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.

An inhibitor of ubiquitin conjugation and aggresome formation

“…39 ABP1 was designed based on previously developed pan-E1 inhibitor Compound I, both of which closely mimic the structure of AMP. 26 The unique feature of ABP1, however, is the presence of an alkyne tag that facilitates the rapid identification of intracellular targets of ABP1 and its intracellular selectivity and potency.…”

Covalent tethering of fragments for covalent probe discovery

“…3, 4 The advent of covalent probes (both nucleophilic and electrophilic) for chemical biology has been assisted by the development of click chemistry methods. 5, 6 In fact, covalent probes are the most convenient probes to use, because their intracellular selectivity and potency, and the covalent labelling of off-target proteins can be easily estimated by conducting click chemistry experiments. 5, 6 Furthermore, click chemistry allows for tracking tissue and organ distribution of covalent probes in vivo .…”

“…5, 6 In fact, covalent probes are the most convenient probes to use, because their intracellular selectivity and potency, and the covalent labelling of off-target proteins can be easily estimated by conducting click chemistry experiments. 5, 6 Furthermore, click chemistry allows for tracking tissue and organ distribution of covalent probes in vivo . 6 Importantly, reversible interactions of covalent inhibitors with the protein target are also essential for their biochemical and cellular potency.…”

Catalytically Important Residues of E6AP Ubiquitin Ligase Identified Using Acid-Cleavable Photo-Cross-Linkers

“…To identify modified E6AP residues, the UbcH7 mutant that most efficiently cross-linked when modified by 1 was equipped with cross-linker 2 , which harbors an acid-cleavable N -acylsulfamate moiety. 12 Cleavage of photo-cross-linked peptides facilitates their subsequent MS and MS/MS analysis (Figure 1C). 10 Thus, cleavage of the UbcH7-E6AP cross-link prior to digestion and mass analysis provided the robust signal of E6AP peptides with a unique butanol covalent modification (+72.06 Da, Spectral Appendix I of the Supporting Information).…”

“…This may be due to deprotonation of the acylsulfamate, which reduces the reactivity of 2 and 3 toward thiol nucleophiles. 12 Therefore, 1 was first used to rapidly survey the proximity of UbcH7 surfaces to an interface with E6AP. To avoid nonspecific alkylation of native UbcH7 cysteine residues Cys 17 , Cys 86 (catalytic cysteine), and Cys 137 , they were mutated to serine (termed UbcH7 CΔS).…”