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1.
Zhonghua Shao Shang Za Zhi ; 37(9): 853-859, 2021 Sep 20.
Artigo em Chinês | MEDLINE | ID: mdl-34645151

RESUMO

Objective: To explore the effects and possible molecular mechanism of histone deacetylase 6 (HDAC6) inhibitor Tubastatin A on the proliferation and movement of human skin fibroblasts (HSFs). Methods: The experimental research method was used. HSFs in logarithmic growth phase were taken and divided into negative control group, 1 µmol/L Tubastatin A group, 5 µmol/L Tubastatin A group, and 10 µmol/L Tubastatin A group according to the random number table. The HSFs in negative control group were added with Dulbecco's modified eagle medium with the final volume fraction of 0.1% dimethyl sulfoxide (hereinafter referred to as the complete medium), and the other three groups were added with the complete medium with the corresponding final molarity of Tubastatin A. After 24 h of conventional culture, the cell proliferation activity was detected using cell counting kit 8 (CCK-8) method and 5-ethynyl-2'-deoxyuridine (EdU) staining; the range of motion of cells within 3 h was observed under the living cell workstation, and the curve movement velocity of the cells was calculated. The protein expressions of extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2) were detected by Western blotting, and the ratio of p-ERK1/2 to ERK1/2 was calculated to represent the activity of ERK1/2. The sample number in cell proliferation activity detection with CCK-8 method was 6, while the sample numbers in other experiments were 3. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: After 24 h of culture, CCK-8 method and EdU staining showed that compared with negative control group, the cell proliferation activities in 1 µmol/L Tubastatin A group, 5 µmol/L Tubastatin A group, and 10 µmol/L Tubastatin A group were significantly decreased (P<0.01). After 24 h of culture, CCK-8 method showed that compared with 1 µmol/L Tubastatin A group, the cell proliferation activity in 10 µmol/L Tubastatin A group was significantly decreased (P<0.05); EdU staining showed that compared with 1 µmol/L Tubastatin A group, the cell proliferation activities in 5 µmol/L Tubastatin A group and 10 µmol/L Tubastatin A group were significantly decreased (P<0.05 or P<0.01). Within 3 h of observation, the ranges of cell motion in 1 µmol/L Tubastatin A group, 5 µmol/L Tubastatin A group, and 10 µmol/L Tubastatin A group were obviously reduced compared with that in negative control group. Within 3 h of observation, the curve movement velocity of cells in negative control group was (0.780±0.028) µm/min, which was obviously faster than (0.594±0.023), (0.469±0.028), and (0.391±0.021) µm/min of 1 µmol/L Tubastatin A group, 5 µmol/L Tubastatin A group, and 10 µmol/L Tubastatin A group (P<0.01); the curve movement velocity of cells in 1 µmol/L Tubastatin A group was obviously faster than those in 5 µmol/L Tubastatin A group and 10 µmol/L Tubastatin A group (P<0.01); the curve movement velocity of cells in 5 µmol/L Tubastatin A group was obviously faster than that in 10 µmol/L Tubastatin A group (P<0.05). After 24 h of culture, compared with negative control group, the activities of ERK1/2 of cells in 1 µmol/L Tubastatin A group, 5 µmol/L Tubastatin A group, and 10 µmol/L Tubastatin A group were decreased significantly (P<0.01); compared with 1 µmol/L Tubastatin A group, the activities of ERK1/2 of cells in 5 µmol/L Tubastatin A group and 10 µmol/L Tubastatin A group were decreased significantly (P<0.01); compared with 5 µmol/L Tubastatin A group, the activity of ERK1/2 of cells in 10 µmol/L Tubastatin A group was decreased significantly (P<0.05). Conclusions: HDAC6 inhibitor Tubastatin A may mediate the inhibitory effect on proliferation and movement of HSFs by inhibiting the activity of ERK1/2.


Assuntos
Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos , Fibroblastos , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis
2.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638868

RESUMO

Mechanical unloading contributes to significant cardiovascular deconditioning. Endothelial dysfunction in the sites of microcirculation may be one of the causes of the cardiovascular degeneration induced by unloading, but the detailed mechanism is still unclear. Here, we first demonstrated that mechanical unloading inhibited brain microvascular endothelial cell proliferation and downregulated histone deacetylase 6 (HDAC6) expression. Furthermore, HDAC6 promoted microvascular endothelial cell proliferation and attenuated the inhibition of proliferation caused by clinorotation unloading. To comprehensively identify microRNAs (miRNAs) that are regulated by HDAC6, we analyzed differential miRNA expression in microvascular endothelial cells after transfection with HDAC6 siRNA and selected miR-155-5p, which was the miRNA with the most significantly increased expression. The ectopic expression of miR-155-5p inhibited microvascular endothelial cell proliferation and directly downregulated Ras homolog enriched in brain (RHEB) expression. Moreover, RHEB expression was downregulated under mechanical unloading and was essential for the miR-155-5p-mediated promotion of microvascular endothelial cell proliferation. Taken together, these results are the first to elucidate the role of HDAC6 in unloading-induced cell growth inhibition through the miR-155-5p/RHEB axis, suggesting that the HDAC6/miR-155-5p/RHEB pathway is a specific target for the preventative treatment of cardiovascular deconditioning.


Assuntos
Proliferação de Células , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Desacetilase 6 de Histona/metabolismo , MicroRNAs/biossíntese , Microvasos/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Animais , Linhagem Celular , Células Endoteliais/citologia , Camundongos , Microvasos/citologia
3.
FASEB J ; 35(11): e22009, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34694026

RESUMO

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), remains a major cause of morbidity and mortality worldwide. Increasing lines of evidence indicate that certain individuals, which are termed resisters, are naturally resistant to TB infection. The resister phenotype has been linked to host efficient innate immune responses, but the underlying mechanisms and the key immune factors remain unclear. Here, we find that upon Mtb infection, monocyte-derived macrophages (MDMs) from TB resisters exhibited distinctly higher production of TNF-α, IL-1ß and IL-6, higher ratio of bacteria in acidic vacuoles, and lower intracellular bacterial loads, as compared to that from the healthy controls, individuals with latent TB infection, and TB patients. Such enhanced anti-Mtb immune capacity of macrophages from resisters largely depends on histone deacetylase 6 (HDAC6), whose expression is specifically maintained in MDMs from TB resisters during Mtb infection. Furthermore, we demonstrate that HDAC6 is required for acidification of Mtb-containing phagosomes in macrophages, thus controlling the intracellular survival of Mtb. Taken together, these findings unravel an indispensable role of HDAC6 in human innate resistance against Mtb infection, suggesting that HDAC6 may serve as a marker for individual TB risk as well as a novel host-directed anti-TB therapeutic target.


Assuntos
Resistência à Doença , Desacetilase 6 de Histona/imunologia , Imunidade Inata , Macrófagos/imunologia , Tuberculose/imunologia , Adulto , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Macrófagos/citologia , Masculino , Pessoa de Meia-Idade
4.
Stem Cell Res Ther ; 12(1): 484, 2021 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-34454588

RESUMO

BACKGROUND: Senile osteoporosis can cause bone fragility and increased risk for fractures and has been one of the most prevalent and severe diseases affecting the elderly population worldwidely. The underlying mechanisms are currently intensive areas of investigation. In age-related bone loss, decreased bone formation overweighs increased bone resorption. The molecular mechanisms underlying defective bone formation in age-related bone loss are not completely understood. In particular, the specific role of histone acetylation in age-related bone loss has not been examined thoroughly. METHODS: We employed 6- and 18-month-old mice to investigate the mechanisms of defective bone formation in age-related bone loss. Bone marrow stromal cells (BMSCs) were induced to undergo in vitro osteogenic differentiation. Chromatin immunoprecipitation (ChIP) was used to investigate the binding of histone deacetylases (HDACs) on Runx2 promoter in BMSCs. Luciferase reporter and transient transfection assay were employed to study Runx2 gene expression modulation by HDAC and androgen receptor (AR). siRNA and HDAC6 inhibitor, Tubastatin A, were used to inhibit HDAC6 in vitro. And systemic administration of Tubastatin A was used to block HDAC6 in vivo. RESULTS: Age-related trabecular bone loss was observed in 18-month-old mice compared with 6-month-old mice. In vitro osteogenic differentiation potential of BMSCs from 18-month-old mice was weaker than 6-month-old mice, in which there was Runx2 expression inactivation in BMSCs of 18-month-old mice compared with 6-month-old mice, which was attributable to HDAC6-mediated histone hypoacetylation in Runx2 promoter. There was competitive binding of HDAC6 and AR on Runx2 promoter to modulate Runx2 expression in BMSCs. More importantly, through siRNA- or specific inhibitor-mediated HDAC6 inhibition, we could activate Runx2 expression, rescue in vitro osteogenesis potential of BMSCs, and alleviate in vivo age-related bone loss of mice. CONCLUSION: HDAC6 accumulation and histone hypoacetylation on Runx2 promoter contributed to the attenuation of in vitro osteogenic differentiation potential of BMSCs from aged mice. Through HDAC6 inhibition, we could activate Runx2 expression and osteogenic differentiation potential of BMSCs from aged mice and alleviate the age-related bone loss of aged mice. Our study will benefit not only for understanding the age-related bone loss, but also for finding new therapies to treat senile osteoporosis.


Assuntos
Células-Tronco Mesenquimais , Osteoporose , Idoso , Animais , Células da Medula Óssea , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Desacetilase 6 de Histona/genética , Humanos , Camundongos , Osteogênese/genética , Osteoporose/genética , Regiões Promotoras Genéticas
5.
Front Immunol ; 12: 680441, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34234781

RESUMO

The objective of this study was to investigate mechanisms of allergic inflammation both in vitro and in vivo in details. For this, RNA sequencing was performed. Early growth response 3 gene (Egr3) was one of the most highly upregulated genes in rat basophilic leukemia (RBL2H3) cells stimulated by antigen. The role of Egr3 in allergic inflammation has not been studied extensively. Egr3 was necessary for passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA). Egr3 promoter sequences contained potential binding site for NF-κB p65. NF-κB p65 directly regulated Egr3 expression and mediated allergic inflammation in vitro. Histone deacetylases (HDACs) is known to be involved in allergic airway inflammation. HDAC6 promoter sequences contained potential binding site for EGR3. EGR3 showed binding to promoter sequences of HDAC6. EGR3 was necessary for increased expression of histone deacetylase 6 (HDAC6) in antigen-stimulated RBL2H3 cells. HDAC6 mediated allergic inflammation in vitro and PSA. TargetScan analysis predicted that miR-182-5p was a negative regulator of EGR3. Luciferase activity assay confirmed that miR-182-5p was a direct regulator of EGR3. MiR-182-5p mimic inhibited allergic inflammation both in vitro and in vivo. Cytokine array showed that HDAC6 was necessary for increased interleukin-27 (IL-27) expression in BALB/C mouse model of PSA. Antigen stimulation did not affect expression of EBI3, another subunit of IL-27 in RBL2H3 cells or BALB/C mouse model of PCA or PSA. IL-27 receptor alpha was shown to be able to bind to HDAC6. IL-27 p28 mediated allergic inflammation in vitro, PCA, and PSA. Mouse recombinant IL-27 protein promoted features of allergic inflammation in an antigen-independent manner. HDAC6 was necessary for tumorigenic and metastatic potential enhanced by PSA. PSA enhanced the metastatic potential of mouse melanoma B16F1 cells in an IL-27-dependent manner. Experiments employing culture medium and mouse recombinant IL-27 protein showed that IL-27 mediated and promoted cellular interactions involving B16F1 cells, lung macrophages, and mast cells during allergic inflammation. IL-27 was present in exosomes of antigen-stimulated RBL2H3 cells. Exosomes from antigen-stimulated RBL2H3 cells enhanced invasion of B16F1 melanoma cells in an IL-27-dependemt manner. These results present evidence that EGR3-HDAC6-IL-27 axis can regulate allergic inflammation by mediating cellular interactions.


Assuntos
Comunicação Celular , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Proteína 3 de Resposta de Crescimento Precoce/metabolismo , Desacetilase 6 de Histona/metabolismo , Interleucina-27/metabolismo , Transdução de Sinais , Animais , Comunicação Celular/genética , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Suscetibilidade a Doenças , Feminino , Hipersensibilidade/complicações , Hipersensibilidade/etiologia , Hipersensibilidade/metabolismo , Melanoma Experimental , Camundongos , MicroRNAs/genética , Ratos
6.
Nucleic Acids Res ; 49(15): 8974-8986, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34329468

RESUMO

Cytosine base editor (CBE) enables targeted C-to-T conversions at single base-pair resolution and thus has potential therapeutic applications in humans. However, the low efficiency of the system limits practical use of this approach. We reported a high-throughput human cells-based reporter system that can be harnessed for quickly measuring editing activity of CBE. Screening of 1813 small-molecule compounds resulted in the identification of Ricolinostat (an HDAC6 inhibitor) that can enhance the efficiency of BE3 in human cells (2.45- to 9.21-fold improvement). Nexturastat A, another HDAC6 inhibitor, could also increase BE3-mediated gene editing by 2.18- to 9.95-fold. Ricolinostat and Nexturastat A also boost base editing activity of the other CBE variants (BE4max, YE1-BE4max, evoAPOBEC1-BE4max and SpRY-CBE4max, up to 8.32-fold). Meanwhile, combined application of BE3 and Ricolinostat led to >3-fold higher efficiency of correcting a pathogenic mutation in ABCA4 gene related to Stargardt disease in human cells. Moreover, we demonstrated that our strategy could be applied for efficient generation of mouse models through direct zygote injection and base editing in primary human T cells. Our study provides a new strategy to improve the activity and specificity of CBE in human cells. Ricolinostat and Nexturastat A augment the effectiveness and applicability of CBE.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Sistemas CRISPR-Cas/genética , Citosina/metabolismo , Desacetilase 6 de Histona/antagonistas & inibidores , Doença de Stargardt/genética , Animais , Edição de Genes/tendências , Células HEK293 , Desacetilase 6 de Histona/genética , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Mutação/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Pirimidinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Doença de Stargardt/tratamento farmacológico , Doença de Stargardt/patologia , Linfócitos T/efeitos dos fármacos , Zigoto/efeitos dos fármacos
7.
Elife ; 102021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34250905

RESUMO

Pathophysiological defects in water homeostasis can lead to renal failure. Likewise, common genetic disorders associated with abnormal cytoskeletal dynamics in the kidney collecting ducts and perturbed calcium and cAMP signaling in the ciliary compartment contribute to chronic kidney failure. We show that collecting ducts in mice lacking the A-Kinase anchoring protein AKAP220 exhibit enhanced development of primary cilia. Mechanistic studies reveal that AKAP220-associated protein phosphatase 1 (PP1) mediates this phenotype by promoting changes in the stability of histone deacetylase 6 (HDAC6) with concomitant defects in actin dynamics. This proceeds through a previously unrecognized adaptor function for PP1 as all ciliogenesis and cytoskeletal phenotypes are recapitulated in mIMCD3 knock-in cells expressing a phosphatase-targeting defective AKAP220-ΔPP1 mutant. Pharmacological blocking of local HDAC6 activity alters cilia development and reduces cystogenesis in kidney-on-chip and organoid models. These findings identify the AKAP220-PPI-HDAC6 pathway as a key effector in primary cilia development.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Cílios/metabolismo , Desacetilase 6 de Histona/metabolismo , Homeostase , Rim/metabolismo , Proteína Fosfatase 1/metabolismo , Actinas/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células HEK293 , Inibidores de Histona Desacetilases/farmacologia , Humanos , Túbulos Renais Coletores , Camundongos , Organoides/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Molecules ; 26(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299636

RESUMO

Age-related macular degeneration (AMD) occurs due to an abnormality of retinal pigment epithelium (RPE) cells that leads to gradual degeneration of the macula. Currently, AMD drug pipelines are endowed with limited options, and anti-VEGF agents stand as the dominantly employed therapy. Despite the proven efficacy of such agents, the evidenced side effects associated with their use underscore the need to elucidate other mechanisms involved and identify additional molecular targets for the sake of therapy improvement. The previous literature provided us with a solid rationale to preliminarily explore the potential of selective HDAC6 and HSP90 inhibitors to treat wet AMD. Rather than furnishing single-target agents (either HDAC6 or HSP90 inhibitor), this study recruited scaffolds endowed with the ability to concomitantly modulate both targets (HDAC6 and HSP90) for exploration. This plan was anticipated to accomplish the important goal of extracting amplified benefits via dual inhibition (HDAC6/HSP90) in wet AMD. As a result, G570 (indoline-based hydroxamate), a dual selective HDAC6-HSP90 inhibitor exerting its effects at micromolar concentrations, was pinpointed in the present endeavor to attenuate blue light-induced cell migration and retinal neovascularization by inhibiting VEGF production. In addition to the identification of a potential chemical tool (G570), the outcome of this study validates the candidate HDAC6-HSP90 as a compelling target for the development of futuristic therapeutics for wet AMD.


Assuntos
Movimento Celular , Células Epiteliais/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Luz , Neovascularização Retiniana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Células Epiteliais/patologia , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/química , Humanos , Masculino , Camundongos , Neovascularização Retiniana/induzido quimicamente , Neovascularização Retiniana/patologia , Epitélio Pigmentado da Retina/irrigação sanguínea , Epitélio Pigmentado da Retina/patologia
9.
J Med Chem ; 64(14): 9960-9988, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34251197

RESUMO

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by a progressive-fibrosing phenotype. IPF has been associated with aberrant HDAC activities confirmed by our immunohistochemistry studies on HDAC6 overexpression in IPF lung tissues. We herein developed a series of novel hHDAC6 inhibitors, having low inhibitory potency over hHDAC1 and hHDAC8, as potential pharmacological tools for IPF treatment. Their inhibitory potency was combined with low in vitro and in vivo toxicity. Structural analysis of 6h and structure-activity relationship studies contributed to the optimization of the binding mode of the new molecules. The best-performing analogues were tested for their efficacy in inhibiting fibrotic sphere formation and cell viability, proving their capability in reverting the IPF phenotype. The efficacy of analogue 6h was also determined in a validated human lung model of TGF-ß1-dependent fibrogenesis. The results highlighted in this manuscript may pave the way for the identification of first-in-class molecules for the treatment of IPF.


Assuntos
Desenho de Fármacos , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Fibrose Pulmonar Idiopática/tratamento farmacológico , Relação Dose-Resposta a Droga , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
10.
Nat Commun ; 12(1): 4482, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301959

RESUMO

Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. PANX1 can be activated by G protein-coupled receptors (GPCRs), including α1-adrenergic receptors (α1-ARs), but how receptor engagement leads to channel opening remains unclear. Here, we show that GPCR-mediated PANX1 activation can occur via channel deacetylation. We find that α1-AR-mediated activation of PANX1 channels requires Gαq but is independent of phospholipase C or intracellular calcium. Instead, α1-AR-mediated PANX1 activation involves RhoA, mammalian diaphanous (mDia)-related formin, and a cytosolic lysine deacetylase activated by mDia - histone deacetylase 6. HDAC6 associates with PANX1 and activates PANX1 channels, even in excised membrane patches, suggesting direct deacetylation of PANX1. Substitution of basally-acetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism.


Assuntos
Conexinas/metabolismo , Desacetilase 6 de Histona/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Acetilação , Animais , Células Cultivadas , Conexinas/genética , Conexinas/fisiologia , Células HEK293 , Desacetilase 6 de Histona/genética , Humanos , Células Jurkat , Lisina/genética , Lisina/metabolismo , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Técnicas de Patch-Clamp , Receptores Adrenérgicos alfa 1/genética , Transdução de Sinais/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
11.
Arthritis Res Ther ; 23(1): 177, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34225810

RESUMO

BACKGROUND: To investigate the effects of inhibiting histone deacetylase (HDAC) 6 on inflammatory responses and tissue-destructive functions of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA). METHODS: FLS from RA patients were activated with interleukin (IL)-1ß in the presence of increasing concentrations of M808, a novel specific HDAC6 inhibitor. Production of ILs, chemokines, and metalloproteinases (MMPs) was measured in ELISAs. Acetylation of tubulin and expression of ICAM-1 and VCAM-1 were assessed by Western blotting. Wound healing and adhesion assays were performed. Cytoskeletal organization was visualized by immunofluorescence. Finally, the impact of HDAC6 inhibition on the severity of arthritis and joint histology was examined in a murine model of adjuvant-induced arthritis (AIA). RESULTS: HDAC6 was selectively inhibited by M808. The HDAC6 inhibitor suppressed the production of MMP-1, MMP-3, IL-6, CCL2, CXCL8, and CXCL10 by RA-FLS in response to IL-1ß. Increased acetylation of tubulin was associated with decreased migration of RA-FLS. Inhibiting HDAC6 induced cytoskeletal reorganization in RA-FLS by suppressing the formation of invadopodia following activation with IL-1ß. In addition, M808 tended to decrease the expression of ICAM-1 and VCAM-1. In the AIA arthritis model, M808 improved the clinical arthritis score in a dose-dependent manner. Also, HDAC6 inhibition was associated with less severe synovial inflammation and joint destruction. CONCLUSION: Inhibiting HDAC6 dampens the inflammatory and destructive activity of RA-FLS and reduces the severity of arthritis. Thus, targeting HDAC6 has therapeutic potential.


Assuntos
Artrite Reumatoide , Desacetilase 6 de Histona/antagonistas & inibidores , Sinoviócitos , Animais , Artrite Reumatoide/tratamento farmacológico , Células Cultivadas , Fibroblastos , Humanos , Camundongos , Membrana Sinovial
12.
Sci Rep ; 11(1): 15423, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326423

RESUMO

Accumulation of tau protein is a key pathology of age-related neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Those diseases are collectively termed tauopathies. Tau pathology is associated with axonal degeneration because tau binds to microtubules (MTs), a component of axon and regulates their stability. The acetylation state of MTs contributes to stability and histone deacetylase 6 (HDAC6) is a major regulator of MT acetylation status, suggesting that pharmacological HDAC6 inhibition could improve axonal function and may slow the progression of tauopathy. Here we characterize N-[(1R,2R)-2-{3-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl]-5-oxo-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl}cyclohexyl]-2,2,3,3,3-pentafluoropropanamide (T-518), a novel, potent, highly selective HDAC6 inhibitor with clinically favorable pharmacodynamics. T-518 shows potent inhibitory activity against HDAC6 and superior selectivity over other HDACs compared with the known HDAC6 inhibitors in the enzyme and cellular assays. T-518 showed brain penetration in an oral dose and blocked HDAC6-dependent tubulin deacetylation at Lys40 in mouse hippocampus. A 2-week treatment restored impaired axonal transport and novel object recognition in the P301S tau Tg mouse, tauopathy model, while a 3-month treatment also decreased RIPA-insoluble tau accumulation. Pharmaceutical inhibition of HDAC6 is a potential therapeutic strategy for tauopathy, and T-518 is a particularly promising drug candidate.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Acetilação , Administração Oral , Animais , Transporte Axonal/efeitos dos fármacos , Axônios/efeitos dos fármacos , Axônios/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Desacetilase 6 de Histona/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transdução de Sinais/genética
13.
Biosci Rep ; 41(8)2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34323266

RESUMO

An imbalance between protein aggregation and protein degradation may induce 'stress' in the functionality of the endoplasmic reticulum (ER). There are quality control (QC) mechanisms to minimize misfolding and to eliminate misfolded proteins before aggregation becomes lethal for the cell. Proper protein folding and maturation is one of the crucial functions of the ER. Chaperones of the ER and folding enzymes guarantee correct conformational maturation of emerging secretory proteins. Histone deacetylase (HDAC) 6 (HDAC6) is a masterpiece coordinating the cell response to protein aggregate formation. The balance between HDAC6 and its partner Valosin-containing protein/p97 determines the fate of polyubiquitinated misfolded proteins. WT161 is a terrific, selective, and bioavailable HDAC6 inhibitor. WT161 selectively inhibits HDAC6 and adequately increases levels of acetylated α-tubulin. This compound induces accumulation of acetylated tubulin and cytotoxicity in multiple myeloma (MM) cells. In this journal, Sun et al. (Biosci. Rep.41, DOI: 10.1042/BSR20203905) identified that WT161 suppresses the cell growth of osteosarcoma cells. This discovery opens the door to future chemotherapeutic regimens of this bone neoplasm.


Assuntos
Inibidores de Histona Desacetilases , Osteossarcoma , Fluoruracila , Desacetilase 6 de Histona/genética , Histona Desacetilases/genética , Humanos , Ácidos Hidroxâmicos , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Compostos de Terfenil , Tubulina (Proteína)
14.
Sci Rep ; 11(1): 14466, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262061

RESUMO

Despite advances in therapeutic strategies for multiple sclerosis (MS), the therapy options remain limited with various adverse effects. Here, the therapeutic potential of CKD-506, a novel HDAC6-selective inhibitor, against MS was evaluated in mice with myelin oligodendrocyte glycoprotein35-55 (MOG35-55)-induced experimental autoimmune encephalitis (EAE) under various treatment regimens. CKD-506 exerted prophylactic and therapeutic effects by regulating peripheral immune responses and maintaining blood-brain barrier (BBB) integrity. In MOG35-55-re-stimulated splenocytes, CKD-506 decreased proliferation and downregulated the expression of IFN-γ and IL-17A. CKD-506 downregulated the levels of pro-inflammatory cytokines in the blood of EAE mice. Additionally, CKD-506 decreased the leakage of intravenously administered Evans blue into the spinal cord; CD4+ T cells and CD4-CD11b+CD45+ macrophage/microglia in the spinal cord was also decreased. Moreover, CKD-506 exhibited therapeutic efficacy against MS, even when drug administration was discontinued from day 15 post-EAE induction. Disease exacerbation was not observed when fingolimod was changed to CKD-506 from day 15 post-EAE induction. CKD-506 alleviated depression-like behavior at the pre-symptomatic stage of EAE. In conclusion, CKD-506 exerts therapeutic effects by regulating T cell- and macrophage-mediated peripheral immune responses and strengthening BBB integrity. Our results suggest that CKD-506 is a potential therapeutic agent for MS.


Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/etiologia , Animais , Antidepressivos/administração & dosagem , Antidepressivos/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/etiologia , Feminino , Cloridrato de Fingolimode/farmacologia , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/administração & dosagem , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/toxicidade , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiopatologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologia
15.
ACS Chem Biol ; 16(8): 1435-1444, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34314149

RESUMO

Histone deacetylase 6 (HDAC6) is upregulated in a variety of tumor cell lines and has been linked to many cellular processes, such as cell signaling, protein degradation, cell survival, and cell motility. HDAC6 is an enzyme that deacetylates the acetyllysine residues of protein substrates, and the discovery of HDAC6 substrates, including tubulin, has revealed many roles of HDAC6 in cell biology. Unfortunately, among the wide variety of acetylated proteins in the cell, only a few are verified as HDAC6 substrates, which limits the full characterization of HDAC6 cellular functions. Substrate trapping mutants were recently established as a tool to discover unanticipated substrates of histone deacetylase 1 (HDAC1). In this study, we applied the trapping approach to identify potential HDAC6 substrates. Among the high confidence protein hits after trapping, protein arginine methyl transferase 5 (PRMT5) was successfully validated as a novel HDAC6 substrate. PRMT5 acetylation enhanced its methyltransferase activity and symmetrical dimethylation of downstream substrates, revealing possible crosstalk between acetylation and methylation. Substrate trapping represents a powerful, systematic, and unbiased approach to discover substrates of HDAC6.


Assuntos
Desacetilase 6 de Histona/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Acetilação , Domínio Catalítico/genética , DNA Helicases/metabolismo , Células HEK293 , Desacetilase 6 de Histona/química , Desacetilase 6 de Histona/genética , Humanos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteína-Arginina N-Metiltransferases/química , Proteômica/métodos , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo
16.
J Med Chem ; 64(14): 10403-10417, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34185525

RESUMO

Epigenetic post-translational modifications are essential for human malaria parasite survival and progression through its life cycle. Here, we present new functionalized suberoylanilide hydroxamic acid (SAHA) derivatives that chemically combine the pan-histone deacetylase inhibitor SAHA with the DNA methyltransferase inhibitor procainamide. A three- or four-step chemical synthesis was designed starting from cheap raw materials. Compared to the single drugs, the combined molecules showed a superior activity in Plasmodium and a potent inhibition against human HDAC6, exerting no cytotoxicity in human cell lines. These new compounds are fully active in multidrug-resistant Plasmodium falciparum Cambodian isolates. They target transmission of the parasite by inducing irreversible morphological changes in gametocytes and inhibiting exflagellation. The compounds are slow-acting and have an additive antimalarial effect in combination with fast-acting epidrugs and dihydroartemisinin. The lead compound decreases parasitemia in mice in a severe malaria model. Taken together, this novel fused molecule offers an affordable alternative to current failing antimalarial therapy.


Assuntos
Antimaláricos/farmacologia , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Procainamida/farmacologia , Antimaláricos/síntese química , Antimaláricos/química , Relação Dose-Resposta a Droga , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Estrutura Molecular , Procainamida/química , Relação Estrutura-Atividade
17.
Ecotoxicol Environ Saf ; 221: 112453, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34186418

RESUMO

Breast cancer (BrCa) as one of the major malignancies threatening women's health worldwide occurs due to the genetic and environmental interactions. Epidemiological studies have suggested that exposure to endocrine disrupting chemicals (EDCs) can elevate the risk of breast cancer. Di-(2-ethylhexyl)-phthalate (DEHP) and bisphenol A (BPA) are known as two typical EDCs. Although several studies have implied that there appear to have adverse effects of exposure to BPA or DEHP alone on breast development, no study to date has demonstrated the exact toxic effect of combined exposure to DEHP and BPA on breast tumorigenesis. In the present study, we performed an in vivo experiment including 160 female Sprague-Dawley (SD) rats, in which 80 rats were randomly allocated to 4 groups including control group given to normal diet, DEHP (150 mg/kg body weight/day), BPA (20 mg/kg body weight/day), and DEHP (150 mg/kg body weight/day) combined with BPA (20 mg/kg body weight/day) by gavage for 30 weeks. Additionally, a DEN/MNU/DHPN (DMD)-induced carcinogenesis animal model was also established to assess their effect on tumor promotion. Namely, the other 80 SD rats were separated into another 4 groups: in addition to DMD initiation each group treated with vehicle, DEHP, BPA and the combination of BPA and DEHP respectively. Our data demonstrated that BPA alone or in combination with DEHP may induce hyperplasia of mammary glands, including the proliferation of ductal epithelial cells and an increase in the number of lobules and acinus after a 30-week exposure. Notably, co-exposure to DEHP and BPA increased the incidence and reduced the latency of mammary tumor, which seemed to enhance the susceptibility of carcinogens-induced tumor. Mechanistically, our results supported the hypothesis that exposure to BPA and DEHP might promote breast cancer dependent on Esr1 and HDAC6 as pivotal factors, and further lead to the activation of oncogene c-Myc. Our study suggested that BPA combined with DEHP facilitate the occurrence of mammary tumors, which contributed to advance our understanding in the complex effects of compound exposure to endocrine disrupting chemicals.


Assuntos
Compostos Benzidrílicos/toxicidade , Dietilexilftalato/toxicidade , Disruptores Endócrinos/toxicidade , Receptor alfa de Estrogênio/metabolismo , Desacetilase 6 de Histona/metabolismo , Neoplasias Mamárias Animais/induzido quimicamente , Fenóis/toxicidade , Animais , Sinergismo Farmacológico , Receptor alfa de Estrogênio/genética , Feminino , Desacetilase 6 de Histona/genética , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Ratos Sprague-Dawley , Ativação Transcricional , Regulação para Cima/efeitos dos fármacos
18.
Nat Commun ; 12(1): 3384, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099674

RESUMO

Despite recent success in computational design of structured cyclic peptides, de novo design of cyclic peptides that bind to any protein functional site remains difficult. To address this challenge, we develop a computational "anchor extension" methodology for targeting protein interfaces by extending a peptide chain around a non-canonical amino acid residue anchor. To test our approach using a well characterized model system, we design cyclic peptides that inhibit histone deacetylases 2 and 6 (HDAC2 and HDAC6) with enhanced potency compared to the original anchor (IC50 values of 9.1 and 4.4 nM for the best binders compared to 5.4 and 0.6 µM for the anchor, respectively). The HDAC6 inhibitor is among the most potent reported so far. These results highlight the potential for de novo design of high-affinity protein-peptide interfaces, as well as the challenges that remain.


Assuntos
Desenho de Fármacos , Inibidores de Histona Desacetilases/farmacologia , Peptídeos Cíclicos/farmacologia , Relação Estrutura-Atividade , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Ensaios Enzimáticos , Histona Desacetilase 2/antagonistas & inibidores , Histona Desacetilase 2/isolamento & purificação , Histona Desacetilase 2/metabolismo , Histona Desacetilase 2/ultraestrutura , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/isolamento & purificação , Desacetilase 6 de Histona/ultraestrutura , Inibidores de Histona Desacetilases/química , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Biblioteca de Peptídeos , Peptídeos Cíclicos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/ultraestrutura
19.
J Med Chem ; 64(12): 8486-8509, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34101461

RESUMO

Epigenetic targeting has emerged as an efficacious therapy for hematological cancers. The rare and incurable T-cell prolymphocytic leukemia (T-PLL) is known for its aggressive clinical course. Current epigenetic agents such as histone deacetylase (HDAC) inhibitors are increasingly used for targeted therapy. Through a structure-activity relationship (SAR) study, we developed an HDAC6 inhibitor KT-531, which exhibited higher potency in T-PLL compared to other hematological cancers. KT-531 displayed strong HDAC6 inhibitory potency and selectivity, on-target biological activity, and a safe therapeutic window in nontransformed cell lines. In primary T-PLL patient cells, where HDAC6 was found to be overexpressed, KT-531 exhibited strong biological responses, and safety in healthy donor samples. Notably, combination studies in T-PLL patient samples demonstrated KT-531 synergizes with approved cancer drugs, bendamustine, idasanutlin, and venetoclax. Our work suggests HDAC inhibition in T-PLL could afford sufficient therapeutic windows to achieve durable remission either as stand-alone or in combination with targeted drugs.


Assuntos
Antineoplásicos/uso terapêutico , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Leucemia Prolinfocítica de Células T/tratamento farmacológico , Sulfonamidas/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Cloridrato de Bendamustina/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/farmacocinética , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacocinética , Masculino , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Pirrolidinas/farmacologia , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , para-Aminobenzoatos/farmacologia
20.
Int J Biochem Cell Biol ; 136: 106000, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33933678

RESUMO

High-risk human papillomavirus (HR-HPV) infection is a major risk factor for the initiation and progression of cervical cancer (CC). This study aimed to explore the role of histone deacetylase 6 (HDAC6) in HPV-positive CC and the molecules implicated. Differentially expressed genes between HPV-positive and HPV-negative tissues, and differentially expressed microRNAs (miRNAs) in cells after HDAC6 downregulation were identified using microarray analyses. The expression profiles of HDAC6 and miR-199a and their cellular functions were investigated via loss-of-function studies. Xenograft tumors were induced in mice for in vivo studies. HDAC6 and Wnt5a were highly expressed, whereas miR-199a was poorly expressed in HPV-positive CC tissues. Downregulation of HDAC6 reduced proliferation, migration, invasion, and resistance to apoptosis of HPV-positive CC cells. HDAC6 suppressed the transcription of miR-199a, and miR-199a targeted Wnt5a to inactivate the Wnt signaling pathway. Further downregulation of miR-199a blocked the inhibitory effect of HDAC6 silencing on CC cell growth both in vivo and in vitro, whereas further artificial inhibition of Wnt5a inactivated Wnt signaling and blocked the malignant behaviors of CC cells. This study showed that HDAC6 suppresses the transcription of miR-199a and enhances the progression of HPV-positive cervical cancer through upregulation of Wnt5a.


Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Desacetilase 6 de Histona/metabolismo , MicroRNAs/genética , Infecções por Papillomavirus/complicações , Neoplasias do Colo do Útero/patologia , Proteína Wnt-5a/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Desacetilase 6 de Histona/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/virologia , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologia , Proteína Wnt-5a/genética , Ensaios Antitumorais Modelo de Xenoenxerto
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