Integrating in silico and in vitro approaches for identifying potential TNF-α inhibitors.

Tumor Necrosis Factor-alpha (TNF-α) is a key pro-inflammatory cytokine that plays a central role in the development and progression of several inflammatory and autoimmune disorders, including psoriasis, rheumatoid arthritis, and inflammatory bowel disease. Targeting TNF-α has become a crucial therapeutic strategy for managing these conditions. In this study, a virtual screening approach was employed to identify potential TNF-α inhibitors from an in-house natural compound library maintained at CSIR-IIIM, Jammu. Molecular docking analyses were performed to evaluate the binding affinities of these natural compounds to the TNF-α protein. The top-ranked compounds were further refined through a similarity search against known natural product TNF-α inhibitors to identify promising candidates. Based on molecular docking and similarity search analyses, 13 potential compounds were identified and subjected to in vitro validation. The experimental results confirmed that compounds IN00569 and IN00007 exhibited strong inhibitory activity by significantly reducing TNF-α secretion in LPS-stimulated RAW 264.7 macrophages, supporting their potential as lead anti-inflammatory agents. Molecular dynamics simulations and MM/GBSA analyses further validated the stability and binding potential of IN00007 and IN00569 with TNF-α, reinforcing their in vitro anti-inflammatory activity. These findings suggest that natural product-based inhibitors may serve as promising candidates for TNF-α-targeted drug development.