RESUMO
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.
RESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Fosforribosilaminoimidazolcarboxamida Formiltransferase/antagonistas & inibidores , Fosforribosilglicinamido Formiltransferase/antagonistas & inibidores , Pirimidinas/farmacologia , Tiofenos/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Células CHO , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetulus , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Receptores de Folato com Âncoras de GPI/metabolismo , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Fosforribosilaminoimidazolcarboxamida Formiltransferase/metabolismo , Fosforribosilglicinamido Formiltransferase/metabolismo , Ligação Proteica , Pirimidinas/síntese química , Pirimidinas/metabolismo , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/metabolismoRESUMO
Folate-dependent one-carbon (C1) metabolism is compartmentalized in the mitochondria and cytosol and is a source of critical metabolites for proliferating tumors. Mitochondrial C1 metabolism including serine hydroxymethyltransferase 2 (SHMT2) generates glycine for de novo purine nucleotide and glutathione biosynthesis and is an important source of NADPH, ATP, and formate, which affords C1 units as 10-formyl-tetrahydrofolate and 5,10-methylene-tetrahydrofolate for nucleotide biosynthesis in the cytosol. We previously discovered novel first-in-class multitargeted pyrrolo[3,2-d]pyrimidine inhibitors of SHMT2 and de novo purine biosynthesis at glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase with potent in vitro and in vivo antitumor efficacy toward pancreatic adenocarcinoma cells. In this report, we extend our findings to an expanded panel of pancreatic cancer models. We used our lead analog AGF347 [(4-(4-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)butyl)-2-fluorobenzoyl)-l-glutamic acid] to characterize pharmacodynamic determinants of antitumor efficacy for this series and demonstrated plasma membrane transport into the cytosol, uptake from cytosol into mitochondria, and metabolism to AGF347 polyglutamates in both cytosol and mitochondria. Antitumor effects of AGF347 downstream of SHMT2 and purine biosynthesis included suppression of mammalian target of rapamycin signaling, and glutathione depletion with increased levels of reactive oxygen species. Our results provide important insights into the cellular pharmacology of novel pyrrolo[3,2-d]pyrimidine inhibitors as antitumor compounds and establish AGF347 as a unique agent for potential clinical application for pancreatic cancer, as well as other malignancies. SIGNIFICANCE STATEMENT: This study establishes the antitumor efficacies of novel inhibitors of serine hydroxymethyltransferase 2 and of cytosolic targets toward a panel of clinically relevant pancreatic cancer cells and demonstrates the important roles of plasma membrane transport, mitochondrial accumulation, and metabolism to polyglutamates of the lead compound AGF347 to drug activity. We also establish that loss of serine catabolism and purine biosynthesis resulting from AGF347 treatment impacts mammalian target of rapamycin signaling, glutathione pools, and reactive oxygen species, contributing to antitumor efficacy.
Assuntos
Antineoplásicos/farmacologia , Citosol/efeitos dos fármacos , Glicina Hidroximetiltransferase/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Pirimidinas/farmacologia , Pirróis/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citosol/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas de Inativação de Genes , Glutationa/biossíntese , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Humanos , Leucovorina/análogos & derivados , Leucovorina/metabolismo , Mitocôndrias/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Nucleotídeos de Purina/biossíntese , Pirimidinas/química , Pirimidinas/uso terapêutico , Pirróis/química , Pirróis/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Serina/metabolismo , Tetra-Hidrofolatos/metabolismoRESUMO
Tumor-targeted 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl compounds based on 2 were isosterically modified at the 4-carbon bridge by replacing the vicinal (C11) carbon by heteroatoms N (4), O (5) or S (6), or with an N-substituted formyl (7), trifluoroacetyl (8) or acetyl (9). Replacement with sulfur (6) afforded the most potent KB tumor cell inhibitor, ~6-fold better than the parent 2. In addition, 6 retained tumor transport selectivity via folate receptor (FR) α and -ß over the ubiquitous reduced folate carrier (RFC). FRα-mediated cell inhibition for 6 was generally equivalent to 2, while the FRß-mediated activity was improved by 16-fold over 2. N (4) and O (5) substitutions afforded similar tumor cell inhibitions as 2, with selectivity for FRα and -ß over RFC. The N-substituted analogs 7-9 also preserved transport selectivity for FRα and -ß over RFC. For FRα-expressing CHO cells, potencies were in the order of 8 > 7 > 9. Whereas 8 and 9 showed similar results with FRß-expressing CHO cells, 7 was ~16-fold more active than 2. By nucleoside rescue experiments, all the compounds inhibited de novo purine biosynthesis, likely at the step catalyzed by glycinamide ribonucleotide formyltransferase. Thus, heteroatom replacements of the CH2 in the bridge of 2 afford analogs with increased tumor cell inhibition that could provide advantages over 2, as well as tumor transport selectivity over clinically used antifolates including methotrexate and pemetrexed.
Assuntos
Antineoplásicos/síntese química , Desenho de Fármacos , Receptor 1 de Folato/metabolismo , Receptor 2 de Folato/metabolismo , Ácido Fólico/metabolismo , Pirimidinas/química , Pirróis/química , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Sítios de Ligação , Células CHO , Domínio Catalítico , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Receptor 1 de Folato/química , Receptor 1 de Folato/genética , Receptor 2 de Folato/química , Receptor 2 de Folato/genética , Ácido Fólico/química , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Humanos , Simulação de Acoplamento Molecular , Fosforribosilglicinamido Formiltransferase/química , Fosforribosilglicinamido Formiltransferase/metabolismo , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Pirróis/metabolismo , Pirróis/farmacologia , Relação Estrutura-AtividadeRESUMO
Tumors can be therapeutically targeted with novel antifolates (e.g. AGF94) that are selectively transported by the human proton-coupled folate transporter (hPCFT). Studies were performed to determine the transcription regulation of hPCFT in tumors and identify possible mechanisms that contribute to the highly disparate levels of hPCFT in HepG2 versus HT1080 tumor cells. Transfection of hPCFT-null HT1080 cells with hPCFT restored transport and sensitivity to AGF94 Progressive deletions of the hPCFT promoter construct (-2005 to +96) and reporter gene assays in HepG2 and HT1080 cells confirmed differences in hPCFT transactivation and localized a minimal promoter to between positions -50 and +96. The minimal promoter included KLF15, GC-Box and NRF-1 cis-binding elements whose functional importance was confirmed by promoter deletions and mutations of core consensus sequences and reporter gene assays. In HepG2 cells, NRF-1, KLF15 and Sp1 transcripts were increased over HT1080 cells by â¼5.1-, â¼44-, and â¼2.4-fold, respectively. In Drosophila SL2 cells, transfection with KLF15 and NRF-1 synergistically activated the hPCFT promoter; Sp1 was modestly activating or inhibitory. Chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA) and supershifts confirmed differential binding of KLF15, Sp1, and NRF-1 to the hPCFT promoter in HepG2 and HT1080 cells that paralleled hPCFT levels. Treatment of HT1080 nuclear extracts (NE) with protein kinase A increased Sp1 binding to its consensus sequence by EMSA, suggesting a role for Sp1 phosphorylation in regulating hPCFT transcription. A better understanding of determinants of hPCFT transcriptional control may identify new therapeutic strategies for cancer by modulating hPCFT levels in combination with hPCFT-targeted antifolates.
Assuntos
Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Transportador de Folato Acoplado a Próton/biossíntese , Elementos de Resposta , Fator de Transcrição Sp1/metabolismo , Animais , Drosophila melanogaster , Células HeLa , Células Hep G2 , Humanos , Fatores de Transcrição Kruppel-Like/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Fator 1 Nuclear Respiratório/genética , Transportador de Folato Acoplado a Próton/genética , Fator de Transcrição Sp1/genéticaRESUMO
We previously showed that classical 6-substituted pyrrolo[2,3-d]pyrimidine antifolates bind to folate receptor (FR) α and the target purine biosynthetic enzyme glycinamide ribonucleotide formyltransferase (GARFTase) with different cis and trans conformations. In this study, we designed novel analogs of this series with an amide moiety in the bridge region that can adopt both the cis and trans lowest energy conformations. This provides entropic benefit, by restricting the number of side-chain conformations of the unbound ligand to those most likely to promote binding to FRα and the target enzyme required for antitumor activity. NMR of the most active compound 7 showed both cis and trans amide bridge conformations in ~1:1 ratio. The bridge amide group in the best docked poses of 7 in the crystal structures of FRα and GARFTase adopted both cis and trans conformations, with the lowest energy conformations predicted by Maestro and evidenced by NMR within 1â¯kcal/mol. Compound 7 showed ~3-fold increased inhibition of FRα-expressing cells over its non-restricted parent analog 1 and was selectively internalized by FRα over the reduced folate carrier (RFC), resulting in significant in vitro antitumor activity toward FRα-expressing KB human tumor cells. Antitumor activity of 7 was abolished by treating cells with adenosine but was incompletely protected by 5-aminoimidazole-4-carboxamide (AICA) at higher drug concentrations, suggesting GARFTase and AICA ribonucleotide formyltransferase (AICARFTase) in de novo purine biosynthesis as the likely intracellular targets. GARFTase inhibition by compound 7 was confirmed by an in situ cell-based activity assay. Our results identify a "first-in-class" classical antifolate with a novel amide linkage between the scaffold and the side chain aryl L-glutamate that affords exclusive selectivity for transport via FRα over RFC and antitumor activity resulting from inhibition of GARFTase and likely AICARFTase. Compound 7 offers significant advantages over clinically used inhibitors of this class that are transported by the ubiquitous RFC, resulting in dose-limiting toxicities.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Piridinas/química , Piridinas/farmacologia , Pirróis/química , Pirróis/farmacologia , Amidas/química , Amidas/farmacocinética , Amidas/farmacologia , Animais , Antineoplásicos/farmacocinética , Vias Biossintéticas/efeitos dos fármacos , Células CHO , Linhagem Celular Tumoral , Cricetulus , Receptor 1 de Folato/metabolismo , Antagonistas do Ácido Fólico/farmacocinética , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Nucleotídeos de Purina/metabolismo , Piridinas/farmacocinética , Pirróis/farmacocinéticaRESUMO
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.
Assuntos
Cisplatino , Citosol , Resistencia a Medicamentos Antineoplásicos , Mitocôndrias , Neoplasias Ovarianas , Humanos , Feminino , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Citosol/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/genética , Animais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Cisplatino/farmacologia , Camundongos , Linhagem Celular Tumoral , Carbono/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Glicina Hidroximetiltransferase/metabolismo , Glicina Hidroximetiltransferase/genética , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Epitelial do Ovário/metabolismo , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/patologia , Carcinoma Epitelial do Ovário/genética , Antagonistas do Ácido Fólico/farmacologiaRESUMO
Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: 1TW7), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded active site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC50: 4.4nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6a against both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of (15)N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV.
Assuntos
Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , HIV-1/enzimologia , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Desenho de Fármacos , Farmacorresistência Viral Múltipla , Infecções por HIV/virologia , Protease de HIV/genética , Inibidores da Protease de HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Simulação de Acoplamento Molecular , Mutagênese , Biblioteca de Peptídeos , Peptídeos/genéticaRESUMO
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.
RESUMO
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.
Assuntos
Antineoplásicos , Antagonistas do Ácido Fólico , Hidroximetil e Formil Transferases , Neoplasias , Humanos , Antineoplásicos/química , Carbono , Citosol , Antagonistas do Ácido Fólico/química , Hidroximetil e Formil Transferases/metabolismo , Mitocôndrias , Neoplasias/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Antineoplásicos , Antagonistas do Ácido Fólico , Neoplasias Ovarianas , Animais , Antineoplásicos/farmacologia , Carcinoma Epitelial do Ovário/tratamento farmacológico , Feminino , Antagonistas do Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/uso terapêutico , Humanos , Camundongos , Neoplasias Ovarianas/tratamento farmacológico , Pirimidinas/metabolismo , Microambiente TumoralRESUMO
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.
Assuntos
Receptor 1 de Folato/genética , Ácido Fólico/metabolismo , Neoplasias/tratamento farmacológico , Transportador de Folato Acoplado a Próton/genética , Proteína Carregadora de Folato Reduzido/genética , Transporte Biológico/genética , Proliferação de Células/efeitos dos fármacos , Receptor 1 de Folato/metabolismo , Ácido Fólico/genética , Antagonistas do Ácido Fólico/farmacologia , Células HeLa , Humanos , Metotrexato/farmacologia , Neoplasias/genética , Neoplasias/metabolismo , Ornitina/análogos & derivados , Ornitina/farmacologia , Pemetrexede/farmacologia , Transportador de Folato Acoplado a Próton/metabolismo , Pterinas/farmacologia , Proteína Carregadora de Folato Reduzido/metabolismoRESUMO
Folate-dependent one-carbon (C1) metabolism is compartmentalized into the mitochondria and cytosol and supports cell growth through nucleotide and amino acid biosynthesis. Mitochondrial C1 metabolism, including serine hydroxymethyltransferase (SHMT) 2, provides glycine, NAD(P)H, ATP, and C1 units for cytosolic biosynthetic reactions, and is implicated in the oncogenic phenotype across a wide range of cancers. Whereas multitargeted inhibitors of cytosolic C1 metabolism, such as pemetrexed, are used clinically, there are currently no anticancer drugs that specifically target mitochondrial C1 metabolism. We used molecular modeling to design novel small-molecule pyrrolo[3,2-d]pyrimidine inhibitors targeting mitochondrial C1 metabolism at SHMT2. In vitro antitumor efficacy was established with the lead compounds (AGF291, AGF320, AGF347) toward lung, colon, and pancreatic cancer cells. Intracellular targets were identified by metabolic rescue with glycine and nucleosides, and by targeted metabolomics using a stable isotope tracer, with confirmation by in vitro assays with purified enzymes. In addition to targeting SHMT2, inhibition of the cytosolic purine biosynthetic enzymes, ß-glycinamide ribonucleotide formyltransferase and/or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase, and SHMT1 was also established. AGF347 generated significant in vivo antitumor efficacy with potential for complete responses against both early-stage and upstage MIA PaCa-2 pancreatic tumor xenografts, providing compelling proof-of-concept for therapeutic targeting of SHMT2 and cytosolic C1 enzymes by this series. Our results establish structure-activity relationships and identify exciting new drug prototypes for further development as multitargeted antitumor agents.
Assuntos
Antineoplásicos/farmacologia , Carbono/metabolismo , Citosol/metabolismo , Mitocôndrias/metabolismo , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Antineoplásicos/química , Vias Biossintéticas/efeitos dos fármacos , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Citosol/efeitos dos fármacos , Feminino , Concentração Inibidora 50 , Metabolômica , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Purinas/biossíntese , Pirimidinas/química , Pirróis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Novel fluorinated 2-amino-4-oxo-6-substituted pyrrolo[2,3- d]pyrimidine analogues 7-12 were synthesized and tested for selective cellular uptake by folate receptors (FRs) α and ß or the proton-coupled folate transporter (PCFT) and for antitumor efficacy. Compounds 8, 9, 11, and 12 showed increased in vitro antiproliferative activities (â¼11-fold) over the nonfluorinated analogues 2, 3, 5, and 6 toward engineered Chinese hamster ovary and HeLa cells expressing FRs or PCFT. Compounds 8, 9, 11, and 12 also inhibited proliferation of IGROV1 and A2780 epithelial ovarian cancer cells; in IGROV1 cells with knockdown of FRα, 9, 11, and 12 showed sustained inhibition associated with uptake by PCFT. All compounds inhibited glycinamide ribonucleotide formyltransferase, a key enzyme in the de novo purine biosynthesis pathway. Molecular modeling studies validated in vitro cell-based results. NMR evidence supports the presence of an intramolecular fluorine-hydrogen bond. Potent in vivo efficacy of 11 was established with IGROV1 xenografts in severe compromised immunodeficient mice.
Assuntos
Flúor/química , Receptor 1 de Folato/metabolismo , Transportador de Folato Acoplado a Próton/metabolismo , Nucleotídeos de Purina/biossíntese , Pirimidinas/química , Pirimidinas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Transporte Biológico , Células CHO , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetulus , Ácido Fólico/metabolismo , Humanos , Camundongos , Pirimidinas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor-targeted specificities of 6-substituted pyrrolo[2,3- d]pyrimidine analogues of 1, where the phenyl side-chain is replaced by 3',6' (5, 8), 2',5' (6, 9), and 2',6' (7, 10) pyridyls, were analyzed. Proliferation inhibition of isogenic Chinese hamster ovary (CHO) cells expressing folate receptors (FRs) α and ß were in rank order, 6 > 9 > 5 > 7 > 8, with 10 showing no activity, and 6 > 9 > 5 > 8, with 10 and 7 being inactive, respectively. Antiproliferative effects toward FRα- and FRß-expressing cells were reflected in competitive binding with [3H]folic acid. Only compound 6 was active against proton-coupled folate receptor (PCFT)-expressing CHO cells (â¼4-fold more potent than 1) and inhibited [3H]methotrexate uptake by PCFT. In KB and IGROV1 tumor cells, 6 showed <1 nM IC50, â¼2-3-fold more potent than 1. Compound 6 inhibited glycinamide ribonucleotide formyltransferase in de novo purine biosynthesis and showed potent in vivo efficacy toward subcutaneous IGROV1 tumor xenografts in SCID mice.
Assuntos
Antineoplásicos/síntese química , Antagonistas do Ácido Fólico/síntese química , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Células CHO , Proliferação de Células/efeitos dos fármacos , Cricetulus , Receptor 1 de Folato/análise , Receptor 1 de Folato/metabolismo , Receptor 2 de Folato/análise , Antagonistas do Ácido Fólico/farmacologia , Xenoenxertos , Humanos , Camundongos , Camundongos SCID , Transportador de Folato Acoplado a Próton/metabolismo , Nucleotídeos de Purina/antagonistas & inibidores , Pirimidinas/química , Pirimidinas/farmacocinética , Pirróis/química , Pirróis/farmacocinéticaRESUMO
Folate uptake in epithelial ovarian cancer (EOC) involves the reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT), both facilitative transporters and folate receptor (FR) α. Although in primary EOC specimens, FRα is widely expressed and increases with tumor stage, PCFT was expressed independent of tumor stage (by real-time RT-PCR and IHC). EOC cell line models, including cisplatin sensitive (IGROV1 and A2780) and resistant (SKOV3 and TOV112D) cells, expressed a 17-fold range of FRα and similar amounts (within â¼2-fold) of PCFT. Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates AGF94 and AGF154 exhibited potent antiproliferative activities toward all of the EOC cell lines, reflecting selective cellular uptake by FRα and/or PCFT over RFC. When IGROV1 cells were pretreated with AGF94 at pH 6.8, clonogenicity was potently inhibited, confirming cell killing. FRα was knocked down in IGROV1 cells with lentiviral shRNAs. Two FRα knockdown clones (KD-4 and KD-10) showed markedly reduced binding and uptake of [3H]folic acid and [3H]AGF154 by FRα, but maintained high levels of [3H]AGF154 uptake by PCFT compared to nontargeted control cells. In proliferation assays, KD-4 and KD-10 cells preserved in vitro inhibition by AGF94 and AGF154, compared to a nontargeted control, attributable to residual FRα- and substantial PCFT-mediated uptake. KD-10 tumor xenografts in severe-compromised immune-deficient mice were likewise sensitive to AGF94 Collectively, our results demonstrate the substantial therapeutic potential of novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with dual targeting of PCFT and FRα toward EOCs that express a range of FRα, along with PCFT, as well as cisplatin resistance. Mol Cancer Ther; 16(5); 819-30. ©2017 AACR.
Assuntos
Receptor 1 de Folato/genética , Antagonistas do Ácido Fólico/administração & dosagem , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Transportador de Folato Acoplado a Próton/genética , Animais , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptor 1 de Folato/antagonistas & inibidores , Ácido Fólico/genética , Antagonistas do Ácido Fólico/síntese química , Humanos , Camundongos , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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 ß 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.
Assuntos
Antineoplásicos/química , Receptor 1 de Folato/metabolismo , Antagonistas do Ácido Fólico/química , Transportador de Folato Acoplado a Próton/metabolismo , Nucleotídeos de Purina/antagonistas & inibidores , Pirimidinas/química , Pirróis/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/farmacologia , Xenoenxertos , Humanos , Camundongos SCID , Simulação de Acoplamento Molecular , Transplante de Neoplasias , Fosforribosilglicinamido Formiltransferase/antagonistas & inibidores , Nucleotídeos de Purina/biossíntese , Pirimidinas/síntese química , Pirimidinas/farmacologia , Pirróis/síntese química , Pirróis/farmacologia , Relação Estrutura-AtividadeRESUMO
OBJECTIVES: To develop mobile-based physical activity promotion text messages, understand users' preferences, and assess feasibility and acceptability in a college student sample. METHODS: Five focus groups (N = 33) were conducted using a participatory approach. An Audience Response System was adopted for data collection to ensure confidentiality and for directing the discussion foci. A framework analysis of transcribed focus group discussions was conducted. Atlas. ti qualitative analysis software was used to manage the data. RESULTS: Participants were uniformly enthusiastic about a text message-based intervention to encourage regular physical activity. They also preferred positive, supportive personally tailored messages. Participants placed a high value on messages related to establishing and monitoring realistic and achievable goals. CONCLUSIONS: mHealth text messaging was well received. The results support the feasibility and acceptance of such an intervention.