Abstract
Purpose :
In macrophages, LPS triggers an inflammatory program of antibacterial defense that causes pyroptosis. Therefore, the inhibition of GSDMD is an important biomarker for inhibiting macrophage pyroptosis. We hypothesized that a small molecule inhibitor that can effectively inhibit GSDMD cleavage could be found by using virtual screening technology.
Methods :
We hydrogenated the crystal structure of Human GSDMD (PDB ID: 6VFE) using the Protein Preparation Wizard module and optimized 12.3K compounds using Schrodinger software. Finally, the Virtual Screening Workflow module was used for virtual screening. The output small molecule compounds were further screened. Lipopolysaccharides and Nigericin were used as models to induce pyroptosis in THP-1 cell, and the optimal small molecule inhibitors of GSDMD were found by High-throughput screening.
Results :
Using an absolute docking score greater than or equal to 3.328 as the screening threshold, 200 compounds with strong binding capacity were output, and 54 compounds were further optimized for high-throughput screening in vitro. We found that Glutathione oxidized (GO) and Raddeanin A (RA) can effectively protect the integrity of the cell membrane (n=3, P<0.05), MST revealed that Glutathione oxidized (Kd=2.747E-05, automatic curve fitting and Amplitude>5) and Raddeanin A (Kd=1.058E-04, automatic curve fitting and Amplitude>5) could directly bind to the GSDMD protein. The GSDMD binding energies of Glutathione oxidized and Raddeanin A with human and mice were -5.8kcal/mol and -6.1kcal/mol, and -7.8kcal/mol and -9.5kcal/mol, respectively.
Conclusions :
We found that GO and RA had good binding ability to human and mouse GSDMD proteins and can inhibit the pyroptosis of THP-1 cells induced by LPS. These results suggest that GO and RA inhibit pyroptosis by inhibiting the cleavage of GSDMD. We hypothesize that GO and RA can effectively inhibit retinal inflammation in animal models of diabetic retinopathy and glaucoma, which cause activation of pyroptosis pathways. Our findings are of great significance and further mechanistic studies will be conducted
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.