Biochemical suppression of small-molecule inhibitors: a strategy to identify inhibitor targets and signaling pathway components

Chem Biol. 2006 Apr;13(4):443-52. doi: 10.1016/j.chembiol.2006.02.009.


Identification of small-molecule targets remains an important challenge for chemical genetics. We report an approach for target identification and protein discovery based on functional suppression of chemical inhibition in vitro. We discovered pirl1, an inhibitor of actin assembly, in a screen conducted with cytoplasmic extracts. Pirl1 was used to partially inhibit actin assembly in the same assay, and concentrated biochemical fractions of cytoplasmic extracts were added to find activities that suppressed pirl1 inhibition. Two activities were detected, separately purified, and identified as Arp2/3 complex and Cdc42/RhoGDI complex, both known regulators of actin assembly. We show that pirl1 directly inhibits activation of Cdc42/RhoGDI, but that Arp2/3 complex represents a downstream suppressor. This work introduces a general method for using low-micromolar chemical inhibitors to identify both inhibitor targets and other components of a signaling pathway.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Actin-Related Protein 2-3 Complex / antagonists & inhibitors
  • Actins / metabolism
  • Animals
  • Cell Surface Extensions / drug effects
  • Drug Evaluation, Preclinical / methods*
  • Female
  • Guanine Nucleotide Dissociation Inhibitors / antagonists & inhibitors
  • In Vitro Techniques
  • Oocytes / drug effects
  • Oocytes / metabolism
  • Phosphatidylinositol 4,5-Diphosphate / metabolism
  • Signal Transduction / drug effects*
  • Tetradecanoylphorbol Acetate / pharmacology
  • Xenopus laevis
  • cdc42 GTP-Binding Protein / antagonists & inhibitors
  • rho-Specific Guanine Nucleotide Dissociation Inhibitors


  • Actin-Related Protein 2-3 Complex
  • Actins
  • Guanine Nucleotide Dissociation Inhibitors
  • Phosphatidylinositol 4,5-Diphosphate
  • rho-Specific Guanine Nucleotide Dissociation Inhibitors
  • cdc42 GTP-Binding Protein
  • Tetradecanoylphorbol Acetate