic features for these compounds distinct from all other known antifolates. The potent inhibitory effects of compounds 3-6 toward cells expressing FRs but not PCFT provide direct evidence that cellular uptake of this series of compounds by FRs does not depend on the presence of PCFT and argues that direct coupling between these transporters is not obligatory.
doi:10.1021/jm8011323
PMCID: PMC2730022  PMID: 19371039
10.  A Humanized Mouse Model for the Reduced Folate Carrier 
The ubiquitously expressed reduced folate carrier (RFC) or SLC19A1 is recognized to be an essential transport system for folates in mammalian cells and tissues. In addition to its generalized role as a folate transporter, RFC provides specialized tissue functions including absorption across intestinal/colonic epithelia, transport across the basolateral membrane of renal proximal tubules, transplacental transport of folates, and folate transport across the blood-brain barrier. The human RFC (hRFC) gene is regulated by 5 major upstream non-coding regions (designated A1/A2, A, B, C, and D), each transcribed from a unique promoter. Altogether, at least 14 distinct hRFC transcripts can be envisaged in which different 5’ untranslated regions (UTRs) are fused to a common splice acceptor region (positions -1 to -49) within the first coding exon with a common 1776 bp coding sequence. The 5’ non-coding regions are characterized by alternate transcription start sites, multiple splice forms, and selective tissue distributions. Alternate 5’UTRs impact mRNA stabilities and translation efficiencies, and result in synthesis of modified hRFC proteins translated from upstream AUGs. In this report, we describe production and characterization of transgenic mice (TghRFC1) containing a functional hRFC gene and of humanized mice in which the mRFC gene is inactivated and an active hRFC gene has been introduced. The mice appear to be healthy and to breed well. Analysis of tissue specificity of expression in both the TghRFC1 and humanized hRFC mice by real-time RT-PCR demonstrates that the hRFC gene is expressed with a specificity closely resembling that seen in human tissues. For the humanized hRFC mice, levels of B and A1/A2 5’UTRs predominated in all mice/tissues, thus resembling results in normal human tissues. Lower levels of A and C 5’UTRs were also detected. The availability of humanized mouse models for hRFC will permit investigators to address critical unanswered questions pertinent to human health and disease. These include the ability to analyze the hRFC gene in vivo, to control dietary and other environmental conditions that may impact levels of gene expression, and to control the genetics of the mice in order to assess the effects of hRFC gene alterations on tissue folate uptake and distribution, none of which can be easily achieved in human populations.
doi:10.1016/j.ymgme.2007.09.014
PMCID: PMC2271060  PMID: 17983788
reduced folate carrier; mouse models; folate homeostasis; promoter usage

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