Cat. # BK122

Product Uses Include

• Screening for new anti-hypertensive drugs
• Screening for new anti-tumor drugs
• Screening for new neuroregenerative drugs
• Screening for new anti-angiogenesis drugs
• Screening for new anti-apoptotic drugs
• Secondary assays for improving specificity
  
  

Introduction
Cytoskeleton, Inc. has introduced a range of in vitro small G-protein / effector interaction assays. These assays are in the same convenient 96-well format as the other cell based G-LISA™ assays, but entirely performed with purified proteins. This G-LISA™ format can identify compounds that disrupt the interaction between a Rho family member and its specific downstream effector. A popular example of this format is the RhoA::ROCK assay, this interaction has been reported to be a possible target for anti-tumor, anti-hypertension drugs and neuroregenerative drugs. It is an upstream target compared to the current target of choice, which is the ROCK kinase domain.

Figure 1. Detection method. Immobilized effector protein binds the activated form of the small G-protein, which is then detected by standard ELISA technology. Inhibitors (i) reduce the binding of activated small G-protein, which leads to a reduction in signal. Signal to noise ratios are generally 8:1 to 12:1.

The following assays are currently available:

• G-LISA™ for RhoA::Rhotekin interaction. For drug discovery applications, see refs. 2 and 3.
• G-LISA™ for RhoB::Rhotekin interaction. For drug discovery applications see refs. 2 and 3.
• G-LISA™ for RhoC::Rhotekin interaction. Possible use in discovering anti-tumor drugs (ref. 4).
• G-LISA™ for RhoA::ROCK interaction. For drug discovery applications see refs 1, 2 and 3.
• G-LISA™ for RhoB::ROCK interaction. For drug discovery applications see refs. 2 and 3.
• G-LISA™ for RhoC::ROCK interaction. Possible use in discovering anti-tumor drugs (ref. 4).
• G-LISA™ for Rac1::PAK interaction. Possible use in discovering anti-angiogenesis drugs (ref. 5)
• G-LISA™ for Rac1::POSH interaction. Possible use in discovering anti-apoptotic drugs (ref. 6)
• G-LISA™ for Cdc42::PAK interaction. Possible use in discovering anti-angiogenesis drugs (ref. 5)
• G-LISA™ for Cdc42::WASP interaction. Possible use in discovering anti-tumor drugs (ref. 7)
• G-LISA™ for H-Ras::Raf Kinase interaction. Possible use in discovering anti-tumor drugs (ref. 8, 9)
• G-LISA™ for K-Ras::Raf Kinase interaction. Possible use in discovering anti-tumor drugs (ref. 8, 9)

For other combinations of small G-proteins and effectors, please inquire.

NOTE: These assays are available on a custom order basis only. There is a 12 week production time to provide the completed batch of assays. Please inquire for a quotation or more information to our technical support.

Kit contents
The kits contain all the reagents needed for the assay. The assays are performed in a 96-well format.

Equipment needed

1. 96-well plate spectrophotometer capable of reading 490 nm wavelength

Example results
The inhibition of Cdc42 binding to WASP, Rac1 binding to POSH, and RhoA binding to ROCK was tested by the addition of competing effector domains. Each G-protein::effector interaction was efficiently inhibited (Fig 2)

Figure 2. Small G-protein::effector inhibiton assay. Effectors for Rho family proteins are covalently bound to wells of a 96-well plate. Compound is added to each well and followed by addition of 10 ng of small G-protein. After washing the wells are developed by a normal ELISA technique with primary and secondary antibodies. Effectors for Cdc42, Rac1 and RhoA are WASP, POSH and ROCK, respectively (Cat. # BK122j, BK122h and BK122d). In this experiment, the inhibitors were 50 nM of PAK, PAK and Rhotekin, respectively.

References

Lee et al. 2004, Hypertension, 44, 796-799
Gomez del Pulgar et al. 2005, BioEssays, 27, 602-613
Mueller et al. 2005, Nat. Rev. Drug Discov., 4, 387-398
van Golen et al. 1999, Clin. Cancer Res., 5, 2511-2519.
Fryer and Field 2005, Cancer Lett., 229, 13-23.
Xu Z. et al. 2003, EMBO J., 22, 252-261.
Rao and Li 2004, Curr. Cancer Drug Targets, 4, 345-354.
Bollag et al. 2003, Curr. Opin. Investig. Drugs., 4, 1436-1441.
Adjei 2001, J. Natl. Cancer Inst. 93, 1062-1074.