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As an emerging therapeutic strategy, proteolysis-targeting chimeras (PROTACs) have been proven to be superior to traditional drugs in many aspects. However, due to their unique mechanism of action, existing methods for evaluating the degradation still have many limitations, which seriously restricts the development of PROTACs. In this methodological study, using direct stochastic optical reconstruction microscopy (dSTORM)-based single-cell protein quantitative analysis, we systematically investigated the dynamic degradation characteristics of FLT3 protein during PROTACs treatment. We found that the distribution of FLT3 varies between FLT3-ITD mutation and FLT3-WT cells. PROTACs had an obvious time-course effect on protein degradation and present two distinct phases; this provided a basis for deciding when to evaluate protein degradation. High concentrations of PROTACs were more effective than long-time administration because a higher D max was achieved. Two-color dSTORM-based colocalization analysis efficiently detected the proportion of ternary complexes, making it very useful in screening PROTACs. Taken together, our findings show that the dSTORM method is an ideal tool for evaluating PROTACs and will accelerate the development of new PROTACs.

Endocrine resistance remains a significant problem in the clinical treatment of estrogen receptor α-positive (ERα + ) breast cancer (BC). In this study, we developed a series of novel dual-functional ERα degraders based on a bridged bicyclic scaffold with selenocyano (SeCN) side chains. These compounds displayed potent ERα degradation and tubulin depolymerization activity. Among them, compounds 35s and 35t exhibited the most promising antiproliferative and ERα degradation activity in multiple ERα + BC cell lines bearing either wild-type or mutant ERα. Meanwhile, compounds 35s and 35t disrupted the microtubule network by restraining tubulin polymerization, evidenced by 35t inducing cell cycle arrest in the G2/M phase. In MCF-7 and LCC2 xenograft models, compounds 35s and 35t remarkably suppressed tumor growth without noticeable poisonousness. Finally, this study provided guidance for developing new dual-target antitumor drug candidates for the ERα + BC therapy, especially for the resistant variant.

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