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1.
Eur J Pharmacol ; 926: 175017, 2022 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-35588870

RESUMO

It has been recently proposed that repeated bladder ischemia/reperfusion induced by chronic pelvic ischemia may lead to detrusor overactivity, followed by lower urinary tract symptoms. Vibegron is a selective ß3-adrenoceptor agonist approved for the treatment of overactive bladder. Several studies have tested ß3-adrenoceptor agonists using animal models with detrusor overactivity related to bladder ischemia/reperfusion. However, whether ß3-adrenoceptor agonists directly affect ischemia/reperfusion-evoked detrusor overactivity is unclear. Therefore, we examined whether bladder anoxia/reoxygenation could enhance spontaneous bladder contractions (SBCs) and investigated the effect of vibegron on enhanced SBCs. Isolated whole bladders from rats were incubated with Krebs solution aerated with 95% N2 + 5% CO2 for 5 h (anoxia). Subsequently, the bathing solution was replaced with an oxygen-saturated solution (reoxygenation). Anoxia/reoxygenation caused enhancement of the amplitude but not the frequency of SBC compared with that before reoxygenation. Vibegron (0.3-30 µM) inhibited this increase in SBC amplitude, but not the frequency, in a dose-dependent manner. The inhibitory effect of vibegron was not affected by pretreatment with the adenylyl cyclase inhibitor SQ22536 (100 µM) or protein kinase A inhibitor KT5720 (1 µM) and was not accompanied by considerable changes in cyclic adenosine monophosphate (cAMP) content in the bladder. In contrast, the large conductance potassium channel inhibitor iberiotoxin (100 nM) suppressed the inhibitory effect of vibegron. These results suggest that bladder ischemia/reperfusion induces SBC enhancement and vibegron directly inhibits detrusor overactivity via the large conductance potassium channel, which involves ß3-adrenoceptor, rather than the cAMP signaling pathway.


Assuntos
Pirimidinonas , Pirrolidinas , Bexiga Urinária Hiperativa , Bexiga Urinária , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Animais , Hipóxia/metabolismo , Canais de Potássio/metabolismo , Pirimidinonas/farmacologia , Pirrolidinas/farmacologia , Ratos , Receptores Adrenérgicos/metabolismo , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo , Bexiga Urinária Hiperativa/tratamento farmacológico , Bexiga Urinária Hiperativa/etiologia , Bexiga Urinária Hiperativa/metabolismo , Urodinâmica
2.
Life Sci ; 298: 120515, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35367243

RESUMO

AIMS: Dipeptidyl peptidase-4 (DPP-4) inhibitors have been extensively used for the treatment of type 2 diabetes mellitus. Nevertheless, side effects like sore throat and diarrhea also occur in DPP-4 inhibitors treatment. The study aims to identify and develop novel DPP-4 inhibitors with better therapeutic profiles. MATERIALS AND METHODS: Here we synthesized a series of vildagliptin analogs, and among which, ZD-2 showed the moderate inhibition of DPP-4 activity compared with vildagliptin. High-fat-diet (HFD) mice were treated with ZD-2 (4.5 and 7.5 mg/kg) or vildagliptin (6 mg/kg) for 7 weeks following the examinations of metabolic index and pancreatic ß-cell function. Mouse pancreatic cell line MIN6 was used to evaluate ß-cell function, and intestinal enteroendocrine cell line STC-1 was used to evaluate the expression of gut hormones. KEY FINDINGS: The IC50 of ZD-2 was over 30-fold higher than vildagliptin. However, both ZD-2 and vildagliptin treatment showed comparable effects on improving glucose tolerance and reducing the steatosis of liver and fat mass in HFD mice. Moreover, ZD-2 exerted ß-cell-protective actions by preserving islet ß-cell mass and increasing the expression of functional ß-cell-related genes. Additionally, ZD-2 also stimulated the expression of gut hormones in STC-1 cells. SIGNIFICANCE: ZD-2 showed comparable anti-diabetic activities in HFD-fed mice although its lower potency on inhibition of DPP-4 compared with vildagliptin. Protection of ß-cell function might contribute to its anti-diabetic effects.


Assuntos
Adamantano , Diabetes Mellitus Tipo 2 , Inibidores da Dipeptidil Peptidase IV , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Inibidores da Dipeptidil Peptidase IV/farmacologia , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Controle Glicêmico , Hormônios/uso terapêutico , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Camundongos , Camundongos Obesos , Nitrilas/farmacologia , Nitrilas/uso terapêutico , Pirrolidinas/farmacologia , Vildagliptina/farmacologia , Vildagliptina/uso terapêutico
3.
Eur J Med Chem ; 233: 114191, 2022 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-35263708

RESUMO

Transient receptor potential vanilloid 1 (TRPV1) antagonists can inhibit the transmission of nociceptive signals from the peripheral to the central nervous system (CNS), providing a new strategy for pain relief. In this work, in order to develop potent, CNS-penetrant, and orally available TRPV1 antagonists, three series of novel molecules based on the key pharmacophore structures of classic TRPV1 ligands SB-705498 and MDR-652 were designed and synthesized. Through systematic in vitro and in vivo bioassays, (S)-N-(3-isopropylphenyl)-2-(5-phenylthiazol-2-yl)pyrrolidine-1-carboxamide (7q) was finally identified, which had enhanced TRPV1 antagonistic activity (IC50 (capsaicin) = 2.66 nM), excellent CNS penetration (brain/plasma ratio = 1.66), favorable mode-selectivity, good bioavailability, and no side effects of hyperthermia. Molecular docking and dynamics studies indicated that the high binding affinity of compound 7q to TRPV1 was related to multiple interactions, which resulted in significant conformational changes of TRPV1. Overall, our findings have led to a potent, mode-selective, and CNS-penetrant TRPV1 antagonist as a valuable lead for development of novel TRPV1 antagonists.


Assuntos
Capsaicina , Canais de Cátion TRPV , Encéfalo/metabolismo , Capsaicina/farmacologia , Simulação de Acoplamento Molecular , Pirrolidinas/farmacologia , Canais de Cátion TRPV/metabolismo
4.
Int J Mol Sci ; 23(6)2022 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-35328345

RESUMO

REV-ERBα (nr1d1, nuclear receptor subfamily 1 group D member 1) is a transcriptional repressor that in mammals regulates nutrient metabolism, and has effects on energy homeostasis, although its role in teleosts is poorly understood. To determine REV-ERBα's involvement in fish energy balance and metabolism, we studied the effects of acute and 7-day administration of its agonist SR9009 on food intake, weight and length gain, locomotor activity, feeding regulators, plasma and hepatic metabolites, and liver enzymatic activity. SR9009 inhibited feeding, lowering body weight and length gain. In addition, the abundance of ghrelin mRNA decreased in the intestine, and abundance of leptin-aI mRNA increased in the liver. Hypocretin, neuropeptide y (npy), and proopiomelanocortin (pomc) mRNA abundance was not modified after acute or subchronic SR9009 administration, while hypothalamic cocaine- and amphetamine-regulated transcript (cartpt-I) was induced in the subchronic treatment, being a possible mediator of the anorectic effects. Moreover, SR9009 decreased plasma glucose, coinciding with increased glycolysis and a decreased gluconeogenesis in the liver. Decreased triglyceride levels and activity of lipogenic enzymes suggest a lipogenesis reduction by SR9009. Energy expenditure by locomotor activity was not significantly affected by SR9009. Overall, this study shows for the first time in fish the effects of REV-ERBα activation via SR9009, promoting a negative energy balance by reducing energetic inputs and regulating lipid and glucose metabolism.


Assuntos
Carpa Dourada , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares , Animais , Metabolismo Energético , Carpa Dourada/genética , Mamíferos/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Pirrolidinas/farmacologia , RNA Mensageiro/metabolismo , Tiofenos
5.
Commun Biol ; 5(1): 154, 2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35194144

RESUMO

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Exonucleases/antagonistas & inibidores , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Anilidas/farmacologia , Animais , Sequência de Bases , Benzimidazóis/farmacologia , COVID-19/virologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Sinergismo Farmacológico , Exonucleases/genética , Exonucleases/metabolismo , Humanos , Prolina/farmacologia , Pirrolidinas/farmacologia , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Valina/farmacologia , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
6.
Eur J Med Chem ; 233: 114208, 2022 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-35220015

RESUMO

A series of novel spiro-quinolinone derivatives were designed and synthesized and their structures were confirmed by spectroscopic methods. The enzymatic experiments showed that all the seventeen synthesized compounds had inhibition potency against chitin synthase, among them five compounds had excellent inhibition potency that equal to that of polyoxin B. The Kinetic parameters of enzymatic assays indicated that these compounds were non-competitive inhibitors of chitin synthase. The antimicrobial experiments displayed that the synthesized compounds had selectively and broad-spectrum antifungal activity in vitro Among them, two compounds had stronger antifungal activity against C. albicans than that of fluconazole meanwhile five others compounds showed antifungal activity against C. albicans being equal to that of fluconazole. Moreover, there are four or five compounds that possessed antifungal activities against C. neoformans, A. fumigatus and A. flavus as high as fluconazole had, respectively. The sorbitol protection assay and evaluation of antifungal activity against micafungin-resistant strain further verified that these compounds possessed antifungal activity through inhibiting the synthesis of chitin of cell wall. The evaluation of antifungal activity against others drug-resistant fungi variants showed these designed compounds had significant antifungal activity against these tested variants. The combination use experiments exhibited that the synthesized compounds had synergistic effects or additive effects with current used drugs in clinic. These results demonstrated that these synthesized compounds were chitin synthase inhibitors and had selective and broad-spectra antifungal activities.


Assuntos
Antifúngicos , Quitina Sintase , Antifúngicos/química , Candida albicans , Quitina/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/química , Testes de Sensibilidade Microbiana , Pirrolidinas/farmacologia , Relação Estrutura-Atividade
7.
Bioorg Chem ; 120: 105650, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35144103

RESUMO

Two libraries of mono- and dimeric pyrrolidine iminosugars were synthesized by CuAAC and (thio)urea-bond-forming reactions from the respective azido/aminohexylpyrrolidine iminosugar precursors. The resulting monomeric and dimeric compounds were screened for inhibition of ß-N-acetylglucosaminidase from Jack beans, the plant ortholog of human lysosomal hexosaminidases. A selection of the best inhibitors of these libraries was then evaluated against human lysosomal ß-N-acetylhexosaminidase B (hHexB) and human nucleocytoplasmic ß-N-acetylglucosaminidase (hOGA). This evaluation identified a potent (nM) and selective monomeric inhibitor of hOGA (compound 7A) that showed a 6770-fold higher affinity for this enzyme than for hHexB. The corresponding dimeric derivative (compound 9D) further remarkably improved the selectivity in the inhibition of hOGA (2.7 × 104 times more selective for hOGA over hHexB) and the inhibition potency (by one order of magnitude). Docking studies were performed to explain the selectivity of inhibition observed in compound 7A.


Assuntos
Imino Açúcares , Acetilglucosaminidase , Inibidores Enzimáticos/farmacologia , Humanos , Imino Açúcares/farmacologia , Pirrolidinas/farmacologia , Relação Estrutura-Atividade , beta-N-Acetil-Hexosaminidases
8.
Int J Mol Sci ; 23(3)2022 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-35163176

RESUMO

Premature infants are born with developing lungs burdened by surfactant deficiency and a dearth of antioxidant defense systems. Survival rate of such infants has significantly improved due to advances in care involving mechanical ventilation and oxygen supplementation. However, a significant subset of such survivors develops the chronic lung disease, Bronchopulmonary dysplasia (BPD), characterized by enlarged, simplified alveoli and deformed airways. Among a host of factors contributing to the pathogenesis is oxidative damage induced by exposure of the developing lungs to hyperoxia. Recent data indicate that hyperoxia induces aberrant sphingolipid signaling, leading to mitochondrial dysfunction and abnormal reactive oxygen species (ROS) formation (ROS). The role of sphingolipids such as ceramides and sphingosine 1-phosphate (S1P), in the development of BPD emerged in the last decade. Both ceramide and S1P are elevated in tracheal aspirates of premature infants of <32 weeks gestational age developing BPD. This was faithfully reflected in the murine models of hyperoxia and BPD, where there is an increased expression of sphingolipid metabolites both in lung tissue and bronchoalveolar lavage. Treatment of neonatal pups with a sphingosine kinase1 specific inhibitor, PF543, resulted in protection against BPD as neonates, accompanied by improved lung function and reduced airway remodeling as adults. This was accompanied by reduced mitochondrial ROS formation. S1P receptor1 induced by hyperoxia also aggravates BPD, revealing another potential druggable target in this pathway for BPD. In this review we aim to provide a detailed description on the role played by sphingolipid signaling in hyperoxia induced lung injury and BPD.


Assuntos
Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/patologia , Lesão Pulmonar/metabolismo , Esfingolipídeos/fisiologia , Remodelação das Vias Aéreas , Animais , Animais Recém-Nascidos , Ceramidas/metabolismo , Modelos Animais de Doenças , Humanos , Hiperóxia/metabolismo , Hiperóxia/fisiopatologia , Lactente , Recém-Nascido , Pulmão/patologia , Lesão Pulmonar/patologia , Lisofosfolipídeos/metabolismo , Metanol/farmacologia , Camundongos , Estresse Oxidativo/fisiologia , Alvéolos Pulmonares/metabolismo , Pirrolidinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Sulfonas/farmacologia
9.
J Exp Anal Behav ; 117(3): 442-456, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35142382

RESUMO

Polysubstance use makes up a majority of drug use, yet relatively few studies investigate the abuse-related effects of drug mixtures. Dose-addition analyses provide a rigorous and quantitative method to determine the nature of the interaction (i.e., supraadditive, additive, or subadditive) between two or more drugs. As briefly reviewed here, studies in rhesus monkeys have applied dose-addition analyses to group level data to characterize the nature of the interaction between the reinforcing effects of stimulants and opioids (e.g., mixtures of cocaine + heroin). Building upon these foundational studies, more recent work has applied dose-addition analyses to better understand the nature of the interaction between caffeine and illicit stimulants such as MDPV and methamphetamine in rats. In addition to utilizing a variety of operant procedures, including drug discrimination, drug self-administration, and drug-primed reinstatement, these studies have incorporated potency and effectiveness ratios as a method for both statistical analysis and visualization of departures from additivity at both the group and individual subject level. As such, dose-addition analyses represent a powerful and underutilized approach to quantify the nature of drug-drug interactions that can be applied to a variety of abuse-related endpoints in order to better understand the behavioral pharmacology of polysubstance use.


Assuntos
Estimulantes do Sistema Nervoso Central , Cocaína , Animais , Benzodioxóis/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Cocaína/farmacologia , Relação Dose-Resposta a Droga , Preparações Farmacêuticas , Pirrolidinas/farmacologia , Ratos , Ratos Sprague-Dawley
10.
J Neuroinflammation ; 19(1): 9, 2022 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-34991625

RESUMO

BACKGROUND: Gangliosides are glycosphingolipids highly enriched in the brain, with important roles in cell signaling, cell-to-cell communication, and immunomodulation. Genetic defects in the ganglioside biosynthetic pathway result in severe neurodegenerative diseases, while a partial decrease in the levels of specific gangliosides was reported in Parkinson's disease and Huntington's disease. In models of both diseases and other conditions, administration of GM1-one of the most abundant gangliosides in the brain-provides neuroprotection. Most studies have focused on the direct neuroprotective effects of gangliosides on neurons, but their role in other brain cells, in particular microglia, is not known. In this study we investigated the effects of exogenous ganglioside administration and modulation of endogenous ganglioside levels on the response of microglia to inflammatory stimuli, which often contributes to initiation or exacerbation of neurodegeneration. METHODS: In vitro studies were performed using BV2 cells, mouse, rat, and human primary microglia cultures. Modulation of microglial ganglioside levels was achieved by administration of exogenous gangliosides, or by treatment with GENZ-123346 and L-t-PDMP, an inhibitor and an activator of glycolipid biosynthesis, respectively. Response of microglia to inflammatory stimuli (LPS, IL-1ß, phagocytosis of latex beads) was measured by analysis of gene expression and/or secretion of pro-inflammatory cytokines. The effects of GM1 administration on microglia activation were also assessed in vivo in C57Bl/6 mice, following intraperitoneal injection of LPS. RESULTS: GM1 decreased inflammatory microglia responses in vitro and in vivo, even when administered after microglia activation. These anti-inflammatory effects depended on the presence of the sialic acid residue in the GM1 glycan headgroup and the presence of a lipid tail. Other gangliosides shared similar anti-inflammatory effects in in vitro models, including GD3, GD1a, GD1b, and GT1b. Conversely, GM3 and GQ1b displayed pro-inflammatory activity. The anti-inflammatory effects of GM1 and other gangliosides were partially reproduced by increasing endogenous ganglioside levels with L-t-PDMP, whereas inhibition of glycolipid biosynthesis exacerbated microglial activation in response to LPS stimulation. CONCLUSIONS: Our data suggest that gangliosides are important modulators of microglia inflammatory responses and reveal that administration of GM1 and other complex gangliosides exerts anti-inflammatory effects on microglia that could be exploited therapeutically.


Assuntos
Anti-Inflamatórios/farmacologia , Gangliosídeo G(M1)/farmacologia , Inflamação/patologia , Microglia/efeitos dos fármacos , Animais , Células Cultivadas , Dioxanos/farmacologia , Humanos , Inflamação/metabolismo , Interleucina-1beta/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/metabolismo , Microglia/patologia , Fagocitose/efeitos dos fármacos , Pirrolidinas/farmacologia , Ratos
11.
Drug Alcohol Depend ; 232: 109299, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35063839

RESUMO

BACKGROUND: Recent evidence suggesting that polysubstance use is the norm rather than the exception highlights the need for a better understanding of interactions amongst the abuse-related effects of commonly co-abused drugs. Synthetic cathinones remain one of the most popular families of novel psychoactive substances and are typically used in preparations containing multiple stimulants. Evaluating the reinforcing effects of drugs under both single-operant procedures and procedures in which alternatives are available can provide a more complete characterization of their reinforcing effects and economic interactions. METHODS: These studies utilized a drug-versus-drug choice procedure in 18 male Sprague-Dawley rats to evaluate economic interactions between the synthetic cathinone, MDPV, and cocaine in addition to how a history of concurrent access impacts reinstatement behavior. RESULTS: When equi-effective doses of MDPV and cocaine were made concurrently available, approximately half of the subjects responded exclusively on the MDPV-reinforced lever whereas the other half responded exclusively on the cocaine-reinforced lever. Allocation of responding was reversed when the cost of the preferred drug increased, or the cost of the non-preferred drug decreased. Drug-paired cues and MDPV, cocaine, and methamphetamine pretreatments reinstated responding on both drug levers, regardless of preference. CONCLUSION: These data demonstrate that MDPV and cocaine act as economic substitutes and suggest that measures of reinforcing effectiveness determined under a progressive ratio schedule of reinforcement can predict drug choice. These data also suggest that environmental stimuli associated with a particular drug might stimulate class-specific drug-seeking, however, further studies are needed to test the generality of this claim.


Assuntos
Cocaína , Animais , Benzodioxóis/farmacologia , Cocaína/farmacologia , Relação Dose-Resposta a Droga , Masculino , Pirrolidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Autoadministração
12.
J Immunol ; 208(1): 38-48, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34862257

RESUMO

RNA-binding protein HuR (ELAVL1) is a master regulator of gene expression in human pathophysiology. Its dysregulation plays an important role in many diseases. We hypothesized that HuR plays an important role in Th2 inflammation in asthma in both mouse and human. To address this, we used a model of airway inflammation in a T cell-specific knockout mouse model, distal lck-Cre HuRfl/fl, as well as small molecule inhibitors in human peripheral blood-derived CD4+ T cells. Peripheral CD4+ T cells were isolated from 26 healthy control subjects and 45 asthmatics (36 type 2 high and 9 non-type 2 high, determined by blood eosinophil levels and fraction of exhaled NO). Our mouse data showed conditional ablation of HuR in T cell-abrogated Th2 differentiation, cytokine production, and lung inflammation. Studies using human T cells showed that HuR protein levels in CD4+ T cells were significantly higher in asthmatics compared with healthy control subjects. The expression and secretion of Th2 cytokines were significantly higher in asthmatics compared with control subjects. AMP-activated protein kinase activator treatment reduced the expression of several cytokines in both type 2 high and non-type 2 high asthma groups. However, the effects of CMLD-2 (a HuR-specific inhibitor) were more specific to endotype-defining cytokines in type 2 high asthmatics. Taken together, these data suggest that HuR plays a permissive role in both allergen and non-allergen-driven airway inflammation by regulating key genes, and that interfering with its function may be a novel method of asthma treatment.


Assuntos
Asma/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Células Th2/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alérgenos/imunologia , Animais , Anti-Inflamatórios/farmacologia , Asma/genética , Asma/terapia , Benzopiranos/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Proteína Semelhante a ELAV 1/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Ovalbumina/imunologia , Pirrolidinas/farmacologia , Adulto Jovem
13.
Neuropharmacology ; 205: 108897, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34822817

RESUMO

Dravet syndrome is a catastrophic childhood epilepsy with multiple seizure types that are refractory to treatment. The endocannabinoid system regulates neuronal excitability so a deficit in endocannabinoid signaling could lead to hyperexcitability and seizures. Thus, we sought to determine whether a deficiency in the endocannabinoid system might contribute to seizure phenotypes in a mouse model of Dravet syndrome and whether enhancing endocannabinoid tone is anticonvulsant. Scn1a+/- mice model the clinical features of Dravet syndrome: hyperthermia-induced seizures, spontaneous seizures and reduced survival. We examined whether Scn1a+/- mice exhibit deficits in the endocannabinoid system by measuring brain cannabinoid receptor expression and endocannabinoid concentrations. Next, we determined whether pharmacologically enhanced endocannabinoid tone was anticonvulsant in Scn1a+/- mice. We used GAT229, a positive allosteric modulator of the cannabinoid (CB1) receptor, and ABX-1431, a compound that inhibits the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). The Scn1a+/- phenotype is strain-dependent with mice on a 129S6/SvEvTac (129) genetic background having no overt phenotype and those on an F1 (129S6/SvEvTac x C57BL/6J) background exhibiting a severe epilepsy phenotype. We observed lower brain cannabinoid CB1 receptor expression in the seizure-susceptible F1 compared to seizure-resistant 129 strain, suggesting an endocannabinoid deficiency might contribute to seizure susceptibility. GAT229 and ABX-1431 were anticonvulsant against hyperthermia-induced seizures. However, subchronic ABX1431 treatment increased spontaneous seizure frequency despite reducing seizure severity. Cnr1 is a putative genetic modifier of epilepsy in the Scn1a+/- mouse model of Dravet syndrome. Compounds that increase endocannabinoid tone could be developed as novel treatments for Dravet syndrome.


Assuntos
Anticonvulsivantes/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Endocanabinoides/antagonistas & inibidores , Endocanabinoides/metabolismo , Epilepsias Mioclônicas/tratamento farmacológico , Epilepsias Mioclônicas/metabolismo , Receptor CB1 de Canabinoide/agonistas , Animais , Modelos Animais de Doenças , Endocanabinoides/deficiência , Indóis/farmacologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Piperazinas/farmacologia , Pirrolidinas/farmacologia
14.
Eur J Med Chem ; 228: 114025, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34871839

RESUMO

Parkinson's disease (PD) is a common neurodegenerative disease among the elderly. Currently, monoamine oxidase B (MAO-B) inhibitors are extensively used for PD in clinics. In this work, a series of novel chiral fluorinated pyrrolidine derivatives were designed and synthesized. In vitro biological evaluations revealed that compound D5 was the most potent, selective MAO-B inhibitor (IC50 = 0.019 µM, MAO-A/MAO-B selectivity index = 2440), which was 10-fold than that of miracle drug safinamide (IC50 = 0.163 µM, MAO-A/MAO-B selectivity index = 172). It was verified that the enhanced hydrophobic interaction of D5 improved the activity against MAO-B in molecular docking study. Besides, D5 exhibited excellent metabolic properties and pharmacokinetic profiles in monkeys and rats. Moreover, D5 displayed more efficacious than safinamide in vivo models. In the MPTP-induced PD mouse model, D5 significantly alleviated DA deficits and increased the effect of levodopa on dopamine concentration in the striatum. Meanwhile, D5 produced a prominent reduction in tremulous jaw movements induced by galantamine. Accordingly, we present D5 as a novel, highly potent, and selective MAO-B inhibitor for PD therapy.


Assuntos
Simulação de Acoplamento Molecular , Inibidores da Monoaminoxidase/farmacologia , Monoaminoxidase/metabolismo , Pirrolidinas/farmacologia , Animais , Cães , Relação Dose-Resposta a Droga , Halogenação , Haplorrinos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Inibidores da Monoaminoxidase/química , Inibidores da Monoaminoxidase/metabolismo , Pirrolidinas/síntese química , Pirrolidinas/química , Ratos , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade
15.
Toxicol Lett ; 355: 127-140, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34863860

RESUMO

In this study, we newly synthesized four α-pyrrolidinononanophenone (α-PNP) derivatives [4'-halogenated derivatives and α-pyrrolidinodecanophenone (α-PDP)], and then performed the structure-cytotoxicity relationship analyses. The results showed the rank order for the cytotoxic effects, α-PNP < α-PDP < 4'-fluoro-α-PNP < 4'-chrolo-α-PNP < 4'-bromo-α-PNP < 4'-iodo-α-PNP (I-α-PNP), and suggest that cytotoxicities of 4'-halogenated derivatives were more intensive than that of elongation of the hydrocarbon chain (α-PDP). We also surveyed the apoptotic mechanism of I-α-PNP in brain microvascular endothelial (HBME) cells that are utilized as the in vitro model of the blood-brain barrier. HBME cell treatment with I-α-PNP facilitated the apoptotic events (caspase-3 activation, externalization of phosphatidylserine, and DNA fragmentation), which were almost completely abolished by pretreating with antioxidants. In addition, the immunofluorescent staining revealed the enhanced production of hydroxyl radical in mitochondria by the I-α-PNP treatment, inferring that the I-α-PNP treatment triggers the apoptotic mechanism dependent on the enhanced ROS production in mitochondria. The treatment with I-α-PNP increased the production of cytotoxic aldehyde 4-hydroxy-2-nonenal and decreased the amount of reduced glutathione. Additionally, the treatment decreased the 26S proteasome-based proteolytic activities and aggresome formation. These results suggest that decrease in the antioxidant properties is also ascribable to HBME cell apoptosis elicited by I-α-PNP.


Assuntos
Antioxidantes/farmacologia , Encéfalo/irrigação sanguínea , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Cetonas/farmacologia , Pirrolidinas/farmacologia , Antioxidantes/química , Apoptose/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Cetonas/síntese química , Estrutura Molecular , Pirrolidinas/síntese química , Relação Estrutura-Atividade
16.
Anal Bioanal Chem ; 414(2): 1151-1162, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34734312

RESUMO

LGD-4033 (ligandrol) is a selective androgen receptor modulator (SARM), which is prohibited in sports by the World Anti-Doping Agency (WADA) and led to 62 adverse analytical findings (AAFs) in 2019. But not only deliberate doping with LGD-4033 constitutes a problem. In the past years, some AAFs that concerned SARMs can be attributed to contaminated dietary supplements (DS). Thus, the urgency to develop methods to differentiate between inadvertent doping and abuse of SARMs to benefit from the performance-enhancing effect of the compound in sports is growing. To gain a better understanding of the metabolism and excretion patterns of LGD-4033, human micro-dose excretion studies at 1, 10, and 50 µg LGD-4033 were conducted. Collected urine samples were prepared for analysis using enzymatic hydrolysis followed by solid-phase extraction and analyzed via LC-HRMS/MS. Including isomers, a total of 15 phase I metabolites were detected in the urine samples. The LC-HRMS/MS method was validated for qualitative detection of LGD-4033, allowing for a limit of detection (LOD) of 8 pg/mL. The metabolite M1, representing the epimer of LGD-4033, was synthesized and the structure elucidated by NMR spectroscopy. As the M1/LGD-4033 ratio changes over time, the ratio and the approximate LGD-4033 concentration can contribute to estimating the time point of drug intake and dose of LGD-4033 in doping control urine samples, which is particularly relevant in anti-doping result management.


Assuntos
Doping nos Esportes/prevenção & controle , Nitrilas/farmacologia , Pirrolidinas/farmacologia , Receptores Androgênicos/efeitos dos fármacos , Cromatografia Líquida/métodos , Humanos , Limite de Detecção , Espectrometria de Massas em Tandem/métodos
17.
Eur J Med Chem ; 228: 113954, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34772527

RESUMO

Modulating the glucose transport in skeletal muscle is a promising strategy for ameliorating glucose homeostasis disorders. However, the complicated mechanisms of glucose transport make it difficult to find compounds therapeutically relevant molecular mechanisms of action, while phenotypic screening is thought to be an alternative approach to mimic the cell state of interest. Here, we report (±)-seneciobipyrrolidine (1a) is first found to enhance glucose uptake in L6 myotubes through phenotype-based screening. Further SAR investigation led to the identfication of compound A27 (EC50 = 2.7 µM). Proteomiic analysis discloses the unique function mechanism of A27 through upregulating the level of the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), subsequently enhancing the Akt and AMPK phosphorylation, thereby promoting the glucose uptake. Chronic oral administration of A27 significantly lowers blood glucose and improves glucose tolerance in db/db mice. This work is new research on seneciobipyrrolidine derivatives, providing a promising avenue for ameliorating glucose homeostasis.


Assuntos
Antipsicóticos/farmacologia , Glicemia/efeitos dos fármacos , Descoberta de Drogas , Transtornos Psicóticos/tratamento farmacológico , Pirrolidinas/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antipsicóticos/síntese química , Antipsicóticos/química , Glicemia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transtornos Psicóticos/metabolismo , Pirrolidinas/síntese química , Pirrolidinas/química , Transdução de Sinais/efeitos dos fármacos , Estereoisomerismo , Relação Estrutura-Atividade
18.
Biochem Biophys Res Commun ; 587: 9-15, 2022 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-34861472

RESUMO

OBJECTIVE: The role of circadian clock in cementogenesis is unclear. This study examines the role of REV-ERBs, one of circadian clock proteins, in proliferation, migration and mineralization of cementoblasts to fill the gap in knowledge. METHODS: Expression pattern of REV-ERBα in cementoblasts was investigated in vivo and in vitro. CCK-8 assay, scratch wound healing assay, alkaline phosphatase (ALP) and alizarin red S (ARS) staining were performed to evaluate the effects of REV-ERBs activation by SR9009 on proliferation, migration and mineralization of OCCM-30, an immortalized cementoblast cell line. Furthermore, mineralization related markers including osterix (OSX), ALP, bone sialoprotein (BSP) and osteocalcin (OCN) were evaluated. RESULTS: Strong expression of REV-ERBα was found in cellular cementum around tooth apex. Rev-erbα mRNA oscillated periodically in OCCM-30 and declined after mineralization induction. REV-ERBs activation by SR9009 inhibited proliferation but promoted migration of OCCM-30 in vitro. Results of ALP and ARS staining suggested that REV-ERBs activation negatively regulated mineralization of OCCM-30. Mechanically, REV-ERBs activation attenuated the expression of OSX and its downstream targets including ALP, BSP and OCN. CONCLUSIONS: REV-ERBs are involved in cementogenesis and negatively regulate mineralization of cementoblasts via inhibiting OSX expression. Our study provides a potential target regarding periodontal and cementum regeneration.


Assuntos
Relógios Biológicos/genética , Calcificação Fisiológica/genética , Cemento Dentário/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Cementogênese/efeitos dos fármacos , Cementogênese/genética , Cemento Dentário/citologia , Cemento Dentário/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Humanos , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Pirrolidinas/farmacologia , Transdução de Sinais , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Tiofenos/farmacologia
19.
J Med Chem ; 65(4): 2848-2865, 2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33891389

RESUMO

The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II, and XII, with each containing a reactive warhead that covalently modifies the catalytic Cys145. Coupling structure-based drug design with the one-pot Ugi four-component reaction, we discovered one of the most potent noncovalent inhibitors, 23R (Jun8-76-3A) that is structurally distinct from the canonical Mpro inhibitor GC376. Significantly, 23R is highly selective compared with covalent inhibitors such as GC376, especially toward host proteases. The cocrystal structure of SARS-CoV-2 Mpro with 23R revealed a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study discovered 23R, one of the most potent and selective noncovalent SARS-CoV-2 Mpro inhibitors reported to date, and a novel binding pocket in Mpro that can be explored for inhibitor design.


Assuntos
Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Desenho de Fármacos , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/síntese química , Antivirais/química , COVID-19/tratamento farmacológico , COVID-19/metabolismo , Chlorocebus aethiops , Proteases 3C de Coronavírus/isolamento & purificação , Proteases 3C de Coronavírus/metabolismo , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Relação Dose-Resposta a Droga , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Prolina/análogos & derivados , Prolina/síntese química , Prolina/química , Prolina/farmacologia , Pirrolidinas/síntese química , Pirrolidinas/química , Pirrolidinas/farmacologia , SARS-CoV-2/enzimologia , Ácidos Sulfônicos/síntese química , Ácidos Sulfônicos/química , Ácidos Sulfônicos/farmacologia , Células Vero
20.
Gut ; 71(3): 627-642, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-33833066

RESUMO

OBJECTIVE: HCV-genotype 4 infections are a major cause of liver diseases in the Middle East/Africa with certain subtypes associated with increased risk of direct-acting antiviral (DAA) treatment failures. We aimed at developing infectious genotype 4 cell culture systems to understand the evolutionary genetic landscapes of antiviral resistance, which can help preserve the future efficacy of DAA-based therapy. DESIGN: HCV recombinants were tested in liver-derived cells. Long-term coculture with DAAs served to induce antiviral-resistance phenotypes. Next-generation sequencing (NGS) of the entire HCV-coding sequence identified mutation networks. Resistance-associated substitutions (RAS) were studied using reverse-genetics. RESULT: The in-vivo infectious ED43(4a) clone was adapted in Huh7.5 cells, using substitutions identified in ED43(Core-NS5A)/JFH1-chimeric viruses combined with selected NS5B-changes. NGS, and linkage analysis, permitted identification of multiple genetic branches emerging during culture adaptation, one of which had 31 substitutions leading to robust replication/propagation. Treatment of culture-adapted ED43 with nine clinically relevant protease-DAA, NS5A-DAA and NS5B-DAA led to complex dynamics of drug-target-specific RAS with coselection of genome-wide substitutions. Approved DAA combinations were efficient against the original virus, but not against variants with RAS in corresponding drug targets. However, retreatment with glecaprevir/pibrentasvir remained efficient against NS5A inhibitor and sofosbuvir resistant variants. Recombinants with specific RAS at NS3-156, NS5A-28, 30, 31 and 93 and NS5B-282 were viable, but NS3-A156M and NS5A-L30Δ (deletion) led to attenuated phenotypes. CONCLUSION: Rapidly emerging complex evolutionary landscapes of mutations define the persistence of HCV-RASs conferring resistance levels leading to treatment failure in genotype 4. The high barrier to resistance of glecaprevir/pibrentasvir could prevent persistence and propagation of antiviral resistance.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/genética , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Hepatócitos/virologia , Mutação/genética , Benzimidazóis/farmacologia , Técnicas de Cultura de Células , Combinação de Medicamentos , Genótipo , Hepacivirus/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Pirrolidinas/farmacologia , Quinoxalinas/farmacologia , Sofosbuvir/farmacologia , Sulfonamidas/farmacologia
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