Studying the Mechanism of Interaction of Doxofylline with Human Lysozyme: A Biophysical and In Silico Approach

In this study, multiple spectroscopic and computational methods were utilized to investigate the binding mechanism of doxofylline with lysozyme. The in vitro methods were used to obtain the binding kinetics and thermodynamics. UV–vis spectroscopy indicated the formation of complex between doxofylline and lysozyme. The Gibb’s free energy and binding constant from UV–vis data was obtained as −7.20 kcal M(−1) and 1.929 × 10(5) M(−1), respectively. Doxofylline successfully quenched the fluorescence of lysozyme, confirming the formation of complex. The k(q) and K(sv) values for the quenching of lysozyme’s fluorescence by doxofylline were 5.74 × 10(11) M(−1) s(−1) and 3.32 × 10(3) M(−1), respectively. These values signified a moderate binding affinity between doxofylline and lysozyme. In synchronous spectroscopy, red shifts were observed for indicating the changes in microenvironment of lysozyme following the binding of doxofylline. The secondary structural analysis was determined using circular dichroism (CD) which revealed an increase in % α-helical as a result of doxofylline interaction. The binding affinity and flexibility of lysozyme upon complexation have been revealed via molecular docking and molecular dynamic (MD) simulations, respectively. According to the many parameters of the MD simulation, the lysozyme–doxofylline complex was stable under physiological conditions. All during the simulation time, hydrogen bonds were continuously present. The MM-PBSA binding energy for lysozyme and doxofylline binding was found to be −30.55 kcal mol(−1).