{"doi":"10.1016/j.apsb.2021.06.010","title":"Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression","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.","journal":"Acta Pharmaceutica Sinica B","year":2021,"id":160610,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":34,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9542,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":335145,"name":"Neelakshi Gohain","orcid":"0000-0003-1370-312X","position":1,"is_corresponding":false},{"id":230438,"name":"Si Chen","orcid":"0000-0002-8202-8634","position":2,"is_corresponding":false},{"id":673845,"name":"Yinghua Li","orcid":"0000-0002-2391-6738","position":3,"is_corresponding":false},{"id":673846,"name":"Xiaoyuan Zhao","orcid":"0000-0002-8628-2023","position":4,"is_corresponding":false},{"id":673847,"name":"Bo Li","orcid":"0000-0002-2820-9098","position":5,"is_corresponding":false},{"id":335146,"name":"William D. Tolbert","orcid":"0000-0001-7549-9596","position":6,"is_corresponding":false},{"id":673848,"name":"Wangxiao He","orcid":"0000-0002-2054-6022","position":7,"is_corresponding":false},{"id":335148,"name":"Marzena Pazgier","orcid":"0000-0003-0594-5057","position":8,"is_corresponding":false},{"id":363442,"name":"Honggang Hu","orcid":"0000-0003-0577-3021","position":9,"is_corresponding":false},{"id":350927,"name":"Wuyuan Lu","orcid":"0000-0003-1318-9968","position":10,"is_corresponding":false},{"id":673844,"name":"Xiang Li","orcid":"0000-0002-9161-1848","position":0,"is_corresponding":true}],"reference_count":77,"raw_metadata":null,"created_at":"2026-07-18T23:44:48.240577Z","pmid":"34589387","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}