Effect of PDD00017273 and GSK2606414 on MZ1-induced cell death

Targeted protein degradation is a groundbreaking modality in drug discovery ; however, further improvement of the degradation efficacy is demanded. Here, we identify small molecules that enhance the targeted degradation of the anticancer target BRD4 and related hard-to-degrade targets BRD2/3 induced by CRL2VHL- or CRL4CRBN -based PROTACs. The chemicals include inhibitors of cellular signaling pathways such as poly-ADP ribosylation (PARG inhibitor PDD00017273), unfolded protein response (PERK inhibitor GSK2606414), and protein stabilization (HSP90 inhibitor luminespib). Mechanistically, PARG inhibition promotes TRIP12-mediated K29/K48-linked branched ubiquitylation of BRD4 by facilitating chromatin dissociation of BRD4 and formation of the BRD4–PROTAC– CRL2VHL ternary complex; by contrast, HSP90 inhibition promotes BRD4 degradation after the ubiquitylation step. Consequently, these signal inhibitors sensitize cells to the PROTAC-induced apoptosis. Combining the treatment with these chemicals further increases the efficacy of a highly potent PROTAC, SIM1. We propose that various cell intrinsic signaling pathways spontaneously counteract chemically induced target degradation, which can be liberated by specific inhibitors. To further investigate the effect of PDD or GSK2606414 on the MZ1-induced apoptosis, we performed RNA-Seq analysis. HCT116 cells were treated with either PDD, GSK, or MZ1 for 24 h, and total RNA was isolated.

靶向蛋白质降解（Targeted Protein Degradation）是药物发现领域极具革命性的技术范式，但其降解效能仍有待进一步提升。本研究中，我们鉴定得到可增强基于CRL2VHL或CRL4CRBN的蛋白水解靶向嵌合体（PROTAC）所诱导的抗癌靶点BRD4，以及相关难降解靶点BRD2/3的靶向降解的小分子化合物。这类化合物涵盖多种细胞信号通路抑制剂，包括多聚ADP核糖基化（poly-ADP ribosylation）相关的PARG抑制剂PDD00017273、未折叠蛋白反应（unfolded protein response）相关的PERK抑制剂GSK2606414，以及参与蛋白稳定调控的HSP90抑制剂luminespib。从机制层面来看，PARG抑制可通过促进BRD4从染色质上解离，并加速BRD4-PROTAC-CRL2VHL三元复合物的形成，从而增强TRIP12介导的BRD4的K29/K48连接型分支泛素化；与之相反，HSP90抑制则在泛素化修饰完成后促进BRD4的降解。因此，这类信号通路抑制剂可使细胞对PROTAC诱导的细胞凋亡更为敏感。将这类化合物与高活性PROTAC SIM1联合施用，可进一步提升其降解效能。我们提出，多种细胞内源性信号通路会自发拮抗化学诱导的靶点降解，而这类特异性抑制剂可解除这种拮抗作用。为进一步探究PDD或GSK2606414对MZ1诱导的细胞凋亡的影响，我们开展了RNA测序（RNA-Seq）分析。将HCT116细胞分别用PDD、GSK或MZ1处理24小时后，提取总RNA。