Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression

Xiang Li; Neelakshi Gohain; Si Chen; Yinghua Li; Xiaoyuan Zhao; Bo Li; William D Tolbert; Wangxiao He; Marzena Pazgier; Honggang Hu; Wuyuan Lu

doi:10.1016/j.apsb.2021.06.010

Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression

Acta Pharm Sin B. 2021 Sep;11(9):2655-2669. doi: 10.1016/j.apsb.2021.06.010. Epub 2021 Jun 18.

Authors

Xiang Li^{1

2}, Neelakshi Gohain², Si Chen³, Yinghua Li⁴, Xiaoyuan Zhao^{4

5}, Bo Li⁵, William D Tolbert², Wangxiao He⁶, Marzena Pazgier², Honggang Hu⁴, Wuyuan Lu⁷

Affiliations

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.

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 K _d 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.

Keywords: Antitumor peptide; FP, fluorescence polarization; ITC, isothermal titration calorimetry; MDM2; MDM2, murine double minute 2; MDMX; MDMX, murine double minute X; P53; PMI, P53‒MDM2/MDMX inhibitor; SAR, structure‒activity relationship; SPR, surface plasmon resonance; Systematic mutational analysis.