Interaction of the lysozyme with anticoagulant drug warfarin: Spectroscopic and computational analyses

Warfarin is a cardiovascular drug, used to treat or inhibit the coagulation of the blood. In this paper, we have studied the interaction of lysozyme with warfarin using several experimental (fluorescence, UV-visible and circular dichroism spectroscopies) and computational (molecular docking, molecular dynamics and DFT) approaches. Experimental studies have suggested that there was a strong interaction between lysozyme and warfarin. Inner filter effect played important role in fluorescence experimental data which show that the emission intensity of lysozyme decreased on the addition of warfarin, however, after inner filter effect correction the actual outcome turned out be the fluorescence enhancement. The extent of binding, increased with temperature rise. The interaction was primarily taken place via the dominance of hydrophobic forces. Small amount of warfarin didn't influence the secondary structure of lysozyme; however, the higher concentration of warfarin caused a decrease in the helicity of the protein and a consequent partial unfolding. Molecular docking studies were also performed which revealed that warfarin binds with lysozyme mainly with hydrophobic forces along with a significant contribution of hydrogen bonding. The flexibility of warfarin played important role in fitting the molecule into the binding pocket of lysozyme. Frontier molecular orbitals of warfarin, using DFT, in free as well as complexed form have also been calculated and discussed. Molecular dynamics simulations of unbound and warfarin bound lysozyme reveal a stable complex with slightly higher RMSD values in the presence of warfarin. Despite slightly increased RMSF values, the overall compactness and folding properties remain consistent, emphasizing strong binding towards lysozyme through the results obtained from intermolecular hydrogen bonding analysis. Essential dynamics analysis suggests warfarin induces slight structural changes without significantly altering the conformation, additionally supported by SASA patterns. Aside from the examination of global and essential motion, the MM/PBSA-based analysis of binding free energy elucidates the significant binding of warfarin to lysozyme, indicating a binding free energy of -13.3471 kcal/mol.