RESUMEN
The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from Candidatus Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (Km, 146.6 µM and Vmax, 0.95 µmol/min/mg) and AICAR (Km, 34.81 µM and Vmax, 0.56 µmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (Km, 1.81 µM and Vmax, 2.87 µmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In-silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 µM and 34.2 µM, respectively) compared to AICAR (Kd, 83.4 µM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing CLas and its unique cell protective, wound-healing properties for future biotechnological applications.
Asunto(s)
Aminoimidazol Carboxamida , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/química , Aminoimidazol Carboxamida/metabolismo , Aminoimidazol Carboxamida/farmacología , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/química , Simulación del Acoplamiento Molecular , Ribonucleótidos/metabolismo , Ribonucleótidos/química , Cinética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/antagonistas & inhibidores , Nucleótido Desaminasas/metabolismo , Nucleótido Desaminasas/química , Nucleótido Desaminasas/genética , Especificidad por Sustrato , Proliferación Celular/efectos de los fármacos , Transferasas de Hidroximetilo y Formilo/metabolismo , Transferasas de Hidroximetilo y Formilo/química , Transferasas de Hidroximetilo y Formilo/genética , Transferasas de Hidroximetilo y Formilo/antagonistas & inhibidores , Complejos MultienzimáticosRESUMEN
We assessed the effects of two levels of calorie restriction (CR; eating either 15% or 35% less than ad libitum, AL, food intake for 8 weeks) by 24-month-old female and male rats on glucose uptake (GU) and phosphorylation of key signaling proteins (Akt; AMP-activated protein kinase, AMPK; Akt substrate of 160 kDa, AS160) measured in isolated skeletal muscles that underwent four incubation conditions (without either insulin or AICAR, an AMPK activator; with AICAR alone; with insulin alone; or with insulin and AICAR). Regardless of sex: (1) neither CR group versus the AL group had greater GU by insulin-stimulated muscles; (2) phosphorylation of Akt in insulin-stimulated muscles was increased in 35% CR versus AL rats; (3) prior AICAR treatment of muscle resulted in greater GU by insulin-stimulated muscles, regardless of diet; and (4) AICAR caused elevated phosphorylation of acetyl CoA carboxylase, an indicator of AMPK activation, in all diet groups. There was a sexually dimorphic diet effect on AS160 phosphorylation, with 35% CR exceeding AL for insulin-stimulated muscles in male rats, but not in female rats. Our working hypothesis is that the lack of a CR-effect on GU by insulin-stimulated muscles was related to the extended duration of the ex vivo incubation period (290 min compared to 40-50 min that was previously reported to be effective). The observed efficacy of prior treatment of muscles with AICAR to improve glucose uptake in insulin-stimulated muscles supports the strategy of targeting AMPK with the goal of improving insulin sensitivity in older females and males.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Aminoimidazol Carboxamida , Restricción Calórica , Glucosa , Insulina , Músculo Esquelético , Proteínas , Proteínas Proto-Oncogénicas c-akt , Ribonucleótidos , Transducción de Señal , Animales , Femenino , Masculino , Ratas , Acetil-CoA Carboxilasa/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Glucosa/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Hipoglucemiantes/farmacología , Músculo Esquelético/metabolismo , Músculo Esquelético/efectos de los fármacos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ribonucleótidos/farmacología , Factores Sexuales , Transducción de Señal/efectos de los fármacos , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismoRESUMEN
The fungal pathogen Cryptococcus neoformans is a leading cause of meningoencephalitis in the immunocompromised. As current antifungal treatments are toxic to the host, costly, limited in their efficacy, and associated with drug resistance, there is an urgent need to identify vulnerabilities in fungal physiology to accelerate antifungal discovery efforts. Rational drug design was pioneered in de novo purine biosynthesis as the end products of the pathway, ATP and GTP, are essential for replication, transcription, and energy metabolism, and the same rationale applies when considering the pathway as an antifungal target. Here, we describe the identification and characterization of C. neoformans 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/5'-inosine monophosphate cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final two enzymatic steps in the formation of the first purine base inosine monophosphate. We demonstrate that mutants lacking the ATIC-encoding ADE16 gene are adenine and histidine auxotrophs that are unable to establish an infection in a murine model of virulence. In addition, our assays employing recombinantly expressed and purified C. neoformans ATIC enzyme revealed Km values for its substrates AICAR and 5-formyl-AICAR are 8-fold and 20-fold higher, respectively, than in the human ortholog. Subsequently, we performed crystallographic studies that enabled the determination of the first fungal ATIC protein structure, revealing a key serine-to-tyrosine substitution in the active site, which has the potential to assist the design of fungus-specific inhibitors. Overall, our results validate ATIC as a promising antifungal drug target.
Asunto(s)
Criptococosis , Cryptococcus neoformans , Transferasas de Hidroximetilo y Formilo , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa , Animales , Humanos , Ratones , Antifúngicos , Cryptococcus neoformans/enzimología , Cryptococcus neoformans/genética , Descubrimiento de Drogas , Inosina Monofosfato , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/química , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/genética , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Purinas , Criptococosis/metabolismoRESUMEN
We discovered 6-substituted thieno[2,3-d]pyrimidine compounds (3-9) with 3-4 bridge carbons and side-chain thiophene or furan rings for dual targeting one-carbon (C1) metabolism in folate receptor- (FR) expressing cancers. Synthesis involved nine steps starting from the bromo-aryl carboxylate. From patterns of growth inhibition toward Chinese hamster ovary cells expressing FRα or FRß, the proton-coupled folate transporter or reduced folate carrier, specificity for uptake by FRs was confirmed. Anti-proliferative activities were demonstrated toward FRα-expressing KB tumor cells and NCI-IGROV1 ovarian cancer cells. Inhibition of de novo purine biosynthesis at both 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and glycinamide ribonucleotide formyltransferase (GARFTase) was confirmed by metabolite rescue, metabolomics and enzyme assays. X-ray crystallographic structures were obtained with compounds 3-5 and human GARFTase. Our studies identify first-in-class C1 inhibitors with selective uptake by FRs and dual inhibition of enzyme targets in de novo purine biosynthesis, resulting in anti-tumor activity. This series affords an exciting new platform for selective multi-targeted anti-tumor agents.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/antagonistas & inhibidores , Fosforribosilglicinamida-Formiltransferasa/antagonistas & inhibidores , Pirimidinas/farmacología , Tiofenos/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Células CHO , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cricetulus , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Receptores de Folato Anclados a GPI/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Fosforribosilglicinamida-Formiltransferasa/metabolismo , Unión Proteica , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/metabolismoRESUMEN
Dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/IMP cyclohydrolase (PurH) play key roles in maintaining folate pools in cells, and are targets of antimicrobial and anticancer drugs. While the activities of bacterial DHFR and PurH on their classical substrates (DHF and 10-CHO-THF, respectively) are known, their activities and kinetic properties of utilisation of 10-CHO-DHF are unknown. We have determined the kinetic properties (kcat/Km) of conversion of 10-CHO-DHF to 10-CHO-THF by DHFR, and to DHF by PurH. We show that DHFR utilises 10-CHO-DHF about one third as efficiently as it utilises DHF. The 10-CHO-DHF is also utilised (as a formyl group donor) by PurH albeit slightly less efficiently than 10-CHO-THF. The utilisation of 10-CHO-DHF by DHFR is ~50 fold more efficient than its utilisation by PurH. A folate deficient Escherichia coli (∆pabA) grows well when supplemented with adenine, glycine, thymine and methionine, the metabolites that arise from the one-carbon metabolic pathway. Notably, when the ∆pabA strain harboured a folate transporter, it grew in the presence of 10-CHO-DHF alone, suggesting that it (10-CHO-DHF) can enter one-carbon metabolic pathway to provide the required metabolites. Thus, our studies reveal that both DHFR and PurH could utilise 10-CHO-DHF for folate homeostasis in E. coli.
Asunto(s)
Escherichia coli/metabolismo , Ácido Fólico/análogos & derivados , Nucleótido Desaminasas/metabolismo , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Ácido 4-Aminobenzoico , Clonación Molecular , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Ácido Fólico/metabolismo , Deficiencia de Ácido Fólico/genética , Homeostasis , Cinética , Redes y Vías Metabólicas , Nucleótido Desaminasas/genética , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/genética , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
A hallmark of cancer is unbridled proliferation that can result in increased demand for de novo synthesis of purine and pyrimidine bases required for DNA and RNA biosynthesis. These synthetic pathways are frequently upregulated in cancer and involve various folate-dependent enzymes. Antifolates have a proven record as clinically used oncolytic agents. Our recent research efforts have produced LSN 3213128 (compound 28a), a novel, selective, nonclassical, orally bioavailable antifolate with potent and specific inhibitory activity for aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), an enzyme in the purine biosynthetic pathway. Inhibition of AICARFT with compound 28a results in dramatic elevation of 5-aminoimidazole 4-carboxamide ribonucleotide (ZMP) and growth inhibition in NCI-H460 and MDA-MB-231met2 cancer cell lines. Treatment with this inhibitor in a murine based xenograft model of triple negative breast cancer (TNBC) resulted in tumor growth inhibition.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/uso terapéutico , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/uso terapéutico , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Mama/efectos de los fármacos , Mama/metabolismo , Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Descubrimiento de Drogas , Femenino , Antagonistas del Ácido Fólico/farmacocinética , Antagonistas del Ácido Fólico/farmacología , Humanos , Masculino , Ratones , Ratones Desnudos , Modelos Moleculares , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Sulfonamidas/química , Sulfonamidas/farmacocinética , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Tiofenos/química , Tiofenos/farmacocinética , Tiofenos/farmacología , Tiofenos/uso terapéutico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
A novel series of 6-substituted benzoyl and non-benzoyl straight chain pyrrolo[2,3-d]pyrimidines were designed and synthesized as potential antitumor agents targeting both thymidylate and purine nucleotide biosynthesis. Starting from the key intermediate 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidin-6-yl-acetic acid, target compounds 1-6 were successfully obtained through two sequential condensation and saponification reactions in decent yield. The newly synthesized compounds showed antiproliferative potencies against a panel of tumor cell lines including KB, SW620 and MCF7. In particular, most compounds of this series exhibited nanomolar to subnanomolar inhibitory activities toward KB tumor cells, significantly more potent than the positive control methotrexate (MTX) and pemetrexed (PMX). Along with the results of nucleoside protection assays, molecular modeling studies suggested that the antitumor activity of compound 6 could be attributed to multitargeted inhibition of folate-dependent enzymes thymidylate synthase (TS), glycinamide ribonucleotide formyltransferase (GARFTase) and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase). Growth inhibition by compound 6 also induced distinct early apoptosis and cell cycle arrest at S-phase, which resulted in cell death.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/antagonistas & inhibidores , Fosforribosilglicinamida-Formiltransferasa/antagonistas & inhibidores , Pirimidinas/farmacología , Pirroles/farmacología , Timidilato Sintasa/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Fosforribosilglicinamida-Formiltransferasa/metabolismo , Pirimidinas/síntesis química , Pirimidinas/química , Pirroles/síntesis química , Pirroles/química , Relación Estructura-Actividad , Timidilato Sintasa/metabolismoRESUMEN
Purine nucleotide biosynthesis de novo (PNB) requires 2 folate-dependent transformylases-5'-phosphoribosyl-glycinamide (GAR) and 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) transformylases-to introduce carbon 8 (C8) and carbon 2 (C2) into the purine ring. Both transformylases utilize 10-formyltetrahydrofolate (10-formyl-H4folate), where the formyl-carbon sources include ring-2-C of histidine, 3-C of serine, 2-C of glycine, and formate. Our findings in human studies indicate that glycine provides the carbon for GAR transformylase (exclusively C8), whereas histidine and formate are the predominant carbon sources for AICAR transformylase (C2). Contrary to the previous notion, these carbon sources may not supply a general 10-formyl-H4folate pool, which was believed to equally provide carbons to C8 and C2. To explain these phenomena, we postulate that GAR transformylase is in a complex with the trifunctional folate-metabolizing enzyme (TFM) and serine hydroxymethyltransferase to channel carbons of glycine and serine to C8. There is no evidence for channeling carbons of histidine and formate to AICAR transformylase (C2). GAR transformylase may require the TFM to furnish 10-formyl-H4folate immediately after its production from serine to protect its oxidation to 10-formyldihydrofolate (10-formyl-H2folate), whereas AICAR transformylase can utilize both 10-formyl-H2folate and 10-formyl-H4folate. Human liver may supply AICAR to AICAR transformylase in erythrocytes/erythroblasts. Incorporation of ring-2-C of histidine and formate into C2 of urinary uric acid presented a circadian rhythm with a peak in the morning, which corresponds to the maximum DNA synthesis in the bone marrow, and it may be useful in the timing of the administration of drugs that block PNB for the treatment of cancer and autoimmune disease.
Asunto(s)
Ácido Fólico/administración & dosificación , Nucleótidos de Purina/biosíntesis , Carbono/metabolismo , Ritmo Circadiano , Formiatos/metabolismo , Glicina/metabolismo , Humanos , Leucovorina/análogos & derivados , Leucovorina/metabolismo , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Fosforribosilglicinamida-Formiltransferasa/metabolismo , Serina/metabolismo , Ácido Úrico/metabolismoRESUMEN
A new series of 5-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 6-11 with varying chain lengths (n = 1-6) were designed and synthesized as hybrids of the clinically used anticancer drug pemetrexed (PMX) and our 6-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 2c and 2d with folate receptor (FR) α and proton-coupled folate transporter (PCFT) uptake specificity over the reduced folate carrier (RFC) and inhibition of de novo purine nucleotide biosynthesis at glycinamide ribonucleotide formyltransferase (GARFTase). Compounds 6-11 inhibited KB human tumor cells in the order 9 = 10 > 8 > 7 > 6 = 11. Compounds 8-10 were variously transported by FRα, PCFT, and RFC and, unlike PMX, inhibited de novo purine nucleotide rather than thymidylate biosynthesis. The antiproliferative effects of 8 and 9 appeared to be due to their dual inhibitions of both GARFTase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase. Our studies identify a unique structure-activity relationship for transport and dual target inhibition.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/antagonistas & inhibidores , Fosforribosilglicinamida-Formiltransferasa/antagonistas & inhibidores , Pirimidinas/farmacología , Pirroles/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Células CHO , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cricetulus , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Células KB , Estructura Molecular , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Fosforribosilglicinamida-Formiltransferasa/metabolismo , Pirimidinas/síntesis química , Pirimidinas/química , Pirroles/síntesis química , Pirroles/química , Relación Estructura-ActividadRESUMEN
Glycinamide ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme in the de novo purine biosynthesis pathway, which has long been considered a potential target for development of anti-neoplastic therapeutics. Here we report the biological and X-ray crystallographic evaluations of both independent C10 diastereomers, 10S- and 10R-methylthio-DDACTHF, bound to human GAR Tfase, including the highest-resolution apo GAR Tfase structure to date (1.52 Å). Both diastereomers are potent inhibitors (Ki = 210 nM for 10R, and Ki = 180 nM for 10S) of GAR Tfase and exhibit effective inhibition of human leukemia cell growth (IC50 = 80 and 50 nM, respectively). Their inhibitory activity was surprisingly high, and these lipophilic C10-substituted analogues show distinct advantages over their hydrophilic counterparts, most strikingly in retaining potency in mutant human leukemia cell lines that lack reduced folate carrier protein activity (IC50 = 70 and 60 nM, respectively). Structural characterization reveals a new binding mode for these diastereoisomers, in which the lipophilic thiomethyl groups penetrate deeper into a hydrophobic pocket within the folate-binding site. In silico docking simulations of three other sulfur-containing folate analogues also indicate that this hydrophobic cleft represents a favorable region for binding lipophilic substituents. Overall, these results suggest sulfur and its substitutions play an important role in not only the binding of anti-folates to GAR Tfase but also the selectivity and cellular activity (growth inhibition), thereby presenting new possibilities for the future design of potent and selective anti-folate drugs that target GAR Tfase.
Asunto(s)
Antimetabolitos Antineoplásicos/química , Ligasas de Carbono-Nitrógeno/química , Inhibidores Enzimáticos/química , Modelos Moleculares , Fosforribosilglicinamida-Formiltransferasa/química , Tetrahidrofolatos/química , Antimetabolitos Antineoplásicos/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Apoproteínas/antagonistas & inhibidores , Apoproteínas/química , Apoproteínas/metabolismo , Sitios de Unión , Ligasas de Carbono-Nitrógeno/antagonistas & inhibidores , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Concentración 50 Inhibidora , Leucemia/tratamiento farmacológico , Leucemia/enzimología , Conformación Molecular , Simulación del Acoplamiento Molecular , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/antagonistas & inhibidores , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/química , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/genética , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Fosforribosilglicinamida-Formiltransferasa/antagonistas & inhibidores , Fosforribosilglicinamida-Formiltransferasa/genética , Fosforribosilglicinamida-Formiltransferasa/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , Tetrahidrofolatos/metabolismo , Tetrahidrofolatos/farmacologíaRESUMEN
BACKGROUND: Malignant mesothelioma is a highly aggressive tumor arising from mesothelial-lined surfaces, most often in the pleura cavities. Antifolates belong to the most effective cytotoxic drugs for malignant pleural mesothelioma (MPM) treatment. Pemetrexed is an antifolate inhibiting different folate pathway genes (thymidylate synthase [TS], dihydrofolate reductase, glycinamide ribonucleotide formyltransferase [GARFT], and aminoimidazole carboxamide ribonucleotide formyltransferase, [AICARFT]). Increased activity of pemetrexed occurs by folylpolyglutamate synthetase (FPGS), intracellular transport by reduced folate carrier (RFC). The aim of the study was to explore potential correlations between TS, GARFT, AICARFT, RFC, and FPGS levels in MPM and associations with clinical benefit from pemetrexed treatment. METHODS: Samples from 63 patients were tested using immunohistochemistry (IHC) and quantitative polymerase chain reaction(qPCR) for expression levels of TS, GARFT, AICARFT, RFC, and FPGS. Clinical data were evaluated to determine associations between efficacy of pemetrexed and enzyme expression levels. Evaluation of expression levels was done through TaqMan-based qPCR, and IHC was evaluated semiquantitatively by using the H-score. RESULTS: qPCR analysis showed no difference in expression pattern of GARFT and AICARFT. IHC analysis revealed a heterogeneous staining pattern for all the enzymes. No significant association was found between TS expression and survival or objective response of the tumors after pemetrexed treatment. FPGS (p = 0.0111) and RFC (p = 0.0088) mRNA expression levels were strongly associated with overall survival in these patients. CONCLUSIONS: Our results reveal that in pemetrexed-treated MPMs TS expression levels have no influence on patient outcome. Furthermore, GARFT and AICARFT were homogeneously expressed in the patient samples. Folate uptake mechanisms by RFC and activation by FPGS were associated with clinical benefit from pemetrexed treatment.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Glutamatos/uso terapéutico , Guanina/análogos & derivados , Mesotelioma/enzimología , Mesotelioma/terapia , Neoplasias Pleurales/enzimología , Neoplasias Pleurales/terapia , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Expresión Génica , Guanina/uso terapéutico , Humanos , Estimación de Kaplan-Meier , Masculino , Mesotelioma/genética , Persona de Mediana Edad , Pemetrexed , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/genética , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Fosforribosilglicinamida-Formiltransferasa/genética , Fosforribosilglicinamida-Formiltransferasa/metabolismo , Neoplasias Pleurales/genética , Modelos de Riesgos Proporcionales , ARN Mensajero/metabolismo , Proteína Portadora de Folato Reducido/genética , Proteína Portadora de Folato Reducido/metabolismo , Timidilato Sintasa/genética , Timidilato Sintasa/metabolismoRESUMEN
Aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is a bifunctional homodimeric enzyme that catalyzes the last two steps of de novo purine biosynthesis. Homodimerization of ATIC, a protein-protein interaction with an interface of over 5000 Å(2), is required for its aminoimidazole carboxamide ribonucleotide (AICAR) transformylase activity, with the active sites forming at the interface of the interacting proteins. Here, we report the development of a small-molecule inhibitor of AICAR transformylase that functions by preventing the homodimerization of ATIC. The compound is derived from a previously reported cyclic hexapeptide inhibitor of AICAR transformylase (with a K(i) of 17 µM), identified by high-throughput screening. The active motif of the cyclic peptide is identified as an arginine-tyrosine dipeptide, a capped analogue of which inhibits AICAR transformylase with a K(i) value of 84 µM. A library of nonnatural analogues of this dipeptide was designed, synthesized, and assayed. The most potent compound inhibits AICAR transformylase with a K(i) value of 685 nM, a 25-fold improvement in activity from the parent cyclic peptide. The potential for this AICAR transformylase inhibitor in cancer therapy was assessed by studying its effect on the proliferation of a model breast cancer cell line. Using a nonradioactive proliferation assay and live cell imaging, a dose-dependent reduction in cell numbers and cell division rates was observed in cells treated with our ATIC dimerization inhibitor.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Inhibidores Enzimáticos/farmacología , Péptidos Cíclicos/farmacología , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/química , Multimerización de Proteína/efectos de los fármacos , Antineoplásicos/síntesis química , Antineoplásicos/química , Dominio Catalítico/efectos de los fármacos , Recuento de Células , División Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Células MCF-7 , Estructura Molecular , Peso Molecular , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/química , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/antagonistas & inhibidores , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Relación Estructura-ActividadRESUMEN
A series of seven 2-amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines with bridge length variations (from 2 to 8 carbon atoms) were synthesized as selective folate receptor (FR) alpha and beta substrates and as antitumor agents. The syntheses were accomplished from appropriate allylalcohols and 4-iodobenzoate to afford the aldehydes, which were converted to the appropriate 2-amino-4-carbethoxy-5-substituted thiophenes 23-29. Cyclization with chloroformamidine afforded the thieno[2,3-d]pyrimidines 30-36, which were hydrolyzed and coupled with diethyl-L-glutamate, followed by saponification, to give the target compounds 2-8. Compounds 3-6 were potent growth inhibitors (IC(50) 4.7-334 nM) of human tumor cells (KB and IGROV1) that express FRs. In addition, compounds 3-6 inhibited the growth of Chinese hamster ovary (CHO) cells that expressed FRs but not the reduced folate carrier (RFC) or proton-coupled folate transporter (PCFT). However, the compounds were inactive toward CHO cells that lacked FRs but contained either the RFC or PCFT. By nucleoside and 5-amino-4-imidazole carboxamide (AICA) protection studies, along with in vitro and in situ enzyme activity assays, the mechanism of antitumor activity was identified as the dual inhibition of glycinamide ribonucleotide formyltransferase and, likely, AICA ribonucleotide formyltransferase. The dual inhibitory activity of the active thieno[2,3-d]pyrimidine antifolates and the FR specificity represent unique mechanistic features for these compounds distinct from all other known antifolates. The potent inhibitory effects of compounds 3-6 toward cells expressing FRs but not PCFT provide direct evidence that cellular uptake of this series of compounds by FRs does not depend on the presence of PCFT and argues that direct coupling between these transporters is not obligatory.
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Proteínas de Transporte de Anión/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Protones , Purinas/biosíntesis , Pirimidinas/síntesis química , Pirimidinas/farmacología , Receptores de Superficie Celular/metabolismo , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Transporte Biológico/efectos de los fármacos , Células CHO , Proliferación Celular/efectos de los fármacos , Cricetinae , Cricetulus , Receptores de Folato Anclados a GPI , Antagonistas del Ácido Fólico/síntesis química , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/farmacología , Regulación de la Expresión Génica , Humanos , Enlace de Hidrógeno , Nucleósidos/farmacología , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Pirimidinas/química , Proteína Portadora de Folato Reducido , Especificidad por Sustrato , TransfecciónRESUMEN
The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is highly conserved throughout evolution and regulates cell size and survival and cell cycle progression. It regulates the latter by stimulating procession through G(1) and the G(1)/S phase transition. Entry into S phase requires an abundant supply of purine nucleotides, but the effect of the PI3K/Akt pathway on purine synthesis has not been studied. We now show that the PI3K/Akt cassette regulates both de novo and salvage purine nucleotide synthesis in insulin-responsive mouse mesenchymal cells. We found that serum and insulin stimulated de novo purine synthesis in serum-starved cells largely through PI3K/Akt signaling, and pharmacologic and genetic inhibition of PI3K/Akt reduced de novo synthesis by 75% in logarithmically growing cells. PI3K/Akt regulated early steps of de novo synthesis by modulating phosphoribosylpyrophosphate production by the non-oxidative pentose phosphate pathway and late steps by modulating activity of the bifunctional enzyme aminoimidazole-carboxamide ribonucleotide transformylase IMP cyclohydrolase, an enzyme not previously known to be regulated. The effects of PI3K/Akt on purine nucleotide salvage were likely through regulating phosphoribosylpyrophosphate availability. These studies define a new mechanism whereby the PI3K/Akt cassette functions as a master regulator of cellular metabolism and a key player in oncogenesis.
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Evolución Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Nucleótidos de Purina/biosíntesis , Transducción de Señal , Animales , Línea Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Fase G1/genética , Humanos , Insulina/fisiología , Ratones , Ratones Mutantes , Nucleótido Desaminasas/genética , Nucleótido Desaminasas/metabolismo , Vía de Pentosa Fosfato/genética , Fosfatidilinositol 3-Quinasas/genética , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/genética , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Nucleótidos de Purina/genética , Fase S/genéticaRESUMEN
Purine biosynthesis requires 10 enzymatic steps in higher organisms, while prokaryotes require an additional enzyme for step 6. In most organisms steps 9 and 10 are catalyzed by the purH gene product, a bifunctional enzyme with both 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) synthase and inosine monophosphate (IMP) cyclohydrolase activity. Recently it was discovered that Archaea utilize different enzymes to catalyze steps 9 and 10. An ATP-dependent FAICAR synthetase is encoded by the purP gene, and IMP cyclohydrolase is encoded by the purO gene. We have determined the X-ray crystal structures of FAICAR synthetase from Methanocaldococcus jannaschii complexed with various ligands, including the tertiary substrate complex and product complex. The enzyme belongs to the ATP grasp superfamily and is predicted to use a formyl phosphate intermediate formed by an ATP-dependent phosphorylation. In addition, we have determined the structures of a PurP orthologue from Pyrococcus furiosus, which is functionally unclassified, in three crystal forms. With approximately 50% sequence identity, P. furiosus PurP is structurally homologous to M. jannaschii PurP. A phylogenetic analysis was performed to explore the possible role of this functionally unclassified PurP.
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Proteínas Arqueales/metabolismo , Methanococcaceae/metabolismo , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Ribonucleótidos/biosíntesis , Proteínas Arqueales/química , Proteínas Arqueales/genética , Sitios de Unión , Cristalografía por Rayos X , Methanococcaceae/clasificación , Methanococcaceae/genética , Modelos Moleculares , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/química , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/genética , Filogenia , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The objectives were: (1) to test the association of methotrexate (MTX) efficacy in rat adjuvant arthritis (rat AA) with interference of purine biosynthesis and adenosine metabolism and (2) to test the efficacy of erythro-9-(2-hydroxynon-3-yl) adenine (EHNA), an inhibitor of adenosine deaminase, and the efficacy of aminoimidazolecarboxamide (AICA) riboside plus MTX in rat AA. Radiographic and histologic examinations of the hind limbs were measures of efficacy. Urinary excretions of AICA and adenosine were markers of AICA ribotide transformylase inhibition (i.e., blockage of purine biosynthesis) and interference with adenosine metabolism, respectively. AICA and adenosine excretions increased during the day of MTX dosing (treatment day) compared to the previous baseline day in animals responding well to MTX (i.e., low radiographic and histologic scores). Based on radiographic and histologic scores, adjuvant injected rats were separated into two disease categories (i.e., no/mild and moderate/severe). Only AICA excretion was significantly elevated on the treatment day in rat AA with no/mild disease (i.e., those responding well to MTX therapy). AICA (not adenosine) excretion was significantly correlated with the above scores. EHNA was not efficacious, even at toxic levels, while AICA riboside potentiated the efficacy of MTX. The data suggests that efficacious MTX therapy in rat AA (1) blocks purine biosynthesis; (2) increases in in vivo AICA levels. Also adenosine accumulation and blockage of adenosine deaminase (i.e., by EHNA) appear to be less critical to MTX efficacy. Increased levels of AICA metabolites may suppress the immune response in rat AA.
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Adenina/análogos & derivados , Aminoimidazol Carboxamida/análogos & derivados , Antirreumáticos/farmacología , Artritis Experimental/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Metotrexato/farmacología , Purinas/metabolismo , Ribonucleósidos/farmacología , Adenina/farmacología , Adenina/uso terapéutico , Adenosina/orina , Adenosina Desaminasa/metabolismo , Inhibidores de la Adenosina Desaminasa , Aminoimidazol Carboxamida/farmacología , Aminoimidazol Carboxamida/uso terapéutico , Aminoimidazol Carboxamida/orina , Animales , Antirreumáticos/uso terapéutico , Artritis Experimental/enzimología , Artritis Experimental/patología , Artritis Experimental/orina , Biomarcadores/orina , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Inhibidores Enzimáticos/uso terapéutico , Metotrexato/uso terapéutico , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/antagonistas & inhibidores , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Purinas/orina , Ratas , Ribonucleósidos/uso terapéuticoRESUMEN
The inosine monophosphate cyclohydrolase (IMPCH) component (residues 1-199) of the bifunctional enzyme aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase, residues 200-593)/IMPCH (ATIC) catalyzes the final step in the de novo purine biosynthesis pathway that produces IMP. As a potential target for antineoplastic intervention, we designed IMPCH inhibitors, 1,5-dihydroimidazo[4,5-c][1,2,6]thiadiazin-4(3H)-one 2,2-dioxide (heterocycle, 1), the corresponding nucleoside (2), and the nucleoside monophosphate (nucleotide) (3), as mimics of the tetrahedral intermediate in the cyclization reaction. All compounds are competitive inhibitors against IMPCH (K(i) values = 0.13-0.23 microm) with the simple heterocycle 1 exhibiting the most potent inhibition (K(i) = 0.13 microm). Crystal structures of bifunctional ATIC in complex with nucleoside 2 and nucleotide 3 revealed IMPCH binding modes similar to that of the IMPCH feedback inhibitor, xanthosine 5'-monophosphate. Surprisingly, the simpler heterocycle 1 had a completely different IMPCH binding mode and was relocated to the phosphate binding pocket that was identified from previous xanthosine 5'-monophosphate structures. The aromatic imidazole ring interacts with a helix dipole, similar to the interaction with the phosphate moiety of 3. The crystal structures not only revealed the mechanism of inhibition of these compounds, but they now serve as a platform for future inhibitor improvements. Importantly, the nucleoside-complexed structure supports the notion that inhibitors lacking a negatively charged phosphate can still inhibit IMPCH activity with comparable potency to phosphate-containing inhibitors. Provocatively, the nucleotide inhibitor 3 also binds to the AICAR Tfase domain of ATIC, which now provides a lead compound for the design of inhibitors that simultaneously target both active sites of this bifunctional enzyme.
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Proteínas Aviares/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/enzimología , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/antagonistas & inhibidores , Animales , Proteínas Aviares/química , Proteínas Aviares/metabolismo , Sitios de Unión , Aves/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Nucleósidos/síntesis química , Nucleósidos/química , Nucleósidos/metabolismo , Nucleótidos/síntesis química , Nucleótidos/química , Nucleótidos/metabolismo , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/química , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Purinas/biosíntesisRESUMEN
Impaired folate-mediated 1-carbon metabolism has been linked to multiple disease outcomes. A better understanding of the nutritional and genetic influences on this complex biochemical pathway is needed to comprehend their impact on human health. To this end, we created a mathematical model of folate-mediated 1-carbon metabolism. The model uses published data on folate enzyme kinetics and regulatory mechanisms to simulate the impact of genetic and nutritional variation on critical aspects of the pathway. We found that the model predictions match experimental data, while providing novel insights into pathway kinetics. Our primary observations were as follows: 1) the inverse association between folate and homocysteine is strongest at very low folate concentrations, but there is no association at high folate concentrations; 2) the DNA methylation reaction rate is relatively insensitive to changes in folate pool size; and 3) as folate concentrations become very high, enzyme velocities decrease. With regard to polymorphisms in 5,10-methylenetetrahydrofolate reductase (MTHFR), the modeling predicts that decrease MTHFR activity reduces concentrations of S-adenosylmethionine and 5-methyltetrahydrofolate, as well as DNA methylation, while modestly increasing S-adenosylhomocysteine and homocysteine concentrations and thymidine or purine synthesis. Decreased folate together with a simulated vitamin B-12 deficiency results in decreases in DNA methylation and purine and thymidine synthesis. Decreased MTHFR activity superimposed on the B-12 deficiency appears to reverse the declines in purine and thymidine synthesis. These mathematical simulations of folate-mediated 1-carbon metabolism provide a cost-efficient approach to in silico experimentation that can complement and help guide laboratory studies.
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Carbono/metabolismo , Ácido Fólico/fisiología , Modelos Biológicos , Biología Molecular , Fenómenos Fisiológicos de la Nutrición , Betaína/análisis , Metilación de ADN , Ácido Fólico/análisis , Formiato-Tetrahidrofolato Ligasa/metabolismo , Homocisteína/análisis , Humanos , Cinética , Matemática , Metionina/administración & dosificación , Metilación , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/metabolismo , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Polimorfismo Genético , S-Adenosilmetionina/análisis , Deficiencia de Vitamina B 12/genética , Deficiencia de Vitamina B 12/metabolismoRESUMEN
Because of its ability to mimic a low energy status of the cell, the cell-permeable nucleoside 5-aminoimidazole-4-carboxamide (AICA) riboside was proposed as an antineoplastic agent switching off major energy-consuming processes associated with the malignant phenotype (lipid production, DNA synthesis, cell proliferation, cell migration, etc.). Key to the antineoplastic action of AICA riboside is its conversion to ZMP, an AMP mimetic that at high concentrations activates the AMP-activated protein kinase (AMPK). Here, in an attempt to increase the efficacy of AICA riboside, we pretreated cancer cells with methotrexate, an antimetabolite blocking the metabolism of ZMP. Methotrexate enhanced the AICA riboside-induced accumulation of ZMP and led to a decrease in the levels of ATP, which functions as an intrasteric inhibitor of AMPK. Consequently, methotrexate markedly sensitized AMPK for activation by AICA riboside and potentiated the inhibitory effects of AICA riboside on tumor-associated processes. As cotreatment elicited antiproliferative effects already at concentrations of compounds that were only marginally effective when used alone, our findings on the cooperation between methotrexate and AICA riboside provide new opportunities both for the application of classic antimetabolic chemotherapeutics, such as methotrexate, and for the exploitation of the energy-sensing machinery as a target for cancer intervention.