Rational Design of Rho GTPase-Targeting Inhibitors

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 928)

Abstract

Rho GTPases have been implicated in diverse cellular functions and are potential therapeutic targets in inflammation, cancer, and neurologic diseases. Virtual screening of compounds that fit into surface grooves of RhoA known to be critical for guanine nucleotide exchange factor (GEF) interaction produced chemical candidates with minimized docking energy. Subsequent screening for inhibitory activity of RhoA binding to the Rho-GEF, LARG, identified a Rho-specific inhibitor as a lead compound capable of blocking RhoA–LARG interaction and RhoA activation by LARG specifically and dose dependently. A microscale thermophoresis analysis was applied to directly quantify the binding interaction of the lead inhibitor with RhoA target. The lead inhibitor highlights the principle that rational targeting of subfamily members of Rho GTPases is feasible and potentially useful in future drug design effort.

Key words

Rho GTPases RhoA Signaling Small molecule Inhibitor Rational drug design Targeting 

References

  1. 1.
    Etienne-Manneville S, Hall A (2002) Rho GTPases in cell biology. Nature 420:629–635PubMedCrossRefGoogle Scholar
  2. 2.
    Ridley AJ (2001) Rho family proteins: coordinating cell responses. Trends Cell Biol 11:471–477PubMedCrossRefGoogle Scholar
  3. 3.
    Zohn IM, Campbell SL, Khosravi-Far R, Rossman KL, Der CL (1998) Rho family proteins and Ras transformation: the RHOad less traveled gets congested. Oncogene 17:1415–1438PubMedCrossRefGoogle Scholar
  4. 4.
    Sahai E, Marshall CJ (2002) RHO-GTPases and cancer. Nat Rev Cancer 2:133–142PubMedCrossRefGoogle Scholar
  5. 5.
    Boettner B, Van Aelst L (2002) The role of Rho GTPases in disease development. Gene 286:155–174PubMedCrossRefGoogle Scholar
  6. 6.
    Nassar N, Cancelas J, Zheng J, Williams DA, Zheng Y (2006) Structure-function based design of small molecule inhibitors targeting Rho family GTPases. Curr Top Med Chem 6:1109–1116PubMedCrossRefGoogle Scholar
  7. 7.
    Marchioni F, Zheng Y (2009) Targeting rho GTPases by peptidic structures. Curr Pharm Des 15:2481–2487PubMedCrossRefGoogle Scholar
  8. 8.
    Sebti SM, Der CJ (2003) Opinion: searching for the elusive targets of farnesyltransferase inhibitors. Nat Rev Cancer 3:945–951PubMedCrossRefGoogle Scholar
  9. 9.
    Genth H, Dreger SC, Huelsenbeck J, Just I (2008) Clostridium difficile toxins: more than mere inhibitors of Rho proteins. Int J Biochem Cell Biol 40:592–597PubMedCrossRefGoogle Scholar
  10. 10.
    Gao Y, Dickerson JB, Guo F, Zheng J, Zheng Y (2004) Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci USA 101:7618–7623PubMedCrossRefGoogle Scholar
  11. 11.
    Onesto C, Shutes A, Picard V, Schweighoffer F, Der CJ (2008) Characterization of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases. Methods Enzymol 439:111–129PubMedCrossRefGoogle Scholar
  12. 12.
    Narumiya S, Ishizaki T, Uehata M (2000) Use and properties of ROCK-specific inhibitor Y-27632. Methods Enzymol 325:273–284PubMedCrossRefGoogle Scholar
  13. 13.
    Evelyn CR et al (2010) Design, synthesis and prostate cancer cell-based studies of analogs of the Rho/MKL1 transcriptional pathway ­inhibitor, CCG-1423. Bioorg Med Chem Lett 20:665–672PubMedCrossRefGoogle Scholar
  14. 14.
    Vetter IR, Wittinghofer A (2001) The guanine nucleotide-binding switch in three dimensions. Science 294:1299–1304PubMedCrossRefGoogle Scholar
  15. 15.
    Rossman KL, Der CJ, Sondek J (2005) GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol 6:167–180PubMedCrossRefGoogle Scholar
  16. 16.
    Kristelly R, Gao G, Tesmer JJ (2004) Structural determinants of RhoA binding and nucleotide exchange in leukemia-associated Rho guanine-nucleotide exchange factor. J Biol Chem 279:47352–47362PubMedCrossRefGoogle Scholar
  17. 17.
    Krieger E, Nielsen JE, Spronk CA, Kollman PA (2006) Fast empirical pKa prediction by Ewald summation. J Mol Graph Model 25:481–486PubMedCrossRefGoogle Scholar
  18. 18.
    Krieger E, Darden T, Nabuurs S, Finkelstein A, Vriend G (2004) Making optimal use of empirical energy functions: force-field parameterization in crystal space. Proteins 57:678–683PubMedCrossRefGoogle Scholar
  19. 19.
    Wienken CJ, Baaske P, Rothbauer U, Braun D, Duhr S (2010) Protein-binding assays in biological liquids using microscale thermophoresis. Nat Commun 19:100CrossRefGoogle Scholar
  20. 20.
    Fukuhara S, Chikumi H, Gutkind JS (2000) Leukemia-associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G(12) family to Rho. FEBS Lett 485:183–188PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  1. 1.Division of Experimental Hematology and Cancer Biology, Children’s Hospital Medical CenterUniversity of CincinnatiCincinnatiUSA
  2. 2.Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical CenterUniversity of CincinnatiCincinnatiUSA

Personalised recommendations