Comprehensive insight into the molecular interaction of an anticancer drug-ifosfamide with human alpha-2-macroglobulin: biophysical and in silico studies.

Ifosfamide is an active alkylating chemotherapeutic drug chemically related to nitrogen mustard. The pharmacokinetics of drugs is affected upon binding with protein, making the studies on drug-protein interaction promising. The present study investigates the interaction between ifosfamide and human antiproteinase-alpha-2-macroglobulin (α2M) by using multi-spectroscopic and in silico techniques. The UV-visible absorption, intrinsic fluorescence and circular dichroism (CD) spectroscopic methods were employed to unveil the mode and mechanism of ifosfamide-α2M interaction. Fluorescence quenching studies performed at three different temperatures indicated that ifosfamide-α2M complex formation involves static quenching. Far UV-CD spectra revealed a minor alteration in the secondary structure of α2M instigated by ifosfamide. The thermodynamic parameters determined by fluorescence quenching experiment and isothermal titration calorimetry (ITC) suggested that the complex between ifosfamide and α2M involves hydrogen bonding and hydrophobic interactions. Molecular docking illustrates that ifosfamide binds with moderate affinity to Lys1240, Asn173, Ser957, Leu955, Asp953, Lys1216 and Thr1236 residues during the interaction. Molecular dynamic (MD) simulation suggested that the ifosfamide forms a stable complex with α2M. Communicated by Ramaswamy H. Sarma.