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1.
J Med Chem ; 67(7): 5233-5258, 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38552030

RESUMO

The salt-inducible kinases (SIKs) SIK1, SIK2, and SIK3 belong to the adenosine monophosphate-activated protein kinase (AMPK) family of serine/threonine kinases. SIK inhibition represents a new therapeutic approach modulating pro-inflammatory and immunoregulatory pathways that holds potential for the treatment of inflammatory diseases. Here, we describe the identification of GLPG3970 (32), a first-in-class dual SIK2/SIK3 inhibitor with selectivity against SIK1 (IC50 of 282.8 nM on SIK1, 7.8 nM on SIK2 and 3.8 nM on SIK3). We outline efforts made to increase selectivity against SIK1 and improve CYP time-dependent inhibition properties through the structure-activity relationship. The dual activity of 32 in modulating the pro-inflammatory cytokine TNFα and the immunoregulatory cytokine IL-10 is demonstrated in vitro in human primary myeloid cells and human whole blood, and in vivo in mice stimulated with lipopolysaccharide. Compound 32 shows dose-dependent activity in disease-relevant mouse pharmacological models.


Assuntos
Proteínas Quinases , Proteínas Serina-Treonina Quinases , Camundongos , Humanos , Animais , Proteínas Quinases/metabolismo , Citocinas , Fator de Necrose Tumoral alfa
2.
J Med Chem ; 67(1): 380-401, 2024 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-38147525

RESUMO

Salt-inducible kinases (SIKs) SIK1, SIK2, and SIK3 are serine/threonine kinases and form a subfamily of the protein kinase AMP-activated protein kinase (AMPK) family. Inhibition of SIKs in stimulated innate immune cells and mouse models has been associated with a dual mechanism of action consisting of a reduction of pro-inflammatory cytokines and an increase of immunoregulatory cytokine production, suggesting a therapeutic potential for inflammatory diseases. Following a high-throughput screening campaign, subsequent hit to lead optimization through synthesis, structure-activity relationship, kinome selectivity, and pharmacokinetic investigations led to the discovery of clinical candidate GLPG3312 (compound 28), a potent and selective pan-SIK inhibitor (IC50: 2.0 nM for SIK1, 0.7 nM for SIK2, and 0.6 nM for SIK3). Characterization of the first human SIK3 crystal structure provided an understanding of the binding mode and kinome selectivity of the chemical series. GLPG3312 demonstrated both anti-inflammatory and immunoregulatory activities in vitro in human primary myeloid cells and in vivo in mouse models.


Assuntos
Proteínas Quinases Ativadas por AMP , Proteínas Serina-Treonina Quinases , Camundongos , Animais , Humanos , Expressão Gênica , Citocinas
3.
Toxicol Sci ; 140(2): 352-63, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24848799

RESUMO

The obesogen hypothesis states that together with an energy imbalance between calories consumed and calories expended, exposure to environmental compounds early in life or throughout lifetime might have an influence on obesity development. In this work, we propose a new approach for obesogen screening, i.e., the use of transcriptomics in the 3T3-L1 pre-adipocyte cell line. Based on the data from a previous study of our group using a lipid accumulation based adipocyte differentiation assay, several human-relevant obesogenic compounds were selected: reference obesogens (Rosiglitazone, Tributyltin), test obesogens (Butylbenzyl phthalate, butylparaben, propylparaben, Bisphenol A), and non-obesogens (Ethylene Brassylate, Bis (2-ethylhexyl)phthalate). The high stability and reproducibility of the 3T3-L1 gene transcription patterns over different experiments and cell batches is demonstrated by this study. Obesogens and non-obesogen gene transcription profiles were clearly distinguished using hierarchical clustering. Furthermore, a gradual distinction corresponding to differences in induction of lipid accumulation could be made between test and reference obesogens based on transcription patterns, indicating the potential use of this strategy for classification of obesogens. Marker genes that are able to distinguish between non, test, and reference obesogens were identified. Well-known genes involved in adipocyte differentiation as well as genes with unknown functions were selected, implying a potential adipocyte-related function of the latter. Cell-physiological lipid accumulation was well estimated based on transcription levels of the marker genes, indicating the biological relevance of omics data. In conclusion, this study shows the high relevance and reproducibility of this 3T3-L1 based in vitro toxicogenomics tool for classification of obesogens and biomarker discovery. Although the results presented here are promising, further confirmation of the predictive value of the set of candidate biomarkers identified as well as the validation of their clinical role will be needed.


Assuntos
Obesidade/induzido quimicamente , Toxicogenética , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Diferenciação Celular , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Reprodutibilidade dos Testes , Transcrição Gênica
4.
PLoS One ; 9(1): e84816, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24427296

RESUMO

INTRODUCTION: Persistent Organic Pollutants (POPs) accumulate in adipose tissue and some are described to possess endocrine disrupting capacities. Therefore, it is important to evaluate their effects on key endocrine pathways in adipose tissue (AT), to further evaluate their potential role in metabolic pathologies such as obesity. OBJECTIVES: THE AIM IS TWOFOLD: (i) evaluate gene expression levels of obesity marker genes, i.e. the adipokines leptin (LEP), adiponectin (ADIPOQ) and Tumor Necrosis Factor α (TNFα) and the nuclear receptor, Peroxisome Proliferator Activated Receptor γ (PPARγ) in paired subcutaneous (SAT) and visceral (VAT) AT of obese subjects (n = 50) and to relate these values to serum concentrations of LEP and ADIPOQ (ii) evaluate the association of expression levels of marker genes in AT and serum with POP concentrations in AT. RESULTS AND CONCLUSIONS: Leptin and adiponectin levels in serum were positively correlated to respectively expression levels of leptin in SAT and adiponectin in VAT. Our study shows more significant correlations between gene expression of obesity marker genes and POP concentrations in VAT compared to SAT. Since VAT is more important than SAT in pathologies associated with obesity, this suggests that POPs are able to influence the association between obesity and the development of associated pathologies. Moreover, this finding reveals the importance of VAT when investigating the obesogen hypothesis. Concerning PPARγ expression in VAT, negative correlations with polychlorinated biphenyls (PCBs) concentrations were found in non T2D patients. LEP serum concentrations correlated with several PCBs in women whereas in men no correlations were found. This strengthens the potential importance of gender differences in obesity and within the obesogen hypothesis.


Assuntos
Tecido Adiposo/metabolismo , Expressão Gênica , Obesidade/genética , Obesidade/metabolismo , Adolescente , Adulto , Biomarcadores/sangue , Biomarcadores/metabolismo , Feminino , Humanos , Gordura Intra-Abdominal/metabolismo , Masculino , Pessoa de Meia-Idade , Gordura Subcutânea/metabolismo , Adulto Jovem
5.
PLoS One ; 8(10): e77481, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24155963

RESUMO

Recently the environmental obesogen hypothesis has been formulated, proposing a role for endocrine disrupting compounds (EDCs) in the development of obesity. To evaluate this hypothesis, a screening system for obesogenic compounds is urgently needed. In this study, we suggest a standardised protocol for obesogen screening based on the 3T3-L1 cell line, a well-characterised adipogenesis model, and direct fluorescent measurement using Nile red lipid staining technique. In a first phase, we characterised the assay using the acknowledged obesogens rosiglitazone and tributyltin. Based on the obtained dose-response curves for these model compounds, a lipid accumulation threshold value was calculated to ensure the biological relevance and reliability of statistically significant effects. This threshold based method was combined with the well described strictly standardized mean difference (SSMD) method for classification of non-, weak- or strong obesogenic compounds. In the next step, a range of EDCs, used in personal and household care products (parabens, musks, phthalates and alkylphenol compounds), were tested to further evaluate the obesogenicity screening assay for its discriminative power and sensitivity. Additionally, the peroxisome proliferator activated receptor γ (PPARγ) dependency of the positive compounds was evaluated using PPARγ activation and antagonist experiments. Our results showed the adipogenic potential of all tested parabens, several musks and phthalate compounds and bisphenol A (BPA). PPARγ activation was associated with adipogenesis for parabens, phthalates and BPA, however not required for obesogenic effects induced by Tonalide, indicating the role of other obesogenic mechanisms for this compound.


Assuntos
Disruptores Endócrinos/efeitos adversos , Disruptores Endócrinos/análise , Obesidade/induzido quimicamente , PPAR gama/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipócitos/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Insulina/farmacologia , Camundongos , Obesidade/genética , PPAR gama/antagonistas & inibidores , PPAR gama/genética , Padrões de Referência , Coloração e Rotulagem , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética
6.
PLoS One ; 8(3): e60030, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23555872

RESUMO

Environmental pollutants have recently emerged as potential risk factors for metabolic diseases, urging systematic investigation of pollutant effects on metabolic disease processes. To enable risk assessment of these so-called metabolic disruptors the use of stable, robust and well-defined cell based screening systems has recently been encouraged. Since beta-cell (dys)functionality is central in diabetes pathophysiology, the need to develop beta-cell based pollutant screening systems is evident. In this context, the present research evaluated the strengths and weaknesses of the INS-1 832/13 pancreatic beta-cell line as diabetogenic pollutant screening system with a focus on beta-cell function. After optimization of exposure conditions, positive (exendin-4, glibenclamide) and negative (diazoxide) control compounds for acute insulin secretion responses were tested and those with the most profound effects were selected to allow potency estimations and ranking of pollutants. This was followed by a first explorative screening of acute bisphenol A and bis(2-ethylhexyl)phthalate effects. The same approach was applied for chronic exposures, focusing primarily on evaluation of acknowledged chronic stimulators (diazoxide, T0901317, exendin-4) or inhibitors (glibenclamide) of insulin secretion responses to select the most responsive ones for use as control compounds in a chronic pollutant testing framework. Our results showed that INS-1 832/13 cells responded conform previous observations regarding acute effects of control compounds on insulin secretion, while bisphenol A and bis(2-ethylhexyl)phthalate had limited acute effects. Furthermore, chronic exposure to known beta-cell reactive compounds resulted in deviating insulin secretion and insulin content profiles compared to previous reports. In conclusion, this INS-1 subclone appears to lack certain characteristics needed to respond appropriately to acute pollutant exposure or long term exposure to known beta-cell reactive compounds and thus seems to be, in our setting, inadequate as a diabetogenic pollutant screening system.


Assuntos
Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Compostos Benzidrílicos/toxicidade , Linhagem Celular , Diazóxido/toxicidade , Exenatida , Glibureto/toxicidade , Humanos , Hidrocarbonetos Fluorados/toxicidade , Insulina/metabolismo , Secreção de Insulina , Peptídeos/toxicidade , Fenóis/toxicidade , Ácidos Ftálicos/toxicidade , Sulfonamidas/toxicidade , Peçonhas/toxicidade
7.
Environ Int ; 55: 33-42, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23518385

RESUMO

There are only few studies defining persistent organic pollutant (POP) concentrations in various fat compartments from living obese individuals. The present study has therefore determined the concentrations of various classes of organohalogenated compounds, such as dichlorodiphenyltrichloroethane and its metabolites (DDTs), chlordane compounds (CHLs), hexachlorocyclohexanes (HCHs), hexachlorobenzene (HCB), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecanes (HBCDs) in visceral fat (VF: n=52) and subcutaneous abdominal fat (SF: n=52) samples collected in 2010-2012 from obese individuals in Belgium. Organohalogen compounds were detected in all fat samples in the decreasing order of their concentrations: PCBs>DDTs>HCHs>CHLs>HCB>HBCDs>PBDEs, suggesting that Belgians have been widely exposed to these contaminants. The levels and the patterns of POP distribution in VF and SF tissue depots were not significantly different. Concentrations of PCBs (VF/SF; median: 285/275ng/g lw) and DDTs (VF/SF; median: 150/155ng/g lw) were the major POPs in all fat samples. Concerning PCBs, PCB 153 (VF/SF: 27/26%) was the most dominant congener, followed by PCB 180 (VF/SF: 17/18%), PCB 138 (VF/SF: 15/14.5%) and PCB 170 (VF/SF: 8.1/8.4%) to the sum PCBs, respectively. Levels of HBCDs (VF/SF; median: 4.0/3.7ng/g lw) and PBDEs (VF/SF; median: 2.6/2.7ng/g lw) were 1-2 orders of magnitude lower than those of PCBs and DDTs. Among PBDEs, BDE 153 (VF/SF: 31/34%) was the dominant congener, followed by BDE 47 (VF/SF: 26/23%), BDE 154 (VF/SF: 16/16%), BDE 100 (VF/SF: 10/11%) and BDE 99 (VF/SF: 9/9%). To our knowledge, this is the first report on HBCD concentrations in Belgian human fat tissues. Total PBDE and HBCD levels in human fat samples could not be correlated with age. In agreement with the literature, a significant correlation (p<0.05) between age and the concentration of PCBs (r=0.828), DDTs (r=0.640), HCHs (r=0.666), CHLs (r=0.534) and HCB (r=0.754), was observed in the present study. Levels of DDTs, HCHs, HCB and CHLs were also significantly correlated to each other, suggesting that they share similar exposure routes. Correlation with computed tomography (CT) scan data revealed that VF and VF/SF ratios are positive for most of the POPs, such as PCBs, PBDEs, p,p'DDE, CHLs, ß-HCH, and HCB. To our knowledge, this study is the first to assess the relationship between POP levels in adipose tissue and markers of abdominal adiposity, determined by CT.


Assuntos
Tecido Adiposo/metabolismo , Poluentes Ambientais/metabolismo , Hidrocarbonetos Clorados/metabolismo , Adolescente , Adulto , Bélgica , DDT/metabolismo , Exposição Ambiental/análise , Exposição Ambiental/estatística & dados numéricos , Feminino , Éteres Difenil Halogenados/metabolismo , Hexaclorobenzeno/metabolismo , Hexaclorocicloexano/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Bifenilos Policlorados/metabolismo , Adulto Jovem
8.
Mol Cell Endocrinol ; 370(1-2): 52-64, 2013 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-23428407

RESUMO

Obesogenic compounds are chemicals that have an influence on obesity development. This study was designed to unravel the molecular mechanisms of the model obesogen TBT, using microarray analysis in the 3T3-L1 in vitro system, and to evaluate the use of toxicogenomics for obesogen screening. The microarray results revealed enrichment of Gene Ontology terms involved in energy and fat metabolism after 10 days of TBT exposure. Pathway analysis unveiled PPAR signalling pathway as the sole pathway significantly enriched after 1 day and the most significantly enriched pathway after 10 days of exposure. To our knowledge, this is the first study delivering an in depth mechanistic outline of the mode of action of TBT as an obesogen, combining effects on both cell physiological and gene expression level. Furthermore, our results show that combining transcriptomics with 3T3-L1 cells is a promising tool for screening of potential obesogenic compounds.


Assuntos
Metabolismo Energético/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Obesidade/induzido quimicamente , Compostos de Trialquitina/farmacologia , Células 3T3 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipocinas/genética , Animais , Linhagem Celular , Colesterol/metabolismo , Estrogênios/metabolismo , Expressão Gênica , Glucocorticoides/genética , Glucocorticoides/metabolismo , Camundongos , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais , Triglicerídeos/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt
9.
Toxicol Lett ; 212(2): 180-9, 2012 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-22652326

RESUMO

This study was designed to evaluate if the rat H4IIE hepatoma cell line is a physiologically relevant model to study hepatic insulin responses to hint at its prospective application in pollutant-related insulin resistance research. DNA microarray analysis, real-time PCR and flow cytometric cell cycle analysis were used to assess the relevance of the insulin response in H4IIE cells. Insulin dose dependently stimulated H4IIE growth and time dependently altered the expression of the known insulin responsive genes: Fasn, Pck1 and Irs2. Microarray analysis performed on cells exposed to insulin (100nM) for 6h and 24h showed that genes related to carbohydrate and lipid metabolism were most profoundly afflicted, in accordance with in vivo hepatic insulin action. Since changes in carbohydrate and lipid metabolism are pivotal in the pathogenesis of insulin resistance, the presence of a physiological relevant insulin response in H4IIE cells pleads for further testing of its potential use in research on pollutant-driven insulin resistance.


Assuntos
Carcinoma Hepatocelular/metabolismo , Resistência à Insulina , Neoplasias Hepáticas/metabolismo , Animais , Metabolismo dos Carboidratos/efeitos dos fármacos , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos
10.
Blood ; 114(21): 4664-74, 2009 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-19726720

RESUMO

Alternatively activated macrophages (AAMs), triggered by interleukin-4 (IL-4) and IL-13, play a modulating role during Th2 cytokine-driven pathologies, but their molecular armament remains poorly characterized. Here, we established E-cadherin (Cdh1) as a selective marker for IL-4/IL-13-exposed mouse and human macrophages, which is STAT6-dependently induced during polarized Th2 responses associated with Taenia crassiceps helminth infections or allergic airway inflammation. The IL-4-dependent, arginase-1/ornithine decarboxylase-mediated production of polyamines is important for maximal Cdh1 induction, unveiling a novel mechanism for IL-4-dependent gene transcription. At the macrophage surface, E-cadherin forms a functional complex with the catenins that accumulates at sites of cell contact. Macrophage-specific deletion of the Cdh1 gene illustrates the implication of E-cadherin in IL-4-driven macrophage fusion and heterotypic interactions with CD103(+) and KLRG1(+) T cells. This study identifies the E-cadherin/catenin complex as a discriminative, partly polyamine-regulated feature of IL-4/IL-13-exposed alternatively activated macrophages that contributes to homotypic and heterotypic cellular interactions.


Assuntos
Caderinas/imunologia , Interleucina-4/imunologia , Macrófagos/imunologia , Poliaminas/imunologia , Transdução de Sinais/imunologia , alfa Catenina/imunologia , Animais , Asma/imunologia , Western Blotting , Caderinas/metabolismo , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Hipersensibilidade/imunologia , Imunoprecipitação , Interleucina-13/imunologia , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Teníase/imunologia , alfa Catenina/metabolismo
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