BRCA status dictates Wnt responsiveness in epithelial ovarian cancer.

The association of BRCA1 and BRCA2 mutations with increased risk for developing epithelial ovarian cancer is well established. However, the observed clinical differences, particularly the improved therapy response and patient survival in BRCA2 mutant (BRCA2mt) patients are unexplained. Our objective is to identify molecular pathways that are differentially regulated upon the loss of BRCA1 and BRCA2 function in ovarian cancer. Transcriptomic and pathway analysis comparing BRCA1 mutant (BRCA1mt), BRCA2mt and homologous recombination wild-type (HRwt) ovarian tumors showed differential regulation of the Wnt/ß-catenin pathway. Using Wnt3A-treated BRCA1/2 wild-type (BRCAwt), BRCA1null and BRCA2null mouse ovarian cancer cells, we observed preferential activation of the canonical Wnt/ß-catenin signaling in BRCAwt ovarian cancer cells while the non-canonical Wnt/ß-catenin signaling was preferentially activated in the BRCA1null cells. Interestingly, BRCA2null mouse ovarian cancer cells, demonstrated a unique response to Wnt3A with the preferential upregulation of the Wnt signaling inhibitor, Axin2. In addition, decreased phosphorylation and enhanced stability of ß-catenin were observed in BRCA2null mouse ovarian cancer cells, which correlated with increased inhibitory phosphorylation on GSK3ß. These findings open venues for the translation of these molecular observations into modalities that can impact patient survival.

Role of mitochondrial and cytosolic folylpolyglutamate synthetase in one-carbon metabolism and antitumor efficacy of mitochondrial-targeted antifolates.

Folate-dependent one-carbon (C1) metabolism encompasses distinct cytosolic and mitochondrial pathways connected by an interchange between serine, glycine and formate. In both the cytosol and mitochondria, folates exist as polyglutamates with polyglutamylation catalyzed by folylpolyglutamate synthetase (FPGS), including cytosolic and mitochondrial isoforms. Serine is metabolized by serine hydroxymethyltransferase (SHMT) 2 in the mitochondria and generates glycine and C1 units for cellular biosynthesis in the cytosol. AGF347 is a novel pyrrolo[3,2-d]pyrimidine antifolate that targets SHMT2 in the mitochondria, and SHMT1 and de novo purine biosynthesis in the cytosol. FPGS is expressed in primary pancreatic cancer specimens and FPGS levels correlate with in vitro efficacies of AGF347 toward human pancreatic cancer cells. MIA PaCa-2 pancreatic cancer cells with CRISPR knockout of FPGS were engineered to express doxycycline-inducible FPGS exclusively in the cytosol (cFPGS) or in both the cytosol and mitochondria (mFPGS). Folate and AGF347 accumulations increased in both the cytosol and mitochondria with increased mFPGS but were restricted to the cytosol with cFPGS. AGF347-Glu5 inhibited SHMT2 ~19-fold greater than AGF347 By metabolomics analysis, mFPGS stimulated the C1 flux from serine in the mitochondria and de novo purine and dTTP synthesis far greater than cFPGS. mFPGS enhanced in vitro inhibition of MIA PaCa-2 cell proliferation by AGF347 (~30-fold) more than cFPGS (~4.9-fold). Similar results were seen with other pyrrolo[3,2-d]pyrimidine antifolates (AGF291, AGF320); however, elevated mFPGS adversely impacted inhibition by the non-classical SHMT2/SHMT1 inhibitor, SHIN1. These results suggest a critical role of mFPGS levels in determining anti-tumor efficacies of mitochondrial-targeted pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer. Significance Statement AGF347 is a novel pyrrolo[3,2-d]pyrimidine antifolate that targets serine hydroxymethyltransferase (SHMT) 2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. AGF347 accumulation increases with folylpolyglutamate synthetase (FPGS) levels in both the cytosol and mitochondria. Increased mitochondrial FPGS stimulated one-carbon metabolic fluxes in the cytosol and mitochondria and substantially enhanced in vitro inhibition of pancreatic cancer cells by AGF347 Mitochondrial FPGS levels play important roles in determining the anti-tumor efficacies of pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer.

Mitochondrial and Cytosolic One-Carbon Metabolism Is a Targetable Metabolic Vulnerability in Cisplatin-Resistant Ovarian Cancer.

One-carbon (C1) metabolism is compartmentalized between the cytosol and mitochondria with the mitochondrial C1 pathway as the major source of glycine and C1 units for cellular biosynthesis. Expression of mitochondrial C1 genes including SLC25A32, serine hydroxymethyl transferase (SHMT) 2, 5,10-methylene tetrahydrofolate dehydrogenase 2, and 5,10-methylene tetrahydrofolate dehydrogenase 1-like was significantly elevated in primary epithelial ovarian cancer (EOC) specimens compared with normal ovaries. 5-Substituted pyrrolo[3,2-d]pyrimidine antifolates (AGF347, AGF359, AGF362) inhibited proliferation of cisplatin-sensitive (A2780, CaOV3, IGROV1) and cisplatin-resistant (A2780-E80, SKOV3) EOC cells. In SKOV3 and A2780-E80 cells, colony formation was inhibited. AGF347 induced apoptosis in SKOV3 cells. In IGROV1 cells, AGF347 was transported by folate receptor (FR) α. AGF347 was also transported into IGROV1 and SKOV3 cells by the proton-coupled folate transporter (SLC46A1) and the reduced folate carrier (SLC19A1). AGF347 accumulated to high levels in the cytosol and mitochondria of SKOV3 cells. By targeted metabolomics with [2,3,3-2H]L-serine, AGF347, AGF359, and AGF362 inhibited SHMT2 in the mitochondria. In the cytosol, SHMT1 and de novo purine biosynthesis (i.e., glycinamide ribonucleotide formyltransferase, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase) were targeted; AGF359 also inhibited thymidylate synthase. Antifolate treatments of SKOV3 cells depleted cellular glycine, mitochondrial NADH and glutathione, and showed synergistic in vitro inhibition toward SKOV3 and A2780-E80 cells when combined with cisplatin. In vivo studies with subcutaneous SKOV3 EOC xenografts in SCID mice confirmed significant antitumor efficacy of AGF347. Collectively, our studies demonstrate a unique metabolic vulnerability in EOC involving mitochondrial and cytosolic C1 metabolism, which offers a promising new platform for therapy.

Structural Characterization of 5-Substituted Pyrrolo[3,2-d]pyrimidine Antifolate Inhibitors in Complex with Human Serine Hydroxymethyl Transferase 2.

We previously discovered first-in-class multitargeted 5-substituted pyrrolo[3,2-d]pyrimidine antifolates that inhibit serine hydroxymethyltransferase 2 (SHMT2), resulting in potent in vitro and in vivo antitumor efficacies. In this report, we present crystallographic structures for SHMT2 in complex with an expanded series of pyrrolo[3,2-d]pyrimidine compounds with variations in bridge length (3-5 carbons) and the side chain aromatic ring (phenyl, thiophene, fluorine-substituted phenyl, and thiophene). We evaluated structural features of the inhibitor-SHMT2 complexes and correlations to inhibitor potencies (i.e., Kis), highlighting conserved polar contacts and identifying 5-carbon bridge lengths as key determinants of inhibitor potency. Based on the analysis of SHMT2 structural data, we investigated the impact of mutation of Tyr105 in SHMT2 kinetic analysis and studies with HCT116 cells with inducible expression of wild-type and Y105F SHMT2. Increased enzyme inhibition potency by the pyrrolo[3,2-d]pyrimidine inhibitors with Phe105 SHMT2 accompanied an increased growth inhibition of Phe105-expressing HCT116 cells compared to wild-type SHMT2. Pyrrolo[3,2-d]pyrimidine inhibitors with polyglutamate modifications were evaluated for potencies against SHMT2. We determined the crystal structures of SHMT2 in complex with our lead antifolate AGF347 lacking L-glutamate, or as a diglutamate and triglutamate, for comparison with parent AGF347. These data provide the first insights into the influence of antifolate polyglutamylation on SHMT2:inhibitor interactions. Collectively, our results provide new insights into the critical structural determinants of SHMT2 binding by pyrrolo[3,2-d]pyrimidine inhibitors as novel antitumor agents, as well as the first structural characterization of human SHMT2 in complex with polyglutamates of an SHMT2-targeted antifolate.

Enhancing anti-AML activity of venetoclax by isoflavone ME-344 through suppression of OXPHOS and/or purine biosynthesis in vitro.

Venetoclax (VEN), in combination with low dose cytarabine (AraC) or a hypomethylating agent, is FDA approved to treat acute myeloid leukemia (AML) in patients who are over the age of 75 or cannot tolerate standard chemotherapy. Despite high response rates to these therapies, most patients succumb to the disease due to relapse and/or drug resistance, providing an unmet clinical need for novel therapies to improve AML patient survival. ME-344 is a potent isoflavone with demonstrated inhibitory activity toward oxidative phosphorylation (OXPHOS) and clinical activity in solid tumors. Given that OXPHOS inhibition enhances VEN antileukemic activity against AML, we hypothesized that ME-344 could enhance the anti-AML activity of VEN. Here we report that ME-344 enhanced VEN to target AML cell lines and primary patient samples while sparing normal hematopoietic cells. Cooperative suppression of OXPHOS was detected in a subset of AML cell lines and primary patient samples. Metabolomics analysis revealed a significant reduction of purine biosynthesis metabolites by ME-344. Further, lometrexol, a purine biosynthesis inhibitor, synergistically enhanced VEN-induced apoptosis in AML cell lines. Interestingly, AML cells with acquired AraC resistance showed significantly increased purine biosynthesis metabolites and sensitivities to ME-344. Furthermore, synergy between ME-344 and VEN was preserved in these AraC-resistant AML cells. In vivo studies revealed significantly prolonged survival upon combination therapy of ME-344 and VEN in NSGS mice bearing parental or AraC-resistant MV4-11 leukemia compared to the vehicle control. This study demonstrates that ME-344 enhances VEN antileukemic activity against preclinical models of AML by suppressing OXPHOS and/or purine biosynthesis.

Discovery of Tumor-Targeted 6-Methyl Substituted Pemetrexed and Related Antifolates with Selective Loss of RFC Transport.

Pemetrexed and related 5-substituted pyrrolo[2,3-d]pyrimidine antifolates are substrates for the ubiquitously expressed reduced folate carrier (RFC), and the proton-coupled folate transporter (PCFT) and folate receptors (FRs) which are more tumor-selective. A long-standing goal has been to discover tumor-targeted therapeutics that draw from one-carbon metabolic vulnerabilities of cancer cells and are selective for transport by FRs and PCFT over RFC. We discovered that a methyl group at the 6-position of the pyrrole ring in the bicyclic scaffold of 5-substituted 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidine antifolates 1-4 (including pemetrexed) abolished transport by RFC with modest impacts on FRs or PCFT. From molecular modeling, loss of RFC transport involves steric repulsion in the scaffold binding site due to the 6-methyl moiety. 6-Methyl substitution preserved antiproliferative activities toward human tumor cells (KB, IGROV3) with selectivity over IOSE 7576 normal ovary cells and inhibition of de novo purine biosynthesis. Thus, adding a 6-methyl moiety to 5-substituted pyrrolo[2,3-d]pyrimidine antifolates affords tumor transport selectivity while preserving antitumor efficacy.

Structure-Based Design of Transport-Specific Multitargeted One-Carbon Metabolism Inhibitors in Cytosol and Mitochondria.

Multitargeted agents provide tumor selectivity with reduced drug resistance and dose-limiting toxicities. We previously described the multitargeted 6-substituted pyrrolo[3,2-d]pyrimidine antifolate 1 with activity against early- and late-stage pancreatic tumors with limited tumor selectivity. Structure-based design with our human serine hydroxymethyl transferase (SHMT) 2 and glycinamide ribonucleotide formyltransferase (GARFTase) structures, and published X-ray crystal structures of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC), SHMT1, and folate receptor (FR) α and ß afforded 11 analogues. Multitargeted inhibition and selective tumor transport were designed by providing promiscuous conformational flexibility in the molecules. Metabolite rescue identified mitochondrial C1 metabolism along with de novo purine biosynthesis as the targeted pathways. We identified analogues with tumor-selective transport via FRs and increased SHMT2, SHMT1, and GARFTase inhibition (28-, 21-, and 11-fold, respectively) compared to 1. These multitargeted agents represent an exciting new structural motif for targeted cancer therapy with substantial advantages of selectivity and potency over clinically used antifolates.

Enhancing anti-AML activity of venetoclax by isoflavone ME-344 through suppression of OXPHOS and/or purine biosynthesis.

Venetoclax (VEN), in combination with low dose cytarabine (AraC) or a hypomethylating agent, is FDA approved to treat acute myeloid leukemia (AML) in patients who are over the age of 75 or cannot tolerate standard chemotherapy. Despite high response rates to these combination therapies, most patients succumb to the disease due to relapse and/or drug resistance, providing an unmet clinical need for novel therapies to improve AML patient survival. ME-344 is a potent isoflavone with demonstrated inhibitory activity toward oxidative phosphorylation (OXPHOS) and clinical activity in solid tumors. Given that OXPHOS inhibition enhances VEN antileukemic activity against AML, we hypothesized that ME-344 could enhance the anti-AML activity of VEN. Here we report that ME-344 synergized with VEN to target AML cell lines and primary patient samples while sparing normal hematopoietic cells. Cooperative suppression of OXPHOS was detected in a subset of AML cell lines and primary patient samples. Metabolomics analysis revealed a significant reduction of purine biosynthesis metabolites by ME-344. Further, lometrexol, an inhibitor of purine biosynthesis, synergistically enhanced VEN-induced apoptosis in AML cell lines. Interestingly, AML cells with acquired resistance to AraC showed significantly increased purine biosynthesis metabolites and sensitivities to ME-344. Furthermore, synergy between ME-344 and VEN was preserved in these AraC-resistant AML cells. These results translated into significantly prolonged survival upon combination of ME-344 and VEN in NSGS mice bearing parental or AraC-resistant MV4-11 leukemia. This study demonstrates that ME-344 enhances VEN antileukemic activity against preclinical models of AML by suppressing OXPHOS and/or purine biosynthesis.

Multitargeted 6-Substituted Thieno[2,3-d]pyrimidines as Folate Receptor-Selective Anticancer Agents that Inhibit Cytosolic and Mitochondrial One-Carbon Metabolism.

Multitargeted agents with tumor selectivity result in reduced drug resistance and dose-limiting toxicities. We report 6-substituted thieno[2,3-d]pyrimidine compounds (3-9) with pyridine (3, 4), fluorine-substituted pyridine (5), phenyl (6, 7), and thiophene side chains (8, 9), for comparison with unsubstituted phenyl (1, 2) and thiophene side chain (10, 11) containing thieno[2,3-d]pyrimidine compounds. Compounds 3-9 inhibited proliferation of Chinese hamster ovary cells (CHO) expressing folate receptors (FRs) α or ß but not the reduced folate carrier (RFC); modest inhibition of CHO cells expressing the proton-coupled folate transporter (PCFT) by 4, 5, 6, and 9 was observed. Replacement of the side-chain 1',4'-phenyl ring with 2',5'-pyridyl, or 2',5'-pyridyl with a fluorine insertion ortho to l-glutamate resulted in increased potency toward FR-expressing CHO cells. Toward KB tumor cells, 4-9 were highly active (IC50's from 2.11 to 7.19 nM). By metabolite rescue in KB cells and in vitro enzyme assays, de novo purine biosynthesis was identified as a targeted pathway (at 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase)). Compound 9 was 17- to 882-fold more potent than previously reported compounds 2, 10, and 11 against GARFTase. By targeted metabolomics and metabolite rescue, 1, 2, and 6 also inhibited mitochondrial serine hydroxymethyl transferase 2 (SHMT2); enzyme assays confirmed inhibition of SHMT2. X-ray crystallographic structures were obtained for 4, 5, 9, and 10 with human GARFTase. This series affords an exciting new structural platform for potent multitargeted antitumor agents with FR transport selectivity.

A Beta Strain-Based Spike Glycoprotein Vaccine Candidate Induces Broad Neutralization and Protection against SARS-CoV-2 Variants of Concern.

The coronavirus disease 2019 (COVID-19) pandemic is still ongoing. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) are circulating worldwide, making it resistant to existing vaccines and antiviral drugs. Therefore, the evaluation of variant-based expanded spectrum vaccines to optimize the immune response and provide broad protectiveness is very important. In this study, we expressed spike trimer protein (S-TM) based on the Beta variant in a GMP-grade workshop using CHO cells. Mice were immunized twice with S-TM protein combined with aluminum hydroxide (Al) and CpG Oligonucleotides (CpG) adjuvant to evaluate its safety and efficacy. BALB/c immunized with S-TM + Al + CpG induced high neutralizing antibody titers against the Wuhan-Hu-1 strain (wild-type, WT), the Beta and Delta variants, and even the Omicron variant. In addition, compared with the S-TM + Al group, the S-TM + Al + CpG group effectively induced a stronger Th1-biased cell immune response in mice. Furthermore, after the second immunization, H11-K18 hACE2 mice were well protected from challenge with the SARS-CoV-2 Beta strain, with a 100% survival rate. The virus load and pathological lesions in the lungs were significantly reduced, and no virus was detected in mouse brain tissue. Our vaccine candidate is practical and effective for current SARS-CoV-2 VOCs, which will support its further clinical development for potential sequential immune and primary immunization. IMPORTANCE Continuous emergence of adaptive mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to challenge the use and development of existing vaccines and drugs. The value of variant-based vaccines that are capable of inducing a higher and broader protection immune response against SARS-CoV-2 variants is currently being evaluated. This article shows that a recombinant prefusion spike protein based on a Beta variant was highly immunogenic and could induced a stronger Th1-biased cell immune response in mice and was effectively protective against challenge with the SARS-CoV-2 Beta variant. Importantly, this Beta-based SARS-CoV-2 vaccine could also offer a robust humoral immune response with effectively broad neutralization ability against the wild type and different variants of concern (VOCs): the Beta, Delta, and Omicron BA.1 variants. To date, the vaccine described here has been produced in a pilot scale (200L), and the development, filling process, and toxicological safety evaluation have also been completed, which provides a timely response to the emerging SARS-CoV-2 variants and vaccine development.

Biology and therapeutic applications of the proton-coupled folate transporter.

INTRODUCTION: The proton-coupled folate transporter (PCFT; SLC46A1) was discovered in 2006 as the principal mechanism by which folates are absorbed in the intestine and the causal basis for hereditary folate malabsorption (HFM). In 2011, it was found that PCFT is highly expressed in many tumors. This stimulated interest in using PCFT for cytotoxic drug targeting, taking advantage of the substantial levels of PCFT transport and acidic pH conditions commonly associated with tumors. AREAS COVERED: We summarize the literature from 2006 to 2022 that explores the role of PCFT in the intestinal absorption of dietary folates and its role in HFM and as a transporter of folates and antifolates such as pemetrexed (Alimta) in relation to cancer. We provide the rationale for the discovery of a new generation of targeted pyrrolo[2,3-d]pyrimidine antifolates with selective PCFT transport and inhibitory activity toward de novo purine biosynthesis in solid tumors. We summarize the benefits of this approach to cancer therapy and exciting new developments in the structural biology of PCFT and its potential to foster refinement of active structures of PCFT-targeted anti-cancer drugs. EXPERT OPINION: We summarize the promising future and potential challenges of implementing PCFT-targeted therapeutics for HFM and a variety of cancers.

Targeted therapy of pyrrolo[2,3-d]pyrimidine antifolates in a syngeneic mouse model of high grade serous ovarian cancer and the impact on the tumor microenvironment.

Novel therapies are urgently needed for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. In addition, therapies that target unique vulnerabilities in the tumor microenvironment (TME) of EOC have largely been unrealized. One strategy to achieve selective drug delivery for EOC therapy involves use of targeted antifolates via their uptake by folate receptor (FR) proteins, resulting in inhibition of essential one-carbon (C1) metabolic pathways. FRα is highly expressed in EOCs, along with the proton-coupled folate transporter (PCFT); FRß is expressed on activated macrophages, a major infiltrating immune population in EOC. Thus, there is great potential for targeting both the tumor and the TME with agents delivered via selective transport by FRs and PCFT. In this report, we investigated the therapeutic potential of a novel cytosolic C1 6-substituted pyrrolo[2,3-d]pyrimidine inhibitor AGF94, with selectivity for uptake by FRs and PCFT and inhibition of de novo purine nucleotide biosynthesis, against a syngeneic model of ovarian cancer (BR-Luc) which recapitulates high-grade serous ovarian cancer in patients. In vitro activity of AGF94 was extended in vivo against orthotopic BR-Luc tumors. With late-stage subcutaneous BR-Luc xenografts, AGF94 treatment resulted in substantial anti-tumor efficacy, accompanied by significantly decreased M2-like FRß-expressing macrophages and increased CD3+ T cells, whereas CD4+ and CD8+ T cells were unaffected. Our studies demonstrate potent anti-tumor efficacy of AGF94 in the therapy of EOC in the context of an intact immune system, and provide a framework for targeting the immunosuppressive TME as an essential component of therapy.

Pattern Analysis of Organellar Maps for Interpretation of Proteomic Data.

Localization of organelle proteins by isotope tagging (LOPIT) maps are a coordinate-directed representation of proteome data that can aid in biological interpretation. Analysis of organellar association for proteins as displayed using LOPIT is evaluated and interpreted for two types of proteomic data sets. First, test and control group protein abundances and fold change data obtained in a proximity labeling experiment are plotted on a LOPIT map to evaluate the likelihood of true protein interactions. Selection of true positives based on co-localization of proteins in the organellar space is shown to be consistent with carboxylase enrichment which serves as a positive control for biotinylation in streptavidin affinity selected proteome data sets. The mapping in organellar space facilitates discrimination between the test and control groups and aids in identification of proteins of interest. The same representation of proteins in organellar space is used in the analysis of extracellular vesicle proteomes for which protein abundance and fold change data are evaluated. Vesicular protein organellar localization patterns provide information about the subcellular origin of the proteins in the samples which are isolates from the extracellular milieu. The organellar localization patterns are indicative of the provenance of the vesicular proteome origin and allow discrimination between proteomes prepared using different enrichment methods. The patterns in LOPIT displays are easy to understand and compare which aids in the biological interpretation of proteome data.

The evolving biology of the proton-coupled folate transporter: New insights into regulation, structure, and mechanism.

The human proton-coupled folate transporter (PCFT; SLC46A1) or hPCFT was identified in 2006 as the principal folate transporter involved in the intestinal absorption of dietary folates. A rare autosomal recessive hereditary folate malabsorption syndrome is attributable to human SLC46A1 variants. The recognition that hPCFT was highly expressed in many tumors stimulated substantial interest in its potential for cytotoxic drug targeting, taking advantage of its high-level transport activity under acidic pH conditions that characterize many tumors and its modest expression in most normal tissues. To better understand the basis for variations in hPCFT levels between tissues including human tumors, studies have examined the transcriptional regulation of hPCFT including the roles of CpG hypermethylation and critical transcription factors and cis elements. Additional focus involved identifying key structural and functional determinants of hPCFT transport that, combined with homology models based on structural homologies to the bacterial transporters GlpT and LacY, have enabled new structural and mechanistic insights. Recently, cryo-electron microscopy structures of chicken PCFT in a substrate-free state and in complex with the antifolate pemetrexed were reported, providing further structural insights into determinants of (anti)folate recognition and the mechanism of pH-regulated (anti)folate transport by PCFT. Like many major facilitator proteins, hPCFT exists as a homo-oligomer, and evidence suggests that homo-oligomerization of hPCFT monomeric proteins may be important for its intracellular trafficking and/or transport function. Better understanding of the structure, function and regulation of hPCFT should facilitate the rational development of new therapeutic strategies for conditions associated with folate deficiency, as well as cancer.

Discovery of 6-substituted thieno[2,3-d]pyrimidine analogs as dual inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis in folate receptor expressing human tumors.

We discovered 6-substituted thieno[2,3-d]pyrimidine compounds (3-9) with 3-4 bridge carbons and side-chain thiophene or furan rings for dual targeting one-carbon (C1) metabolism in folate receptor- (FR) expressing cancers. Synthesis involved nine steps starting from the bromo-aryl carboxylate. From patterns of growth inhibition toward Chinese hamster ovary cells expressing FRα or FRß, the proton-coupled folate transporter or reduced folate carrier, specificity for uptake by FRs was confirmed. Anti-proliferative activities were demonstrated toward FRα-expressing KB tumor cells and NCI-IGROV1 ovarian cancer cells. Inhibition of de novo purine biosynthesis at both 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and glycinamide ribonucleotide formyltransferase (GARFTase) was confirmed by metabolite rescue, metabolomics and enzyme assays. X-ray crystallographic structures were obtained with compounds 3-5 and human GARFTase. Our studies identify first-in-class C1 inhibitors with selective uptake by FRs and dual inhibition of enzyme targets in de novo purine biosynthesis, resulting in anti-tumor activity. This series affords an exciting new platform for selective multi-targeted anti-tumor agents.

Folate transporter dynamics and therapy with classic and tumor-targeted antifolates.

There are three major folate uptake systems in human tissues and tumors, including the reduced folate carrier (RFC), folate receptors (FRs) and proton-coupled folate transporter (PCFT). We studied the functional interrelationships among these systems for the novel tumor-targeted antifolates AGF94 (transported by PCFT and FRs but not RFC) and AGF102 (selective for FRs) versus the classic antifolates pemetrexed, methotrexate and PT523 (variously transported by FRs, PCFT and RFC). We engineered HeLa cell models to express FRα or RFC under control of a tetracycline-inducible promoter with or without constitutive PCFT. We showed that cellular accumulations of extracellular folates were determined by the type and levels of the major folate transporters, with PCFT and RFC prevailing over FRα, depending on expression levels and pH. Based on patterns of cell proliferation in the presence of the inhibitors, we established transport redundancy for RFC and PCFT in pemetrexed uptake, and for PCFT and FRα in AGF94 uptake; uptake by PCFT predominated for pemetrexed and FRα for AGF94. For methotrexate and PT523, uptake by RFC predominated even in the presence of PCFT or FRα. For both classic (methotrexate, PT523) and FRα-targeted (AGF102) antifolates, anti-proliferative activities were antagonized by PCFT, likely due to its robust activity in mediating folate accumulation. Collectively, our findings describe a previously unrecognized interplay among the major folate transport systems that depends on transporter levels and extracellular pH, and that determines their contributions to the uptake and anti-tumor efficacies of targeted and untargeted antifolates.

Folate Transport and One-Carbon Metabolism in Targeted Therapies of Epithelial Ovarian Cancer.

New therapies are urgently needed for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. To identify new approaches for targeting EOC, metabolic vulnerabilities must be discovered and strategies for the selective delivery of therapeutic agents must be established. Folate receptor (FR) α and the proton-coupled folate transporter (PCFT) are expressed in the majority of EOCs. FRß is expressed on tumor-associated macrophages, a major infiltrating immune population in EOC. One-carbon (C1) metabolism is partitioned between the cytosol and mitochondria and is important for the synthesis of nucleotides, amino acids, glutathione, and other critical metabolites. Novel inhibitors are being developed with the potential for therapeutic targeting of tumors via FRs and the PCFT, as well as for inhibiting C1 metabolism. In this review, we summarize these exciting new developments in targeted therapies for both tumors and the tumor microenvironment in EOC.

Therapeutic Targeting of Mitochondrial One-Carbon Metabolism in Cancer.

One-carbon (1C) metabolism encompasses folate-mediated 1C transfer reactions and related processes, including nucleotide and amino acid biosynthesis, antioxidant regeneration, and epigenetic regulation. 1C pathways are compartmentalized in the cytosol, mitochondria, and nucleus. 1C metabolism in the cytosol has been an important therapeutic target for cancer since the inception of modern chemotherapy, and "antifolates" targeting cytosolic 1C pathways continue to be a mainstay of the chemotherapy armamentarium for cancer. Recent insights into the complexities of 1C metabolism in cancer cells, including the critical role of the mitochondrial 1C pathway as a source of 1C units, glycine, reducing equivalents, and ATP, have spurred the discovery of novel compounds that target these reactions, with particular focus on 5,10-methylene tetrahydrofolate dehydrogenase 2 and serine hydroxymethyltransferase 2. In this review, we discuss key aspects of 1C metabolism, with emphasis on the importance of mitochondrial 1C metabolism to metabolic homeostasis, its relationship with the oncogenic phenotype, and its therapeutic potential for cancer.