title: A phenotypic screening assay for modulators of huntingtin-induced transcriptional dysregulation.
abstract: 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.
authors.affiliation: 1Biomolecular Screening Unit, Siena Biotech Spa, Siena, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Lazzeroni
authors.forename: Tiziana
authors.initials: T
authors.lastname: Benicchi
authors.forename: Freddy
authors.initials: F
authors.lastname: Heitz
authors.forename: Letizia
authors.initials: L
authors.lastname: Magnoni
authors.forename: Daniela
authors.initials: D
authors.lastname: Diamanti
authors.forename: Lara
authors.initials: L
authors.lastname: Rossini
authors.forename: Luisa
authors.initials: L
authors.lastname: Massai
authors.forename: Cesare
authors.initials: C
authors.lastname: Federico
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Fecke
authors.forename: Andrea
authors.initials: A
authors.lastname: Caricasole
authors.forename: Salvatore
authors.initials: S
authors.lastname: La Rosa
authors.forename: Valentina
authors.initials: V
authors.lastname: Porcari
mesh_terms: D000577:Amides / Q000737:chemistry / Q000494:pharmacology; D001681:Biological Assay; D050882:CREB-Binding Protein / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018696:Neuroprotective Agents / Q000737:chemistry / Q000494:pharmacology*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011725:Pyridines / Q000737:chemistry / Q000494:pharmacology; D020218:Response Elements; D015398:Signal Transduction; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D000577:Amides; C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D018696:Neuroprotective Agents; D010455:Peptides; D011725:Pyridines; D054852:Small Molecule Libraries; C108830:Y 27632; C097188:polyglutamine; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; D054460:rho-Associated Kinases
keywords: cell-based assays; gene expression; phenotypic drug discovery; reporter gene assays