RESUMO
Pyruvate kinase (PK) is an essential component of cellular metabolism, converting ADP and phosphoenolpyruvate (PEP) to pyruvate in the final step of glycolysis. Of the four unique isoforms of pyruvate kinase, R (PKR) is expressed exclusively in red blood cells and is a tetrameric enzyme that depends on fructose-1,6-bisphosphate (FBP) for activation. PKR deficiency leads to hemolysis of red blood cells resulting in anemia. Activation of PKR in both sickle cell disease and beta-thalassemia patients could lead to improved red blood cell fitness and survival. The discovery of a novel series of substituted urea PKR activators, via the serendipitous identification and diligent characterization of a minor impurity in an High Throughput Screening (HTS) hit will be discussed.
Assuntos
Ensaios de Triagem em Larga Escala , Piruvato Quinase , Piruvato Quinase/metabolismo , Piruvato Quinase/antagonistas & inibidores , Humanos , Descoberta de Drogas , Relação Estrutura-Atividade , Ureia/química , Ureia/farmacologia , Ativadores de Enzimas/farmacologia , Ativadores de Enzimas/química , Ativadores de Enzimas/síntese química , Estrutura Molecular , AnimaisRESUMO
Two protoberberine alkaloids with a unique C28 skeleton, named xanthiumines A (1) and B (2), respectively, were isolated from the fruits of Xanthium sibiricum Patr. Their structures including absolute configurations were unequivocally established by the comprehensive NMR and MS spectroscopic data analysis together with gauge-independent atomic orbital (GIAO) NMR calculations, and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 are the first examples of natural protoberberine alkaloid with a phenolic acid group at C-13a. Their plausible biosynthetic pathway was proposed on the basis of the coexisting alkaloid monomer as the precursor. Furthermore, the effects and related molecular mechanism of compound 1 on hepatic lipid accumulation were also investigated in oleic acid (OA)-treated HepG2 cells.
Assuntos
Proteínas Quinases Ativadas por AMP , Alcaloides de Berberina , Frutas , Xanthium , Humanos , Frutas/química , Xanthium/química , Alcaloides de Berberina/química , Alcaloides de Berberina/farmacologia , Alcaloides de Berberina/isolamento & purificação , Células Hep G2 , Estrutura Molecular , Proteínas Quinases Ativadas por AMP/metabolismo , Relação Estrutura-Atividade , Relação Dose-Resposta a Droga , Descoberta de Drogas , Ativadores de Enzimas/farmacologia , Ativadores de Enzimas/química , Ativadores de Enzimas/isolamento & purificaçãoRESUMO
Alzheimer's disease (AD) is a devastating neurodegenerative disorder, one of the main characteristics of which is the abnormal accumulation of amyloid peptide (Aß) in the brain. Whereas ß-secretase supports Aß formation along the amyloidogenic processing of the ß-amyloid precursor protein (ßAPP), α-secretase counterbalances this pathway by both preventing Aß production and triggering the release of the neuroprotective sAPPα metabolite. Therefore, stimulating α-secretase and/or inhibiting ß-secretase can be considered a promising anti-AD therapeutic track. In this context, we tested andrographolide, a labdane diterpene derived from the plant Andrographis paniculata, as well as 24 synthesized derivatives, for their ability to induce sAPPα production in cultured SH-SY5Y human neuroblastoma cells. Following several rounds of screening, we identified three hits that were subjected to full characterization. Interestingly, andrographolide (8,17-olefinic) and its close derivative 14α-(5',7'-dichloro-8'-quinolyloxy)-3,19-acetonylidene (compound 9) behave as moderate α-secretase activators, while 14α-(2'-methyl-5',7'-dichloro-8'-quinolyloxy)-8,9-olefinic compounds 31 (3,19-acetonylidene) and 37 (3,19-diol), whose two structures are quite similar although distant from that of andrographolide and 9, stand as ß-secretase inhibitors. Importantly, these results were confirmed in human HEK293 cells and these compounds do not trigger toxicity in either cell line. Altogether, these findings may represent an encouraging starting point for the future development of andrographolide-based compounds aimed at both activating α-secretase and inhibiting ß-secretase that could prove useful in our quest for the therapeutic treatment of AD.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Diterpenos , Ativadores de Enzimas , Diterpenos/síntese química , Diterpenos/química , Diterpenos/farmacologia , Ativadores de Enzimas/síntese química , Ativadores de Enzimas/química , Ativadores de Enzimas/farmacologia , Células HEK293 , HumanosRESUMO
As a vital kinase in the glycolysis system, PKM2 is extensively expressed in colorectal cancer (CRC) to support the energy and biosynthetic needs. In this study, we designed a series of parthenolide (PTL) derivatives through a stepwise structure optimization, and an excellent derivate 29e showed good activity on PKM2 (AC50 = 86.29 nM) and displayed significant antiproliferative activity against HT29 (IC50 = 0.66 µM) and SW480 (IC50 = 0.22 µM) cells. 29e decreased the expression of total PKM2, prevented nucleus translocation of PKM2 dimer, and inhibited PKM2/STAT3 signaling pathway. 29e remarkably increased OCR and decreased the extracellular acidification rate (ECAR). The antiproliferative effect of 29e depended on PKM2, and the Cys424 of PKM2 was the key binding site. Furthermore, 29e significantly suppressed tumor growth in the HT29 xenograft model without obvious toxicity. These outcomes demonstrate that 29e is a promising drug candidate for the treatment of CRC.
Assuntos
Neoplasias Colorretais/patologia , Ativadores de Enzimas/farmacologia , Proteína Quinase C/efeitos dos fármacos , Sesquiterpenos/farmacologia , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/enzimologia , Dimerização , Ativadores de Enzimas/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fator de Transcrição STAT3/metabolismo , Sesquiterpenos/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mitochondrial oxidative phosphorylation (OXPHOS) has become an attractive target in anti-cancer studies in recent years. In this study, we found that a small molecule phenylbutenoid dimer NMac1 (Nm23-H1 activator 1), (±)-trans-3-(3,4-dimethoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene, a previously identified anti-metastatic agent, has novel anti-proliferative effect only under glucose starvation in metastatic breast cancer cells. NMac1 causes significant activation of AMPK by decreasing ATP synthesis, lowers mitochondrial membrane potential (MMP, ΔΨm), and inhibits oxygen consumption rate (OCR) under glucose starvation. These effects of NMac1 are provoked by a consequence of OXPHOS complex I inhibition. Through the structure-activity relationship (SAR) study of NMac1 derivatives, NMac24 was identified as the most effective compound in anti-proliferation. NMac1 and NMac24 effectively suppress cancer cell proliferation in 3D-spheroid in vivo-like models only under glucose starvation. These results suggest that NMac1 and NMac24 have the potential as anti-cancer agents having cytotoxic effects selectively in glucose restricted cells.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Cicloexenos/farmacologia , Nucleosídeo NM23 Difosfato Quinases/efeitos dos fármacos , Estirenos/farmacologia , Trifosfato de Adenosina/biossíntese , Antineoplásicos/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cicloexenos/química , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Ativadores de Enzimas/química , Ativadores de Enzimas/farmacologia , Feminino , Redes Reguladoras de Genes/efeitos dos fármacos , Glucose/metabolismo , Humanos , Células MCF-7 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Estirenos/químicaRESUMO
A previous study showed that 2'-3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) was a weak allosteric activator of Rhizobium etli pyruvate carboxylase (RePC) in the absence of acetyl-CoA. On the other hand, TNP-ATP inhibited the allosteric activation of RePC by acetyl-CoA. Here, we aimed to study the role of triphosphate group of TNP-ATP on its allosteric activation of the enzyme and inhibition of acetyl-CoA-dependent activation of RePC using TNP-ATP and its derivatives, including TNP-ADP, TNP-AMP and TNP-adenosine. The pyruvate carboxylation activity was assayed to determine the effect of reducing the number of phosphate groups in TNP-ATP derivatives on allosteric activation and inhibition of acetyl-CoA activation of RePC and chicken liver pyruvate carboxylase (CLPC). Reducing the number of phosphate groups in TNP-ATP derivatives decreased the activation efficacy for both RePC and CLPC compared to TNP-ATP. The apparent binding affinity and inhibition of activation of the enzymes by acetyl-CoA were also diminished when the number of phosphate groups in the TNP-ATP derivatives was reduced. Whilst TNP-AMP activated RePC, it did not activate CLPC, but it did inhibit acetyl-CoA activation of both RePC and CLPC. Similarly, TNP-adenosine did not activate RePC; however, it did inhibit acetyl-CoA activation using a different mechanism compared to phosphorylated TNP-derivatives. These findings indicate that mechanisms of PC activation and inhibition of acetyl-CoA activation by TNP-ATP and its derivatives are different. This study provides the basis for possible drug development for treatment of metabolic diseases and cancers with aberrant expression of PC.
Assuntos
Acetilcoenzima A/química , Trifosfato de Adenosina/análogos & derivados , Regulação Alostérica/efeitos dos fármacos , Ativadores de Enzimas/química , Piruvato Carboxilase/química , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/química , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Animais , Galinhas , Ensaios Enzimáticos , Cinética , Fígado/enzimologia , Estrutura MolecularRESUMO
The presented research concerns the triple activity of trans-cinnamic (tCA), ferulic (FA) and syringic acids (SA). They act as thyroid peroxidase (TPO) activators, lipoxygenase (LOX) inhibitors and show antiradical activity. All compounds showed a dose-dependent TPO activatory effect, thus the AC50 value (the concentration resulting in 50% activation) was determined. The tested compounds can be ranked as follows: tCA > FA > SA with AC50 = 0.10, 0.39, 0.69 mM, respectively. Strong synergism was found between FA and SA. The activatory effects of all tested compounds may result from interaction with the TPO allosteric site. It was proposed that conformational change resulting from activator binding to TPO allosteric pocket results from the flexibility of a nearby loop formed by residues Val352-Tyr363. All compounds act as uncompetitive LOX inhibitors. The most effective were tCA and SA, whereas the weakest was FA (IC50 = 0.009 mM and IC50 0.027 mM, respectively). In all cases, an interaction between the inhibitors carboxylic groups and side-chain atoms of Arg102 and Arg139 in an allosteric pocket of LOX was suggested. FA/tCA and FA/SA acted synergistically, whereas tCA/SA demonstrated antagonism. The highest antiradical activity was found in the case of SA (IC50 = 0.22 mM). FA/tCA and tCA/SA acted synergistically, whereas antagonism was found for the SA/FA mixture.
Assuntos
Autoantígenos/metabolismo , Ativadores de Enzimas/farmacologia , Iodeto Peroxidase/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Inibidores de Lipoxigenase/farmacologia , Compostos Fitoquímicos/farmacologia , Proteína-Lisina 6-Oxidase/metabolismo , Autoantígenos/química , Cinamatos/química , Cinamatos/farmacologia , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacologia , Relação Dose-Resposta a Droga , Ativadores de Enzimas/química , Ácido Gálico/análogos & derivados , Ácido Gálico/química , Ácido Gálico/farmacologia , Humanos , Concentração Inibidora 50 , Iodeto Peroxidase/química , Proteínas de Ligação ao Ferro/química , Inibidores de Lipoxigenase/química , Modelos Moleculares , Compostos Fitoquímicos/química , Proteína-Lisina 6-Oxidase/química , Relação Estrutura-AtividadeRESUMO
Selective modulation of retinaldehyde dehydrogenases (RALDHs)-the main aldehyde dehydrogenase (ALDH) enzymes converting retinal into retinoic acid (RA), is very important not only in the RA signaling pathway but also for the potential regulatory effects on RALDH isozyme-specific processes and RALDH-related cancers. However, very few selective modulators for RALDHs have been identified, partly due to variable overexpression protocols of RALDHs and insensitive activity assay that needs to be addressed. In the present study, deletion of the N-terminal disordered regions is found to enable simple preparation of all RALDHs and their closest paralog ALDH2 using a single protocol. Fluorescence-based activity assay was employed for enzymatic activity investigation and screening for RALDH-specific modulators from extracts of various spices and herbs that are well-known for containing many phyto-derived anti-cancer constituents. Under the established conditions, spice and herb extracts exhibited differential regulatory effects on RALDHs/ALDH2 with several extracts showing potential selective inhibition of the activity of RALDHs. In addition, the presence of magnesium ions was shown to significantly increase the activity for the natural substrate retinal of RALDH3 but not the others, while His-tag cleavage considerably increased the activity of ALDH2 for the non-specific substrate retinal. Altogether we propose a readily reproducible workflow to find selective modulators for RALDHs and suggest potential sources of selective modulators from spices and herbs.
Assuntos
Ensaios Enzimáticos/métodos , Extratos Vegetais/farmacologia , Retinal Desidrogenase/metabolismo , Ativadores de Enzimas/química , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli , Humanos , Extratos Vegetais/química , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retinal Desidrogenase/química , Retinal Desidrogenase/efeitos dos fármacos , Retinal Desidrogenase/genética , Homologia de SequênciaRESUMO
Adenosine monophosphate-activated protein kinase (AMPK) plays a key role in maintaining whole-body homeostasis and has been regarded as a therapeutic target for the treatment of diabetic nephropathy (DN). Herein, a series of 1,2,4-oxadiazole-containing pyrazolo[3,4-b]pyridinone derivatives is reported as AMPKÉ1ß1γ1 activators. The in vitro biological assay demonstrated that compounds 12k (EC50 [AMPKα1γ1ß1] = 180 nM) and 13q (EC50 [AMPKα1γ1ß1] = 2 nM) displayed significant enzyme activation. Mechanism studies indicated that both compounds reduced the levels of reactive oxygen species in a rat kidney fibroblast cell line (NRK-49F) stimulated by transforming growth factor-ß and induced early apoptosis of NRK-49F cells at 10 µM. Molecular docking studies suggested that 13q exhibited critical hydrogen-bond interactions with the critical amino acid residues Lys29, Lys31, Asn111, and Asp88 at the binding site of the AMPK protein. These results enrich the structure pool of AMPK activators and provide novel lead compounds for the subsequent development of compounds with a promising therapeutic potential against DN.
Assuntos
Ativadores de Enzimas/farmacologia , Oxidiazóis/farmacologia , Pirazóis/farmacologia , Piridonas/farmacologia , Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Desenho de Fármacos , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/síntese química , Ativadores de Enzimas/química , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Simulação de Acoplamento Molecular , Oxidiazóis/síntese química , Oxidiazóis/química , Pirazóis/síntese química , Pirazóis/química , Piridonas/síntese química , Piridonas/química , Ratos , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-AtividadeRESUMO
GPR55 is a GPCR of the non-CB1/CB2 cannabinoid receptor family, which is activated by lysophosphatidylinositol (LPI) and stimulates the proliferation of cancer cells. Anandamide, a bioactive lipid endocannabinoid, acts as a biased agonist of GPR55 and induces cancer cell death, but is unstable and psychoactive. We hypothesized that other endocannabinoids and structurally similar compounds, which are more hydrolytically stable, could also induce cancer cell death via GPR55 activation. We chemically synthesized and tested a set of fatty acid amides and esters for cell death induction via GPR55 activation. The most active compounds appeared to be N-acyl dopamines, especially N-docosahexaenoyl dopamine (DHA-DA). Using a panel of cancer cell lines and a set of receptor and intracellular signal transduction machinery inhibitors together with cell viability, Ca2+, NO, ROS (reactive oxygen species) and gene expression measurement, we showed for the first time that for these compounds, the mechanism of cell death induction differed from that published for anandamide and included neuronal nitric oxide synthase (nNOS) overstimulation with concomitant oxidative stress induction. The combination of DHA-DA with LPI, which normally stimulates cancer proliferation and is increased in cancer setting, had an increased cytotoxicity for the cancer cells indicating a therapeutic potential.
Assuntos
Antineoplásicos/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Dopamina/análogos & derivados , Ativadores de Enzimas/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Receptores de Canabinoides/metabolismo , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Agonistas de Receptores de Canabinoides/química , Linhagem Celular Tumoral , Dopamina/química , Dopamina/farmacologia , Ativadores de Enzimas/química , Ácidos Graxos/química , Ácidos Graxos/farmacologia , Humanos , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Células PC12 , RatosRESUMO
The adenosine monophosphateactivated protein kinase (AMPK) is a promising target in drug development for various metabolic diseases. In the present study, the aim was to discover natural direct AMPK activators from natural sources, thus a virtual screening for direct AMPK activators was conducted by combining ligandbased and structurebased screening. A commonfeature pharmacophore model (HipHop1) was generated with two hydrogen bond acceptor lipid features and one hydrophobic region feature. A total of 1,235 natural products were screened using the HipHop1 hypothesis and CDOCKER protocol successively. According to the docking score, seven hit compounds were selected for AMPK activation assays. Ultimately, ()catechin (compound 522) and licochalcone A (compound 1148) exhibited the highest AMPK activation activity. These findings may contribute to the development of AMPK activators from medicinal plants.
Assuntos
Proteínas Quinases Ativadas por AMP/química , Ativadores de Enzimas/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Desenho de Fármacos , Humanos , Ligantes , Relação Quantitativa Estrutura-AtividadeRESUMO
An activatable photosensitizer based on methylene blue was developed and validated for its efficacy in the selective killing of γ-glutamyl transpeptidase expressing cells. The 1O2 deactivation via bond breakage, rapid in situ enzymatic photosensitivity conversion, long lysosomal retention, and nucleus relocation collectively contribute to its strong and selective photodynamic effects.
Assuntos
Antineoplásicos/farmacologia , Ativadores de Enzimas/farmacologia , Glioma/tratamento farmacológico , Azul de Metileno/farmacologia , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , gama-Glutamiltransferase/metabolismo , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Ativadores de Enzimas/química , Glioma/metabolismo , Glioma/patologia , Humanos , Azul de Metileno/química , Estrutura Molecular , Imagem Óptica , Fármacos Fotossensibilizantes/química , RatosRESUMO
Janus kinases (JAKs) are non-receptor tyrosine kinases that are essential components of the JAK-STAT signaling pathway. Associated aberrant signaling is responsible for many forms of cancer and disorders of the immune system. The present focus is on the discovery of molecules that may regulate the activity of JAK2 by selective binding to the JAK2 pseudokinase domain, JH2. Specifically, the Val617Phe mutation in JH2 stimulates the activity of the adjacent kinase domain (JH1) resulting in myeloproliferative disorders. Starting from a non-selective screening hit, we have achieved the goal of discovering molecules that preferentially bind to the ATP binding site in JH2 instead of JH1. We report the design and synthesis of the compounds and binding results for the JH1, JH2, and JH2 V617F domains, as well as five crystal structures for JH2 complexes. Testing with a selective and non-selective JH2 binder on the autophosphorylation of wild-type and V617F JAK2 is also contrasted.
Assuntos
Amitrol (Herbicida)/química , Amitrol (Herbicida)/metabolismo , Ativadores de Enzimas/química , Ativadores de Enzimas/metabolismo , Janus Quinase 2/química , Janus Quinase 2/metabolismo , Animais , Células HEK293 , Humanos , Ligantes , Ligação Proteica/fisiologia , Células Sf9 , Difração de Raios X/métodosRESUMO
ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that generates antigenic peptides and is an emerging target for cancer immunotherapy and the control of autoimmunity. ERAP1 inhibitors described previously target the active site and are limited in selectivity, minimizing their clinical potential. To address this, we targeted the regulatory site of ERAP1 using a high-throughput screen and discovered a small molecule hit that is highly selective for ERAP1. (4aR,5S,6R,8S,8aR)-5-(2-(Furan-3-yl)ethyl)-8-hydroxy-5,6,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carboxylic acid is a natural product found in Dodonaea viscosa that constitutes a submicromolar, highly selective, and cell-active modulator of ERAP1. Although the compound activates hydrolysis of small model substrates, it is a competitive inhibitor for physiologically relevant longer peptides. Crystallographic analysis confirmed that the compound targets the regulatory site of the enzyme that normally binds the C-terminus of the peptide substrate. Our findings constitute a novel starting point for the development of selective ERAP1 modulators that have potential for further clinical development.
Assuntos
Aminopeptidases/antagonistas & inibidores , Apresentação de Antígeno/efeitos dos fármacos , Diterpenos Clerodânicos/farmacologia , Epitopos/metabolismo , Peptídeos/metabolismo , Inibidores de Proteases/farmacologia , Sítio Alostérico , Aminopeptidases/química , Aminopeptidases/metabolismo , Animais , Domínio Catalítico , Cristalografia por Raios X , Diterpenos Clerodânicos/química , Diterpenos Clerodânicos/metabolismo , Ativadores de Enzimas/química , Ativadores de Enzimas/metabolismo , Ativadores de Enzimas/farmacologia , Epitopos/química , Células HeLa , Humanos , Camundongos , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/metabolismo , Peptídeos/química , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Ligação Proteica , Proteólise/efeitos dos fármacosRESUMO
Acer okamotoanum is reported to have various antioxidant, antiinflammatory and beneficial immune system effects. The antiadipocyte differentiation effects and mechanisms of the ethyl acetate (EtOAc) fraction of an A. okamotoanum extraction was investigated in 3T3L1 adipocyte cells. Treatment with differentiation inducers increased the level of triglycerides (TGs) in 3T3L1 adipocyte cells compared with an untreated control. However, the EtOAc fraction of A. okamotoanum significantly decreased TGs. Treatment with 1, 2.5 and 5 µg/ml showed weak activity, but TG production was inhibited at 10 µg/ml compared with the control. In addition, A. okamotoanum caused a significant downregulation of proteins related to adipogenesis, such as γcytidinecytidineadenosineadenosinethymidine/enhancer binding proteinα, ß and peroxisome proliferatoractivated receptorγ, compared with the untreated control. Furthermore, A. okamotoanum significantly upregulated lipolysis related protein, hormonesensitive lipase and the phosphorylation of adenosine monophosphateactivated protein kinase (AMPK). Therefore, these results indicate that A. okamotoanum suppressed adipogenesis and increased lipolysis and the activation of AMPK, suggesting a protective role in adipocyte differentiation.
Assuntos
Acer , Adipogenia/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Lipólise/efeitos dos fármacos , Extratos Vegetais/farmacologia , Células 3T3-L1 , Quinases Proteína-Quinases Ativadas por AMP , Acer/química , Animais , Ativadores de Enzimas/química , Camundongos , Extratos Vegetais/química , Proteínas Quinases/metabolismoRESUMO
The importance of upregulated Wnt signaling in colorectal cancers led to efforts to develop inhibitors that target ß-catenin in this pathway. We now report that several "Wnt inhibitors" that allegedly target ß-catenin actually function as mitochondrial proton uncouplers that independently activate AMPK and concomitantly inhibit Wnt signaling. As expected for a process in which mitochondrial uncoupling diminishes ATP production, a mitochondrial proton uncoupler, FCCP, and a glucose metabolic inhibitor, 2-DG, activated AMPK and inhibited Wnt signaling. Also consistent with these findings, a well-known "Wnt inhibitor", FH535, functioned as a proton uncoupler, and in support of this finding, the N-methylated analog, 2,5-dichloro-N-methyl-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535-M), was inactive as an uncoupler and Wnt inhibitor. Apart from suggesting an opportunity to develop dual Wnt inhibitors and AMPK activators, these findings provide a cautionary tale that claims for Wnt inhibition alone require scrutiny as possible mitochondrial proton uncouplers or inhibitors of the electron transport chain.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Encéfalo/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Ativadores de Enzimas/farmacologia , Hidrocarbonetos Fluorados/farmacologia , Mitocôndrias/efeitos dos fármacos , Ureia/farmacologia , Proteínas Wnt/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Animais , Encéfalo/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Metabolismo Energético , Ativação Enzimática , Ativadores de Enzimas/química , Regulação Neoplásica da Expressão Gênica , Humanos , Hidrocarbonetos Fluorados/química , Mitocôndrias/metabolismo , Consumo de Oxigênio , Sulfonamidas/química , Sulfonamidas/farmacologia , Células Tumorais Cultivadas , Ureia/análogos & derivados , Ureia/químicaRESUMO
BACKGROUND: The incidence of non-small cell lung cancer (NSCLC) accounts for approximately 85-90% of lung cancer, which has been shown to be challenging for treatment owing to poorly understanding of pathological mechanisms. Natural products serve as a source of almost all pharmaceutical preparations or offer guidance for those chemicals that have entered clinical trials, especially in NSCLC. PURPOSE: We investigated the effect of B10G5, a natural products isolated from the Croton tiglium, in human non-small cell lung canceras as a protein kinase C (PKC) activator. METHODS: The cell viability assay was evaluated by the MTT assay. The apoptosis and cell cycle distribution were assessed by flow cytometry. Reactive oxygen species (ROS) production was determined by using the fluorescent probe DCFDA. Cell migration ability of H1975 cells was analyzed by using the wound healing assay. The inhibiting effect of B10G5 against the phosphorylation level of the substrate by PKCs was assessed by using homogeneous time-resolved fluorescence (HTRF) technology. The correlation between PKCs and overall survival (OS) of Lung Adenocarcinoma (LUAD) patients was analysis by TCGA portal. The binding mode between B10G5 and the PKC isoforms was explored by molecular docking. Protein expression was detected by western blotting analysis. RESULTS: B10G5 suppressed cell proliferation and colony formation, as well as migration ability of NSCLC cells, without significant toxic effect on normal lung cells. B10G5 induced the cell apoptosis through the development of PARP cleavage, which is evidenced by means of the production of mitochondrial ROS. In addition, the B10G5 inhibitory effect was also related to the cell cycle arrest at G2/M phase. Mechanistically, molecular modelling technology suggested that the potential target of B10G5 was associated with PKC family. In vitro PKC kinase assay indicated that B10G5 effectively activated the PKC activity. Western blotting data revealed that B10G5 upregulated PKC to activate PKC-mediated RAF/MEK/ERK pathway. CONCLUSION: Our results showed that B10G5, a naturally occurring phorbol ester, considered to be a potential and a valuable therapeutic chemical in the treatment of NSCLC.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Croton/química , Ativadores de Enzimas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteína Quinase C/metabolismo , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Ativadores de Enzimas/química , Humanos , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Simulação de Acoplamento Molecular , Fosforilação/efeitos dos fármacos , Proteína Quinase C/química , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.
Assuntos
Ativadores de Enzimas/química , Fosfopeptídeos/química , Proteína Fosfatase 2C/química , Bibliotecas de Moléculas Pequenas/química , Ativadores de Enzimas/isolamento & purificação , Ativadores de Enzimas/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Proteína Fosfatase 2C/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato , Proteína Supressora de Tumor p53/químicaRESUMO
The active components of Malus pumila flowers on antioxidant and tyrosinase activity were investigated with the method of spectrum-effect relationship and knock-out. Some compounds were identified by UPLC-MS/MS method. The chemical fingerprints were established by HPLC and the activity of antioxidant and tyrosinase were assayed in vitro. Chromatographic peaks P34, P35, P39, P44, P45 and P49 were identified as phloridzin, hyperoside, astragalin, afzelin, quercetin and kaempferol by UPLC-MS/MS method. Hyperoside and kaempferol were identified in M. pumila flowers for the first time. When the concentration was 1â¯g/mL of sample (equivalent to raw material), the scavenging capacity of P35 (hyperoside) on DPPH free radicals were consistent with the spectrum-effect relationship. The scavenging capacity of P34 (phloridzin) and P45 (quercetin) on ABTS free radicals were consistent with the spectrum-effect relationship. The activation effect of P45 (quercetin) on tyrosinase was consistent with the spectrum-effect relationship. The inhibitory effect of P34 (phloridzin), P35 (hyperoside) on tyrosinase were consistent with the spectrum-effect relationship.
Assuntos
Ativadores de Enzimas/química , Inibidores Enzimáticos/química , Flavonoides/química , Sequestradores de Radicais Livres/química , Malus/química , Monofenol Mono-Oxigenase/antagonistas & inibidores , Cromatografia Líquida de Alta Pressão/métodos , Ativadores de Enzimas/isolamento & purificação , Inibidores Enzimáticos/isolamento & purificação , Flavonoides/isolamento & purificação , Flores/química , Sequestradores de Radicais Livres/isolamento & purificação , Espectrometria de Massas em Tandem/métodosRESUMO
Type 2 diabetes mellitus is a complex metabolic disorder requiring polypharmacology approaches for effective treatment. Combinatorial library of fifteen new tricyclic benzimidazole derivatives have been designed and synthesized to combine fragments commonly found in allosteric AMPK activators and AT1 receptor antagonists. It was found that 2'-cyanobiphenyl serves as the pharmacophore of AMPK-activating activity, which also increases with the expansion of the external hydrogenated cycle. Also, pronounced antiplatelet activity is characteristic of the studied compounds. One of derivatives was identified as a potent inhibitor of the formation of advanced protein glycation end-products with reactive dicarbonyl scavenging activity. Two submicromolar AMPK activators 2b and 3b prevents inflammatory activation of murine macrophages. Along with good water solubility and synthetic availability, these results render biphenyl derivatives of fused benzimidazoles as a valuable starting point for the development of AMPK activators with multi-target antidiabetic activity.