Specificity of Protein Covalent Modification by the Electrophilic Proteasome Inhibitor Carfilzomib in Human Cells

Abstract

Carfilzomib (CFZ) is a second-generation proteasome inhibitor that is Food and Drug Administration and European Commission approved for the treatment of relapsed or refractory multiple myeloma. CFZ is an epoxomicin derivative with an epoxyketone electrophilic warhead that irreversibly adducts the catalytic threonine residue of the β5 subunit of the proteasome. Although CFZ produces a highly potent, sustained inactivation of the proteasome, the electrophilic nature of the drug could potentially produce off-target protein adduction. To address this possibility, we synthesized an alkynyl analog of CFZ and investigated protein adduction by this analog in HepG2 cells. Using click chemistry coupled with streptavidin based IP and shotgun tandem mass spectrometry (MS/MS), we identified two off-target proteins, cytochrome P450 27A1 (CYP27A1) and glutathione S-transferase omega 1 (GSTO1), as targets of the alkynyl CFZ probe. We confirmed the adduction of CYP27A1 and GSTO1 by streptavidin capture and immunoblotting methodology and then site-specifically mapped the adducts with targeted MS/MS methods. Although CFZ adduction of CYP27A1 and GSTO1 in vitro decreased the activities of these enzymes, the small fraction of these proteins modified by CFZ in intact cells should limit the impact of these off-target modifications. The data support the high selectivity of CFZ for covalent modification of its therapeutic targets, despite the presence of a reactive electrophile. The approach we describe offers a generalizable method to evaluate the safety profile of covalent protein-modifying therapeutics.

Fig. 1.

Carfilzomib off-target identification strategy. A, The structures of the parent carfilzomib compound and three clickable analogs of it. B, Metabolic stability and activity assays comparing the performance of the click analog compounds to the parent compound. C, Workflow for the click capture and LC-MS/MS of the OP-829 analog in HepG2 cells.

Fig. 2.

Verification of CFZ adducted proteins. A, Plots of spectral counts for the proteasome and two off-target proteins from the shotgun study demonstrating concentration-dependent increases. B, Immunoblot validation of MS data. See text for discussion of lack of a positive signal for CYP27A1.

Fig. 3.

Adduction of CYP27A1 and GTSO1 in vitro. Concentration dependent adduction of (A) CYP27A1 and (B) GSTO1 by OP-829 in vitro as assessed by streptavidin-based immunoblots.

Fig. 4.

Functional effect of protein adduction by CFZ. (A) CYP27A1 and (B) GSTO1 were adducted by CFZ in vitro and activity assays were performed to assess the consequences of adduction. Both proteins show concentration-dependent decreases in activity in response to adduction.