Novel Methods: Development of a Split Luciferase Complementation Assay for Rapid Screening of Pharmacological Inhibitors of Protein: Protein Interactions

Principal Investigators: Kathryn Cunningham, PhD and Fernanda Laezza, MD, PhD

Acrylamide gel is used to separate proteins - Diane A. Reid, PhotographerDevelopment of small molecule inhibitors targeting the protein: protein interface will enable the disruption of large protein:protein complexes and, theoretically, provide a rich source of vastly overlooked targets for drug discovery. A key component in the development of novel disrupters of protein:protein interactions is the capability to identify, detect and characterize these interactions. Here, we propose to optimize a novel cellular assay, the split luciferase complementation assay (LCA), to explore the biology of neuronal protein:protein interactions, in particular, those that occur between membrane-localized receptors and intracellular signaling molecules that control neuronal function, as a means to discover new pharmacological disruptors of these interactions. The LCA assay is a powerful tool to provide quantitative, real-time readout of protein:protein interactions in vitro and in vivo.

We propose to create and optimize the LCA assay to explore the biology of the protein:protein interactions of the serotonin (5-HT) 5-HT2C receptor (5-HT2CR) macromolecular complex. Recently, disruption of coupling between the 5-HT2CR and its protein partner PTEN (phosphatase and tensin homologue deleted on chromosome 10) was shown to result in agonist-like effects in vivo, suggesting that a pathway-specific inhibition of the 5-HT2CR:PTEN interface provides a new way to enhance the efficiency of 5-HT2CR signaling and may prove therapeutically useful for a variety of physiological and psychological conditions. We will focus on optimizing the conditions for detecting the 5-HT2CR:PTEN assembly and assessing functional activity of new cellular models. We will then build on these results to develop a robust detection method suitable for screening of small molecules that interfere with the 5-HT2CR:PTEN interaction. We will engineer a new stable cell line for the 5-HT2CR:PTEN interaction for use in screening for peptides/small molecule inhibitors of the 5-HT2CR:PTEN interaction using the LCA assay.

The proposed studies will result in the development of the LCA technology and the creation of a stable cell line needed for screening assays geared to discover small molecule inhibitors of the 5-HT2CR:PTEN interaction. Further, this assay model will provide the foundation for subsequent adaptation of this novel approach to probe the involvement of protein:protein interactions in multiple arenas.