BiFC in Drug Discovery
Contrary to the wide applications of BiFC assay for molecular interactions in cells and in various model systems, the use of BiFC in drug discovery has not really taken off. Part of the reason is due to the low success for screening of small molecule inhibitors targeting protein-protein interactions. However, recent success along with the development of new HTS assays such as BiFC-based assays will reinvigorate the interest of the pharmaceutical industry to search for small molecules targeting protein-protein interactions. A proof-of-principle system was reported using a YFP-based fluorescence complementation⁽⁶¹⁾. The use of BiFC assay for HTS for small molecule inhibitors in living cells overcomes the limitations discussed above for the current methods and allows screening of inhibitors under physiological conditions. More importantly, the assay also allows the screening of small molecule inhibitors for inducible interactions. Because multicolor BiFC assay allows visualization of multiple interactions, we are currently using our improved multicolor BiFC system to develop a multicolor BiFC-based HTS assay to screen small molecule inhibitors for protein-protein interactions (Fig. 2A)⁽³⁶⁾. Compared with current HTS assays for the screening of small molecule inhibitors of protein-protein interactions^{(7, 62-64)}, the multicolor BiFC-based screening system offers several advantages.


Shyu et al. Figure 2



Figure 2.A. Nonfluorescent N-terminal fragments of Venus (VN) and Cerulean (CrN) and C-terminal fragment of CFP (CC) are fused to proteins A, B and C, respectively. The interaction between proteins A and C brings VN and CC together and reconstitutes a fluorescent protein VN-CC (Venus-like), and the interaction between proteins B and C reconstitutes a fluorescent protein CrN-CC (Cerulean-like).



Figure 2.B. The binding of a specific inhibitor to protein A inhibits the interactions between proteins A and C, leading to the inhibition of Venus reconstitution.



Figure 2.C. The binding of a specific inhibitor to protein C inhibits the interactions between proteins B and C, leading to the inhibition of Cerulean reconstitution.



Figure 2.D. The binding of a nonspecific inhibitor to either the shared CC, or homologous VN or CrN (left), or the shared binding surface for proteins A and B on protein C (right) inhibits the reconstitution of both CC-VN and CC-CrN or inhibits both A-C and B-C interactions. Hence, this assay can be used to screen for inhibitors specific for two interactions simultaneously and the lack of both Venus and Cerulean fluorescence is likely caused by non-specific inhibitors.

First, the screening is specific. Since two closely related interactions are simultaneously screened, one interaction serves as an internal control of the other. Hence, the lack of Venus fluorescence indicates a specific inhibitor for proteins A-C interaction (Fig. 2B), whereas the lack of Cerulean fluorescence indicates a specific inhibitor for proteins B-C interaction (Fig. 2C). Any inhibitor targeting the interaction interface on the protein C, which is shared by both proteins A and B, results in the loss of both Venus and Cerulean fluorescence (Fig. 2D). Because CrN173 and VN173 are homologous in sequences and structures, any inhibitors targeting the VN173 fragment will have a high probability of targeting the CrN173 fragment, or vice versa (Fig. 2D, left). Therefore, the formation of complex between VN173 or CrN173 and CC155 will be similarly inhibited. Likewise, any inhibitors targeting CC155 will affect the formation of both complexes. In one interaction-based screening assays^{(7, 61-64)}, these kinds of non-specific inhibitors can be eliminated only in the secondary screening. Second, the screening is cost-effective. We propose to screen for two interactions in the same screening. In principle, three or even more interactions can be screened in the same assay if different combinations of fluorescent proteins are used^{(28, 40)}. Third, the multicolor BiFC assay can be used for screening small molecule inhibitors that target inducible interactions through a physiological stimulus. Fourth, the screening can be also performed in living animals such as C. elegans.

Future Perspectives
Although BiFC technology is still in its infancy, its wide applications discussed above clearly indicate that BiFC assay is one of the most powerful tools for visualization and identification of protein-protein interactions in living cells and in living animals. Despite the fact that its current use in industry is very limited, we anticipate that BiFC-based technologies for drug discovery will play an important part in the screening of small molecule or peptide inhibitors of protein-protein interactions. In addition, the BiFC-based technologies will be extremely useful for validation of hits in vivo. However, further development is still needed. One of these is the irreversibility of BiFC system. Once the BiFC complex is formed in vitro, it is essentially irreversible in vitro⁽³⁵⁾. While irreversibility of the BiFC system allows for identification of weak and transient interactions, it may limit its use for the screening of small molecule inhibitors that can also disrupt protein interactions. Thus, the availability of a reversible BiFC system will increase the flexibility for the design of screening on an individual basis. It would be also interesting to test whether fragments derived from mRFP1 can complement the C-terminal fragment of CFP (CC155), which is used for multicolor BiFC analysis. This will lead to the development of simultaneous screening for multiple interactions. Obviously, identification of new fluorescent protein fragments that can complement fragments derived from different fluorescent proteins will increase the number of target interactions in one screening. In addition, development of a corresponding expression system, such as tricistronic and inducible expression system, to meet these needs will facilitate the assay development. Given the rapid growth of a variety of BiFC-based technologies and applications, the BiFC-based technologies will undoubtedly have a colorful future in drug discovery.

Note: BiFC is used in this article to avoid confusion to readers. The protein fragment complementation assay (PCA) and split GFP are used in publications from the Michnick and Regan labs, respectively.

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