靶向Rpn13Pru的小分子诱导乳腺癌细胞有丝分裂阻滞

Proteolysis-targeting chimeras are heterobifunctional molecules that link a protein of interest (POI) to an E3 ubiquitin ligase, promoting ubiquitylation and degradation of the POI via the ubiquitin- proteasome system (UPS). XL44 is a structurally modified small molecule previously identified as a potent hRpn13 Pru degrader, that induces ubiquitin-dependent apoptosis in myeloma cells. and ubiquitin-independent cell cycle defects. Recently, we evaluated two more potent derivatives; XL69 and XL80 with a similar cell cycle arrest mechanism. Here, we evaluated that the anticancer activity of XL69 and XL80 mediated disruption of UPS function in the MCF-7 cell line. MCF-7 cells were treated with XL69 and XL80 for 24 h and the IC 50 values were determined by MTT assay. For cell cycle analysis, cells were treated with 10 µM of each compound for 24 h and analyzed via flow cytometry. The spindle assembly checkpoint (SAC), and anaphase promoting complex/ cyclosome (APC/C) activity were assessed through western blotting and immunofluorescence microscopy. The MTT assay for XL69 and XL80 showed increased cytotoxicity at concentrations above 30 µM upon treatment but cells exhibited rapid detachment at lower concentrations. Detached, floating cells remained viable for the next 48-72 h post-treatment before cell death and exhibited marked variation in nuclear morphology and cytoskeleton organization. XL69 and XL80 treated MCF-7 cells accumulated in G2/M (65.7% and 54.1%, respectively vs DMSO- (25.2%; p < 0.001). In addition, elevated levels of phospho-histone 3 were detected 24 h post-treatment (31.5%, 32.5%, vs DMSO 10.7%, p-value <0.01) indicating robust mitotic arrest. Immunofluorescent detection of aurora B with DAPI nuclear staining confirmed prometaphase-metaphase arrest in treated cells. Western blot analysis showed upregulation of Bub1 and BubR1, confirming SAC activation post mitotic arrest. Activation of the SAC led to formation of the mitotic checkpoint complex, subsequently inhibiting the APC/C, as evidenced by the accumulation of cyclin B and securin. Furthermore, the inhibition of APC/C also prevented degradation of aurora B kinase, resulting in impaired tubulin assembly. Immunofluorescent images showed disordered organization of tubulin with XL69 and XL80 treatment, similar to Nocodazole, a microtubule polymerization inhibitor. These findings suggest that XL69 and XL80 induce mitotic arrest by targeting the APC/C pathway and disrupt microtubule dynamics. These studies demonstrate that the small molecules XL69 and XL80 effectively induce mitotic arrest, impair microtubule dynamics, disrupt cellular attachment leading to cell death in adherent MCF-7 cells. The treatment led to sustained cell cycle arrest, SAC activation, APC/C pathway inhibition and impaired microtubule organization. As such, the study highlights XL69 and XL80 as promising anti-cancer agents that target mitotic progression.