Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor

Chem Biol. 2005 Mar;12(3):385-95. doi: 10.1016/j.chembiol.2005.01.015.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • BALB 3T3 Cells
  • Cell Movement / drug effects*
  • Cell Movement / physiology
  • Dose-Response Relationship, Drug
  • Intracellular Signaling Peptides and Proteins
  • Mice
  • Microscopy, Confocal / methods
  • Protein Kinase Inhibitors / chemistry
  • Protein Kinase Inhibitors / pharmacology*
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors*
  • Protein-Serine-Threonine Kinases / metabolism
  • rho-Associated Kinases


  • Intracellular Signaling Peptides and Proteins
  • Protein Kinase Inhibitors
  • Protein-Serine-Threonine Kinases
  • rho-Associated Kinases