RESUMO
Patients with triple-negative breast cancer have a poor prognosis as only a few efficient targeted therapies are available. Cancer cells are characterized by their unregulated proliferation and require large amounts of nucleotides to replicate their DNA. One-carbon metabolism contributes to purine and pyrimidine nucleotide synthesis by supplying one carbon atom. Although mitochondrial one-carbon metabolism has recently been focused on as an important target for cancer treatment, few specific inhibitors have been reported. In this study, we aimed to examine the effects of DS18561882 (DS18), a novel, orally active, specific inhibitor of methylenetetrahydrofolate dehydrogenase (MTHFD2), a mitochondrial enzyme involved in one-carbon metabolism. Treatment with DS18 led to a marked reduction in cancer-cell proliferation; however, it did not induce cell death. Combinatorial treatment with DS18 and inhibitors of checkpoint kinase 1 (Chk1), an activator of the S phase checkpoint pathway, efficiently induced apoptotic cell death in breast cancer cells and suppressed tumorigenesis in a triple-negative breast cancer patient-derived xenograft model. Mechanistically, MTHFD2 inhibition led to cell cycle arrest and slowed nucleotide synthesis. This finding suggests that DNA replication stress occurs due to nucleotide shortage and that the S-phase checkpoint pathway is activated, leading to cell-cycle arrest. Combinatorial treatment with both inhibitors released cell-cycle arrest, but induced accumulation of DNA double-strand breaks, leading to apoptotic cell death. Collectively, a combination of MTHFD2 and Chk1 inhibitors would be a rational treatment option for patients with triple-negative breast cancer.
Assuntos
Aminoidrolases/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Inibidores Enzimáticos/uso terapêutico , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Enzimas Multifuncionais/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Administração Oral , Aminoidrolases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/metabolismo , Quimioterapia Combinada , Inibidores Enzimáticos/administração & dosagem , Feminino , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Enzimas Multifuncionais/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) plays an important role in one-carbon metabolism. The MTHFD2 gene is upregulated in various cancers but very low or undetectable in normal proliferating cells, and therefore a potential target for cancer treatment. In this study, we present the structure of MTHFD2 in complex with xanthine derivative 15, which allosterically binds to MTHFD2 and coexists with the substrate analogue. A kinetic study demonstrated the uncompetitive inhibition of MTHFD2 by 15. Allosteric inhibitors often provide good selectivity and, indeed, xanthine derivatives are highly selective for MTHFD2. Moreover, several conformational changes were observed upon the binding of 15, which impeded the binding of the cofactor and phosphate to MTHFD2. To the best of our knowledge, this is the first study to identify allosteric inhibitors targeting the MTHFD family and our results would provide insights on the inhibition mechanism of MTHFD proteins and the development of novel inhibitors.
Assuntos
Aminoidrolases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Enzimas Multifuncionais/antagonistas & inibidores , Xantina/farmacologia , Sítio Alostérico/efeitos dos fármacos , Aminoidrolases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Modelos Moleculares , Estrutura Molecular , Enzimas Multifuncionais/metabolismo , Relação Estrutura-Atividade , Xantina/síntese química , Xantina/químicaRESUMO
Metabolic enzymes and metabolites display non-metabolic functions in immune cell signalling that modulate immune attack ability. However, whether and how a tumour's metabolic remodelling contributes to its immune resistance remain to be clarified. Here we perform a functional screen of metabolic genes that rescue tumour cells from effector T cell cytotoxicity, and identify the embryo- and tumour-specific folate cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2). Mechanistically, MTHFD2 promotes basal and IFN-γ-stimulated PD-L1 expression, which is necessary for tumourigenesis in vivo. Moreover, IFN-γ stimulates MTHFD2 through the AKT-mTORC1 pathway. Meanwhile, MTHFD2 drives the folate cycle to sustain sufficient uridine-related metabolites including UDP-GlcNAc, which promotes the global O-GlcNAcylation of proteins including cMYC, resulting in increased cMYC stability and PD-L1 transcription. Consistently, the O-GlcNAcylation level positively correlates with MTHFD2 and PD-L1 in pancreatic cancer patients. These findings uncover a non-metabolic role for MTHFD2 in cell signalling and cancer biology.
Assuntos
Aminoidrolases/genética , Antígeno B7-H1/genética , Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Enzimas Multifuncionais/genética , Neoplasias Pancreáticas/genética , Processamento de Proteína Pós-Traducional , Linfócitos T Citotóxicos/imunologia , Aminoidrolases/antagonistas & inibidores , Aminoidrolases/imunologia , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Carcinogênese/imunologia , Carcinogênese/patologia , Linhagem Celular Tumoral , Embrião de Mamíferos , Fibroblastos/imunologia , Fibroblastos/patologia , Ácido Fólico/imunologia , Ácido Fólico/metabolismo , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Metilenotetra-Hidrofolato Desidrogenase (NADP)/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Enzimas Multifuncionais/antagonistas & inibidores , Enzimas Multifuncionais/imunologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Transdução de Sinais , Linfócitos T Citotóxicos/patologia , Carga Tumoral , Evasão Tumoral , Uridina Difosfato N-Acetilglicosamina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Rationale: Lung adenocarcinoma (LUAD) is an aggressive disease with high propensity of metastasis. Among patients with early-stage disease, more than 30% of them may relapse or develop metastasis. There is an unmet medical need to stratify patients with early-stage LUAD according to their risk of relapse/metastasis to guide preventive or therapeutic approaches. In this study, we identified 4 genes that can serve both therapeutic and diagnostic (theranostic) purposes. Methods: Three independent datasets (GEO, TCGA, and KMPlotter) were used to evaluate gene expression profile of patients with LUAD by unbiased screening approach. Upon significant genes uncovered, functional enrichment analysis was carried out. The predictive power of their expression on patient prognosis were evaluated. Once confirmed their theranostic roles by integrated bioinformatics, we further conducted in vitro and in vivo validation. Results: We found that four genes (ADAM9, MTHFD2, RRM2, and SLC2A1) were associated with poor patient outcomes with an increased hazard ratio in LUAD. Knockdown of them, both separately and simultaneously, suppressed lung cancer cell proliferation and migration ability in vitro and prolonged survival time in metastatic tumor mouse models. Moreover, these four biomarkers were found to be overexpressed in tumor tissues from LUAD patients, and the total immunohistochemical staining scores correlated with poor prognosis. Conclusions: These results suggest that these four identified genes could be theranostic biomarkers for stratifying high-risk patients who develop relapse/metastasis in early-stage LUAD. Developing therapeutic approaches for the four biomarkers may benefit early-stage LUAD patients after surgery.
Assuntos
Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/secundário , Biomarcadores Tumorais/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Células A549 , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/genética , Adenocarcinoma de Pulmão/cirurgia , Aminoidrolases/antagonistas & inibidores , Aminoidrolases/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Técnicas de Silenciamento de Genes , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/genética , Células HEK293 , Humanos , Neoplasias Pulmonares/cirurgia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Camundongos , Camundongos SCID , Enzimas Multifuncionais/antagonistas & inibidores , Enzimas Multifuncionais/genética , Medicina de Precisão , Prognóstico , Ribonucleosídeo Difosfato Redutase/antagonistas & inibidores , Ribonucleosídeo Difosfato Redutase/genética , Fatores de Risco , Transcriptoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Folate-mediated one-carbon metabolism (FOCM) supports vital events for the growth and survival of proliferating cells. Nucleotide synthesis and DNA methylation are the biochemical bases of cancers that are highly dependent on FOCM. Recent studies revealed that FOCM is connected with redox homeostasis and epigenetics in cancer. Furthermore, folate-metabolizing enzymes, such as serine hydroxymethyltransferase 2 (SHMT2) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), are associated with the development of cancers, including breast cancer, highlighting their potential application in tumor-targeted therapy. Therefore, targeting metabolizing enzymes, especially SHMT2 and MTHFD2, provides a novel strategy for cancer treatment. In this review, we outline current understanding of the functions of SHMT2 and MTHFD2, discussing their expression, potential functions, and regulatory mechanism in cancers. Furthermore, we discuss examples of inhibitors of SHMT2 and MTHFD2.
Assuntos
Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Aminoidrolases/antagonistas & inibidores , Aminoidrolases/metabolismo , Animais , Carbono/metabolismo , Sobrevivência Celular/fisiologia , Metilação de DNA/genética , Ácido Fólico/metabolismo , Glicina Hidroximetiltransferase/antagonistas & inibidores , Glicina Hidroximetiltransferase/metabolismo , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Enzimas Multifuncionais/antagonistas & inibidores , Enzimas Multifuncionais/metabolismo , Neoplasias/patologiaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Terapia de Alvo Molecular , Neoplasias/metabolismo , Aminoidrolases/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Biomarcadores , Carbono/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicina Hidroximetiltransferase/antagonistas & inibidores , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Terapia de Alvo Molecular/métodos , Enzimas Multifuncionais/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/etiologia , Neoplasias/patologia , Serina/biossínteseRESUMO
We report the discovery of a potent and isozyme-selective MTHFD2 inhibitor, DS18561882 (2). Through investigation of the substituents on our tricyclic coumarin scaffold (1,2,3,4-tetrahydrochromeno[3,4-c]pyridin-5-one), MTHFD2 inhibitory activity was shown to be elevated by incorporating an amine moiety at the 8-position and a methyl group at the 7-position of the initial lead 1. X-ray structure analysis revealed that a key interaction for enhanced potency was salt bridge formation between the amine moiety and the diphosphate linker of an NAD+ cofactor. Furthermore, ortho-substituted sulfonamide in place of benzoic acid of 1 significantly improved cell permeability and cell-based growth inhibition against a human breast cancer cell line. The thus-optimized DS18561882 showed the strongest cell-based activity (GI50 = 140 nM) in the class, a good oral pharmacokinetic profile, and thereby tumor growth inhibition in a mouse xenograft model upon oral administration.
Assuntos
Aminoidrolases/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Enzimas Multifuncionais/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cristalografia por Raios X , Feminino , Humanos , Masculino , Camundongos Endogâmicos BALB C , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Overcoming oxidative stress is a critical step for tumor progression; however, the underlying mechanisms in colorectal cancer (CRC) remain unclear. METHODS: We investigated nicotinamide adenine dinucleotide (phosphate) (NAD(P))-dependent enzyme methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) expression, clinical relevance, redox modification, and molecular mechanisms using the CRC cells and tissues (n = 462 paired samples). The antitumor effects of MTHFD2 inhibitor LY345899 on CRC tumorigenesis and metastasis were evaluated in vitro and in vivo. Data analysis used Kaplan-Meier, Pearson's correlation, and Student t test where appropriate. All statistical tests were two-sided. RESULTS: Here, we report that the patients with high expression of MTHFD2 have a shorter overall survival (HR = 1.62, 95% CI = 1.12 to 2.36, P = .01) and disease-free survival (HR = 1.55, 95% CI = 1.07 to 2.27, P = .02) than patients with low MTHFD2 expression. Suppression of MTHFD2 disturbs NADPH and redox homeostasis and accelerates cell death under oxidative stress, such as hypoxia or anchorage independence (P ≤ .01 for all). Also, genetic or pharmacological inhibition of MTHFD2 suppresses CRC cell growth and lung and peritoneal metastasis in cell-based xenografts (n = 5-8 mice per group). Importantly, LY345899 treatment statistically significantly suppresses tumor growth and decreases the tumor weight in CRC patient-derived xenograft models (n = 10 mice per group, mean [SD] tumor weight of the vehicle-treated group was 1.83 [0.19] mg vs 0.74 [0.30] mg for the LY345899-treated group, P < .001). CONCLUSIONS: Our study presents evidence that MTHFD2 confers redox homeostasis and promotes CRC cell growth and metastasis. The folate analog LY345899 as MTHFD2 inhibitor displays therapeutic activity against CRC and warrants further clinical investigation for CRC treatment.
Assuntos
Aminoidrolases/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Inibidores Enzimáticos/farmacologia , Glutamatos/farmacologia , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Enzimas Multifuncionais/antagonistas & inibidores , Aminoidrolases/genética , Aminoidrolases/metabolismo , Animais , Anoikis/efeitos dos fármacos , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Feminino , Humanos , Neoplasias Pulmonares/secundário , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Distribuição Aleatória , Transdução de Sinais , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND/AIMS: To investigate the role of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in the clinical prognosis and cell biology of renal cell carcinoma (RCC). METHODS: A total of 137 RCC tissues were evaluated by immunohistochemistry. The relationship between MTHFD2 overexpression and clinical parameters and vimentin expression was assessed. Kaplan-Meier curves and the log-rank test were applied for survival analysis according to MTHFD2 and vimentin expression in RCC tissues. The expression of MTHFD2 mRNA and protein was examined by quantitative reverse transcription PCR and western blotting, respectively. To determine further the biological activity of MTHFD2 in RCC, 786-O cells were transfected with short hairpin RNA specifically targeting MTHFD2 (shMTHFD2) with or without tumor necrosis factor (TNF)-α stimulation. Cell proliferation, cell migration and invasion and drug sensitivity were subsequently assessed using Cell Counting Kit-8, wound healing, and Transwell assays. RESULTS: Immunohistochemical analysis demonstrated that both MTHFD2 and vimentin overexpression was positively associated with clinical staging, pathological grade, and poor overall survival (all P < 0.05). MTHFD2 expression was closely correlated with vimentin overexpression in RCC (r = 0.402, P < 0.001). After knocking down MTHFD2 expression in 786-O cells, decreased cell proliferation, migration, and invasion were observed and accompanied by the reduced expression of vimentin. The effects of MTHFD2 down-regulation could be partially restrained by TNF-α treatment. Vimentin expression and cell migration and invasion, but not cell proliferation, were reversed by TNF-α stimulation. Furthermore, treatment of 786-O cells with shMTHFD2 increased their sensitivity to chemotherapy drugs. CONCLUSION: The current results demonstrated that MTHFD2 was overexpressed in RCC and associated with poor clinical characteristics, vimentin expression, and cellular features connected to malignant disease, thus, implicating MTHFD2 as a potential target for RCC therapy.
Assuntos
Aminoidrolases/metabolismo , Carcinoma de Células Renais/patologia , Proliferação de Células , Neoplasias Renais/patologia , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Enzimas Multifuncionais/metabolismo , Vimentina/metabolismo , Idoso , Aminoidrolases/antagonistas & inibidores , Aminoidrolases/genética , Antineoplásicos/farmacologia , Carcinoma de Células Renais/mortalidade , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/mortalidade , Masculino , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Pessoa de Meia-Idade , Enzimas Multifuncionais/antagonistas & inibidores , Enzimas Multifuncionais/genética , Gradação de Tumores , Prognóstico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Vimentina/genéticaRESUMO
Arsenic exposure increases risk for cancers and is teratogenic in animal models. Here we demonstrate that small ubiquitin-like modifier (SUMO)- and folate-dependent nuclear de novo thymidylate (dTMP) biosynthesis is a sensitive target of arsenic trioxide (As2O3), leading to uracil misincorporation into DNA and genome instability. Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) and serine hydroxymethyltransferase (SHMT) generate 5,10-methylenetetrahydrofolate for de novo dTMP biosynthesis and translocate to the nucleus during S-phase, where they form a multienzyme complex with thymidylate synthase (TYMS) and dihydrofolate reductase (DHFR), as well as the components of the DNA replication machinery. As2O3 exposure increased MTHFD1 SUMOylation in cultured cells and in in vitro SUMOylation reactions, and increased MTHFD1 ubiquitination and MTHFD1 and SHMT1 degradation. As2O3 inhibited de novo dTMP biosynthesis in a dose-dependent manner, increased uracil levels in nuclear DNA, and increased genome instability. These results demonstrate that MTHFD1 and SHMT1, which are key enzymes providing one-carbon units for dTMP biosynthesis in the form of 5,10-methylenetetrahydrofolate, are direct targets of As2O3-induced proteolytic degradation, providing a mechanism for arsenic in the etiology of cancer and developmental anomalies.
Assuntos
Aminoidrolases/antagonistas & inibidores , Núcleo Celular/metabolismo , Formiato-Tetra-Hidrofolato Ligase/antagonistas & inibidores , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Complexos Multienzimáticos/antagonistas & inibidores , Óxidos/toxicidade , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/antagonistas & inibidores , Timidina Monofosfato/biossíntese , Aminoidrolases/genética , Aminoidrolases/metabolismo , Animais , Trióxido de Arsênio , Arsenicais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Núcleo Celular/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Formiato-Tetra-Hidrofolato Ligase/genética , Formiato-Tetra-Hidrofolato Ligase/metabolismo , Instabilidade Genômica/efeitos dos fármacos , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Camundongos , Camundongos Knockout , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Proteólise , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Uracila/metabolismoRESUMO
Pterin deaminase is an amidohydrolase enzyme hydrolyzing pteridines to form lumazine derivatives and ammonia. The enzyme captured the attention of scientists as early as 1959 and had been patented for its application as an anticancer agent. It is ubiquitously present in prokaryotes and has been reported in some eukaryotes such as honey bee, silkworm and rats. The enzyme has been observed to have a spectrum of substrates with the formation of respective lumazines. The role of the substrates of the enzyme in various metabolic pathways warrants a significant role in the biological activity of both prokaryotes and eukaryotes. Even though the functions of the enzyme have been explored in prokaryotes, their niche in the eukaryotic system is not clear. There is very few information on the structural and functional properties of the enzyme. This review has been congregated to emphasize the significance of pterin deaminase and analyzes the lacunae in understanding the biological characters of the enzyme.
Assuntos
Amidoidrolases/metabolismo , Aminoidrolases/metabolismo , Aminoidrolases/antagonistas & inibidores , Animais , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Células Eucarióticas/enzimologia , Células Procarióticas/enzimologia , Pteridinas/químicaRESUMO
C-Alkyl amidine analogues of asymmetric N(ω),N(ω)-dimethyl-L-arginine are dual-targeted inhibitors of both human DDAH-1 and nitric oxide (NO) synthase, and provide a promising scaffold for the development of therapeutics to control NO overproduction in a variety of pathologies including septic shock and some cancers. Using a two-part click-chemistry-mediated activity probe, a homologated series of C-alkyl amidines were ranked for their ability to inhibit DDAH-1 within cultured HEK 293T cells. N5-(1-Iminopentyl)-L-ornithine was determined to be the most potent compound inâ vitro (K(d)=7â µM) as well as in cultured cells, and the binding conformation and covalent reversible mode of inhibition was investigated by comparison of interactions made with DDAH-1 and a catalytically inactive C274S variant, as gauged by X-ray crystallography and isothermal titration calorimetry. By interrupting the ability of the inhibitor to form a covalent bond, the contribution of this interaction could be estimated. These results suggest that further stabilization of the covalent adduct is a promising strategy for lead optimization in the design of effective reagents to block NO synthesis.
Assuntos
Amidinas , Aminoidrolases/antagonistas & inibidores , Arginina/análogos & derivados , Inibidores Enzimáticos , Óxido Nítrico Sintase/antagonistas & inibidores , Amidinas/farmacologia , Aminoidrolases/genética , Aminoidrolases/metabolismo , Arginina/metabolismo , Disponibilidade Biológica , Varredura Diferencial de Calorimetria , Linhagem Celular , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismoRESUMO
Acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] was investigated as an inhibitor of the triad glutamine amidotransferases, IGP synthase and GMP synthetase. Nucleophilic substitution of the chlorine atom in acivicin results in the formation of an imine-thioether adduct at the active site cysteine. Cys 77 was identified as the site of modification in the heterodimeric IGPS from Escherichia coli (HisHF) by tryptic digest and FABMS. Distinctions in the glutaminase domains of IGPS from E. coli, the bifunctional protein from Saccharomyces cerevisiae (HIS7), and E. coli GMPS were revealed by the differential rates of inactivation. While the ammonia-dependent turnover was unaffected by acivicin, the glutamine-dependent reaction was inhibited with unit stoichiometry. In analogy to the conditional glutaminase activity seen in IGPS and GMPS, the rates of inactivation were accelerated > or =25-fold when a nucleotide substrate (or analogue) was present. The specificity (k(inact)/K(i)app) for acivicin is on the same order of magnitude as the natural substrate glutamine in all three enzymes. The (alphaS,5R) diastereomer of acivicin was tested under identical conditions as acivicin and showed little inhibitory effect on the enzymes indicating that acivicin binds in the glutamine reactive site in a specific conformation. The data indicate that acivicin undergoes a glutamine amidotransferase mechanism-based covalent bond formation in the presence of nucleotide substrates or products. Acivicin and its (alphaS,5R) diastereomer were modeled in the glutaminase active site of GMPS and CPS to confirm that the binding orientation of the dihydroisoxazole ring is identical in all three triad glutamine amidotransferases. Stabilization of the imine-thioether intermediate by the oxyanion hole in triad glutamine amidotransferases appears to confer the high degree of specificity for acivicin inhibition and relates to a common mechanism for inactivation.
Assuntos
Aminoidrolases/antagonistas & inibidores , Antranilato Sintase , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/antagonistas & inibidores , Inibidores Enzimáticos/química , Isoxazóis/química , Complexos Multienzimáticos/antagonistas & inibidores , Transferases de Grupos Nitrogenados/antagonistas & inibidores , Sequência de Aminoácidos , Aminoidrolases/química , Azasserina/química , Ligação Competitiva , Carbono-Nitrogênio Ligases , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/química , Diazo-Oxo-Norleucina/química , Escherichia coli/enzimologia , Glutamina/análogos & derivados , Glutamina/química , Cinética , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Transferases de Grupos Nitrogenados/química , Ribonucleotídeos/química , Saccharomyces cerevisiae/enzimologia , Eletricidade Estática , Estereoisomerismo , Especificidade por SubstratoRESUMO
A stability study of adenosine receptor agonists in rat and human whole blood was performed. The compounds were incubated at 37 degrees in fresh blood, and aliquots of the incubation mixture were hemolyzed at regular time intervals and analyzed with HPLC. N6-cyclopentyladenosine (CPA) and N6-cyclobutyladenosine (CBA) were degraded, whereas N6-cyclohexyladenosine, N6-cycloheptyladenosine and N6-sulfophenyladenosine were not. 2-Chloroadenosine had a half-life very similar to that of CPA. However, the 2'-, 3'-, and 5'-deoxyribose derivatives of CPA remained intact. The nucleoside transport inhibitor nitrobenzylthioinosine attenuated CBA and CPA metabolism in rat blood as did the inhibitor of adenosine deaminase erythro-9-(2-hydroxy-3-nonyl)adenine, albeit at relatively high concentrations. Complete blockade of CBA and CPA degradation was achieved by a preincubation of rat and human blood with the adenosine kinase (AK) inhibitor 5'-amino-5'-deoxyadenosine. We conclude that the two adenosine analogues are metabolized by AK both in rat and in human whole blood.
Assuntos
Adenosina Quinase/metabolismo , Aminoidrolases/metabolismo , Agonistas do Receptor Purinérgico P1 , Adenina/análogos & derivados , Adenina/farmacologia , Adenosina/análogos & derivados , Adenosina Quinase/antagonistas & inibidores , Aminoidrolases/antagonistas & inibidores , Animais , Sangue , Desoxiadenosinas/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Ratos , Tioinosina/análogos & derivados , Tioinosina/farmacologiaRESUMO
N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl ]-benzoyl]-L-glutamic acid (LY231514) is a novel pyrrolo[2,3-d]pyrimidine-based antifolate currently undergoing extensive Phase II clinical trials. Previous studies have established that LY231514 and its synthetic gamma-polyglutamates (glu3 and glu5) exert potent inhibition against thymidylate synthase (TS). We now report that LY231514 and its polyglutamates also markedly inhibit other key folate-requiring enzymes, including dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). For example, the Ki values of the pentaglutamate of LY231514 are 1.3, 7.2, and 65 nM for inhibition against TS, DHFR, and GARFT, respectively. In contrast, although a similar high level of inhibitory potency was observed for the parent monoglutamate against DHFR (7.0 nM), the inhibition constants (Ki) for the parent monoglutamate are significantly weaker for TS (109 nM) and GARFT (9,300 nM). The effects of LY231514 and its polyglutamates on aminoimidazole carboxamide ribonucleotide formyltransferase, 5,10-methylenetetrahydrofolate dehydrogenase, and 10-formyltetrahydrofolate synthetase were also evaluated. The end product reversal studies conducted in human cell lines further support the concept that multiple enzyme-inhibitory mechanisms are involved in cytotoxicity. The reversal pattern of LY231514 suggests that although TS may be a major site of action for LY231514 at concentrations near the IC50, higher concentrations can lead to inhibition of DHFR and/or other enzymes along the purine de novo pathway. Studies with mutant cell lines demonstrated that LY231514 requires polyglutamation and transport via the reduced folate carrier for cytotoxic potency. Therefore, our data suggest that LY231514 is a novel classical antifolate, the antitumor activity of which may result from simultaneous and multiple inhibition of several key folate-requiring enzymes via its polyglutamated metabolites.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Glutamatos/farmacologia , Guanina/análogos & derivados , Hidroximetil e Formil Transferases , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , 5,10-Metilenotetra-Hidrofolato Redutase (FADH2) , Aciltransferases/antagonistas & inibidores , Aminoidrolases/antagonistas & inibidores , Formiato-Tetra-Hidrofolato Ligase/antagonistas & inibidores , Glutamatos/química , Guanina/química , Guanina/farmacologia , Humanos , Metotrexato/farmacologia , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Metilenotetra-Hidrofolato Redutase (NADPH2) , Estrutura Molecular , Complexos Multienzimáticos/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Pemetrexede , Fosforribosilaminoimidazolcarboxamida Formiltransferase , Fosforribosilglicinamido Formiltransferase , Ácido Poliglutâmico/farmacologia , Quinazolinas/farmacologia , Tetra-Hidrofolatos/farmacologia , Tiofenos/farmacologia , Timidilato Sintase/antagonistas & inibidores , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The bifunctional dehydrogenase/cyclohydrolase domain of the human trifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase/formyltetrahydrofolate synthetase catalyzes two sequential reactions with significant channeling of the intermediate, methenyltetrahydrofolate. Equilibrium dialysis established that a single, high-affinity NADP+ binding site exists per monomer of the dimeric enzyme. Kinetic characterization of NADP+ binding to the dehydrogenase using analogs as inhibitors demonstrated that affinity for this substrate is due almost exclusively to binding at the 2',5'-ADP subsite. The same structural specificities for binding are exhibited by these analogs in their effects on the cyclohydrolase. Both NADP+ and its 3-aminopyridine analog AADP partially inhibit the activity of the cyclohydrolase when assayed with added methenyltetrahydrofolate as substrate. However, under the same conditions, the cyclohydrolase is actually activated by 2',5'-ADP; activation requires the presence of the 5'-phosphate since 2'-AMP binds but does not activate. Nicotinamide ribose monophosphate (NMN) has no detectable effect either alone or in combination with 2',5'-ADP. The results are consistent with the existence of a shared dehydrogenase/cyclohydrolase active site proximal to the 2',5'-ADP subsite. NADP+ reduces the rate of the fully activated cyclohydrolase by 2-fold. Inhibition appears to be due to the loosely bound nicotinamide ring interacting with the common folate subsite, resulting in only partial inhibition by NADP+. The interaction of 2',5'-ADP with the cyclohydrolase suggests a potential role for this portion of the molecule in promoting the efficiency of the channeling of endogenously generated methenyltetrahydrofolate.
Assuntos
Difosfato de Adenosina/metabolismo , Aminoidrolases/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , NADP/farmacologia , Nucleotídeos de Adenina/farmacologia , Monofosfato de Adenosina/metabolismo , Aminoidrolases/antagonistas & inibidores , Sítios de Ligação , Ligação Competitiva , Corantes Fluorescentes , Humanos , Cinética , Meteniltetra-Hidrofolato Cicloidrolase , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , NADP/metabolismo , Fenilglioxal/metabolismo , Tetra-Hidrofolatos/metabolismoRESUMO
An enzyme can enhance the rate of a reaction only to the extent that it binds the altered substrate in the transition state (S not equal to) more tightly than it binds the substrate in the ground state. Inhibitors that resemble S not equal to can be used to stop an enzyme from working, probe its mechanism of action and obtain exact structural information about intermediates in catalysis. In S not equal to analog inhibitors of adenosine and cytidine deaminases, a single hydroxyl group appears to make extremely large contributions to binding affinity. The magnitude of this contribution becomes even more striking when differences in free energy of solvation by water are taken into account. Other results, obtained by deleting individual binding determinants, indicate the operation of remarkable levels of cooperativity and suggest that if every group is in exactly the right position and is part of an inflexible structure, then a single substituent or H-bond can produce very large increases in binding affinity. Some implications for inhibitor design are considered.
Assuntos
Aminoidrolases/química , Aminoidrolases/metabolismo , Citidina Desaminase/química , Citidina Desaminase/metabolismo , Inibidores Enzimáticos/metabolismo , Aminoidrolases/antagonistas & inibidores , Animais , Ligação Competitiva/efeitos dos fármacos , Quelantes/farmacologia , Citidina Desaminase/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Relação Estrutura-AtividadeRESUMO
Rhodococcus ATCC 39484 produced a nitrilase when induced with isovaleronitrile. The enzyme was obtainable pure in milligram amounts, had a subunit Mr of 40 kDa, and demonstrated a substrate-induced activation related to aggregation of subunits to form a 560-kDa complex. The enzyme had a broad substrate specificity, had a pH optimum of 7.5, was stable up to 40 degrees C, and had one disulfide bridge and two free cysteine residues, one of which appeared to be catalytically essential. The N-terminal sequence was determined and found to have 78.3% homology, in a 23-residue overlap, with Klebsiella ozaenae nitrilase. The enzyme was inhibited competitively by benzylamine and benzaldehyde and irreversibly by benzyl bromide. However, benzyl bromide was shown to be nonspecific, causing multiple alkylation. Acid quenching of enzyme-substrate mixtures allowed for the detection of covalent enzyme-substrate complexes using mass spectrometry. The covalent intermediate is suggested to be either a thioimidate or an acylenzyme and a reaction mechanism consistent with this observation and also the inhibitor results is proposed. The rate of breakdown of the covalent intermediates was found to be rate limiting even for substrates with undetectable rates of hydrolysis or those with very slow rates of intermediate formation. For phenylacetonitrile, a poor substrate, in addition to acid, approximately 2% of the product was the corresponding amide. This result suggests that a tetrahedral intermediate is formed which, for selected substrates, can break down anomalously to produce amide in place of the normal acid product. Under the conditions used in this study all other substrates tested were converted to acid.
Assuntos
Aminoidrolases/metabolismo , Rhodococcus/enzimologia , Sequência de Aminoácidos , Aminoidrolases/antagonistas & inibidores , Aminoidrolases/química , Concentração de Íons de Hidrogênio , Cinética , Klebsiella/enzimologia , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Conformação Proteica , Homologia de Sequência , Especificidade da Espécie , Especificidade por Substrato , Compostos de Sulfidrila/análise , TemperaturaRESUMO
Nitrilase was purified from an extract of isovaleronitrile-induced cells of Rhodococcus rhodochrous J1 in seven steps. In the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous by polyacrylamide electrophoresis, ampholyte electrofocusing and double immunodiffusion in agarose. The enzyme has a molecular mass of about 78 kDa and consists of two subunits identical in molecular mass. The purified enzyme exhibits a pH optimum of 7.6 and a temperature optimum of 45 degrees C. The enzyme catalyzed stoichiometrically the hydrolysis of benzonitrile to benzoic acid and ammonia, and no formation of amide was detected. The enzyme required thiol compounds such as dithiothreitol, L-cysteine or reduced glutathione to exhibit maximum activity. The enzyme was specific for nitrile groups attached to an aromatic or heteroaromatic ring, e.g. benzonitrile, 3-chlorobenzonitrile, 4-tolunitrile, 2-furonitrile and 2-thiophenecarbonitrile. The comparison of the properties of the enzyme with other nitrilases and nitrile hydratases has been also discussed.
Assuntos
Aminoidrolases/isolamento & purificação , Rhodococcus/enzimologia , Aminoácidos/análise , Aminoidrolases/antagonistas & inibidores , Aminoidrolases/imunologia , Cromatografia/métodos , Estabilidade Enzimática , Hidroliases/análise , Concentração de Íons de Hidrogênio , Cinética , Oxirredução , Especificidade por Substrato , TemperaturaRESUMO
Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate resulting in the decomposition of cyanate to ammonia and bicarbonate. In this study, the role of the single sulfhydryl group in each of the eight identical subunits of cyanase was investigated. Tetranitromethane, methyl methanethiosulfonate, N-ethylmaleimide, and Hg2+ all reacted with the sulfhydryl group to give derivatives which had reduced activities and which dissociated reversibly to inactive dimer. Association of inactive dimer to active octamer was facilitated by the presence of azide (cyanate analog) and bicarbonate, increased temperature and enzyme concentration, and presence of phosphate. Nitration of tyrosine residues by tetranitromethane occurred only in the absence of azide and bicarbonate, suggesting that at least some of the tyrosine residues become exposed when octamer dissociates to dimer. Site-directed mutagenesis was used to prepare a mutant enzyme in which serine was substituted for cysteine. The mutant enzyme was catalytically active and had properties very similar to native enzyme, except that it was less stable to treatment with urea and to high temperatures. These results establish that in native cyanase the sulfhydryl group per se is not required for catalytic activity, but it may play a role in stabilizing octameric structure, and that octameric structure is required for catalytic activity.