Ligands for cereblon, a component of a functional E3 ligase complex that targets proteins for proteolysis, are critical for developing molecular glues and proteolysis-targeting chimeras (PROTACs), which have therapeutic implications for various diseases. However, the lack of sensitivity of previously reported assays limits characterization of cereblon ligands. To address this shortcoming, we developed BODIPY FL thalidomide (10) as a high-affinity fluorescent probe for the human cereblon protein, with a K_d value of 3.6 nM. We then used BODIPY FL thalidomide (10) to develop a cereblon time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay. The IC₅₀ values of the cereblon ligand pomalidomide (8) were 6.4 nM in our cereblon TR-FRET binding assay, 264.8 nM in a previously reported Cy5-conjugated thalidomide (7)-mediated fluorescence polarization (FP) assay, and 1.2 μM in a previously reported Cy5-conjugated cereblon modulator (compound 7) (9)-mediated TR-FRET assay, indicating that our cereblon TR-FRET binding assay is 41- and 187-fold more sensitive than these two previously published assays. With our cereblon TR-FRET binding assay, we detected binding of cereblon ligands but not binding of bromodomain-containing protein 4 or von Hippel–Lindau ligands, thereby demonstrating its selectivity. Our cereblon TR-FRET binding assay was very stable and detected changes in phthalimide activity due to thalidomide isomerization. Therefore, the BODIPY FL thalidomide (10)-mediated cereblon TR-FRET binding assay we designed is highly sensitive, selective, and stable and will aid the development and characterization of novel cereblon ligands.

中文翻译：

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开发 BODIPY FL 沙利度胺作为时间分辨荧光共振能量转移测定中 Cereblon 的高亲和力荧光探针

cereblon 的配体是功能性 E3 连接酶复合物的一个组成部分，该复合物靶向蛋白质进行蛋白水解，对于开发分子胶和蛋白水解靶向嵌合体 (PROTAC) 至关重要，这些嵌合体对各种疾病具有治疗意义。然而，先前报道的检测缺乏敏感性限制了 cereblon 配体的表征。为了解决这个缺点，我们开发了 BODIPY FL 沙利度胺 ( 10 ) 作为人类 cereblon 蛋白的高亲和力荧光探针，K _d值为 3.6 nM。然后，我们使用 BODIPY FL 沙利度胺 ( 10 ) 开发了脑时间分辨荧光共振能量转移 (TR-FRET) 结合测定。cereblon 配体泊马度胺的 IC ₅₀值（8 ) 在我们的 cereblon TR-FRET 结合测定中为 6.4 nM，在先前报道的 Cy5 偶联的沙利度胺 ( 7 ) 介导的荧光偏振 (FP) 测定中为 264.8 nM，在先前报道的 Cy5 偶联的 cereblon 调节剂（化合物7) ( 9 ) 介导的 TR-FRET 测定，表明我们的 cereblon TR-FRET 结合测定比这两个先前发表的测定灵敏 41 倍和 187 倍。通过我们的 cereblon TR-FRET 结合测定，我们检测到 cereblon 配体的结合，但未检测到含溴结构域的蛋白质 4 或 von Hippel-Lindau 配体的结合，从而证明了其选择性。我们的 cereblon TR-FRET 结合测定非常稳定，并且检测到由于沙利度胺异构化引起的邻苯二甲酰亚胺活性的变化。因此，BODIPY FL沙利度胺（10 ) 介导的 cereblon TR-FRET 结合测定是高度灵敏、选择性和稳定的，将有助于新型 cereblon 配体的开发和表征。