RESUMO
A three-dimensional pharmacophore model was generated utilizing a set of known inhibitors of c-Myc-Max heterodimer formation. The model successfully identified a set of structurally diverse compounds with potential inhibitory activity against c-Myc. Nine compounds were tested in vitro, and four displayed affinities in the micromolar range and growth inhibitory activity against c-Myc-overexpressing cells. These studies demonstrate the applicability of pharmacophore modeling to the identification of novel and potentially more puissant inhibitors of the c-Myc oncoprotein.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Modelos Moleculares , Proteínas Proto-Oncogênicas c-myc/química , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Benzofuranos/química , Benzofuranos/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Ensaio de Desvio de Mobilidade Eletroforética , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Células HL-60 , Proteína HMGA1b/biossíntese , Proteína HMGA1b/genética , Humanos , Ligação Proteica , Multimerização Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piridinas/química , Piridinas/farmacologia , Pirrolidinas/química , Pirrolidinas/farmacologia , Ratos , Tiazóis/química , Tiazóis/farmacologiaRESUMO
To assess whether retinoblastoma formation is associated with the expression of high mobility group (HMG) A2 protein, a transcription factor that is highly expressed during embryogenesis and completely repressed in normal adult tissues, we performed Northern and Western blots and RT-PCR analyses, and immunohistochemistry to test for HMGA2 expression. We used established retinoblastoma cell lines in tumors grown in nude mice and clinical retinoblastoma specimens, and contrasted these tumors with normal embryonic and adult retina. Adenoviral-mediated antisense experiments were conducted on the retinoblastoma cell lines to suppress HMGA2 expression and determine if cell proliferation is HMGA2-dependent. We also transfected a retinoblastoma cell line to identify cis-regulatory elements and transcription initiation sites on the HMGA2 gene promoter. HMGA2 gene expression was silenced in terminally differentiated retina of 6-wk-old mice, but it was detected in retina of a 13.5-d postcoitum embryo. Reactivation of HMGA2 gene expression was observed in the retinoblastoma cell lines Y79, WERI-Rb1, and TOTL-1, in tumors derived from some of these cells propagated in nude mice, and in a high frequency of retinoblastomas excised from human patients. This suggests that expression of HMGA2 gene in retinoblastoma cells involves a derepression process. By using an antisense approach to block HMGA2 expression, we observed a decrease in the number of proliferating retinoblastoma cells. As a 1st step toward understanding HMGA2 gene reactivation in retinoblastoma, we mapped the 2 transcription initiation sites and associated positive regulatory elements within the WERI-Rb1 cells. Our discovery of derepression of HMGA2 gene expression in retinoblastoma provides the 1st evidence that this protein might contribute to neoplastic transformation of retina cells.
