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.
Highlights
From the ‡Department of Biochemistry, Vanderbilt University School of MedicineNashville, Tennessee; §Onyx Pharmaceuticals, an Amgen subsidiary, San Francisco, California 94080
To identify cellular proteins modified by CFZ, we employed a click chemistry-based strategy, which has been used in several recent studies to identify targets of covalent protein modification (23, 24, 28 –31, 50)
An important problem presented by the emergence of electrophilic covalent protein-modifying drugs is the scope and impact of off-target protein binding
Summary
From the ‡Department of Biochemistry, Vanderbilt University School of MedicineNashville, Tennessee; §Onyx Pharmaceuticals, an Amgen subsidiary, San Francisco, California 94080. This study confirms the high specificity of CFZ for its therapeutic targets and provides a useful approach to assess the safety of covalent protein-modifying drugs. Comparison of the spectral counts of GSTO1 and CYP27A1 to the known carfilzomib target protein (PSMB5) showed similar concentration-dependent increases in spectral counts with OP-829 treatment concentration (Fig. 2A).
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