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1.
Mol Syst Biol ; 17(8): e10239, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34339582

RESUMO

Understanding the mechanism of SARS-CoV-2 infection and identifying potential therapeutics are global imperatives. Using a quantitative systems pharmacology approach, we identified a set of repurposable and investigational drugs as potential therapeutics against COVID-19. These were deduced from the gene expression signature of SARS-CoV-2-infected A549 cells screened against Connectivity Map and prioritized by network proximity analysis with respect to disease modules in the viral-host interactome. We also identified immuno-modulating compounds aiming at suppressing hyperinflammatory responses in severe COVID-19 patients, based on the transcriptome of ACE2-overexpressing A549 cells. Experiments with Vero-E6 cells infected by SARS-CoV-2, as well as independent syncytia formation assays for probing ACE2/SARS-CoV-2 spike protein-mediated cell fusion using HEK293T and Calu-3 cells, showed that several predicted compounds had inhibitory activities. Among them, salmeterol, rottlerin, and mTOR inhibitors exhibited antiviral activities in Vero-E6 cells; imipramine, linsitinib, hexylresorcinol, ezetimibe, and brompheniramine impaired viral entry. These novel findings provide new paths for broadening the repertoire of compounds pursued as therapeutics against COVID-19.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos/métodos , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , COVID-19/genética , COVID-19/virologia , Chlorocebus aethiops , Reposicionamento de Medicamentos , Células HEK293 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Imidazóis/farmacologia , Pirazinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/patogenicidade , Xinafoato de Salmeterol/farmacologia , Células Vero
2.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360605

RESUMO

Piezo1/2 are mechanosensitive calcium-permeable channels that can be activated by various modes of membrane deformation. The identification of the small molecule Yoda1, a synthetic Piezo1 agonist, revealed the possibility of chemical activation of the channel. Stimulating effects of Yoda1 on Piezo1 have been mainly documented using over-expressing cellular systems or channel proteins incorporated in artificial lipid bilayers. However, the activating effect of Yoda1 on native Piezo1 channels in the plasma membrane of living cells remains generally undefined, despite the increasing number of studies in which the agonist is utilized as a functional tool to reveal the contribution of Piezo1 to cellular reactions. In the current study, we used the human myeloid leukemia K562 cell line as a suitable model to examine chemically induced Piezo1 activity with the use of the patch-clamp technique in various specific modes. The functional expression of Piezo1 in leukemia cells was evidenced using a combinative approach, including single channel patch-clamp measurements. Utilizing our established single-current whole-cell assay on K562 cells, we have shown, for the first time, the selective real-time chemical activation of endogenously expressed Piezo1. Extracellular application of 0.5-1 µM Yoda1 effectively stimulated single Piezo1 currents in the cell membrane.


Assuntos
Membrana Celular/metabolismo , Canais Iônicos/efeitos dos fármacos , Leucemia/tratamento farmacológico , Mecanotransdução Celular , Pirazinas/farmacologia , Análise de Célula Única/métodos , Tiadiazóis/farmacologia , Membrana Celular/efeitos dos fármacos , Humanos , Canais Iônicos/agonistas , Canais Iônicos/metabolismo , Leucemia/metabolismo , Leucemia/patologia
3.
Viruses ; 13(7)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34372559

RESUMO

The human BK polyomavirus (BKPyV) is latent in the kidneys of most adults, but can be reactivated in immunosuppressed states, such as following renal transplantation. If left unchecked, BK polyomavirus nephropathy (PyVAN) and possible graft loss may result from viral destruction of tubular epithelial cells and interstitial fibrosis. When coupled with regular post-transplant screening, immunosuppression reduction has been effective in limiting BKPyV viremia and the development of PyVAN. Antiviral drugs that are safe and effective in combating BKPyV have not been identified but would be a benefit in complementing or replacing immunosuppression reduction. The present study explores inhibition of the host DNA damage response (DDR) as an antiviral strategy. Immunohistochemical and immunofluorescent analyses of PyVAN biopsies provide evidence for stimulation of a DDR in vivo. DDR pathways were also stimulated in vitro following BKPyV infection of low-passage human renal proximal tubule epithelial cells. The role of Chk1, a protein kinase known to be involved in the replication stress-induced DDR, was examined by inhibition with the small molecule LY2603618 and by siRNA-mediated knockdown. Inhibition of Chk1 resulted in decreased replication of BKPyV DNA and viral spread. Activation of mitotic pathways was associated with the reduction in BKPyV replication. Chk1 inhibitors that are found to be safe and effective in clinical trials for cancer should also be evaluated for antiviral activity against BKPyV.


Assuntos
Vírus BK/genética , Quinase 1 do Ponto de Checagem/metabolismo , Infecções por Polyomavirus/tratamento farmacológico , Vírus BK/patogenicidade , Células Cultivadas , Quinase 1 do Ponto de Checagem/fisiologia , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Humanos , Rim/patologia , Rim/virologia , Transplante de Rim , Compostos de Fenilureia/farmacologia , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/imunologia , Pirazinas/farmacologia , Infecções Tumorais por Vírus/tratamento farmacológico , Infecções Tumorais por Vírus/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia
4.
Molecules ; 26(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34443411

RESUMO

Several novel methyl 7-[(hetero)arylamino]thieno[2,3-b]pyrazine-6-carboxylates were synthesized by Pd-catalyzed C-N Buchwald-Hartwig cross-coupling of either methyl 7-aminothieno[3,2-b]pyrazine-6-carboxylate with (hetero)arylhalides or 7-bromothieno[2,3-b]pyrazine-6-carboxylate with (hetero)arylamines in good-to-excellent yields (50% quantitative yield), using different reaction conditions, namely ligands and solvents, due to the different electronic character of the substrates. The antitumoral potential of these compounds was evaluated in four human tumor cell lines: gastric adenocarcinoma (AGS), colorectal adenocarcinoma (CaCo-2), breast carcinoma (MCF7), and non-small-cell lung carcinoma (NCI-H460) using the SRB assay, and it was possible to establish some structure-activity relationships. Furthermore, they did not show relevant toxicity against a non-tumor cell line culture from the African green monkey kidney (Vero). The most promising compounds (GI50 ≤ 11 µM), showed some selectivity either against AGS or CaCo-2 cell lines without toxicity at their GI50 values. The effects of the methoxylated compounds 2b (2-OMeC6H4), 2f and 2g (3,4- or 3,5-diOMeC6H3, respectively) on the cell cycle profile and induction of apoptosis were further studied in the AGS cell line. Nevertheless, even for the most active (GI50 = 7.8 µM) and selective compound (2g) against this cell line, it was observed that a huge number of dead cells gave rise to an atypical distribution on the cell cycle profile and that these cells were not apoptotic, which points to a different mechanism of action for the AGS cell growth inhibition.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Apoptose , Pontos de Checagem do Ciclo Celular , Pirazinas/síntese química , Pirazinas/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Pirazinas/química
6.
Nat Commun ; 12(1): 4360, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272384

RESUMO

The glucocorticoid receptor (GR) regulates gene expression, governing aspects of homeostasis, but is also involved in cancer. Pharmacological GR activation is frequently used to alleviate therapy-related side-effects. While prior studies have shown GR activation might also have anti-proliferative action on tumours, the underpinnings of glucocorticoid action and its direct effectors in non-lymphoid solid cancers remain elusive. Here, we study the mechanisms of glucocorticoid response, focusing on lung cancer. We show that GR activation induces reversible cancer cell dormancy characterised by anticancer drug tolerance, and activation of growth factor survival signalling accompanied by vulnerability to inhibitors. GR-induced dormancy is dependent on a single GR-target gene, CDKN1C, regulated through chromatin looping of a GR-occupied upstream distal enhancer in a SWI/SNF-dependent fashion. These insights illustrate the importance of GR signalling in non-lymphoid solid cancer biology, particularly in lung cancer, and warrant caution for use of glucocorticoids in treatment of anticancer therapy related side-effects.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Cromatina/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Glucocorticoides/farmacologia , Neoplasias Pulmonares/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/genética , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imidazóis/farmacologia , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Camundongos , Proteômica , Pirazinas/farmacologia , RNA Interferente Pequeno , RNA-Seq , Receptor IGF Tipo 1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
J Gen Virol ; 102(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34242156

RESUMO

Bactrian camel hepatitis E virus (HEV) is a novel HEV belonging to genotype 8 (HEV-8) in the Orthohepevirus A species of the genus Hepevirus in the family Hepeviridae. HEV-8 cross-transmits to cynomolgus monkeys and has a potential risk for zoonotic infection. Until now, neither a cell-culture system to grow the virus nor a reverse genetics system to generate the virus has been developed. To generate replication-competent HEV-8 and to establish a cell-culture system, we synthesized capped genomic HEV-8 RNAs by in vitro transcription and used them to transfect into PLC/PRF/5 cells. A HEV-8 strain, HEV-8M2, was recovered from the capped HEV-8 RNA-transfected cell-culture supernatants and subsequently passaged in the cells, demonstrating that PLC/PRF/5 cells were capable of supporting the replication of the HEV-8, and that a cell-culture system for HEV-8 was successfully established. In addition to PLC/PRF/5 cells, A549 and Caco-2 cells appeared to be competent for the replication, but HepG2 C3/A, Vero, Hela S3, HEp-2C, 293T and GL37 cells were incompetent. The HEV-8M2 strain was capable of infecting cynomolgus monkeys by an intravenous inoculation, indicating that HEV-8 was infectious and again carried a risk for zoonotic infection. In contrast, HEV-8 did not infect nude rats and BALB/c nude mice, suggesting that the reservoir of HEV-8 was limited. In addition, the replication of the HEV-8M2 strain was efficiently abrogated by ribavirin but not by favipiravir, suggesting that ribavirin is a drug candidate for therapeutic treatment of HEV-8-induced hepatitis. The infectious HEV-8 produced by a reverse genetics system would be useful to elucidate the mechanisms of HEV replication and the pathogenesis of type E hepatitis.


Assuntos
Vírus da Hepatite E/genética , Vírus da Hepatite E/fisiologia , Hepatite E/virologia , Genética Reversa , Amidas/farmacologia , Animais , Antivirais/farmacologia , Proteínas do Capsídeo/análise , Linhagem Celular , Feminino , Genoma Viral , Vírus da Hepatite E/efeitos dos fármacos , Vírus da Hepatite E/patogenicidade , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Nus , Pirazinas/farmacologia , RNA Viral/genética , Ratos , Ribavirina/farmacologia , Transfecção , Replicação Viral/efeitos dos fármacos
8.
Fitoterapia ; 153: 104962, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34139315

RESUMO

Infectious diseases are reported to be one of the major causes of death in the world. The World Health Organization (WHO) warns of an increase in the deaths number because of antibacterial resistance. Lately, a trend towards searching for new active antibacterial compounds in plants has been observed. Ilex paraguariensis, known as Yerba Mate, is a plant known to be rich in numerous bioactive compounds that have an important role in human health. In this study, Yerba Mate was extracted with acetone: water (1:1) and further fractionated with hexane, chloroform and ethyl acetate. The obtained fractions were tested for antibacterial activity against Staphylococcus aureus and Salmonella species. The minimum inhibitory concentration (MIC) values on S. aureus ranged from 1.56 to 3.12 mg/mL for both the chloroform and ethyl acetate fractions. Whereas for the water fraction, the MIC values ranged from 0.78 to 3.12 mg/mL on S. aureus and ranged from 1.56 mg/mL to 3.12 mg/mL on Salmonella species. The aqueous fraction was further treated with different enzymes to mimic in vivo digestion and the fractions obtained were then tested for antibacterial activity. Furthermore, the Yerba Mate aqueous fraction was run on High Performance Liquid Chromatography (HPLC) and collected fractions were tested for antibacterial activity, to identify the active metabolite. Fraction 3 was tested on different strains of S. aureus and the MIC values ranged from 0.19 to 1.56 µg/mL. A novel pyrazinone, Libanstin, from Ilex paraguariensis was identified using NMR spectroscopy.


Assuntos
Antibacterianos/farmacologia , Ilex paraguariensis/química , Pirazinas/farmacologia , Antibacterianos/isolamento & purificação , Testes de Sensibilidade Microbiana , Estrutura Molecular , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Folhas de Planta/química , Pirazinas/isolamento & purificação , Salmonella/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos
9.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34071893

RESUMO

Insulin-like growth factor 1 receptor (IGF1R), a receptor-type tyrosine kinase, transduces signals related to cell proliferation, survival, and differentiation. We recently reported that OSI-906, an IGF1R inhibitor, in combination with the Aurora B inhibitor ZM447439 suppresses cell proliferation. However, the mechanism underlying this suppressive effect is yet to be elucidated. In this study, we examined the effects of combination treatment with OSI-906 and ZM447439 on cell division, so as to understand how cell proliferation was suppressed. Morphological analysis showed that the combination treatment generated enlarged cells with aberrant nuclei, whereas neither OSI-906 nor ZM447439 treatment alone caused this morphological change. Flow cytometry analysis indicated that over-replicated cells were generated by the combination treatment, but not by the lone treatment with either inhibitors. Time-lapse imaging showed mitotic slippage following a severe delay in chromosome alignment and cytokinesis failure with furrow regression. Furthermore, in S-trityl-l-cysteine-treated cells, cyclin B1 was precociously degraded. These results suggest that the combination treatment caused severe defect in the chromosome alignment and spindle assembly checkpoint, which resulted in the generation of over-replicated cells. The generation of over-replicated cells with massive aneuploidy may be the cause of reduction of cell viability and cell death. This study provides new possibilities of cancer chemotherapy.


Assuntos
Aurora Quinase B/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Ciclina B1/metabolismo , Imidazóis/farmacologia , Mitose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/farmacologia , Benzamidas/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteólise , Quinazolinas/farmacologia , Receptor IGF Tipo 1/metabolismo
10.
Eur J Med Chem ; 221: 113515, 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-33984806

RESUMO

Cation-π interaction is a type of noncovalent interaction formed between the π-electron system and the positively charged ion or moieties. In this study, we designed a series of novel NQO1 substrates by introducing aliphatic nitrogen-containing side chains to fit with the L-shaped pocket of NQO1 by the formation of cation-π interactions. Molecular dynamics (MD) simulation indicated that the basic N atom in the side chain of NQO1 substrates, which is prone to be protonated under physiological conditions, can form cation-π interactions with the Phe232 and Phe236 residues of the NQO1 enzyme. Compound 4 with a methylpiperazinyl substituent was identified as the most efficient substrate for NQO1 with the reduction rate and catalytic efficiency of 1263 ± 61 µmol NADPH/min/µmol NQO1 and 2.8 ± 0.3 × 106 M-1s-1, respectively. Notably, compound 4 exhibited increased water solubility (110 µg/mL) compared to that of ß-lap (43 µg/mL), especially under acidic condition (pH = 3, solubility > 1000 µg/mL). Compound 4 (IC50/A549 = 2.4 ± 0.6 µM) showed potent antitumor activity against NQO1-rich cancer cells through ROS generation via NQO1-mediated redox cycling. These results emphasized that the application of cation-π interactions by introducing basic aliphatic amine moiety is beneficial for both the water solubility and the NQO1-substrate binding, leading to promising NQO1-targeting antitumor candidates with improved druglike properties.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Simulação de Dinâmica Molecular , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , Pirazinas/farmacologia , Apoptose/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Cátions/síntese química , Cátions/química , Cátions/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , NAD(P)H Desidrogenase (Quinona)/química , NAD(P)H Desidrogenase (Quinona)/metabolismo , Pirazinas/síntese química , Pirazinas/química , Relação Estrutura-Atividade , Especificidade por Substrato
11.
Viruses ; 13(4)2021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33805122

RESUMO

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that causes an important disease in ruminants, with great economic losses. The infection can be also transmitted to humans; therefore, it is considered a major threat to both human and animal health. In a previous work, we described a novel RVFV variant selected in cell culture in the presence of the antiviral agent favipiravir that was highly attenuated in vivo. This variant displayed 24 amino acid substitutions in different viral proteins when compared to its parental viral strain, two of them located in the NSs protein that is known to be the major virulence factor of RVFV. By means of a reverse genetics system, in this work we have analyzed the effect that one of these substitutions, P82L, has in viral attenuation in vivo. Rescued viruses carrying this single amino acid change were clearly attenuated in BALB/c mice while their growth in an interferon (IFN)-competent cell line as well as the production of interferon beta (IFN-ß) did not seem to be affected. However, the pattern of nuclear NSs accumulation was modified in cells infected with the mutant viruses. These results highlight the key role of the NSs protein in the modulation of viral infectivity.


Assuntos
Substituição de Aminoácidos , Febre do Vale de Rift/prevenção & controle , Vírus da Febre do Vale do Rift/química , Vírus da Febre do Vale do Rift/imunologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Amidas/farmacologia , Animais , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Células HEK293 , Humanos , Rim/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pirazinas/farmacologia , Genética Reversa , Febre do Vale de Rift/imunologia , Vírus da Febre do Vale do Rift/efeitos dos fármacos , Vírus da Febre do Vale do Rift/genética , Células Vero , Virulência , Fatores de Virulência/genética
12.
Commun Biol ; 4(1): 370, 2021 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-33854168

RESUMO

Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencing in aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer. We previously reported a key role for the oncogene BMI-1 in adenocarcinomas. We therefore investigated the effects of in vivo PTC596 treatment, which affects BMI-1 activity, in our murine model. Post-treatment, MRI analysis showed decreased tumor size, while single cell transcriptomics concomitantly detected near complete ablation of the mutant-KRAS-associated subpopulation, signifying the presence of a pharmacologically targetable, tumor-associated subpopulation. Our findings therefore hold promise for the development of a targeted therapy for KRAS-mutant adenocarcinomas.


Assuntos
Benzimidazóis/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Células Epiteliais/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirazinas/farmacologia , Células A549 , Animais , Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Terapia de Alvo Molecular , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA-Seq , Análise de Célula Única , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Aging (Albany NY) ; 13(7): 9991-10014, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33795523

RESUMO

Human Mesenchymal stem cells (hMSCs) are multi-potential cells which are widely used in cell therapy. However, the frequently emerged senescence and decrease of differentiation capabilities limited the broad applications of MSC. Several strategies such as small molecules treatment have been widely studied and used to improve the stem characteristics bypassing the senescence but the exact mechanisms for them to reduce senescence have not been fully studied. In this study, hMSCs were treated by rapamycin, oltipraz, metformin, and vitamin C for the indicated time and these cells were subjected to senescence evaluation and trilineage differentiation. Furthermore, transcriptomics and lipidomics datasets of hMSCs after drug treatment were analyzed to interpret biological pathways responsible for their anti-senescence effects. Although four drugs exhibited significant activities in promoting MSC osteogenic differentiation, metformin is the optimal drug to promote trilineage differentiation. GO terms illustrated that the anti-aging effects of drugs were mainly associated with cellular senescence, mitotic and meiosis process. Biosynthesis of phosphatidylcholines (PC) and phosphatidylethanolamine (PE) were inhibited whereas production of phosphatidylinositols (PIs) and saturated fatty acids (SFA)/ mono-unsaturated fatty acids (MUFA) conversion was activated. Medium free fatty acids (FFA) was increased in hMSCs with different anti-aging phenotypes. Therefore, we established a comprehensive method in assessing drug intervention based on the results of transcriptomics and lipidomics. The method can be used to study different biological phenotypes upon drug intervention in MSC which will extend the clinical application of hMSCs.


Assuntos
Diferenciação Celular/fisiologia , Senescência Celular/fisiologia , Células-Tronco Mesenquimais/metabolismo , Transcriptoma , Ácido Ascórbico/farmacologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Lipidômica , Células-Tronco Mesenquimais/efeitos dos fármacos , Metformina/farmacologia , Pirazinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Sirolimo/farmacologia , Tionas/farmacologia , Tiofenos/farmacologia
14.
Nat Chem ; 13(5): 451-457, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33875818

RESUMO

Recent advances in end-to-end continuous-flow synthesis are rapidly expanding the capabilities of automated customized syntheses of small-molecule pharmacophores, resulting in considerable industrial and societal impacts; however, many hurdles persist that limit the number of sequential steps that can be achieved in such systems, including solvent and reagent incompatibility between individual steps, cumulated by-product formation, risk of clogging and mismatch of timescales between steps in a processing chain. To address these limitations, herein we report a strategy that merges solid-phase synthesis and continuous-flow operation, enabling push-button automated multistep syntheses of active pharmaceutical ingredients. We demonstrate our platform with a six-step synthesis of prexasertib in 65% isolated yield after 32 h of continuous execution. As there are no interactions between individual synthetic steps in the sequence, the established chemical recipe file was directly adopted or slightly modified for the synthesis of twenty-three prexasertib derivatives, enabling both automated early and late-stage diversification.


Assuntos
Técnicas de Química Sintética/métodos , Pirazinas/uso terapêutico , Pirazóis/uso terapêutico , Técnicas de Síntese em Fase Sólida/métodos , Humanos , Pirazinas/farmacologia , Pirazóis/farmacologia
15.
Theranostics ; 11(11): 5127-5142, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33859738

RESUMO

Hypoxic microenvironment is a hallmark of solid tumors, especially glioblastoma. The strong reliance of glioma-propagating cells (GPCs) on hypoxia-induced survival advantages is potentially exploitable for drug development. Methods: To identify key signaling pathways for hypoxia adaptation by patient-derived GPCs, we performed a kinase inhibitor profiling by screening 188 small molecule inhibitors against 130 different kinases in normoxia and hypoxia. Potential kinase candidates were prioritized for in vitro and in vivo investigations using a ranking algorithm that integrated information from the kinome connectivity network and estimated patients' survival based on expression status. Results: Hypoxic drug screen highlighted extensive modifications of kinomic landscape and a crucial functionality of c-MET-PI3K. c-MET inhibitors diminished phosphorylation of c-MET and PI3K in GPCs subjected to hypoxia, suggesting its role in the hypoxic adaptation of GPCs. Mechanistically, the inhibition of c-MET and PI3K impaired antioxidant defense, leading to oxidative catastrophe and apoptosis. Repurposed c-MET inhibitors PF04217903 and tivantinib exhibited hypoxic-dependent drug synergism with temozolomide, resulting in reduced tumor load and growth of GPC xenografts. Detailed analysis of bulk and single-cell glioblastoma transcriptomes associates the cellular subpopulation over-expressing c-MET with inflamed, hypoxic, metastatic, and stem-like phenotypes. Conclusions: Thus, our "bench to bedside (the use of patient-derived GPCs and xenografts for basic research) and back (validation with independent glioblastoma transcriptome databases)" analysis unravels the novel therapeutic indications of c-MET and PI3K/Akt inhibitors for the treatment of glioblastoma, and potentially other cancers, in the hypoxic tumor microenvironment.


Assuntos
Glioma/genética , Hipóxia/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-met/genética , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioma/tratamento farmacológico , Humanos , Hipóxia/tratamento farmacológico , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Pirazinas/farmacologia , Pirrolidinonas/farmacologia , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Temozolomida/farmacologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Triazóis/farmacologia
16.
Expert Opin Drug Saf ; 20(7): 791-799, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33853481

RESUMO

INTRODUCTION: FLT3 inhibitors are important drugs in the therapy of FLT3 positive acute myeloid leukemia (AML). Midostaurin was registered in combination with chemotherapy to treat newly diagnosed AML. Gilteritinib and quizartinib demonstrate effectiveness in a randomized trial in relapsed/refractory AML. Several promising FLT3 inhibitors are being evaluated in clinical research. AREAS COVERED: This review will report the safety of FLT3 inhibitors that are registered for acute myeloid leukemia induction and rescue therapy. EXPERT OPINION: In the near future, it is possible that all the FLT3 positive non M3-AML patients will receive a FLT3 inhibitor. Therapy adherence and strategies to mitigate adverse events must be pursued. The treatment with FLT3 inhibitors may be optimized in terms of toxicities with a rational evaluation of antifungal prophylaxis and concomitant therapy, cardiology monitoring, and keeping in mind rare adverse events. Future studies on unfit patients, special populations, and maintenance settings are warranted, together with post-market studies and real-life experiences. Whenever new FLT3 inhibitors will come to the clinic, we could face a scenario in which profound knowledge of effectiveness, toxicities, and off-target effects will be relevant to choose the best drug for each patient.


Assuntos
Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Compostos de Anilina/administração & dosagem , Compostos de Anilina/efeitos adversos , Compostos de Anilina/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Benzotiazóis/administração & dosagem , Benzotiazóis/efeitos adversos , Benzotiazóis/farmacologia , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/patologia , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/efeitos adversos , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/administração & dosagem , Pirazinas/efeitos adversos , Pirazinas/farmacologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Estaurosporina/administração & dosagem , Estaurosporina/efeitos adversos , Estaurosporina/análogos & derivados , Estaurosporina/farmacologia
17.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802742

RESUMO

New anti-inflammatory treatments are needed for CF airway disease. Studies have implicated the endoplasmic reticulum stress transducer inositol requiring enzyme 1α (IRE1α) in CF airway inflammation. The activation of IRE1α promotes activation of its cytoplasmic kinase and RNase, resulting in mRNA splicing of X-box binding protein-1 (XBP-1s), a transcription factor required for cytokine production. We tested whether IRE1α kinase and RNase inhibition decreases cytokine production induced by the exposure of primary cultures of homozygous F508del CF human bronchial epithelia (HBE) to supernatant of mucopurulent material (SMM) from CF airways. We evaluated whether IRE1α expression is increased in freshly isolated and native CF HBE, and couples with increased XBP-1s levels. A FRET assay confirmed binding of the IRE1α kinase and RNase inhibitor, KIRA6, to the IRE1α kinase. F508del HBE cultures were exposed to SMM with or without KIRA6, and we evaluated the mRNA levels of XBP-1s, IL-6, and IL-8, and the secretion of IL-6 and IL-8. IRE1α mRNA levels were up-regulated in freshly isolated CF vs. normal HBE and coupled to increased XBP-1s mRNA levels. SMM increased XBP-1s, IL-6, and IL-8 mRNA levels and up-regulated IL-6 and IL-8 secretion, and KIRA6 blunted these responses in a dose-dependent manner. Moreover, a triple combination of CFTR modulators currently used in the clinic had no effect on SMM-increased XBP-1s levels coupled with increased cytokine production in presence or absence of KIRA6. These findings indicate that IRE1α mediates cytokine production in CF airways. Small molecule IRE1α kinase inhibitors that allosterically reduce RNase-dependent XBP-1s may represent a new therapeutic strategy for CF airway inflammation.


Assuntos
Fibrose Cística/tratamento farmacológico , Fibrose Cística/patologia , Endorribonucleases/metabolismo , Inflamação/tratamento farmacológico , Inflamação/patologia , Pulmão/patologia , Terapia de Alvo Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Células Cultivadas , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citocinas/biossíntese , Endorribonucleases/genética , Epitélio/efeitos dos fármacos , Epitélio/patologia , Humanos , Imidazóis/química , Imidazóis/farmacologia , Inflamação/genética , Modelos Biológicos , Naftalenos/química , Naftalenos/farmacologia , Proteínas Serina-Treonina Quinases/genética , Pirazinas/química , Pirazinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Proteína 1 de Ligação a X-Box/metabolismo
18.
Molecules ; 26(9)2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33919319

RESUMO

A radical approach to late-stage functionalization using photoredox and Diversinate™ chemistry on the Open Source Malaria (OSM) triazolopyrazine scaffold (Series 4) resulted in the synthesis of 12 new analogues, which were characterized by NMR, UV, and MS data analysis. The structures of four triazolopyrazines were confirmed by X-ray crystal structure analysis. Several minor and unexpected side products were generated during these studies, including two resulting from a possible disproportionation reaction. All compounds were tested for their ability to inhibit the growth of the malaria parasite Plasmodium falciparum (3D7 and Dd2 strains) and for cytotoxicity against a human embryonic kidney (HEK293) cell line. Moderate antimalarial activity was observed for some of the compounds, with IC50 values ranging from 0.3 to >20 µM; none of the compounds displayed any toxicity against HEK293 at 80 µM.


Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Técnicas de Química Sintética , Pirazinas/química , Pirazinas/farmacologia , Álcoois/química , Antimaláricos/síntese química , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Plasmodium falciparum/efeitos dos fármacos , Pirazinas/síntese química , Relação Estrutura-Atividade
19.
Int J Mol Sci ; 22(8)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33923996

RESUMO

The majority of patients with testicular germ cell tumors (GCTs) can be cured with cisplatin-based chemotherapy. However, for a subset of patients present with cisplatin-refractory disease, which confers a poor prognosis, the treatment options are limited. Novel therapies are therefore urgently needed to improve outcomes in this challenging patient population. It has previously been shown that Wnt/ß-catenin signaling is active in GCTs suggesting that its inhibitors LGK974 and PRI-724 may show promise in the management of cisplatin-refractory GCTs. We herein investigated whether LGK-974 and PRI-724 provide a treatment effect in cisplatin-resistant GCT cell lines. Taking a genoproteomic approach and utilizing xenograft models we found the increased level of ß-catenin in 2 of 4 cisplatin-resistant (CisR) cell lines (TCam-2 CisR and NCCIT CisR) and the decreased level of ß-catenin and cyclin D1 in cisplatin-resistant NTERA-2 CisR cell line. While the effect of treatment with LGK974 was limited or none, the NTERA-2 CisR exhibited the increased sensitivity to PRI-724 in comparison with parental cell line. Furthermore, the pro-apoptotic effect of PRI-724 was documented in all cell lines. Our data strongly suggests that a Wnt/ß-catenin signaling is altered in cisplatin-resistant GCT cell lines and the inhibition with PRI-724 is effective in NTERA-2 CisR cells. Further evaluation of Wnt/ß-catenin pathway inhibition in GCTs is therefore warranted.


Assuntos
Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Neoplasias Embrionárias de Células Germinativas/tratamento farmacológico , Neoplasias Embrionárias de Células Germinativas/metabolismo , Pirazinas/uso terapêutico , Piridinas/uso terapêutico , Pirimidinonas/uso terapêutico , Neoplasias Testiculares/tratamento farmacológico , Neoplasias Testiculares/metabolismo , beta Catenina/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos SCID , Pirazinas/farmacologia , Piridinas/farmacologia , Pirimidinonas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos
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