title: 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.
authors: From the ‡Department of Biochemistry, Vanderbilt University School of MedicineNashville, Tennessee.
authors: Joel D
authors: JD
authors: Federspiel
authors: From the ‡Department of Biochemistry, Vanderbilt University School of MedicineNashville, Tennessee.
authors: Simona G
authors: SG
authors: Codreanu
authors: From the ‡Department of Biochemistry, Vanderbilt University School of MedicineNashville, Tennessee.
authors: Sandeep
authors: S
authors: Goyal
authors: From the ‡Department of Biochemistry, Vanderbilt University School of MedicineNashville, Tennessee.
authors: Matthew E
authors: ME
authors: Albertolle
authors: §Onyx Pharmaceuticals, an Amgen subsidiary, San Francisco, California 94080.
authors: Eric
authors: E
authors: Lowe
authors: §Onyx Pharmaceuticals, an Amgen subsidiary, San Francisco, California 94080.
authors: Juli
authors: J
authors: Teague
authors: §Onyx Pharmaceuticals, an Amgen subsidiary, San Francisco, California 94080.
authors: Hansen
authors: H
authors: Wong
authors: From the ‡Department of Biochemistry, Vanderbilt University School of MedicineNashville, Tennessee.
authors: F Peter
authors: FP
authors: Guengerich
authors: From the ‡Department of Biochemistry, Vanderbilt University School of MedicineNashville, Tennessee; daniel.liebler@vanderbilt.edu.
authors: Daniel C
authors: DC
authors: Liebler
mesh_terms: D045744:Cell Line, Tumor; D053493:Cholestanetriol 26-Monooxygenase / Q000378:metabolism*; D057930:Click Chemistry; D005982:Glutathione Transferase / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D015394:Molecular Structure; D009842:Oligopeptides / Q000737:chemistry*; D061988:Proteasome Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D053719:Tandem Mass Spectrometry
chemical_list: D009842:Oligopeptides; D061988:Proteasome Inhibitors; C524865:carfilzomib; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; C490476:GSTO1 protein, human; D005982:Glutathione Transferase