STAT3を分解するPROTAC/SNIPERの分子設計、合成と活性評価

蛋白水解靶向嵌合体（Proteolysis targeting chimera， PROTAC）是一种利用嵌合化学物质结合感兴趣蛋白（POI）和E3连接酶，通过泛素-蛋白酶体系统诱导POI降解的技术。在这个项目中，我们打算开发针对STAT3的新型PROTAC降解剂。首先，我们采用磺胺衍生物作为小分子战斗部与STAT3的SH2结构域结合，并通过烷基连接剂与沙利度胺（一种小脑E3连接酶结合剂）偶联合成PROTACs。所得protac的分子量约为900。然后，在MCF7和SU-DHL-1细胞中测试了这些PROTACs及其亲本STAT3结合物的STAT3降解活性。western blot分析表明，PS314 （PROTAC，以PS313为战斗部）在两种细胞系中均表现出显著的降低STAT3蛋白水平的活性，而PS312 （PROTAC，以PS311为战斗部）没有降低STAT3蛋白水平。我们还开发了针对STAT3的寡核苷酸弹头PROTAC分子。将STAT3结合的诱饵寡核苷酸与3种不同的E3配体偶联，分别为LCL-STAT3诱饵、POM-STAT3诱饵和VH-STAT3诱饵。在MCF7细胞中测试，发现POM-STAT3诱饵比VH-STAT3诱饵更能降解STAT3，而LCL-STAT3诱饵没有降解活性。

Proteolysis targeting chimera (PROTAC) is a technology that uses chimeric chemicals to bind to a protein of interest (POI) and an E3 ligase so as to induce POI degradation through the ubiquitin-proteasome system. In this project, we intended to develop novel PROTAC degraders targeting STAT3.First, we employed sulfonamide derivatives that are believed to bind to the SH2 domain of STAT3 as small molecule-warheads and synthesized PROTACs by conjugating to thalidomide (a cereblon E3 ligase binder) via alkyl linkers. The molecular weights of the resulting PROTACs are approximately 900. Then, the STAT3 degradation activity of these PROTACs were tested along with their parental STAT3 binders in MCF7 and SU-DHL-1 cells. The western blot analysis indicated that PS314 (PROTAC, using PS313 as a warhead) showed a significant activity to reduce STAT3 protein level in both cell lines, while PS312 (PROTAC, using PS311 as a warhead) did not reduce the STAT3 protein level.We also developed oligonucleotide-warheaded PROTAC molecules against STAT3. A decoy oligonucleotide to which STAT3 binds was conjugated to three different E3 ligands to be LCL-STAT3 decoy, POM-STAT3 decoy and VH-STAT3 decoy. Tested in MCF7 cells, POM-STAT3 decoy was found to be more able to degrade STAT3 than VH-STAT3 decoy, and LCL-STAT3 decoy showed no degradation activity.