RESUMO
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.
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
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
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
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
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.
RESUMO
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.