A BromoTag-Based Toolkit for Rapid Modulation of Chemically Induced Proximity

Abstract

Mammalian cells control a wide range of essential processes via protein–protein interactions (PPIs), including signal transduction, gene expression, cell cycle regulation, and metabolism. Pathologic alterations in PPIs have been linked to many human diseases. Thus, the ability to rapidly and selectively induce PPIs in cultured cells is crucial for many fields of biological research. One proven approach is the use of synthetic bivalent chemical ligands to selectively recruit protein domains with engineered mutations (i.e., "bump-and-hole"). Developing new PPI chemical inducers with unique protein domains enables multiplexing and potentially expands the breadth of recruitable proteins. Herein, we describe the design and characterization of a BromoTag- and dTAG-based toolkit to induce protein homo- and heterodimerization. Using a NanoBiT split-luciferase assay, we characterized a library of BromoTag-based homo- (BromoTag/BromoTag) and heterobivalent (BromoTag/dTAG) compounds for their ability to induce neo-PPIs. Compounds were evaluated for activity across multiple cellular applications, including caspase-9 and c-Kit tyrosine kinase activation, with comparable activity to other dimerizer systems. This BromoTag-based protein dimerization toolkit provides a useful alternative to current controlled dimerization systems that will enable robust prototyping of chemically induced proximity.