GRIP is a universal protein interaction discovery system that can be used to generate truly HTS compatible cellular assays to be used to screen for inhibitors of protein interactions. The technology uses bait and prey principle, and the advantages of the assay include:

• Modular format adaptable to protein interaction partners of choice
• High-throughput screening of protein-protein interaction in a cell based physiological relevant environment
• Universal assay control compound is available
• Can be read on high content imager or regular plate reader depending on assay
• Configurable with different anchor and localization formats

One example of GRIP assays is based on the distinct translocation behaviour of the human cAMP phosphodiesterase PDE4A4.

P53-HDM2 assay:
The p53-HDM2 GRIP assay is designed to measure the interaction between p53 and HDM2, and the principle of the different assay components are illustrated in figure 1. In short:

• HDM2 is fused to PDE4A4
• p53 is fused to GFP
• - In growing cells p53 and Hdm2 interact (figure 1A)
• When anchor stimulus (RS25344) is applied to cells PDE4A4 is recruited to spots and the interacting proteins follows resulting in GFP intense foci (Figure 1B).

Figure 1. Components of the GRIP P53-HDM2 protein-protein interaction assay; generation of foci.

The GFP foci can be dispersed by two different mechanisms; either by breaking the p53-HDM2 interaction or by disrupting the PDE4A4 anchor. The two different scenarios can be distinguished by carefully interpreting the response features (Figure 2):

• The PDE4A4 inhibitor RP73401 causes foci dispersal by disrupting the anchor (Figure 2A) resulting in cytoplasmic GFP fluorescence
• The p53-HDM2 interaction inhibitor Nutlin3 disrupts the p53-HDM2 interaction, and the GFP-p53 fusion protein translocates to the nucleus (Figure 2B).

Figure 2. Components of the GRIP P53-HDM2 protein-protein interaction assay; dispersal of foci.

The assay is quantitative and can be used to discover inhibitors of the p53-HDM2 interaction. Figure 3 shows concentration response of the two classes of inhibitors identified in by the assay. Taking advantage of the high content information extracted from the images the two hit classes can be distinguished:

• Nutlin3 and RP73401 have an EC₅₀ of 1.5 µM and 0.13 µM respectively (Figure 3A)
• Nutlin3 - that disrupts the p53-HDM2 interaction - causes p53-GFP to translocate to the nucleus and causes an increase in nucleus to cytoplasm ratio (Figure 3B).
• RP73401 - a PDE4A4 inhibitor causes dissociating of the anchor - remains in the cytoplasm (Figure 3B).

Figure 3. Quantification of the p53-hdm2 interaction (A), and separation of the two types of hit compounds dissociating the p53-hdm2 interaction (Nutlin3) translocating to the nucleus and the anchor dissociating compound PR73401 remaining in the cytoplasm (B).

See this technology application note for more details

Biological basis for GRIP technology
As mentioned above, the GRIP technology uses a bait and prey principle, and is based on the distinct translocation behaviour of the human cAMP phosphodiesterase PDE4A4. Treatment with the PDE4A4 inhibitor RS25344 or Rolipram leads to a sublocalization of PDE4A4 into compact foci and the interaction between bait and prey constructs is revealed by a redistribution of GFP fluorescence to these same foci (Figure 4):

Figure 4. Biological basis for GRIP technology

References:
1. Terry B. et al., Cell Signal 15, 955-971, 2003.