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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.

Targeted antifolates with heteroatom replacements of the carbon vicinal to the phenyl ring in 1 by N (4), O (8), or S (9), or with N-substituted formyl (5), acetyl (6), or trifluoroacetyl (7) moieties, were synthesized and tested for selective cellular uptake by folate receptor (FR) α and β or the proton-coupled folate transporter. Results show increased in vitro antiproliferative activity toward engineered Chinese hamster ovary cells expressing FRs by 4-9 over the CH2 analogue 1. Compounds 4-9 inhibited de novo purine biosynthesis and glycinamide ribonucleotide formyltransferase (GARFTase). X-ray crystal structures for 4 with FRα and GARFTase showed that the bound conformations of 4 required flexibility for attachment to both FRα and GARFTase. In mice bearing IGROV1 ovarian tumor xenografts, 4 was highly efficacious. Our results establish that heteroatom substitutions in the 3-atom bridge region of 6-substituted pyrrolo[2,3-d]pyrimidines related to 1 provide targeted antifolates that warrant further evaluation as anticancer agents.

Pemetrexed and methotrexate are antifolates used for cancer chemotherapy and inflammatory diseases. These agents have toxic side effects resulting, in part, from nonspecific cellular transport by the reduced folate carrier (RFC), a ubiquitously expressed facilitative transporter. We previously described 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with modifications of the side chain linker and aromatic ring that are poor substrates for RFC but are efficiently transported via folate receptors (FRs) and the proton-coupled folate transporter (PCFT). These targeted antifolates are cytotoxic in vitro toward FR- and PCFT-expressing tumor cells and in vivo with human tumor xenografts in immune-compromised mice, reflecting selective cellular uptake. Antitumor efficacy is due to inhibition of glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) activity in de novo synthesis of purine nucleotides. This study used purified human GARFTase (formyltransferase domain) to assess in vitro inhibition by eight novel thieno- and pyrrolo[2,3-d]pyrimidine antifolates. Seven analogues (AGF23, AGF71, AGF94, AGF117, AGF118, AGF145, and AGF147) inhibited GARFTase with Ki values in the low- to mid-nanomolar concentration range, whereas AGF50 inhibited GARFTase with micromolar potency similar to that of PMX. On the basis of crystal structures of ternary complexes with GARFTase, β-GAR, and the monoglutamyl antifolates, differences in inhibitory potencies correlated well with antifolate binding and the positions of the terminal carboxylates. Our data provide a mechanistic basis for differences in inhibitory potencies between these novel antifolates and a framework for future structure-based drug design. These analogues could be more efficacious than clinically used antifolates, reflecting their selective cellular uptake by FRs and PCFT and potent GARFTase inhibition.

Structure–activity relationships for cellular uptake and inhibition of cell proliferation were studied for 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates in which the terminal l-glutamate of the parent structure (7) was replaced by natural or unnatural amino acids. Compounds 7 and 10–13 were selectively inhibitory toward folate receptor (FR) α-expressing Chinese hamster ovary (CHO) cells. Antiproliferative effects of compounds 7 and 9–13 toward FRα- and FRβ-expressing CHO cells were only partly reflected in binding affinities to FRα and FRβ or in the docking scores with molecular models of FRα and FRβ. Compounds 7 and 11 were potent inhibitors of glycinamide ribonucleotide formyltransferase in de novo purine biosynthesis in KB human tumor cells. These studies establish for the first time the importance of the α- and γ-carboxylic acid groups, the length of the amino acid, and the conformation of the side chain for transporter binding and biological activity of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates.

The proton-coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum. By real-time reverse transcription-polymerase chain reaction, PCFT was expressed in the majority of 53 human tumor cell lines, with the highest levels in Caco-2 (colorectal adenocarcinoma), SKOV3 (ovarian), and HepG2 (hepatoma) cells. A novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate (compound 1) was used to establish whether PCFT can deliver cytotoxic drug under pH conditions that mimic the tumor microenvironment. Both 1 and pemetrexed (Pmx) inhibited proliferation of R1-11-PCFT4 HeLa cells engineered to express PCFT without the reduced folate carrier (RFC) and of HepG2 cells expressing both PCFT and RFC. Unlike Pmx, 1 did not inhibit proliferation of R1-11-RFC6 HeLa cells, which express RFC without PCFT. Treatment of R1-11-PCFT4 cells at pH 6.8 with 1 or Pmx inhibited colony formation with dose and time dependence. Transport of [(3)H]compound 1 into R1-11-PCFT4 and HepG2 cells was optimal at pH 5.5 but appreciable at pH 6.8. At pH 6.8, [(3)H]compound 1 was metabolized to (3)H-labeled polyglutamates. Glycinamide ribonucleotide formyltransferase (GARFTase) in R1-11-PCFT4 cells was inhibited by 1 at pH 6.8, as measured by an in situ GARFTase assay, and was accompanied by substantially reduced ATP levels. Compound 1 caused S-phase accumulation and a modest level of apoptosis. An in vivo efficacy trial with severe combined immunodeficient mice implanted with subcutaneous HepG2 tumors showed that compound 1 was active. Our findings suggest exciting new therapeutic possibilities to selectively deliver novel antifolate drugs via transport by PCFT over RFC by exploiting the acidic tumor microenvironment.

Tumor-specific folate receptor (FR) targeting over the ubiquitous reduced folate carrier (RFC) allows for specific tumor targeting without the attending dose-limiting toxicity of all clinically used cytotoxic agents. In an attempt to provide selectivity for FRα and/or β, folic acid has been conjugated with a variety of cytotoxic payloads to alleviate dose-limiting toxicities since folic acid is not transported via RFC (the major tissue folate transporter) to any significant extent. These conjugates have been unsuccessful in clinical trials likely in part due to the instability of the linkers used for conjugation. Premature cleavage results in the cytotoxic component being released prior to tumor penetration, thus precluding any possibility of selective tumor targeting. We designed novel pyrrolo[3,2-d]pyrimidine compounds in which both the targeting component and the cytotoxic component are in the same molecule. These compounds do not require any conjugation or linkers and hence do not suffer any premature cleavage. In this report, we synthesized and evaluated a “first-in-class” series of fluorinated analogues that target FRα/β and have limited transport via RFC, thus providing selectivity for tumors expressing FRα and/or FRβ. Two lead molecules, AGF347 and AGF355, were potent inhibitors of KB human tumor cell proliferation in vitro (IC50 = 1.05 nM and 6.25 nM respectively). Based on protection experiments with glycine, thymidine, adenosine and 5-aminoimidazole-4-carboxamide (AICA), AGF347 and AGF355 were identified as dual inhibitors of serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in mitochondrial one-carbon metabolism and reported oncodriver, and AICA ribonucleotide formyltransferase (AICARFTase), the 2nd folate-dependent step in de novo purine biosynthesis. These compounds are currently being evaluated in preclinical studies as potential candidates for clinical trials. Citation Format: Md Junayed Nayeen, Khushbu Shah, Aleem Gangjee, Aamod Dekhne, Zhanjun Hou, Larry H. Matherly. First-in-class, fluorinated folate receptor specific agents that target tumor cells via inhibition of serine hydroxymethyltransferase 2 (SHMT2) and 5-aminoimidazole-4-carboxamide formyltransferase (AICARFTase) [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 1656.

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 Frühauf, Khushbu Shah, Md. Junayed Nayeen, Adrianne Wallace-Povirk, Carrie O'Connor, 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.

Design, modeling and synthesis of pyrrolo[3,2-d]pyrimidine analogs were carried out based on the concept of dual mechanisms-of-action for achieving maximal antitumor efficacy. Tumor-targeted delivery via agents that are selectively transported by the folate receptor (FRα/β) and/or PCFT, overexpressed in tumor cells such as epithelial ovarian cancer and non-small cell lung cancer, over the ubiquitously expressed reduced folate carrier (RFC) would afford tumor selectivity. Gangjee, Matherly and coworkers previously reported 5-substituted pyrrolo[2,3-d]pyrimidines as inhibitors of de novo purine nucleotide biosynthesis glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase). To synthesize potential dual inhibitors of de novo purine biosynthesis at cytosolic AICARFTase and mitochondrial one-carbon metabolism at serine hydroxymethyl transferase (SHMT) 2, we generated structural hybrids of these inhibitors and 5,10-methylenetetrahydrofolate (the SHMT2 cofactor), 5-substituted pyrrolo[3,2-d]pyrimidine analogs AGF291, AGF299, AGF300, AGF318, AGF320, and AGF331. Molecular modeling of these compounds utilizing X-ray crystal structures of the transporters (FRα/β) and enzyme targets (AICARFTase, SHMT2) predicted excellent activities. The influence of bridge lengths and the aromatic moieties of these novel compounds were evaluated by testing their anti-proliferative activities in Chinese hamster ovary cells expressing human FRα or PCFT, and in FRα- and/or PCFT expressing nasopharyngeal carcinoma (KB) and lung cancer (H460) cells. All the pyrrolo[3,2-d]pyrimidine analogs were inhibitory toward FR- and/or PCFT-expressing cells. Compared to its pyrrolo[2,3-d]pyrimidine analog, AGF300 increased inhibition of cells via FR uptake from IC50 8.6 nM to 2.1 nM (by 4-fold) and decreased uptake through RFC from 56.5 nM to 516 nM (by 10-fold), suggesting a FR-targeted delivery of the agent. Toward H460 xenografts in SCID mice, AGF291 was efficacious. Protection studies with thymidine, glycine, adenosine and AICA and radiotracer studies with [3-14C]serine identified the likely intracellular targets of the pyrrolo[3,2-d]pyrimidine analogs as SHMT2 and AICARFTase. Mitochondrial one-carbon metabolism originating with SHMT2 is a critical source of reducing equivalents and one-carbon units for cytosolic biosynthesis and SHMT2 has been implicated as an oncodriver in several tumor types. While inhibition of SHMT2 was the subject of exploratory studies, our novel pyrrolo[3,2-d]pyrimidine series are “first-in-class” in terms of their in vitro and in vivo antitumor efficacies attributable to dual targeting SHMT2 and AICARFTase. Citation Format: Khushbu Shah, Aamod Dekhne, Zhanjun Hou, Larry Matherly, Aleem Gangjee. 5N-substituted pyrrolo[3,2-d]pyrimidines: Tumor-targeted agents with first-in-class dual inhibition of serine hydroxymethyl transferase 2 and 5-amino-4-imidazolecarboxamide ribonucleotide formyl transferase enzymes [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 1658.

Epithelial ovarian cancer (EOC) is the deadliest gynecologic cancer, with limited progress in targeted therapy or in combating drug resistance. Reflecting this, there is an urgent need to develop novel inhibitors with minimal toxicity. EOC is associated with increasing expression of folate receptor alpha (FRα) with increasing stage and grade of disease. Of particular interest are targeted therapeutics toward FRα which spare transport by the ubiquitously expressed reduced folate carrier (RFC). FRα is a viable target for tumor selectivity with limited expression in normal tissues, where it is only expressed on apical membranes. We previously described 6-substituted thieno[2,3-d]pyrimidine (6TP) benzoyl antifolates with bridge lengths of 2-8 carbons as potent and selective inhibitors of tumor cells including EOCs that express FRs. The most potent compound (AGF50) included a 4-carbon bridge and showed FRα selectivity and inhibition of de novo purine biosynthesis. We synthesized AGF50 analogs with various aryl side-chain modifications (e.g., thiophene, furan, pyridine) including 2'fluorine substitutions. Toward isogenic Chinese hamster ovary cell lines individually expressing FRα, RFC and the proton-coupled folate transporter (PCFT), the 6TP analogs were completely selective toward FRα over RFC and PCFT, manifested as inhibition of cell proliferation with nmolar IC50s. Activity of this series extended to KB human tumor cells and human EOC cell lines (e.g., IGROV1). Drug effects were abolished with excess folic acid, confirming FRα uptake. Adenosine completely protected cells from the inhibitory effects of 6TPs in vitro, suggesting inhibition of one-carbon transfers mediated by glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) and/or 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase), leading to synthesis of purine nucleotides. Protection from 6TPs by AICA was incomplete (in contrast to a “pure” GARFTase inhibitor AGF94), suggesting inhibition of AICARFTase. Whereas AGF94 potently inhibited GARFTase (>98%) in KB cells, reflected in [14C]glycine incorporation into [14C]formyl GAR, inhibition by the 6TPs was incomplete (75-90%) up to 1 μM drug. AICARFTase inhibition was confirmed by measuring accumulation of AICA ribonucleotide (ZMP), although this varied for different 6TP analogs. Our studies establish an important structure-activity relationship for novel FRα-targeted 6TPs with aromatic side-chain modifications and imply the therapeutic potential of targeting AICARFTase for EOC. Citation Format: Adrianne Wallace-Povirk, Nian Tong, Carrie O'Connor, Zhanjun Hou, Aleem Gangjee, Larry Matherly, Xilin Zhou. Tumor-targeting with novel dual-targeted 6-substituted thieno[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α, and inhibition of de novo purine nucleotide biosynthesis [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 3983.

The reduced folate carrier (RFC) is the major transport system for folates in mammals. We previously demonstrated the existence of human RFC (hRFC) homo-oligomers and established the importance of these higher order structures to intracellular trafficking and carrier function. In this report, we examined the operational significance of hRFC oligomerization and the minimal functional unit for transport. In negative dominance experiments, multimeric transporters composed of different ratios of active (either wild type (WT) or cysteine-less (CLFL)) and inactive (either inherently inactive (Y281L and R373A) due to mutation, or resulting from inactivation of the Y126C mutant by (2-sulfonatoethyl) methanethiosulfonate (MTSES)) hRFC monomers were expressed in hRFC-null HeLa (R5) cells, and residual WT or CLFL activity was measured. In either case, residual transport activity with increasing levels of inactive mutant correlated linearly with the fraction of WT or CLFL hRFC in plasma membranes. When active covalent hRFC dimers, generated by fusing CLFL and Y126C monomers, were expressed in R5 cells and treated with MTSES, transport activity of the CLFL-CLFL dimer was unaffected, whereas Y126C-Y126C was potently (64%) inhibited; heterodimeric CLFL-Y126C and Y126C-CLFL were only partly (27 and 23%, respectively) inhibited by MTSES. In contrast to Y126C-Y126C, trans-stimulation of methotrexate uptake by intracellular folates for Y126C-CLFL and CLFL-Y126C was nominally affected by MTSES. Collectively, these results strongly support the notion that each hRFC monomer comprises a single translocation pathway for anionic folate substrates and functions independently of other monomers (i.e. despite an oligomeric structure, hRFC functions as a monomer).

The ubiquitously expressed reduced folate carrier (RFC) is the major transport system for folate cofactors in mammalian cells and tissues. Previous considerations of RFC structure and mechanism were based on the notion that RFC monomers were sufficient to mediate transport of folate and antifolate substrates. The present study examines the possibility that human RFC (hRFC) exists as higher order homo-oligomers. By chemical cross-linking, transiently expressed hRFC in hRFC-null HeLa (R5) cells with the homobifunctional cross-linker 1,3-propanediyl bis-methanethiosulfonate and Western blotting, hRFC species with molecular masses of hRFC homo-oligomers were identified. Hemagglutinin- and Myc epitope-tagged hRFC proteins expressed in R5 cells were co-immunoprecipitated from both membrane particulate and surface-enriched membrane fractions, indicating that oligomeric hRFC is expressed at the cell surface. By co-expression of wild type and inactive mutant S138C hRFCs, combined with surface biotinylation and confocal microscopy, a dominant-negative phenotype was demonstrated involving greatly decreased cell surface expression of both mutant and wild type carrier caused by impaired intracellular trafficking. For another hRFC mutant (R373A), expression of oligomeric wild type-mutant hRFC was accompanied by a significant and disproportionate loss of wild type activity unrelated to the level of surface carrier. Collectively, our results demonstrate the existence of hRFC homo-oligomers. They also establish the likely importance of these higher order hRFC structures to intracellular trafficking and carrier function.

6-Substituted classical pyrrolo[2,3-d]pyrimidine antifolates with a three- to six-carbon bridge between the heterocycle and the benzoyl-L-glutamate (compounds 2-5, respectively) were synthesized starting from methyl 4-formylbenzoate and a Wittig reaction with the appropriate triphenylphosphonium bromide, followed by reduction and conversion to the alpha-bromomethylketones. Cyclocondensation of 2,4-diamino-4-oxopyrimidine with the alpha-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 FRalpha or FRbeta and toward FRalpha-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 analogues as antitumor agents.

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)-[(3)H]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 K411A RFC was not inhibited by NHS-MTX. By radioaffinity labeling with NHS-[(3)H]MTX, wild-type (wt) RFC was labeled; for K411A RFC, radiolabeling was abolished. When Lys411 was replaced with Ala, Arg, Gln, Glu, Leu, and Met, only K411E RFC showed substantially decreased transport. Nine classic diamino furo[2,3-d]pyrimidine antifolates with unsubstituted alpha- and gamma-carboxylates (1), hydrogen- or methyl-substituted alpha-(2,3) or gamma-(4,5) carboxylates, or substitutions of both alpha- and gamma-carboxylates (6-9) were used to inhibit [(3)H]MTX transport with RFC-null K562 cells expressing wt and K411A RFCs. For wt and K411A RFCs, inhibitory potencies were in the order 45132; 6 to 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 214; inhibition was abolished for K411A RFC. These results establish that Lys411 participates in substrate binding via an ionic association with the substrate gamma-carboxylate; however, this is not essential for transport. An unmodified alpha-carboxylate is required for high-affinity substrate binding to RFC, whereas the gamma-carboxyl is not essential.

2-Amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine antifolate thiophene regioisomers of AGF94 (4) with a thienoyl side chain and three-carbon bridge lengths [AGF150 (5) and AGF154 (7)] were synthesized as potential antitumor agents. These analogues inhibited proliferation of Chinese hamster ovary (CHO) sublines expressing folate receptors (FRs) α or β (IC50s < 1 nM) or the proton-coupled folate transporter (PCFT) (IC50 < 7 nM). Compounds 5 and 7 inhibited KB, IGROV1, and SKOV3 human tumor cells at subnanomolar concentrations, reflecting both FRα and PCFT uptake. AGF152 (6) and AGF163 (8), 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine thiophene regioisomers, also inhibited growth of FR-expressing CHO and KB cells. All four analogues inhibited glycinamide ribonucleotide formyltransferase (GARFTase). Crystal structures of human GARFTase complexed with 5 and 7 were reported. In severe combined immunodeficient mice bearing SKOV3 tumors, 7 was efficacious. The selectivity of these compounds for PCFT and for FRα and β over the ubiquitously expressed reduced folate carrier is a paradigm for selective tumor targeting.

The human proton-coupled folate transporter (hPCFT) is expressed in solid tumours and is active at pHs characterizing the tumour microenvironment. Recent attention focused on exploiting hPCFT for targeting solid tumours with novel cytotoxic anti-folates. hPCFT has 12 transmembrane domains (TMDs) and forms homo-oligomers with functional significance. The hPCFT primary sequence includes GXXXG motifs in TMD2 (G93XXXG97) and TMD4 (G155XXXG159). To investigate roles of these motifs in hPCFT function, stability and surface expression, we mutated glycine to leucine to generate single or multiple substitution mutants. Only the G93L and G159L mutants preserved substantial [3H]methotrexate (Mtx) transport when expressed in hPCFT-null (R1-11) HeLa cells. Transport activity of the glycine-to-leucine mutants correlated with surface hPCFT by surface biotinylation and confocal microscopy with ECFP*-tagged hPCFTs, suggesting a role for GXXXG in hPCFT stability and intracellular trafficking. When co-expressed in R1-11 cells, haemagglutinin-tagged glycine-to-leucine mutants and His10-tagged wild-type (WT) hPCFT co-associated on nickel affinity columns, suggesting that the GXXXG motifs are not directly involved in hPCFT oligomerization. This was substantiated by in situ FRET experiments with co-expressed ECFP*- and YFP-tagged hPCFT. Molecular modelling of dimeric hPCFT structures showed juxtaposed TMDs 2, 3, 4 and 6 as potential structural interfaces between monomers. hPCFT cysteine insertion mutants in TMD3 (Q136C and L137C) and TMD6 (W213C, L214C, L224C, A227C, F228C, F230C and G231C) were expressed in R1-11 cells and cross-linked with 1,6-hexanediyl bis methanethiosulfonate, confirming TMD juxtapositions. Altogether, our results imply that TMDs 3 and 6 provide critical interfaces for formation of hPCFT oligomers, which might be facilitated by the GXXXG motifs in TMD2 and TMD4. * cl , : cysteine-less; ECFP*, : enhanced cyan fluorescent protein with K26R/N164H mutation; HA, : haemagglutinin; hPCFT, : human PCFT; MFS, : major facilitator superfamily; MTS-1-MTS, : 1,1-methanediyl bis methanethiosulfonate; MTS-6-MTS, : 1,6-hexanediyl bis methanethiosulfonate; Mtx, : methotrexate; PCFT, : proton-coupled folate transporter; Pmx, : pemetrexed; RFC, : reduced folate carrier; sulfo-NHS-SS-biotin, : sulfo- N -hydroxysuccinimide-SS-biotin; TMD, : transmembrane domain; WT, : wild-type; YPet, : FRET-optimized yellow fluorescent protein

The human reduced folate carrier (hRFC) facilitates membrane transport of folates and antifolates. hRFC is characterized by 12 transmembrane domains (TMDs). To identify residues or domains involved in folate binding, we used substituted cysteine (Cys) accessibility methods (SCAM) with sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES). We previously showed that residues in TMD11 of hRFC were involved in substrate binding, whereas those in TMD12 were not (Hou, Z., Stapels, S. E., Haska, C. L., and Matherly, L. H. (2005) J. Biol. Chem. 280, 36206-36213). In this study, 232 Cys-substituted mutants spanning TMDs 1-10 and conserved stretches within the TMD6-7 (residues 204-217) and TMD10-11 connecting loop domains were transiently expressed in hRFC-null HeLa cells. All Cys-substituted mutants showed moderate to high levels of expression on Western blots, and only nine mutants including R133C, I134C, A135C, Y136C, S138C, G163C, Y281C, R373C, and S313C were inactive for methotrexate transport. MTSES did not inhibit transport by any of the mutants in TMDs 1, 3, 6, and 9 or for positions 204-217. Whereas most of the mutants in TMDs 2, 4, 5, 7, 8, and 10, and in the TMD10-11 connecting loop were insensitive to MTSES, this reagent inhibited methotrexate transport (25-75%) by 26 mutants in these TMDs. For 13 of these (Y126C, S137C, V160C, S168C, W274C, S278C, V284C, V288C, A311C, T314C, Y376C, Q377C, and V380C), inhibition was prevented by leucovorin, another hRFC substrate. Combined with our previous findings, these results implicate amino acids in TMDs 4, 5, 7, 8, 10, and 11, but not in TMDs 1, 2, 3, 6, 9, or 12, as important structural or functional components of the putative hydrophilic cavity for binding of anionic folate substrates.

ZntA from Escherichia coli is a member of the P1B-type ATPase family that confers resistance specifically to Pb2+, Zn2+, and Cd2 salts by active efflux across the cytoplasmic membrane. P1B-type ATPases are important for homeostasis of metal ions such as Cu+, Ag+, Pb2+, Zn2+, Cd2+ Cu2+, and Co2+, with different subgroups showing specificity for different metal ions. Sequence alignments of P1B-type ATPases show that ZntA and close homologues have a strictly conserved Asp714 in the eighth transmembrane domain that is not conserved in other subgroups of P1B-type ATPases. However, in the sarcoplasmic reticulum Ca2+-ATPase, a structurally characterized P-type ATPase, the residue corresponding to Asp714 is a metal-binding residue. Four site-specific mutants at Asp714, D714E, D714H, D714A, and D714P, were characterized. A comparison of their metal-binding affinity with that of wtZntA revealed that Asp714 is a ligand for the metal ion in the transmembrane site. Thus, Asp714 is one of the residues that determine metal ion specificity in ZntA homologues. All four substitutions at Asp714 in ZntA resulted in complete loss of in vivo resistance activity and complete or large reductions in ATPase activity, though D714E and D714H retained the ability to bind metal ions with high affinity at the transmembrane site. Thus, the ability to bind metal ions with high affinity did not correlate with high activity. The metal-binding affinity of the N-terminal site remained unchanged in all four mutants. The affinities of the two metal-binding sites in wtZntA determined in this study are similar to values reported previously for the individual sites in isolated ZntA fragments.

The majority of folate uptake into tissues and tumors involves facilitated carriers, the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). PCFT is highly expressed in a wide range of human tumor cell lines and primary tumor specimens from a variety of lineages, with modest expression in most normal tissues. PCFT transports optimally at pHs characterizing the tumor microenvironment. We discovered novel 6-substituted pyrrolo[2,3-d]pyrimidine folate analogs with substantial selectivity for PCFT over RFC, resulting in potent antitumor efficacy in vitro and in vivo. However, in some cases PCFT levels varied substantially. Studies were performed to characterize the basis for these dramatic differences in PCFT gene expression between HepG2 (elevated) and HT1080 (negligible) tumor cells. Low levels of PCFT gene expression in HT1080 cells were not likely due to methylation of CpG islands in the PCFT promoter since 5-aza-2’-deoxycytidine treatment did not restore PCFT gene expression. A PCFT promoter reporter construct -2005/+96 in pGL3 (transcription starts at +1) was generated and showed high levels of activity in HeLa cells by luciferase reporter assays. For HepG2 and HT1080 cells, wide variations in PCFT transcript levels by real-time RT-PCR were paralleled by reporter gene activities with the -2005/+96 construct, suggesting that these were primarily due to PCFT transcriptional regulation. Progressive deletion analysis of the -2005/+96 construct and reporter assays in HepG2 and HT1080 cells localized the minimal promoter region to between -50 and +96. Additional deletion constructs (-35/+96, -20/+96, -15/+96 and -10/+96) were generated and studied. Whereas the -35/+96, -20/+96, -15/+96 promoter constructs retained ~50% activity for HepG2 cells and ~10-15% activity for HT1080 cells relative to the -50/+96 construct, in both cases the -10/+96 construct was essentially inactive. Two critical cis elements were identified in the PCFT minimal promoter by deletion analysis located between positions -50 and -35, and -15 and -10. Bioinformatics analysis localized putative cis elements for KLF-15 (-50 to -35) and NRF1 (-15 to -10) binding. The functional importance of these elements was confirmed by mutations of core consensus sequences in the -50/+96 construct and reporter gene assays. Binding of NRF1 and KLF15 to the endogenous PCFT promoter in HepG2 and HT1080 cells was confirmed by ChIP assays which paralleled patterns of differential PCFT gene expression. Collectively, our results suggest that NRF1 and KLF15 contribute to dramatic variations in PCFT gene expression between tumor cells. Better understanding the key determinants of PCFT transcriptional control may lead to strategies for modulating PCFT levels in combination with PCFT-targeted 6-pyrrolo[2,3-d]pyrimidine folate analogs for treating solid tumors. Citation Format: Zhanjun Hou, Carrie O'Connor, Steve Orr, Larry H. Matherly. Identification of transcriptional controls responsible for differential gene expression of the proton-coupled folate transporter in human solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5491. doi:10.1158/1538-7445.AM2017-5491

The reduced folate carrier (RFC) is ubiquitously expressed in tissues and tumors and is the major tissue transporter for folate cofactors. Folate receptor (FR) α and β, as well as the proton-coupled folate transporter (PCFT), exhibit narrower patterns of tissue expression and are likely to serve more specialized physiologic roles. FRs are expressed in a subset of cancer cells (e.g., ovarian and non-small cell lung cancer for FRα, acute myelogenous leukemia for FRβ), whereas PCFT is expressed in a wide range of human solid tumors but not leukemias. Earlier generations of glycinamide ribonucleotide formyltransferase (GARFTase) and de novo purine nucleotide biosynthesis (e.g., Lometrexol) have shown promise as antitumor drugs. However, these compounds are excellent substrates for RFC and thus are non-selective for tumors, leading to dose-limiting toxicities. We previously reported a novel class of 6-substituted thieno[2,3-d] pyrimidines with a phenyl side chain and 3- and 4-carbon bridge lengths (AGF17 and AGF23, respectively) which selectively targeted FRs but not RFC or PCFT. To increase antitumor efficacy, we synthesized fluorinated analogs of AGF17 and AGF23, designated as AGF309 and AGF304, respectively. AGF309 and AGF304 potently inhibited proliferation of Chinese hamster ovary (CHO) cell lines engineered to individually express human FRα (IC50s of 1.08 and 2.27 nM, respectively) or FRβ (IC50s 0.48 and 1.14 nM, respectively). AGF304 and AGF309 showed nominal activity toward RFC- and PCFT-expressing CHO cells. Both AGF309 and AGF304 were also potently inhibitory toward FRα-expressing human KB human tumor cells (IC50s of 7.19 and 5.27nM, respectively). By analogy with previous iterations of 6-substituted thieno [2,3-d] pyrimidine compounds, growth inhibitory activity by AGF309 and AGF304 likely involves inhibition of de novo purine biosynthesis at GARFTase and potentially a secondary intracellular target. Collectively, our in vitro findings of the potent and selective antitumor activity and FR selectivity suggest that further preclinical evaluation of AGF309 and AGF304 as potential antitumor agents is warranted. Citation Format: Nian Tong, Aleem Gangjee, Adrianne Wallace Povrik, Carrie O’Connor, Aamod Aamod Dekhneb, Zhanjun Hou, Larry H. Matherly. Targeted thieno[2,3-d]pyrimidines with fluorinated phenyl side chains as antitumor agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5123. doi:10.1158/1538-7445.AM2017-5123

Three specialized systems exist for membrane transport of folates and antifolates across biological membranes. The reduced folate carrier (RFC) is ubiquitously expressed in tumors and normal tissues. Folate receptors (FR) α and β, and the proton-coupled folate receptor (PCFT) exhibit narrow patterns of tissue expression and serve specialized physiologic roles. FRs are expressed in a number of cancer cells (e.g., FRα in ovarian cancer and non-small cell lung cancer; FRβ in acute myelogenous leukemia), whereas PCFT is expressed in a large number of human solid tumors but not leukemias. FRs are either inaccessible to the circulation (FRα) or are nonfunctional (FRβ) in normal tissues. PCFT transport is limited in most normal tissues, given the requirement of acidic pH for optimal activity. These properties facilitate tumor targeting of cytotoxic compounds with specificities for PCFT or/and FR transport. Clinically used antifolates such as methotrexate and pemetrexed are substrates for RFC and their uptake by both normal tissues and tumors confers dose-limiting toxicity due to limited tumor selectivity. We previously reported a 5-substituted pyrrolo[2,3-d]pyrimidine antifolate with a 4-carbon bridge and a phenyl glutamate side chain (AGF127). Previous studies of related 6-substituted pyrrolo[2,3-d]pyrimidines established that the nature and length of the bridge region plays an important role in determining tumor cell potency and transport selectivity. Thus, it was of interest to vary the length and insert heteroatoms into the side chain -(CH2)4- of AGF127, including -(CH2)n-S- (compound 1, n= 3 and compound 2, n = 4) and -(CH2)n-O- (compound 3, n= 3 and compound 4, n = 4), to determine the impact on drug potency and transport selectivity. The novel analogs were tested as growth inhibitors against engineered Chinese hamster ovary (CHO) cells expressing human FRα (RT16), RFC (PC43-10), and PCFT (R2/PCFT4). AGF127 showed potent inhibition of CHO cells expressing FRα (IC50 = 8.6 nM), and reduced activity toward CHO cells expressing RFC (57 nM) or PCFT (840 nM). Compounds 1, 2, and 3, showed reduced inhibitions toward FRα-expressing RT16 cells (4-35-fold) compared to AGF127; however, compound 4 was equipotent to AGF127. Like AGF127, none of the series were effective PCFT substrates. Compound 3, like AGF127, inhibited RFC-expressing PC43-10 cells (IC50 = 37 nM); RFC activity was reduced 6- to >20-fold for compounds 1, 2, and 4. Toward FRα-, RFC-, and PCFT-expressing KB human tumor cells, the order of potency was 4 = AGF127 > 2 = 3 = 1. Compound 4 was also more active (15-fold) than pemetrexed with KB cells. Collectively, the potent and selective activity of compound 4 toward FRα-expressing tumor cells and limited activity toward RFC-expressing cells offers significant advantages over AGF127 and clinically used non-targeted antifolates, suggesting that further preclinical evaluation is warranted. Citation Format: Aleem Gangjee, Rishabh Mohan, Manasa Ravindra, Adrianne Wallace-Povirk, Carrie O’Connor, Aamod Dekhne, Zhanjun Hou, Larry H. Matherly. Selective tumor targeting with 5-substituted pyrrolo[2,3-d]pyrimidines with heteroatom bridge substitution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3239. doi:10.1158/1538-7445.AM2017-3239

The RFC (reduced folate carrier) is the principal mechanism by which folates and clinically used antifolates are delivered to mammalian cells. hRFC (human RFC) is subject to complex transcriptional controls and exists as homo-oligomer. To explore the post-transcriptional regulation of hRFC by exogenous folates, hRFC-null HeLa cells were stably transfected with hRFC under control of a constitutive promoter. hRFC transcripts and the total membrane protein increased with increasing LCV [(6R,S)5-formyl tetrahydrofolate (leucovorin)] with a maximum at 20 nM LCV, attributable to reduced turnover of hRFC transcripts. hRFC homo-oligomerization was unaffected by increasing LCV. Cell surface hRFC paralleled [3H]methotrexate transport and increased from 0.5 to 2 nM LCV, and then decreased (~2-fold) with increasing LCV up to 20 nM. hRFC was localized to the cell surface at low LCV concentrations (0.5–1.5 nM). However, at higher LCV concentrations, significant intracellular hRFC was localized to the ER (endoplasmic reticulum), such that at 20 nM LCV, intracellular hRFC was predominated. Our results demonstrate a novel post-transcriptional regulation of hRFC involving: (i) increased hRFC transcripts and proteins, accompanying increased extracellular folates, attributable to differences in hRFC transcript stabilities; and (ii) increased retention of hRFC in the ER under conditions of folate excess, because of impaired intracellular trafficking and plasma membrane targeting., A novel regulation of the physiologically/pharmacologically important human reduced folate carrier was demonstrated in response to increasing extracellular folates, involving: (i) increased transcripts and total protein, reflecting increased transcript stabilities; and (ii) increased endoplasmic reticulum trapping, due to impaired intracellular trafficking.

Wheat yellow mosaic virus (WYMV) isolate HC was used for viral cDNA synthesis and sequencing. The results show that the viral RNAl is 7 629 nucleotides encoding a polyprotein with 2 407 amino acids, from which seven putative proteins may be produced by an autolytic cleavage processing besides the viral coat protein. The RNA2 is 3 639 nucleotides and codes for a polyprotein of 903 amino acids, which may contain two putative non-structural proteins. Although WYMV shares a similarity in genetic organization to wheat spindle streak mosaic virus (WSSMV), the identities in their nucleotide sequences or deduced amino acid sequences are as low as 70 % and 75 % respectively. Based on this result, it is confirmed that WYMV and WSSMV are different species withinBymmirus.

This review summarizes the biology of the major facilitative membrane transporters, the reduced folate carrier (RFC) (Solute Carrier 19A1) and the proton-coupled folate transporter (PCFT) (Solute Carrier 46A1). Folates are essential vitamins, and folate deficiency contributes to a variety of health disorders. RFC is ubiquitously expressed and is the major folate transporter in mammalian cells and tissues. PCFT mediates the intestinal absorption of dietary folates and appears to be important for transport of folates into the central nervous system. Clinically relevant antifolates for cancer, such as methotrexate and pralatrexate, are transported by RFC, and loss of RFC transport is an important mechanism of methotrexate resistance in cancer cell lines and in patients. PCFT is expressed in human tumors, and is active at pH conditions associated with the tumor microenvironment. Pemetrexed is an excellent substrate for both RFC and PCFT. Novel tumor-targeted antifolates related to pemetrexed with selective membrane transport by PCFT over RFC are being developed. In recent years, there have been major advances in understanding the structural and functional properties and the regulation of RFC and PCFT. The molecular bases for methotrexate resistance associated with loss of RFC transport and for hereditary folate malabsorption, attributable to mutant PCFT, were determined. Future studies should continue to translate molecular insights from basic studies of RFC and PCFT biology into new therapeutic strategies for cancer and other diseases.

This chapter focuses on the biology of the major facilitative membrane folate transporters, the reduced folate carrier (RFC), and the proton-coupled folate transporter (PCFT). Folates are essential vitamins, and folate deficiency contributes to a variety of heath disorders. RFC is ubiquitously expressed and is the major folate transporter in mammalian cells and tissues. PCFT mediates intestinal absorption of dietary folates. Clinically relevant antifolates such as methotrexate (MTX) are transported by RFC, and the loss of RFC transport is an important mechanism of MTX resistance. PCFT is abundantly expressed in human tumors and is active under pH conditions associated with the tumor microenvironment. Pemetrexed (PMX) is an excellent substrate for PCFT as well as for RFC. Novel tumor-targeted antifolates related to PMX with selective membrane transport by PCFT over RFC are being developed. The molecular picture of RFC and PCFT continues to evolve relating to membrane topology, N-glycosylation, energetics, and identification of structurally and functionally important domains and amino acids. The molecular bases for MTX resistance associated with loss of RFC function, and for the rare autosomal recessive condition, hereditary folate malabsorption (HFM), attributable to mutant PCFT, have been established. From structural homologies to the bacterial transporters GlpT and LacY, homology models were developed for RFC and PCFT, enabling new mechanistic insights and experimentally testable hypotheses. RFC and PCFT exist as homo-oligomers, and evidence suggests that homo-oligomerization of RFC and PCFT monomeric proteins may be important for intracellular trafficking and/or transport function. Better understanding of the structure and function of RFC and PCFT should facilitate the rational development of new therapeutic strategies for cancer as well as for HFM.

Pemetrexed and methotrexate are antifolates used for cancer chemotherapy and inflammatory diseases. These agents have toxic side effects resulting, in part, from nonspecific cellular transport by the reduced folate carrier (RFC), a ubiquitously expressed facilitative transporter. We previously described 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with modifications of the side chain linker and aromatic ring that are poor substrates for RFC but are efficiently transported via folate receptors (FRs) and the proton-coupled folate transporter (PCFT). These targeted antifolates are cytotoxic in vitro toward FR- and PCFT-expressing tumor cells and in vivo with human tumor xenografts in immune-compromised mice, reflecting selective cellular uptake. Antitumor efficacy is due to inhibition of glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) activity in de novo synthesis of purine nucleotides. This study used purified human GARFTase (formyltransferase domain) to assess in vitro inhibition by eight novel thieno- and pyrrolo[2,3-d]pyrimidine antifolates. Seven analogues (AGF23, AGF71, AGF94, AGF117, AGF118, AGF145, and AGF147) inhibited GARFTase with Ki values in the low- to mid-nanomolar concentration range, whereas AGF50 inhibited GARFTase with micromolar potency similar to that of PMX. On the basis of crystal structures of ternary complexes with GARFTase, β-GAR, and the monoglutamyl antifolates, differences in inhibitory potencies correlated well with antifolate binding and the positions of the terminal carboxylates. Our data provide a mechanistic basis for differences in inhibitory potencies between these novel antifolates and a framework for future structure-based drug design. These analogues could be more efficacious than clinically used antifolates, reflecting their selective cellular uptake by FRs and PCFT and potent GARFTase inhibition.

The proton coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum, while folate receptor (FR) á delivers folate substrates into the cell via endocytosis. FRá and PCFT are highly expressed in ovarian cancer. PCFT transport is favored by acidic pH, including pHs associated with the acidic tumor microenvironment. We previously synthesized a novel class of 6-substituted pyrrolo[2,3-d]pyrimidine thiophene antifolates with 4- and 3-carbon bridge lengths, designated AGF117 and AGF150, targeted to FRá and PCFT, but not the ubiquitously expressed reduced folate carrier (RFC). To increase antitumor specificity and activity, reflecting multi-polar interactions with cellular targets, we synthesized fluorinated analogs of AGF117 and AGF150, designated AGF278 and AGF283. In the presence of a physiologic concentration of reduced folate, AGF278 and AGF283 potently inhibited proliferation of Chinese hamster ovary (CHO) cells engineered to express PCFT, but not RFC or FRá (R2/PCFT4) (IC50s of 6 and 1.5 nM, respectively), or FRá without PCFT or RFC (RT16) (IC50s of 4.8 and 5.4 nM). AGF278 and AGF283 also inhibited growth of IGROV1 (IC50s of 1.5 and 11 nM, respectively) and SKOV3 (IC50s of 37.8 and 30.2 nM, respectively) ovarian cancer cells. We measured [3H]methotrexate uptake in R2/PCFT4 CHO cells in the presence of AGF278 and AGF283; AGF278 and AGF283 potently inhibited PCFT transport with increased binding over AGF117 and AGF150, respectively, as reflected in Ki values. Analogous to AGF117 and AGF150, glycinamide ribonucleotide (GAR) formyltransferase (GARFTase), the first folate-dependent enzyme in de novo purine biosynthesis, was implicated as the cellular target for AGF278 and AGF283 as adenosine (10 ìM) and 5-aminoimidazole-4-carboxamide (320 ìM) protected IGROV1 cells from growth inhibition. GARFTase inhibition by AGF278 and AGF283 was confirmed by an in situ GARFTase assay which measures incorporation of [14C]glycine into formyl GAR, the GARFTase product, with potencies similar to those of the parent molecules. Preliminary in vivo studies with AGF278 using SKOV3 tumor xenografts in SCID mice established antitumor efficacy. Our results suggest that novel fluorinated 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates may offer significant promise for treating ovarian cancer, reflecting their selective cellular uptake by PCFT and FRá and potent inhibition of de novo purine biosynthesis. Additional utility of our fluorine-substituted analogs lies in their potential for positron emission tomography imaging of PCFT-expressing tumors. Citation Format: Mike R. Wilson, Manasa P. Ravindraa, Adrianne Wallace-Povirk, Lisa Polin, Zhanjun Hou, Aleem Gangjee, Larry H. Matherly. Targeting ovarian cancer with novel 6-substituted pyrrolo [2,3-d] pyrimidine fluorinated antifolates via cellular uptake by folate receptor á and the proton-coupled folate transporter. [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 4791.

Drugs usually bind to different targets in different conformations. As antitumor agents, antifolates first need to be selectively transported by tumor cells via the reduced folate carrier (RFC), the proton-coupled folate transporter (PCFT) or folate receptors (FRs). The second step is the binding of antifolates with the intracellular targets. We are systematically developing novel folate analogs with selective membrane transport for FRs and PCFT over the ubiquitously expressed RFC. These 6-substituted pyrrolo[2,3-d]pyrimidine antifolates are principally inhibitors of de novo purine biosynthesis at the steps catalyzed by glycinamide ribonucleotide formyl transferase (GARFTase) and 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase). We previously demonstrated that classical antifolates bind to FRα and GARFTase in different conformations. An amide in the bridge region of classical antifolates has two interchangeable lowest energy conformers, pseudo-cis and pseudo-trans. We reported that a novel antifolate (AGF238) with an amide in the bridge region binds to FRα in a pseudo-trans conformation and binds to GARFTase in a pseudo-cis conformation. As part of our continued efforts to pursue targeted antifolates as antitumor agents, we designed an expand series of amide-bridged classical antifolates with different energy barriers between the pseudo-cis and pseudo-trans conformers. Among these, AGF266 was exclusively transported by Chinese hamster ovary (CHO) cells engineered to express human FRα (IC50 = 0.42 nM) or FRβ (IC50 = 0.88 nM) over CHO expressing human RFC (IC50 > 1000 nM). AGF266 was also found to exhibit highly potent antitumor activity (IC50 = 0.79 nM in KB human tumor cells). AGF266 inhibited GARFTase as its principal cellular target, based on patterns of protection from growth inhibition by adenosine and AICA. Our in vitro findings of antitumor activity associated with FR selectivity suggest that further preclinical evaluation of AGF266 as a potential antitumor agent is warranted. Citation Format: Aleem Gangjee, Weiguo Xiang, Aamod Dehkne, Mike R. Wilson, Adrianne Wallace-Povrik, Carrie O’Connor, Zhanjun Hou, Larry H. Matherly. Pseudo-cis and pseudo-trans amide conformations lead to potent targeted antifolates that are selectively transported by FR over RFC. [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 1365.

Folates are important single carbon transferring cofactors required for the biosynthesis of purine and pyrimidine nitrogen bases and DNA. Antifolates inhibit synthesis of these nitrogenous bases and function as antineopastic agents. The use of currently marketed antifolates such as methotrexate (MTX and pemetrexed (PMX) is limited by dose-limiting toxicity due to non-selective transport by the ubiquitously expressed reduced folate carrier (RFC). We previously reported a series of 6-substituted pyrrolo[2,3-d]pyrimidine classical antifolates that are selectively transported by folate receptors (FR) and/or by the proton-coupled folate transporter (PCFT) over RFC and inhibit FR/PCFT expressing tumor cells (KB and IGROV1) at sub-nanomolar IC50 values. These antifolates are comparatively more potent and circumvent the important limitation of currently marketed clinical antifolates that lack selective uptake into tumors. As an extension of the SAR, we now report on the role of fluorine atom(s) in improving selectivity as well potency. Fluorine was incorporated into our 6-substituted pyrrolo[2,3-d]pyrimidine antifolates to improve pharmacodynamics by improving binding affinity to target proteins via a number of well documented electrostatic and hydrophobic interactions of fluorine. In addition, fluorines were strategically placed on the side chain of our targeted antifolates for improved potency and selectivity through induced conformational restriction due to a plausible intramolecular fluorine-hydrogen bonding interaction. The fluorinated analogs had IC50s in the subnanomolar range (IC50 = 0.33 to 0.29 nM) for KB tumor cells and nanomolar (IC50 = 2.77 to 3.47 nM) for IGROV-1 cells. In addition, these analogs showed 600- to 1000-fold selectivity for FRα expressing cells over RFC. Protection studies suggest that glycinamide ribonucleotide formyl transferase (GARFTase) is the intracellular targets. In vivo activity was established toward the SKOV3 tumor xenograft model. These preclinical studies suggest that these fluorinated analogs are candidates for possible clinical trial as antitumor agents. Citation Format: Aleem Gangjee, Manasa P. Ravindra, Mike R. Wilson, Adrianne W. Povrik, Lisa P. Hou, Carrie O’Connor, Zhanjun Hou, Larry H. Matherly. Design, synthesis and evaluation of fluorinated antifolates for improved selectivity and potency against tumor cells. [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 1359.

Folates function as cofactors in one-carbon transfer reactions including de novo biosynthesis of nucleotides and play a key in DNA and RNA synthesis, epigenetic processes, cellular proliferation and survival. Cellular folate uptake is mediated by three distinct transporters. Among these, the reduced folate carrier (RFC) is the predominant folate transporter and is critical for antitumor efficacy of currently used classical antifolates such as methotrexate (MTX), pemetrexed (PMX), and raltitrexed (RTX). RFC is ubiquitously expressed in tumor cells and in normal tissues, resulting in dose-limiting toxicity with classical antifolates. We previously reported a series of 6-substituted pyrrolo[2,3-d]pyrimidine antifolates that are selectively transported by folate receptors (FR) and/or by the proton-coupled folate transporter (PCFT) over RFC and inhibit FR/PCFT-expressing human tumor cells (KB and IGROV1) at sub-nanomolar IC50 values. These antifolates are comparatively more potent and reduce the toxicity of clinically available antifolates which have non-selective uptake. As an extension of the SAR, we focused on a novel isosteric series of 6-substituted thieno[2,3-d]pyrimidine analogs, with a 3- or 4- carbon bridge between the thieno[2,3-d]pyrimidine and the thienoyl regioisomeric side chain. This simple isosteric replacement of pyrrole with thiophene in the scaffold, combined with varying the positions of attachment of 3- and 4-carbon bridge and the L-glutamic acid on the thiophene ring, provides compounds with different chain length, conformations and extra hydrogen bond donors and/or acceptors compared to the previously reported compounds. All the analogs (AGF 102, AGF131, AGF 271, AGF 275, AGF 276 and AGF 282) are potently inhibitory toward Chinese hamster ovary (CHO) cells (RT16) expressing human FRá (IC50s of 3.1, 0.33, 0.45, 1.1, 0.83 and 5.6 nM, respectively). All the analogs were inactive in the CHO cell line expressing RFC indicating absolute selectivity for FRá. AGF271, AGF275, and AGF282 showed modest (IC50s from 95-460 nM) with PCFT-expressing CHO cells (R2/PCFT4). AGF102, AGF 131 and AGF 271 AGF 275 AGF 276 AGF 282 were all potently inhibitory toward KB tumor cells (IC50 of 2.10, 1.70, 1.48, 1.81, 5.74 and 7.61nM, respectively). Collectively, our results suggest that AGF102, AGF 131, AGF 271, AGF 275, AGF 276 and AGF 282 are targeted antifolates with activity profiles that suggest that further preclinical studies and optimization are warranted. Citation Format: Aleem Gangjee, Nian Tong, Adrianne Wallace Povrik, Zhanjun Hou, Larry H. Matherly. Targeted antifolates with thienoyl regioisomers in the side chain result in improved selectivity and potency against KB human tumor cells. [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 4836.

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]pyrimidine antifolates that derive their cytotoxic effects by targeting de novo purine nucleotide biosynthesis.

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.

The proton-coupled folate transporter (PCFT; SLC46A1) is a proton-folate symporter that is abundantly expressed in solid tumors and normal tissues, such as duodenum. The acidic pH optimum for PCFT is relevant to intestinal absorption of folates and could afford a means of selectively targeting tumors with novel cytotoxic antifolates. PCFT is a member of the major facilitator superfamily of transporters. Because major facilitator superfamily members exist as homo-oligomers, we tested this for PCFT because such structures could be significant to PCFT mechanism and regulation. By transiently expressing PCFT in reduced folate carrier- and PCFT-null HeLa (R1-11) cells and chemical cross-linking with 1,1-methanediyl bismethanethiosulfonate and Western blotting, PCFT species with molecular masses approximating those of the PCFT dimer and higher order oligomers were detected. Blue native polyacrylamide gel electrophoresis identified PCFT dimer, trimer, and tetramer forms. PCFT monomers with hemagglutinin and His(10) epitope tags were co-expressed in R1-11 cells, solubilized, and bound to nickel affinity columns, establishing their physical associations. Co-expressing YPet and ECFP*-tagged PCFT monomers enabled transport and fluorescence resonance energy transfer in plasma membranes of R1-11 cells. Combined wild-type (WT) and inactive mutant P425R PCFTs were targeted to the cell surface by surface biotinylation/Western blots and confocal microscopy and functionally exhibited a "dominant-positive" phenotype, implying positive cooperativity between PCFT monomers and functional rescue of mutant by WT PCFT. Our results demonstrate the existence of PCFT homo-oligomers and imply their functional and regulatory impact. Better understanding of these higher order PCFT structures may lead to therapeutic applications related to folate uptake in hereditary folate malabsorption, and delivery of PCFT-targeted chemotherapy drugs for cancer.

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 the proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including KB and IGROV1 human tumor cells, 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.

The proton-coupled folate transporter (PCFT) is a folate-proton symporter with an acidic pH optimum, approximating the microenvironments of solid tumors. We tested 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with one to six carbons in the bridge region for inhibition of proliferation in isogenic Chinese hamster ovary (CHO) and HeLa cells expressing PCFT or reduced folate carrier (RFC). Only analogs with three and four bridge carbons (N-{4-[3-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)propyl]benzoyl}-l-glutamic acid (compound 2) and N-{4-[4-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)butyl]benzoyl}*-l-glutamic acid (compound 3), respectively) were inhibitory, with 2 ≫ 3. Activity toward RFC-expressing cells was negligible. Compound 2 and pemetrexed (Pmx) competed with [3H]methotrexate for PCFT transport in PCFT-expressing CHO (R2/hPCFT4) cells from pH 5.5 to 7.2; inhibition increased with decreasing pH. In Xenopus laevis oocytes microinjected with PCFT cRNA, uptake of 2, like that of Pmx, was electrogenic. Cytotoxicity of 2 toward R2/hPCFT4 cells was abolished in the presence of adenosine or 5-amino-4-imidazolecarboxamide, suggesting that glycinamide ribonucleotide formyltransferase (GARFTase) in de novo purine biosynthesis was the primary target. Compound 2 decreased GTP and ATP pools by ∼50 and 75%, respectively. By an in situ GARFTase assay, 2 was ∼20-fold more inhibitory toward intracellular GARFTase than toward cell growth or colony formation. Compound 2 irreversibly inhibited clonogenicity, although this required at least 4 h of exposure. Our results document the potent antiproliferative activity of compound 2, attributable to its efficient cellular uptake by PCFT, resulting in inhibition of GARFTase and de novo purine biosynthesis. Furthermore, they establish the feasibility of selective chemotherapy drug delivery via PCFT over RFC, a process that takes advantage of a unique biological feature of solid tumors.

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.

A series of seven 2-amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines with bridge length variations (from 2 to 8 carbon atoms) were synthesized as selective folate receptor (FR) alpha and beta 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 chloroformamidine 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 (IC(50) 4.7-334 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.

The ubiquitously expressed reduced folate carrier (RFC) is the major transport system for folate cofactors in mammalian cells and tissues. Previous considerations of RFC structure and mechanism were based on the notion that RFC monomers were sufficient to mediate transport of folate and antifolate substrates. The present study examines the possibility that human RFC (hRFC) exists as higher order homo-oligomers. By chemical cross-linking, transiently expressed hRFC in hRFC-null HeLa (R5) cells with the homobifunctional cross-linker 1,3-propanediyl bis-methanethiosulfonate and Western blotting, hRFC species with molecular masses of hRFC homo-oligomers were identified. Hemagglutinin- and Myc epitope-tagged hRFC proteins expressed in R5 cells were co-immunoprecipitated from both membrane particulate and surface-enriched membrane fractions, indicating that oligomeric hRFC is expressed at the cell surface. By co-expression of wild type and inactive mutant S138C hRFCs, combined with surface biotinylation and confocal microscopy, a dominant-negative phenotype was demonstrated involving greatly decreased cell surface expression of both mutant and wild type carrier caused by impaired intracellular trafficking. For another hRFC mutant (R373A), expression of oligomeric wild type-mutant hRFC was accompanied by a significant and disproportionate loss of wild type activity unrelated to the level of surface carrier. Collectively, our results demonstrate the existence of hRFC homo-oligomers. They also establish the likely importance of these higher order hRFC structures to intracellular trafficking and carrier function.

Folates are essential for life and folate deficiency contributes to a host of health problems including cardiovascular disease, fetal abnormalities, neurological disorders, and cancer. Antifolates, represented by methotrexate, continue to occupy a unique niche among the modern day pharmacopoeia for cancer along with other pathological conditions. This article focuses on the biology of the membrane transport system termed the "reduced folate carrier" or RFC with a particular emphasis on RFC structure and function. The ubiquitously expressed RFC is the major transporter for folates in mammalian cells and tissues. Loss of RFC expression or function portends potentially profound physiological or developmental consequences. For chemotherapeutic antifolates used for cancer, loss of RFC expression or synthesis of mutant RFC protein with impaired function results in antifolate resistance due to incomplete inhibition of cellular enzyme targets and low levels of substrate for polyglutamate synthesis. The functional properties for RFC were first documented nearly 40 years ago in murine leukemia cells. Since 1994, when RFC was first cloned, tremendous advances in the molecular biology of RFC and biochemical approaches for studying the structure of polytopic membrane proteins have led to an increasingly detailed picture of the molecular structure of the carrier, including its membrane topology, its N-glycosylation, identification of functionally and structurally important domains and amino acids, and helix packing associations. Although no crystal structure for RFC is yet available, biochemical and molecular studies, combined with homology modeling, based on homologous bacterial major facilitator superfamily transporters such as LacY, now permit the development of experimentally testable hypotheses designed to establish RFC structure and mechanism.

This paper focuses on the application of reinforcement learning to obstacle avoidance in dynamic environments. Behavior-based control architecture is more robust and better in real-time performance than conventional model based architecture in the control of mobile robot. An intelligent controller is proposed by integrating reinforcement learning with the behavior-based control architecture and applied to the obstacle avoidance. Neural network is used to approximate the Q-function to store the Q-value. By using the reinforcement learning, the mobile robot can learn to select proper behavior online without knowing the exact model of the system. In experiments, dynamic and static obstacles are placed in the environments separately. Experiment results show that the mobile robot can get to the target point without colliding with any obstacle after a period of learning.

Folates are essential for life and folate deficiency contributes to a host of health problems including cardiovascular disease, fetal abnormalities, neurological disorders, and cancer. Antifolates, represented by methotrexate, continue to occupy a unique niche among the modern day pharmacopoeia for cancer along with other pathological conditions. This article focuses on the biology of the membrane transport system termed the "reduced folate carrier" or RFC with a particular emphasis on RFC structure and function. The ubiquitously expressed RFC is the major transporter for folates in mammalian cells and tissues. Loss of RFC expression or function portends potentially profound physiological or developmental consequences. For chemotherapeutic antifolates used for cancer, loss of RFC expression or synthesis of mutant RFC protein with impaired function results in antifolate resistance due to incomplete inhibition of cellular enzyme targets and low levels of substrate for polyglutamate synthesis. The functional properties for RFC were first documented nearly 40 years ago in murine leukemia cells. Since 1994, when RFC was first cloned, tremendous advances in the molecular biology of RFC and biochemical approaches for studying the structure of polytopic membrane proteins have led to an increasingly detailed picture of the molecular structure of the carrier, including its membrane topology, its N-glycosylation, identification of functionally and structurally important domains and amino acids, and helix packing associations. Although no crystal structure for RFC is yet available, biochemical and molecular studies, combined with homology modeling, based on homologous bacterial major facilitator superfamily transporters such as LacY, now permit the development of experimentally testable hypotheses designed to establish RFC structure and mechanism.

This paper focuses on the learning action selection in behavior-based autonomous mobile robot. Autonomous mobile robot needs a large space to store the state-action pair in the application of tabular Q-learning. Neural network has a good ability of generalization, so in this paper Q-learning based on neural network is developed which has a good ability to approximate to Q-function. The Q-learning based on neural network is applied to autonomous mobile robot for goal directed obstacle avoidance. Results of simulation show that the mobile robot can learn to select proper actions itself to accomplish the task autonomously.

This review attempts to provide a comprehensive overview of the biology of the physiologically and pharmacologically important transport system termed the “reduced folate carrier” (RFC). The ubiquitously expressed RFC has unequivocally established itself as the major transport system in mammalian cells and tissues for a group of compounds including folate cofactors and classical antifolate therapeutics. Loss of RFC expression or function may have potentially profound pathophysiologic consequences including cancer. For chemotherapeutic antifolates used for cancer such as methotrexate or pemetrexed, synthesis of mutant RFCs or loss of RFC transcripts and proteins results in antifolate resistance due to incomplete inhibition of cellular enzyme targets and insufficient substrate for polyglutamate synthesis. Since RFC was first cloned in 1994, tremendous advances have been made in understanding the complex transcriptional and posttranscriptional regulation of RFC, in identifying structurally and functionally important domains and amino acids in the RFC molecule as a prelude to establishing the mechanism of transport, and in characterizing the molecular defects in RFC associated with loss of transport in antifolate resistant cell line models. Many of the insights gained from laboratory models of RFC portend opportunities for modulating carrier expression in drug resistant tumors, and for designing a new generation of agents with improved transport by RFC or substantially enhanced transport by other folate transporters over RFC. Many of the advances in the basic biology of RFC in cell line models are now being directly applied to human cancers in the clinical setting, most notably pediatric acute lymphoblastic leukemia and osteogenic sarcoma.

Major folate uptake mechanisms include the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT), and folate receptors α and β. Antifolates such as methotrexate or pemetrexed are excellent substrates for human RFC (hRFC) and variously for human PCFT (hPCFT) and human folate receptor α (hFRα). Recent attention has focused on developing tumor-targeted folate-based therapies, reflecting tumor-selective uptake by hFRα and/or hPCFT. Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers AGF94 (2′,5′) and AGF154 (2′,4′) were synthesized and characterized as potent inhibitors of cell proliferation of engineered Chinese hamster ovary (CHO) cells expressing hPCFT or hFRα, but not for CHO cells expressing hRFC. In 41 ovarian cancer specimens from patients, hFRα, hPCFT and hRFC were all expressed. FRα transcript levels increased with stage of disease, although hPCFT levels were constant. IGROV1 is a clinically relevant ovarian cancer cell line that expresses both hFRα and hPCFT. IGROV1 cells were highly sensitive to both AGF94 and AGF154, as reflected in inhibition of cell proliferation in vitro. Growth inhibition by AGF94 or AGF154 in IGROV1 cells was partially reversed by excess folic acid, likely reflecting drug uptake by hFRα, as well as by hPCFT. To evaluate the impact of loss of hFRα on AGF94 and AGF154 in vitro inhibition of IGROV1 cell proliferation, FRα was knocked down (>95%) by lentivirus shRNAs. Clonal isolates were generated (n = 17) and profiled by RT-PCR for expression of the folate transporters and key enzymes likely to impact antifolate sensitivities (e.g., folylpolyglutamyl synthetase, β-glycinamide ribonucleotide formyltransferase), relative to a non-targeted control (NTC) cells. Gene expression profiles were similar for IGROV1 hFRα knockdown (KD) clones and NTC cells. Two representative hFRα KD clones were assayed for inhibition of cell proliferation by AGF94 and AGF154, with IC50s for IGROV1 KD clones of 26±1.8 and 26±9 nM for AGF94, and 65±1.2 and 81±2.4 nM for AGF154. For comparison, IC50s in the IGROV1 NTC cells expressing both hFRα and hPCFT were 0.6±0.1 nM for AGF94 and 2.2±0.8 nM for AGF154. In contrast, the hFRα-specific antifolate, AGF102 showed IC50s of > 1000 nM and ∼2.5 nM for KD and NTC cells. The preservation of substantial inhibitory activity for AGF94 and AGF154, accompanying the nearly complete loss of FRα expression in the KD clones is likely due to the sustained uptake of these novel antifolates by hPCFT. For AGF154, this was confirmed by direct uptake assays with [3H]-AGF154. Our results suggest that 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with substantial substrate activities for hFRα and hPCFT may offer significant advantages in treating tumors that express both these transport systems. Citation Format: Zhanjun Hou, Steve Orr, Christina George, Adrianne Wallace, Larry H. Matherly, Lei Wang, Si Yang, Aleem Gandjee. Impact of loss of folate receptor alpha on antitumor effects of novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with substrate activities for both folate receptors and the proton-coupled folate transporter. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4483. doi:10.1158/1538-7445.AM2015-4483

Recent interest has focused on folate receptor (FR) α in delivering folate-based therapeutics to solid tumors, as FRα is expressed in human tumors, notably ovarian and non-small cell lung cancers. While FRα is present in normal epithelial tissues, this occurs at apical membranes where it is inaccessible to the circulation. Selectivity of FR-targeted agents would be enhanced if these were poor substrates for the ubiquitous reduced folate carrier (RFC). We previously described 6-substituted thieno[2,3-d]pyrimidine benzoyl antifolates with varying bridge lengths (n = 2-8 carbons) as potent and selective inhibitors of tumor cells that express FRs. The most potent compound (AGF50) included a 4-carbon bridge and showed FRα selectivity and inhibition of de novo purine biosynthesis. To investigate the role of the side-chain aromatic ring in transport and pathway inhibition, we synthesized AGF50 analogs with a variety of aryl side-chain modifications (AGF102, AGF103, AGF55, AGF65, and AGF132). The various 6-substituted thienoyl[2,3-d]pyrimidine antifolates were tested in proliferation assays with isogenic Chinese hamster ovary (CHO) sublines expressing RFC, proton-coupled folate transporter (PCFT), or FRα, and with KB tumor cells. The analogs were inert toward RFC- and PCFT-expressing cells. Toward FRα-expressing CHO and KB cells, inhibitory potencies were in the order, AGF102 = AGF132 = AGF103 > AGF50 > AGF64 >> AGF55, with IC50s ranging from ∼ 1 nM to 182 nM. Drug effects were abolished with excess folic acid, confirming FR uptake. Inhibition of cell proliferation was reflected in binding affinities to FRα, measured by competition with [3H]folic acid. While PCFT-targeted activity was nominal, all drugs still bound to PCFT, as reflected in inhibition of [3H]methotrexate uptake at pH 5.5 and 6.8. By protection experiments with thymidine or adenosine, de novo purine biosynthesis was confirmed as the principal targeted pathway for the active analogs. While protection by 5-aminoimidazole-4-carboxamide (AICA) was complete with AGF64 (suggesting glycinamide ribonucleotide formyltransferase (GARFTase) as the primary target), for AGF50, AGF102, AGF103 and AGF132, protection was incomplete, implying a dual targeting of GARFTase and AICA ribonucleotide formyltransferase (AICARFTase). Our studies establish an important structure-activity relationship for FRα-targeted antifolates, involving modifications of the side-chain aromatic group that are well tolerated for FRα binding and transport. 6-Substituted thienoyl[2,3-d]pyrimidine analogs were all inhibitors of de novo purine biosynthesis, although relative targeting of GARFTase versus AICARFTase varied with the nature of the aromatic side-chain. Citation Format: Larry H. Matherly, Xin Zhang, Adrianne Wallace, Zhanjun Hou, Christina George, Xilin Zhou, Aleem Gangjee. Tumor-targeting with novel 6-substituted thienoyl[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α, and inhibition of de novo purine nucleotide biosynthesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4481. doi:10.1158/1538-7445.AM2015-4481

Background: Approximately 40% of malignant pleural mesothelioma (MPM) patients respond to the combination treatment with the antifolate pemetrexed (PMX) and platinum compounds. Several studies highlighted the predictive role of thymidylate synthase (TS), but additional resistance mechanisms are poorly understood. This study aimed to evaluate the role of the reduced folate carrier (RFC/SLC19A1) and proton-coupled folate transporter (PCFT/SLC46A1) as potential biological markers of intrinsic PMX-resistance in MPM. Materials and methods: PCFT promoter methylation was evaluated by combined bisulfite restriction analysis as well as MethyLight PCR in 14 cell lines and 10 MPM tissues, while PCFT and RFC mRNA levels were studied by Real-Time-PCR in MPM specimens from a test and a validation cohort of 73 and 51 PMX-treated patients, respectively. PCFT protein expression was evaluated by immunohistochemistry in a tissue microarray with representative MPM core biopsies from 35 patients. Data were analyzed by t-test, Fisher/log-rank test and Cox proportional hazards models. Results: PCFT expression inversely correlated with promoter methylation and treatment with 5-Aza-2′-deoxycytidine (5-Aza-dC) induced its marked restoration in MPM cells. Patients with low PCFT mRNA levels had significantly shorter overall survival (OS), in both the test (median OS, 11.3 vs. 17.6 months, P = 0.01) and in the validation cohort (OS, 12.6 vs. 30.3 months, P = 0.02). Multivariate analysis, including age, sex, EORTC prognostic score and histology, confirmed PCFT prognostic relevance (HR, 2.1 and 2.4 in the test and validation cohorts, respectively). Low PCFT protein expression was also associated with significantly shorter OS (11.8 vs. 30.3 months, P = 0.02). Remarkably, the subgroup of patients with both low PCFT and high TS mRNA levels (30% of the patients in the test cohort) had the worse prognosis (median OS, 5.5 months, P Conclusions: These results define PCFT as a novel prognostic factor for MPM patients treated with PMX. Prospective trials for the validation of the prognostic/predictive role of PCFT in MPM patients treated with PMX-based regimens are warranted. Furthermore preclinical data suggest that exposure to 5-Aza-dC might restore PCFT expression and result in efficient eradication of antifolate-resistant cells. Citation Format: Elisa Giovannetti, Paolo A. Zucali, Yehuda G. Assaraf, Niccola Funel, Maria Gemelli, Michal Stark, Leticia G. Leon, Zhanjun Hou, Matteo Perrino, Larry H. Matherly, Godefridus J. Peters. Role of proton-coupled folate transporter expression in resistance of mesothelioma patients treated with pemetrexed. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4335. doi:10.1158/1538-7445.AM2015-4335

Pemetrexed (PMX) is a 5-substituted pyrrolo[2,3-d]pyrimidine antifolate used for first-line and maintenance therapy of NSCLC. Whereas correlations were reported between clinical responses in NSCLC and levels of folate receptor (FR) α, FRα is not a pharmacologically relevant PMX transporter. Rather, PMX is primarily transported by the reduced folate carrier (RFC) and PCFT. RFC is ubiquitously expressed, whereas PCFT shows more limited expression in normal tissues, although both are broadly expressed in tumor cells. Unlike RFC, PCFT is active at acidic pH, with significant levels at pH 6.8. Using patient cDNAs from normal lung (n = 8) and NSCLC samples (n = 37), we measured transcripts for RFC and PCFT, and for intracellular targets for PMX, including thymidylate synthase (TS) and glycinamide ribonucleotide formyltransferase (GARFTase), by real-time RT-PCR (qPCR). PCFT was detected at similar levels in normal lung and NSCLC specimens, while RFC was decreased in tumors compared to normal lung. Expression of TS and GARFTase were increased in NSCLC samples, compared to normal tissue. In NSCLC cell lines A549, CRL5872, H460, H1650 and H2030, PCFT and RFC, but not FRα, were detected by qPCR. We synthesized novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers related to PMX [AGF94 (2′,5′) and AGF154 (2′,4′)] as potential antitumor agents. Based on patterns of inhibition of proliferation of engineered Chinese hamster ovary cells expressing human RFC or PCFT, AGF94 and AGF154, unlike PMX, are not transported by RFC but are excellent substrates for PCFT. AGF94 and AGF154 showed potencies comparable to PMX toward all the NSCLC cell lines. We found that [3H]PMX and [3H]AGF154 were transported by PCFT into the NSCLC cell lines at pH 5.5 and pH 6.8 and uptake could be blocked by non-radioactive AGF94. AGF154 accumulation over sustained exposure at pH 5.5 was greater than that of PMX, whereas accumulations of AGF154 and PMX were nearly identical at pH 6.8. In H460 cells treated with PMX, thymidine (10 μM) partially protected cells from growth inhibition, whereas neither adenosine (10 μM) nor 5-aminoimidazole-4-carboxamide (AICA) (320 μM) were protective. In cells treated with AGF94 or AGF154, adenosine and AICA, but not thymidine, were completely protective, consistent with inhibition of de novo purine biosynthesis at GARFTase. Potent GARFTase inhibition by AGF94 and AGF154 was confirmed by an in situ assay which measures incorporation of [14C]glycine into formyl GAR (FGAR), the GARFTase product. Our results suggest that novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates may offer significant promise for treating NSCLC, reflecting their selective membrane transport by PCFT over RFC and potent inhibition of de novo purine biosynthesis. Citation Format: Mike R. Wilson, Zhanjun Hou, Lei Wang, Si Wang, Manohar Ratnam, Aleem Gangjee, Larry Matherly. Targeted therapy of non-small cell lung cancer (NSCLC) by novel 6-pyrrolo[2,3-d]pyrimidine thienoyl antifolates with selective transport by the proton-coupled folate transporter (PCFT). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5403. doi:10.1158/1538-7445.AM2015-5403

The human reduced folate carrier (hRFC) mediates the membrane transport of reduced folates and classical anti-folates into mammalian cells. RFC is characterized by 12 transmembrane domains (TMDs), internally oriented N and C termini, and a large central linker connecting TMDs 1-6 and 7-12. By co-expression and N-hydroxysuccinimide methotrexate (Mtx) radioaffinity labeling of hRFC TMD 1-6 and TMD 7-12 half-molecules, combined with endoproteinase GluC digestion, a substrate binding domain was previously localized to within TMDs 8-12 (Witt, T. L., Stapels, S. E., and Matherly, L. H. (2004) J. Biol. Chem. 279, 46755-46763). In this report, this region was further refined to TMDs 11-12 by digestion with 2-nitro-5-thiocyanatobenzoic acid. A transportcompetent cysteine-less hRFC was used as a template to prepare single cysteine-replacement mutant constructs in which each residue from Glu-394 to Asp-420 of TMD 11 and Tyr-435 to His-457 of TMD 12 was replaced individually by a cysteine. The mutant constructs were transfected into hRFC-null HeLa cells. Most of the 50 single cysteine-substituted constructs were expressed at high levels on Western blots. With the exception of G401C hRFC, all mutants were active for Mtx transport. Treatment with sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) had no effect on hRFC activity for all of the cysteine mutants within TMD 12 and for the majority of the cysteine mutants within TMD 11. However, MTSES inhibited Mtx uptake by the T404C, A407C, T408C, T412C, F416C, I417C, V418C, and S419C mutants by 25-65%. Losses of activity by MTSES treatment for T404C, A407C, T412C, and I417C hRFCs were appreciably reversed in the presence of excess leucovorin, a hRFC substrate. Our results strongly suggest that residues within TMD 11 are likely critical structural and/or functional components of the putative hRFC transmembrane channel for anionic folate and anti-folate substrates.