Structural and anti-colorectal cancer modulation of tumor necrosis factor-alpha (TNF-α) protein by gaillardin: Experimental and theoretical analyses.

This study aimed to elucidate how gaillardin modulates the structural stability and functional activity of tumor necrosis factor-alpha (TNF-α), a key cytokine whose conformation governs its biological efficacy. Spectroscopic, molecular docking, 150 ns molecular dynamics (MD), and cellular analyses were performed to characterize TNF-α behavior in the free and ligand-bound states. Fluorescence and docking results revealed moderate, static interaction stabilized by hydrogen bonding and van der Waals contacts with GLN47 and ASP45. Far and near circular dichroism spectra indicated that gaillardin binding induced minor conformational adjustments, slightly increasing the β-sheet fraction relative to native TNF-α. ANS and intrinsic fluorescence studies showed that this interaction enhanced TNF-α resistance to induced unfolding. MD simulations confirmed these observations, showing modulated fluctuation, compactness, solvent exposure, and 3-8 persistent hydrogen bonds. Cellular assays in SW-480 human colorectal cancer (CRC) cells further revealed that TNF-α in the complexed form significantly enhanced apoptotic activity, lowering the IC₅₀ from 64.7 ± 3.98 ng/mL to 24.1 ± 2.39 ng/mL, thereby effectively enhancing the sensitivity of these cells to TNF-α treatment. In conclusion, gaillardin modulates TNF-α's β-sheet architecture and tertiary dynamics, maintaining its active conformation, which is consistent with the observed enhancement of TNF-α-mediated apoptotic activity while potentially ameliorating TNF-α hyporesponsiveness.