STAT3を分解するPROTAC/SNIPERの分子設計、合成と活性評価

蛋白质水解靶向嵌合体（Proteolysis targeting chimera，PROTAC）是一种利用嵌合化学物质与目标蛋白（POI）和E3连接酶结合，从而通过泛素-蛋白酶体系统诱导POI降解的技术。本课题以STAT 3为靶点，以磺胺类化合物为小分子弹头，通过烷基连接体与沙利度胺（一种cereblon E3连接酶结合剂）偶联，合成了新型的STAT 3降解剂PROTAC。所得PROTAC的分子量约为900。然后，在MCF 7和SU-DHL-1细胞中沿着测试这些PROTAC与其亲本STAT 3结合剂的STAT 3降解活性。蛋白质印迹分析表明，PS314（PROTAC，使用PS313作为弹头）在两种细胞系中显示出显著的降低STAT 3蛋白水平的活性，而PS312（PROTAC，使用PS311作为弹头）不降低STAT 3蛋白水平。将STAT 3结合的诱饵寡核苷酸与三种不同的E3配体缀合，成为LCL-STAT 3诱饵、POM-STAT 3诱饵和VH-STAT 3诱饵。在MCF 7细胞中测试，发现POM-STAT 3诱饵比VH-STAT 3诱饵更能够降解STAT 3，并且LCL-STAT 3诱饵没有显示出降解活性。

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.