title: Small-molecule fluorophores to detect cell-state switching in the context of high-throughput screening.
abstract: A small molecule capable of distinguishing the distinct states resulting from cellular differentiation would be of enormous value, for example, in efforts aimed at regenerative medicine. We screened a collection of fluorescent small molecules for the ability to distinguish the differentiated state of a mouse skeletal muscle cell line. High-throughput fluorescence-based screening of C2C12 myoblasts and myotubes resulted in the identification of six compounds with the desired selectivity, which was confirmed by high-content screening in the same cell states. The compound that resulted in the greatest fluorescence intensity difference between the cell states was used as the screening agent in a pilot screen of 84 kinase inhibitors, each present in four doses, for inhibition of myogenesis. Of the kinase inhibitors, 17 resulted in reduction of fluorescence at one or more concentrations; among the "hits" included known inhibitors of myogenesis, confirming that this compound is capable of detecting the differentiated myotube state. We suggest that the strategy of screening for screening agents reported here may be extended more broadly in the future.
authors.affiliation: Chemical Biology Program, Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02142, USA. bwagner@broad.harvard.edu
authors.forename: Bridget K
authors.initials: BK
authors.lastname: Wagner
authors.forename: Hyman A
authors.initials: HA
authors.lastname: Carrinski
authors.forename: Young-Hoon
authors.initials: YH
authors.lastname: Ahn
authors.forename: Yun Kyung
authors.initials: YK
authors.lastname: Kim
authors.forename: Tamara J
authors.initials: TJ
authors.lastname: Gilbert
authors.forename: Dina A
authors.initials: DA
authors.lastname: Fomina
authors.forename: Stuart L
authors.initials: SL
authors.lastname: Schreiber
authors.forename: Young-Tae
authors.initials: YT
authors.lastname: Chang
authors.forename: Paul A
authors.initials: PA
authors.lastname: Clemons
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D004353:Drug Evaluation, Preclinical; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D015335:Molecular Probes / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D008970:Molecular Weight; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D032446:Myoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010770:Phosphotransferases / Q000037:antagonists & inhibitors; D011237:Predictive Value of Tests; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D015335:Molecular Probes; D010770:Phosphotransferases