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

## TL;DR

Researchers designed a fluorescent probe called FPy1 to track pyroptosis, a type of cell death linked to inflammation, and used it to monitor disease processes and screen drugs.

## Contribution

The novel probe FPy1 enables multiscale pyroptosis imaging and high-throughput screening of caspase-1 inhibitors.

## Key findings

- FPy1, based on the GSDMD cleavage site, shows the best performance for detecting caspase-1 activity.
- FPy1 was successfully applied to monitor pyroptosis in cellular, spheroid, and in vivo models.
- FPy1 was used to create a high-content screening platform for caspase-1 modulators.

## Abstract

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.

A novel caspase-1-activatable probe FPy1 has been successfully used for the imaging of caspase-1 or pyroptosis across multiple biological scenarios, encompassing multiscale pyroptosis imaging and high-throughput screening of caspase-1 inhibitors.