Re-Evaluating the Mechanism of Action of α,β-Unsaturated Carbonyl DUB Inhibitors b-AP15 and VLX1570: A Paradigmatic Example of Unspecific Protein Cross-linking with Michael Acceptor Motif-Containing Drugs.

Jennifer A Ward,Laura Díaz-Sáez,Kilian V M Huber,Adan Pinto-Fernandez,Olivier Riant,Loïc Cornelissen,Edward W Tate,Sarah Bonham,Benedikt M Kessler,Olivier Feron

doi:10.1021/acs.jmedchem.0c00144

Abstract

Deubiquitinating enzymes (DUBs) are a growing target class across multiple disease states, with several inhibitors now reported. b-AP15 and VLX1570 are two structurally related USP14/UCH-37 inhibitors. Through a proteomic approach, we demonstrate that these compounds target a diverse range of proteins, resulting in the formation of higher molecular weight (MW) complexes. Activity-based proteome profiling identified CIAPIN1 as a submicromolar covalent target of VLX1570, and further analysis demonstrated that high MW complex formation leads to aggregation of CIAPIN1 in intact cells. Our results suggest that in addition to DUB inhibition, these compounds induce nonspecific protein aggregation, providing molecular explanation for general cellular toxicity.

Highlights

Ubiquitination, the covalent addition of 76 amino acid protein ubiquitin (Ub) to protein substrates, is a widespread protein post-translational modification in eukaryotic cells.[1]
The results obtained by D’Arcy and colleagues demonstrate that b-AP15 is an inhibitor of USP14/ UCH-37,11 two unrelated cysteine protease enzymes from different deubiquitinating enzymes (DUBs) families, the chemical structure of b-AP15 suggests additional proteins may be targeted by this compound
B-AP15 possesses higher potency in intact cells than that in biophysical assays against USP14 and UCH-37.11 In support of compound promiscuity, another study describing the chemical synthesis of active-site-directed DUB probes showed data compatible with a nonspecific DUB inhibition profile upon increasing concentrations of b-AP15.13 In our hands, b-AP15 inhibits the cleavage of the DUB substrate Ub-AML by a number of purified recombinant DUBs (Figure 1B)

Summary

■ INTRODUCTION

Ubiquitination, the covalent addition of 76 amino acid protein ubiquitin (Ub) to protein substrates, is a widespread protein post-translational modification in eukaryotic cells.[1]. With the help of advanced activitybased protein profiling (ABPP)[7−9] assays that allow the profiling of DUBs in a cellular context, highly selective DUB inhibitors have been reported.[4,5,10] Here, we describe the proteomic investigation of two structurally related DUB inhibitors b-AP15 and VLX1570 These inhibitors share a reactive α,β-unsaturated carbonyl substructure motif capable of covalent interaction with nucleophilic residues. Taken forward into a phase I/II clinical trial for refractory multiple myeloma, VLX1570 has been put on full clinical hold because of dose-limiting toxicity We demonstrate that these inhibitors target a diverse range of proteins beyond their reported targets, resulting in the formation of higher molecular weight (MW) complexes. Through a quantitative chemical proteomic approach, we identify CIAPIN1, known as anamorsin, as a potent covalent target of VLX1570 which forms high MW complexes upon reaction with VLX1570, leading to aggregation of CIAPIN1 in intact cells

■ RESULTS AND DISCUSSION

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES