Dstorm Based Single-Molecule Quantitative Analysis Can Effectively Evaluate Protacs Degradation Ability

Proteolysis targeting chimeras (PROTACs) as an emerging therapeutic strategy has proved its advantages in degrading intractable target proteins which traditional tools are incompetent. However, due to its unique mechanism of action, the existing degradation evaluation methods still have many limitations. Herein, we applied single-molecule quantitative analysis by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the distribution characterization of FLT3 protein before and during PROTACs treatment. We find that the distribution of FLT3 protein varies between FLT3-ITD mutation and FLT3-WT cells. PROTACs have obvious time-course effect on protein degradation and present two distinct phases, which provides a basis for deciding when to evaluating protein degradation. Compared with long time administration, high concentration PROTACs are more effective, because it could achieve a greater Dmax (the maximum degradation efficacy) value. We also find that two-color dSTORM based colocalization analysis could efficiently detect the proportion of ternary complexes, which is important for PROTACs. Our findings reveal the changing profile of protein degraded by PROTACs, and verify that single-molecule localization dSTORM is an ideal tool for evaluating PROTACs and will accelerate the development of new PROTACs.

No relevant conflicts of interest to declare.