RESUMO
Successful drug discovery requires accurate decision making in order to advance the best candidates from initial lead identification to final approval. Chemogenomics, the use of genomic tools in pharmacology and toxicology, offers a promising enhancement to traditional methods of target identification/validation, lead identification, efficacy evaluation, and toxicity assessment. To realize the value of chemogenomics information, a contextual database is needed to relate the physiological outcomes induced by diverse compounds to the gene expression patterns measured in the same animals. Massively parallel gene expression characterization coupled with traditional assessments of drug candidates provides additional, important mechanistic information, and therefore a means to increase the accuracy of critical decisions. A large-scale chemogenomics database developed from in vivo treated rats provides the context and supporting data to enhance and accelerate accurate interpretation of mechanisms of toxicity and pharmacology of chemicals and drugs. To date, approximately 600 different compounds, including more than 400 FDA approved drugs, 60 drugs approved in Europe and Japan, 25 withdrawn drugs, and 100 toxicants, have been profiled in up to 7 different tissues of rats (representing over 3200 different drug-dose-time-tissue combinations). Accomplishing this task required evaluating and improving a number of in vivo and microarray protocols, including over 80 rigorous quality control steps. The utility of pairing clinical pathology assessments with gene expression data is illustrated using three anti-neoplastic drugs: carmustine, methotrexate, and thioguanine, which had similar effects on the blood compartment, but diverse effects on hepatotoxicity. We will demonstrate that gene expression events monitored in the liver can be used to predict pathological events occurring in that tissue as well as in hematopoietic tissues.
Assuntos
Biotecnologia/métodos , Desenho de Fármacos , Indústria Farmacêutica/métodos , 5-Aminolevulinato Sintetase/biossíntese , Animais , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Automação , Ductos Biliares/patologia , Carmustina/toxicidade , Biologia Computacional , Bases de Dados como Assunto , Relação Dose-Resposta a Droga , Regulação para Baixo , Expressão Gênica , Humanos , Hiperplasia/etiologia , Fígado/efeitos dos fármacos , Masculino , Metotrexato/toxicidade , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Tamanho do Órgão , Farmacologia/métodos , RNA/química , RNA Complementar/metabolismo , Ratos , Ratos Sprague-Dawley , Reticulócitos/citologia , Reticulócitos/metabolismo , Tioguanina/toxicidade , Fatores de Tempo , Distribuição Tecidual , Toxicologia/métodosAssuntos
Desenho de Fármacos , RNA Interferente Pequeno , RNA/antagonistas & inibidores , Animais , Células Cultivadas , DNA/genética , Sistemas de Liberação de Medicamentos , Indústria Farmacêutica , Inativação Gênica/efeitos dos fármacos , Terapia Genética , Humanos , RNA Complementar/metabolismo , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/fisiologia , Apoio à Pesquisa como Assunto , Tecnologia FarmacêuticaRESUMO
Transforming growth factor-beta (TGF-beta) and TGF-beta-related factors regulate cell growth, differentiation, and apoptosis, and play key roles in normal development and tumorigenesis. TGF-beta family-induced changes in gene expression are mediated by serine/threonine kinase receptors at the cell surface and Smads as intracellular effectors. Receptor-activated Smads combine with a common Smad4 to translocate into the nucleus where they cooperate with other transcription factors to activate or repress transcription. The activities of the receptor-activated Smads are controlled by post-translational modifications such as phosphorylation and ubiquitylation. Here we show that Smad4 is modified by sumoylation. Sumoylation of Smad4 was enhanced by the conjugating enzyme Ubc9 and members of the PIAS family of SUMO ligases. A major sumoylation site in Smad4 was localized to Lys-159 in its linker segment with an additional site at Lys-113 in the MH-1 domain. Increased sumoylation in the presence of the PIASy E3 ligase correlated with targeting of Smad4 to subnuclear speckles that contain SUMO-1 and PIASy. Replacement of lysines 159 and 113 by arginines or increased sumoylation enhanced the stability of Smad4, and transcription in mammalian cells and Xenopus embryos. These observations suggest a role for Smad4 sumoylation in the regulation of TGF-beta signaling through Smads.