Development of Activity-Based Probes for Ubiquitin and Ubiquitin-like Protein Signaling Pathways

Abstract

Ubiquitin and ubiquitin-like (UBL) proteins regulate a vast variety of cellular functions. Some UBL proteins are present in all cell types, while others are expressed only in certain cells or under certain environmental conditions. This highlights the central role of UBL systems in regulation of ubiquitous as well as specific cellular functions. UBL proteins share little amino acid sequence identity to each other, yet they share similar 3D shapes, which is exemplified by the β-grasp fold. Central to UBL protein signaling pathways are UBL protein-activating E1 enzymes that activate the C-terminus of UBL proteins for subsequent conjugation to the protein substrates. Due to their critical roles in biology, E1 enzymes have been recognized as emerging drug targets to treat human diseases. In spite of their biological significance, however, methods to discover UBL proteins and to monitor the intracellular activity of E1 enzymes are lacking. Thus, there is a critical need for methods to evaluate the intracellular mechanisms of action of E1 enzyme inhibitors. Here we describe the development of a mechanism-based small-molecule probe, ABP1, that can be used to discover and to detect active UBL proteins, and to monitor the intracellular activity of E1 enzymes inside intact cells. The developed probe can also be used to profile the selectivity of E1 enzyme-targeting drugs in vitro and inside intact cells.