Effective cell-free drug screening protocol for protein-protein interaction

Specific protein-protein interaction (PPI) is an essential feature of many cellular processes however, targeting these interactions by small molecules is highly challenging due to the nature of the interaction interface. Thus, screening for PPI inhibitors requires enormous number of compounds. Here we describe a simple and improved protocol designed for a search of direct PPI inhibitors. We engineered a bacterial expression system for the split-Renilla luciferase (RL) complementation assay that monitors PPI. This enables production of large quantities of the RL fusion proteins in a simple and cost effective manner that is suitable for very large screens. Subsequently, inhibitory compounds are analyzed in a similar complementation assay in living cultured mammalian cells to select for those that can penetrate cells. We applied this method to NF-κB, a family of dimeric transcription factors that plays central roles in immune responses, cell survival and aging, and its dysregulation is linked to many pathological states. This strategy led to the identification of several direct NF-κB inhibitors. As the described protocol is very straightforward and robust it may be suitable for many pairs of interacting proteins.