Enabling systematic interrogation of protein-protein interactions in live cells with a versatile ultra-high-throughput biosensor platform.

Large-scale genomics studies have generated vast resources for in-depth understanding of vital biological and pathological processes. A rising challenge is to leverage such enormous information to rapidly decipher the intricate protein-protein interactions (PPIs) for functional characterization and therapeutic interventions. While a number of powerful technologies have been employed to detect PPIs, a singular PPI biosensor platform with both high sensitivity and robustness in a mammalian cell environment remains to be established. Here we describe the development and integration of a highly sensitive NanoLuc luciferase-based bioluminescence resonance energy transfer technology, termed BRET(n), which enables ultra-high-throughput (uHTS) PPI detection in live cells with streamlined co-expression of biosensors in a miniaturized format. We further demonstrate the application of BRET(n) in uHTS format in chemical biology research, including the discovery of chemical probes that disrupt PRAS40 dimerization and pathway connectivity profiling among core members of the Hippo signaling pathway. Such hippo pathway profiling not only confirmed previously reported PPIs, but also revealed two novel interactions, suggesting new mechanisms for regulation of Hippo signaling. Our BRET(n) biosensor platform with uHTS capability is expected to accelerate systematic PPI network mapping and PPI modulator-based drug discovery.