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1.
J Chem Theory Comput ; 15(11): 6504-6512, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31584802

RESUMO

We present an explicit solvent alchemical free-energy method for optimizing the partial charges of a ligand to maximize the binding affinity with a receptor. This methodology can be applied to known ligand-protein complexes to determine an optimized set of ligand partial atomic changes. Three protein-ligand complexes have been optimized in this work: FXa, P38, and the androgen receptor. The sets of optimized charges can be used to identify design principles for chemical changes to the ligands which improve the binding affinity for all three systems. In this work, beneficial chemical mutations are generated from these principles and the resulting molecules tested using free-energy perturbation calculations. We show that three quarters of our chemical changes are predicted to improve the binding affinity, with an average improvement for the beneficial mutations of approximately 1 kcal/mol. In the cases where experimental data are available, the agreement between prediction and experiment is also good. The results demonstrate that charge optimization in explicit solvent is a useful tool for predicting beneficial chemical changes such as pyridinations, fluorinations, and oxygen to sulfur mutations.


Assuntos
Fator Xa/química , Ligantes , Receptores Androgênicos/química , Proteínas Quinases p38 Ativadas por Mitógeno/química , Sítios de Ligação , Fator Xa/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Receptores Androgênicos/metabolismo , Eletricidade Estática , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
2.
Colloids Surf B Biointerfaces ; 177: 496-505, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30807964

RESUMO

Excellent biocompatibility and inflammatory regulation ability are essential to bone repair materials. Herein, Rod-like HAP with a diameter of 0.1 µm and Flake-like HAP with a width of 0.5-1 µm were synthesized by hydrothermal method, and then combined with two kinds of biomolecules, Icariin and Kaempferol. Two kinds of HAPs have similar crystal structure, but different zeta potentials and specific surface area. Rod-like HAP possesses stronger loading capacity and internalization efficiency than Flake-like one. in vitro inflammation assay reveals that HAP particles up-regulate the expression of IL-1ß, TNF-α, IL-6, IL-10, IFN-γ and IL-2 cytokines. HAP particles loaded with Icariin or Kaempferol biomolecules up-regulate anti-inflammatory cytokines and down-regulate the expression of inflammatory cytokines.


Assuntos
Fluoresceína/química , Hidroxiapatitas/química , Inflamação/metabolismo , Ovalbumina/química , Caspases/química , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fluoresceína/metabolismo , Humanos , Hidroxiapatitas/farmacologia , Estrutura Molecular , Ovalbumina/metabolismo , Tamanho da Partícula , Propriedades de Superfície , Células THP-1 , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
3.
J Insect Sci ; 19(1)2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30715437

RESUMO

Proteins p38 map kinase and ribosomal S6 kinase (S6K) as members of mitogen-activated protein kinases (MAPKs) play important roles against pathogens. In this study, Bmp38 and BmS6K were identified as differentially expressed proteins from iTRAQ database. Bmp38 and BmS6K were expressed, and recombinant proteins were purified. The bioinformatics analysis showed that both proteins have serine/threonine-protein kinases, catalytic domain (S_TKc) with 360 and 753 amino acids, respectively. The real-time quantitative polymerase chain reaction (RT-qPCR) results suggest that Bmp38 and BmS6K had high expression in the midgut and hemolymph. The comparative expression level of Bmp38 and BmS6K in BC9 was upregulated than in P50 in the midgut after Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Western bolt results showed a positive correlation between RT-qPCR and iTRAQ data for Bmp38, but BmS6K data showed partial correlation with iTRAQ. Injection of anti-Bmp38 and anti-BmS6K serum suggested that Bmp38 may be involved against BmNPV infection, whereas BmS6K may require phosphorylation modification to inhibit BmNPV infection. Taken together, our results suggest that Bmp38 and BmS6k might play an important role in innate immunity of silkworm against BmNPV.


Assuntos
Bombyx/genética , Proteínas de Insetos/genética , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bombyx/crescimento & desenvolvimento , Bombyx/imunologia , Bombyx/virologia , Imunidade Inata/genética , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/imunologia , Larva/virologia , Filogenia , Proteínas Quinases S6 Ribossômicas/química , Proteínas Quinases S6 Ribossômicas/metabolismo , Alinhamento de Sequência , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
4.
Nat Commun ; 10(1): 131, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30631068

RESUMO

PROteolysis-TArgeting Chimeras (PROTACs) are hetero-bifunctional molecules that recruit an E3 ubiquitin ligase to a given substrate protein resulting in its targeted degradation. Many potent PROTACs with specificity for dissimilar targets have been developed; however, the factors governing degradation selectivity within closely-related protein families remain elusive. Here, we generate isoform-selective PROTACs for the p38 MAPK family using a single warhead (foretinib) and recruited E3 ligase (von Hippel-Lindau). Based on their distinct linker attachments and lengths, these two PROTACs differentially recruit VHL, resulting in degradation of p38α or p38δ. We characterize the role of ternary complex formation in driving selectivity, showing that it is necessary, but insufficient, for PROTAC-induced substrate ubiquitination. Lastly, we explore the p38δ:PROTAC:VHL complex to explain the different selectivity profiles of these PROTACs. Our work attributes the selective degradation of two closely-related proteins using the same warhead and E3 ligase to heretofore underappreciated aspects of the ternary complex model.


Assuntos
Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Domínios Proteicos , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Especificidade por Substrato , Ubiquitina-Proteína Ligases/química , Proteína Supressora de Tumor Von Hippel-Lindau/química , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
5.
Fish Shellfish Immunol ; 84: 848-856, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30381267

RESUMO

p38 mitogen-activated protein kinase (MAPK) is an important protein which plays a key role in regulating the innate immunity, so exploring its molecular characterization is helpful in understanding the resistance against microbial infections in cultured fish. Here, a full-length cDNA of p38 MAPK was cloned from liver of blunt snout bream (Megalobrama amblycephala) which covered 2419 bp with an open reading frame of 1086 bp encoding 361 amino acids. p38 MAPK contained the characteristic structures of Thr-Gly-Tyr (TGY) motif and substrate binding site Ala-Thr-Arg-Trp (ATRW), which are conserved in MAPK family. To investigate p38 MAPK functions, two in vivo experiments were carried out to examine its expression following ammonia exposure and bacterial challenge. Also, an in vitro experiment was conducted to assess the role of p38 MAPK in inflammation of primary hepatocytes induced by lipopolysaccharide (LPS). The results showed the ubiquitous expression of p38 MAPK in all the tested tissues with varying levels. p38 MAPK mRNA expression was significantly up-regulated by ammonia stress and Aeromonas hydrophila challenge, and altered in a time-dependent manner. Moreover, the results indicated that the inflammatory response induced by LPS in hepatocytes is p38 MAPK dependent as knockdown of p38 MAPK using siRNA technology depressed the expression of IL-1ß and IL-6. The findings in this study showed that p38 MAPK has anti-stress property, and plays key role in protection against bacterial infection and inflammation in blunt snout bream.


Assuntos
Cyprinidae/genética , Cyprinidae/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Amônia/efeitos adversos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas/imunologia , Imunidade Celular/genética , Lipopolissacarídeos/farmacologia , Filogenia , Distribuição Aleatória , Alinhamento de Sequência/veterinária , Proteínas Quinases p38 Ativadas por Mitógeno/química
6.
Pharmacol Rep ; 71(1): 13-23, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30343043

RESUMO

BACKGROUND: Berberine is an alkaloid plant-based DNA intercalator that affects gene regulation, particularly expression of oncogenic and tumor suppressor proteins. The effects of berberine on different signaling proteins remains to be elucidated. The present study aimed to identify the effects of berberine against key oncogenic proteins in breast cancer cells. METHODS: Molecular docking and molecular dynamics simulations were used for EGFR, p38, ERK1/2, and AKT. The effects of berberine and lapatinib on MAPK and PI3K pathways in MDA-MB231 and MCF-7 cells were evaluated using immunoflorescence assays, and the amounts of phosphorylated kinases were compared to total kinases after treating with different concentrations of berberine. RESULTS: Simulations showed berberine accurately interacted with EGFR, AKT, P38, and ERK1/2 active sites in silico (scores = -7.57 to -7.92 Kcal/mol) and decreased the levels of active forms of corresponding enzymes in both cell lines; however, berberine binding to p38 showed less stability. Cytotoxicity analysis indicated that MDA-MB231 cells were resistant to berberine compared to MCF-7 cells [72 h IC50 = 50 versus 15 µM, respectively). Also, lapatinib strongly activated AKT but suppressed EGFR in MDA-MB231 cells. The activity of EGFR, AKT, P38, and ERK1/2 were affected by berberine; however, berberine dramatically reduced EGFR and AKT phosphorylation. CONCLUSION: By way of its multikinase inhibitory effects, berberine might be a useful replacement for lapatinib, an EGFR inhibitor which can cause acquired drug resistance in patients.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Berberina/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/metabolismo , Berberina/química , Berberina/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Domínio Catalítico , Estabilidade Enzimática , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Receptores ErbB/metabolismo , Feminino , Humanos , Lapatinib/farmacologia , Células MCF-7 , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
7.
J Med Chem ; 61(20): 9316-9334, 2018 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-30253095

RESUMO

As regulators of transcription, epigenetic proteins that interpret post-translational modifications to N-terminal histone tails are essential for maintaining cellular homeostasis. When dysregulated, "reader" proteins become drivers of disease. In the case of bromodomains, which recognize N-ε-acetylated lysine, selective inhibition of individual bromodomain-and-extra-terminal (BET)-family bromodomains has proven challenging. We describe the >55-fold N-terminal-BET bromodomain selectivity of 1,4,5-trisubstituted-imidazole dual kinase-bromodomain inhibitors. Selectivity for the BRD4 N-terminal bromodomain (BRD4(1)) over its second bromodomain (BRD4(2)) arises from the displacement of ordered waters and the conformational flexibility of lysine-141 in BRD4(1). Cellular efficacy was demonstrated via reduction of c-Myc expression, inhibition of NF-κB signaling, and suppression of IL-8 production through potential synergistic inhibition of BRD4(1) and p38α. These dual inhibitors provide a new scaffold for domain-selective inhibition of BRD4, the aberrant function of which plays a key role in cancer and inflammatory signaling.


Assuntos
Imidazóis/química , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Células A549 , Humanos , Domínios Proteicos , Água/química , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/química
8.
Int J Mol Sci ; 19(9)2018 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-30135361

RESUMO

The p38 mitogen activated protein kinase (MAPK) signaling pathway has been suggested to play a significant role in the gastric mucosal inflammatory response to chronic Helicobacter pylori (H. pylori) infection. Nuclear translocation is thought to be important for p38 function, but no nuclear translocation signals have been found in the protein and no nuclear carrier proteins have been identified for p38. We have investigated the role of small ubiquitin-related modifier (SUMO) in the nuclear transfer of p38 in response to H. pylori infection. Exposure of human AGS cells to H. pylori induced the activation of p38 and the expression of SUMOs, especially SUMO-2. SUMO knockdown counteracted the effect of H. pylori infection by decreasing the resulting p38 mediated cellular apoptosis through a reduction in the nuclear fraction of phosphorylated p38. We identified a non-covalent interaction between SUMOs and p38 via SUMO interaction motifs (SIMs), and showed that SUMO-dependent nuclear transfer of p38 was decreased upon mutation of its SIMs. This study has identified a new pathway of p38 nuclear translocation, in response to H. pylori infection. We conclude that in the presence of H. pylori SUMO-2 has a major role in regulating nuclear levels of p38, through non-covalent SUMO-p38 interactions, independent of the p38 phosphorylation state.


Assuntos
Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter pylori , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Transporte Ativo do Núcleo Celular , Apoptose , Linhagem Celular , Humanos , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Quinases p38 Ativadas por Mitógeno/química
9.
Nanomedicine (Lond) ; 13(13): 1607-1621, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30028250

RESUMO

AIM: To investigate whether p38 small-interfering RNA-loaded nanoparticles (p38 siRNA NPs) attenuate spinal nerve ligation (SNL)-induced neuropathic pain in rats by suppressing spinal microglia activation via p38 targeting. MATERIALS & METHODS: After synthesizing p38 siRNA NPs with sonication, physical characteristics were measured for size and zeta potential. p38 siRNA NPs were then administrated intrathecally into SNL rats if they could reduce pain behavior excellently. RESULTS: p38 siRNA NPs significantly reduced mechanical allodynia as well as microgliosis in the spinal dorsal horns of SNL rats, consistent with a downregulation of p38-related proinflammatory mediators. CONCLUSION: As p38 in the spinal microglia plays a critical role in neuropathic pain, we expect that p38 siRNA NPs could be a promising tool for the treatment of neuropathic pain.


Assuntos
Nanopartículas/administração & dosagem , Neuralgia/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , Proteínas Quinases p38 Ativadas por Mitógeno/administração & dosagem , Animais , Humanos , Ligadura , Microglia/efeitos dos fármacos , Nanopartículas/química , Neuralgia/genética , Neuralgia/fisiopatologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/administração & dosagem , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Nervos Espinhais/efeitos dos fármacos , Nervos Espinhais/lesões , Nervos Espinhais/fisiopatologia , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/genética
10.
Proc Natl Acad Sci U S A ; 115(18): 4655-4660, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29666261

RESUMO

Mitogen-activated protein kinases, which include p38, are essential for cell differentiation and autophagy. The current model for p38 activation involves activation-loop phosphorylation with subsequent substrate binding leading to substrate phosphorylation. Despite extensive efforts, the molecular mechanism of activation remains unclear. Here, using NMR spectroscopy, we show how the modulation of protein dynamics across timescales activates p38. We find that activation-loop phosphorylation does not change the average conformation of p38; rather it quenches the loop ps-ns dynamics. We then show that substrate binding to nonphosphorylated and phosphorylated p38 results in uniform µs-ms backbone dynamics at catalytically essential regions and across the entire molecule, respectively. Together, these results show that phosphorylation and substrate binding flatten the energy landscape of the protein, making essential elements of allostery and activation dynamically accessible. The high degree of structural conservation among ser/thr kinases suggests that elements of this mechanism may be conserved across the kinase family.


Assuntos
Simulação de Dinâmica Molecular , Proteínas Quinases p38 Ativadas por Mitógeno/química , Regulação Alostérica/fisiologia , Ativação Enzimática/fisiologia , Humanos , Ressonância Magnética Nuclear Biomolecular , Fosforilação/fisiologia , Estrutura Secundária de Proteína , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
11.
J Chem Inf Model ; 58(5): 968-981, 2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29620886

RESUMO

Understanding the governing factors of fast or slow inhibitor binding/unbinding assists in developing drugs with preferred kinetic properties. For inhibitors with the same binding affinity targeting different binding sites of the same protein, the kinetic behavior can profoundly differ. In this study, we investigated unbinding kinetics and mechanisms of fast (type-I) and slow (type-II/III) binders of p38α mitogen-activated protein kinase, where the crystal structures showed that type-I and type-II/III inhibitors bind to pockets with different conformations of the Asp-Phe-Gly (DFG) motif. The work used methods that combine conventional molecular dynamics (MD), accelerated molecular dynamics (AMD) simulations, and the newly developed pathway search guided by internal motions (PSIM) method to find dissociation pathways. The study focuses on revealing key interactions and molecular rearrangements that hinder ligand dissociation by using umbrella sampling and post-MD processing to examine changes in free energy during ligand unbinding. As anticipated, the initial dissociation steps all require breaking interactions that appeared in crystal structures of the bound complexes. Interestingly, for type-I inhibitors such as SB2, p38α keeps barrier-free conformational fluctuation in the ligand-bound complex and during ligand dissociation. In contrast, with a type-II/III inhibitor such as BIRB796, with the rearrangements of p38α in its bound state, ligand unbinding features energetically unfavorable protein-ligand concerted movement. Our results also show that the type-II/III inhibitors preferred dissociation pathways through the allosteric channel, which is consistent with an existing publication. The study suggests that the level of required protein rearrangement is one major determining factor of drug binding kinetics in p38α systems, providing useful information for development of inhibitors.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Inibidores de Proteínas Quinases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/química
12.
J Biol Chem ; 293(15): 5447-5461, 2018 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-29414778

RESUMO

Caspase-3 activation and function have been well-defined during programmed cell death, but caspase activity, at low levels, is also required for developmental processes such as lymphoid proliferation and erythroid differentiation. Post-translational modification of caspase-3 is one method used by cells to fine-tune activity below the threshold required for apoptosis, but the allosteric mechanism that reduces activity is unknown. Phosphorylation of caspase-3 at a conserved allosteric site by p38-MAPK (mitogen-activated protein kinase) promotes survival in human neutrophils, and the modification of the loop is thought to be a key regulator in many developmental processes. We utilized phylogenetic, structural, and biophysical studies to define the interaction networks that facilitate the allosteric mechanism in caspase-3. We show that, within the modified loop, Ser150 evolved with the apoptotic caspases, whereas Thr152 is a more recent evolutionary event in mammalian caspase-3. Substitutions at Ser150 result in a pH-dependent decrease in dimer stability, and localized changes in the modified loop propagate to the active site of the same protomer through a connecting surface helix. Likewise, a cluster of hydrophobic amino acids connects the conserved loop to the active site of the second protomer. The presence of Thr152 in the conserved loop introduces a "kill switch" in mammalian caspase-3, whereas the more ancient Ser150 reduces without abolishing enzyme activity. These data reveal how evolutionary changes in a conserved allosteric site result in a common pathway for lowering activity during development or a more recent cluster-specific switch to abolish activity.


Assuntos
Caspase 3 , Evolução Molecular , Regulação Alostérica/genética , Substituição de Aminoácidos , Animais , Caspase 3/química , Caspase 3/genética , Humanos , Mutação de Sentido Incorreto , Fosforilação/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/genética
13.
J Diet Suppl ; 15(3): 269-284, 2018 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-28800275

RESUMO

Parquetina nigrescens is commonly used to treat diseases in humans and animals in developing countries, including Nigeria. This study evaluates the effects of its polyphenol-rich fraction (prf) on dichlorvos-induced cardio- and renal toxicity. There were several factors assessed during this study, including cardiac and renal markers, serum myeloperoxidase and xanthine oxidase, and electrocardiograph (ECG) changes. The changes in electrocardiograph (ECG) were recorded. Immunohistochemistry of cardiac and renal p38 and nitrotyrosine was determined. Dichlorvos exposure caused a significant decrease in L-glutathione (reduced glutathione) and other antioxidant enzymes with increases in malondialdehyde, myeloperoxidase, advanced oxidation protein products, and protein carbonyl levels. It also brought about alterations in microanatomy of the heart and kidneys accompanied by increases in serum creatinine and urea levels. Exposure to dichlorvos induced prolonged QRS interval and shortened QT durations in rats. Immunohistochemistry revealed lower expressions of cardiac nitrotyrosine and renal p38 (mitogen-activated protein kinase; MAPK) in rats treated with prf of P. nigrescens. Combining all, prf of P. nigrescens demonstrated antioxidant as well as protective properties in the heart and kidneys of rats exposed to dichlorvos. It ameliorated dichlorvos-induced cardio- and nephrotoxicity giving credence to its use in ethnomedicine.


Assuntos
Cryptolepis/química , Suplementos Nutricionais , Intoxicação por Organofosfatos/prevenção & controle , Componentes Aéreos da Planta/química , Extratos Vegetais/uso terapêutico , Polifenóis/uso terapêutico , Substâncias Protetoras/uso terapêutico , Administração Oral , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Cryptolepis/crescimento & desenvolvimento , Diclorvós/administração & dosagem , Diclorvós/antagonistas & inibidores , Diclorvós/toxicidade , Suplementos Nutricionais/análise , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Inseticidas/administração & dosagem , Inseticidas/antagonistas & inibidores , Inseticidas/toxicidade , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Rim/fisiopatologia , Masculino , Nigéria , Intoxicação por Organofosfatos/metabolismo , Intoxicação por Organofosfatos/patologia , Intoxicação por Organofosfatos/fisiopatologia , Componentes Aéreos da Planta/crescimento & desenvolvimento , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Polifenóis/administração & dosagem , Polifenóis/análise , Polifenóis/isolamento & purificação , Substâncias Protetoras/administração & dosagem , Substâncias Protetoras/química , Substâncias Protetoras/isolamento & purificação , Distribuição Aleatória , Ratos Wistar , Insuficiência Renal/etiologia , Insuficiência Renal/prevenção & controle , Tirosina/agonistas , Tirosina/análogos & derivados , Tirosina/antagonistas & inibidores , Tirosina/metabolismo , Disfunção Ventricular/etiologia , Disfunção Ventricular/prevenção & controle , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
Front Immunol ; 9: 3102, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30671063

RESUMO

Objective: Plumbago zeylanica L. (with plumbagin as its active ingredients) has been used for centuries to treat conditions such as joint swelling, fractures, and bacterial infections, suggesting that it possesses anti-inflammatory and immunosuppressive properties. In the present study, we evaluated the potential anti-arthritic activity and related mechanisms of plumbagin. Methods: Collagen-induced arthritis (CIA) was initiated in Wistar rats with collagen type II. Plumbagin (2 and 6 mg/kg) was orally administered to rats with CIA from day 12 to day 32 post immunization. The effects of plumbagin on arthritis progression were assessed by paw swelling, clinical scoring, and histologic analysis. The percentage of Treg and Th17 were defined by flow cytometry or immunofluorescence (IF) staining. Bone erosion and resorption were assessed by micro-CT and histomorphometric analysis. Osteoclast differentiation was further determined by in vitro osteoclastogenesis assay. The molecular docking assay was used to determine the potential binding site of plumbagin. Results: Treatment with plumbagin significantly inhibited arthritis development, as well as suppressed the local and systemic inflammation. Plumbagin reciprocally regulated pro-inflammatory Th17 cell and immunosuppressive Treg cell populations. In addition, plumbagin protected inflammation-induced bone loss by inhibiting osteoclast formation and activity. Plumbagin markedly suppressed RANKL-stimulated osteoclast-specific gene expression by repressing NF-κB signaling activation and MAP kinase phosphorylation. Further study via molecular docking assay demonstrated that plumbagin bound to MET169 of JNK kinase and LYS138 and SER183 of p38 kinase. Conclusion: Plumbagin not only attenuates the immune-induced arthritis by inhibiting inflammation, but also protects bone erosion by directly inhibiting osteoclast formation and activity. These data suggest plumbagin is a promising new candidate drug for treating inflammatory joint diseases.


Assuntos
Artrite Experimental/tratamento farmacológico , Naftoquinonas/uso terapêutico , Osteogênese/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Análise de Variância , Animais , Artrite Experimental/induzido quimicamente , Sítios de Ligação , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno/efeitos adversos , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , MAP Quinase Quinase 4/química , Masculino , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Naftoquinonas/administração & dosagem , Naftoquinonas/química , Osteoclastos/fisiologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
J Comput Chem ; 39(7): 361-372, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29178493

RESUMO

We have studied whether calculations of the binding free energy of small ligands to a protein by the MM/GBSA approach (molecular mechanics combined with generalized Born and surface area solvation) can be sped up by including only a restricted number of atoms close to the ligand. If the protein is truncated before the molecular dynamics (MD) simulations, quite large changes are observed for the calculated binding energies, for example, 4 kJ/mol average difference for a radius of 19 Å for the binding of nine phenol derivatives to ferritin. The results are improved if no atoms are fixed in the simulations, with average and maximum errors of 2 and 3 kJ/mol at 19 Å and 3 and 6 kJ/mol at 7 Å. Similar results are obtained for two additional proteins, p38α MAP kinase and factor Xa. On the other hand, if energies are calculated on snapshots that are truncated after the MD simulation, all residues more than 8.5 Å from the ligand can be omitted without changing the energies by more than 1 kJ/mol on average (maximum error 1.4 kJ/mol). At the molecular mechanics level, the gain in computer time for such an approach is small. However, it shows what size of system should be used if the energies instead are calculated with a more demanding method, for example, quantum-mechanics. © 2017 Wiley Periodicals, Inc.


Assuntos
Fator Xa/química , Ferritinas/química , Simulação de Dinâmica Molecular , Fenóis/química , Proteínas Quinases p38 Ativadas por Mitógeno/química , Ligantes , Propriedades de Superfície , Termodinâmica , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
16.
J Am Chem Soc ; 140(3): 1148-1158, 2018 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-29276882

RESUMO

Intrinsically disordered proteins (IDPs) display a large number of interaction modes including folding-upon-binding, binding without major structural transitions, or binding through highly dynamic, so-called fuzzy, complexes. The vast majority of experimental information about IDP binding modes have been inferred from crystal structures of proteins in complex with short peptides of IDPs. However, crystal structures provide a mainly static view of the complexes and do not give information about the conformational dynamics experienced by the IDP in the bound state. Knowledge of the dynamics of IDP complexes is of fundamental importance to understand how IDPs engage in highly specific interactions without concomitantly high binding affinity. Here, we combine rotating-frame R1ρ, Carr-Purcell-Meiboom Gill relaxation dispersion as well as chemical exchange saturation transfer to decipher the dynamic interaction profile of an IDP in complex with its partner. We apply the approach to the dynamic signaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disordered regulatory domain of the MAPK kinase MKK4. Our study demonstrates that MKK4 employs a subtle combination of interaction modes in order to bind to p38α, leading to a complex displaying significantly different dynamics across the bound regions.


Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , MAP Quinase Quinase 4/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Humanos , Proteínas Intrinsicamente Desordenadas/química , MAP Quinase Quinase 4/química , Camundongos , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Quinases p38 Ativadas por Mitógeno/química
17.
J Med Chem ; 60(20): 8552-8564, 2017 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-28945083

RESUMO

To explore novel kinase hinge-binding scaffolds, we carried out structure-based virtual screening against p38α MAPK as a model system. With the assistance of developed kinase-specific structural filters, we identify a novel lead compound that selectively inhibits a panel of kinases with threonine as the gatekeeper residue, including BTK and LCK. These kinases play important roles in lymphocyte activation, which encouraged us to design novel kinase inhibitors as drug candidates for ameliorating inflammatory diseases and cancers. Therefore, we chemically modified our substituted triazole-class lead compound to improve the binding affinity and selectivity via a "minimal decoration" strategy, which resulted in potent and selective kinase inhibitors against LCK (18 nM) and BTK (8 nM). Subsequent crystallographic experiments validated our design. These rationally designed compounds exhibit potent on-target inhibition against BTK in B cells or LCK in T cells, respectively. Our work demonstrates that structure-based virtual screening can be applied to facilitate the development of novel chemical entities in crowded chemical space in the field of kinase inhibitor discovery.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Descoberta de Drogas , Humanos , Ativação Linfocitária/efeitos dos fármacos , Espectrometria de Massas , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Espectroscopia de Prótons por Ressonância Magnética , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/química
18.
PLoS One ; 12(9): e0184627, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28892510

RESUMO

In protein kinase research, identifying and addressing small molecule binding sites other than the highly conserved ATP-pocket are of intense interest because this line of investigation extends our understanding of kinase function beyond the catalytic phosphotransfer. Such alternative binding sites may be involved in altering the activation state through subtle conformational changes, control cellular enzyme localization, or in mediating and disrupting protein-protein interactions. Small organic molecules that target these less conserved regions might serve as tools for chemical biology research and to probe alternative strategies in targeting protein kinases in disease settings. Here, we present the structure-based design and synthesis of a focused library of 2-arylquinazoline derivatives to target the lipophilic C-terminal binding pocket in p38α MAPK, for which a clear biological function has yet to be identified. The interactions of the ligands with p38α MAPK was analyzed by SPR measurements and validated by protein X-ray crystallography.


Assuntos
Desenho de Drogas , Modelos Moleculares , Quinazolinas/química , Proteínas Quinases p38 Ativadas por Mitógeno/química , Sítios de Ligação , Domínio Catalítico , Cristalização , Ligantes , Conformação Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Relação Quantitativa Estrutura-Atividade , Quinazolinas/síntese química , Quinazolinas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
19.
Eur J Pharmacol ; 810: 149-155, 2017 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-28690191

RESUMO

Methyl (E)-(3-(3,4-dihydroxyphenyl)acryloyl)tryptophanate (MHAT) is an O-methyl ester of javamide-II showing strong anti-inflammatory activity. Therefore, in this study, MHAT was chemically synthesized, and its effects on p38 MAP kinase, NF-κB, and monocyte chemotactic factor-1 (MCP-1) expression were investigated in LPS-stimulated differentiated THP-1 cells. MHAT inhibited p38 MAP kinase with an IC50 of 12µM, and the inhibition was supported by an in silico model showing that its binding to p38 MAP kinase was stronger than that of SB203580. At the concentration of 20µM, the p38 inhibition reduced ATF-2 phosphorylation by 55% (P < 0.05). Additionally, MHAT inhibited NF-κB (p65) phosphorylation by 30% (P < 0.05) at the same concentration, suggesting that MHAT was able to reduce NF-κB transcriptional activity. This supposition was confirmed by the NF-κB reporter assay, demonstrating that MHAT (20µM) could suppress NF-κB transcriptional activity by 29% (P < 0.05) in the NF-κB reporter (Luc)-HEK293 cell line. As expected, the treatment with MHAT (5-40µM) significantly inhibited MCP-1 mRNA expression by 9-73% (P < 0.05) and the production of MCP-1 protein by 10-70% (P < 0.05) in the THP-1 cells. Furthermore, MHAT was found to inhibit RANTES expression as well in the same THP-1 cells, supporting its purported inhibition of p38 MAP kinase and NF-κB. All these data suggest that MHAT is a potent compound that can inhibit MCP-1 production by suppressing p38 kinase/ATF-2 phosphorylation and NF-κB in the differentiated THP-1 cells.


Assuntos
Acrilamidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Quimiocina CCL2/genética , Regulação da Expressão Gênica/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Triptofano/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Linhagem Celular , Humanos , Lipopolissacarídeos , Simulação de Acoplamento Molecular , Conformação Proteica , Triptofano/análogos & derivados , Triptofano/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
Fish Shellfish Immunol ; 67: 459-466, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28602680

RESUMO

P38 mitogen-activated protein kinases (MAPKs) are one of the most important central regulatory proteins response to extra environmental stresses. In this study, two novel p38 MAPKs, Ec-P38γ and Ec-P38δ, were identified from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. Both of Ec-p38γ and Ec-p38δ sequences contain a serine/threonine protein kinase (S_TKc) domain and a highly conserved Thr-Gly-Tyr (TGY) motif. Analysis of phylogenetic relationships illustrated that p38 amino acid sequences were conserved between different species indicating that the functions may be similar. The four subtypes of p38 (α, ß, γ, and δ) mRNA can be detected in all thirteen tissues examined, but the expression level is different in these tissues. The expression patterns of the four Ec-p38 subtypes in E. coioides were also detected response to Cryptocaryon irritans infection, one of the most important protozoan pathogens of marine fish. The expression of four p38 subtypes was up-regulated in the tissues examined, with the highest expressions of Ec-p38α (5.2 times) and Ec-p38δ (4.2 times) occurring in the skin, while Ec-p38ß (24.8 times) and γ (16.6 times) occurred in the spleen. There was no significantly correlation between the expression of Ec-p38γ/Ec-p38δ and the expression of nuclear factor kappaB (NF-kB). The results indicated the sequences and the characters of Ec-p38γ and Ec-p38δ were conserved, the p38 subtypes showed tissue-specific expression patterns in healthy grouper, and their expressions were significantly up-regulated post C. irritans infection, suggesting these p38 MAPKs may play important roles in these tissues during pathogen-caused inflammation.


Assuntos
Bass , Doenças dos Peixes/imunologia , Imunidade Inata/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Sequência de Aminoácidos , Animais , Cilióforos/fisiologia , Infecções por Cilióforos/imunologia , Infecções por Cilióforos/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , Alinhamento de Sequência/veterinária , Proteínas Quinases p38 Ativadas por Mitógeno/química
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