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1.
Pharmacol Res ; 176: 106046, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35007708

RESUMO

Ischemic stroke remains one of the leading causes of death worldwide, thereby highlighting the urgent necessary to identify new therapeutic targets. Deoxyhypusine hydroxylase (DOHH) is a fundamental enzyme catalyzing a unique posttranslational hypusination modification of eukaryotic translation initiation factor 5A (eIF5A) and is highly involved in the progression of several human diseases, including HIV-1 infection, cancer, malaria, and diabetes. However, the potential therapeutic role of pharmacological regulation of DOHH in ischemic stroke is still poorly understood. Our study first discovered a natural small-molecule brazilin (BZ) with an obvious neuroprotective effect against oxygen-glucose deprivation/reperfusion insult. Then, DOHH was identified as a crucial cellular target of BZ using HuProt™ human proteome microarray. By selectively binding to the Cys232 residue, BZ induced a previously undisclosed allosteric effect to significantly increase DOHH catalytic activity. Furthermore, BZ-mediated DOHH activation amplified mitophagy for mitochondrial function and morphology maintenance via DOHH/eIF5A hypusination signaling pathway, thereby protecting against ischemic neuronal injury in vitro and in vivo. Collectively, our study first identified DOHH as a previously unreported therapeutic target for ischemic stroke, and provided a future drug design direction for DOHH allosteric activators using BZ as a novel molecular template.


Assuntos
Benzopiranos/uso terapêutico , Infarto da Artéria Cerebral Média/tratamento farmacológico , AVC Isquêmico/tratamento farmacológico , Oxigenases de Função Mista/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Animais , Benzopiranos/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Feminino , Humanos , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , AVC Isquêmico/metabolismo , AVC Isquêmico/patologia , Masculino , Camundongos Endogâmicos ICR , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Gravidez , Processamento de Proteína Pós-Traducional , Ratos Wistar , Peixe-Zebra
2.
Acta Pharmacol Sin ; 41(2): 173-180, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31506572

RESUMO

Microglia-mediated neuroinflammation is a crucial risk factor for neurological disorders. Recently, dopamine receptors have been found to be involved in multiple immunopathological processes and considered as valuable therapeutic targets for inflammation-associated neurologic diseases. In this study we investigated the anti-neuroinflammation effect of isosibiricin, a natural coumarin compound isolated from medicinal plant Murraya exotica. We showed that isosibiricin (10-50 µM) dose-dependently inhibited lipopolysaccharide (LPS)-induced BV-2 microglia activation, evidenced by the decreased expression of inflammatory mediators, including nitrite oxide (NO), tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and interleukin-18 (IL-18). By using transcriptomics coupled with bioinformatics analysis, we revealed that isosibiricin treatment mainly affect dopamine receptor signalling pathway. We further demonstrated that isosibiricin upregulated the expression of dopamine D1/2 receptors in LPS-treated BV-2 cells, resulting in inhibitory effect on nucleotide binding domain-like receptor protein 3 (NLRP3)/caspase-1 inflammasome pathway. Treatment with dopamine D1/2 receptor antagonists SCH 23390 (1 µM) or sultopride (1 µM) could reverse the inhibitory effects of isosibiricin on NLRP3 expression as well as the cleavages of caspase-1 and IL-1ß. Collectively, this study demonstrates a promising therapeutic strategy for neuroinflammation by targeting dopamine D1/2 receptors.


Assuntos
Inflamassomos/efeitos dos fármacos , Inflamação/tratamento farmacológico , Microglia/efeitos dos fármacos , Animais , Caspase 1/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Inflamassomos/metabolismo , Inflamação/patologia , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos BALB C , Microglia/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Transdução de Sinais/efeitos dos fármacos
3.
Zhongguo Zhong Yao Za Zhi ; 44(13): 2657-2661, 2019 Jul.
Artigo em Zh | MEDLINE | ID: mdl-31359673

RESUMO

Target identification is an important prerequisite for the study of medicine action mechanism. Currently,drug target identification is mostly based on various cell models in vitro. However,the growth microenvironment,nutrition metabolism,biological properties as well as functions are quite different between in vitro cell culture and physiological environment in vivo; wherefore,it is a challenging scientific issue to establish an effective method for identifying drug targets in vivo condition. In this study,we successfully prepared a kind of magnetic nanoparticles( MNPs) which can be chemically modified by the hydroxyl structure of natural bioactive compound echinacoside( ECH) via the epoxy group label on the surface of MNPs. Therefore,organ-selective and recoverable nanoscale target-recognizing particles were prepared. We then intravenously injected the ECH-binding MNPs into rats and distributed them to specific organs in vivo. After cell endocytosis,ECH-binding MNPs captured target proteins in situ for further analysis. Based on this method,we discovered several potential target proteins in the spleen lysates for ECH,and preliminarily clarified the immuno-regulation mechanism of ECH. Collectively,our strategy developed a proof-of-concept technology using nanoparticles for in vivo target identification,and also provided a feasible approach for drug target prediction and pharmacological mechanism exploration.


Assuntos
Sistemas de Liberação de Medicamentos , Nanopartículas de Magnetita , Medicina Tradicional Chinesa , Animais , Endocitose , Glicosídeos/análise , Magnetismo , Estudo de Prova de Conceito , Ratos
4.
Eur J Pharmacol ; 972: 176551, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38570082

RESUMO

Fibroblast-like synoviocytes (FLS) play an important role in rheumatoid arthritis (RA)-related swelling and bone damage. Therefore, novel targets for RA therapy in FLS are urgently discovered for improving pathologic phenomenon, especially joint damage and dyskinesia. Here, we suggested that pyruvate kinase M2 (PKM2) in FLS represented a pharmacological target for RA treatment by antimalarial drug artemisinin (ART). We demonstrated that ART selectively inhibited human RA-FLS and rat collagen-induced arthritis (CIA)-FLS proliferation and migration without observed toxic effects. In particular, the identification of targets revealed that PKM2 played a crucial role as a primary regulator of the cell cycle, leading to the heightened proliferation of RA-FLS. ART exhibited a direct interaction with PKM2, resulting in an allosteric modulation that enhances the lactylation modification of PKM2. This interaction further promoted the binding of p300, ultimately preventing the nuclear translocation of PKM2 and inducing cell cycle arrest at the S phase. In vivo, ART obviously suppressed RA-mediated synovial hyperplasia, bone damage and inflammatory response to further improve motor behavior in CIA-rats. Taken together, these findings indicate that directing interventions towards PKM2 in FLS could offer a hopeful avenue for pharmaceutical treatments of RA through the regulation of cell cycle via PKM2 lactylation.


Assuntos
Artrite Reumatoide , Proliferação de Células , Sinoviócitos , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Artrite Reumatoide/patologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Humanos , Ratos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Piruvato Quinase/metabolismo , Proteínas de Ligação a Hormônio da Tireoide , Masculino , Hormônios Tireóideos/metabolismo , Artrite Experimental/patologia , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Movimento Celular/efeitos dos fármacos , Terapia de Alvo Molecular , Proteínas de Membrana/metabolismo , Proteínas de Transporte/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química
5.
Theranostics ; 14(3): 988-1009, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38250049

RESUMO

The hypothalamus plays a fundamental role in controlling lipid metabolism through neuroendocrine signals. However, there are currently no available drug targets in the hypothalamus that can effectively improve human lipid metabolism. In this study, we found that the antimalarial drug artemether (ART) significantly improved lipid metabolism by specifically inhibiting microglial activation in the hypothalamus of high-fat diet-induced mice. Mechanically, ART protects the thyrotropin-releasing hormone (TRH) neurons surrounding microglial cells from inflammatory damage and promotes the release of TRH into the peripheral circulation. As a result, TRH stimulates the synthesis of thyroid hormone (TH), leading to a significant improvement in hepatic lipid disorders. Subsequently, we employed a biotin-labeled ART chemical probe to identify the direct cellular target in microglial cells as protein kinase Cδ (PKCδ). Importantly, ART directly targeted PKCδ to inhibit its palmitoylation modification by blocking the binding of zinc finger DHHC-type palmitoyltransferase 5 (ZDHHC5), which resulted in the inhibition of downstream neuroinflammation signaling. In vivo, hypothalamic microglia-specific PKCδ knockdown markedly impaired ART-dependent neuroendocrine regulation and lipid metabolism improvement in mice. Furthermore, single-cell transcriptomics analysis in human brain tissues revealed that the level of PKCδ in microglia positively correlated with individuals who had hyperlipemia, thereby highlighting a clinical translational value. Collectively, these data suggest that the palmitoylation of microglial PKCδ in the hypothalamus plays a role in modulating peripheral lipid metabolism through hypothalamus-liver communication, and provides a promising therapeutic target for fatty liver diseases.


Assuntos
Lipoilação , Hepatopatia Gordurosa não Alcoólica , Humanos , Animais , Camundongos , Microglia , Hipotálamo , Metabolismo dos Lipídeos , Artemeter
6.
Adv Sci (Weinh) ; 10(18): e2206533, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37088726

RESUMO

Osteoblasts play an important role in the regulation of bone homeostasis throughout life. Thus, the damage of osteoblasts can lead to serious skeletal diseases, highlighting the urgent need for novel pharmacological targets. This study introduces chemical genetics strategy by using small molecule forskolin (FSK) as a probe to explore the druggable targets for osteoporosis. Here, this work reveals that transglutaminase 2 (TGM2) served as a major cellular target of FSK to obviously induce osteoblast differentiation. Then, this work identifies a previously undisclosed allosteric site in the catalytic core of TGM2. In particular, FSK formed multiple hydrogen bonds in a saddle-like domain to induce an "open" conformation of the ß-sandwich domain in TGM2, thereby promoting the substrate protein crosslinks by incorporating polyamine. Furthermore, this work finds that TGM2 interacted with several mitochondrial homeostasis-associated proteins to improve mitochondrial dynamics and ATP production for osteoblast differentiation. Finally, this work observes that FSK effectively ameliorated osteoporosis in the ovariectomy mice model. Taken together, these findings show a previously undescribed pharmacological allosteric site on TGM2 for osteoporosis treatment, and also provide an available chemical tool for interrogating TGM2 biology and developing bone anabolic agent.


Assuntos
Osteoporose , Proteína 2 Glutamina gama-Glutamiltransferase , Camundongos , Animais , Feminino , Regulação Alostérica , Osteogênese , Osteoblastos/metabolismo , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo
7.
Sci Adv ; 8(32): eabo0789, 2022 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-35947662

RESUMO

Neuroinflammation is a fundamental contributor to progressive neuronal damage, which arouses a heightened interest in neurodegenerative disease therapy. Ubiquitin-specific protease 7 (USP7) has a crucial role in regulating protein stability in multiple biological processes; however, the potential role of USP7 in neurodegenerative progression is poorly understood. Here, we discover the natural small molecule eupalinolide B (EB), which targets USP7 to inhibit microglia activation. Cocrystal structure reveals a previously undisclosed covalent allosteric site, Cys576, in a unique noncatalytic HUBL domain. By selectively modifying Cys576, EB allosterically inhibits USP7 to cause a ubiquitination-dependent degradation of Keap1. Keap1 function loss further results in an Nrf2-dependent transcription activation of anti-neuroinflammation genes in microglia. In vivo, pharmacological USP7 inhibition attenuates microglia activation and resultant neuron injury, thereby notably improving behavioral deficits in dementia and Parkinson's disease mouse models. Collectively, our findings provide an attractive future direction for neurodegenerative disease therapy by inhibiting microglia-mediated neuroinflammation by targeting USP7.


Assuntos
Doenças Neurodegenerativas , Ubiquitina Tiolesterase , Animais , Proteína 1 Associada a ECH Semelhante a Kelch , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Bibliotecas de Moléculas Pequenas , Ubiquitina Tiolesterase/genética , Peptidase 7 Específica de Ubiquitina/metabolismo
8.
Eur J Pharmacol ; 891: 173723, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33159933

RESUMO

Endoplasmic reticulum (ER) stress has been considered as a promising strategy in developing novel therapeutic agents for cardiovascular diseases through inhibiting cardiomyocyte apoptosis. Protocatechualdehyde (PCA) is a natural phenolic compound from medicinal plant Salvia miltiorrhiza with cardiomyocyte protection. However, the potential mechanism of PCA on cardiovascular ischemic injury is largely unexplored. Here, we found that PCA exerted markedly anti-apoptotic effect in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced H9c2 cells (Rat embryonic ventricular H9c2 cardiomyocytes), which was detected by 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), lactate dehydrogenase (LDH), Hoechst 33258 and acridine orange/ethidium bromide (AO/EB) assays. PCA also obviously protected cardiomyocytes in myocardial fibrosis model of mice, which was determined by hematoxylin-eosin (HE) staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining. Transcriptomics coupled with bioinformatics analysis revealed a complex pharmacological signaling network especially for PCA-mediated ER stress on cardiomyocytes. Further mechanism study suggested that PCA suppressed ER stress via inhibiting protein kinase R-like ER kinase (PERK), inositol-requiring enzyme1α (IRE1α), and transcription factor 6α (ATF6α) signaling pathway through Western blot, DIOC6 and ER-Tracker Red staining, leading to a protective effect against ER stress-mediated cardiomyocyte apoptosis. Taken together, our observations suggest that PCA is a major component from Salvia miltiorrhiza against cardiovascular ischemic injury by suppressing ER stress-associated PERK, IRE1α and ATF6α signaling pathways.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Benzaldeídos/farmacologia , Catecóis/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/metabolismo , Complexos Multienzimáticos/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , eIF-2 Quinase/metabolismo , Fator 6 Ativador da Transcrição/genética , Animais , Hipóxia Celular , Linhagem Celular , Modelos Animais de Doenças , Endorribonucleases/genética , Fibrose , Glucose/deficiência , Masculino , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/genética , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Proteínas Serina-Treonina Quinases/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Transcriptoma , eIF-2 Quinase/genética
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