Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression |
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Authors: | Xiang Li Neelakshi Gohain Si Chen Yinghua Li Xiaoyuan Zhao Bo Li William D. Tolbert Wangxiao He Marzena Pazgier Honggang Hu Wuyuan Lu |
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Affiliation: | 1. School of Pharmacy, Second Military Medical University, Shanghai 200433, China;2. Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA;3. School of Medicine, Shanghai University, Shanghai 200444, China;4. Institute of Translational Medicine, Shanghai University, Shanghai 200444, China;5. State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China;6. Department of Talent Highland, the First Affiliated Hospital of Xi''an Jiaotong University, Xi''an 710061, China;7. Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS) of School of Basic Medical Sciences and Shanghai Institute of Infectious Disease and Biosecurity of School of Public Health, Fudan University, Shanghai 200032, China |
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Abstract: | Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53 in vitro and in vivo, representing a viable therapeutic strategy for cancer treatment. Using phage display techniques, we previously identified a potent peptide activator of P53, termed PMI (TSFAEYWNLLSP), with binding affinities for both MDM2 and MDMX in the low nanomolar concentration range. Here we report an ultrahigh affinity, dual-specificity peptide antagonist of MDM2 and MDMX obtained through systematic mutational analysis and additivity-based molecular design. Functional assays of over 100 peptide analogs of PMI using surface plasmon resonance and fluorescence polarization techniques yielded a dodecameric peptide termed PMI-M3 (LTFLEYWAQLMQ) that bound to MDM2 and MDMX with Kd values in the low picomolar concentration range as verified by isothermal titration calorimetry. Co-crystal structures of MDM2 and of MDMX in complex with PMI-M3 were solved at 1.65 and 3.0 Å resolution, respectively. Similar to PMI, PMI-M3 occupied the P53-binding pocket of MDM2/MDMX, which was dominated energetically by intermolecular interactions involving Phe3, Tyr6, Trp7, and Leu10. Notable differences in binding between PMI-M3 and PMI were observed at other positions such as Leu4 and Met11 with MDM2, and Leu1 and Met11 with MDMX, collectively contributing to a significantly enhanced binding affinity of PMI-M3 for both proteins. By adding lysine residues to both ends of PMI and PMI-M3 to improve their cellular uptake, we obtained modified peptides termed PMI-2K (KTSFAEYWNLLSPK) and M3-2K (KLTFLEYWAQLMQK). Compared with PMI-2K, M3-2K exhibited significantly improved antitumor activities in vitro and in vivo in a P53-dependent manner. This super-strong peptide inhibitor of the P53-MDM2/MDMX interactions may become, in its own right, a powerful lead compound for anticancer drug development, and can aid molecular design of other classes of P53 activators as well for anticancer therapy. |
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Keywords: | MDM2 MDMX P53 Antitumor peptide Systematic mutational analysis FP" },{" #name" :" keyword" ," $" :{" id" :" kwrd0045" }," $$" :[{" #name" :" text" ," _" :" fluorescence polarization ITC" },{" #name" :" keyword" ," $" :{" id" :" kwrd0055" }," $$" :[{" #name" :" text" ," _" :" isothermal titration calorimetry MDM2" },{" #name" :" keyword" ," $" :{" id" :" kwrd0065" }," $$" :[{" #name" :" text" ," _" :" murine double minute 2 MDMX" },{" #name" :" keyword" ," $" :{" id" :" kwrd0075" }," $$" :[{" #name" :" text" ," _" :" murine double minute X PMI" },{" #name" :" keyword" ," $" :{" id" :" kwrd0085" }," $$" :[{" #name" :" text" ," _" :" P53‒MDM2/MDMX inhibitor SAR" },{" #name" :" keyword" ," $" :{" id" :" kwrd0095" }," $$" :[{" #name" :" text" ," _" :" structure‒activity relationship SPR" },{" #name" :" keyword" ," $" :{" id" :" kwrd0105" }," $$" :[{" #name" :" text" ," _" :" surface plasmon resonance |
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