Shih Po-Chang, Naganuma Miyako, Tsuji Genichiro, Demizu Yosuke, Naito Mikihiko
Institute of BioPharmaceutical Sciences, National Sun Yat-sen University, Kaohsiung 804201, Taiwan; Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan.
National Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki, Kanagawa 210-9501, Japan; Graduate School of Medical Life Science, Yokohama City University, 1-7-29, Yokohama, Kanagawa 230-0045, Japan.
Bioorg Med Chem. 2023 Nov 15;95:117507. doi: 10.1016/j.bmc.2023.117507. Epub 2023 Oct 21.
Proteolysis-targeting chimera (PROTAC) technology is a disruptive innovation in the drug development community, and over 20 PROTAC molecules are currently under clinical evaluation. These PROTAC molecules contain small-molecule warheads that bind to target proteins. Recently, oligonucleotide-warheaded PROTACs have emerged as a promising new tool to degrade DNA-binding proteins such as transcription factors. In this study, we applied an oligonucleotide-warheaded PROTAC technology to induce the degradation of signal transducer and activator of transcription 3 (STAT3), which is a hard-to-target protein. A double-stranded decoy oligonucleotide specific to STAT3 was conjugated to E3 binders (pomalidomide, VH032, and LCL161) to generate PROTAC molecules that recruited different E3 ubiquitin ligases cereblon (CRBN), von Hippel-Lindau (VHL), and inhibitor of apoptosis protein (IAP), respectively. One of the resulting PROTAC molecules, POM-STAT3, which recruits CRBN, potently induces STAT3 degradation. STAT3 degradation by POM-STAT3 was abolished by scrambling the oligonucleotide sequences of POM-STAT3 and by adding a double-stranded decoy oligonucleotide against STAT3 in a competitive manner, suggesting the significance of oligonucleotide sequences in STAT3 degradation. Moreover, POM-STAT3-induced STAT3 degradation was suppressed by the CRBN binder thalidomide, proteasome inhibitor bortezomib, E1 inhibitor MLN7243, and siRNA-mediated depletion of CRBN, indicating that STAT3 degradation is mediated by the ubiquitin-proteasome system, which involves CRBN as the responsible E3 ubiquitin ligase. Consistent with STAT3 degradation, NCI-H2087 cell viability was severely reduced following POM-STAT3 treatment. Thus, POM-STAT3 is a STAT3 degrader that potentially has cytocidal activity against cancer cells that are highly dependent on STAT3 signaling, which implies that inducing protein degradation by decoy oligonucleotide-warheaded PROTAC molecules could be harnessed to be therapeutic against oncogenic transcription factors.
蛋白酶靶向嵌合体（PROTAC）技术是药物研发领域的一项颠覆性创新，目前有20多种PROTAC分子正在进行临床评估。这些PROTAC分子包含与靶蛋白结合的小分子弹头。最近，寡核苷酸弹头的PROTAC作为一种有前景的新工具出现，可用于降解诸如转录因子等DNA结合蛋白。在本研究中，我们应用寡核苷酸弹头的PROTAC技术来诱导信号转导和转录激活因子3（STAT3）的降解，STAT3是一种难以靶向的蛋白。将针对STAT3的双链诱饵寡核苷酸与E3结合剂（泊马度胺、VH032和LCL161）偶联，以生成分别招募不同E3泛素连接酶脑啡肽（CRBN）、冯·希佩尔-林道（VHL）和凋亡抑制蛋白（IAP）的PROTAC分子。所得到的PROTAC分子之一，招募CRBN的POM-STAT3，能有效诱导STAT3降解。通过打乱POM-STAT3的寡核苷酸序列以及以竞争方式添加针对STAT3的双链诱饵寡核苷酸，可消除POM-STAT3对STAT3的降解作用，这表明寡核苷酸序列在STAT3降解中具有重要意义。此外，CRBN结合剂沙利度胺、蛋白酶体抑制剂硼替佐米、E1抑制剂MLN7243以及siRNA介导的CRBN缺失可抑制POM-STAT3诱导的STAT3降解，表明STAT3降解是由泛素-蛋白酶体系统介导的，其中CRBN作为负责的E3泛素连接酶。与STAT3降解一致，POM-STAT3处理后NCI-H2087细胞活力严重降低。因此，POM-STAT3是一种STAT3降解剂，对高度依赖STAT3信号传导的癌细胞可能具有杀细胞活性，这意味着利用诱饵寡核苷酸弹头的PROTAC分子诱导蛋白降解可用于治疗致癌转录因子。
