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1.
J Vis Exp ; (163)2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32986023

RESUMO

Parkinson's disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Neurônios Dopaminérgicos/citologia , Mesencéfalo/citologia , Animais , Caspase 3/metabolismo , Células Cultivadas , Dependovirus/genética , Neurônios Dopaminérgicos/efeitos dos fármacos , Embrião de Mamíferos/citologia , Vetores Genéticos/metabolismo , Glutamatos/farmacologia , Processamento de Imagem Assistida por Computador , Camundongos , Quinoxalinas/farmacologia , Receptores de AMPA/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo
2.
PLoS One ; 15(8): e0236839, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32780746

RESUMO

The majority of chronic myeloid leukemia (CML) cases are caused by a chromosomal translocation linking the breakpoint cluster region (BCR) gene to the Abelson murine leukemia viral oncogene-1 (ABL1), creating the mutant fusion protein BCR-ABL1. Downstream of BCR-ABL1 is growth factor receptor-bound protein-2 (GRB2), an intracellular adapter protein that binds to BCR-ABL1 via its src-homology-2 (SH2) domain. This binding constitutively activates growth pathways, downregulates apoptosis, and leads to an over proliferation of immature and dysfunctional myeloid cells. Utilizing novel synthetic methods, we developed four furo-quinoxaline compounds as GRB2 SH2 domain antagonists with the goal of disrupting this leukemogenic signaling. One of the four antagonists, NHD2-15, showed a significant reduction in proliferation of K562 cells, a human BCR-ABL1+ leukemic cell line. To elucidate the mode of action of these compounds, various biophysical, in vitro, and in vivo assays were performed. Surface plasmon resonance (SPR) assays indicated that NHD2-15 antagonized GRB2, binding with a KD value of 119 ± 2 µM. Cellulose nitrate (CN) assays indicated that the compound selectively bound the SH2 domain of GRB2. Western blot assays suggested the antagonist downregulated proteins involved in leukemic transformation. Finally, NHD2-15 was nontoxic to primary cells and adult zebrafish, indicating that it may be an effective clinical treatment for CML.


Assuntos
Proliferação de Células/efeitos dos fármacos , Proteína Adaptadora GRB2/antagonistas & inibidores , Quinoxalinas/farmacologia , Animais , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/metabolismo , Proteína Adaptadora GRB2/química , Proteína Adaptadora GRB2/metabolismo , Humanos , Células K562 , Rim/citologia , Cinética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Ligação Proteica , Quinoxalinas/química , Quinoxalinas/metabolismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Ressonância de Plasmônio de Superfície , Peixe-Zebra , Domínios de Homologia de src
3.
Comput Biol Med ; 122: 103848, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658735

RESUMO

The recent outbreak of coronavirus disease-19 (COVID-19) continues to drastically affect healthcare throughout the world. To date, no approved treatment regimen or vaccine is available to effectively attenuate or prevent the infection. Therefore, collective and multidisciplinary efforts are needed to identify new therapeutics or to explore effectiveness of existing drugs and drug-like small molecules against SARS-CoV-2 for lead identification and repurposing prospects. This study addresses the identification of small molecules that specifically bind to any of the three essential proteins (RdRp, 3CL-protease and helicase) of SARS-CoV-2. By applying computational approaches we screened a library of 4574 compounds also containing FDA-approved drugs against these viral proteins. Shortlisted hits from initial screening were subjected to iterative docking with the respective proteins. Ranking score on the basis of binding energy, clustering score, shape complementarity and functional significance of the binding pocket was applied to identify the binding compounds. Finally, to minimize chances of false positives, we performed docking of the identified molecules with 100 irrelevant proteins of diverse classes thereby ruling out the non-specific binding. Three FDA-approved drugs showed binding to 3CL-protease either at the catalytic pocket or at an allosteric site related to functionally important dimer formation. A drug-like molecule showed binding to RdRp in its catalytic pocket blocking the key catalytic residues. Two other drug-like molecules showed specific interactions with helicase at a key domain involved in catalysis. This study provides lead drugs or drug-like molecules for further in vitro and clinical investigation for drug repurposing and new drug development prospects.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Reposicionamento de Medicamentos , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Domínio Catalítico , Simulação por Computador , Dimerização , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Pandemias , Inibidores de Proteases/química , Quinoxalinas/farmacologia , Rimantadina/farmacologia , Proteínas Virais/química
4.
Infect Genet Evol ; 84: 104451, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32640381

RESUMO

WHO has declared the outbreak of COVID-19 as a public health emergency of international concern. The ever-growing new cases have called for an urgent emergency for specific anti-COVID-19 drugs. Three structural proteins (Membrane, Envelope and Nucleocapsid protein) play an essential role in the assembly and formation of the infectious virion particles. Thus, the present study was designed to identify potential drug candidates from the unique collection of 548 anti-viral compounds (natural and synthetic anti-viral), which target SARS-CoV-2 structural proteins. High-end molecular docking analysis was performed to characterize the binding affinity of the selected drugs-the ligand, with the SARS-CoV-2 structural proteins, while high-level Simulation studies analyzed the stability of drug-protein interactions. The present study identified rutin, a bioflavonoid and the antibiotic, doxycycline, as the most potent inhibitor of SARS-CoV-2 envelope protein. Caffeic acid and ferulic acid were found to inhibit SARS-CoV-2 membrane protein while the anti-viral agent's simeprevir and grazoprevir showed a high binding affinity for nucleocapsid protein. All these compounds not only showed excellent pharmacokinetic properties, absorption, metabolism, minimal toxicity and bioavailability but were also remain stabilized at the active site of proteins during the MD simulation. Thus, the identified lead compounds may act as potential molecules for the development of effective drugs against SARS-CoV-2 by inhibiting the envelope formation, virion assembly and viral pathogenesis.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Proteínas do Nucleocapsídeo/química , Proteínas do Envelope Viral/química , Proteínas da Matriz Viral/química , Vírion/efeitos dos fármacos , Sequência de Aminoácidos , Antivirais/química , Betacoronavirus/genética , Betacoronavirus/metabolismo , Sítios de Ligação , Ácidos Cafeicos/química , Ácidos Cafeicos/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacologia , Doxiciclina/química , Doxiciclina/farmacologia , Expressão Gênica , Humanos , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas do Nucleocapsídeo/antagonistas & inibidores , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Quinoxalinas/química , Quinoxalinas/farmacologia , Rutina/química , Rutina/farmacologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Simeprevir/química , Simeprevir/farmacologia , Termodinâmica , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Vírion/genética , Vírion/metabolismo
5.
Cancer Immunol Immunother ; 69(11): 2259-2273, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32504246

RESUMO

AKT-inhibition is a promising approach to improve T cell therapies; however, its effect on CD4+ T cells is insufficiently explored. Previously, we and others showed that AKT-inhibition during ex vivo CD8+ T cell expansion facilitates the generation of polyfunctional T cells with stem cell memory-like traits. However, most therapeutic T cell products are generated from lymphocytes, containing CD4+ T cells that can affect CD8+ T cells dependent on the Th-subset. Here, we investigated the effect of AKT-inhibition on CD4+ T cells, during separate as well as total T cell expansions. Interestingly, ex vivo AKT-inhibition preserved the early memory phenotype of CD4+ T cells based on higher CD62L, CXCR4 and CCR7 expression. However, in the presence of AKT-inhibition, Th-differentiation was skewed toward more Th2-associated at the expense of Th1-associated cells. Importantly, the favorable effect of AKT-inhibition on the functionality of CD8+ T cells drastically diminished in the presence of CD4+ T cells. Moreover, also the expansion method influenced the effect of AKT-inhibition on CD8+ T cells. These findings indicate that the effect of AKT-inhibition on CD8+ T cells is dependent on cell composition and expansion strategy, where presence of CD4+ T cells as well as polyclonal stimulation impede the favorable effect of AKT-inhibition.


Assuntos
Benzimidazóis/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Quinoxalinas/farmacologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/imunologia , Células Cultivadas , Humanos
6.
Exp Anim ; 69(4): 388-394, 2020 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-32507787

RESUMO

We recently demonstrated that aspartoacylase (Aspa) and hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (Hcn1) genes were causative of essential tremor (ET) in rats. This finding was obtained using Aspaem34Kyo/Hcn1A354V double-mutant rats, but they were bred on a heterogeneous genetic background of two strains, F344 and WTC. Here, we developed an Aspaem34Kyo/Hcn1em1Kyo double-knockout rat strain with a homogenous F344 genetic background and studied the ability of glutamate receptor antagonists to suppress ET. The F344-Aspa/Hcn1 double-knockout rats exhibited spontaneous, intense body tremor equivalent to that in the double-mutant rats. N-acetyl-aspartate (NAA), a substrate of ASPA, showed accumulation in all brain regions and in the spinal cord. However, N-acetyl-aspartyl-glutamate (NAAG), which is derived from NAA and interacts with glutamatergic receptors, was decreased in the medulla oblongata of the double-knockout rats. The tremor was suppressed by 3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid, an N-methyl-D-aspartate (NMDA) receptor antagonist, in F344-Aspa/Hcn1 double-knockout rats. The non-NMDA glutamate receptor antagonist NBQX weakly inhibited the tremor, while the metabotropic glutamate receptor antagonist LY341495 showed no effect. In addition, both NR2B subunit-specific (Ro 25-6981) and NR2C/NR2D subunit-specific (cis-piperidine dicarboxylic acid) NMDA receptor antagonists suppressed the tremor. These data indicated that the pathogenesis of tremor in Aspa/Hcn1 double-knockout rats involved ionotropic glutamate receptors, particularly NMDA receptors.


Assuntos
Amidoidrolases/genética , Tremor Essencial/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais de Potássio/genética , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/fisiologia , Amidoidrolases/metabolismo , Animais , Encéfalo/metabolismo , Tremor Essencial/tratamento farmacológico , Técnicas de Inativação de Genes , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Terapia de Alvo Molecular , Fenóis/farmacologia , Fenóis/uso terapêutico , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Canais de Potássio/metabolismo , Quinoxalinas/farmacologia , Quinoxalinas/uso terapêutico , Ratos Endogâmicos F344 , Ratos Mutantes , Medula Espinal/metabolismo
7.
Biosci Rep ; 40(6)2020 06 26.
Artigo em Inglês | MEDLINE | ID: covidwho-459137

RESUMO

Due to the lack of efficient therapeutic options and clinical trial limitations, the FDA-approved drugs can be a good choice to handle Coronavirus disease (COVID-19). Many reports have enough evidence for the use of FDA-approved drugs which have inhibitory potential against target proteins of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we utilized a structure-based drug design approach to find possible drug candidates from the existing pool of FDA-approved drugs and checked their effectiveness against the SARS-CoV-2. We performed virtual screening of the FDA-approved drugs against the main protease (Mpro) of SARS-CoV-2, an essential enzyme, and a potential drug target. Using well-defined computational methods, we identified Glecaprevir and Maraviroc (MVC) as the best inhibitors of SARS-CoV-2 Mpro. Both drugs bind to the substrate-binding pocket of SARS-CoV-2 Mpro and form a significant number of non-covalent interactions. Glecaprevir and MVC bind to the conserved residues of substrate-binding pocket of SARS-CoV-2 Mpro. This work provides sufficient evidence for the use of Glecaprevir and MVC for the therapeutic management of COVID-19 after experimental validation and clinical manifestations.


Assuntos
Betacoronavirus/enzimologia , Maraviroc/farmacologia , Inibidores de Proteases/farmacologia , Quinoxalinas/farmacologia , Sulfonamidas/farmacologia , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos/métodos , Maraviroc/química , Maraviroc/metabolismo , Estrutura Molecular , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Quinoxalinas/química , Quinoxalinas/metabolismo , Sulfonamidas/química , Sulfonamidas/metabolismo
8.
Biosci Rep ; 40(6)2020 06 26.
Artigo em Inglês | MEDLINE | ID: covidwho-343226

RESUMO

Due to the lack of efficient therapeutic options and clinical trial limitations, the FDA-approved drugs can be a good choice to handle Coronavirus disease (COVID-19). Many reports have enough evidence for the use of FDA-approved drugs which have inhibitory potential against target proteins of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we utilized a structure-based drug design approach to find possible drug candidates from the existing pool of FDA-approved drugs and checked their effectiveness against the SARS-CoV-2. We performed virtual screening of the FDA-approved drugs against the main protease (Mpro) of SARS-CoV-2, an essential enzyme, and a potential drug target. Using well-defined computational methods, we identified Glecaprevir and Maraviroc (MVC) as the best inhibitors of SARS-CoV-2 Mpro. Both drugs bind to the substrate-binding pocket of SARS-CoV-2 Mpro and form a significant number of non-covalent interactions. Glecaprevir and MVC bind to the conserved residues of substrate-binding pocket of SARS-CoV-2 Mpro. This work provides sufficient evidence for the use of Glecaprevir and MVC for the therapeutic management of COVID-19 after experimental validation and clinical manifestations.


Assuntos
Betacoronavirus/enzimologia , Maraviroc/farmacologia , Inibidores de Proteases/farmacologia , Quinoxalinas/farmacologia , Sulfonamidas/farmacologia , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos/métodos , Maraviroc/química , Maraviroc/metabolismo , Estrutura Molecular , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Quinoxalinas/química , Quinoxalinas/metabolismo , Sulfonamidas/química , Sulfonamidas/metabolismo
9.
Biosci Rep ; 40(6)2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32441299

RESUMO

Due to the lack of efficient therapeutic options and clinical trial limitations, the FDA-approved drugs can be a good choice to handle Coronavirus disease (COVID-19). Many reports have enough evidence for the use of FDA-approved drugs which have inhibitory potential against target proteins of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we utilized a structure-based drug design approach to find possible drug candidates from the existing pool of FDA-approved drugs and checked their effectiveness against the SARS-CoV-2. We performed virtual screening of the FDA-approved drugs against the main protease (Mpro) of SARS-CoV-2, an essential enzyme, and a potential drug target. Using well-defined computational methods, we identified Glecaprevir and Maraviroc (MVC) as the best inhibitors of SARS-CoV-2 Mpro. Both drugs bind to the substrate-binding pocket of SARS-CoV-2 Mpro and form a significant number of non-covalent interactions. Glecaprevir and MVC bind to the conserved residues of substrate-binding pocket of SARS-CoV-2 Mpro. This work provides sufficient evidence for the use of Glecaprevir and MVC for the therapeutic management of COVID-19 after experimental validation and clinical manifestations.


Assuntos
Betacoronavirus/enzimologia , Maraviroc/farmacologia , Inibidores de Proteases/farmacologia , Quinoxalinas/farmacologia , Sulfonamidas/farmacologia , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos/métodos , Maraviroc/química , Maraviroc/metabolismo , Estrutura Molecular , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Quinoxalinas/química , Quinoxalinas/metabolismo , Sulfonamidas/química , Sulfonamidas/metabolismo
10.
Life Sci ; 254: 117819, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32442451

RESUMO

AIMS: Vascular dysfunction plays a key role in sepsis but the role of perivascular adipose tissue (PVAT) in this condition is relatively unknown. MAIN METHODS: Sepsis was induced by cecal ligation and puncture (CLP). The responses of the aorta and superior mesenteric artery to norepinephrine in the presence or absence of PVAT were evaluated. Fluorescent probes measured the production of nitric oxide (NO) and reactive oxygen species (ROS). NO synthases (NOS) and ß3-adrenoceptor expression were detected by immunofluorescence and S-nitrosylation by the biotin switch assay. KEY FINDINGS: Aorta and superior mesenteric arteries from septic animals with intact PVAT showed a worsened response to the vasoconstrictor compared to vessels without PVAT. PVAT from the aorta (APVAT) produced NO and ROS whereas PVAT from the superior mesenteric artery (MPVAT) produced only ROS. Septic APVAT exhibited a higher density of NOS-1 and NOS-3. S-nitrosylation was found in APVAT. Donor (PVAT obtained from normal or septic rats):Host (normal vessel without PVAT) experiments showed that L-NAME, ODQ and ß3-adrenergic receptor antagonist blocked the septic APVAT anti-contractile effect. None of these compounds affected MPVAT; tempol, but not apocynin, blocked its anti-contractile effect. SIGNIFICANCE: PVAT contributes to the anti-contractile effect in the aorta and mesenteric artery of septic rats through different pathways. ß3-Adrenergic receptor and NO appear to be key mediators of this effect in APVAT, but not in MPVAT where ROS seem to be a relevant mediator. Therefore, PVAT is a relevant player of sepsis vascular dysfunction.


Assuntos
Aorta/metabolismo , Artérias Mesentéricas/metabolismo , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Adrenérgicos beta 3/fisiologia , Sepse/fisiopatologia , Acetofenonas/farmacologia , Tecido Adiposo/metabolismo , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Animais , Óxidos N-Cíclicos/farmacologia , Feminino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/metabolismo , Norepinefrina/farmacologia , Oxidiazóis/farmacologia , Fenótipo , Quinoxalinas/farmacologia , Ratos , Receptores Adrenérgicos beta 3/biossíntese , Marcadores de Spin , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia
11.
Res Vet Sci ; 131: 232-243, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32417693

RESUMO

Cyadox, a new antibacterial agent as the quinoxaline-1, 4-dioxides, has a good antibacterial and growth-promoting effect, and has the advantages of lower toxicity, adequate safety and faster absorption. Seven differential expressed genes (DEGs) induced by cyadox were screened in swine liver tissues, including Insulin-like Growth Factor-1 (IGF-1), Epidermal Growth Factor (EGF), Poly ADP-ribose polymerase (PARP), the Defender Against Apoptotic Death 1 (DAD1), Complement Component 3 (C3), Transketolase (TK) and cyadox-related novel gene (CRNG). To elucidate the signal mechanism that cyadox altered these genes expression, the time-effect relationship and signaling pathways related to 7 DEGs induced by cyadox were determined in Porcine Kidney-15 (PK-15) cells by RT-qPCR and the application of various signal pathway inhibitors. The phosphorylation levels of signal factors in PK-15 cells were detected by Western blot. The analyses demonstrated that, the mRNA expressions of 7 DEGs were significantly enhanced by cyadox mainly through the phosphoinositide 3-kinase (PI3K) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) signaling pathways in PK-15 cells. Furthermore, EGF might be the early response gene of cyadox to activate downstream signaling pathways and regulates the expression of other related genes or directly exerting biological effects. In brief, cyadox mainly regulates the expression of these 7 genes by PI3K and NF-кB signaling pathways to exert it's antibacterial and growth-promoting activity in PK-15 cells.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transcrição Genética/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Linhagem Celular , Fator de Crescimento Epidérmico/farmacologia , NF-kappa B/genética , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Quinoxalinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Suínos
12.
Klin Lab Diagn ; 65(4): 244-250, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32227731

RESUMO

The study is devoted to the study of the antimicrobial activity of the antioxidant dioxidin and the complex dioxin-containing preparation Nosolin-ultra, nasal drops against planktonic and biofilm cultures of pathogens of ENT infections, the dynamics of the formation of microbial resistance to dioxidine. 11 reference strains and 9 clinical strains of microorganisms were used in the study: Streptococcus spp., Staphylococcus spp., Micrococcus luteus, Haemophilus influenzae, Acinetobacter pittii, Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa. The antimicrobial activity of preparations against planktonic cultures was determined by serial dilution in broth and spot method on solid nutrient media, against biofilms by the applicator method. The dynamics of dioxidine resistance formation was studied by passaging cultures in a liquid nutrient medium with increasing concentrations of antiseptic. Based on the study, it was found that Dioxidin showed antimicrobial activity against plankton cells of all strains (MBC=0.08-5 mg/ml), except S. pyogenes SN345 (MBC>5 mg/ml), inhibited the growth of formed biofilms (MBC=0.08-2.5 mg/ml) of all strains except S. pyogenes SN345 (MBC>5 mg/ml). The drug «Nosolin-ultra, nasal drops¼ was highly active against plankton cells (MBC=0.04-0.63 mg/ml) and biofilms (MBC=0.02-0.31 mg/ml) of gram-negative bacteria, except A. pittii (MBC>2.5 mg/ml), less active against plankton cells (MBC=1.25-2.5 mg/ml) and biofilms (MBC=0.02-0.31 mg/ml) of gram-positive bacteria and C. albicans. One strain (S. aureus) formed a variant resistant to dioxidine at a concentration of 20 mg/ml, which exceeded the concentration of dioxidine in the complex preparation; other strains (P. aeruginosa, K. pneumoniae, C. albicans) did not form such variants. The data obtained indicate that the drug «Nosolin-ultra, nasal drops¼ can be effectively used against most pathogens of ENT infections. It is worth noting that with prolonged use of the drug for some types of ENT pathogens in the future, a slight decrease in effectiveness may be noted.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Quinoxalinas/farmacologia , Administração Intranasal , Testes de Sensibilidade Microbiana
13.
Artigo em Inglês | MEDLINE | ID: mdl-32292063

RESUMO

Both reactive nitrogen and oxygen species (RNS and ROS), such as nitric oxide, peroxynitrite, and hydrogen peroxide, have been implicated as mediators of pancreatic ß-cell damage during the pathogenesis of autoimmune diabetes. While ß-cells are thought to be vulnerable to oxidative damage due to reportedly low levels of antioxidant enzymes, such as catalase and glutathione peroxidase, we have shown that they use thioredoxin reductase to detoxify hydrogen peroxide. Thioredoxin reductase is an enzyme that participates in the peroxiredoxin antioxidant cycle. Peroxiredoxins are expressed in ß-cells and, when overexpressed, protect against oxidative stress, but the endogenous roles of peroxiredoxins in the protection of ß-cells from oxidative damage are unclear. Here, using either glucose oxidase or menadione to continuously deliver hydrogen peroxide, or the combination of dipropylenetriamine NONOate and menadione to continuously deliver peroxynitrite, we tested the hypothesis that ß-cells use peroxiredoxins to detoxify both of these reactive species. Either pharmacological peroxiredoxin inhibition with conoidin A or specific depletion of cytoplasmic peroxiredoxin 1 (Prdx1) using siRNAs sensitizes INS 832/13 cells and rat islets to DNA damage and death induced by hydrogen peroxide or peroxynitrite. Interestingly, depletion of peroxiredoxin 2 (Prdx2) had no effect. Together, these results suggest that ß-cells use cytoplasmic Prdx1 as a primary defense mechanism against both ROS and RNS.


Assuntos
Dano ao DNA , Peróxido de Hidrogênio/toxicidade , Células Secretoras de Insulina/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas/metabolismo , Ácido Peroxinitroso/toxicidade , Animais , Morte Celular , Linhagem Celular Tumoral , Citoplasma/enzimologia , Citoproteção , Inibidores Enzimáticos/farmacologia , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/patologia , Masculino , Peroxirredoxinas/antagonistas & inibidores , Peroxirredoxinas/genética , Quinoxalinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , Tiorredoxina Redutase 1/metabolismo
14.
Stroke ; 51(5): 1578-1586, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32279622

RESUMO

Background and Purpose- Our recent study demonstrated that release of Prx2 (peroxiredoxin 2) from red blood cells (RBCs) is involved in the inflammatory response and brain injury after intracerebral hemorrhage. The current study investigated the role of extracellular Prx2 in hydrocephalus development after experimental intraventricular hemorrhage. Methods- There were 4 parts in this study. First, Sprague-Dawley rats received an intraventricular injection of lysed RBC or saline and were euthanized at 1 hour for Prx2 measurements. Second, rats received an intraventricular injection of Prx2, deactivated Prx2, or saline. Third, lysed RBC was coinjected with conoidin A, a Prx2 inhibitor, or vehicle. Fourth, rats received Prx2 injection and were treated with minocycline or saline (i.p.). The effects of Prx2 and the inhibitors were examined using magnetic resonance imaging assessing ventriculomegaly, histology assessing ventricular wall damage, and immunohistochemistry to assess inflammation, particularly at the choroid plexus. Results- Intraventricular injection of lysed RBC resulted in increased brain Prx2 and hydrocephalus. Intraventricular injection of Prx2 alone caused hydrocephalus, ventricular wall damage, activation of choroid plexus epiplexus cells (macrophages), and an accumulation of neutrophils. Conoidin A attenuated lysed RBC-induced injury. Systemic minocycline treatment reduced the epiplexus cell activation and hydrocephalus induced by Prx2. Conclusions- Prx2 contributed to the intraventricular hemorrhage-induced hydrocephalus, probably by inducing inflammatory responses in choroid plexus and ventricular wall damage.


Assuntos
Hemorragia Cerebral Intraventricular/metabolismo , Plexo Corióideo/metabolismo , Hidrocefalia/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Peroxirredoxinas/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Hemorragia Cerebral Intraventricular/complicações , Plexo Corióideo/efeitos dos fármacos , Plexo Corióideo/patologia , Modelos Animais de Doenças , Epêndima/efeitos dos fármacos , Epêndima/patologia , Feminino , Hidrocefalia/etiologia , Hylobatidae , Inflamação/patologia , Injeções Intraventriculares , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Minociclina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Peroxirredoxinas/antagonistas & inibidores , Peroxirredoxinas/farmacologia , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley
15.
World Neurosurg ; 139: e455-e462, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32311563

RESUMO

OBJECTIVE: A large body of evidence has suggested that the disruptions of neural plasticity in the brain play a pivotal role in major depressive disorder (MDD). Electroacupuncture (EA) therapy has been shown to be an effective treatment modality for MDD. However, the mechanism underling the antidepressive effect of EA treatment has not been clearly elucidated. This study aimed to investigate the antidepressant-like effects of EA associated with its protection effect of synaptic structural plasticity. METHODS: An MDD model was induced by exposing Sprague Dawley rats to chronic unpredictable mild stress (CUMS). EA stimulation (Hegu and Taichong) and AMPA receptor (AMPAR) antagonist NBQX intrahippocampal injection were used to treat the depressed rats. RESULTS: We found EA improved behavioral performance, enhanced synaptic structural plasticity, and upregulated gene and protein levels of GluR1, GluR2, Stargazin, Pick1, SYP, PSD-95, and GAP-43. AMPAR antagonist NBQX had the opposite effect on behavioral performance, synaptic plasticity, and the aforementioned genes and proteins. CONCLUSIONS: These results suggest that EA has a potent antidepressant effect, likely through upregulated expression of the AMPAR and protected neural plasticity in CUMS-treated rats.


Assuntos
Depressão/terapia , Eletroacupuntura/métodos , Hipocampo , Receptores de AMPA , Estresse Psicológico/complicações , Sinapses/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Doença Crônica , Depressão/etiologia , Depressão/psicologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Masculino , Plasticidade Neuronal , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/antagonistas & inibidores , Estresse Psicológico/psicologia
16.
PLoS One ; 15(3): e0230230, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32214328

RESUMO

OBJECTIVE: To investigate the effect and mechanism of SB525334 on self-renewal, migration and invasion of ovarian cancer stem cells. METHODS: ALDHhigh-expressing cancer stem cells (CSCs) were isolated from human ovarian cancer cell line SKOV-3 by flow cytometry and treated with 2µg/mL SB525334 for 6h. The sphere forming assay was used to detect the ability of self-renewal of CSCs and the colony formation assay was used to detect the tumorigenicity in vitro. Transwell migration and invasion assay were used to detect the migration and invasion ability of CSCs. To further explore the mechanism, real-time quantitative PCR and flow cytometry were used to detect the mRNA and protein expression of TGF-ß, Smad2, Smad3, phosphorylated Smad2, phosphorylated Smad3 and Smad4, respectively. Expressions of epithelial-mesenchymal transition (EMT)-related genes E-cadherin, Snail, Vimentin were also assessed. RESULTS: The self-renewal ability, tumorigenicity in vitro, migration and invasion ability of CSCs were significantly attenuated after SB525334 treatment. The expressions of TGF-ß, phosphorylated Smad2, phosphorylated Smad3, Snail, and Vimentin were decreased, while Smad4 and E-cadherin expressions were increased. CONCLUSION: SB525334 may inhibit the self-renewal, invasion and migration of ovarian CSCs by blocking the TGF-ß/Smad/EMT pathway.


Assuntos
Movimento Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Imidazóis/farmacologia , Invasividade Neoplásica/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Quinoxalinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas Smad/metabolismo , Vimentina/metabolismo
17.
Int J Mol Sci ; 21(3)2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32046095

RESUMO

The JAK-STAT signalling pathway regulates cellular processes like cell division, cell death and immune regulation. Dysregulation has been identified in solid tumours and STAT3 activation is a marker for poor outcome. The aim of this study was to explore potential therapeutic strategies by targeting this pathway in bladder cancer (BC). High STAT3 expression was detected in 51.3% from 149 patient specimens with invasive bladder cancer by immunohistochemistry. Protein expression of JAK, STAT and downstream targets were confirmed in 10 cell lines. Effects of the JAK inhibitors Ruxolitinib and BSK-805, and STAT3/5 inhibitors Stattic, Nifuroxazide and SH-4-54 were analysed by cell viability assays, immunoblotting, apoptosis and cell cycle progression. Treatment with STAT3/5 but not JAK1/2 inhibitors reduced survival, levels of phosphorylated STAT3 and Cyclin-D1 and increased apoptosis. Tumour xenografts, using the chicken chorioallantoic membrane (CAM) model responded to Stattic monotherapy. Combination of Stattic with Cisplatin, Docetaxel, Gemcitabine, Paclitaxel and CDK4/6 inhibitors showed additive effects. The combination of Stattic with the oncolytic adenovirus XVir-N-31 increased viral replication and cell lysis. Our results provide evidence that inhibitors against STAT3/5 are promising as novel mono- and combination therapy in bladder cancer.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Terapia Viral Oncolítica/métodos , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT6/antagonistas & inibidores , Neoplasias da Bexiga Urinária/terapia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Embrião de Galinha , Terapia Combinada/métodos , Óxidos S-Cíclicos/farmacologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Humanos , Hidroxibenzoatos/farmacologia , Janus Quinases/antagonistas & inibidores , Nitrofuranos/farmacologia , Pirazóis/farmacologia , Quinoxalinas/farmacologia , Neoplasias da Bexiga Urinária/metabolismo
18.
Am J Health Syst Pharm ; 77(5): 346-351, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32073123

RESUMO

PURPOSE: To provide an overview of fibroblast growth factor receptor (FGFR) gene alterations and the pharmacology, clinical effectiveness, dosage and administration, cost, and place in therapy of erdafitinib in bladder cancer. SUMMARY: Erdafitinib (Balversa, Janssen Pharmaceuticals) is a novel pan-FGFR inhibitor recently approved for the treatment of patients with advanced urothelial cancer with specific FGFR genetic alterations who have received at least one prior platinum-containing regimen. Erdafitinib binding to the FGFR2 and FGFR3 receptors inhibits FGF activity, resulting in cell death. Erdafitinib is available in tablet form, and the current recommended daily dosing is 8 mg, with dose escalation to 9 mg after 14 to 21 days of therapy if tolerated. A phase 2 clinical trial demonstrated that patients who received erdafitinib experienced on average 5.5 months of progression-free survival (95% confidence interval [CI], 4.2-6.0 months). In addition, 40% (95% CI, 31-50%) of patients responded to erdafitinib therapy. Patients receiving erdafitinib therapy should be monitored specifically for elevations in serum phosphate levels and changes in vision. Other adverse effects include anemia, thrombocytopenia, and electrolyte abnormalities. CONCLUSION: Erdafitinib is the first small-molecule FGFR inhibitor approved for use in advanced bladder cancer.


Assuntos
Antineoplásicos/administração & dosagem , Pirazóis/administração & dosagem , Quinoxalinas/administração & dosagem , Neoplasias da Bexiga Urinária/tratamento farmacológico , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Carcinoma de Células de Transição/tratamento farmacológico , Carcinoma de Células de Transição/patologia , Humanos , Intervalo Livre de Progressão , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/efeitos adversos , Pirazóis/farmacologia , Quinoxalinas/efeitos adversos , Quinoxalinas/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Neoplasias da Bexiga Urinária/patologia
19.
Int J Mol Sci ; 21(4)2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-32070042

RESUMO

Cortical spreading depression (CSD) is a propagating wave of depolarization followed by depression of cortical activity. CSD triggers neuroinflammation via the pannexin-1 (Panx1) channel opening, which may eventually cause migraine headaches. However, the regulatory mechanism of Panx1 is unknown. This study investigates whether sarcoma family kinases (SFK) are involved in transmitting CSD-induced Panx1 activation, which is mediated by the NR2A-containing N-methyl-D-aspartate receptor. CSD was induced by topical application of K+ to cerebral cortices of rats and mouse brain slices. SFK inhibitor, PP2, or NR2A-receptor antagonist, NVP-AAM077, was perfused into contralateral cerebral ventricles (i.c.v.) of rats prior to CSD induction. Co-immunoprecipitation and Western blot were used for detecting protein interactions, and histofluorescence for addressing Panx1 activation. The results demonstrated that PP2 attenuated CSD-induced Panx1 activation in rat ipsilateral cortices. Cortical susceptibility to CSD was reduced by PP2 in rats and by TAT-Panx308 that disrupts SFK-Panx1 interaction in mouse brain slices. Furthermore, CSD promoted activated SFK coupling with Panx1 in rat ipsilateral cortices. Moreover, inhibition of NR2A by NVP-AAM077 reduced elevation of ipsilateral SFK-Panx1 interaction, Panx1 activation induced by CSD and cortical susceptibility to CSD in rats. These data suggest NR2A-regulated, SFK-dependent Panx1 activity plays an important role in migraine aura pathogenesis.


Assuntos
Conexinas/genética , Transtornos de Enxaqueca/tratamento farmacológico , Proteínas do Tecido Nervoso/genética , Fosfotransferases/genética , Receptores de N-Metil-D-Aspartato/genética , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Conexinas/antagonistas & inibidores , Depressão Alastrante da Atividade Elétrica Cortical/efeitos dos fármacos , Humanos , Masculino , Camundongos , Transtornos de Enxaqueca/genética , Transtornos de Enxaqueca/patologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Pirimidinas/farmacologia , Quinoxalinas/farmacologia , Ratos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores
20.
J Neurosci ; 40(13): 2737-2752, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32075899

RESUMO

Microinjections of a glutamate AMPA antagonist (DNQX) in medial shell of nucleus accumbens (NAc) can cause either intense appetitive motivation (i.e., 'desire') or intense defensive motivation (i.e., 'dread'), depending on site along a flexible rostrocaudal gradient and on environmental ambience. DNQX, by blocking excitatory AMPA glutamate inputs, is hypothesized to produce relative inhibitions of NAc neurons. However, given potential alternative explanations, it is not known whether neuronal inhibition is in fact necessary for NAc DNQX microinjections to generate motivations. Here we provide a direct test of whether local neuronal inhibition in NAc is necessary for DNQX microinjections to produce either desire or dread. We used optogenetic channelrhodopsin (ChR2) excitations at the same local sites in NAc as DNQX microinjections to oppose relative neuronal inhibitions induced by DNQX in female and male rats. We found that same-site ChR2 excitation effectively reversed the ability of NAc DNQX microinjections to generate appetitive motivation, and similarly reversed ability of DNQX microinjections to generate defensive motivation. Same-site NAc optogenetic excitations also attenuated recruitment of Fos expression in other limbic structures throughout the brain, which was otherwise elevated by NAc DNQX microinjections that generated motivation. However, to successfully reverse motivation generation, an optic fiber tip for ChR2 illumination needed to be located within <1 mm of the corresponding DNQX microinjector tip; that is, both truly at the same NAc site. Thus, we confirm that localized NAc neuronal inhibition is required for AMPA-blocking microinjections in medial shell to induce either positively-valenced 'desire' or negatively-valenced 'dread'.SIGNIFICANCE STATEMENT A major hypothesis posits neuronal inhibitions in nucleus accumbens generate intense motivation. Microinjections in nucleus accumbens of glutamate antagonist, DNQX, which might suppress local neuronal firing, generate either appetitive or defensive motivation, depending on site and environmental factors. Is neuronal inhibition in nucleus accumbens required for such pharmacologically-induced motivations? Here we demonstrate that neuronal inhibition is necessary to generate appetitive or defensive motivations, using local optogenetic excitations to oppose putative DNQX-induced inhibitions. We show that excitation at the same site prevents DNQX microinjections from recruiting downstream limbic structures into neurobiological activation, and simultaneously prevents generation of either appetitive or defensive motivated behaviors. These results may be relevant to roles of nucleus accumbens mechanisms in pathological motivations, including addiction and paranoia.


Assuntos
Inibição Psicológica , Motivação/fisiologia , Núcleo Accumbens/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Masculino , Microinjeções , Motivação/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos , Optogenética , Quinoxalinas/farmacologia , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley
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