RESUMO
Since the efficiency in the transcription of the HIV genome contributes to the success of viral replication and infectivity, we investigated the downregulating effects of the spirobisindole alkaloids globospiramine (1), deoxyvobtusine (2), and vobtusine lactone (3) from the endemic Philippine medicinal plant, Voacanga globosa, during HIV gene transcription. Alkaloids 1-3 were explored for their inhibitory activity on TNF-α-induced viral replication in two latently HIV-infected cell lines, OM10.1 and J-Lat. The induction of HIV replication from OM10.1 and J-Lat cells elicited by TNF-α was blocked by globospiramine (1) within noncytotoxic concentrations. Furthermore, globospiramine (1) was found to target the NF-ĸB activation cascade in a dose-dependent manner when the transcriptional step at which inhibitory activity is exerted was examined in TNF-α-induced 293 human cells using transient reporter (luciferase) gene expression systems (HIV LTR-luc, ĸB-luc, and mutant ĸB-luc). Interrogation through molecular docking against the NF-ĸB p50/p65 heterodimer and target sites of the subunits comprising the IKK complex revealed high binding affinities of globospiramine (1) against the S281 pocket of the p65 subunit (BE = -9.2 kcal/mol) and the IKKα activation loop (BE = -9.1 kcal/mol). These findings suggest globospiramine (1) as a molecular inspiration to discover new alkaloid-based anti-HIV derivatives.
Assuntos
Alcaloides/farmacologia , Infecções por HIV/metabolismo , HIV-1/fisiologia , Quinase I-kappa B/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Fator de Transcrição RelA/metabolismo , Voacanga/química , Alcaloides/química , Linhagem Celular , Relação Dose-Resposta a Droga , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Células HL-60 , Humanos , Quinase I-kappa B/química , Alcaloides Indólicos/farmacologia , Modelos Biológicos , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/química , Extratos Vegetais/química , Transdução de Sinais/efeitos dos fármacos , Compostos de Espiro/farmacologia , Fator de Transcrição RelA/química , Fator de Necrose Tumoral alfa/farmacologia , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
The benefits associated with resveratrol (Resv; 3,4',5-trihydroxy-trans-stilbene) are known for a long time. The therapeutic properties of Resv are observed in diseases like cancer, neurological disorders, atherosclerosis, aging, inflammation, etc. Multiple studies suggest that the beneficial properties of Resv are due to its binding to targets in multiple pathways. The same has been reflected in inflammation, where Resv has been shown to inhibit nuclear factor κ light-chain enhancer of activated B cells in the toll-like receptor 4 (TLR4) pathway. There are multiple cellular targets which bind to Resv, however the mode and the key interactions involved remain elusive for many of them. In the current work, we have investigated the structural insights of Resv with three of its binding partners involved in the inflammatory TLR4 signaling pathway. Through a structure-based modelling and molecular dynamics study, we have unraveled the molecular and atomic interactions involved in the Resv-binary complexes of inhibitor of κB kinase, cyclooxygeanse-2, and tank-binding kinase I, all three of which are key players in TLR4 inflammatory signaling. This study is the latest addition to the investigations of the structural partners of Resv and its molecular interactions.
Assuntos
Ciclo-Oxigenase 2/química , Quinase I-kappa B/química , Extratos Vegetais/química , Proteínas Serina-Treonina Quinases/química , Resveratrol/química , Receptor 4 Toll-Like/metabolismo , Sítios de Ligação , Proteínas de Transporte/química , Cristalização , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Quinase I-kappa B/antagonistas & inibidores , Inflamação/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Compostos Fitoquímicos/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Estrutura Secundária de Proteína , Projetos de Pesquisa , Sesquiterpenos/química , Vitis/química , FitoalexinasRESUMO
The nuclear factor-κB (NF-κB) plays an important role in inflammatory and immune responses. Aberrant NF-κB signaling is implicated in multiple disorders, including cancer. Targeting the regulatory scaffold subunit IκB kinase γ (IKKγ/NEMO) as therapeutic interventions could be promising due to its specific involvement in canonical NF-κB activation without interfering with non-canonical signaling. In this study, the use of unnatural amino acid substituted IKKß with unique photophysical activity to sense water environment changes upon interaction with NEMO provides a powerful in vitro screening platform that would greatly facilitate the identification of compounds having the potential to disrupt IKKß-NEMO interaction, and thus specifically modulate the canonical NF-κB pathway. We then utilized a competitive binding platform to screen the binding ability of a number of potential molecules being synthesized. Our results suggest that a lead compound (-)-PDC-099 is a potent agent with ascertained potency to disrupt IKKß-NEMO complex for modulating NF-κB canonical pathway.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Corantes Fluorescentes/química , Quinase I-kappa B/metabolismo , Peptídeos/química , Mapas de Interação de Proteínas/efeitos dos fármacos , Triptofano/análogos & derivados , Compostos Aza/química , Compostos Aza/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/química , Modelos Moleculares , Peptídeos/metabolismo , Triptofano/metabolismoRESUMO
The iridoids of Hedyotis diffusa Willd play an important role in the anti-inflammatory process, but the specific iridoid with anti-inflammatory effect and its mechanism has not be thoroughly studied. An iridoid compound named scandoside (SCA) was isolated from H. diffusa and its anti-inflammatory effect was investigated in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Its anti-inflammatory mechanism was confirmed by in intro experiments and molecular docking analyses. As results, SCA significantly decreased the productions of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and inhibited the levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α and IL-6 messenger RNA (mRNA) expression in LPS-induced RAW 264.7 macrophages. SCA treatment suppressed the phosphorylation of inhibitor of nuclear transcription factor kappa-B alpaha (IκB-α), p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). The docking data suggested that SCA had great binding abilities to COX-2, iNOS and IκB. Taken together, the results indicated that the anti-inflammatory effect of SCA is due to inhibition of pro-inflammatory cytokines and mediators via suppressing the nuclear transcription factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, which provided useful information for its application and development.
Assuntos
Anti-Inflamatórios/farmacologia , Hedyotis/química , Iridoides/farmacologia , Lipopolissacarídeos/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/antagonistas & inibidores , Dinoprostona/biossíntese , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/química , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Iridoides/química , Iridoides/isolamento & purificação , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , NF-kappa B/genética , NF-kappa B/metabolismo , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Extratos Vegetais/química , Células RAW 264.7 , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The aim of this study was to investigate the effects of resolvin D1 (RvD1), as well as the combined treatment of docosahexaenoic acid monoglyceride (MAG-DHA) and acetylsalicylic acid (ASA), on the resolution of inflammation markers and Ca(2+) sensitivity in IL-13-pretreated human bronchi (HB). Tension measurements performed with 300 nM RvD1 largely abolished (50%) the over-reactivity triggered by 10 ng/ml IL-13 pretreatment and reversed hyper Ca(2+) sensitivity. Addition of 300 nM 17(S)-HpDoHE, the metabolic intermediate between DHA and RvD1, displayed similar effects. In the presence of 100 µM ASA (a COX inhibitor), the inhibitory effect of 1 µM MAG-DHA on muscarinic tone was further amplified, but not in the presence of Ibuprofen. Western blot analysis revealed that the combined treatment of MAG-DHA and ASA upregulated GPR-32 expression and downregulated cytosolic TNFα detection, hence preventing IκBα degradation and p65-NFκB phosphorylation. The Ca(2+) sensitivity of HB was also quantified on ß-escin permeabilized preparations. The presence of ASA potentiated the inhibitory effects of MAG-DHA in reducing the Ca(2+) hypersensitivity triggered by IL-13 by decreasing the phosphorylation levels of the PKC-potentiated inhibitor protein-17 regulatory protein (CPI-17). In summary, MAG-DHA combined with ASA, as well as exogenously added RvD1, may represent valuable assets against critical AHR disorder.
Assuntos
Brônquios/efeitos dos fármacos , Bronquite/tratamento farmacológico , Broncodilatadores/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacologia , Ácidos Docosa-Hexaenoicos/metabolismo , Monoglicerídeos/farmacologia , Resistência das Vias Respiratórias/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Brônquios/imunologia , Brônquios/metabolismo , Bronquite/imunologia , Bronquite/metabolismo , Broncodilatadores/agonistas , Sinergismo Farmacológico , Ácidos Graxos Ômega-3/antagonistas & inibidores , Ácidos Graxos Ômega-3/metabolismo , Humanos , Quinase I-kappa B/química , Quinase I-kappa B/metabolismo , Técnicas In Vitro , Interleucina-13/antagonistas & inibidores , Interleucina-13/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Monoglicerídeos/agonistas , Proteínas Musculares , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Bergenin (1), a unique fused C-glycoside isolated from Bergenia species, possesses interesting anti-inflammatory and antipain activities. To study SAR of this scaffold, first-generation derivatives were synthesized and evaluated for inhibition of lymphocyte proliferation and production of pro-inflammatory cytokines. The C-7 substituted derivatives showed inhibition of IL-6 as well as TNF-α production. Bergenin and its most potent IL-6 inhibitor derivatives 4e and 4f were then investigated in a panel of in vitro and in vivo inflammation/arthritis models. These compounds significantly decreased the expression of NF-kB and IKK-ß in THP-1 cells. In in vivo study in BALB/c mice, a dose-dependent inhibition of SRBC-induced cytokines, reduction in humoral/cell-mediated immunity, and antibody titer was observed. The CIA study in DBA/1J mice indicated that compounds led to reduction in swelling of paws, cytokine levels, and anticollagen IgG1/IgG2a levels. The significant in vivo immunosuppressive efficacy of pyrano-isochromanones demonstrates the promise of this scaffold for development of next-generation antiarthritic drugs.
Assuntos
Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Artrite Experimental/tratamento farmacológico , Interleucina-6/antagonistas & inibidores , Animais , Anti-Inflamatórios não Esteroides/síntese química , Benzopiranos/química , Proteínas de Ligação ao Cálcio/efeitos dos fármacos , Cromanos/química , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Citocinas/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Quinase I-kappa B/química , Quinase I-kappa B/metabolismo , Imunidade Humoral/efeitos dos fármacos , Imunoglobulina G/metabolismo , Linfócitos/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , NF-kappa B/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição , Fator de Necrose Tumoral alfa/biossínteseRESUMO
IκB kinase ß (IKKß), an attractive anti-inflammation and anti-cancer target, plays a crucial role in the activation of NF-κB signalling pathway. To identify novel IKKß inhibitors, we combined structure-based and ligand-based methods based on the co-crystal structure of IKKß. According to the chemical similarity, 162 reported IKKß inhibitors were divided into five classes. For each class, a 3D pharmacophore model was established based on the binding conformations of the compounds. The validated models were further used in virtual screening. Twelve drugable compounds were retained for biological test, resulting in two novel inhibitors with IC50 values lower than 10 µM. Compared to other models, our method considers the crystal structure of IKKß for the first time.
Assuntos
Quinase I-kappa B/química , Conformação Molecular , Inibidores de Proteínas Quinases/química , Estrutura Terciária de Proteína , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/metabolismo , Ligantes , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-AtividadeRESUMO
GA (gambogic acid) is a polyprenylated xanthone abundant in the resin of Garcinia morella and Garcinia hanburyi with a long history of use as a complementary and alternative medicine. The antitumour activity of GA has been well demonstrated and is thought to arise partly from the associated anti-inflammatory activity. Recent studies have indicated that the antitumour activity of GA is mediated by its ligation of TfR1 (transferrin receptor-1). Since the cellular expression of TfR1 is down-regulated by LPS (lipopolysaccharide), we hypothesized that an alternative pathway exists in immune cells, such as macrophages, where GA could mitigate the expression of pro-inflammatory genes. Here we demonstrate that GA inhibits the LPS-dependent expression of NF-kappaB (nuclear factor kappaB) target pro-inflammatory genes in macrophages. Western immunoblot, NF-kappaB-luciferase reporter and gel-shift analyses revealed that GA strongly blocked the activation of NF-kappaB induced by LPS, whereas 9,10-dihydro-GA, which lacks the reactive alpha,beta-unsaturated carbonyl group, was ineffective. Moreover, GA was able to decrease nuclear p65 levels in RAW264.7 macrophages, where the expression of TfR1 was down-regulated by RNA interference. in vitro kinase assays coupled with interaction studies using biotinylated GA as well as proteomic analysis demonstrated that IKKbeta [IkappaB (inhibitory kappaB) kinase-beta], a key kinase of the NF-kappaB signalling axis, was covalently modified by GA at Cys-179, causing significant inhibition of its kinase activity. Taken together, these results demonstrate the potent anti-inflammatory activity of GA.
Assuntos
Quinase I-kappa B/química , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo , Xantonas/química , Xantonas/farmacologia , Animais , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Humanos , Imunoprecipitação , Lipopolissacarídeos/farmacologia , Camundongos , Interferência de RNARESUMO
Berberine, an isoquinoline alkaloid derived from a plant used traditionally in Chinese and Ayurvedic medicine, has been reported to exhibit chemopreventive and anti-inflammatory activities through unknown mechanism. Because of the critical role of the transcription factor nuclear factor-kappaB (NF-kappaB) in these processes, we investigated the effect of berberine on this pathway. We found that berberine suppressed NF-kappaB activation induced by various inflammatory agents and carcinogens. This alkaloid also suppressed constitutive NF-kappaB activation found in certain tumor cells. Suppression of NF-kappaB activation occurred through the inhibition of phosphorylation and degradation of IkappaBalpha by the inhibition of IkappaB kinase (IKK) activation, leading to suppression of phosphorylation and nuclear translocation of p65, and finally to inhibition of NF-kappaB reporter activity. Inhibition of IKK by berbeine was direct and could be reversed by reducing agents. Site-specific mutagenesis suggested the involvement of cysteine residue 179 in IKK. Berberine also suppressed the expression of NF-kappaB-regulated gene products involved in antiapoptosis (Bcl-xL, Survivin, IAP1, IAP2, and cFLIP), proliferation (cyclin D1), inflammation (cyclooxygenase-2), and invasion (matrix metalloproteinase-9). Suppression of antiapoptotic gene products correlated with enhancement of apoptosis induced by tumor necrosis factor (TNF)-alpha and chemotherapeutic agents and with inhibition of TNF-induced cellular invasion. Overall, our results indicate that chemopreventive, apoptotic, and anti-inflammatory activities displayed by berberine may be mediated in part through the suppression of the NF-kappaB activation pathway. This may provide the molecular basis for the ability of berberine to act as an anticancer and anti-inflammatory agent.