Abstract
Mdm2 and MdmX are highly homologous and Mdm2 inhibitors exhibit weak affinity for MdmX. The structural mechanism underlying how Mdm2/MdmX distinguish ligand binding affinity remains elusive. Guided by NMR study, we have identified three flexible regions on N-MdmX that affected inhibitor-binding affinity. Furthermore, using disulfide staple, we found that the Phe19-binding site on MdmX was one of the most flexible regions weakening nutlin-3a binding. On the other hand, we continued to pursue high-resolution structure of Mdm2 in complex with ntutin-3a. An X-ray crystallographic structure of Mdm2/nutlin-3a at 1.10 Å identified that His72 had two conformations, a bound state and an unbound state. Molecular dynamics simulation also revealed that His72 in MdmX existed two states. Thus, our work started to search fragments that could rigidify Phe19-binding site. We designed a fusion protein model that p5315-29 was fused with MdmX through a flexible linker, i.e., p5315-29-MdmX, for fragment screening. By use of the intrinsic fluorescence signal of p5315-29 to monitor high-throughput screening, we found that the imidazoindole was a bona fide scaffold for template-based design of MdmX inhibitors. Next the p5315-29-MdmX fusion protein was modified to leave Phe19-binding site unoccupied, i.e., p5323-29-MdmX. By screening a fragment library, a small fragment, CPD-01, could bind to Phe19-binding site in an allosteric mode that enhanced the binding of p5323-29 to MdmX. Furthermore, CPD-01 was conjugated with p5323-29 to synthesize p53 peptide analogues with different linkers. These peptide analogues exhibited enhanced binding affinity for MdmX, compared with p5323-29. Then, CPD-01 was integrated into nutlin-3a to replace Phe19-binding-site-pharmacophore. The new nutlin analogue exhibited high-affinity binding MdmX and Mdm2 as well. Taken together, our work provided a promising strategy to design inhibitors of MdmX/Mdm2. This strategy should be applicable for lead optimization in drug discovery.
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