Design and optimization of caspase-1-responsive fluorescent probes for pyroptosis imaging and anti-pyroptosis drug screening

Pyroptosis is a recently-identified form of inflammatory caspase-dependent programmed cell death that is closely associated with many diseases. Real-time imaging of pyroptosis is crucial for monitoring the inflammatory pathological process. Caspase-1, a representative of inflammatory caspase, plays a pivotal role in pyroptosis and inflammatory diseases. Therefore, caspase-1 activity can reflect pyroptosis and related inflammatory states. Herein, we report on a variety of caspase-1 activatable probes based on potential hydrolytic peptides of caspase-1. Through systematic performance evaluation, we identified that FPy1 designed based on the cleavage of pyroptosis-related protein GSDMD exhibits the best detection performance. Thus, the specific peptide –FLTDG– from GSDMD could serve as a potential responsive element for the design of caspase-1 or pyroptosis-related probes. Owing to the outstanding performance of FPy1, we further applied it to monitor pyroptosis processes in three distinct biological contexts, i.e. cellular, cell spheroid, and in vivo models, using degenerative bone and joint diseases, i.e. intervertebral disc degeneration and osteoarthritis. Moreover, we combined FPy1 with high-content analysis to establish a screening platform for caspase-1 modulators, based on the classic NLRP3 inflammasome-mediated caspase-1 activation model in primary macrophages. Collectively, these results illustrated the potential of FPy1 as a versatile tool for tracking the progression of pyroptosis and monitoring caspase-1 activity across various application scenarios.