Increased Flexibility of Local Conformation in the N-Terminal Domain of MdmX Enhances Ligand-Binding Affinity

Abstract

The overexpression of Mdm2 and MdmX ultimately impairs the cancer suppression of p53 function in approximately half of human cancers. Discovery of Mdm2 inhibitors has been made tremendous progress and some potent prodrugs have been considered for clinical investigation. Although MdmX is homologous to Mdm2 regarding their amino acid sequences and three-dimensional structures, the Mdm2 inhibitors exhibit weak affinity binding to MdmX. In order to aid in designing of MdmX inhibitors based on the Mdm2 inhibitor scaffolds, we engineered a disulfide bond to tie up the N-terminus and the C-terminus of the N-terminal domain of MdmX (N-MdmX) so that the pre-defined ligand-binding pocket on N-MdmX would be rigidified for docking an inhibitor. The resultant N-MdmX mutant was defined as N-MdmX2C and its interaction with nutlin-3a (a potent Mdm2 inhibitor) was characterized with isothermal titration calorimetry (ITC), circular dichroism spectroscopy (CD) and nuclear magnetic resonance (NMR) spectroscopy. The ITC data reveal that N-MdmX2C exhibits relatively-strong affinity binding to nutlin-3a compared to N-MdmX. The CD analysis indicates that both N-MdmX2C and N-MdmX have similar secondary structure content while their tertiary structures show difference. However, the 15N-1H HSQC NMR spectra reveal that the conformation of N-MdmX2C becomes more flexible compared to N-MdmX unexpectedly. Nevertheless, the 15N-1H HSQC NMR titration confirms that N-MdmX2C has higher binding affinity for nutlin-3a than N-MdmX. On the other hand, both N-MdmX2C and N-MdmX exhibit the same binding affinity to p53p investigated with ITC and NMR titration. Taken together, the current results suggest the confirmation of N-MdmX2C resembles a binding intermediate state for ligand binding, providing an ideal model for anticancer drug design targeting MdmX/Mdm2. (∗Corresponding authors)