A Phenotypic Screening Assay for Modulators of Huntingtin-Induced Transcriptional Dysregulation
| Autor: | Cesare Federico, Andrea Caricasole, Letizia Magnoni, Wolfgang Fecke, Valentina Porcari, Freddy Heitz, Giulia Lazzeroni, Salvatore La Rosa, Luisa Massai, Tiziana Benicchi, Lara Rossini, Daniela Diamanti |
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| Rok vydání: | 2013 |
| Předmět: |
Huntingtin
Transcription Genetic Pyridines Phenotypic screening Nerve Tissue Proteins Response Elements CREB Biochemistry Analytical Chemistry Small Molecule Libraries Genes Reporter Cell Line Tumor Gene expression Huntingtin Protein Humans Viability assay CREB-binding protein Neurons rho-Associated Kinases Reporter gene biology Amides CREB-Binding Protein Molecular biology Huntington Disease Neuroprotective Agents Gene Expression Regulation biology.protein Molecular Medicine Biological Assay Peptides Protein Binding Signal Transduction Biotechnology |
| Zdroj: | SLAS Discovery. 18:984-996 |
| ISSN: | 2472-5552 |
| Popis: | Huntington's Disease is a rare neurodegenerative disease caused by an abnormal expansion of CAG repeats encoding polyglutamine in the first exon of the huntingtin gene. N-terminal fragments containing polyglutamine (polyQ) sequences aggregate and can bind to cellular proteins, resulting in several pathophysiological consequences for affected neurons such as changes in gene transcription. One transcriptional pathway that has been implicated in HD pathogenesis is the CREB binding protein (CBP)/cAMP responsive element binding (CREB) pathway. We developed a phenotypic assay to screen for compounds that can reverse the transcriptional dysregulation of the pathway caused by induced mutated huntingtin protein (µHtt). 293/T-REx cells were stably co-transfected with an inducible full-length mutated huntingtin gene containing 138 glutamine repeats and with a reporter gene under control of the cAMP responsive element (CRE). One clone, which showed reversible inhibition of µHtt-induced reporter activity upon treatment with the neuroprotective Rho kinase inhibitor Y27632, was used for the development of a high-throughput phenotypic assay suitable for a primary screening campaign, which was performed on a library of 24,000 compounds. Several hit compounds were identified and validated further in a cell viability adenosine triphosphate assay. The assay has the potential for finding new drug candidates for the treatment of HD. |
| Databáze: | OpenAIRE |
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