Identification of a small GTPase inhibitor using a high-throughput flow cytometry bead-based multiplex assay

J Biomol Screen. 2010 Jan;15(1):10-20. doi: 10.1177/1087057109352240. Epub 2009 Dec 11.


Small GTPases are key regulators of cellular activity and represent novel targets for the treatment of human diseases using small-molecule inhibitors. The authors describe a multiplex, flow cytometry bead-based assay for the identification and characterization of inhibitors or activators of small GTPases. Six different glutathione-S-transferase (GST)-tagged small GTPases were bound to glutathione beads, each labeled with a different red fluorescence intensity. Subsequently, beads bearing different GTPase were mixed and dispensed into 384-well plates with test compounds, and fluorescent-guanosine triphosphate (GTP) binding was used as the readout. This novel multiplex assay allowed the authors to screen a library of almost 200,000 compounds and identify more than 1200 positive compounds, which were further verified by dose-response analyses, using 6- to 8-plex assays. After the elimination of false-positive and false-negative compounds, several small-molecule families with opposing effects on GTP binding activity were identified. The authors detail the characterization of MLS000532223, a general inhibitor that prevents GTP binding to several GTPases in a dose-dependent manner and is active in biochemical and cell-based secondary assays. Live-cell imaging and confocal microscopy studies revealed the inhibitor-induced actin reorganization and cell morphology changes, characteristic of Rho GTPases inhibition. Thus, high-throughput screening via flow cytometry provides a strategy for identifying novel compounds that are active against small GTPases.

Publication types

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

MeSH terms

  • Actins / metabolism
  • Animals
  • Cell Line
  • Cell Shape / drug effects
  • Dose-Response Relationship, Drug
  • Enzyme Activation / drug effects
  • Enzyme Inhibitors / analysis*
  • Enzyme Inhibitors / pharmacology*
  • Epidermal Growth Factor / pharmacology
  • Flow Cytometry / methods*
  • High-Throughput Screening Assays / methods*
  • Immunoglobulin E / pharmacology
  • Kinetics
  • Ligands
  • Mast Cells / cytology
  • Mast Cells / drug effects
  • Mice
  • Microspheres*
  • Monomeric GTP-Binding Proteins / antagonists & inhibitors*
  • Monomeric GTP-Binding Proteins / metabolism
  • Rats
  • Reproducibility of Results
  • beta-N-Acetylhexosaminidases / metabolism
  • rac1 GTP-Binding Protein / metabolism
  • rho GTP-Binding Proteins / antagonists & inhibitors
  • rho GTP-Binding Proteins / metabolism


  • Actins
  • Enzyme Inhibitors
  • Ligands
  • Immunoglobulin E
  • Epidermal Growth Factor
  • beta-N-Acetylhexosaminidases
  • Monomeric GTP-Binding Proteins
  • rac1 GTP-Binding Protein
  • rho GTP-Binding Proteins