Bioinformatic design of ASC PYD-targeting peptides and preliminary cellular evaluation of their encoding mRNAs

Inflammasome activation is central to innate immune signaling and inflammatory disorders. The adaptor protein ASC is a component of the inflammasome, and its Pyrin domain (PYD) mediates homotypic interactions involved in ASC oligomerization. Therefore, targeting the ASC PYD may modulate inflammasome-associated responses. In the present study, a structure-guided computational strategy was employed to design two candidate peptides, ASC1 and ASC2, targeting Interface I of the ASC PYD. The cryo-electron microscopy structure of the ASC PYD filament (PDB ID: 3J63) was used to identify an α-helical native template. Candidate sequence modifications were evaluated using ProDA and mmCSM-PPI servers, and the final peptide structures were generated using YASARA software. Peptide-ASC PYD complexes were assessed by 100-ns molecular dynamics simulations in GROMACS, MM/PBSA binding free energy calculations, and interaction analysis using Discovery Studio Visualizer. The peptide-encoding sequences were transcribed in vitro into mRNA and transfected into Jurkat T-cells. Cellular responses following LPS/SiO₂ stimulation were evaluated using Annexin V-FITC/PI staining, DCFH-DA-based intracellular oxidative activity assays, and ELISA quantification of IL-1β and IL-18. The predicted ΔTOTAL values for ASC1, ASC2, and the Native template were -27.07, -22.97, and -9.16 kcal/mol, respectively, with ASC1 showing the most favorable predicted binding free energy. In Jurkat T-cells, ASC1- and ASC2-encoding mRNAs were associated with reduced apoptotic populations, decreased DCFH-associated intracellular oxidative activity, and lower IL-1β and IL-18 production following LPS/SiO₂ stimulation. ASC1 produced greater reductions in both cytokines than ASC2. ASC1 exhibited the most favorable computational and cytokine profiles and could be considered for further mechanistic investigation.