title: An image-based biosensor assay strategy to screen for modulators of the microRNA 21 biogenesis pathway.
abstract: microRNAs (miRNAs) are evolutionary conserved, small endogenous non-coding, RNA molecules. Although their mode of action has been extensively studied, little is known about their biogenesis. As their altered expression has been implicated in many diseases, small molecules that would modulate their expression are sought after. They are generated through the concerted action of several complexes which promote their transcription, maturation, export, trafficking, and loading of mature miRNA into silencing complexes. An increasing number of studies have suggested that each of these steps serves as a regulatory junction in the process, and therefore provides an intervention point. For this purpose, we have developed a simple image-based assay strategy to screen for such modulators. Here, we describe its successful implementation which combines the use of a microRNA 21 (miR-21) synthetic mimic together with an EGFP based reporter cell line, where its expression is under the control of miR-21, to monitor EGFP expression in a format suitable for HTS. The strategy was further validated using a small panel of known gene modulators of the miRNA pathway. A screen was performed in duplicate against a library of 6,912 compounds and identified 48 initial positives exhibiting enhanced EGFP fluorescence intensity. 42 compounds were found to be inherently fluorescent in the green channel leaving the remaining 6 as potential inhibitors and with a positive rate of 0.09%. Taken together, this validated strategy offers the opportunity to discover novel and specific inhibitors of the pathway through the screening of diverse chemical libraries.
authors.affiliation: HTS Core Facility, Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
authors.forename: David
authors.initials: D
authors.lastname: Shum
authors.forename: Bhavneet
authors.initials: B
authors.lastname: Bhinder
authors.forename: Constantin
authors.initials: C
authors.lastname: Radu
authors.forename: Thalia
authors.initials: T
authors.lastname: Farazi
authors.forename: Markus
authors.initials: M
authors.lastname: Landthaler
authors.forename: Thomas
authors.initials: T
authors.lastname: Tuschl
authors.forename: Paul
authors.initials: P
authors.lastname: Calder
authors.forename: Christina N
authors.initials: CN
authors.lastname: Ramirez
authors.forename: Hakim
authors.initials: H
authors.lastname: Djaballah
mesh_terms: D015374:Biosensing Techniques; D002478:Cells, Cultured; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D035683:MicroRNAs / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*
chemical_list: C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D054852:Small Molecule Libraries