RESUMO
Several recent preclinical studies have demonstrated that simultaneously blocking exogenous and endogenous sources of serine in malignant cells mediates superior anticancer effects as compared with limiting either source alone. Here, we critically summarize key developments in targeting serine to treat cancer and discuss persisting challenges for implementing such a therapeutic approach in patients.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Dieta com Restrição de Proteínas , Neoplasias/terapia , Serina/antagonistas & inibidores , Antimetabólitos Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Terapia Combinada/métodos , Proteínas Alimentares/efeitos adversos , Proteínas Alimentares/metabolismo , Humanos , Neoplasias/metabolismo , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Fosfoglicerato Desidrogenase/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Serina/biossíntese , Transaminases/antagonistas & inibidores , Transaminases/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cytarabine is a key chemotherapy drug for treating leukemia; however, chemotherapyinduced multidrug resistance is a major cause of therapy failure or tumor recurrence. Current medical treatment strategies still cannot address the issue of multidrug resistance phenotypes in the treatment of leukemia. Curcumin counteracts tumor development by inducing apoptosis in cytarabineresistant acute myeloid leukemia cells. Branchedchain amino acid transaminase 1 (BCAT1), an aminotransferase enzyme, acts on branchedchain amino acids. Moreover, the aberrant expression of BCAT1 has been observed in numerous cancer cells, and BCAT1 serves a critical role in the progression of myeloid leukemia. BCAT1 can interfere with cancer cell proliferation by regulating mTORmediated mitochondrial biogenesis and function. The present study aimed to investigate whether curcumin induces apoptosis by regulating BCAT1 expression and mTOR signaling in cytarabineresistant myeloid leukemia cells. Four leukemia cell lines and three primary myeloid leukemia cells were treated with curcumin, and the expression and activity of BCAT1 and mTOR were investigated by reverse transcriptionquantitative PCR, western blotting and αKG quantification assay. The results demonstrated that curcumin inhibited BCAT1 expression in Kasumi1, KG1, HL60, cytarabineresistant HL60, and cytarabineresistant primary myeloid leukemia cells. Notably, tetrahydrocurcumin, a major metabolite of curcumin, and cytarabine had no inhibitory effect on BCAT1 expression. Furthermore, BCAT1 and mTOR signaling may modulate each other in cytarabineresistant HL60 cells. The present results indicated that curcumin may induce apoptosis by inhibiting the BCAT1 and mTOR pathways. Thus, understanding the mechanism underlying curcumininduced apoptosis in cytarabineresistant cells can support the development of novel drugs for leukemia.
Assuntos
Curcumina/farmacologia , Citarabina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Transaminases/antagonistas & inibidores , Adolescente , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Criança , Feminino , Humanos , Indóis/farmacologia , Ácidos Cetoglutáricos/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Purinas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Transaminases/genética , Transaminases/metabolismoRESUMO
AIMS: Gastric cancer stem cells (GCSCs) have been used as a therapeutic target. This study aims to estimate the role of miR-98-5p (termed miR-98) in the development of GCSCs. MAIN METHODS: The expression of miR-98 in CD44+ GCSCs was verified by RT-PCR. The miR-98 was overexpressed in CD44+ GCSCs by Lentivirus. The ability of self-renewal, invasion, chemoresistance and tumorigenicity was detected in vitro or in vivo after overexpression of miR-98. The target genes of miR-98 were predicted and verified by luciferase reporter assays. The effects miR-98/BCAT1 signaling on the chemoresistance and tumorigenicity of CD44+ GCSCs were investigated in a xenograft model by rescue experiments. KEY FINDINGS: We have shown that miR-98 was decreased in CD44+ GCSCs. The overexpression of miR-98 could inhibit the expression of stem-related genes and the ability of self-renewal, invasion, and tumorigenicity of GCSCs. Also, we found that miR-98 overexpression enhances the sensitivity to cisplatin treatment in vitro. Using a xenograft model, we showed that miR-98 overexpression reversed paclitaxel resistance to CD44+ GCSCs. Finally, we found that branched-chain aminotransferases 1 (BCAT1) is a target gene of miR-98. Overexpressed BCAT1 reversed xenograft tumor formation ability and attenuated the paclitaxel chemosensitivity induced by miR-98 downregulation. Furthermore, BCAT1 restoration affected the expression of invasion and drug resistance-related genes. SIGNIFICANCE: This study revealed miR-98 inhibits gastric cancer cell stemness and chemoresistance by targeting BCAT1, suggesting that this miR-98/BCAT1 axis represents a potential therapeutic target in gastric cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Transaminases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Cisplatino/farmacologia , Humanos , Receptores de Hialuronatos/metabolismo , Masculino , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Prognóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Transaminases/genética , Transaminases/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hypoxia-inducible factors (HIFs) mediate metabolic reprogramming in response to hypoxia. However, the role of HIFs in branched-chain amino acid (BCAA) metabolism remains unknown. Here we show that hypoxia upregulates mRNA and protein levels of the BCAA transporter LAT1 and the BCAA metabolic enzyme BCAT1, but not their paralogs LAT2-4 and BCAT2, in human glioblastoma (GBM) cell lines as well as primary GBM cells. Hypoxia-induced LAT1 protein upregulation is mediated by both HIF-1 and HIF-2 in GBM cells. Although both HIF-1α and HIF-2α directly bind to the hypoxia response element at the first intron of the human BCAT1 gene, HIF-1α is exclusively responsible for hypoxia-induced BCAT1 expression in GBM cells. Knockout of HIF-1α and HIF-2α significantly reduces glutamate labeling from BCAAs in GBM cells under hypoxia, which provides functional evidence for HIF-mediated reprogramming of BCAA metabolism. Genetic or pharmacological inhibition of BCAT1 inhibits GBM cell growth under hypoxia. Together, these findings uncover a previously unrecognized HIF-dependent metabolic pathway that increases GBM cell growth under conditions of hypoxic stress.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Sistemas CRISPR-Cas/genética , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Glioblastoma/metabolismo , Glioblastoma/patologia , Ácido Glutâmico/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Ligação Proteica , Transaminases/antagonistas & inibidores , Transaminases/genética , Transaminases/metabolismoRESUMO
Uncontrolled proliferation and altered metabolic reprogramming are hallmarks of cancer. Active glycolysis and glutaminolysis are characteristic features of these hallmarks and required for tumorigenesis. A fine balance between cancer metabolism and autophagy is a prerequisite of homeostasis within cancer cells. Here we show that glutamate pyruvate transaminase 2 (GPT2), which serves as a pivot between glycolysis and glutaminolysis, is highly upregulated in aggressive breast cancers, particularly the triple-negative breast cancer subtype. Abrogation of this enzyme results in decreased tricarboxylic acid cycle intermediates, which promotes the rewiring of glucose carbon atoms and alterations in nutrient levels. Concordantly, loss of GPT2 results in an impairment of mechanistic target of rapamycin complex 1 activity as well as the induction of autophagy. Furthermore, in vivo xenograft studies have shown that autophagy induction correlates with decreased tumor growth and that markers of induced autophagy correlate with low GPT2 levels in patient samples. Taken together, these findings indicate that cancer cells have a close network between metabolic and nutrient sensing pathways necessary to sustain tumorigenesis and that aminotransferase reactions play an important role in maintaining this balance.
Assuntos
Autofagia/genética , Regulação Neoplásica da Expressão Gênica , Transaminases/genética , Neoplasias de Mama Triplo Negativas/genética , Carga Tumoral/genética , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Feminino , Técnicas de Inativação de Genes , Humanos , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Interferência de RNA , Análise de Sobrevida , Transaminases/antagonistas & inibidores , Transaminases/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/terapia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Esophageal squamous cell carcinoma (ESCC) is the main subtype of esophageal cancer in China, and the prognosis of patients remains poor mainly due to the occurrence of lymph node and distant metastasis. The long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) has been shown to have tumorsuppressive properties and to play an important role in epithelialtomesenchymal transition (EMT) in some solid tumors. However, whether MEG3 is involved in EMT in ESCC remains unclear. In the present study, the MEG3 expression level and its association with tumorigenesis were determined in 43 tumor tissues of patients with ESCC and in ESCC cells using reverse transcriptionquantitative PCR analysis. Gene microarray analysis was performed to detect differentially expressed genes (DEGs). Based on the functional annotation results, the effects of ectopic expression of MEG3 on cell growth, migration, invasion and EMT were assessed. MEG3 expression level was found to be markedly lower in tumor tissues and cells. Statistical analysis revealed that MEG3 expression was significantly negatively associated with lymph node metastasis and TNM stage in ESCC. Fluorescence in situ hybridization assay demonstrated that MEG3 was expressed mainly in the nucleus. Ectopic expression of MEG3 inhibited cell proliferation, migration, invasion and cell cycle progression in EC109 cells. Gene microarray results demonstrated that 177 genes were differentially expressed ≥2.0 fold in MEG3overexpressing cells, including 23 upregulated and 154 downregulated genes. Functional annotation revealed that the DEGs were mainly involved in amino acid biosynthetic process, mitogenactivated protein kinase signaling, and serine and glycine metabolism. Further experiments indicated that the ectopic expression of MEG3 significantly suppressed cell proliferation, migration, invasion and EMT by downregulating phosphoserine aminotransferase 1 (PSAT1). In pathological tissues, PSAT1 and MEG3 were significantly negatively correlated, and high expression of PSAT1 predicted poor survival. Taken together, these results suggest that MEG3 may be a useful prognostic biomarker and may suppress EMT by inhibiting the PSAT1dependent glycogen synthase kinase3ß/Snail signaling pathway in ESCC.
Assuntos
Transição Epitelial-Mesenquimal/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , Fatores de Transcrição da Família Snail/antagonistas & inibidores , Transaminases/antagonistas & inibidores , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Regulação para Baixo , Transição Epitelial-Mesenquimal/fisiologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Xenoenxertos , Humanos , Metástase Linfática , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , RNA Longo não Codificante/genética , Transdução de Sinais , Fatores de Transcrição da Família Snail/metabolismo , Taxa de Sobrevida , Transaminases/metabolismoRESUMO
The targeting of glutamine metabolism specifically via pharmacological inhibition of glutaminase 1 (GLS1) has been translated into clinical trials as a novel therapy for several cancers. The results, though encouraging, show room for improvement in terms of tumor reduction. In this study, the glutaminase II pathway is found to be upregulated for glutamate production upon GLS1 inhibition in pancreatic tumors. Moreover, genetic suppression of glutamine transaminase K (GTK), a key enzyme of the glutaminase II pathway, leads to the complete inhibition of pancreatic tumorigenesis in vivo unveiling GTK as a new metabolic target for cancer therapy. These results suggest that current trials using GLS1 inhibition as a therapeutic approach targeting glutamine metabolism in cancer should take into account the upregulation of other metabolic pathways that can lead to glutamate production; one such pathway is the glutaminase II pathway via GTK.
Assuntos
Inibidores Enzimáticos/farmacologia , Glutaminase/genética , Liases/genética , Neoplasias Pancreáticas/tratamento farmacológico , Transaminases/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Glutaminase/antagonistas & inibidores , Glutamina/genética , Glutamina/metabolismo , Humanos , Liases/antagonistas & inibidores , Redes e Vias Metabólicas/efeitos dos fármacos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Transaminases/antagonistas & inibidoresRESUMO
Neurodegeneration in diabetic retina has been widely considered as initiating factor that may lead to vascular damage, the classical hallmark of diabetic retinopathy. Diabetes induced altered glutamate metabolism in the retina, especially through glutamate excitotoxicity might play a major role in the neurodegeneration. Increased level of branched chain amino acids (BCAAs) measured in diabetic retina might cause an increase in the neurotoxic level of glutamate by transamination of citric acid cycle intermediates. In order to analyze the transamination of BCAAs and their influence on neurodegenerative factors, we treated streptozotocin-induced diabetic rats with gabapentin, a leucine analogue and an inhibitor of branched chain amino transferase (BCATc). Interestingly, gabapentin lowered the retinal level of BCAAs in diabetic rats. Furthermore, gabapentin treatments ameliorated the reduced antioxidant glutathione level and increased malondialdehyde (MDA), the marker of lipid peroxidation in diabetic rat retinas. In addition, gabapentin also reduced the expression of proapoptotic caspase-3, a marker of apoptosis and increased anti-apoptotic marker Bcl-2 in diabetic retinas. Thus, these results suggest that gabapentin stimulates glutamate disposal, and ameliorates apoptosis and oxidative stress in diabetic rat retina. The influence of gabapentin may be due to its capacity to increase the ratio of BCKA to BCAA which in turn would reduce glutamate excitotoxicity in diabetic retina.
Assuntos
Apoptose/efeitos dos fármacos , Retinopatia Diabética/metabolismo , Gabapentina/administração & dosagem , Ácido Glutâmico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Aminoácidos/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Glutationa/metabolismo , Peroxidação de Lipídeos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Estreptozocina/administração & dosagem , Transaminases/antagonistas & inibidoresRESUMO
IDH1 mutations are common in low-grade gliomas and secondary glioblastomas and cause overproduction of (R)-2HG. (R)-2HG modulates the activity of many enzymes, including some that are linked to transformation and some that are probably bystanders. Although prior work on (R)-2HG targets focused on 2OG-dependent dioxygenases, we found that (R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect, thereby decreasing glutamate levels and increasing dependence on glutaminase for the biosynthesis of glutamate and one of its products, glutathione. Inhibiting glutaminase specifically sensitized IDH mutant glioma cells to oxidative stress in vitro and to radiation in vitro and in vivo. These findings highlight the complementary roles for BCATs and glutaminase in glutamate biosynthesis, explain the sensitivity of IDH mutant cells to glutaminase inhibitors, and suggest a strategy for maximizing the effectiveness of such inhibitors against IDH mutant gliomas.
Assuntos
Glioma/metabolismo , Ácido Glutâmico/biossíntese , Transaminases/fisiologia , Linhagem Celular Tumoral , Glioma/fisiopatologia , Ácido Glutâmico/efeitos dos fármacos , Glutaratos/metabolismo , Glutaratos/farmacologia , Homeostase/efeitos dos fármacos , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/fisiologia , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/fisiologia , Mutação , Oxirredução/efeitos dos fármacos , Proteínas da Gravidez/genética , Proteínas da Gravidez/fisiologia , Transaminases/antagonistas & inibidores , Transaminases/genéticaRESUMO
BACKGROUND: Resistance to radiotherapy is one of the main obstacles to improving cancer prognoses. To effectively destroy cancer cells, novel radiation sensitizers are needed. Recently, several natural products have been shown to exhibit promising tumor-killing properties. However, little is known about the specific mechanisms of these natural compounds on cancer treatment. In this study, after screening a high-throughput natural product library, we identified tanshinone I (Tan I) as a potential radiation sensitizer in lung cancer cells. METHODS: Lung cancer radioresistant cell lines, H358-IR and H157-IR, were first established to confirm the radioresistant phenotypes. After that, a natural product library was used to screen the potential radiation sensitizer. We further examined the inhibition functions of Tan I on radioresistant cancer cells via a series of experiments. RESULTS: Tan I significantly inhibited cell proliferation and clone formation, consequently enhancing radiosensitivity in radioresistant lung cancer cells, H358-IR and H157-IR. Stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics indicated that Tan I downregulates expression of pro-oncogenic protein phosphoribosyl pyrophosphate aminotransferase (PPAT) in both H358-IR and H157-IR cells. Further analysis of molecular docking showed that Tan I is well-docked into the active pocket of the structure of PPAT, serving as a potential PPAT inhibitor. CONCLUSIONS: Taken together, these findings suggest that inhibition of the tumor promoter PPAT by Tan I exerts marked inhibitory effects on radioresistant lung cancer cells, improving radiation efficacy.
Assuntos
Abietanos/farmacologia , Neoplasias Pulmonares/terapia , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Transaminases/antagonistas & inibidores , Abietanos/química , Abietanos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Quimiorradioterapia/métodos , Regulação para Baixo , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Neoplasias Pulmonares/patologia , Simulação de Acoplamento Molecular , Radiossensibilizantes/química , Radiossensibilizantes/uso terapêutico , Transaminases/química , Transaminases/metabolismoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is extremely refractory to the therapeutic approaches that have been evaluated to date. Recently, it has been demonstrated that PDAC tumors are dependent upon a metabolic pathway involving aspartate aminotransferase 1, also known as glutamate-oxaloacetate transaminase 1 (GOT1), for the maintenance of redox homeostasis and sustained proliferation. As such, small molecule inhibitors targeting this metabolic pathway may provide a novel therapeutic approach for the treatment of this devastating disease. To this end, from a high throughput screen of â¼800,000 molecules, 4-(1H-indol-4-yl)-N-phenylpiperazine-1-carboxamide was identified as an inhibitor of GOT1. Mouse pharmacokinetic studies revealed that potency, rather than inherent metabolic instability, would limit immediate cell- and rodent xenograft-based experiments aimed at validating this potential cancer metabolism-related target. Medicinal chemistry-based optimization resulted in the identification of multiple derivatives with >10-fold improvements in potency, as well as the identification of a tryptamine-based series of GOT1 inhibitors.
Assuntos
Aspartato Aminotransferases/antagonistas & inibidores , Inibidores Enzimáticos/uso terapêutico , Indóis/uso terapêutico , Compostos de Fenilureia/uso terapêutico , Piperazinas/uso terapêutico , Transaminases/antagonistas & inibidores , Animais , Aspartato Aminotransferase Citoplasmática , Carcinoma Ductal Pancreático/tratamento farmacológico , Descoberta de Drogas , Estabilidade de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Feminino , Indóis/síntese química , Indóis/química , Indóis/farmacocinética , Camundongos , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacocinética , Piperazinas/síntese química , Piperazinas/química , Piperazinas/farmacocinética , Relação Estrutura-AtividadeRESUMO
The mammalian kynurenine aminotransferase (KAT) enzymes are a family of related isoforms that are pyridoxal 5'-phosphate-dependent, responsible for the irreversible transamination of kynurenine to kynurenic acid. Kynurenic acid is implicated in human diseases such as schizophrenia where it is found in elevated levels and consequently KAT-II, as the isoform predominantly responsible for kynurenic acid production in the brain, has been targeted for the development of specific inhibitors. One class of compounds that have also shown inhibitory activity towards the KAT enzymes are estrogens and their sulfate esters. Estradiol disulfate in particular is very strongly inhibitory and it appears that the 17-sulfate makes a significant contribution to its potency. The work here demonstrates that the effect of this moiety can be mirrored in existing KAT-II inhibitors, from the development of two novel inhibitors, JN-01 and JN-02. Both inhibitors were based on NS-1502 (IC50: 315 µM), but the deliberate placement of a sulfonamide group significantly improved the potency of JN-01 (IC50: 73.8 µM) and JN-02 (IC50: 112.8 µM) in comparison to the parent compound. This 3-4 fold increase in potency shows the potential of these moieties to be accommodated in the KAT-II active site and the effect they can have on improving inhibitors, and the environments in the KAT-II have been suitably modelled using docking calculations.
Assuntos
Inibidores Enzimáticos/síntese química , Ésteres/síntese química , Estradiol/análogos & derivados , Sulfatos/síntese química , Transaminases/antagonistas & inibidores , Domínio Catalítico , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Ésteres/química , Ésteres/metabolismo , Ésteres/farmacologia , Estradiol/química , Estradiol/farmacologia , Ácido Cinurênico/química , Ácido Cinurênico/metabolismo , Cinurenina/química , Cinurenina/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Mimetismo Molecular , Sulfatos/química , Sulfatos/metabolismo , Sulfatos/farmacologia , Transaminases/química , Transaminases/metabolismoRESUMO
Aromatic amino acids are protein building blocks and precursors to a number of plant natural products, such as the structural polymer lignin and a variety of medicinally relevant compounds. Plants make tyrosine and phenylalanine by a different pathway from many microbes; this pathway requires prephenate aminotransferase (PAT) as the key enzyme. Prephenate aminotransferase produces arogenate, the unique and immediate precursor for both tyrosine and phenylalanine in plants, and also has aspartate aminotransferase (AAT) activity. The molecular mechanisms governing the substrate specificity and activation or inhibition of PAT are currently unknown. Here we present the X-ray crystal structures of the wild-type and various mutants of PAT from Arabidopsis thaliana (AtPAT). Steady-state kinetic and ligand-binding analyses identified key residues, such as Glu108, that are involved in both keto acid and amino acid substrate specificities and probably contributed to the evolution of PAT activity among class Ib AAT enzymes. Structures of AtPAT mutants co-crystallized with either α-ketoglutarate or pyridoxamine 5'-phosphate and glutamate further define the molecular mechanisms underlying recognition of keto acid and amino acid substrates. Furthermore, cysteine was identified as an inhibitor of PAT from A. thaliana and Antirrhinum majus plants as well as the bacterium Chlorobium tepidum, uncovering a potential new effector of PAT.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Transaminases/metabolismo , Aminoácidos/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/antagonistas & inibidores , Domínio Catalítico , Cristalografia por Raios X , Cisteína/farmacologia , Cetoácidos/metabolismo , Redes e Vias Metabólicas , Conformação Proteica , Especificidade por Substrato , Transaminases/antagonistas & inibidoresRESUMO
The kynurenine aminotransferase (KAT) enzymes are pyridoxal 5'-phosphate-dependent homodimers that catalyse the irreversible transamination of kynurenine into kynurenic acid (KYNA) in the tryptophan metabolic pathway. Kynurenic acid is implicated in cognitive diseases such as schizophrenia, and several inhibitors have been reported that selectively target KAT-II as it is primarily responsible for kynurenic acid production in the human brain. Not only is schizophrenia a sexually dimorphic condition, but women that have schizophrenia have reduced estrogen levels in their serum. Estrogens are also known to interact in the kynurenine pathway therefore exploring these interactions can yield a better understanding of the condition and improve approaches in ameliorating its effects. Enzyme inhibitory assays and binding studies showed that estradiol disulfate is a strong inhibitor of KAT-I and KAT-II (IC50: 291.5 µM and 26.3 µM, respectively), with estradiol, estradiol 3-sulfate and estrone sulfate being much weaker (IC50 > 2 mM). Therefore it is possible that estrogen levels can dictate the balance of kynurenic acid in the brain. Inhibition assay results and modelling suggests that the 17-sulfate moiety in estradiol disulfate is very important in improving its potency as an inhibitor, increasing the inhibition by approximately 10-100 fold compared to estradiol.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Estrogênios/química , Estrogênios/farmacologia , Transaminases/antagonistas & inibidores , Domínio Catalítico , Desenho de Fármacos , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Modelos Moleculares , Transaminases/químicaRESUMO
Disrupted kynurenine pathway (KP) metabolism has been implicated in the progression of neurodegenerative disease, psychiatric disorders and cancer. Modulation of enzyme activity along this pathway may therefore offer potential new therapeutic strategies for these conditions. Considering their prominent positions in the KP, the enzymes indoleamine 2,3-dioxygenase, kynurenine 3-monooxygenase and kynurenine aminotransferase, appear the most attractive targets. Already, increasing interest in this pathway has led to the identification of a number of potent and selective enzyme inhibitors with promising pre-clinical data and the elucidation of several enzyme crystal structures provides scope to rationalize the molecular mechanisms of inhibitor activity. The field seems poised to yield one or more inhibitors that should find clinical utility.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Quinurenina 3-Mono-Oxigenase/antagonistas & inibidores , Cinurenina/metabolismo , Transaminases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Quinurenina 3-Mono-Oxigenase/metabolismo , Transaminases/metabolismoRESUMO
MTB lysine-É-aminotransferase (LAT) was found to play a crucial role in persistence and antibiotic tolerance. LAT serves as a potential target in the management of latent tuberculosis. In present work we attempted to derivatize the benzothiazole lead identified through high throughput virtual screening of Birla Institute of Technology and Science in house database. For Structure activity relationship purpose 22 derivatives were synthesized and characterized. Among synthesized compounds, eight compounds were found to be more efficacious in terms of LAT inhibition when compared to lead compound (IC50 10.38±1.21µM). Compound 22 exhibits bactericidal action against nutrient starved Mycobacterium tuberculosis (MTB). It also exhibits significant activity in nutrient starvation model (2.9log folds) and biofilm model (2.3log folds).
Assuntos
Antituberculosos/química , Proteínas de Bactérias/antagonistas & inibidores , Benzotiazóis/química , Inibidores Enzimáticos/química , Mycobacterium tuberculosis/metabolismo , Transaminases/antagonistas & inibidores , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Benzotiazóis/metabolismo , Benzotiazóis/farmacologia , Sítios de Ligação , Domínio Catalítico , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Relação Estrutura-Atividade , Transaminases/metabolismoRESUMO
Antiestrogen-resistant and triple-negative breast tumors pose a serious clinical challenge because of limited treatment options. We assessed global gene expression changes in antiestrogen-sensitive compared with antiestrogen-resistant (two tamoxifen resistant and two fulvestrant resistant) MCF-7 breast cancer cell lines. The branched-chain amino acid transaminase 1 (BCAT1), which catalyzes the first step in the breakdown of branched-chain amino acids, was among the most upregulated transcripts in antiestrogen-resistant cells. Elevated BCAT1 expression was confirmed in relapsed tamoxifen-resistant breast tumor specimens. High intratumoral BCAT1 levels were associated with a reduced relapse-free survival in adjuvant tamoxifen-treated patients and overall survival in unselected patients. On a tissue microarray (n=1421), BCAT1 expression was detectable in 58% of unselected primary breast carcinomas and linked to a higher Ki-67 proliferation index, as well as histological grade. Interestingly, BCAT1 was predominantly expressed in estrogen receptor-α-negative/human epidermal growth factor receptor-2-positive (ERα-negative/HER-2-positive) and triple-negative breast cancers in independent patient cohorts. The inverse relationship between BCAT1 and ERα was corroborated in various breast cancer cell lines and pharmacological long-term depletion of ERα induced BCAT1 expression in vitro. Mechanistically, BCAT1 indirectly controlled expression of the cell cycle inhibitor p27Kip1 thereby affecting pRB. Correspondingly, phenotypic analyses using a lentiviral-mediated BCAT1 short hairpin RNA knockdown revealed that BCAT1 sustains proliferation in addition to migration and invasion and that its overexpression enhanced the capacity of antiestrogen-sensitive cells to grow in the presence of antiestrogens. Importantly, silencing of BCAT1 in an orthotopic triple-negative xenograft model resulted in a massive reduction of tumor volume in vivo, supporting our findings that BCAT1 is necessary for the growth of hormone-independent breast tumors.
Assuntos
Neoplasias da Mama/metabolismo , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/metabolismo , Transaminases/genética , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Tamoxifeno/farmacologia , Transaminases/antagonistas & inibidores , Transaminases/biossíntese , Transaminases/metabolismo , Regulação para CimaRESUMO
Ovarian cancer is one of the leading causes of death in the world, which is linked to its resistance to chemotherapy. Strategies to overcome chemoresistance have been keenly investigated. Culturing cancer cells in suspension, which results in formation of spheroids, is a more accurate reflection of clinical cancer behavior in vitro than conventional adherent cultures. By performing RNA-seq analysis, we found that the focal adhesion pathway was essential in spheroids. The phosphorylation of focal adhesion kinase (FAK) was increased in spheroids compared to adherent cells, and inhibition of FAK in spheroids resulted in inhibition of the downstream mammalian target of the rapamycin (mTOR) pathway in ovarian clear cell carcinomas. This result also suggested that only using a FAK inhibitor might have limitations because the phosphorylation level of FAK could not be reduced to the level in adherent cells, and it appeared that some combination therapies might be necessary. We previously reported that glutamine and glutamate concentrations were higher in spheroids than adherent cells, and we investigated a synergistic effect targeting glutamine metabolism with FAK inhibition on the mTOR pathway. The combination of AOA, a pan-transaminase inhibitor, and PF 573228, a FAK inhibitor, additively inhibited the mTOR pathway in spheroids from ovarian clear cell carcinomas. Our in vitro study proposed a rationale for the positive and negative effects of using FAK inhibitors in ovarian clear cell carcinomas and suggested that targeting glutamine metabolism could overcome the limitation of FAK inhibitors by additively inhibiting the mTOR pathway.
Assuntos
Adenocarcinoma de Células Claras/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Quinase 1 de Adesão Focal/metabolismo , Glutamina/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Ácido Amino-Oxiacético/uso terapêutico , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Quimioterapia Combinada , Feminino , Quinase 1 de Adesão Focal/antagonistas & inibidores , Humanos , Fosforilação , Quinolonas/uso terapêutico , RNA Mensageiro/genética , Análise de Sequência de RNA , Esferoides Celulares , Sulfonas/uso terapêutico , Transaminases/antagonistas & inibidoresRESUMO
Branched-chain amino acids (BCAAs) are important nutrient signals that have direct and indirect effects. BCAA catabolism is a conserved regulator of physiological aging and participates in diverse physiological and pathological processes, including carcinoma development. The roles of BCAA catabolism in human breast cancer remains unknown. Here we provide evidence that BCAA catabolism is involved in human breast cancer. The plasma and tissue levels of BCAAs are increased in breast cancer, which is accompanied by the elevated expression of the catabolic enzymes, including branched-chain amino acid transaminase 1 (BCAT1). Knockdown of BCAT1 represses the growth rate and colony formation capacity of breast cancer cells, opposing results are observed when BCAT1 is overexpressed. BCAT1 can promote mitochondrial biogenesis, ATP production and repress mitochondrial ROS in breast cancer cells by regulating the expression of related genes. Mechanism study reveals that BCAT1 activates the mTOR, but not AMPK or SIRT1, signaling to promote mitochondrial biogenesis and function, and subsequently facilitates growth and colony formation of breast cancer cells. Taken together, we demonstrate that BCAA catabolism is activated in human breast cancer, and abolishment of BCAA catabolism by knocking down BCAT1 inhibits breast cancer cell growth by repressing mTOR-mediated mitochondrial biogenesis and function.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Neoplasias da Mama/genética , Carcinoma/genética , Regulação Neoplásica da Expressão Gênica , Serina-Treonina Quinases TOR/genética , Transaminases/genética , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Células MCF-7 , Mitocôndrias/metabolismo , Biogênese de Organelas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transaminases/antagonistas & inibidores , Transaminases/metabolismoRESUMO
BACKGROUND: Phosphoserine aminotransferase (PSAT) catalyses the second reversible step of the phosphoserine biosynthetic pathway in Trichomonas vaginalis, which is crucial for the synthesis of serine and cysteine. METHODS: PSAT from T. vaginalis (TvPSAT) was analysed using X-ray crystallography, enzyme kinetics, and molecular dynamics simulations. RESULTS: The crystal structure of TvPSAT was determined to 2.15Å resolution, and is the first protozoan PSAT structure to be reported. The active site of TvPSAT structure was found to be in a closed conformation, and at the active site PLP formed an internal aldimine linkage to Lys 202. In TvPSAT, Val 340 near the active site while it is Arg in most other members of the PSAT family, might be responsible in closing the active site. Kinetic studies yielded Km values of 54 µM and 202 µM for TvPSAT with OPLS and AKG, respectively. Only iodine inhibited the TvPSAT activity while smaller halides could not inhibit. CONCLUSION: Results from the structure, comparative molecular dynamics simulations, and the inhibition studies suggest that iodine is the only halide that can bind TvPSAT strongly and may thus inhibit the activity of TvPSAT. The long loop between ß8 and α8 at the opening of the TvPSAT active site cleft compared to other PSATs, suggests that this loop may help control the access of substrates to the TvPSAT active site and thus influences the enzyme kinetics. GENERAL SIGNIFICANCE: Our structural and functional studies have improved our understanding of how PSAT helps this organism persists in the environment.