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/fisiologiaRESUMO
An indazole based series of glucocorticoid receptor agonists is reported. The SAR exploration of this scaffold yielded compounds with nanomolar affinity for the glucocorticoid receptor with indications of selectivity for the preferred transrepression mechanism; in vivo efficacy was observed in the mouse LPS induced TNFalpha model for compound 28.
Assuntos
Anti-Inflamatórios/química , Indazóis/química , Receptores de Glucocorticoides/agonistas , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacologia , Indazóis/síntese química , Indazóis/farmacologia , Camundongos , Receptores de Glucocorticoides/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The introduction of an aryl ring onto the 4-position of the C-6 benzyl amino group of the Cdk inhibitor roscovitine (2), maintained the potent Cdk inhibition demonstrated by roscovitine (2) as well as greatly improving the antiproliferative activity. A series of C-6 biarylmethylamino derivatives was prepared addressing modifications on the C-6 biaryl rings, N-9 and C-2 positions to provide compounds that displayed potent cytotoxic activity against tumor cell lines. In particular, derivative 21h demonstrated a >750-fold improvement in the growth inhibition of HeLa cells compared to roscovitine (2).
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Purinas/síntese química , Purinas/química , Roscovitina , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
C-6 Biarylmethylamino purine derivatives of roscovitine (1) inhibit cyclin dependent kinases and demonstrate potent antiproliferative activity. Replacement of the aryl rings of the C-6 biarylmethylamino group with heterobiaryl rings has provided compounds with significantly improved activity. In particular, derivatives 18 g and 9 c demonstrated 1000-fold and 1250-fold improvements, respectively, in the growth inhibition of HeLa cells compared to roscovitine (1).
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Purinas/síntese química , Purinas/química , Roscovitina , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A series of A-ring and E-ring analogues of the natural product luotonin A, a known topoisomerase I poison, was evaluated for growth inhibition in human carcinoma and leukemia cell lines. Rational design of structures was based on analogues of the related alkaloid camptothecin, which has been demonstrated to exert cytotoxic effects by the same mechanism of action. When compared to luotonin A, several compounds exhibited an improved topoisomerase I-dependent growth inhibition of a human leukemia cell line.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Pirróis/síntese química , Pirróis/farmacologia , Quinonas/síntese química , Quinonas/farmacologia , Inibidores da Topoisomerase I , Inibidores da Topoisomerase II , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The roles of the HIV1 protein Vpr in virus replication and pathogenesis remain unclear. Expression of Vpr in dividing cells causes cell cycle arrest in G(2). Vpr also facilitates low titer infection of terminally differentiated macrophages, enhances transcription, promotes apoptosis, and targets cellular uracil N-glycosylase for degradation. Using co-immunoprecipitation and tandem mass spectroscopy, we found that HIV1 Vpr engages a DDB1- and cullin4A-containing ubiquitin-ligase complex through VprBP/DCAF1. HIV2 Vpr has two Vpr-like proteins, Vpr and Vpx, which cause G(2) arrest and facilitate macrophage infection, respectively. HIV2 Vpr, but not Vpx, engages the same set of proteins. We further demonstrate that the interaction between Vpr and the ubiquitin-ligase components as well as further assembly of the ubiquitin-ligase are necessary for Vpr-mediated G(2) arrest. Our data support a model in which Vpr engages the ubiquitin ligase to deplete a cellular factor that is required for cell cycle progression into mitosis. Vpr, thus, functions like the HIV1 proteins Vif and Vpu to usurp cellular ubiquitin ligases for viral functions.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas Culina/fisiologia , Proteínas de Ligação a DNA/fisiologia , Fase G2 , Produtos do Gene vpr/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Células Cultivadas , Humanos , Imunoprecipitação , Uracila-DNA Glicosidase/metabolismoRESUMO
BACKGROUND: Simian Virus 40 (SV40) infection of growth-arrested monkey kidney cells stimulates S phase entry and the continued synthesis of both viral and cellular DNA. Infected cells can attain total DNA contents as high as DNA Index, DI = 5.0-6.0 (10-12C), with host cell DNA representing 70-80% of the total. In this study, SV40-infected and uninfected control cells were compared to determine whether continued DNA replication beyond DI = 2.0 was associated with rebinding of the minichromosome maintenance (MCM) hexamer, the putative replicative helicase, to chromatin. METHOD: Laser scanning cytometry was used to measure the total expression per cell and the chromatin/matrix-association of two MCM subunits in relation to DNA content. RESULTS: MCM2 and MCM3 proteins that were associated with the chromatin/matrix fraction in G1 phase of both uninfected and SV40-infected cells were gradually released during progression through S phase. However, in SV40-infected cells that progressed beyond DI = 2.0, chromatin/matrix-associated MCM2 and MCM3 remained at the low levels observed at the end of S phase. Rereplication was not preceded by an obvious rebinding of MCM proteins to chromatin, as was observed in G1 phase. CONCLUSIONS: The rereplication of host cell DNA in the absence of the reassociation of MCM proteins with chromatin indicates that SV40 infection induces a novel mechanism of licensing cellular DNA replication.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Fase S/fisiologia , Animais , Antígenos Virais de Tumores/metabolismo , Western Blotting , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Linhagem Celular , Chlorocebus aethiops , Cromatina/metabolismo , DNA/genética , DNA/metabolismo , Células Eucarióticas/metabolismo , Células Eucarióticas/virologia , Imuno-Histoquímica , Citometria de Varredura a Laser , Microscopia de Fluorescência , Componente 2 do Complexo de Manutenção de Minicromossomo , Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Vírus 40 dos Símios/crescimento & desenvolvimento , Vírus 40 dos Símios/imunologia , Fatores de Tempo , Replicação Viral/fisiologiaRESUMO
Lytic infection of African green monkey kidney (CV-1) cells by simian virus 40 (SV40) is characterized by stimulation of DNA synthesis leading to bypass of mitosis and replication of cellular and viral DNA beyond a 4C DNA content. To define mechanisms underlying the absence of mitosis, the expression levels of upstream regulatory molecules of mitosis-promoting factor (MPF) were compared in parallel synchronized cultures of SV40-infected and uninfected CV-1 cells. The DNA replication/damage checkpoint kinase Chk1 was phosphorylated in both uninfected and SV40-infected cultures arrested at G(1)/S by mimosine, consistent with checkpoint activation. Following release of uninfected cultures from G(1)/S, Chk1 phosphorylation was lost even though Chk1 protein levels were retained. In contrast, G(1)/S-released SV40-infected cultures exhibited dephosphorylation of Chk1 in S phase, followed by an increase in Chk1 phosphorylation coinciding with entry of infected cells into >G(2). Inhibitors of Chk1, UCN-01 and caffeine, induced mitosis and abnormal nuclear condensation and increased the protein kinase activity of MPF in SV40-infected CV-1 cells. These results demonstrate that SV40 lytic infection triggers components of a DNA damage checkpoint pathway. In addition, chemical inhibition of Chk1 activity suggests that Chk1 contributes to the absence of mitosis during SV40 lytic infection.
Assuntos
Proteínas Quinases/fisiologia , Vírus 40 dos Símios/fisiologia , Proteínas Virais/fisiologia , Alcaloides/farmacologia , Animais , Cafeína/farmacologia , Linhagem Celular , Quinase 1 do Ponto de Checagem , Chlorocebus aethiops , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Imunofluorescência , Mitose/efeitos dos fármacos , Nocodazol/farmacologia , Fosforilação , Inibidores de Proteínas Quinases , Estaurosporina/análogos & derivadosRESUMO
Livers from wild-type and p53-deficient mice were analyzed for the expression of cell-cycle regulatory proteins in an attempt to determine the mechanism for the increased proliferation of liver cells in p53-deficient mice associated with enhanced susceptibility to aflatoxin-induced liver cancer. The most striking difference found was a significant reduction of the cyclin-dependent kinase inhibitor p27(kip1) in the livers of 3-mo-old p53-/- mice, whereas only small changes were found in the expression of cyclins, cyclin-dependent kinases, and the inhibitors p21(cip1) and p16(ink4a). Relative to wild-type liver, the amounts of p27(kip1) mRNA were reduced at both 1 and 3 mo, whereas the levels of p27(kip1) protein were decreased only at 3 mo. These results identify an uncharacterized link between the expression of p53 and p27(kip1) that may involve both transcriptional and post-transcriptional regulation and allow hepatocytes to continue to proliferate after 3 wk of age. We postulate that this increased proliferation leads to increased susceptibility to aflatoxin-induced hepatocarcinogenesis.