A split intein and split luciferase-coupled system for detecting protein-protein interactions

Elucidation of protein-protein interactions (PPIs) represents one of the most important methods in biomedical research. Recently, PPIs have started to be exploited for drug discovery purposes and have thus attracted much attention from both the academic and pharmaceutical sectors. We previously developed a sensitive method, Split Intein-Mediated Protein Ligation (SIMPL), for detecting binary PPIs via irreversible splicing of the interacting proteins being investigated. Here, we incorporated tripart nanoluciferase (tNLuc) into the system, providing a luminescence signal which, in conjunction with homogenous liquid phase operation, improves the quantifiability and operability of the assay. Using a reference PPI set, we demonstrated an improvement in both sensitivity and specificity over the original SIMPL assay. Moreover, we designed the new SIMPL-tNLuc ('SIMPL2') platform with an inherent modularity allowing for flexible measurement of molecular modulators of target PPIs, including inhibitors, molecular glues and PROTACs. Our results demonstrate that SIMPL2 is a sensitive, cost- and labor-effective tool suitable for high-throughput screening (HTS) in both PPI mapping and drug discovery applications.