RESUMO
Treatment with a nicotinamide N-methyltransferase inhibitor (NNMTi; 5-amino-1-methylquinolinium) combined with low-fat diet (LD) promoted dramatic whole-body adiposity and weight loss in diet-induced obese (DIO) mice, rapidly normalizing these measures to age-matched lean animals, while LD switch alone was unable to restore these measures to age-matched controls in the same time frame. Since mouse microbiome profiles often highly correlate with body weight and fat composition, this study was designed to test whether the cecal microbiomes of DIO mice treated with NNMTi and LD were comparable to the microbiomes of age-matched lean counterparts and distinct from microbiomes of DIO mice maintained on a high-fat Western diet (WD) or subjected to LD switch alone. There were minimal microbiome differences between lean and obese controls, suggesting that diet composition and adiposity had limited effects. However, DIO mice switched from an obesity-promoting WD to an LD (regardless of treatment status) displayed several genera and phyla differences compared to obese and lean controls. While alpha diversity measures did not significantly differ between groups, beta diversity principal coordinates analyses suggested that mice from the same treatment group were the most similar. K-means clustering analysis of amplicon sequence variants by animal demonstrated that NNMTi-treated DIO mice switched to LD had a distinct microbiome pattern that was highlighted by decreased Erysipelatoclostridium and increased Lactobacillus relative abundances compared to vehicle counterparts; these genera are tied to body weight and metabolic regulation. Additionally, Parasutterella relative abundance, which was increased in both the vehicle- and NNMTi-treated LD-switched groups relative to the controls, significantly correlated with several adipose tissue metabolites' abundances. Collectively, these results provide a novel foundation for future investigations.
Assuntos
Inibidores Enzimáticos/administração & dosagem , Microbioma Gastrointestinal/efeitos dos fármacos , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Nicotinamida N-Metiltransferase/metabolismo , Obesidade/dietoterapia , Obesidade/tratamento farmacológico , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Adiposidade/efeitos dos fármacos , Animais , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/isolamento & purificação , Peso Corporal/efeitos dos fármacos , Terapia Combinada , Dieta com Restrição de Gorduras , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Nicotinamida N-Metiltransferase/genética , Obesidade/metabolismo , Obesidade/microbiologia , Compostos de Quinolínio/administração & dosagemRESUMO
A recently discovered bisubstrate inhibitor of Nicotinamide N-methyltransferase (NNMT) was found to be highly potent in biochemical assays with a single digit nanomolar IC50 value but lacking in cellular activity. We, here, report a prodrug strategy designed to translate the observed potent biochemical inhibitory activity of this inhibitor into strong cellular activity. This prodrug strategy relies on the temporary protection of the amine and carboxylic acid moieties of the highly polar amino acid side chain present in the bisubstrate inhibitor. The modification of the carboxylic acid into a range of esters in the absence or presence of a trimethyl-lock (TML) amine protecting group yielded a range of candidate prodrugs. Based on the stability in an aqueous buffer, and the confirmed esterase-dependent conversion to the parent compound, the isopropyl ester was selected as the preferred acid prodrug. The isopropyl ester and isopropyl ester-TML prodrugs exhibit improved cell permeability, which also translates to significantly enhanced cellular activity as established using assays designed to measure the enzymatic activity of NNMT in live cells.
Assuntos
Inibidores Enzimáticos/farmacologia , Esterases/metabolismo , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Pró-Fármacos/farmacologia , Bioensaio , Soluções Tampão , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Nicotinamida N-Metiltransferase/metabolismo , Pró-Fármacos/síntese química , Pró-Fármacos/química , Especificidade por Substrato/efeitos dos fármacosRESUMO
Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide (vitamin B3) to generate 1-methylnicotinamide (MNA). NNMT overexpression has been linked to a variety of diseases, most prominently human cancers, indicating its potential as a therapeutic target. The development of small-molecule NNMT inhibitors has gained interest in recent years, with the most potent inhibitors sharing structural features based on elements of the nicotinamide substrate and the S-adenosyl-l-methionine (SAM) cofactor. We here report the development of new bisubstrate inhibitors that include electron-deficient aromatic groups to mimic the nicotinamide moiety. In addition, a trans-alkene linker was found to be optimal for connecting the substrate and cofactor mimics in these inhibitors. The most potent NNMT inhibitor identified exhibits an IC50 value of 3.7 nM, placing it among the most active NNMT inhibitors reported to date. Complementary analytical techniques, modeling studies, and cell-based assays provide insights into the binding mode, affinity, and selectivity of these inhibitors.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Regulação Enzimológica da Expressão Gênica , Humanos , Estrutura Molecular , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide (NA) to generate 1-methyl nicotinamide. Since its discovery 70â¯years ago, the appreciation of the role of NNMT in human health has evolved from serving only metabolic functions to also being a driving force in diseases, including a variety of cancers. Despite the increasing evidence indicating NNMT as a viable therapeutic target, the development of cell-active inhibitors against this enzyme is lacking. In this review, we provide an overview of the current status of NNMT inhibitor development, relevant in vitro and in vivo studies, and a discussion of the challenges faced in the development of NNMT inhibitors.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Doenças Metabólicas/metabolismo , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Nicotinamida N-Metiltransferase/metabolismo , Humanos , Doenças Metabólicas/tratamento farmacológico , Neoplasias/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Nicotinamida N-Metiltransferase/antagonistas & inibidores , S-Adenosilmetionina/metabolismoRESUMO
Chemoresistance is the main cause of poor prognosis in colorectal cancer (CRC). Nicotinamide Nmethyltransferase (NNMT) is a metabolic enzyme that is upregulated in various tumor types. It has been reported that NNMT inhibits apoptosis and enhances resistance to 5fluorouracil (5Fu) via inhibition of the apoptosis signal regulating kinase 1 (ASK1)p38 MAPK pathway in CRC cells. A natural product library was screened, and it was found that vanillin, also known as 4hydroxy3methoxybenzaldehyde, a plant secondary metabolite found in several essential plant oils, mainly Vanilla planifolia, Vanilla tahitensis, and Vanilla pompon, may be a promising anticancer compound targeted to NNMT. The aim of the present study was to explore the effect of vanillin on promoting apoptosis and attenuating NNMTinduced resistance to 5Fu in CRC. Lentiviral vectors of short hairpin RNA and small interfering RNA were transfected into HT29 cells to construct NNMTknockdown HT29 cell lines. Vectors containing an open reading frame of NNMT were stably transfected into SW480 cells to induce NNMT overexpression in SW480 cell lines. Vanillin was found to inhibit the mRNA and protein expression levels of NNMT following the inhibition of NNMT activity in HT29 cell lines. Vanillin was able to reverse NNMTinduced increased cell proliferation, decreased cell apoptosis and resistance to 5Fu by inhibiting NNMT expression. Furthermore, it increased cell apoptosis by activating the ASK1p38 MAPK pathway, which could be inhibited by NNMT. In addition, vanillin increased cell apoptosis by promoting mitochondrial damage and reactive oxygen species. In vivo, the combination of vanillin with 5Fu yielded a notable synergy in inhibiting tumor growth and inducing apoptosis. Considering that vanillin is an important flavor and aromatic component used in foods worldwide, vanillin is deemed to be a promising anticancer candidate by inhibiting NNMT and may attenuate NNMTinduced resistance to 5Fu in human CRC therapy with few side effects.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzaldeídos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Fluoruracila/farmacologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Benzaldeídos/uso terapêutico , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Fluoruracila/uso terapêutico , Humanos , Nicotinamida N-Metiltransferase/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The anti-proliferative effects of 5-methylquinolinium (5MQ) of nicotinamide N-methyltransferase (NNMT) have not been previously investigated on a cervical cancer cell line. NNMT is a metabolic enzyme that is correlated with tumour progression and metastasis. 5MQ is a small molecule inhibitor of NNMT. 0.1-500 µM of 5MQ was tested on the HeLa epithelial cervical cancer cell line. Cell viability was assessed with the MTT test. TWIST, ZEB1, SERPIN1, SIRT1, CD16, mRNA and various protein expression levels were analysed with Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Western Blotting, respectively. 5MQ significantly inhibited HeLa cell proliferation in a concentration and time-dependent manner. Increased cell shrinkage, loss of cellular adhesions and apoptotic bodies were observed in HeLa cells after 5MQ treatment. Following treatment with 5MQ, ZEB1, SIRT1, CD16 mRNA levels were increased while TWIST and SERPIN1 mRNA levels were reduced. Expressions of oncogenic proteins phospho-Akt and SIRT1 were decreased. 5MQ can effectively inhibit HeLa cell proliferation without apparently affecting HEK-293 cell proliferation.IMPACT STATEMENTWhat is already known on this subject? NNMT is a cytosolic enzyme involved in tumour progression, metastasis and treatment resistance. It was overexpressed in many human malignancies. 5-amino-1-methylquinolinium (5MQ) is a novel small molecule inhibitor of NNMT that has shown promising results in the treatment of obesity and in senescent muscle regeneration. 5MQ has not been tested on the HeLa cervical cancer cell line, previously.What do the results of this study add? In this study, 5MQ was tested on the HeLa cervical cancer cell line for the first time and the molecular changes associated with 5MQ treatment were analysed. 5MQ demonstrated significant anti-proliferative activity on HeLa cells, which displayed morphological signs of apoptosis. Treatment of HeLa cells with 5MQ led to an increase in ZEB1, SIRT1 mRNA while TWIST mRNA was decreased. Phospho-Akt and Sirtuin1 protein expressions were decreased.What are the implications of these findings for clinical practice and/or further research? 5MQ can effectively inhibit HeLa cell proliferation without apparently affecting HEK-293 cell proliferation. 5MQ treatment was associated with a decrease in the expression of phospho-Akt and Sirtuin1 proteins, both of which have been reported to maintain tumour progression. 5MQ can further be investigated and modified for anti-cancer therapy.
Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Compostos de Quinolínio/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Compostos de Quinolínio/químicaRESUMO
Obesity is a large and growing global health problem with few effective therapies. The present study investigated metabolic and physiological benefits of nicotinamide N-methyltransferase inhibitor (NNMTi) treatment combined with a lean diet substitution in diet-induced obese mice. NNMTi treatment combined with lean diet substitution accelerated and improved body weight and fat loss, increased whole-body lean mass to body weight ratio, reduced liver and epididymal white adipose tissue weights, decreased liver adiposity, and improved hepatic steatosis, relative to a lean diet substitution alone. Importantly, combined lean diet and NNMTi treatment normalized body composition and liver adiposity parameters to levels observed in age-matched lean diet control mice. NNMTi treatment produced a unique metabolomic signature in adipose tissue, with predominant increases in ketogenic amino acid abundance and alterations to metabolites linked to energy metabolic pathways. Taken together, NNMTi treatment's modulation of body weight, adiposity, liver physiology, and the adipose tissue metabolome strongly support it as a promising therapeutic for obesity and obesity-driven comorbidities.
Assuntos
Composição Corporal , Restrição Calórica , Inibidores Enzimáticos/farmacologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Tecido Adiposo Branco/patologia , Adiposidade/efeitos dos fármacos , Animais , Biomarcadores/sangue , Composição Corporal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Epididimo/patologia , Fígado Gorduroso/sangue , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Metaboloma/efeitos dos fármacos , Metabolômica , Camundongos Endogâmicos C57BL , Camundongos Obesos , Nicotinamida N-Metiltransferase/metabolismo , Magreza/patologiaRESUMO
Nicotinamide-N-methyltransferase (NNMT) is a cytosolic enzyme catalyzing the transfer of a methyl group from S-adenosyl-methionine (SAM) to nicotinamide (Nam). It is expressed in many tissues including the liver, adipose tissue, and skeletal muscle. Its expression in several cancer cell lines has been widely discussed in the literature, and recent work established a link between NNMT expression and metabolic diseases. Here we describe our approach to identify potent small molecule inhibitors of NNMT featuring different binding modes as elucidated by X-ray crystallographic studies.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Doenças Metabólicas/tratamento farmacológico , Doenças Metabólicas/enzimologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Animais , Sítios de Ligação , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Modelos Moleculares , Niacinamida/metabolismo , Nicotinamida N-Metiltransferase/metabolismo , Ratos , Especificidade por Substrato/efeitos dos fármacosRESUMO
Peritoneal metastasis (PM) is the major cause of recurrence in patients with gastric cancer (GC) and is associated with poor prognosis. The oncogenic role of Nicotinamide N-methyltransferase (NNMT) in GC has been reported, but the role of secreted NNMT that is transported by exosomes remains unknown. In this study, exosomes were isolated from GC patients with or without PM and from GC cell line, including GC-114, GC-026, MKN45, and SNU-16 cells. The contents of NNMT were significantly enhanced in exosomes isolated from GC patients with PM compared with those from GC patients without PM. Furthermore, the levels of NNMT were significantly enhanced in exosomes from GC cell lines relative to those from normal human gastric epithelial cell line GES-1 cells. These data indicate that NNMT may be involved in intercellular communication for peritoneal dissemination. Moreover, colocalization of GC-derived exosomal NNMT was found in human peritoneal mesothelial cell line HMrSV5 cells. Additionally, relative to GES-1 exosomes, SNU-16 exosomes significantly activated TGF-ß/smad2 signaling in HMrSV5 cells. However, when NNMT was silenced, the activation of TGF-ß/smad2 by SNU-16 exosomes was abolished in HMrSV5 cells. We propose that NNMT-containing exosomes derived from GC cells could promote peritoneal metastasis via TGF-ß/smad2 signaling.
Assuntos
Regulação Neoplásica da Expressão Gênica , Nicotinamida N-Metiltransferase/genética , Neoplasias Peritoneais/genética , Proteína Smad2/genética , Neoplasias Gástricas/genética , Fator de Crescimento Transformador beta1/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Exossomos/metabolismo , Exossomos/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Nicotinamida N-Metiltransferase/metabolismo , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/patologia , Neoplasias Peritoneais/cirurgia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgia , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Methylation-mediated pathways play important roles in the progression of various diseases. Thus, targeting methyltransferases has proven to be a promising strategy for developing novel therapies. Nicotinamide N-methyltransferase (NNMT) is a major metabolic enzyme involved in epigenetic regulation through catalysis of methyl transfer from the cofactor S-adenosyl-l-methionine onto nicotinamide and other pyridines. Accumulating evidence infers that NNMT is a novel therapeutic target for a variety of diseases such as cancer, diabetes, obesity, cardiovascular and neurodegenerative diseases. Therefore, there is an urgent need to discover potent and specific inhibitors for NNMT to assess its therapeutical potential. Herein, we reported the design and synthesis of a fluorescent probe II138, exhibiting a Kd value of 369 ± 14 nM for NNMT. We also established a fluorescence polarization (FP)-based competition assay for evaluation of NNMT inhibitors. Importantly, the unique feature of this FP competition assay is its capability to identify inhibitors that interfere with the interaction of the NNMT active site directly or allosterically. In addition, this assay performance is robust with a Z'factor of 0.76, indicating its applicability in high-throughput screening for NNMT inhibitors.
Assuntos
Técnicas Biossensoriais/métodos , Inibidores Enzimáticos , Polarização de Fluorescência/métodos , Corantes Fluorescentes , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Domínio Catalítico , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/metabolismo , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Ligação ProteicaRESUMO
Ovarian cancer is an aggressive epithelial tumor that remains a major cause of cancer morbidity and mortality in women. Epigenetic alterations including DNA methylation and histone modifications are being characterized in ovarian cancer and have been functionally linked to processes involved in tumor initiation, chemotherapy resistance, cancer stem cell survival, and tumor metastasis. The epigenetic traits of cancer cells and of associated tumor microenvironment components have been shown to promote an immunosuppressive tumor milieu. However, DNA methylation and histone modifications are reversible, and therapies targeting the epigenome have been implicated in potential reinvigoration of the antitumor immunity. In this review, we provide an overview specifically of DNA methylation and histone modifications as "clothes of the ovarian cancer genome" in relationship to their functional effects and highlight recent developments in the field. We also address the clinical implications of therapeutic strategies to remove or alter specific articles of genomic "clothing" and restore normal cellular function. As the clothes of the genome continue to be deciphered, we envision that the epigenome will become an important therapeutic target for cancer.
Assuntos
Metilação de DNA/genética , Epigênese Genética , Histonas/genética , Neoplasias Ovarianas/genética , Microambiente Tumoral/genética , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Feminino , Genes BRCA1 , Inibidores de Histona Desacetilases/uso terapêutico , Histonas/metabolismo , Humanos , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/terapiaRESUMO
BACKGROUND/OBJECTIVES: Nicotinamide N-methyltransferase (NNMT) is a novel regulator of energy homeostasis in adipocytes. NNMT expression in adipose tissue is increased in obesity and diabetes. Knockdown of NNMT prevents mice from developing diet-induced obesity, which is closely linked to insulin resistance. An early sign of systemic insulin resistance is reduced expression of glucose transporter 4 (GLUT4) selectively in adipose tissue. Adipose tissue-specific knockout and overexpression of GLUT4 cause reciprocal changes in NNMT expression. The aim of the current study was to elucidate the mechanism that regulates NNMT expression in adipocytes. METHODS: 3T3-L1 adipocytes were cultured in media with varying glucose concentrations or activators and inhibitors of intracellular pathways. NNMT mRNA and protein levels were measured with quantitative polymerase chain reaction and Western blotting. RESULTS: Glucose deprivation of 3T3-L1 adipocytes induced a 2-fold increase in NNMT mRNA and protein expression. This effect was mimicked by inhibition of glucose transport with phloretin, and by inhibition of glycolysis with the phosphoglucose isomerase inhibitor 2-deoxyglucose. Conversely, inhibition of the pentose phosphate pathway did not affect NNMT expression. Pharmacological activation of the cellular energy sensor AMP-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin (mTOR) pathway caused an increase in NNMT levels that was similar to the effect of glucose deprivation. Activation of mTOR with MHY1485 prevented the effect of glucose deprivation on NNMT expression. Furthermore, upregulation of NNMT levels depended on functional autophagy and protein translation. CONCLUSION: Glucose availability regulates NNMT expression via an mTOR-dependent mechanism.
Assuntos
Adipócitos/efeitos dos fármacos , Transportador de Glucose Tipo 4/genética , Glucose/farmacologia , Nicotinamida N-Metiltransferase/genética , Serina-Treonina Quinases TOR/genética , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Transporte Biológico/efeitos dos fármacos , Diferenciação Celular , Desoxiglucose/farmacologia , Metabolismo Energético/genética , Regulação da Expressão Gênica , Glucose/metabolismo , Transportador de Glucose Tipo 4/antagonistas & inibidores , Transportador de Glucose Tipo 4/metabolismo , Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Homeostase/genética , Camundongos , Morfolinas/farmacologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Nicotinamida N-Metiltransferase/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/genética , Floretina/farmacologia , Biossíntese de Proteínas , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Triazinas/farmacologiaRESUMO
Nicotinamide N-methyltransferase (NNMT) catalyzes the methyl transfer from the cofactor S-adenosylmethionine to nicotinamide and other pyridine-containing compounds. NNMT is an important regulator for nicotinamide metabolism and methylation potential. Aberrant expression levels of NNMT have been implicated in cancer, metabolic, and neurodegenerative diseases, which makes NNMT a potential therapeutic target. Therefore, potent and selective NNMT inhibitors can serve as valuable tools to investigate the roles of NNMT in its mediated diseases. Here, we applied a rational strategy to design and synthesize the tight-binding bisubstrate inhibitor LL320 through a novel propargyl linker. LL320 demonstrates a Ki value of 1.6 ± 0.3 nM, which is the most potent inhibitor to date. The cocrystal structure of LL320 confirms its interaction with both the substrate and cofactor binding sites on NNMT. Importantly, this is the first example of using the propargyl linker to construct potent methyltransferase inhibitors, which will expand our understanding of the transition state of methyl transfer.
Assuntos
Ácido Aspártico/análogos & derivados , Benzamidas/farmacologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Ácido Aspártico/química , Ácido Aspártico/farmacologia , Benzamidas/química , Desenho de Fármacos , Células HCT116 , Humanos , Nicotinamida N-Metiltransferase/metabolismo , Permeabilidade , Ligação ProteicaRESUMO
Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that methylates nicotinamide (NAM) using cofactor S-adenosylmethionine (SAM). NNMT overexpression has been linked to diabetes, obesity, and various cancers. In this work, structure-based rational design led to the development of potent and selective alkynyl bisubstrate inhibitors of NNMT. The reported nicotinamide-SAM conjugate (named NS1) features an alkyne as a key design element that closely mimics the linear, 180° transition state geometry found in the NNMT-catalyzed SAM â NAM methyl transfer reaction. NS1 was synthesized in 14 steps and found to be a high-affinity, subnanomolar NNMT inhibitor. An X-ray cocrystal structure and SAR study revealed the ability of an alkynyl linker to span the methyl transfer tunnel of NNMT with ideal shape complementarity. The compounds reported in this work represent the most potent and selective NNMT inhibitors reported to date. The rational design principle described herein could potentially be extended to other methyltransferase enzymes.
Assuntos
Alcinos/química , Alcinos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Nicotinamida N-Metiltransferase/metabolismo , Alcanos/química , Alcinos/metabolismo , Inibidores Enzimáticos/metabolismo , Estabilidade Enzimática , Humanos , Células K562 , Simulação de Acoplamento Molecular , Nicotinamida N-Metiltransferase/química , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , TemperaturaRESUMO
Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide to form N-methylnicotinamide. Overexpression of NNMT is associated with a variety of diseases, including a number of cancers and metabolic disorders, suggesting a role for NNMT as a potential therapeutic target. By structural modification of a lead NNMT inhibitor previously developed in our group, we prepared a diverse library of inhibitors to probe the different regions of the enzyme's active site. This investigation revealed that incorporation of a naphthalene moiety, intended to bind the hydrophobic nicotinamide binding pocket via π-π stacking interactions, significantly increases the activity of bisubstrate-like NNMT inhibitors (half-maximal inhibitory concentration 1.41 µM). These findings are further supported by isothermal titration calorimetry binding assays as well as modeling studies. The most active NNMT inhibitor identified in the present study demonstrated a dose-dependent inhibitory effect on the cell proliferation of the HSC-2 human oral cancer cell line.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Modelos Moleculares , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/metabolismo , Naftalenos/química , Naftalenos/farmacologia , Niacinamida/metabolismo , Nicotinamida N-Metiltransferase/metabolismoRESUMO
Nicotinamide N-methyltransferase (NNMT) catalyzes the S-adenosyl-l-methionine-dependent methylation of nicotinamide to form N-methylnicotinamide. This enzyme detoxifies xenobiotics and regulates NAD+ biosynthesis. Additionally, NNMT is overexpressed in various cancers. Herein, we describe the first NNMT-targeted suicide substrates. These compounds, which include 4-chloropyridine and 4-chloronicotinamide, exploit the broad substrate scope of NNMT; methylation of the pyridine nitrogen enhances the electrophilicity of the C4 position, thereby promoting an aromatic nucleophilic substitution by C159, a noncatalytic cysteine. On the basis of this activity, we developed a suicide inhibition-based protein labeling strategy using an alkyne-substituted 4-chloropyridine that selectively labels NNMT in vitro and in cells. In total, this study describes the first NNMT-directed activity-based probes.
Assuntos
Nicotinamida N-Metiltransferase/metabolismo , Proteínas/metabolismo , Catálise , Células HEK293 , Humanos , Cinética , Metilação , Nicotinamida N-Metiltransferase/antagonistas & inibidoresRESUMO
Aging is accompanied by progressive declines in skeletal muscle mass and strength and impaired regenerative capacity, predisposing older adults to debilitating age-related muscle deteriorations and severe morbidity. Muscle stem cells (muSCs) that proliferate, differentiate to fusion-competent myoblasts, and facilitate muscle regeneration are increasingly dysfunctional upon aging, impairing muscle recovery after injury. While regulators of muSC activity can offer novel therapeutics to improve recovery and reduce morbidity among aged adults, there are no known muSC regenerative small molecule therapeutics. We recently developed small molecule inhibitors of nicotinamide N-methyltransferase (NNMT), an enzyme overexpressed with aging in skeletal muscles and linked to impairment of the NAD+ salvage pathway, dysregulated sirtuin 1 activity, and increased muSC senescence. We hypothesized that NNMT inhibitor (NNMTi) treatment will rescue age-related deficits in muSC activity to promote superior regeneration post-injury in aging muscle. 24-month old mice were treated with saline (control), and low and high dose NNMTi (5 and 10â¯mg/kg) for 1-week post-injury, or control and high dose NNMTi for 3-weeks post-injury. All mice underwent an acute muscle injury (barium chloride injection) locally to the tibialis anterior (TA) muscle, and received 5-ethynyl-2'-deoxyuridine systemically to analyze muSC activity. In vivo contractile function measurements were conducted on the injured TA muscle and tissues collected for ex-vivo analyses, including myofiber cross-sectional area (CSA) measurements to assess muscle recovery. Results revealed that muscle stem cell proliferation and subsequent fusion were elevated in NNMTi-treated mice, supporting nearly 2-fold greater CSA and shifts in fiber size distribution to greater proportions of larger sized myofibers and fewer smaller sized fibers in NNMTi-treated mice compared to controls. Prolonged NNMTi treatment post-injury further augmented myofiber regeneration evinced by increasingly larger fiber CSA. Importantly, improved muSC activity translated not only to larger myofibers after injury but also to greater contractile function, with the peak torque of the TA increased by â¼70% in NNMTi-treated mice compared to controls. Similar results were recapitulated in vitro with C2C12 myoblasts, where NNMTi treatment promoted and enhanced myoblast differentiation with supporting changes in the cellular NAD+/NADH redox states. Taken together, these results provide the first clear evidence that NNMT inhibitors constitute a viable pharmacological approach to enhance aged muscle regeneration by rescuing muSC function, supporting the development of NNMTi as novel mechanism-of-action therapeutic to improve skeletal muscle regenerative capacity and functional recovery after musculoskeletal injury in older adults.
Assuntos
Envelhecimento/fisiologia , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Mioblastos , Distribuição AleatóriaRESUMO
Nicotinamide N-methyltransferase (NNMT) catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to nicotinamide, pyridine, and other structural analogues. Aberrantly increased NNMT activity results in the depletion of SAM, nicotinamide (NAM), and nicotinamide adenine dinucleotide (NAD+); NAM is required for NAD+ biosynthesis. SAM depletion impairs the methylation potential of the cell, resulting in hypomethylated histones and an altered epigenetic profile. In addition, decreased NAD+ levels negatively affect energy metabolism by disrupting oxidative phosphorylation. Because of its impact on epigenetic states and NAD+ levels, NNMT is implicated in cancer, neurodegenerative diseases, and metabolic diseases, making it an appealing target for therapeutic intervention. To gain insights that would guide the design of inhibitors and activity-based probes, we performed detailed kinetic studies of human NNMT. Herein, we report the kinetic mechanism of NNMT. Our initial velocity, product inhibition, and dead-end analogue inhibition studies collectively indicate that NNMT uses a rapid equilibrium ordered mechanism, where NNMT first binds SAM, which is followed by NAM. Methyl transfer occurs, and methylated NAM and S-adenosylhomocysteine are released consecutively.
Assuntos
NAD/metabolismo , Niacinamida/metabolismo , Nicotinamida N-Metiltransferase/antagonistas & inibidores , S-Adenosilmetionina/metabolismo , Humanos , Cinética , Metilação , Nicotinamida N-Metiltransferase/metabolismoRESUMO
Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide using S-adenosyl-L-methionine (SAM) as a methyl donor and, through doing so, can modulate cellular methylation potential to impact diverse epigenetic processes. NNMT has been implicated in a range of diseases, including cancer and metabolic disorders. Potent, selective, and cell-active inhibitors would constitute valuable probes to study the biological functions and therapeutic potential of NNMT. We previously reported the discovery of electrophilic small molecules that inhibit NNMT by reacting with an active-site cysteine residue in the SAM-binding pocket. Here, we have used activity-based protein profiling (ABPP)-guided medicinal chemistry to optimize the potency and selectivity of NNMT inhibitors, culminating in the discovery of multiple alpha-chloroacetamide (αCA) compounds with sub-µM IC50 values in vitro and excellent proteomic selectivity in cell lysates. However, these compounds showed much weaker inhibition of NNMT in cells, a feature that was not shared by off-targets of the αCAs. Our results show the potential for developing potent and selective covalent inhibitors of NNMT, but also highlight challenges that may be faced in targeting this enzyme in cellular systems.
Assuntos
Acetamidas/farmacologia , Inibidores Enzimáticos/farmacologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Acetamidas/síntese química , Domínio Catalítico , Linhagem Celular Tumoral , Cisteína/química , Inibidores Enzimáticos/síntese química , Humanos , Nicotinamida N-Metiltransferase/químicaRESUMO
Nicotinamide N-methyltransferase (NNMT) has been linked to obesity and diabetes. We have identified a novel nicotinamide (NA) analog, compound 12 that inhibited NNMT enzymatic activity and reduced the formation of 1-methyl-nicotinamide (MNA), the primary metabolite of NA by â¼80% at 2â¯h when dosed in mice orally at 50â¯mg/kg.