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1.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445223

RESUMO

Increasing evidence suggests that systemic inflammation triggers a neuroinflammatory response that involves sustained microglia activation. This response has deleterious consequences on memory and learning capability in experimental animal models and in patients. However, the mechanisms connecting systemic inflammation and microglia activation remain poorly understood. Here, we identify the autotaxin (ATX)/lysophosphatidic acid (LPA)/LPA-receptor axis as a potential pharmacological target to modulate the LPS-mediated neuroinflammatory response in vitro (the murine BV-2 microglia cell line) and in vivo (C57BL/6J mice receiving a single i.p. LPS injection). In LPS-stimulated (20 ng/mL) BV-2 cells, we observed increased phosphorylation of transcription factors (STAT1, p65, and c-Jun) that are known to induce a proinflammatory microglia phenotype. LPS upregulated ATX, TLR4, and COX2 expression, amplified NO production, increased neurotoxicity of microglia conditioned medium, and augmented cyto-/chemokine concentrations in the cellular supernatants. PF8380 (a type I ATX inhibitor, used at 10 and 1 µM) and AS2717638 (an LPA5 antagonist, used at 1 and 0.1 µM) attenuated these proinflammatory responses, at non-toxic concentrations, in BV-2 cells. In vivo, we demonstrate accumulation of PF8380 in the mouse brain and an accompanying decrease in LPA concentrations. In vivo, co-injection of LPS (5 mg/kg body weight) and PF8380 (30 mg/kg body weight), or LPS/AS2717638 (10 mg/kg body weight), significantly attenuated LPS-induced iNOS, TNFα, IL-1ß, IL-6, and CXCL2 mRNA expression in the mouse brain. On the protein level, PF8380 and AS2717638 significantly reduced TLR4, Iba1, GFAP and COX2 expression, as compared to LPS-only injected animals. In terms of the communication between systemic inflammation and neuroinflammation, both inhibitors significantly attenuated LPS-mediated systemic TNFα and IL-6 synthesis, while IL-1ß was only reduced by PF8380. Inhibition of ATX and LPA5 may thus provide an opportunity to protect the brain from the toxic effects that are provoked by systemic endotoxemia.


Assuntos
Benzoxazóis/farmacologia , Encéfalo/metabolismo , Endotoxemia , Isoquinolinas/farmacologia , Lipopolissacarídeos/toxicidade , Microglia/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Piperazinas/farmacologia , Piperidinas/farmacologia , Receptores de Ácidos Lisofosfatídicos , Animais , Encéfalo/patologia , Linhagem Celular , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , Endotoxemia/patologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Microglia/patologia , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/metabolismo
2.
Gene ; 800: 145842, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34274479

RESUMO

Treatment of serum-starved quiescent human cells with fetal bovine serum (FBS), epidermal growth factor (EGF), or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) activates the RAS-MAPK pathway which initiates a transcriptional program which drives cells toward proliferation. Stimulation of the RAS-MAPK pathway activates mitogen- and stress-activated kinases (MSK) 1 and 2, which phosphorylate histone H3 at S10 (H3S10ph) or S28 (H3S28ph) (nucleosomal response) located at the regulatory regions of immediate-early genes, setting in motion a series of chromatin remodeling events that result in transcription initiation. To investigate immediate-early genes regulated by the MSK, we have completed transcriptome analyses (RNA sequencing) of human normal fibroblast cells (CCD-1070Sk) stimulated with EGF or TPA ± H89, a potent MSK/PKA inhibitor. The induction of many immediate-early genes was independent of MSK activity. However, the induction of immediate-early genes attenuated with H89 also had reduced induction with the PKA inhibitor, Rp-cAMPS. Several EGF-induced genes, coding for transcriptional repressors, were further upregulated with H89 but not with Rp-cAMPS, suggesting a role for MSK in modulating the induction level of these genes.


Assuntos
Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Mitógenos/farmacologia , Linhagem Celular , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Fibroblastos/fisiologia , Perfilação da Expressão Gênica , Genes Precoces/efeitos dos fármacos , Humanos , Isoquinolinas/farmacologia , Reprodutibilidade dos Testes , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Sulfonamidas/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Tionucleotídeos/farmacologia
3.
Molecules ; 26(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207059

RESUMO

Diclinanona calycina R. E. Fries popularly known as "envira", is a species of the Annonaceae family endemic to Brazil. In our ongoing search for bioactive compounds from Annonaceae Amazon plants, the bark of D. calycina was investigated by classical chromatography techniques that yielded thirteen compounds (alkaloids and flavonoids) described for the first time in D. calycina as well as in the genus Diclinanona. The structure of these isolated compounds were established by extensive analysis using 1D/2D-NMR spectroscopy in combination with MS. The isolated alkaloids were identified as belonging to the subclasses: simple isoquinoline, thalifoline (1); aporphine, anonaine (2); oxoaporphine, liriodenine (3); benzyltetrahydroisoquinolines, (S)-(+)-reticuline (4); dehydro-oxonorreticuline (3,4-dihydro-7-hydroxy-6-methoxy-1-isoquinolinyl)(3-hydroxy-4-methoxyphenyl)-methanone) (5); (+)-1S,2R-reticuline Nß-oxide (6); and (+)-1S,2S-reticuline Nα-oxide (7); tetrahydroprotoberberine, coreximine (8); and pavine, bisnorargemonine (9). While the flavonoids belong to the benzylated dihydroflavones, isochamanetin (10), dichamanetin (11), and a mixture of uvarinol (12) and isouvarinol (13). Compound 5 is described for the first time in the literature as a natural product. The cytotoxic activity of the main isolated compounds was evaluated against cancer and non-cancerous cell lines. Among the tested compounds, the most promising results were found for the benzylated dihydroflavones dichamanetin (10), and the mixture of uvarinol (12) and isouvarinol (13), which presented moderate cytotoxic activity against the tested cancer cell lines (<20.0 µg·mL-1) and low cytotoxicity against the non-cancerous cell line MRC-5 (>25.0 µg·mL-1). Dichamanetin (11) showed cytotoxic activity against HL-60 and HCT116 with IC50 values of 15.78 µg·mL-1 (33.70 µmol·L-1) and 18.99 µg·mL-1 (40.56 µmol·L-1), respectively while the mixture of uvarinol (12) and isouvarinol (13) demonstrated cytotoxic activity against HL-60, with an IC50 value of 9.74 µg·mL-1, and HCT116, with an IC50 value of 17.31 µg·mL-1. These cytotoxic activities can be attributed to the presence of one or more hydroxybenzyl groups present in these molecules as well as the position in which these groups are linked. The cytotoxic activities of reticuline, anonaine and liriodenine have been previously established, with liriodenine being the most potent compound.


Assuntos
Alcaloides/química , Annonaceae/química , Flavonas/química , Isoquinolinas/química , Casca de Planta/química , Alcaloides/farmacologia , Aporfinas/química , Aporfinas/farmacologia , Brasil , Linhagem Celular Tumoral , Dioxóis/química , Dioxóis/farmacologia , Flavanonas/farmacologia , Flavonas/farmacologia , Células HCT116 , Células HL-60 , Células Hep G2 , Humanos , Isoquinolinas/farmacologia , Células MCF-7 , Extratos Vegetais , Folhas de Planta/química
4.
Phytochemistry ; 189: 112828, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34174637

RESUMO

Simple isoquinoline alkaloids (heliamine, dehydroheliamine), a phthalide isoquinoline alkaloid noscapine, and an aporphine alkaloid boldine are biosynthetically derived from an amino acid tyrosine. These substrates and a simple synthetic isoquinoline alkaloid (2-acetyl-7-amino-1,2,3,4-tetrahydroisoquinoline) contain an isoquinoline nucleus. The biotransformation of these substrates via reduction, oxidation, hydroxylation, and N-oxidation reactions with different microorganism produced nine metabolites, namely: N-(2-acetyl-1,2,3,4-tetrahydroisoquinolin-7-yl) acetamide (Metabolite 1), heliamine N-oxide (Metabolite 2), 6,7-dimethoxyisoquinoline (Metabolite 3), 3,4-dihydro-6,7-dimethoxy isoquinolin-1-one (Metabolite 4), heliamine (Metabolite 5), dehydroheliamine N-oxide (Metabolite 6), cotarnine (Metabolite 7), 5-hydroxy cotarnine (Metabolite 8), and boldine N-oxide (Metabolite 9). Primarily, the metabolites are structurally elucidated by one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) analyses, and high-resolution electrospray ionization mass spectrometry (HR-ESIMS). Furthermore, the substrates and their isolated metabolites are evaluated in vitro for their anti-inflammatory, antimicrobial, cytotoxicity, and anticancer activities. The in vitro studies reveal that some of the isolated compounds are potential as anti-inflammatory, antitumor, and antimicrobial leads.


Assuntos
Alcaloides , Benzilisoquinolinas , Alcaloides/farmacologia , Benzilisoquinolinas/farmacologia , Isoquinolinas/farmacologia , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray
5.
Mol Cell Biol ; 41(9): e0008521, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34124936

RESUMO

Immune health requires innate and adaptive immune cells to engage precisely balanced pro- and anti-inflammatory forces. We employ the concept of chemical immunophenotypes to classify small molecules functionally or mechanistically according to their patterns of effects on primary innate and adaptive immune cells. The high-specificity, low-toxicity cyclin-dependent kinase 8 (CDK8) inhibitor 16-didehydro-cortistatin A (DCA) exerts a distinct tolerogenic profile in both innate and adaptive immune cells. DCA promotes regulatory T cells (Treg) and Th2 differentiation while inhibiting Th1 and Th17 differentiation in both murine and human cells. This unique chemical immunophenotype led to mechanistic studies showing that DCA promotes Treg differentiation in part by regulating a previously undescribed CDK8-GATA3-FOXP3 pathway that regulates early pathways of Foxp3 expression. These results highlight previously unappreciated links between Treg and Th2 differentiation and extend our understanding of the transcription factors that regulate Treg differentiation and their temporal sequencing. These findings have significant implications for future mechanistic and translational studies of CDK8 and CDK8 inhibitors.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Fatores de Transcrição Forkhead/metabolismo , Fator de Transcrição GATA3/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Imunofenotipagem , Isoquinolinas/farmacologia , Adolescente , Adulto , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Quinase 8 Dependente de Ciclina/metabolismo , Humanos , Imunidade Inata/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adulto Jovem
6.
J Leukoc Biol ; 110(3): 475-484, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34184309

RESUMO

Neutrophil plays a critical role in the progression of periodontitis. In general, its chemotaxis and activation are benefit for the host defense of bacterial infection and inflammation. However, previous studies have reported that the hyperactive and reactive neutrophils appear to be one of the reasons for tissue destruction in periodontitis tissues. In this study, we investigated an isoquinoline alkaloid Litcubanine A (LA), which from the Traditional Chinese medicinal plant, Litsea cubeba. We found LA showed significant activity in inhibiting neutrophils chemotaxis in the zebrafish yolk sac microinjection model in vivo and in mouse neutrophils in vitro. Further investigation proved that LA could inhibit the expression levels of neutrophil respiratory burst-related and inflammation-related genes CYBB and NCF2, as well as inhibit the activation of MAPK signaling pathway. Moreover, using LA, we successfully achieved the effect of reducing periodontitis bone loss by regulating neutrophil chemotaxis and related functions in a mouse ligature-induced periodontitis model.


Assuntos
Alcaloides/uso terapêutico , Quimiotaxia , Isoquinolinas/uso terapêutico , Neutrófilos/patologia , Periodontite/tratamento farmacológico , Alcaloides/farmacologia , Animais , Reabsorção Óssea/patologia , Quimiotaxia/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-8/metabolismo , Isoquinolinas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Microinjeções , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Periodontite/diagnóstico por imagem , Periodontite/patologia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Explosão Respiratória/efeitos dos fármacos , Saco Vitelino/efeitos dos fármacos , Saco Vitelino/metabolismo , Peixe-Zebra
7.
J Med Virol ; 93(10): 5825-5832, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34061377

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has focused attention on the need to develop effective therapeutics against the causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and also against other pathogenic coronaviruses. In this study, we report on a kind of bisbenzylisoquinoline alkaloid, neferine, as a pan-coronavirus entry inhibitor. Neferine effectively protected HEK293/hACE2 and HuH7 cell lines from infection by different coronaviruses pseudovirus particles (SARS-CoV-2, SARS-CoV-2 [D614G, N501Y/D614G, 501Y.V1, 501Y.V2, 501Y.V3 variants], SARS-CoV, MERS-CoV) in vitro, with median effect concentration (EC50 ) of 0.13-0.41 µM. Neferine blocked host calcium channels, thus inhibiting Ca2+ -dependent membrane fusion and suppressing virus entry. This study provides experimental data to support the fact that neferine may be a promising lead for pan-coronaviruses therapeutic drug development.


Assuntos
Antivirais/farmacologia , Benzilisoquinolinas/farmacologia , Cálcio/metabolismo , SARS-CoV-2/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , COVID-19/virologia , Linhagem Celular , Coronavirus/efeitos dos fármacos , Coronavirus/fisiologia , Células HEK293 , Humanos , Isoquinolinas/farmacologia , Fenóis/farmacologia , SARS-CoV-2/fisiologia
8.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069280

RESUMO

The use of human dental pulp stromal cells (hDPSCs) has gained increasing attention as an alternative stem cell source for bone tissue engineering. The modification of the cells' epigenetics has been found to play an important role in regulating differentiation, with the inhibition of histone deacetylases 3 (HDAC3) being linked to increased osteogenic differentiation. This study aimed to induce epigenetic reprogramming using the HDAC2 and 3 selective inhibitor, MI192 to promote hDPSCs osteogenic capacity for bone regeneration. MI192 treatment caused a time-dose-dependent change in hDPSC morphology and reduction in viability. Additionally, MI192 successfully augmented hDPSC epigenetic functionality, which resulted in increased histone acetylation and cell cycle arrest at the G2/M phase. MI192 pre-treatment exhibited a dose-dependent effect on hDPSCs alkaline phosphatase activity. Quantitative PCR and In-Cell Western further demonstrated that MI192 pre-treatment significantly upregulated hDPSCs osteoblast-related gene and protein expression (alkaline phosphatase, bone morphogenic protein 2, type I collagen and osteocalcin) during osteogenic differentiation. Importantly, MI192 pre-treatment significantly increased hDPSCs extracellular matrix collagen production and mineralisation. As such, for the first time, our findings show that epigenetic reprogramming with the HDAC2 and 3 selective inhibitor MI192 accelerates the osteogenic differentiation of hDPSCs, demonstrating the considerable utility of this MSCs engineering approach for bone augmentation strategies.


Assuntos
Benzamidas/farmacologia , Polpa Dentária/citologia , Inibidores de Histona Desacetilases/farmacologia , Isoquinolinas/farmacologia , Osteogênese/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Benzamidas/administração & dosagem , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/administração & dosagem , Histonas/metabolismo , Humanos , Isoquinolinas/administração & dosagem , Dente Serotino/citologia , Osteogênese/fisiologia , Células Estromais/metabolismo
9.
J Virol ; 95(15): e0242520, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980597

RESUMO

HIV persists, despite immune responses and antiretroviral therapy, in viral reservoirs that seed rebound viremia if therapy is interrupted. Previously, we showed that the BCL-2 protein contributes to HIV persistence by conferring a survival advantage to reservoir-harboring cells. Here, we demonstrate that many of the BCL-2 family members are overexpressed in HIV-infected CD4+ T cells, indicating increased tension between proapoptotic and prosurvival family members-and suggesting that inhibition of prosurvival members may disproportionately affect the survival of HIV-infected cells. Based on these results, we chose to study BCL-XL due to its consistent overexpression and the availability of selective antagonists. Infection of primary CD4+ T cells with HIV resulted in increased BCL-XL protein expression, and treatment with two selective BCL-XL antagonists, A-1155463 and A-1551852, led to selective death of productively infected CD4+ T cells. In a primary cell model of latency, both BCL-XL antagonists drove reductions in HIV DNA and in infectious cell frequencies both alone and in combination with the latency reversing agent bryostatin-1, with little off-target cytotoxicity. However, these antagonists, with or without bryostatin-1 or in combination with the highly potent latency reversing agent combination phorbol myristate acetate (PMA) + ionomycin, failed to reduce total HIV DNA and infectious reservoirs in ex vivo CD4+ T cells from antiretroviral therapy (ART)-suppressed donors. Our results add to growing evidence that bona fide reservoir-harboring cells are resistant to multiple "kick and kill" modalities-relative to latency models. We also interpret our results as encouraging further exploration of BCL-XL antagonists for cure, where combination approaches, including with immune effectors, may unlock the ability to eliminate ex vivo reservoirs. IMPORTANCE Although antiretroviral therapy (ART) has transformed HIV infection into a manageable chronic condition, there is no safe or scalable cure. HIV persists in "reservoirs" of infected cells that reinitiate disease progression if ART is interrupted. Whereas most efforts to eliminate this reservoir have focused on exposing these cells to immune-mediated clearance by reversing viral latency, recent work shows that these cells also resist being killed. Here, we identify a "prosurvival" factor, BCL-XL, that is overexpressed in HIV-infected cells, and demonstrate selective toxicity to these cells by BCL-XL antagonists. These antagonists also reduced reservoirs in a primary-cell latency model but were insufficient to reduce "natural" reservoirs in ex vivo CD4+ T cells-adding to growing evidence that the latter are resilient in a way that is not reflected in models. We nonetheless suggest that the selective toxicity of BCL-XL antagonists to HIV-infected cells supports their prioritization for testing in combinations aimed at reducing ex vivo reservoirs.


Assuntos
Benzotiazóis/farmacologia , Briostatinas/farmacologia , Reservatórios de Doenças/virologia , Isoquinolinas/farmacologia , Latência Viral/efeitos dos fármacos , Proteína bcl-X/antagonistas & inibidores , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Infecções por HIV/prevenção & controle , HIV-1/crescimento & desenvolvimento , Humanos , Replicação Viral/efeitos dos fármacos , Proteína bcl-X/metabolismo
10.
Front Immunol ; 12: 633586, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34054802

RESUMO

Myeloid cell interactions with cells of the adaptive immune system are an essential aspect of immunity. A key aspect of that interrelationship is its modulation by the microenvironment. Oxygen is known to influence myelosuppression of T cell activation in part via the Hypoxia inducible (HIF) transcription factors. A number of drugs that act on the HIF pathway are currently in clinical use and it is important to evaluate how they act on immune cell function as part of a better understanding of how they will influence patient outcomes. We show here that increased activation of the HIF pathway, either through deletion of the negative regulator of HIF, the von Hippel-Lindau (VHL) gene, in myeloid cells, or through pharmacological inhibitors of VHL-mediated degradation of HIF, potently suppresses T cell proliferation in myeloid cell/T cell culture. These data demonstrate that both pharmacological and genetic activation of HIF in myeloid cells can suppress adaptive cell immune response.


Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Glicina/análogos & derivados , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isoquinolinas/farmacologia , Macrófagos/efeitos dos fármacos , Oxigênio/metabolismo , Imunidade Adaptativa , Animais , Linfócitos T CD8-Positivos/fisiologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Técnicas de Cocultura , Glicina/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Camundongos , Camundongos Transgênicos , Oxigênio/farmacologia , Proteína Supressora de Tumor Von Hippel-Lindau/genética
11.
Int J Mol Sci ; 22(8)2021 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-33920737

RESUMO

Alcoholism is a chronically relapsing disorder characterized by high alcohol intake and a negative emotional state during abstinence, which contributes to excessive drinking and susceptibility to relapse. Stress, dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and alterations in glucocorticoid receptor (GR) function have been linked to transition from recreational consumption to alcohol use disorder (AUD). Here, we investigated the effect of pharmacological antagonisms of GR on alcohol self-administration (SA) using male and female Wistar and Marchigian Sardinian alcohol-preferring (msP) rats, a rodent line genetically selected for excessive alcohol drinking and highly sensitive to stress. Animals were trained to self-administer 10% (v/v) alcohol. Once a stable alcohol SA baseline was reached, we tested the effect of the GR antagonists mifepristone (0.0, 10, 30 and 60 mg/kg; i.p.) and CORT113176 (0.0, 10, 30 and 60 mg/kg) on alcohol SA. To evaluate whether the effects of the two compounds were specific for alcohol, the two drugs were tested on a similar saccharin SA regimen. Finally, basal blood corticosterone (CORT) levels before and after alcohol SA were determined. Systemic injection with mifepristone dose-dependently reduced alcohol SA in male and female Wistars but not in msPs. Administration of CORT113176 decreased alcohol SA in male and female Wistars as well as in female msPs but not in male msP rats. At the highest dose, mifepristone also reduced saccharin SA in male Wistars and female msPs, suggesting the occurrence of some nonspecific effects at 60 mg/kg of the drug. Similarly, the highest dose of CORT113176 (60 mg/kg) decreased saccharin intake in male Wistars. Analysis of CORT levels revealed that females of both rat lines had higher blood levels of CORT compared to males. Alcohol consumption reduced CORT in females but not in males. Overall, these findings indicate that selective blockade of GR selectively reduces alcohol SA, and genetically selected msP rats are less sensitive to this pharmacological manipulation compared to heterogeneous Wistars. Moreover, results suggest sex differences in response to GR antagonism and the ability of alcohol to regulate GR transmission.


Assuntos
Alcoolismo/tratamento farmacológico , Antagonistas de Hormônios/uso terapêutico , Receptores de Glucocorticoides/antagonistas & inibidores , Alcoolismo/genética , Animais , Feminino , Antagonistas de Hormônios/farmacologia , Isoquinolinas/farmacologia , Isoquinolinas/uso terapêutico , Masculino , Mifepristona/farmacologia , Mifepristona/uso terapêutico , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Ratos , Ratos Wistar
12.
Eur J Med Chem ; 220: 113458, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33901901

RESUMO

The development of new molecules for the treatment of leishmaniasis is, a neglected parasitic disease, is urgent as current anti-leishmanial therapeutics are hampered by drug toxicity and resistance. The pyrrolo[1,2-b]isoquinoline core was selected as starting point, and palladium-catalyzed Heck-initiated cascade reactions were developed for the synthesis of a series of C-10 substituted derivatives. Their in vitro leishmanicidal activity against visceral (L. donovani) and cutaneous (L. amazonensis) leishmaniasis was evaluated. The best activity was found, in general, for the 10-arylmethyl substituted pyrroloisoquinolines. In particular, 2ad (IC50 = 3.30 µM, SI > 77.01) and 2bb (IC50 = 3.93 µM, SI > 58.77) were approximately 10-fold more potent and selective than the drug of reference (miltefosine), against L. amazonensis on in vitro promastigote assays, while 2ae was the more active compound in the in vitro amastigote assays (IC50 = 33.59 µM, SI > 8.93). Notably, almost all compounds showed low cytotoxicity, CC50 > 100 µg/mL in J774 cells, highest tested dose. In addition, we have developed the first Perturbation Theory Machine Learning (PTML) algorithm able to predict simultaneously multiple biological activity parameters (IC50, Ki, etc.) vs. any Leishmania species and target protein, with high values of specificity (>98%) and sensitivity (>90%) in both training and validation series. Therefore, this model may be useful to reduce time and assay costs (material and human resources) in the drug discovery process.


Assuntos
Antiprotozoários/farmacologia , Isoquinolinas/farmacologia , Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Paládio/química , Algoritmos , Antiprotozoários/síntese química , Antiprotozoários/química , Relação Dose-Resposta a Droga , Isoquinolinas/síntese química , Isoquinolinas/química , Leishmaniose/parasitologia , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade
13.
Infect Immun ; 89(7): e0072920, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-33820812

RESUMO

Chlamydia is an obligate intracellular bacterium and the most common reportable cause of human infection in the United States. This pathogen proliferates inside a eukaryotic host cell, where it resides within a membrane-bound compartment called the chlamydial inclusion. It has an unusual developmental cycle, marked by conversion between a replicating form, the reticulate body (RB), and an infectious form, the elementary body (EB). We found that the small molecule H89 slowed inclusion growth and decreased overall RB replication by 2-fold but caused a 25-fold reduction in infectious EBs. This disproportionate effect on EB production was mainly due to a defect in RB-to-EB conversion and not to the induction of chlamydial persistence, which is an altered growth state. Although H89 is a known inhibitor of specific protein kinases and vesicular transport to and from the Golgi apparatus, it did not cause these anti-chlamydial effects by blocking protein kinase A or C or by inhibiting protein or lipid transport. Thus, H89 is a novel anti-chlamydial compound that has a unique combination of effects on an intracellular Chlamydia infection.


Assuntos
Antibacterianos/farmacologia , Infecções por Chlamydia/microbiologia , Chlamydia/efeitos dos fármacos , Isoquinolinas/farmacologia , Sulfonamidas/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Chlamydia/tratamento farmacológico , Relação Dose-Resposta a Droga , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos
14.
Mol Cell ; 81(9): 2041-2052.e6, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33823141

RESUMO

Cellular senescence is a state of stable proliferative arrest triggered by damaging signals. Senescent cells persist during aging and promote age-related pathologies via the pro-inflammatory senescence-associated secretory phenotype (SASP), whose regulation depends on environmental factors. In vivo, a major environmental variable is oxygenation, which varies among and within tissues. Here, we demonstrate that senescent cells express lower levels of detrimental pro-inflammatory SASP factors in physiologically hypoxic environments, as measured in culture and in tissues. Mechanistically, exposure of senescent cells to low-oxygen conditions leads to AMPK activation and AMPK-mediated suppression of the mTOR-NF-κB signaling loop. Finally, we demonstrate that treatment with hypoxia-mimetic compounds reduces SASP in cells and tissues and improves strength in chemotherapy-treated and aged mice. Our findings highlight the importance of oxygen as a determinant for pro-inflammatory SASP expression and offer a potential new strategy to reduce detrimental paracrine effects of senescent cells.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proliferação de Células , Senescência Celular , Hipóxia/enzimologia , Serina-Treonina Quinases TOR/metabolismo , Fatores Etários , Animais , Antibióticos Antineoplásicos/farmacologia , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Hidroxibenzoatos/farmacologia , Hipóxia/patologia , Hipóxia/fisiopatologia , Mediadores da Inflamação/metabolismo , Isoquinolinas/farmacologia , Camundongos Endogâmicos C57BL , Força Muscular , NF-kappa B/metabolismo , Comunicação Parácrina , Fenótipo , Transdução de Sinais
15.
Int J Mol Sci ; 22(9)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33925005

RESUMO

To elucidate the additive effects of an EP2 agonist, omidenepag (OMD) or butaprost (Buta) on the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) on adipose tissue, two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells were analyzed by lipid staining, the mRNA expression of adipogenesis-related genes, extracellular matrix (ECM) molecules including collagen (Col) -1, -4 and -6, and fibronectin (Fn), and the sizes and physical properties of 3D organoids, as measured by a micro-squeezer. The results indicate that adipogenesis induced (1) an enlargement of the 3D organoids; (2) a substantial enhancement in lipid staining as well as the expression of the Pparγ, Ap2 and Leptin genes; (3) a significant softening of the 3D organoids, the effects of which were all enhanced by Rip except for Pparγ expression; and (4) a significant downregulation in Col1 and Fn, and a significant upregulation in Col4, Col6, the effects of which were unchanged by Rip. When adding the EP2 agonist to Rip, (1) the sizes of the 3D organoids were reduced substantially; (2) lipid staining was increased (OMD), or decreased (Buta); (3) the stiffness of the 3D organoids was substantially increased in Buta; (4-1) the expression of Pparγ was suppressed (2D, OMD) or increased (2D, Buta), and the expressions of Ap2 were downregulated (2D, 3D) and Leptin was increased (2D) or decreased (3D), (4-2) all the expressions of four ECM molecules were upregulated in 2D (2D), and in 3D, the expression of Col1, Col4 was upregulated. The collective findings reported herein indicate that the addition of an EP2 agonist, OMD or Buta significantly but differently modulate the Rip-induced effects on adipogenesis and the physical properties of 2D and 3D cultured 3T3-L1 cells.


Assuntos
Adipogenia/efeitos dos fármacos , Alprostadil/análogos & derivados , Glicina/análogos & derivados , Isoquinolinas/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Sulfonamidas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Células 3T3-L1 , Alprostadil/farmacologia , Animais , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Glicina/farmacologia , Camundongos , Organoides , Receptores de Prostaglandina E Subtipo EP2/agonistas
16.
Eur J Pharmacol ; 901: 174097, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33848540

RESUMO

Renal fibrosis is the common pathological hallmark of chronic kidney disease, and SET domain containing lysine methyltransferase 7 (SETD7) promote considerably renal fibrosis. However, the signaling mechanisms underlying SETD7 driving renal fibrosis are not fully understood. Here, we investigated the role of SETD7 in M2 macrophages-myofibroblasts transition and the myeloid fibroblasts activation in folic acid and obstruction-induced renal fibrosis. Mice treated with PFI-2, an inhibitor of SETD7, presented less bone marrow-derived myofibroblasts, fewer CD206+/α-smooth muscle actin + cells and developed less renal fibrosis (P<0.01). Furthermore, SETD7 inhibition reduced the infiltration of inflammatory cells and decreased the production of pro-inflammatory cytokines and chemokines in the kidneys after folic acid treatment (P<0.01). Finally, SETD7 inhibition suppressed the accumulation of NF-κB p65+ cells in folic acid nephropathy (P<0.01). Taken together, SETD7 mediates M2 macrophages-myofibroblasts transition, bone marrow-derived myofibroblasts activation, and inflammation response in the development of renal fibrosis.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Ácido Fólico/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Isoquinolinas/farmacologia , Nefropatias/tratamento farmacológico , Rim/patologia , Sulfonamidas/farmacologia , Animais , Fibroblastos/efeitos dos fármacos , Fibrose , Nefropatias/induzido quimicamente , Nefropatias/patologia , Testes de Função Renal , Lectinas Tipo C/metabolismo , Macrófagos/efeitos dos fármacos , Masculino , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/metabolismo , Fator de Transcrição RelA/efeitos dos fármacos
17.
Molecules ; 26(7)2021 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-33916806

RESUMO

Several new cyano-substituted derivatives with pyrrolo[1,2-a]quinoline and pyrrolo[2,1-a]isoquinoline scaffolds were synthesized by the [3 + 2] cycloaddition of (iso)quinolinium ylides to fumaronitrile. The cycloimmonium ylides reacted in situ as 1,3-dipoles with fumaronitrile to selectively form distinct final compounds, depending on the structure of the (iso)quinolinium salt. Eleven compounds were evaluated for their anticancer activity against a panel of 60 human cancer cell lines. The most potent compound 9a showed a broad spectrum of antiproliferative activity against cancer cell lines representing leukemia, melanoma and cancer of lung, colon, central nervous system, ovary, kidney, breast and prostate cancer. In vitro assays and molecular docking revealed tubulin interaction properties of compound 9a.


Assuntos
Isoquinolinas/análise , Isoquinolinas/síntese química , Nitrilas/química , Pirróis/química , Benzofenonas/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Isoquinolinas/farmacologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Simulação de Acoplamento Molecular , Paclitaxel/farmacologia , Prótons
18.
Cell Rep ; 35(3): 109020, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33852916

RESUMO

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment, and one important factor to consider is oxygen tension, in which hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via an HIF-1α-dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication, showing that post-entry steps in the viral life cycle are oxygen sensitive. This study highlights the importance of HIF signaling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19.


Assuntos
COVID-19/metabolismo , Células Epiteliais/metabolismo , Glicina/análogos & derivados , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isoquinolinas/farmacologia , Pulmão/metabolismo , SARS-CoV-2/fisiologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Células A549 , Animais , COVID-19/tratamento farmacológico , COVID-19/patologia , Células CACO-2 , Hipóxia Celular/efeitos dos fármacos , Chlorocebus aethiops , Células Epiteliais/virologia , Glicina/farmacologia , Humanos , Pulmão/virologia , Camundongos , Células Vero
19.
Biomed Pharmacother ; 137: 111348, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33578237

RESUMO

The ergosterol pathway is a prime antifungal target as it is required for fungal survival, yet is not involved in human homeostasis. Methods to study the ergosterol pathway, however, are often time-consuming. The minimum inhibitory concentration (MIC) assay is a simple research tool that determines the lowest concentration at which a novel antimicrobial is active in vitro with limited scope to determine the mechanism of action for a drug. In this study, we show that by adding hydrogen peroxide, an oxidative stressor, or glutathione (GSH), an antioxidant, to modify a commonly performed MIC assay allowed us to screen selectively for new antifungal drugs that target ergosterol biosynthesis in fungi. A human pathogen and dermatophyte, Microsporum gypseum, was used as a test organism. When exposed to ergosterol targeting drugs, the hydrogen peroxide treatment significantly decreased fungal survival by reducing ergosterol in the cell wall, whereas GSH increased survival of M. gypseum. Further, by performing a series of experiments with M. gypseum and Trichophyton rubrum, it was determined that the oxidative stress from hydrogen peroxide causes cell death at different developmental stages based on fungal species. These findings allow us to describe a simple, high-throughput method for simultaneously screening new antifungal drugs for activity and effects on the ergosterol pathway. By using this tool, two isoquinoline alkaloids were discovered to be potent inhibitors of ergosterol biosynthesis in vitro by reducing the amount of ergosterol without affecting the expression of 1,3-ß-glucan. Both compounds also significantly reduced the severity of acanthosis, hyperkeratosis, spongiosis and dermal edema in vivo.


Assuntos
Alcaloides/farmacologia , Antifúngicos/farmacologia , Ergosterol/biossíntese , Ensaios de Triagem em Larga Escala/métodos , Isoquinolinas/farmacologia , Alcaloides/uso terapêutico , Animais , Antifúngicos/uso terapêutico , Arthrodermataceae/citologia , Arthrodermataceae/efeitos dos fármacos , Benzofenantridinas/farmacologia , Benzofenantridinas/uso terapêutico , Modelos Animais de Doenças , Ergosterol/análise , Feminino , Glutationa/farmacologia , Cobaias , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/farmacologia , Isoquinolinas/uso terapêutico , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Micélio/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Tinha/tratamento farmacológico , Tinha/patologia
20.
Int J Mol Sci ; 22(4)2021 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-33562110

RESUMO

Isoquinoline alkaloids-enriched herbal plants have been used as traditional folk medicine for their anti-inflammatory, antimicrobial, and analgesic effects. They induce cell cycle arrest, apoptosis, and autophagy, leading to cell death. While the molecular mechanisms of these effects are not fully understood, it has been suggested that binding to nucleic acids or proteins, enzyme inhibition, and epigenetic modulation by isoquinoline alkaloids may play a role in the effects. This review discusses recent evidence on the molecular mechanisms by which the isoquinoline alkaloids can be a therapeutic target of cancer treatment.


Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Isoquinolinas/farmacologia , Neoplasias/tratamento farmacológico , Analgésicos/farmacologia , Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Medicina Tradicional , Ácidos Nucleicos/química
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