A Phenstatin-based indole-chalcone hybrid as novel anti-inflammatory agent targeting the NLRP3 Inflammasome: Leads from computational and preclinical studies.

Dysregulated activation of NLRP3 inflammasome contributes to tumor development and progression. We previously identified and characterized 9a, a phenstatin-based indole-chalcone hybrid exhibiting potent anti-oral cancer activity. This study was aimed at understanding the mechanistic role of hybrid 9a in regulating the NLRP3 inflammasome pathway, using an array of computational and experimental approaches. We established an inflammasome activation model using oral squamous carcinoma cell line AW13516 and evaluated effect of 9a. Transcriptome profiling of these cells revealed, 9a distinctly downregulated the expression of NLRP3, GSDMD, IL-1β, and mitochondrial proteins, closely resembling the inhibitory effects of NLRP3-specific inhibitor MCC950. Molecular Dynamics simulation further demonstrated stable association of 9a with NLRP3, supporting direct molecular interaction. These observations were validated by co-localization studies using immunofluorescence and confocal microscopy, which confirmed attenuation of inflammasome activation in AW13516 cells, as evidenced by reduced puncta formation and vesicle exocytosis. Real-time PCR, immunohistochemistry and western blotting analyses further confirmed the inhibitory effect of 9a on inflammasome-associated gene and protein expression. Functional assays in NLRP3 knockdown cells (AW13516NLRP3kd) demonstrated reduced migration, transmigration and tumorigenicity potential in immunocompromised mice compared to AW13516wt cells, establishing a direct link between NLRP3 over expression and cancer progression. Intriguingly, hybrid 9a exhibited cytocompatibility with healthy immune cells while enhancing immune-mediated cytotoxicity against tumor cells. Altogether, these findings establish compound 9a as a modulator of innate immune signaling through selective targeting of the NLRP3 inflammasome, demonstrating its potential as an immunomodulatory therapeutic lead for oral cancer.