Cell-based high-throughput screening using a target-NanoLuc fusion construct to identify molecular glue degraders of c-Myc oncoprotein.

IF 3.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Muyu Xu, Jinying Qiu, Lin Tan, Jiayu Xu, Yi Wang, Wenyue Kong, Hongda Liao, Anran Chen, Xiaolan Chen, Jiying Zhang, Cookson K C Chiu, Meiying Zhang, Yingying Tian, Caohui Li, Biao Ma, Leiming Wang, Jingpeng Fu, Seung H Choi, Jeffrey Hill, Weijun Shen
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Abstract

Oncoprotein c-Myc (Myc) plays a critical role in regulating cellular gene expression. Although Myc dysregulation is found in more than 70% of cancers and can facilitate tumor initiation and progression, it is still considered to be an "undruggable" oncotarget years after its first discovery. Recent advances in the field of targeted protein degradation provide alternative Myc-targeting strategies. Here, we develop the first Myc-NanoLuc fusion plasmid transfected cell-based high-throughput screening assay to identify Myc-downregulating small molecules. We verified the effectiveness of our assay by demonstrating that previously known Myc-downregulating compounds (G9 and SY-1365) were successfully identified from a library of bioactive compounds with established biological function. Next, we screened another 108 800 compounds from the diverse ChemDiv library collection, and 14 novel Myc-downregulating compounds were identified after cherry-pick triplicate confirmation, counter-screening, dose-response and western blotting experiments. A cellular thermal shift assay further demonstrated that five out of the 14 Myc-downregulating compounds bound to endogenous Myc protein in crude 293T whole-cell lysate. Subsequently, compound C1 was shown to selectively degrade Myc protein at a DC50 value of around 5 μM. Further characterization showed that C1 killed cancer cells with high Myc expression at a lower dose than it killed cancer cells with low Myc expression. Moreover, C1 selectively reduced the expression of various Myc-target genes. Intriguingly, co-immunoprecipitation showed that C1 functionally acted like a molecular glue to aggregate Myc proteins and block Myc/Max interaction. The self-aggregation of Myc and the dissociation of the Myc/Max dimer by C1 promoted Myc degradation. Using a target-NanoLuc fusion strategy in our novel cell-based high-throughput screening system, we identified a molecular glue-like small molecule degrader of Myc.

基于细胞的高通量筛选,使用靶标- nanoluc融合构建鉴定c-Myc癌蛋白的分子胶降解剂。
癌蛋白c-Myc (Myc)在调节细胞基因表达中起关键作用。尽管在超过70%的癌症中发现了Myc失调,并且可以促进肿瘤的发生和进展,但在首次发现多年后,它仍然被认为是一种“不可药物”的肿瘤靶点。靶向蛋白降解领域的最新进展提供了替代的myc靶向策略。在这里,我们开发了第一个基于Myc-NanoLuc融合质粒转染细胞的高通量筛选实验,以鉴定myc -下调小分子。我们通过证明先前已知的myc下调化合物(G9和SY-1365)成功地从具有既定生物学功能的生物活性化合物库中鉴定出来,验证了我们实验的有效性。接下来,我们从ChemDiv文库中筛选了另外10800个化合物,并通过樱桃挑选三次确认、反筛选、剂量反应和western blotting实验鉴定了14个新的myc下调化合物。细胞热移实验进一步证明,在粗293T全细胞裂解液中,14种Myc下调化合物中有5种与内源性Myc蛋白结合。随后,化合物C1在5 μM左右的DC50值下选择性地降解Myc蛋白。进一步表征表明,C1杀死Myc高表达癌细胞的剂量低于杀死Myc低表达癌细胞的剂量。此外,C1选择性地降低了各种myc靶基因的表达。有趣的是,共免疫沉淀显示C1在功能上像分子胶一样聚集Myc蛋白并阻断Myc/Max相互作用。Myc的自聚集和Myc/Max二聚体被C1解离促进了Myc的降解。在我们新颖的基于细胞的高通量筛选系统中,使用靶标- nanoluc融合策略,我们鉴定了Myc的分子胶样小分子降解物。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
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