Invisible State of MdmX and Design of its Inhibitors

Mdm2 and MdmX are highly homologousand Mdm2 inhibitors exhibit weak affinity for MdmX. It is fundamentally interesting to understand how Mdm2 and MdmX distinguish binding affinity of thesame inhibitor. Previously, we identified three flexible regions on MdmX and definedan invisible state, facilitated by NMR study (Qin et al. Biochemistry 2017, 56,44, 5943–5954). Recently, we found that the Phe19‐binding site on MdmX is one of the most flexible regions weakening nutlin‐3a binding. Our 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 (Pi et al, Biochemistry, 2019,58, 27, 3005‐3015). Molecular dynamics simulation confirmed that His72 in MdmXexists two states. In this work, through a high‐throughput screening of fragment library, we found that a small fragment, CPD‐01, binds to Phe19‐binding site in an allosteric manner so that enhances binding of p53 23–29 to MdmX. Furthermore we used CPD‐01 to modify p53 23–29 peptide and nutlin‐3a. Our binding assay and cell biological study indicated that p53 23–29 – CPD‐01 peptide analogue and nutlin‐3a‐CPD‐01 analogue exhibited high‐affinity binding MdmX and Mdm2. 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. Support or Funding Information Hubei University of Technology (ZSD) and National Natural Science Foundation of China (YQH, 21603121).Two configurations of His72 in Mdm2 and MdmX