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1.
Chem Biol Interact ; 330: 109236, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32866467

RESUMO

A series of novel pyrrolopyrimidine urea derivatives were synthesized and evaluated for their anticancer activity against colon cancer cell lines. Compounds showed the remarkable cytotoxic activity on HCT-116 wt cell line. The most potent compound 4c (IC50 = 0.14 µM) induced apoptosis in HCT-116 wt and HCT-116 p53-/- cell lines. Otherwise, treatment of HCT-116 BAX-/-BAK-/- cells with compound 4c didn't lead to activation of apoptosis, suggesting that compound 4c induces apoptotic cell death by activating BAX/BAK-dependent pathway. Moreover, while the compound 4c increase the activation of caspase-3 and caspase-9 levels in HCT-116 wt and HCT-116 p53-/- cells, caspase-3 or caspase-9 activation was not observed in HCT-116 BAX-/-BAK-/- cells. In addition, compound 4c induced mitochondrial apoptosis in cells grown as oncospheroids, which better mimic the in vivo milieu of tumors. 4c treatment also activated JNK along with inhibition of prosurvival kinases such as Akt and ERK 1/2 in HCT-116 wt and HCT-116 p53 -/- cells as well as in HCT-116 BAX-/-BAK-/- cells. Notably, our results indicated that compound 4c induced mitochondrial apoptosis through activation p53-independent apoptotic signaling pathways.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Pirimidinas/farmacologia , Pirróis/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Caspase 3/genética , Caspase 9/genética , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Mitocôndrias/metabolismo , Pirimidinas/síntese química , Pirimidinas/química , Pirróis/síntese química , Pirróis/química , Proteína Supressora de Tumor p53/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genética
2.
Nat Commun ; 11(1): 4527, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913197

RESUMO

Evasion of programmed cell death represents a critical form of oncogene addiction in cancer cells. Understanding the molecular mechanisms underpinning cancer cell survival despite the oncogenic stress could provide a molecular basis for potential therapeutic interventions. Here we explore the role of pro-survival genes in cancer cell integrity during clonal evolution in non-small cell lung cancer (NSCLC). We identify gains of MCL-1 at high frequency in multiple independent NSCLC cohorts, occurring both clonally and subclonally. Clonal loss of functional TP53 is significantly associated with subclonal gains of MCL-1. In mice, tumour progression is delayed upon pharmacologic or genetic inhibition of MCL-1. These findings reveal that MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Evolução Clonal , Variações do Número de Cópias de DNA , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Progressão da Doença , Humanos , Pulmão/diagnóstico por imagem , Pulmão/patologia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Cultura Primária de Células , Estudos Prospectivos , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , RNA-Seq , Estudos Retrospectivos , Esferoides Celulares , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Proteína Supressora de Tumor p53/genética , Microtomografia por Raio-X
3.
Gene ; 763: 144997, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-32783992

RESUMO

The CRISPR-Cas system currently stands as one of the best multifaceted tools for site-specific genome engineering in mammals. An important aspect of research in this field focusses on improving the specificity and efficacy of precise genome editing in multiple model systems. The cornerstone of this mini-review is one of the extensively investigated small molecule inhibitor, SCR7, which abrogates NHEJ, a Ligase IV-dependent DSB repair pathway, thus guiding integration of the foreign DNA fragment via the more precise homology directed repair during genome editing. One of our recent studies sheds light on properties of different forms of SCR7. Here, we give a succinct account on the use of SCR7 and its different forms in CRISPR-Cas system, highlighting their chemical properties and biological relevance as potent efficiency-enhancing CRISPR tools.


Assuntos
Sistemas CRISPR-Cas , Inibidores Enzimáticos/farmacologia , Edição de Genes/métodos , Pirimidinas/farmacologia , Reparo de DNA por Recombinação/efeitos dos fármacos , Bases de Schiff/farmacologia , Animais , DNA Ligase Dependente de ATP/antagonistas & inibidores , Inibidores Enzimáticos/química , Humanos , Pirimidinas/química , Bases de Schiff/química
4.
J Cancer Res Clin Oncol ; 146(11): 2871-2883, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32770382

RESUMO

PURPOSE: Polo-like kinase 4 (PLK4) inhibitors, such as CFI-400945 and centrinone, are emerging as promising antineoplastic agents. However, their effectiveness against Ewing's sarcoma, a highly aggressive childhood cancer, remains to be established. METHODS: CFI-400945 and centrinone were tested in three Ewing's sarcoma cell lines with different TP53 status. Effects were assessed by flow-cytometric analyses of cell death, dissipation of the mitochondrial transmembrane potential and cell cycle distribution, by cell viability assay as well as by caspase 3/7 activity measurement, by immunoblotting and by immunofluorescence microscopy. RESULTS: CFI-400945 and centrinone elicited cell death in p53 wild-type and mutant Ewing's sarcoma cells. Both agents induced mitochondrial membrane depolarisation, caspase 3/7 activation, PARP1 cleavage and DNA fragmentation, indicating an apoptotic form of cell death. In addition, the PLK4 inhibitors induced a G2/M cell cycle arrest, particularly when cell killing was attenuated by the pan-caspase inhibitor z-VAD-fmk. Moreover, CFI-400945 treatment produced polyploidy. CONCLUSION: Our findings show that PLK4 inhibitors were effective against Ewing's sarcoma cells in vitro and thus provide a rationale for their evaluation in vivo.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Ósseas/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sarcoma de Ewing/patologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Indazóis/farmacologia , Indóis/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Pirimidinas/farmacologia , Sulfonas/farmacologia
5.
Anticancer Res ; 40(7): 3847-3855, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32620624

RESUMO

BACKGROUND/AIM: The effects of tyrosine kinase inhibitors (TKI) in head and neck squamous cell cancer (HNSCC) are not fully understood. We investigated the effects of selective TKIs erlotinib, gefitinib, nilotinib, and dasatinib and the mTOR-inhibitor everolimus on the expression of insulin-like growth factor 1 receptor (IGF1R) in HPV-positive and HPV-negative squamous cancer cell lines. MATERIALS AND METHODS: HPV-negative UMSCC-11A and UMSCC-14C cells and HPV-positive CERV196 cells were treated with TKIs or everolimus. Protein concentration of IGF1R was measured using ELISA. RESULTS: IGF1R expression was significantly reduced by all tested TKIs and everolimus in both HPV-negative cancer cell lines. In HPV-positive squamous cancer cells we observed significant protein inhibition. CONCLUSION: The crosstalk between epidermal growth factor receptors and IGF1R could be of central interest for the development of novel medical approaches for individualized therapy.


Assuntos
Everolimo/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Papillomavirus Humano 16/isolamento & purificação , Infecções por Papillomavirus/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptor IGF Tipo 1/biossíntese , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Dasatinibe/farmacologia , Cloridrato de Erlotinib/farmacologia , Gefitinibe/farmacologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Infecções por Papillomavirus/virologia , Pirimidinas/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia
6.
Nat Commun ; 11(1): 3288, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620753

RESUMO

The prognostic and therapeutic relevance of molecular subtypes for the most aggressive isocitrate dehydrogenase 1/2 (IDH) wild-type glioblastoma (GBM) is currently limited due to high molecular heterogeneity of the tumors that impedes patient stratification. Here, we describe a distinct binary classification of IDH wild-type GBM tumors derived from a quantitative proteomic analysis of 39 IDH wild-type GBMs as well as IDH mutant and low-grade glioma controls. Specifically, GBM proteomic cluster 1 (GPC1) tumors exhibit Warburg-like features, neural stem-cell markers, immune checkpoint ligands, and a poor prognostic biomarker, FKBP prolyl isomerase 9 (FKBP9). Meanwhile, GPC2 tumors show elevated oxidative phosphorylation-related proteins, differentiated oligodendrocyte and astrocyte markers, and a favorable prognostic biomarker, phosphoglycerate dehydrogenase (PHGDH). Integrating these proteomic features with the pharmacological profiles of matched patient-derived cells (PDCs) reveals that the mTORC1/2 dual inhibitor AZD2014 is cytotoxic to the poor prognostic PDCs. Our analyses will guide GBM prognosis and precision treatment strategies.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Isocitrato Desidrogenase/genética , Proteogenômica/métodos , Proteômica/métodos , Benzamidas/farmacologia , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Glioblastoma/genética , Glioblastoma/terapia , Humanos , Isocitrato Desidrogenase/classificação , Isocitrato Desidrogenase/metabolismo , Estimativa de Kaplan-Meier , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Morfolinas/farmacologia , Mutação , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia
7.
Leukemia ; 34(9): 2354-2363, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647324

RESUMO

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, characterized by immunodeficiency. Hence, patients with CLL might be considered more susceptible to severe complications from COVID-19. We undertook this retrospective international multicenter study to characterize the course of COVID-19 in patients with CLL and identify potential predictors of outcome. Of 190 patients with CLL and confirmed COVID-19 diagnosed between 28/03/2020 and 22/05/2020, 151 (79%) presented with severe COVID-19 (need of oxygen and/or intensive care admission). Severe COVID-19 was associated with more advanced age (≥65 years) (odds ratio 3.72 [95% CI 1.79-7.71]). Only 60 patients (39.7%) with severe COVID-19 were receiving or had recent (≤12 months) treatment for CLL at the time of COVID-19 versus 30/39 (76.9%) patients with mild disease. Hospitalization rate for severe COVID-19 was lower (p < 0.05) for patients on ibrutinib versus those on other regimens or off treatment. Of 151 patients with severe disease, 55 (36.4%) succumbed versus only 1/38 (2.6%) with mild disease; age and comorbidities did not impact on mortality. In CLL, (1) COVID-19 severity increases with age; (2) antileukemic treatment (particularly BTK inhibitors) appears to exert a protective effect; (3) age and comorbidities did not impact on mortality, alluding to a relevant role of CLL and immunodeficiency.


Assuntos
Betacoronavirus , Infecções por Coronavirus/patologia , Leucemia Linfocítica Crônica de Células B/complicações , Pneumonia Viral/patologia , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Comorbidade , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/mortalidade , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/mortalidade , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Estudos Retrospectivos , Índice de Gravidade de Doença , Inquéritos e Questionários
9.
Toxicology ; 441: 152521, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32534105

RESUMO

Homeobox (Hox) genes encode homeodomain proteins, which play important roles in the development and morphological diversification of organisms including plants and animals. Perfluorinated chemicals (PFCs), which are well recognized industrial pollutants and universally detected in human and wildlife, interfere with animal development. In addition, PFCs produce a number of hepatic adverse effects, such as hepatomegaly and dyslipidemia. Homeodomain proteins profoundly contribute to liver regeneration. Hox genes serve as either oncogenes or tumor suppressor genes during target organ carcinogenesis. However, to date, no study investigated whether PFCs regulate expression of Hox genes. This study was designed to determine the regulation of Hox (including Hox-a to -d subfamily members) and paraHox [including GS homeobox (Gsx), pancreatic and duodenal homeobox (Pdx), and caudal-related homeobox (Cdx) family members] genes by PFCs including perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) in mouse liver. 46.4 mg/kg PFNA induced mRNA expression of Hoxa5, b7, c5, d10 and Pdx1 in wild-type and CAR-null mouse livers, but not in PPARα-null mouse livers, indicating a PPARα-dependent manner. PFOA, PFNA, and PFDA all induced mRNA expression of Hoxa5, b7, c5, d10, Pdx1 and Zeb2 in wild-type but not PPARα-null mouse livers. In addition, in Nrf2-null mouse livers, PFNA continued to increase mRNA expression of Hoxa5 and Pdx1, but not Hoxb7, c5 or d10. Furthermore, Wy14643, a classical PPARα agonist, induced mRNA expression of Hoxb7 and c5 in wild-type but not PPARα-null mouse livers. However, Wy14643 did not induce mRNA expression of Hoxa5, d10 or Pdx1 in either wild-type or PPARα-null mouse livers. TCPOBOP, a classical mouse CAR agonist, increased mRNA expression of Hoxb7, c5 and d10 but not Hoxa5 or Pdx1 in mouse livers. Moreover, PFNA decreased cytoplasmic and nuclear Hoxb7 protein levels in mouse livers. However, PFNA increased cytoplasmic Hoxc5 protein level but decreased nuclear Hoxc5 protein level in mouse livers. In conclusion, PFCs induced mRNA expression of several Hox genes such as Hoxb7, c5 and d10, mostly through the activation of PPARα and/or Nrf2 signaling.


Assuntos
Caprilatos/toxicidade , Ácidos Decanoicos/toxicidade , Fluorcarbonetos/toxicidade , Genes Homeobox/efeitos dos fármacos , Fígado/efeitos dos fármacos , Animais , Western Blotting , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , PPAR alfa/antagonistas & inibidores , PPAR alfa/metabolismo , Pirimidinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real
10.
Life Sci ; 256: 117972, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32544464

RESUMO

Acute kidney injury (AKI) has a high morbidity and mortality, and there is no targeted treatment yet. One of the main causes of AKI is ischemia-reperfusion (IR). Increased release of adenosine under stress and hypoxia exerts anti-inflammatory and antioxidant effects. Adenosine kinase (ADK) is an important enzyme that eliminates adenosine in cells, and can maintain low adenosine concentration in cells. Our previous studies have shown that pretreatment of adenosine kinase inhibitor ABT-702 could markedly attenuate cisplatin-induced nephrotoxicity both in vivo and in vitro. This study is designed to investigate the effect of ADK inhibition on IR-induced AKI. The results showed that ADK expression was positively correlated with the degree of renal tubular injury, which suggested that the degree of ADK inhibition reflected the severity of acute tubular necrosis. In vivo, ADK inhibitor could reduce IR-induced renal injury, which might play a protective role by increasing tissue adenosine level, inhibiting oxidative stress, and reducing cell apoptosis. In HK2 cells, cobaltous dichloride (CoCl2) increased the level of oxidative stress, up-regulated the production of pro-inflammatory factor, and induced apoptosis, ADK inhibition could alleviate the above damaging effects. Moreover, the anti-apoptotic effect exerted by ADK inhibition was independent of inosine. In summary, our results support the idea that ADK inhibition has protective effects on IR-induced AKI. Adenosine kinase inhibition might provide a new target for AKI prevention and treatment.


Assuntos
Lesão Renal Aguda/tratamento farmacológico , Lesão Renal Aguda/etiologia , Adenosina Quinase/antagonistas & inibidores , Morfolinas/uso terapêutico , Pirimidinas/uso terapêutico , Traumatismo por Reperfusão/complicações , Adenosina Quinase/metabolismo , Adulto , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Cobalto , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Inflamação/patologia , Inosina/farmacologia , Túbulos Renais/enzimologia , Túbulos Renais/patologia , Masculino , Camundongos Endogâmicos C57BL , Morfolinas/farmacologia , Necrose , Estresse Oxidativo/efeitos dos fármacos , Pirimidinas/farmacologia
11.
PLoS One ; 15(6): e0234708, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555680

RESUMO

Fibroblast growth factor receptors (FGFRs) are frequently altered in a variety of human cancer cells and are overexpressed in hepatocellular carcinoma (HCC). Several literatures have proven that they are efficacious for HCC therapy, however, the underlying mechanism remains unclear. Here, we found FGFR4 was overexpressed in HCC cell lines HepG2 and Hep3B and we used PD173074, an FGFR4 inhibitor, to explore the role of FGFR4 and its underlying mechanism in these cell lines. The results showed that PD173074 significantly arrested HepG2 and Hep3B cells in G1 phase and inhibited cell proliferation. Furthermore, Western blot analysis revealed that PD173074 decreased the levels of P-FRS2α, P-ERK, CDK2, cyclin E and NF-κB (p65) in the nucleus while it increased the levels of ubiquitin and CUL3, an E3 ubiquitin ligase which involves in cyclin E degradation. Meanwhile, the data from RT-qPCR showed that PD173074 also decreased miR-141 level. In conclusion, these results suggest that FGFR4 is involved in HCC by ERK/CUL3/cyclin E signaling pathway, and the finding may provide a potential theoretical basis for treatment by targeting FGFR4 in HCC.


Assuntos
Proteínas Culina/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pirimidinas/farmacologia , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas Culina/antagonistas & inibidores , Proteínas Culina/genética , Ciclina E/metabolismo , Regulação para Baixo/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
12.
Anticancer Res ; 40(5): 2667-2673, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366411

RESUMO

BACKGROUND/AIM: The therapeutic strategy for patients with non-small-cell lung cancer (NSCLC) harboring the BRAF non-V600E mutation has not been established. LY3009120, a newly discovered pan-RAF inhibitor, has shown strong antitumor effects in cancers with various BRAF genotypes. This study investigated the antitumor effects of LY3009120 in NSCLC cells harboring the BRAF non-V600E mutation. MATERIALS AND METHODS: We examined the antitumor effects of LY3009120 by MTS assay and flow cytometry. We analyzed the expression status of proteins by western blot. The mouse xenograft models were used for the in vivo experiments. RESULTS: LY3009120 suppressed BRAF-related downstream pathway molecules and induced cleavage of poly ADP-ribose polymerase in all examined NSCLC cell lines. LY3009120 also inhibited in vivo tumor growth in NSCLC cells harboring the BRAF non-V600E mutation. CONCLUSION: LY3009120 is a potent therapeutic agent for patients with BRAF non-V600E mutant NSCLC.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Pulmonares/genética , Mutação/genética , Oncogenes , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Pirimidinas/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Concentração Inibidora 50 , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Cancer Immunol Immunother ; 69(9): 1929-1936, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32435850

RESUMO

Myeloid-derived suppressor cells (MDSCs) are widely implicated in negative regulation of immune responses in cancer. Inhibition of class I histone deacetylases (HDAC) with entinostat has anti-MDSC activity. However, as single agent, it did not delay tumor growth in EL4 and LLC tumor models. Here, we found that entinostat reduced immune suppressive activity of only one type of MDSC-polymorphonuclear, PMN-MDSC, whereas it had no effect on monocytic M-MDSC or macrophages. M-MDSC had high amount of class II HDAC-HDAC6, which was further increased after the treatment of mice with entinostat. Inhibition of HDAC6 with ricolinostat reduced suppressive activity of M-MDSC, but did not affect PMN-MDSC or delayed tumor growth. However, combination of entinostat and ricolinostat abrogated suppressive activity of both populations of MDSC and substantially delayed tumor progression. Thus, inactivation of MDSC required targeting of both major subsets of these cells via inhibitors of class I and class II HDAC.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Células Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/efeitos dos fármacos , Animais , Benzamidas/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Piridinas/farmacologia , Pirimidinas/farmacologia
14.
PLoS One ; 15(5): e0233089, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32459810

RESUMO

Many drugs are promiscuous and bind to multiple targets. On the one hand, these targets may be linked to unwanted side effects, but on the other, they may achieve a combined desired effect (polypharmacology) or represent multiple diseases (drug repositioning). With the growth of 3D structures of drug-target complexes, it is today possible to study drug promiscuity at the structural level and to screen vast amounts of drug-target interactions to predict side effects, polypharmacological potential, and repositioning opportunities. Here, we pursue such an approach to identify drugs inactivating B-cells, whose dysregulation can function as a driver of autoimmune diseases. Screening over 500 kinases, we identified 22 candidate targets, whose knock out impeded the activation of B-cells. Among these 22 is the gene KDR, whose gene product VEGFR2 is a prominent cancer target with anti-VEGFR2 drugs on the market for over a decade. The main result of this paper is that structure-based drug repositioning for the identified kinase targets identified the cancer drug ibrutinib as micromolar VEGFR2 inhibitor with a very high therapeutic index in B-cell inactivation. These findings prove that ibrutinib is not only acting on the Bruton's tyrosine kinase BTK, against which it was designed. Instead, it may be a polypharmacological drug, which additionally targets angiogenesis via inhibition of VEGFR2. Therefore ibrutinib carries potential to treat other VEGFR2 associated disease. Structure-based drug repositioning explains ibrutinib's anti VEGFR2 action through the conservation of a specific pattern of interactions of the drug with BTK and VEGFR2. Overall, structure-based drug repositioning was able to predict these findings at a fraction of the time and cost of a conventional screen.


Assuntos
Reposicionamento de Medicamentos/métodos , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/metabolismo , Linfócitos B/metabolismo , Humanos , Células Jurkat , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Suramina/química , Suramina/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
15.
PLoS One ; 15(5): e0229630, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32401759

RESUMO

Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by traumatic implantation of many species of black fungi. Due to the refractoriness of some cases and common recurrence of CBM, a more effective and less time-consuming treatment is mandatory. The aim of this study was to identify compounds with in vitro antifungal activity in the Pathogen Box® compound collection against different CBM agents. Synergism of these compounds with drugs currently used to treat CBM was also assessed. An initial screening of the drugs present in this collection at 1 µM was performed with a Fonsecaea pedrosoi clinical strain according to the EUCAST protocol. The compounds with activity against this fungus were also tested against other seven etiologic agents of CBM (Cladophialophora carrionii, Phialophora verrucosa, Exophiala jeanselmei, Exophiala dermatitidis, Fonsecaea monophora, Fonsecaea nubica, and Rhinocladiella similis) at concentrations ranging from 0.039 to 10 µM. The analysis of potential synergism of these compounds with itraconazole and terbinafine was performed by the checkerboard method. Eight compounds inhibited more than 60% of the F. pedrosoi growth: difenoconazole, bitertanol, iodoquinol, azoxystrobin, MMV688179, MMV021013, trifloxystrobin, and auranofin. Iodoquinol produced the lowest MIC values (1.25-2.5 µM) and MMV688179 showed MICs that were higher than all compounds tested (5 - >10 µM). When auranofin and itraconazole were tested in combination, a synergistic interaction (FICI = 0.37) was observed against the C. carrionii isolate. Toxicity analysis revealed that MMV021013 showed high selectivity indices (SI ≥ 10) against the fungi tested. In summary, auranofin, iodoquinol, and MMV021013 were identified as promising compounds to be tested in CBM models of infection.


Assuntos
Antifúngicos/farmacologia , Cromoblastomicose/tratamento farmacológico , Sinergismo Farmacológico , Fungos/patogenicidade , Acetatos/farmacologia , Ascomicetos/efeitos dos fármacos , Ascomicetos/patogenicidade , Auranofina/farmacologia , Compostos de Bifenilo/farmacologia , Cromoblastomicose/microbiologia , Cromoblastomicose/patologia , Dioxolanos/farmacologia , Exophiala/efeitos dos fármacos , Exophiala/patogenicidade , Fungos/efeitos dos fármacos , Humanos , Iminas/farmacologia , Iodoquinol/farmacologia , Pirimidinas/farmacologia , Estrobilurinas/farmacologia , Triazóis/farmacologia
16.
Mol Pharmacol ; 98(1): 61-71, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32358165

RESUMO

The blood-brain barrier (BBB) is essential for the maintenance of homeostasis in the brain. Brain capillary endothelial cells (BCECs) comprise the BBB, and thus a delicate balance between their proliferation and death is required. Although the activity of ion channels in BCECs is involved in BBB functions, the underlying molecular mechanisms remain unclear. In the present study, the molecular components of Ca2+-activated Cl- (ClCa) channels and their physiological roles were examined using mouse BCECs (mBCECs) and a cell line derived from bovine BCECs, t-BBEC117. Expression analyses revealed that TMEM16A was strongly expressed in mBCECs and t-BBEC117 cells. In t-BBEC117 cells, whole-cell Cl- currents were sensitive to the ClCa channel blockers, 100 µM niflumic acid and 10 µM T16Ainh-A01, and were also reduced markedly by small-interfering RNA (siRNA) knockdown of TMEM16A. Importantly, block of ClCa currents with ClCa channel blockers or TMEM16A siRNA induced membrane hyperpolarization. Moreover, treatment with TMEM16A siRNA caused an increase in resting cytosolic Ca2+ concentration ([Ca2+]cyt). T16Ainh-A01 reduced cell viability in a concentration-dependent manner. Either ClCa channel blockers or TMEM16A siRNA also curtailed cell proliferation and migration. Furthermore, ClCa channel blockers attenuated the trans-endothelial permeability. In combination, these results strongly suggest that TMEM16A contributes to ClCa channel conductance and can regulate both the resting membrane potential and [Ca2+]cyt in BCECs. Our data also reveal how these BCECs may be involved in the maintenance of BBB functions, as both the proliferation and migration are altered following changes in channel activity. SIGNIFICANCE STATEMENT: In brain capillary endothelial cells (BCECs) of the blood-brain barrier (BBB), TMEM16A is responsible for Ca2+-activated Cl- channels and can regulate both the resting membrane potential and cytosolic Ca2+ concentration, contributing to the proliferation and migration of BCECs. The present study provides novel information on the molecular mechanisms underlying the physiological functions of BCECs in the BBB and a novel target for therapeutic drugs for disorders associated with dysfunctions in the BBB.


Assuntos
Anoctamina-1/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/citologia , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Animais , Anoctamina-1/antagonistas & inibidores , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Bovinos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Ácido Niflúmico/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia
17.
Inflamm Res ; 69(7): 657-666, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32394143

RESUMO

OBJECTIVES: This study aimed to explore the effects and relative mechanism of JMJD3 on knee osteoarthritis (OA). METHODS: In this study, we first analyzed the expression of JMJD3 in OA cartilage using western blot and immunohistochemistry. In an in vitro study, the effects of GSK-J4, JMJD3 inhibitor, on ATDC-5 chondrocytes were evaluated by CCK-8 assay. Real-time PCR and western blot were used to examine the inhibitory effect of GSK-J4 on the inflammation and ECM degradation of chondrocytes. NF-κB p65 phosphorylation and nuclear translocation were measured by western blot and immunofluorescence. In the animal study, twenty mice were randomized into four experimental groups: sham group, DMM-induced OA + DMSO group, OA + low-dose GSK-J4 group, and OA + high-dose GSK-J4 group. After the treatment, hematoxylin-eosin and safranin O/fast green staining were used to evaluate cartilage degradation of knee joint, with OARSI scores for quantitative assessment of cartilage damage. RESULTS: Our results revealed that JMJD3 was overexpressed in OA cartilage and GSK-J4 could suppress the IL-1ß-induced production of pro-inflammatory cytokines and catabolic enzymes, including IL-6, IL-8, MMP-9 and ADAMTS-5. Consistent with these findings, GSK-J4 could inhibit IL-1ß-induced degradation of collagen II and aggrecan. Mechanistically, GSK-J4 dramatically suppressed IL-1ß-stimulated NF-κB signal pathway activation. In vivo, GSK-J4 prevented cartilage damage in mouse DMM-induced OA model. CONCLUSIONS: This study elucidates the important role of JMJD3 in cartilage degeneration in OA, and our results indicate that JDJM3 may become a novel therapeutic target in OA therapy.


Assuntos
Benzazepinas/farmacologia , Cartilagem/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Osteoartrite/prevenção & controle , Pirimidinas/farmacologia , Agrecanas/metabolismo , Animais , Cartilagem/fisiopatologia , Linhagem Celular , Condrócitos/fisiologia , Colágeno/metabolismo , Expressão Gênica , Humanos , Inflamação/prevenção & controle , Interleucina-1beta/farmacologia , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Osteoartrite/fisiopatologia , Ratos , Transdução de Sinais/fisiologia
18.
Adv Pharmacol ; 88: 173-191, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32416867

RESUMO

The glucagon-like peptide-1 receptor (GLP-1R) is a significant therapeutic target for small molecule drug discovery given the therapeutic impact of peptide agonists in the diabetes sphere. We review the discovery and subsequent characterization of the small molecule GLP-1R allosteric modulator 4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP). BETP is a covalent modulator of the GLP-1R, and we discuss the pharmacological implications and possible structural basis of this novel mode of action. We highlight the insights into class B G-protein coupled receptor pharmacology and biology provided by studies conducted with BETP. These include the descriptions of exquisite allosteric modulator probe dependence and biased signaling in vitro and in vivo. We conclude with an analysis of the utility of BETP as a chemical probe for the GLP-1R.


Assuntos
Descoberta de Drogas , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Pirimidinas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Sequência de Aminoácidos , Animais , AMP Cíclico/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Humanos , Pirimidinas/química , Bibliotecas de Moléculas Pequenas/farmacologia
19.
Mol Pharmacol ; 97(6): 392-401, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32234810

RESUMO

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in terminating signals initiated by agonist-bound GPCRs. However, chronic stimulation of GPCRs, such as that which occurs during heart failure, leads to the overexpression of GRKs and maladaptive downregulation of GPCRs on the cell surface. We previously reported the discovery of potent and selective families of GRK inhibitors based on either the paroxetine or GSK180736A scaffold. A new inhibitor, CCG258747, which is based on paroxetine, demonstrates increased potency against the GRK2 subfamily and favorable pharmacokinetic parameters in mice. CCG258747 and the closely related compound CCG258208 also showed high selectivity for the GRK2 subfamily in a kinome panel of 104 kinases. We developed a cell-based assay to screen the ability of CCG258747 and 10 other inhibitors with different GRK subfamily selectivities and with either the paroxetine or GSK180736A scaffold to block internalization of the µ-opioid receptor (MOR). CCG258747 showed the best efficacy in blocking MOR internalization among the compounds tested. Furthermore, we show that compounds based on paroxetine had much better cell permeability than those based on GSK180736A, which explains why GSK180736A-based inhibitors, although being potent in vitro, do not always show efficacy in cell-based assays. This study validates the paroxetine scaffold as the most effective for GRK inhibition in living cells, confirming that GRK2 predominantly drives internalization of MOR in the cell lines we tested and underscores the utility of high-resolution cell-based assays for assessment of compound efficacy. SIGNIFICANCE STATEMENT: G protein-coupled receptor kinases (GRKs) are attractive targets for developing therapeutics for heart failure. We have synthesized a new GRK2 subfamily-selective inhibitor, CCG258747, which has nanomolar potency against GRK2 and excellent selectivity over other kinases. A live-cell receptor internalization assay was used to test the ability of GRK2 inhibitors to impart efficacy on a GRK-dependent process in cells. Our data indicate that CCG258747 blocked the internalization of the µ-opioid receptor most efficaciously because it has the ability to cross cell membranes.


Assuntos
Indazóis/química , Paroxetina/química , Pirimidinas/química , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/metabolismo , Animais , Western Blotting , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Feminino , Células HEK293 , Humanos , Indazóis/farmacologia , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Pirimidinas/farmacologia
20.
Nat Commun ; 11(1): 2019, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332747

RESUMO

Retinoblastoma protein (Rb) is a tumor suppressor that binds and represses E2F transcription factors. In cervical cancer cells, human papilloma virus (HPV) protein E7 binds to Rb, releasing it from E2F to promote cell cycle progression, and inducing ubiquitination of Rb. E7-mediated proteasomal degradation of Rb requires action by another protease, calpain, which cleaves Rb after Lys 810. However, it is not clear why cleavage is required for Rb degradation. Here, we report that the proteasome cannot initiate degradation efficiently on full-length Rb. Calpain cleavage exposes a region that is recognized by the proteasome, leading to rapid proteolysis of Rb. These findings identify a mechanism for regulating protein stability by controlling initiation and provide a better understanding of the molecular mechanism underlying transformation by HPV.


Assuntos
Calpaína/metabolismo , Fatores de Transcrição E2F/genética , Regulação Neoplásica da Expressão Gênica , Proteínas E7 de Papillomavirus/metabolismo , Proteínas de Ligação a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias do Colo do Útero/genética , Acrilatos/farmacologia , Calpaína/antagonistas & inibidores , Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Ciclopentanos/farmacologia , Fatores de Transcrição E2F/metabolismo , Feminino , Células HEK293 , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/patogenicidade , Humanos , Proteína NEDD8/antagonistas & inibidores , Proteína NEDD8/metabolismo , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Pirimidinas/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia
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