Chemical Development of Intracellular Protein Heterodimerizers

SummaryCell activation initiated by receptor ligands or oncogenes triggers complex and convoluted intracellular signaling. Techniques initiating signals at defined starting points and cellular locations are attractive to elucidate the output of selected pathways. Here, we present the development and validation of a protein heterodimerization system based on small molecules cross-linking fusion proteins derived from HaloTags and SNAP-tags. Chemical dimerizers of HaloTag and SNAP-tag (HaXS) show excellent selectivity and have been optimized for intracellular reactivity. HaXS force protein-protein interactions and can translocate proteins to various cellular compartments. Due to the covalent nature of the HaloTag-HaXS-SNAP-tag complex, intracellular dimerization can be easily monitored. First applications include protein targeting to cytoskeleton, to the plasma membrane, to lysosomes, the initiation of the PI3K/mTOR pathway, and multiplexed protein complex formation in combination with the rapamycin dimerization system.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (344 K)Download as PowerPoint slideHighlights► Design of cell-permeable, SNAP-tag and HaloTag reactive, covalent dimerizers (HaXS) ► HaXS dimerizers force rapid intracellular dimerization of tagged proteins of interest ► In contrast to rapalogs, HaXS8 does not interfere with PI3K/mTOR signaling ► HaXS8 is compatible with multiplexed approaches using other CIDs