Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor.
Small-molecule kinase inhibitors are predominantly discovered in pure protein assays. We have discovered an inhibitor of Rho-kinase (ROCK) through an image-based, high-throughput screen of cell monolayer wound healing. Using automated microscopy, we screened a library of approximately 16,000 compounds finding many that affected cell migration or cell morphology as well as compounds that blocked mitotic progression. We tested approximately 200 compounds in a series of subassays and chose one, 3-(4-pyridyl)indole (Rockout), for more detailed characterization. Rockout inhibits blebbing and causes dissolution of actin stress fibers, phenocopying Rho-kinase inhibitors. Testing Rho-kinase activity in vitro, Rockout inhibits with an IC50 of 25 microM ( approximately 5-fold less potent than Y-27632) but has a similar specificity profile. We also profile the wound healing assay with a library of compounds with known bioactivities, revealing multiple pathways involved in the biology.
Yarrow
Justin C
JC
Department of Systems Biology, Boston, Massachusetts 02115, USA. jyarrow@post.harvard.edu
Totsukawa
Go
G
Charras
Guillaume T
GT
Mitchison
Timothy J
TJ
D000818:Animals; D041702:BALB 3T3 Cells; D002465:Cell Movement / Q000187:drug effects* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054460:rho-Associated Kinases
D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases