Insight into the molecular interaction between the anticancer drug, enzalutamide and human alpha-2-macroglobulin: Biochemical and biophysical approach

The drug pharmacokinetics is affected upon binding with proteins, thus making drug-protein interactions crucial. This study investigated the interaction between enzalutamide and human major antiproteinase alpha-2-macroglobulin (α₂M) by using multi spectroscopic and calorimetric techniques. The spectroscopic techniques such as circular dichroism (CD), intrinsic fluorescence, and UV-visible absorption were used to determine the mechanism of enzalutamide-α₂M interaction. Studies on the quenching of fluorescence at three different temperatures showed that the enzalutamide-α₂M complex is formed through static quenching mechanism. The change in microenvironment around tyrosine residues in protein was detected through synchronised fluorescence. The secondary structure of α₂M was slightly altered by enzalutamide according to far UV-CD spectral analysis. Changes in position of amide I band in FTIR spectra further confirm the secondary structural alteration in α₂M. According to thermodynamic characteristics such as fluorescence quenching and isothermal titration calorimetry (ITC), hydrogen bonds and hydrophobic interactions were involved in the interaction machanism. The ITC reiterated the exothermic and spontaneous nature of the interaction. The lower proteinase inhibitory activity of the α₂M-enzalutamide conjugate as reflects the disruption of the native α₂M structure upon interaction with enzalutamide.