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1.
J Med Chem ; 64(4): 1904-1929, 2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33626870

RESUMO

The modulation of melatonin signaling in peripheral tissues holds promise for treating metabolic diseases like obesity, diabetes, and nonalcoholic steatohepatitis. Here, several benzimidazole derivatives have been identified as novel agonists of the melatonin receptors MT1 and MT2. The lead compounds 10b, 15a, and 19a demonstrated subnanomolar potency at MT1/MT2 receptors, high oral bioavailability in rodents, peripherally preferred exposure, and excellent selectivity in a broad panel of targets. Two-month oral administration of 10b in high-fat diet rats led to a reduction in body weight gain similar to dapagliflozin with superior results on hepatic steatosis and triglyceride levels. An early toxicological assessment indicated that 10b (also codified as ACH-000143) was devoid of hERG binding, genotoxicity, and behavioral alterations at doses up to 100 mg/kg p.o., supporting further investigation of this compound as a drug candidate.

2.
Eur J Pharm Sci ; 159: 105731, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33493668

RESUMO

The multifactorial etiology of hypertension has promoted the research of blood pressure-lowering agents with multitarget actions to achieve better clinical outcomes. We describe here the discovery of novel dual-acting antihypertensive codrugs combining pharmacophores with angiotensin type 1 (AT1) receptor antagonism and neprilysin (NEP) inhibition. Specifically, the codrugs combine the AT1 antagonists losartan or its carboxylic acid active metabolite (E-3174) with selected monocarboxylic acid NEP inhibitors through a cleavable linker. The resulting codrugs exhibited high rates of in vitro conversion into the active molecules upon incubation with human/rat liver S9 fractions and in vivo conversion after oral administration in rodents. Moreover, the acute effects of one of the designed codrugs (3b) was confirmed at the doses of 10, 30 and 60 mg/kg p.o. in the spontaneous hypertensive rat (SHR) model, showing better antihypertensive response over 24 hours than the administration of an equivalent fixed-dose combination of 15 mg/kg of losartan and 14 mg/kg of the same NEP inhibitor used in 3b. The results demonstrate that the codrug approach is a plausible strategy to develop a single molecular entity with combined AT1 and NEP activities, aiming at achieving improved pharmacokinetics, efficacy and dosage convenience, as well as reduced drug-drug interaction for hypertension patients. In addition, the developability of the codrug should be comparable to the one of marketed AT1 antagonists, most of them prodrugs, but bearing only the AT1 pharmacophore.

3.
Clin Epigenetics ; 12(1): 127, 2020 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-32831131

RESUMO

BACKGROUND: We have previously developed a murine cellular system that models the transformation from melanocytes to metastatic melanoma cells. This model was established by cycles of anchorage impediment of melanocytes and consists of four cell lines: differentiated melanocytes (melan-a), pre-malignant melanocytes (4C), malignant (4C11-), and metastasis-prone (4C11+) melanoma cells. Here, we searched for transcriptional and epigenetic signatures associated with melanoma progression and metastasis by performing a gene co-expression analysis of transcriptome data and a mass-spectrometry-based profiling of histone modifications in this model. RESULTS: Eighteen modules of co-expressed genes were identified, and some of them were associated with melanoma progression, epithelial-to-mesenchymal transition (EMT), and metastasis. The genes in these modules participate in biological processes like focal adhesion, cell migration, extracellular matrix organization, endocytosis, cell cycle, DNA repair, protein ubiquitination, and autophagy. Modules and hub signatures related to EMT and metastasis (turquoise, green yellow, and yellow) were significantly enriched in genes associated to patient survival in two independent melanoma cohorts (TCGA and Leeds), suggesting they could be sources of novel prognostic biomarkers. Clusters of histone modifications were also linked to melanoma progression, EMT, and metastasis. Reduced levels of H4K5ac and H4K8ac marks were seen in the pre-malignant and tumorigenic cell lines, whereas the methylation patterns of H3K4, H3K56, and H4K20 were related to EMT. Moreover, the metastatic 4C11+ cell line showed higher H3K9me2 and H3K36me3 methylation, lower H3K18me1, H3K23me1, H3K79me2, and H3K36me2 marks and, in agreement, downregulation of the H3K36me2 methyltransferase Nsd1. CONCLUSIONS: We uncovered transcriptional and histone modification signatures that may be molecular events driving melanoma progression and metastasis, which can aid in the identification of novel prognostic genes and drug targets for treating the disease.

4.
J Proteomics ; 229: 103951, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32860965

RESUMO

Bladder cancer (BC) is classified into non-muscle (NMIBC) and muscle invasive (MIBC) diseases. Several molecular alterations were previously associated with NMIBC and MIBC, but few studies have systematically compared the molecular differences between these subtypes. Here, we analyzed prognostic and differentially expressed genes in NMIBC and MIBC, using an integrative bioinformatics approach. These genes were used in functional enrichment and co-expression protein interaction (COPI) network analyses to reveal common and exclusive biological functions involved in NMIBC and MIBC. In NMIBC, the enriched functions were related to oxidative stress response, cell cycle, glutathione metabolism, ubiquitination and protein translation. Conversely, enriched functions in MIBC were extracellular matrix organization, cell migration and actin cytoskeleton. Several genes in NMIBC did not overlap with those reported to MIBC, suggesting these subtypes may have distinct underlying mechanisms. Particularly, MIBC genes were enriched for functions involved in cell migration and invasion, which could help to molecularly differentiate NMIBC and MIBC. The analysis of COPI networks disclosed high centrality nodes that may be essential for NMIBC and MIBC. Further research will determine to which extent NMIBC and MIBC share common biological functions and identify potential candidates for the differential diagnosis, prognosis and treatment of NMIBC and MIBC. SIGNIFICANCE: This study has systematically compared prognostic and differentially expressed genes between non-muscle (NMIBC) and muscle invasive (MIBC) bladder cancer, using an integrative bioinformatics approach. Many genes and biological functions were exclusively associated with either NMIBC or MIBC, suggesting that these disease subtypes could be driven by distinct molecular mechanisms. Particularly, prognostic and differentially expressed genes in MIBC were involved in cell migration and invasion, which can help to molecularly differentiate the NMIBC and MIBC subtypes. Moreover, the analysis of co-expression protein interaction networks identified high centrality nodes that could be potential candidates for the prognosis and treatment of NMIBC and MIBC.

5.
Neurobiol Stress ; 13: 100226, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32478146

RESUMO

Post-traumatic stress disorder (PTSD) is a mental health condition that is triggered by a stressful event, with symptoms including exaggerated startle response, intrusive traumatic memories and nightmares. The single prolonged stress (SPS) is a multimodal stress protocol that comprises a sequential exposure to physical restraint, forced swimming, predator scent and ether anesthesia. This procedure generates behavioral and neurobiological alterations that resemble clinical findings of PTSD, and thus it is commonly used to model the disease in rodents. Here, we applied c-fos mapping to produce a comprehensive view of stress-activated brain regions in mice exposed to SPS alone or to SPS after oral pretreatment with the serotonin-noradrenaline receptor dual modulator ACH-000029 or the α1-adrenergic blocker prazosin. The SPS protocol evoked c-fos expression in several brain regions that control the stress-anxiety response, including the central and medial amygdala, the bed nucleus of the stria terminalis, the pallidum, the paraventricular hypothalamus, the intermediodorsal, paraventricular and central medial thalamic nuclei, the periaqueductal gray, the lateral habenula and the cuneiform nucleus. These effects were partially blocked by pretreatment with prazosin but completely prevented by ACH-000029. Collectively, these findings contribute to the brain-wide characterization of neural circuits involved in PTSD-related stress responses. Furthermore, the identification of brain areas regulated by ACH-000029 and prazosin revealed regions in which SPS-induced activation may depend on the combined or isolated action of the noradrenergic and serotonergic systems. Finally, the dual regulation of serotonin and α1 receptors by ACH-000029 might represent a potential pharmacotherapy that can be applied in the peri-trauma or early post-trauma period to mitigate the development of symptoms in PTSD patients.

6.
Int Braz J Urol ; 462020 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-32459456

RESUMO

PURPOSE: Proteomic biomarkers have been emerging as alternative methods to the gold standard procedures of cystoscopy and urine cytology in the diagnosis and surveillance of bladder cancer (BC). This review aims to update the state of the art of proteomics research and diagnosis in BC. MATERIALS AND METHODS: We reviewed the current literature related to BC research on urinary, tissue, blood and cell line proteomics, using the Pubmed database. FINDINGS: Two urinary protein biomarkers are FDA-approved (NMP22® and BTA® tests), only if performed along with cystoscopy for surveillance after initial diagnosis, but not in the primary diagnostic setting due to high false-positive rates in case of infections, stones and hematuria. There are a great number of non-FDA approved proteins being studied, with good preliminary results; panels of proteins seem valuable tools to be refined in ongoing trials. Blood proteins are a bigger challenge, because of the complexity of the serum protein profile and the scarcity of blood proteomic studies in BC. Previous studies with the BC tissue proteome do not correlate well with the urinary proteome, likely due to the tumor heterogeneity. Cell line proteomic research helps in the understanding of basic mechanisms that drive BC development and progression; the main difficulty is culturing low-grade tumors in vitro, which represents the majority of BC tumors in clinical practice. CONCLUSION: Protein biomarkers have promising value in the diagnosis, surveillance and prognostic of BC. Urine is the most appropriate body fluid for biomarker research in BC due to its easiness of sampling, stability and enrichment of shed and secreted tumor-specific proteins. Panels of biomarkers may exhibit higher sensitivity than single proteins in the diagnosis of BC at larger populations due to clinical and tumor heterogeneity. Prospective clinical trials are warranted to validate the relevance of proteomic data in the clinical management of BC. Available at. https://www.intbrazjurol.com.br/pdf/aop/2021-99_02RW.pdf.

7.
Neuropharmacology ; 164: 107912, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31843397

RESUMO

Post-traumatic stress disorder (PTSD) is a severe chronic mental illness that develops in individuals exposed to life-threatening trauma and is characterized by hyperarousal, flashbacks and nightmares. The serotonergic (5-HT) and noradrenergic (NE) systems are deeply involved in the pathogenesis of PTSD. We have previously reported a novel anxiolytic compound, ACH-000029, that modulates 5-HT and α1-adrenergic receptors and induces acute anxiolytic-like effects in rodents. Here, we investigated the potential of ACH-000029 to prevent anxiety-like behavior in the single prolonged stress (SPS) PTSD model. Mice were subjected to the SPS procedure, followed by a 7-day treatment with ACH-000029 and, for comparison, with the α1-adrenergic antagonist prazosin. Animals were behaviorally assessed using social interaction, elevated plus maze and open field tests. Interestingly, treatment with ACH-000029 but not with prazosin ameliorated the SPS-induced sociability impairment and anxiety-like behavior. The brain-wide c-fos mapping, used as a surrogate for brain activity, indicated the brain structures that were altered by SPS and putatively involved in the anxiolytic-like effect of ACH-000029. The SPS protocol produced long-lasting impairment of regions involved in stress-anxiety response, such as the amygdala, prefrontal cortex, globus pallidus and superior colliculus. ACH-000029 treatment reversed the SPS-induced c-fos changes in the globus pallidus, lateral septum and entorhinal cortex and exclusively modulated c-fos levels in subregions from the retrosplenial cortex, cerebellum, superior colliculus and ventromedial hypothalamus. These results support the hypothesis that the dual regulation of 5-HT and α1-adrenergic receptors is required to alleviate PTSD symptoms and suggest a possible role of ACH-000029 as a PTSD treatment.

8.
Clin. Epigenetics, v. 12, 127, ago. 2020
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-3143

RESUMO

Background We have previously developed a murine cellular system that models the transformation from melanocytes to metastatic melanoma cells. This model was established by cycles of anchorage impediment of melanocytes and consists of four cell lines: differentiated melanocytes (melan-a), pre-malignant melanocytes (4C), malignant (4C11−), and metastasis-prone (4C11+) melanoma cells. Here, we searched for transcriptional and epigenetic signatures associated with melanoma progression and metastasis by performing a gene co-expression analysis of transcriptome data and a mass-spectrometry-based profiling of histone modifications in this model. Results Eighteen modules of co-expressed genes were identified, and some of them were associated with melanoma progression, epithelial-to-mesenchymal transition (EMT), and metastasis. The genes in these modules participate in biological processes like focal adhesion, cell migration, extracellular matrix organization, endocytosis, cell cycle, DNA repair, protein ubiquitination, and autophagy. Modules and hub signatures related to EMT and metastasis (turquoise, green yellow, and yellow) were significantly enriched in genes associated to patient survival in two independent melanoma cohorts (TCGA and Leeds), suggesting they could be sources of novel prognostic biomarkers. Clusters of histone modifications were also linked to melanoma progression, EMT, and metastasis. Reduced levels of H4K5ac and H4K8ac marks were seen in the pre-malignant and tumorigenic cell lines, whereas the methylation patterns of H3K4, H3K56, and H4K20 were related to EMT. Moreover, the metastatic 4C11+ cell line showed higher H3K9me2 and H3K36me3 methylation, lower H3K18me1, H3K23me1, H3K79me2, and H3K36me2 marks and, in agreement, downregulation of the H3K36me2 methyltransferase Nsd1. Conclusions We uncovered transcriptional and histone modification signatures that may be molecular events driving melanoma progression and metastasis, which can aid in the identification of novel prognostic genes and drug targets for treating the disease.

9.
Clin Sci (Lond) ; 133(17): 1901-1916, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31471462

RESUMO

NLRP3 inflammasome [NLR (nucleotide-binding domain, leucine-rich repeat containing protein) Pyrin-domain-containing 3 ] functions as an innate sensor of several PAMPs and DAMPs (pathogen- and damage-associated molecular patterns). It has been also reported as a transcription factor related to Th2 pattern, although its role in the adaptive immunity has been controversial, mainly because the studies were performed using gene deletion approaches. In the present study, we have investigated the NLRP3 gain-of-function in the context of encephalomyelitis autoimmune disease (EAE), considered to be a Th1- and Th17-mediated disease. We took advantage of an animal model with NLRP3 gain-of-function exclusively to T CD4+ lymphocytes (CD4CreNLRP3fl/fl). These mice presented reduced clinical score, accompanied by less infiltrating T CD4+ cells expressing both IFN-γ and IL-17 at the central nervous system (CNS) during the peak of the disease. However, besides NLRP3 gain-of-function in lymphocytes, these mice lack NLRP3 expression in non-T CD4+ cells. Therefore, in order to circumvent this deficiency, we transferred naive CD4+ T cells from WT, NLRP3-/- or CD4CreNLRP3fl/fl into Rag-1-/- mice and immunized them with MOG35-55 Likewise, the animals repopulated with CD4CreNLRP3fl/fl T CD4+ cells presented reduced clinical score and decreased IFN-γ production at the peak of the disease. Additionally, primary effector CD4+ T cells derived from these mice presented reduced glycolytic profile, a metabolic profile compatible with Th2 cells. Finally, naive CD4+ T cells from CD4CreNLRP3fl/fl mice under a Th2-related cytokine milieu cocktail exhibited in vitro an increased IL-4 and IL-13 production. Conversely, naive CD4+ T cells from CD4CreNLRP3fl/fl mice under Th1 differentiation produced less IFN-γ and T-bet. Altogether, our data evidence that the NLRP3 gain-of-function promotes a Th2-related response, a pathway that could be better explored in the treatment of multiple sclerosis.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologia , Células Th17/metabolismo
10.
ACS Med Chem Lett ; 10(9): 1266-1271, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31531195

RESUMO

Vaccinia-related kinases 1 and 2 (VRK1 and VRK2) are human Ser/Thr protein kinases associated with increased cell division and neurological disorders. Nevertheless, the cellular functions of these proteins are not fully understood. Despite their therapeutic potential, there are no potent and specific inhibitors available for VRK1 or VRK2. We report here the discovery and elaboration of an aminopyridine scaffold as a basis for VRK1 and VRK2 inhibitors. The most potent compound for VRK1 (26) displayed an IC50 value of 150 nM and was fairly selective in a panel of 48 human kinases (selectivity score S(50%) of 0.04). Differences in compound binding mode and substituent preferences between the two VRKs were identified by the structure-activity relationship combined with the crystallographic analysis of key compounds. We expect our results to serve as a starting point for the design of more specific and potent inhibitors against each of the two VRKs.

11.
Artigo em Inglês | MEDLINE | ID: mdl-31330215

RESUMO

Anxiety disorders are serious and common mental diseases, yet there is still a need for the development of more effective anxiolytics with better safety profiles than benzodiazepines and serotonin reuptake inhibitors. The serotonergic and noradrenergic systems have reciprocal interactions and are intricately related to the pathogenesis of anxiety. In this study, the anxiolytic-like effect of the novel compound ACH-000029, 3-(2-(4-(2-methoxyphenyl) piperazine-1-yl) ethyl) quinazoline-4(3H)-one, is reported. This compound acts at selected serotonergic (5-HT1A and 5-HT1D partial agonism and 5-HT2A antagonism) and α-adrenergic (α-1A, 1B and 1D antagonism) receptors, with good selectivity over other G-protein-coupled receptors. ACH-000029 exhibited high blood-brain barrier permeation and acute anxiolytic effects in the marble burying (MB) and light-dark box (LDB) models of anxiety over the dose ranges of 8-32 mg/kg i.p. and 16-30 mg/kg p.o. The anxiolytic activity was comparable to that observed for serotonin reuptake inhibitors (paroxetine and fluoxetine) and benzodiazepines (alprazolam, diazepam and clobazam). The analysis of the whole-brain c-fos expression following oral dosing showed that ACH-000029 regulated regions highly associated with the processing of environmental stimuli and anxiety behavior, such as the amygdala, paraventricular nucleus of the thalamus, retrosplenial dorsal, pallidum, bed nuclei of the stria terminalis, and locus ceruleus. No safety concerns were identified for ACH-000029 in the functional observational battery up to 50 mg/kg i.p. and in the nonprecipitated withdrawal test up to 30 mg/kg p.o. twice daily for 20 days. This work supports the further development of ACH-000029 as a drug candidate for the treatment of anxiety disorders. The analysis of the in vitro pharmacology and brain regions regulated by this compound may also lead to the exploration of other indications within the psychiatry field.


Assuntos
Adrenérgicos/farmacologia , Ansiolíticos/farmacologia , Ansiedade/tratamento farmacológico , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Serotoninérgicos/farmacologia , Adrenérgicos/uso terapêutico , Animais , Ansiolíticos/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Masculino , Camundongos , Serotoninérgicos/uso terapêutico
12.
Sci Rep ; 9(1): 1056, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30705326

RESUMO

A growing body of evidence suggests a key role of tumor microenvironment, especially for bone marrow mesenchymal stem cells (MSC), in the maintenance and progression of multiple myeloma (MM), through direct and indirect interactions with tumor plasma cells. Thus, this study aimed to investigate the gene expression and functional alterations of MSC from MM patients (MM-MSC) in comparison with their normal counterparts from normal donors (ND-MSC). Gene expression analysis (Affymetrix) was performed in MM-MSC and ND-MSC after in vitro expansion. To validate these findings, some genes were selected to be evaluated by quantitative real time PCR (RT-qPCR), and also functional in vitro analyses were performed. We demonstrated that MM-MSC have a distinct gene expression profile than ND-MSC, with 485 differentially expressed genes (DEG) - 280 upregulated and 205 downregulated. Bioinformatics analyses revealed that the main enriched functions among downregulated DEG were related to cell cycle progression, immune response activation and bone metabolism. Four genes were validated by qPCR - ZNF521 and SEMA3A, which are involved in bone metabolism, and HLA-DRA and CHIRL1, which are implicated in the activation of immune response. Taken together, our results suggest that MM-MSC have constitutive abnormalities that remain present even in the absence of tumors cells. The alterations found in cell cycle progression, immune system activation, and osteoblastogenesis suggest, respectively, that MM-MSC are permanently dependent of tumor cells, might contribute to immune evasion and play an essential role in bone lesions frequently found in MM patients.


Assuntos
Osso e Ossos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Mieloma Múltiplo/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/metabolismo , Divisão Celular/genética , Divisão Celular/fisiologia , Feminino , Perfilação da Expressão Gênica/métodos , Cadeias alfa de HLA-DR/genética , Cadeias alfa de HLA-DR/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Microambiente Tumoral/genética , Microambiente Tumoral/fisiologia
13.
Dis Model Mech ; 11(1)2018 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-29196444

RESUMO

Complex febrile seizures during infancy constitute an important risk factor for development of epilepsy. However, little is known about the alterations induced by febrile seizures that make the brain susceptible to epileptic activity. In this context, the use of animal models of hyperthermic seizures (HS) could allow the temporal analysis of brain molecular changes that arise after febrile seizures. Here, we investigated temporal changes in hippocampal gene coexpression networks during the development of rats submitted to HS. Total RNA samples were obtained from the ventral hippocampal CA3 region at four time points after HS at postnatal day (P) 11 and later used for gene expression profiling. Temporal endpoints were selected for investigating the acute (P12), latent (P30 and P60) and chronic (P120) stages of the HS model. A weighted gene coexpression network analysis was used to characterize modules of coexpressed genes, as these modules might contain genes with similar functions. The transcriptome analysis pipeline consisted of building gene coexpression networks, identifying network modules and hubs, performing gene-trait correlations and examining changes in module connectivity. Modules were functionally enriched to identify functions associated with HS. Our data showed that HS induce changes in developmental, cell adhesion and immune pathways, such as Wnt, Hippo, Notch, Jak-Stat and Mapk. Interestingly, modules involved in cell adhesion, neuronal differentiation and synaptic transmission were activated as early as 1 day after HS. These results suggest that HS trigger transcriptional alterations that could lead to persistent neurogenesis, tissue remodeling and inflammation in the CA3 hippocampus, making the brain prone to epileptic activity.


Assuntos
Região CA3 Hipocampal/metabolismo , Redes Reguladoras de Genes , Convulsões Febris/genética , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Hipertermia Induzida , Ratos Wistar , Fatores de Tempo
14.
Sci Rep ; 7(1): 7501, 2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28790404

RESUMO

The human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2.


Assuntos
Antineoplásicos/química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pteridinas/química , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Genoma Humano , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pteridinas/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Vírus Vaccinia/genética , Vírus Vaccinia/metabolismo
15.
Sci Rep ; 7(1): 3192, 2017 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-28600543

RESUMO

The mechanisms triggering renal inflammation in chronic kidney disease (CKD) are unclear. We performed a detailed analysis of the time course of innate and adaptive immunity activation in the 5/6 renal ablation (Nx) model. Munich-Wistar rats undergoing Nx were studied 15, 60 and 120 days after ablation. Hypertension, albuminuria, creatinine retention, interstitial expansion and infiltration by macrophages and T-lymphocytes were already evident 15 days after Nx. PCR-array was used to screen for altered gene expression, whereas gene and protein expressions of TLR4, CASP1, IL-1ß and NLRP3 were individually assessed. Tlr4, Tlr5, Lbp, Nlrp3, Casp1, Irf7 and Il1b were already upregulated 15 days after Nx, while activation of Tlr2, Tlr7, Tlr9, Nod2, Tnf and Il6 was seen after 60 days post-ablation. The number of genes related to innate or adaptive immunity grew steadily with time. These observations indicate that parallel activation of innate and adaptive immunity antecedes glomerular injury and involves a growing number of intricate signaling pathways, helping to explain the difficulty in detaining renal injury in Nx as CKD advances, and, stressing the need for early treatment. Additionally, these findings may contribute to the search of therapeutic targets specific for advanced phases of CKD.


Assuntos
Lesão Renal Aguda/genética , Imunidade Adaptativa/genética , Hipertensão/imunologia , Imunidade Inata/genética , Lesão Renal Aguda/imunologia , Lesão Renal Aguda/patologia , Animais , Ablação por Cateter/efeitos adversos , Creatinina/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/genética , Rim/imunologia , Rim/lesões , Rim/cirurgia , Losartan/farmacologia , Macrófagos/imunologia , Macrófagos/patologia , Nefrectomia/efeitos adversos , Ratos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
16.
Clin Endocrinol (Oxf) ; 86(5): 688-697, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28177124

RESUMO

OBJECTIVE: This study aimed to explore the role of acute exercise on skeletal muscle gene expression related to insulin resistance in patients with polycystic ovary syndrome (PCOS) and controls. METHODS: Four obese women with PCOS and four body mass index (BMI)-matched controls (CTRL) participated in this study. After an overnight fast, the subjects underwent a single 40-min bout of aerobic exercise. Muscle samples were obtained from vastus lateralis at baseline and 60 min after exercise. The expression of a panel of insulin resistance genes was evaluated by a quantitative PCR array system. Network-based analyses were performed to interpret transcriptional changes occurring before and after the exercise challenge. RESULTS: Overall, differentially expressed genes associated with mitochondria function and peroxisome proliferator-activated receptor signalling were identified. At baseline, there was a significant upregulation of six genes exclusively in PCOS (i.e. NFKBIA, MAPK3, PPARGC1A, GAPDH, ACTB and PPARA). Twelve genes were upregulated in CTRL after a single bout of aerobic exercise (i.e. LEPR, CXCR4, CCR5, IL-18R1, CRLF2, ACACA, CEBPA, PPARGC1A, UCP1, TNFRSF1B, TLR4 and IKBKB). After the exercise session, three genes were upregulated in PCOS (i.e. SOCS3, NAMPT and IL-8), whilst IL-6 was upregulated in both groups after exercise. CONCLUSIONS: This study provides novel evidence on the effects of acute exercise on insulin resistance genes in skeletal muscle of PCOS. The differentially expressed genes reported herein could be further investigated as targets for therapeutic interventions aimed at improving insulin resistance in this syndrome.


Assuntos
Terapia por Exercício/métodos , Expressão Gênica/genética , Resistência à Insulina/genética , Músculo Esquelético/metabolismo , Obesidade/terapia , Síndrome do Ovário Policístico/terapia , Adulto , Feminino , Humanos , Adulto Jovem
17.
Front. Immunol. ; 7: 645, 2017.
Artigo | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: but-ib13595

RESUMO

Mesenchymal stromal cells (MSCs) orchestrate tissue repair by releasing cell-derived microvesicles (MVs), which, presumably by small RNA species, modulate global gene expression. The knowledge of miRNA/mRNA signatures linked to a reparative status may elucidate some of the molecular events associated with MSC protection. Here, we used a model of cisplatin-induced kidney injury (acute kidney injury) to assess how MSCs or MVs could restore tissue function. MSCs and MVs presented similar protective effects, which were evidenced in vivo and in vitro by modulating apoptosis, inflammation, oxidative stress, and a set of prosurvival molecules. In addition, we observed that miRNAs (i.e., miR-880, miR-141, miR-377, and miR-21) were modulated, thereby showing active participation on regenerative process. Subsequently, we identified that MSC regulates a particular miRNA subset which mRNA targets are associated with Wnt/TGF-beta fibrosis, and epithelial-mesenchymal transition signaling pathways. Our results suggest that MSCs release MVs that transcriptionally reprogram injured cells, thereby modulating a specific miRNA-mRNA network.

18.
Immun Inflamm Dis ; 4(3): 300-14, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27621813

RESUMO

INTRODUCTION: Macrophages are heterogeneous and thus can be correlated with distinct tissue outcomes after injury. Conflicting data have indicated that the M2-related phenotype directly triggers fibrosis. Conversely, we hypothesize here that the inflammatory milieu provided by early infiltration of pro-inflammatory macrophages dictates tissue scarring after injury. METHODS AND RESULTS: We first determined that tissue-localized macrophages exhibit a pro-inflammatory phenotype (p40IL12(+)CCR7(+)CD11b(+)) during the early phase of a chronic injury model, in contrast to a pro-resolving phenotype (Arg1(+)IL10(+)CD206(+)CD11b(+)) at a later stage. Then, we evaluated the effects of injecting macrophages differentiated in vitro in the presence of IFNγ + LPS or IL4 + IL13 or non-differentiated macrophages (hereafter, M0) on promoting inflammation and progression of chronic injury in macrophage-depleted mice. In addition to enhancing the expression of pro-inflammatory cytokines, the injection of M (IFNγ + LPS), but not M (IL4 + IL13) or M0, accentuated fibrosis while augmenting levels of anti-inflammatory molecules, increasing collagen deposition and impairing organ function. We observed a similar profile after injection of sorted CCR7(+)CD11b(+) cells and a more pronounced effect of M (IFNγ + LPS) cells originated from Stat6(-/-) mice. The injection of M (IFNγ + LPS) cells was associated with the up-regulation of inflammation- and fibrosis-related proteins (Thbs1, Mmp7, Mmp8, and Mmp13). CONCLUSIONS: Our results suggest that pro-inflammatory macrophages promote microenvironmental changes that may lead to fibrogenesis by inducing an inflammatory milieu that alters a network of extracellular-related genes, culminating in tissue fibrosis.

19.
Hum Genomics ; 10: 2, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26742487

RESUMO

BACKGROUND: Proximal tubular dysfunction (PTD) is associated with a decreased long-term graft survival in renal transplant patients and can be detected by the elevation of urinary tubular proteins. This study investigated transcriptional changes in biopsies from renal transplant patients with PTD to disclose molecular mechanisms underlying graft injury and functional recovery. METHODS: Thirty-three renal transplant patients with high urinary levels of retinol-binding protein, a biomarker of PTD, were enrolled in the study. The initial immunosuppressive scheme included azathioprine, cyclosporine, and steroids. After randomization, 18 patients (group 2) had their treatment modified by reducing cyclosporine dosage and substituting azathioprine for mycophenolate mofetil, while the other 15 patients (group 1) remained under the initial scheme. Patients were biopsied at enrollment and after 12 months of follow-up, and paired comparisons were performed between their intragraft gene expression profiles. The differential transcriptome profiles were analyzed by constructing gene co-expression networks and identifying enriched functions and central nodes in each network. RESULTS: Only the alternative immunosuppressive scheme used in group 2 ameliorated renal function and tubular proteinuria after 12 months of follow-up. Intragraft molecular changes observed in group 2 were linked to autophagy, extracellular matrix, and adaptive immunity. Conversely, gene expression changes in group 1 were related to fibrosis, endocytosis, ubiquitination, and endoplasmic reticulum stress. CONCLUSION: These results suggest that molecular networks associated with the control of endocytosis, autophagy, protein overload, fibrosis, and adaptive immunity may be involved in improvement of graft function.


Assuntos
Síndrome de Fanconi/tratamento farmacológico , Síndrome de Fanconi/genética , Imunossupressão/métodos , Transplante de Rim/efeitos adversos , Transcriptoma/genética , Adulto , Idoso , Azatioprina/administração & dosagem , Ciclosporina/administração & dosagem , Síndrome de Fanconi/imunologia , Síndrome de Fanconi/urina , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genômica , Sobrevivência de Enxerto/efeitos dos fármacos , Sobrevivência de Enxerto/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Distribuição Aleatória , Proteínas Celulares de Ligação ao Retinol/urina , Esteroides/administração & dosagem
20.
Front Immunol ; 7: 645, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28096802

RESUMO

Mesenchymal stromal cells (MSCs) orchestrate tissue repair by releasing cell-derived microvesicles (MVs), which, presumably by small RNA species, modulate global gene expression. The knowledge of miRNA/mRNA signatures linked to a reparative status may elucidate some of the molecular events associated with MSC protection. Here, we used a model of cisplatin-induced kidney injury (acute kidney injury) to assess how MSCs or MVs could restore tissue function. MSCs and MVs presented similar protective effects, which were evidenced in vivo and in vitro by modulating apoptosis, inflammation, oxidative stress, and a set of prosurvival molecules. In addition, we observed that miRNAs (i.e., miR-880, miR-141, miR-377, and miR-21) were modulated, thereby showing active participation on regenerative process. Subsequently, we identified that MSC regulates a particular miRNA subset which mRNA targets are associated with Wnt/TGF-ß, fibrosis, and epithelial-mesenchymal transition signaling pathways. Our results suggest that MSCs release MVs that transcriptionally reprogram injured cells, thereby modulating a specific miRNA-mRNA network.

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