ABPA3

ABPA3 is an activity-based probe that covalently inhibits the ubiquitin- and Nedd8-activating E1 enzymes (UBA1/UBA3), resulting in inhibition of both Nedd8-dependent and Nedd8-independent ubiquitin conjugation and protein degradation. Importantly, in A549 cells, ABPA3 selectively decreases ubiquitin and Nedd8 conjugates, but not SUMO1-3, Ufm1, ISG15, or LC3 conjugates. However, more data is needed in order for ABPA3 to be recommended as a useful probe for inhibition of UBA1/UBA3 in general since: 1) the data available are derived from cell-based studies in only one cell line (A549), 2) no broad, biochemical selectivity profile of ABPA3 is available, and 3) preclinical properties for ABPA3 have not been reported.

The published characterization of ABPA3 rests largely on phenotypic, cell-based proteomics in A549 cells. The results, showing selective formation of covalent complexes between ABPA3 and ubiquitin and NEDD8 relative to SUMO1/2/3 or UFM1, are intriguing, as is the selective inhibition of ubiquitin and NEDD8 conjugation of native proteins in A549 cells. A variety of other experiments carried out in A549 cells demonstrate phenotypic and antiproliferative effects that are consistent with the proposed mechanism of action; however, a key limitation to this probe is its inadequate biochemical characterization. MLN-7243, whose structure has now been disclosed (please see above reference), is a compound with a similar sulfamate nucleotide-like structure with reported inhibitory activity at 1 nM on ubiquitin--protein ligase E1 and at 25 nM NEDD8-activating enzyme E1 (UAB3). IABPA3 would be a more useful probe if comparative data between it and ML-7243 were available. Also, all of the cell-based work was done in one cell line, so inferring intrinsic selectivity from only A549 cells is risky when these experiments may be context dependent. Finally, in addition to the above considerations, in the absence of PK data (which have not been reported), ML-7243 should not be considered for use in intact organisms.

This compound is a non-specific, covalent inhibitor of ubiquitinylation and Nedd8 and should therefore be used with caution as a single agent, as this polypharmacolgy will almost certainly confound any attempt to use the tool to dissect specific actions of single E1 enzymes. As such, experiments with this compound must be carefully designed with appropriate follow up in order to have any meaningful consequence. However, one possible use, suggested by the authors, is to use this probe to investigate selectivity of other, next-generation E1 inhibitors - by dosing first with a putatively selective E1 inhibitor, then adding in an appropriate dose of ABPB3. Any non-bound E1 proteins will be covalently modified with the probe and can be detected by mass spectroscopy. The conclusion would then be that any non-labeled E1 enzymes were bound by the putative new selective ligands, whereas any labeled proteins were not bound by the test probe. This experiment assumes binding at competing sites and again needs to be treated with caution and validated further but may offer some outline utility in the preliminary assessment of selective E1 inhibitors.