Rho GTPases pp 253-270 | Cite as

High-Throughput Flow Cytometry Bead-Based Multiplex Assay for Identification of Rho GTPase Inhibitors

  • Zurab SurviladzeEmail author
  • Susan M. Young
  • Larry A. Sklar
Part of the Methods in Molecular Biology book series (MIMB, volume 827)


Rho family GTPases and their effector proteins regulate a wide range of cell signaling pathways. In normal physiological conditions, their activity is tightly controlled and it is not surprising that their aberrant activation contributes to tumorigenesis or other diseases. For this reason, the identification of small, cell permeable molecules capable of inhibition of Rho GTPases can be extraordinarily useful, particularly if they are specific and act reversibly.

Herein, we describe a flow cytometric assay, which allows us to measure the activity of six small GTPases simultaneously. GST-tagged small GTPases are bound to six glutathione bead sets each set having a different intensity of red fluorescence at a fixed wavelength. The coated bead sets were washed, combined, and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out.

This multiplex bead-based assay was successfully used for to identify both general and selective inhibitors of Rho family GTPases.

Key words

Rho GTPases Cdc42 Bead-based multiplex assay Fluorescent GTP binding Screen Flow cytometry 



This work was supported by NIH grants U54 MH074425 and U54 MH084690.


  1. 1.
    Chimini, G., and Chavrier, P. (2000) Function of Rho family proteins in actin dynamics during phagocytosis and engulfment. Nature Cell Biol 2, E191–196.Google Scholar
  2. 2.
    Etienne-Manneville, S., and Hall, A. (2002) Rho GTPases in cell biology. Nature 420, 629–635.Google Scholar
  3. 3.
    Raftopoulou, M., and Hall, A. (2004) Cell migration: Rho GTPases lead the way. Dev Biol 265, 23–32.Google Scholar
  4. 4.
    Boettner, B., and Van Aelst, L. (2002) The role of Rho GTPases in disease development. Gene 286, 155–174.Google Scholar
  5. 5.
    Gomez del Pulgar, T., Benitah, S.A., Valeron, P.F., Espina, C., and Lacal, J.C. (2005) Rho GTPase expression in tumourigenesis: evidence for a significant link. Bioessays 27, 602–613.Google Scholar
  6. 6.
    Sahai, E., and Marshall, C.J. (2002) RHO-GTPases and cancer. Nat Rev Cancer 2, 133–142.Google Scholar
  7. 7.
    Vega, F.M., and Ridley, A.J. (2008) Rho GTPases in cancer cell biology. FEBS Lett 582, 2093–2101.Google Scholar
  8. 8.
    Pelish, H.E., Peterson, J.R., Salvarezza, S.B., Rodriguez-Boulan, E., Chen, J.L., Stamnes, M., Macia, E., Feng, Y., Shair, M.D., and Kirchhausen, T. (2006) Secramine inhibits Cdc42-dependent functions in cells and Cdc42 activation in vitro. Nature Chem Biol 2, 39–46.Google Scholar
  9. 9.
    Gao, Y., Dickerson, J.B., Guo, F., Zheng, J., and Zheng, Y. (2004) Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci USA 101, 7618–7623.Google Scholar
  10. 10.
    Shutes, A., Onesto, C., Picard, V., Leblond, B., Schweighoffer, F., and Der, C.J. (2007) Specificity and mechanism of action of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases. J Biol Chem 282, 35666–35678.Google Scholar
  11. 11.
    Surviladze, Z., Waller, A., Wu, Y., Romero, E., Edwards, B.S., Wandinger-Ness, A., and Sklar, L.A. (2010) Identification of a small GTPase inhibitor using a high-throughput flow cytometry bead-based multiplex assay. J Biomol Screen 15, 10–20.Google Scholar
  12. 12.
    McEwen, D.P., Gee, K.R., Kang, H.C., and Neubig, R.R. (2001) Fluorescent BODIPY-GTP analogs: real-time measurement of nucleotide binding to G proteins. Anal Biochem 291, 109–117.Google Scholar
  13. 13.
    Jameson, E.E., Roof, R.A., Whorton, M.R., Mosberg, H.I., Sunahara, R.K., Neubig, R.R., and Kennedy, R.T. (2005) Real-time detection of basal and stimulated G protein GTPase activity using fluorescent GTP analogues. J Biol Chem 280, 7712–7719.Google Scholar
  14. 14.
    Evelyn, C.R., Ferng, T., Rojas, R.J., Larsen, M.J., Sondek, J., and Neubig, R.R. (2009) High-throughput screening for small-molecule inhibitors of LARG-stimulated RhoA nucleotide binding via a novel fluorescence polarization assay. J Biomol Screen 14, 161–172.Google Scholar
  15. 15.
    Salas, V.M., Edwards, B.S., and Sklar, L.A. (2008) Advances in multiple analyte profiling. Adv Clin Chem 45, 47–74.Google Scholar
  16. 16.
    Edwards, B.S., Young, S.M., Oprea, T.I., Bologa, C.G., Prossnitz, E.R., and Sklar, L.A. (2006) Biomolecular screening of formylpeptide receptor ligands with a sensitive, quantitative, high-throughput flow cytometry platform. Nature Protocols 1, 59–66.Google Scholar
  17. 17.
    Saunders, M.J., Edwards, B.S., Zhu, J., Sklar, L.A., and Graves, S.W. (2010) Microsphere-based flow cytometry protease assays for use in protease activity detection and high-throughput screening. Curr Protoc in Cytom Unit 13.12.1–17.Google Scholar
  18. 18.
    Schwartz, S.L., Tessema, M., Buranda, T., Pylypenko, O., Rak, A., Simons, P.C., Surviladze, Z., Sklar, L.A., and Wandinger-Ness, A. (2008) Flow cytometry for real-time measurement of guanine nucleotide binding and exchange by Ras-like GTPases. Anal Biochem 381, 258–266.Google Scholar
  19. 19.
    Zhang, J. H., Chung, T. D., and Oldenburg, K. R. (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4, 67–73.Google Scholar
  20. 20.
    Malo, N., Hanley, J.A., Cerquozzi, S., Pelletier, J., and Nadon, R. (2006) Statistical practice in high-throughput screening data analysis. Nature Biotech 24, 167–175.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Zurab Surviladze
    • 1
    • 2
    Email author
  • Susan M. Young
    • 1
    • 2
  • Larry A. Sklar
    • 1
    • 3
  1. 1.New Mexico Molecular Libraries Screening CenterAlbuquerqueUSA
  2. 2.Cancer Research and Treatment CenterUniversity of New Mexico School of MedicineAlbuquerqueUSA
  3. 3.Department of Pathology, Cancer Research and Treatment CenterUniversity of New Mexico School of MedicineAlbuquerqueUSA

Personalised recommendations