RESUMO
N-Pyridinylthiophene carboxamide (compound 21) displays activity against peripheral nerve sheath cancer cells and mouse xenografts by an unknown mechanism. Through medicinal chemistry, we identified a more active derivative, compound 9, and found that only analogues with structures similar to nicotinamide retained activity. Genetic screens using compound 9 found that both NAMPT and NMNAT1, enzymes in the NAD salvage pathway, are necessary for activity. Compound 9 is metabolized by NAMPT and NMNAT1 into an adenine dinucleotide (AD) derivative in a cell-free system, cultured cells, and mice, and inhibition of this metabolism blocked compound activity. AD analogues derived from compound 9 inhibit IMPDH in vitro and cause cell death by inhibiting IMPDH in cells. These findings nominate these compounds as preclinical candidates for the development of tumor-activated IMPDH inhibitors to treat neuronal cancers.
Assuntos
NAD , Niacinamida , Tiofenos , Animais , NAD/metabolismo , Humanos , Camundongos , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Niacinamida/farmacologia , Niacinamida/química , Tiofenos/farmacologia , Tiofenos/química , Tiofenos/metabolismo , Linhagem Celular Tumoral , IMP Desidrogenase/antagonistas & inibidores , IMP Desidrogenase/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Nicotinamida Fosforribosiltransferase/metabolismo , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Neoplasias de Bainha Neural/tratamento farmacológico , Neoplasias de Bainha Neural/metabolismo , Neoplasias de Bainha Neural/patologia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidoresRESUMO
Osteosarcoma is a frequent and extremely aggressive type of pediatric cancer. New therapeutic approaches are needed to improve the overall survival of osteosarcoma patients. Our previous results suggest that NMNAT1, a key enzyme in nuclear NAD+ synthesis, facilitates the survival of cisplatin-treated osteosarcoma cells. A high-throughput cytotoxicity screening was performed to identify novel pathways or compounds linked to the cancer-promoting role of NMNAT1. Nine compounds caused higher toxicity in the NMNAT1 KO U2OS cells compared to their wild type counterparts, and actinomycin D (ActD) was the most potent. ActD-treatment of NMNAT1 KO cells increased caspase activity and secondary necrosis. The reduced NAD+ content in NMNAT1 KO cells was further decreased by ActD, which partially inhibited NAD+-dependent enzymes, including the DNA nick sensor enzyme PARP1 and the NAD+-dependent deacetylase SIRT1. Impaired PARP1 activity increased DNA damage in ActD-treated NMNAT1 knockout cells, while SIRT1 impairment increased acetylation of the p53 protein, causing the upregulation of pro-apoptotic proteins (NOXA, BAX). Proliferation was decreased through both PARP- and SIRT-dependent pathways. On the one hand, PARP inhibitors sensitized wild type but not NMNAT1 KO cells to ActD-induced anti-clonogenic effects; on the other hand, over-acetylated p53 induced the expression of the anti-proliferative p21 protein leading to cell cycle arrest. Based on our results, NMNAT1 acts as a survival factor in ActD-treated osteosarcoma cells. By inhibiting both PARP1- and SIRT1-dependent cellular pathways, NMNAT1 inhibition can be a promising new tool in osteosarcoma chemotherapy.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/prevenção & controle , Dactinomicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Osteossarcoma/prevenção & controle , Antibióticos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proliferação de Células , Humanos , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Células Tumorais CultivadasRESUMO
The programmed axon degeneration pathway has emerged as an important process contributing to the pathogenesis of several neurological diseases. The most crucial events in this pathway include activation of the central executioner SARM1 and NAD+ depletion, which leads to an energetic failure and ultimately axon destruction. Given the prevalence of this pathway, it is not surprising that inhibitory therapies are currently being developed in order to treat multiple neurological diseases with the same therapy. Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of neurological diseases that may also benefit from this therapeutic approach. To evaluate the appropriateness of this strategy, the contribution of the programmed axon degeneration pathway to the pathogenesis of different CMT subtypes is being actively investigated. The subtypes CMT1A, CMT1B and CMT2D are the first to have been examined. Based on the results from these studies and advances in developing therapies to block the programmed axon degeneration pathway, promising therapeutics for CMT are now on the horizon.
Assuntos
Axônios/metabolismo , Doença de Charcot-Marie-Tooth/terapia , Terapia Genética , Terapia de Alvo Molecular , Proteínas do Domínio Armadillo/antagonistas & inibidores , Proteínas do Domínio Armadillo/genética , Axônios/patologia , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Nicotinamida-Nucleotídeo Adenililtransferase/genéticaRESUMO
Conventional treatments to combat the tuberculosis (TB) epidemic are falling short, thus encouraging the search for novel antitubercular drugs acting on unexplored molecular targets. Several whole-cell phenotypic screenings have delivered bioactive compounds with potent antitubercular activity. However, their cellular target and mechanism of action remain largely unknown. Further evaluation of these compounds may include their screening in search for known antitubercular drug targets hits. Here, a collection of nearly 1400 mycobactericidal compounds was screened against Mycobacterium tuberculosis NaMN adenylyltransferase ( MtNadD), a key enzyme in the biogenesis of NAD cofactor that was recently validated as a new drug target for dormant and active tuberculosis. We found three chemotypes that efficiently inhibit MtNadD in the low micromolar range in vitro. SAR and cheminformatics studies of commercially available analogues point to a series of benzimidazolium derivatives, here named N2, with bactericidal activity on different mycobacteria, including M. abscessus, multidrug-resistant M. tuberculosis, and dormant M. smegmatis. The on-target activity was supported by the increased resistance of an M. smegmatis strain overexpressing the target and by a rapid decline in NAD(H) levels. A cocrystal structure of MtNadD with N2-8 inhibitor reveals that the binding of the inhibitor induced the formation of a new quaternary structure, a dimer-of-dimers where two copies of the inhibitor occupy symmetrical positions in the dimer interface, thus paving the way for the development of a new generation of selective MtNadD bioactive inhibitors. All these results strongly suggest that pharmacological inhibition of MtNadD is an effective strategy to combat dormant and resistant Mtb strains.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , NAD/antagonistas & inibidores , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , NAD/biossíntese , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Relação Estrutura-AtividadeRESUMO
Golgi fragmentation and loss of Nicotinamide Mononucleotide Adenylyltransferase 2 (NMNAT2) are the early key features of many neurodegenerative disorders. We investigated the link between NMNAT2 loss, Golgi fragmentation and axon degeneration. Golgi fragmentation in the cultured dorsal root ganglion (DRG) neurons resulted in caspase dependent axon degeneration and neuronal cell death. NMNAT2 depletion in the DRG neurons caused Golgi fragmentation and caspase dependent axon degeneration. NMNAT2 depletion did not cause ATP loss in the axons. These results indicate that NMNAT2 is required for maintenance of Golgi structure. Loss of Golgi structure or Nmnat2 depletion causes caspase dependent neurodegeneration. cytNmnat1 overexpression inhibited the axon degeneration induced by Golgi fragmentation or NMNAT2 depletion. These results also suggest that these degeneration signals converge on a common cytNmnat1 mediated axon protective program and are distinct from the SARM1 mediated caspase independent axon degeneration.
Assuntos
Gânglios Espinais/enzimologia , Complexo de Golgi/enzimologia , Neurônios/enzimologia , Nicotinamida-Nucleotídeo Adenililtransferase/deficiência , Animais , Apoptose/fisiologia , Células Cultivadas , Gânglios Espinais/patologia , Complexo de Golgi/patologia , Camundongos , Neurônios/patologia , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Nicotinamida-Nucleotídeo Adenililtransferase/genética , RNA Interferente Pequeno/administração & dosagemRESUMO
Axon injury can lead to several cell survival responses including increased stability and axon regeneration. Using an accessible Drosophila model system, we investigated the regulation of injury responses and their relationship. Axon injury stabilizes the rest of the cell, including the entire dendrite arbor. After axon injury we found mitochondrial fission in dendrites was upregulated, and that reducing fission increased stabilization or neuroprotection (NP). Thus axon injury seems to both turn on NP, but also dampen it by activating mitochondrial fission. We also identified caspases as negative regulators of axon injury-mediated NP, so mitochondrial fission could control NP through caspase activation. In addition to negative regulators of NP, we found that nicotinamide mononucleotide adenylyltransferase (Nmnat) is absolutely required for this type of NP. Increased microtubule dynamics, which has previously been associated with NP, required Nmnat. Indeed Nmnat overexpression was sufficient to induce NP and increase microtubule dynamics in the absence of axon injury. DLK, JNK and fos were also required for NP. Because NP occurs before axon regeneration, and NP seems to be actively downregulated, we tested whether excessive NP might inhibit regeneration. Indeed both Nmnat overexpression and caspase reduction reduced regeneration. In addition, overexpression of fos or JNK extended the timecourse of NP and dampened regeneration in a Nmnat-dependent manner. These data suggest that NP and regeneration are conflicting responses to axon injury, and that therapeutic strategies that boost NP may reduce regeneration.
Assuntos
Axônios/metabolismo , Drosophila melanogaster/genética , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Degeneração Walleriana/genética , Animais , Axônios/patologia , Caspases/biossíntese , Caspases/genética , Dendritos/metabolismo , Dendritos/patologia , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Humanos , MAP Quinase Quinase 4/biossíntese , MAP Quinase Quinase 4/genética , Microtúbulos/genética , Microtúbulos/patologia , Dinâmica Mitocondrial/genética , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Nicotinamida-Nucleotídeo Adenililtransferase/biossíntese , RNA Interferente Pequeno/genética , Degeneração Walleriana/patologiaRESUMO
Nicotinamide adenine dinucleotide (NAD(+)) is an essential substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases (PARPs), which are NAD(+)-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD(+) concentrations within these subcellular compartments are thought to regulate the activity of NAD(+)-consuming enzymes; however, the challenge in measuring compartmentalized NAD(+) in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD(+) concentrations in subcellular compartments. We found that the concentrations of free NAD(+) in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD(+) biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD(+) concentrations.
Assuntos
Técnicas Biossensoriais , Mitocôndrias/metabolismo , NAD/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Núcleo Celular/química , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Citosol/química , Citosol/metabolismo , DNA Ligases/genética , DNA Ligases/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mitocôndrias/química , NAD/análise , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Mutação Puntual , Poli(ADP-Ribose) Polimerases/metabolismo , Sirtuínas/metabolismoRESUMO
Nicotinate mononucleotide adenylyltransferase NadD is an essential enzyme in the biosynthesis of the NAD cofactor, which has been implicated as a target for developing new antimycobacterial therapies. Here we report the crystal structure of Mycobacterium tuberculosis NadD (MtNadD) at a resolution of 2.4 Å. A remarkable new feature of the MtNadD structure, compared with other members of this enzyme family, is a 310 helix that locks the active site in an over-closed conformation. As a result, MtNadD is rendered inactive as it is topologically incompatible with substrate binding and catalysis. Directed mutagenesis was also used to further dissect the structural elements that contribute to the interactions of the two MtNadD substrates, i.e. ATP and nicotinic acid mononucleotide (NaMN). For inhibitory profiling of partially active mutants and wild type MtNadD, we used a small molecule inhibitor of MtNadD with moderate affinity (Ki â¼ 25 µM) and antimycobacterial activity (MIC80) â¼ 40-80 µM). This analysis revealed interferences with some of the residues in the NaMN binding subsite consistent with the competitive inhibition observed for the NaMN substrate (but not ATP). A detailed steady-state kinetic analysis of MtNadD suggests that ATP must first bind to allow efficient NaMN binding and catalysis. This sequential mechanism is consistent with the requirement of transition to catalytically competent (open) conformation hypothesized from structural modeling. A possible physiological significance of this mechanism is to enable the down-regulation of NAD synthesis under ATP-limiting dormancy conditions. These findings point to a possible new strategy for designing inhibitors that lock the enzyme in the inactive over-closed conformation.
Assuntos
Antituberculosos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/enzimologia , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Sequência de Aminoácidos , Antituberculosos/química , Cristalografia por Raios X , Inibidores Enzimáticos/química , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mycobacterium tuberculosis/efeitos dos fármacos , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Nicotinamida-Nucleotídeo Adenililtransferase/química , Conformação Proteica , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite utilized as a redox cofactor and enzyme substrate in numerous cellular processes. Elevated NAD+ levels have been observed in red blood cells infected with the malaria parasite Plasmodium falciparum, but little is known regarding how the parasite generates NAD+. Here, we employed a mass spectrometry-based metabolomic approach to confirm that P. falciparum lacks the ability to synthesize NAD+ de novo and is reliant on the uptake of exogenous niacin. We characterized several enzymes in the NAD+ pathway and demonstrate cytoplasmic localization for all except the parasite nicotinamidase, which concentrates in the nucleus. One of these enzymes, the P. falciparum nicotinate mononucleotide adenylyltransferase (PfNMNAT), is essential for NAD+ metabolism and is highly diverged from the human homolog, but genetically similar to bacterial NMNATs. Our results demonstrate the enzymatic activity of PfNMNAT in vitro and demonstrate its ability to genetically complement the closely related Escherichia coli NMNAT. Due to the similarity of PfNMNAT to the bacterial enzyme, we tested a panel of previously identified bacterial NMNAT inhibitors and synthesized and screened twenty new derivatives, which demonstrate a range of potency against live parasite culture. These results highlight the importance of the parasite NAD+ metabolic pathway and provide both novel therapeutic targets and promising lead antimalarial compounds.
Assuntos
NAD/metabolismo , Plasmodium falciparum/metabolismo , Transporte Biológico , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Humanos , Metabolômica , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
UNLABELLED: Mycobacterium tuberculosis remains a major cause of death due to the lack of treatment accessibility, HIV coinfection, and drug resistance. Development of new drugs targeting previously unexplored pathways is essential to shorten treatment time and eliminate persistent M. tuberculosis. A promising biochemical pathway which may be targeted to kill both replicating and nonreplicating M. tuberculosis is the biosynthesis of NAD(H), an essential cofactor in multiple reactions crucial for respiration, redox balance, and biosynthesis of major building blocks. NaMN adenylyltransferase (NadD) and NAD synthetase (NadE), the key enzymes of NAD biosynthesis, were selected as promising candidate drug targets for M. tuberculosis. Here we report for the first time kinetic characterization of the recombinant purified NadD enzyme, setting the stage for its structural analysis and inhibitor development. A protein knockdown approach was applied to validate bothNadD and NadE as target enzymes. Induced degradation of either target enzyme showed a strong bactericidal effect which coincided with anticipated changes in relative levels of NaMN and NaAD intermediates (substrates of NadD and NadE, respectively) and ultimate depletion of the NAD(H) pool. A metabolic catastrophe predicted as a likely result of NAD(H) deprivation of cellular metabolism was confirmed by (13)C biosynthetic labeling followed by gas chromatography-mass spectrometry (GC-MS) analysis. A sharp suppression of metabolic flux was observed in multiple NAD(P)(H)-dependent pathways, including synthesis of many amino acids (serine, proline, aromatic amino acids) and fatty acids. Overall, these results provide strong validation of the essential NAD biosynthetic enzymes, NadD and NadE, as antimycobacterial drug targets. IMPORTANCE: To address the problems of M. tuberculosis drug resistance and persistence of tuberculosis, new classes of drug targets need to be explored. The biogenesis of NAD cofactors was selected for target validation because of their indispensable role in driving hundreds of biochemical transformations. We hypothesized that the disruption of NAD production in the cell via genetic suppression of the essential enzymes (NadD and NadE) involved in the last two steps of NAD biogenesis would lead to cell death, even under dormancy conditions. In this study, we confirmed the hypothesis using a protein knockdown approach in the model system of Mycobacterium smegmatis. We showed that induced proteolytic degradation of either target enzyme leads to depletion of the NAD cofactor pool, which suppresses metabolic flux through numerous NAD(P)-dependent pathways of central metabolism of carbon and energy production. Remarkably, bactericidal effect was observed even for nondividing bacteria cultivated under carbon starvation conditions.
Assuntos
Amida Sintases/antagonistas & inibidores , Antituberculosos/farmacologia , Inibidores Enzimáticos/farmacologia , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/enzimologia , NAD/biossíntese , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Descoberta de Drogas/métodos , Técnicas de Silenciamento de Genes , Genes Essenciais , Viabilidade Microbiana , NAD/antagonistas & inibidoresRESUMO
Axon degeneration is an evolutionarily conserved process that drives the loss of damaged axons and is an early event in many neurological disorders, so it is important to identify the molecular constituents of this poorly understood mechanism. Here, we demonstrate that the Phr1 E3 ubiquitin ligase is a central component of this axon degeneration program. Loss of Phr1 results in prolonged survival of severed axons in both the peripheral and central nervous systems, as well as preservation of motor and sensory nerve terminals. Phr1 depletion increases the axonal level of the axon survival molecule nicotinamide mononucleotide adenyltransferase 2 (NMNAT2), and NMNAT2 is necessary for Phr1-dependent axon stability. The profound long-term protection of peripheral and central mammalian axons following Phr1 deletion suggests that pharmacological inhibition of Phr1 function may be an attractive therapeutic candidate for amelioration of axon loss in neurological disease.
Assuntos
Axônios/fisiologia , Proteínas de Transporte/metabolismo , Animais , Axônios/ultraestrutura , Proteínas de Transporte/genética , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Knockout , Degeneração Neural , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ubiquitina-Proteína Ligases , Vincristina/farmacologiaRESUMO
Active zones are specialized presynaptic structures critical for neurotransmission. We show that a neuronal maintenance factor, nicotinamide mononucleotide adenylyltransferase (NMNAT), is required for maintaining active zone structural integrity in Drosophila by interacting with the active zone protein, Bruchpilot (BRP), and shielding it from activity-induced ubiquitin-proteasome-mediated degradation. NMNAT localizes to the peri-active zone and interacts biochemically with BRP in an activity-dependent manner. Loss of NMNAT results in ubiquitination, mislocalization and aggregation of BRP, and subsequent active zone degeneration. We propose that, as a neuronal maintenance factor, NMNAT specifically maintains active zone structure by direct protein-protein interaction.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Terminações Pré-Sinápticas/enzimologia , Animais , Drosophila/enzimologia , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Proteólise , RNA Interferente Pequeno/genética , Transdução de Sinais , Transmissão Sináptica/fisiologia , Ubiquitina/genética , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Current studies in plants suggest that the content of the coenzyme NAD is variable and potentially important in determining cell fate. In cases that implicate NAD consumption, re-synthesis must occur to maintain dinucleotide pools. Despite information on the pathways involved in NAD synthesis in plants, the existence of a mitochondrial nicotinamide mononucleotide adenylyltransferase (NMNAT) activity which catalyses NAD synthesis from nicotinamide mononucleotide (NMN) and ATP has not been reported. To verify the latter assumed pathway, experiments with purified and bioenergetically active mitochondria prepared from tubers of Jerusalem artichoke (Helianthus tuberosus L.) were performed. To determine whether NAD biosynthesis might occur, NMN was added to Jerusalem artichoke mitochondria (JAM) and NAD biosynthesis was tested by means of HPLC and spectroscopically. Our results indicate that JAM contain a specific NMNAT inhibited by Na-pyrophosphate, AMP and ADP-ribose. The dependence of NAD synthesis rate on NMN concentration shows saturation kinetics with K (m) and V (max) values of 82 ± 1.05 µM and 4.20 ± 0.20 nmol min(-1) mg(-1) protein, respectively. The enzyme's pH and temperature dependence were also investigated. Fractionation studies revealed that mitochondrial NMNAT activity was present in the soluble matrix fraction. The NAD pool needed constant replenishment that might be modulated by environmental inputs. Thus, the mitochondrion in heterotrophic plant tissues ensures NAD biosynthesis by NMNAT activity and helps to orchestrate NAD metabolic network in implementing the survival strategy of cells.
Assuntos
Helianthus/enzimologia , Mitocôndrias/enzimologia , NAD/biossíntese , Mononucleotídeo de Nicotinamida/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Adenosina Difosfato Ribose/farmacologia , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Fracionamento Celular , Cromatografia Líquida de Alta Pressão , Difosfatos/farmacologia , Helianthus/metabolismo , Processos Heterotróficos , Concentração de Íons de Hidrogênio , Cinética , Mitocôndrias/metabolismo , NADP/biossíntese , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Oxigênio/análise , Oxigênio/metabolismo , Tubérculos/enzimologia , Tubérculos/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
Nicotinamide adenine dinucleotide (NAD(+)) has a crucial role in many cellular processes, both as a coenzyme for redox reactions and as a substrate to donate ADP-ribose units. Thus, enzymes involved in NAD(+) metabolism are attractive targets for drug discovery against a variety of human diseases. Herein we focus on two of them: NMN/NaMN adenylyltransferase (NMNAT) and NAD kinase (NADK). NMNAT is a key enzyme in all organisms catalyzing coupling of ATP and NMN or NaMN yielding NAD or NaAD, respectively. NADKs are ubiquitous enzymes involved in the last step of the biosynthesis of NADP. They phosphorylate NAD to produce NADP using ATP (or inorganic polyphosphates) in the presence of Mg(2+). No other pathway of NADP biosynthesis has been found in prokaryotic or eukaryotic cells. In this review we provide a comprehensive summary of NMNAT and NADK inhibitors highlighting their chemical modifications by different synthetic approaches, and structure-activity relationships depending on their potential therapeutic applications.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Animais , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/uso terapêutico , Humanos , Nicotinamida-Nucleotídeo Adenililtransferase/química , Fosfotransferases (Aceptor do Grupo Álcool)/químicaRESUMO
Nicotinamide mononucleotide adenylyltransferease (NMNAT), a rate-limiting enzyme present in all organisms, reversibly catalyzes the important step in the biosynthesis of NAD from ATP and NMN. NAD and NADP are used reversibly in anabolic and catabolic reactions. NAD is necessary for cell survival in oxidative stress and DNA damage. Based on their localization, three different NMNAT's have been recognized, NMNAT-1 (homohexamer) in the nucleus (chromosome 1 p32-35), NMNAT-2 (homodimer) in the cytoplasm (chromosome 1q25) and NMNAT-3 (homotetramer) in the mitochondria. NMNAT also catalyzes the metabolic conversion of potent antitumor prodrugs like tiazofurin and benzamide riboside to their active forms which are analogs of NAD. NAD synthase-NMNAT acts as a chaperone to protect against neurodegeneration, injury-induced axonal degeneration and also correlates with DNA synthesis during cell cycle. Since its activity is rather low in tumor cells it can be exploited as a source for therapeutic targeting. Steps involved in NAD synthesis are being utilized as targets for chemoprevention, radiosensitization and therapy of wide range of diseases, such as cancer, multiple sclerosis, neurodegeneration and Huntington's disease.
Assuntos
Regulação Enzimológica da Expressão Gênica , NAD/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Envelhecimento/metabolismo , Sequência de Aminoácidos , Animais , Doença , Inibidores Enzimáticos/farmacologia , Humanos , Dados de Sequência Molecular , NAD/biossíntese , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Nicotinamida-Nucleotídeo Adenililtransferase/químicaRESUMO
Bacterial nicotinate mononucleotide adenylyltransferase encoded by the essential gene nadD plays a central role in the synthesis of the redox cofactor NAD(+). The NadD enzyme is conserved in the majority of bacterial species and has been recognized as a novel target for developing new and potentially broad-spectrum antibacterial therapeutics. Here we report the crystal structures of Bacillus anthracis NadD in complex with three NadD inhibitors, including two analogues synthesized in the present study. These structures revealed a common binding site shared by different classes of NadD inhibitors and explored the chemical environment surrounding this site. The structural data obtained here also showed that the subtle changes in ligand structure can lead to significant changes in the binding mode, information that will be useful for future structure-based optimization and design of high affinity inhibitors.
Assuntos
Bacillus anthracis/enzimologia , Proteínas de Bactérias/química , Inibidores Enzimáticos/química , Nicotinamida-Nucleotídeo Adenililtransferase/química , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Hidrazinas/síntese química , Hidrazinas/química , Hidrazonas/síntese química , Hidrazonas/química , Modelos Moleculares , Naftalenos/síntese química , Naftalenos/química , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Propionatos/síntese química , Propionatos/químicaRESUMO
The emergence of multidrug-resistant pathogens necessitates the search for new antibiotics acting on previously unexplored targets. Nicotinate mononucleotide adenylyltransferase of the NadD family, an essential enzyme of NAD biosynthesis in most bacteria, was selected as a target for structure-based inhibitor development. Using iterative in silico and in vitro screens, we identified small molecule compounds that efficiently inhibited target enzymes from Escherichia coli (ecNadD) and Bacillus anthracis (baNadD) but had no effect on functionally equivalent human enzymes. On-target antibacterial activity was demonstrated for some of the selected inhibitors. A 3D structure of baNadD was solved in complex with one of these inhibitors (3_02), providing mechanistic insights and guidelines for further improvement. Most importantly, the results of this study help validate NadD as a target for the development of antibacterial agents with potential broad-spectrum activity.
Assuntos
Antibacterianos/química , Inibidores Enzimáticos/química , NAD/biossíntese , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Antibacterianos/farmacologia , Bacillus anthracis/enzimologia , Sítios de Ligação , Biologia Computacional , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Cinética , NAD/química , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Estrutura Terciária de ProteínaRESUMO
A new lead class of antibacterial drug-like NAD synthetase (NADs) inhibitors was previously identified from a virtual screening study. Here a solution-phase synthetic library of 76 compounds, analogs of the urea-sulfonamide 5838, was synthesized in parallel to explore SAR on the sulfonamide aryl group. All library members were tested for enzyme inhibition against NADs and nicotinic acid mononucleotide adenylyltransferase (NaMNAT), the last two enzymes in the biosynthesis of NAD, and for growth inhibition in a Bacillus anthracis antibacterial assay. Most compounds that inhibited bacterial growth also showed inhibition against one of the enzymes tested. While only modest enhancements in the enzyme inhibition potency against NADs were observed, of significance was the observation that the antibacterial urea-sulfonamides more consistently inhibited NaMNAT.
Assuntos
Amida Sintases/antagonistas & inibidores , Antibacterianos/química , Bacillus anthracis/efeitos dos fármacos , Bacillus anthracis/enzimologia , Sulfonamidas/química , Ureia/química , Amida Sintases/metabolismo , Antraz/tratamento farmacológico , Antibacterianos/síntese química , Antibacterianos/farmacologia , Humanos , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Ureia/síntese química , Ureia/farmacologiaRESUMO
NAD(P) biosynthetic pathways can be considered a generous source of enzymatic targets for drug development. Key reactions for NAD(P) biosynthesis in all organisms, common to both de novo and salvage routes, are catalyzed by NMN/NaMN adenylyltransferase (NMNAT), NAD synthetase (NADS), and NAD kinase (NADK). These reactions represent a three-step pathway, present in the vast majority of living organisms, which is responsible for the generation of both NAD and NADP cellular pools. The validation of these enzymes as drug targets is based on their essentiality and conservation among a large variety of pathogenic microorganisms, as well as on their differential structural features or their differential metabolic contribution to NAD(P) homeostasis between microbial and human cell types. This review describes the structural and functional properties of eubacterial and human enzymes endowed with NMNAT, NADS, and NADK activities, as well as with nicotinamide phosphoribosyltransferase (NamPRT) and nicotinamide riboside kinase (NRK) activities, highlighting the species-related differences, with emphasis on their relevance for drug design. In addition, since the overall NMNAT activity in humans is accounted by multiple isozymes differentially involved in the metabolic activation of antineoplastic compounds, their individual diagnostic value for early therapy optimization is outlined. The involvement of human NMNAT in neurodegenerative disorders and its role in neuroprotection is also discussed.
Assuntos
Amida Sintases/metabolismo , NADP/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Amida Sintases/antagonistas & inibidores , Amida Sintases/química , Bactérias/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Desenho de Fármacos , Humanos , NADP/análogos & derivados , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/química , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Nicotinamida-Nucleotídeo Adenililtransferase/química , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/químicaRESUMO
Nicotinamide adenine dinucleotide (NAD), generally considered a key component involved in redox reactions, has been found to participate in an increasingly diverse range of cellular processes, including signal transduction, DNA repair, and post-translational protein modifications. In recent years, medicinal chemists have become interested in the therapeutic potential of molecules affecting interactions of NAD with NAD-dependent enzymes. Also, enzymes involved in de novo biosynthesis, salvage pathways, and down-stream utilization of NAD have been extensively investigated and implicated in a wide variety of diseases. These studies have bolstered NAD-based therapeutics as a new avenue for the discovery and development of novel treatments for medical conditions ranging from cancer to aging. Industrial and academic groups have produced structurally diverse molecules which target NAD metabolic pathways, with some candidates advancing into clinical trials. However, further intensive structural, biological, and medical studies are needed to facilitate the design and evaluation of new generations of NAD-based therapeutics. At this time, the field of NAD-therapeutics is most likely at a stage similar to that of the early successful development of protein kinase inhibitors, where analogs of ATP (a more widely utilized metabolite than NAD) began to show selectivity against target enzymes. This review focuses on key representative opportunities for research in this area, which extends beyond the scope of this article.