Specificity of Protein Covalent Modification by the Electrophilic Proteasome Inhibitor Carfilzomib in Human Cells
Specificity of Protein Covalent Modification by the Electrophilic Proteasome Inhibitor Carfilzomib in Human Cells
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DOI：
10.1074/mcp.m116.059709
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发表时间：
2016-10-01
影响因子：
7
通讯作者：
Liebler, Daniel C.
中科院分区：
文献类型：
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作者：
Federspiel, Joel D.;Codreanu, Simona G.;Liebler, Daniel C.
卡菲佐米(CFZ)是美国食品和药物管理局和欧盟委员会批准的第二代蛋白酶体抑制剂，用于治疗复发或难治性多发性骨髓瘤。CFZ是一种带有环氧酮亲电弹头的环氧米星衍生物，该弹头不可逆地加成蛋白酶体的β5亚单位的催化苏氨酸残基。尽管CFZ产生了一种高度有效的、持续的蛋白酶体失活，但该药物的亲电性质可能会产生目标外的蛋白质加成。为了解决这种可能性，我们合成了一种炔基CFZ类似物，并研究了这种类似物在HepG2细胞中对蛋白质的加合作用。利用点击化学结合链霉亲和素为基础的IP和鸟枪串联质谱(MS/MS)，我们确定了两个非靶蛋白，细胞色素P45027A1(细胞色素P45027A1)和谷胱甘肽S转移酶omega1(GSTO1)，作为炔基CFZ探针的目标。我们通过链霉亲和素捕获和免疫印迹方法证实了CYP27A1和GSTO1的加合物，然后用靶向MS/MS方法对加合物进行了定点定位。尽管CFZ在体外加入CYP27A1和GSTO1会降低这些酶的活性，但在完整细胞中，CFZ修饰的这些蛋白质中的一小部分应该会限制这些非靶标修饰的影响。这些数据支持CFZ对其治疗靶点进行共价修饰的高选择性，尽管存在活性电泳体。我们描述的方法提供了一种可推广的方法来评估共价蛋白质修饰疗法的安全性。
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 beta 5 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.