Potent Caspase-1 Antagonists From Dihydropyridine Derivatives: In Vitro Fluorescence Assays and Cell Imaging

Caspase-1 is a key marker in pyroptosis and inflammation, playing a central role in the pathogenesis of various acute and chronic inflammatory diseases. While some caspase-1 sensing and inhibition studies have been reported, the use of DHPs for these applications remains unexplored. To address this challenge, a novel fluorescent compound, zWEHD-NH-DHP, was synthesized and subsequently developed as a real-time caspase-1 detection in vitro and in cells. Furthermore, a new class of potential caspase-1 inhibitors was investigated through the screening of a series of nonfluorogenic DHPs, utilizing our established caspase-1 fluorescent sensing system. Among the tested compounds, THD and FD showed caspase-1 inhibition with IC₅₀ values of 2.8 ± 0.1 nM and 3.2 ± 0.1 µM, respectively. THD, which exhibited comparable potency to the well-known inhibitor VX-765 (3.68 nM), was selected for further investigation. Moreover, a competitive inhibition mechanism of THD at the enzyme's active site was supported by molecular docking and MD simulations. Western blot analysis of PMA-differentiated THP-1 cells further supported this observation by showing reduced cleaved IL-1β levels in the presence of THD. This inhibitory effect of THD on caspase-1 activity in a complex cell system was further confirmed by the concentration-dependent morphological changes in BMDMs.