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1
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Zhang XL, Yue HW, Liu YJ, Wang JY, Duan HT, Liu YH, Jiang LL, Hu HY. Designer polyQ fusion proteins sequester USP7/HDM2 for modulating P53 functionality. iScience 2025; 28:112025. [PMID: 40104064 PMCID: PMC11914518 DOI: 10.1016/j.isci.2025.112025] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2024] [Revised: 11/05/2024] [Accepted: 02/11/2025] [Indexed: 03/20/2025] Open
Abstract
Overexpression of USP7 and HDM2 inactivates P53 signaling in tumor cells and facilitates their progression, but suppression of these targets by conventional strategies to reactivate P53 function remains a challenge. We applied polyQ sequences and target-interacting peptides to engineer polyQ fusion proteins that specifically sequester the targets, hence depleting their availabilities and modulating the P53 functionality. We have revealed that the designer fusion Atx793Q-N172-IRF (IRF sequence: SPGEGPSGTG) sequesters USP7 and/or HDM2 into aggregates and thereby increases the P53 level, but it depends on the IRF repeats fused, suggesting that depletion of the USP7 availability plays a dual role in controlling P53 stability. Direct sequestration of HDM2 by Atx793Q-N172-PMI (PMI: TSFAEYWNLLSP) remarkably reduces the protein level of soluble HDM2 and hence increases the P53 level, which consequently up-regulates expression of the downstream genes. The polyQ-fusion strategy is feasible to modulate the P53 stability and functionality, furnishing a therapeutic potential for cancers.
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Affiliation(s)
- Xiang-Le Zhang
- Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, P.R. China
- University of Chinese Academy of Sciences, Beijing 100049, P.R. China
| | - Hong-Wei Yue
- Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, P.R. China
- University of Chinese Academy of Sciences, Beijing 100049, P.R. China
| | - Ya-Jun Liu
- Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, P.R. China
- University of Chinese Academy of Sciences, Beijing 100049, P.R. China
| | - Jian-Yang Wang
- Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, P.R. China
- University of Chinese Academy of Sciences, Beijing 100049, P.R. China
| | - Heng-Tong Duan
- Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, P.R. China
- University of Chinese Academy of Sciences, Beijing 100049, P.R. China
| | - Yin-Hu Liu
- Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, P.R. China
- University of Chinese Academy of Sciences, Beijing 100049, P.R. China
| | - Lei-Lei Jiang
- Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, P.R. China
| | - Hong-Yu Hu
- Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, P.R. China
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2
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Sardina F, Polverino F, Valentini S, Carsetti C, Falvo E, Tisci G, Soddu S, Moretti F, Paiardini A, Rinaldo C. Targeting MDM2 affects spastin protein levels and functions: implications for HSP treatment. Cell Death Discov 2025; 11:53. [PMID: 39920118 PMCID: PMC11806007 DOI: 10.1038/s41420-025-02333-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2024] [Revised: 01/10/2025] [Accepted: 01/28/2025] [Indexed: 02/09/2025] Open
Abstract
Spastin is a microtubule (MT) severing enzyme that regulates several cell functions associated with MT dynamics. A reduction in spastin protein levels is responsible for approximately 40% of cases of Hereditary Spastic Paraplegia (HSP), a neurodegenerative disease. Currently, there is no cure for HSP but strategies to induce a recovery of spastin levels are emerging as potential therapeutic approaches. Here, we show that MDM2 interacts with spastin MT-interacting and trafficking (MIT) domain. By biochemical and functional experiments, we demonstrate that MDM2 binds spastin and regulates its levels in a post-transcriptional manner independently of the E3 ubiquitin ligase activity. Of relevance, treatment of spastin-deficient cells with the MDM2 inhibitor Nutlin-3a can restore spastin levels and functions, such as cytokinetic abscission and sorting of transferrin receptor. These findings identify MDM2 as a novel interactor of spastin and a potential druggable regulator of its protein levels.
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Affiliation(s)
- Francesca Sardina
- Institute of Molecular Biology and Pathology, Italian National Research Council, c/o Sapienza University, Rome, Italy.
| | - Federica Polverino
- Institute of Molecular Biology and Pathology, Italian National Research Council, c/o Sapienza University, Rome, Italy
| | - Sonia Valentini
- Institute of Biochemistry and Cell Biology, Italian National Research Council, Rome, Italy
| | - Claudia Carsetti
- Institute of Molecular Biology and Pathology, Italian National Research Council, c/o Sapienza University, Rome, Italy
- Department of Biology and Biotechnology "Charles Darwin", Sapienza University, Rome, Italy
| | - Elisabetta Falvo
- Institute of Molecular Biology and Pathology, Italian National Research Council, c/o Sapienza University, Rome, Italy
| | - Giada Tisci
- Institute of Molecular Biology and Pathology, Italian National Research Council, c/o Sapienza University, Rome, Italy
- Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University, Rome, Italy
| | - Silvia Soddu
- Unit of Cellular Networks and Molecular Therapeutic Targets, Department of Research and Advanced Technologies, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Fabiola Moretti
- Institute of Biochemistry and Cell Biology, Italian National Research Council, Rome, Italy
| | - Alessandro Paiardini
- Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University, Rome, Italy
| | - Cinzia Rinaldo
- Institute of Molecular Biology and Pathology, Italian National Research Council, c/o Sapienza University, Rome, Italy.
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3
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Neuville M, Bourgeais M, Li B, Varajao L, Hallé F, Goudreau SR, Thinon E, Pasco M, Khatib AM, Guichard G. Cell-Permeable Peptide Inhibitors of the p53-hDM2 Interaction via Foldamer Helix Mimicry and Bis-Thioether Stapling. J Med Chem 2025; 68:236-246. [PMID: 39719869 DOI: 10.1021/acs.jmedchem.4c01762] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2024]
Abstract
Combining helical foldamers with α-peptides can produce α-helix mimetics with a reduced peptide character and enhanced resistance to proteolysis. Previously, we engineered a hybrid peptide-oligourea sequence replicating the N-terminal α-helical domain of p53 to achieve high affinity binding to hDM2. Here, we further advance this strategy by combining the foldamer approach with side chain cross-linking to create more constrained cell-permeable inhibitors capable of effectively engaging the target within cells. Starting from the crystal structure of the foldamer-hDM2 complex, we identified specific sites suitable for stapling, and generated a small library of macrocyclic foldamer-peptide hybrids. The most promising binders were subsequently optimized for cellular uptake and tested in a cellular assay. We observed that the introduction of a short segment of positively charged residues at the N-terminus of the sequence led to inhibitors that exhibited cytotoxic activity independently of p53. In contrast, neutral acetylated peptide-foldamer macrocycles demonstrated activity in a p53-dependent manner.
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Affiliation(s)
- Maxime Neuville
- Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, IECB, F-33607 Pessac, France
- IMMUPHARMA BIOTECH SAS, 15 rue de Bruxelles, 75009 Paris, France
| | - Mathieu Bourgeais
- Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, IECB, F-33607 Pessac, France
- Univ. Bordeaux, INSERM, BRIC, U 1312, F-33000 Bordeaux, France
| | - Bo Li
- Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, IECB, F-33607 Pessac, France
| | - Laetitia Varajao
- Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, IECB, F-33607 Pessac, France
| | - François Hallé
- Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, IECB, F-33607 Pessac, France
| | | | - Emmanuelle Thinon
- Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, IECB, F-33607 Pessac, France
| | - Morgane Pasco
- Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, IECB, F-33607 Pessac, France
| | | | - Gilles Guichard
- Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, IECB, F-33607 Pessac, France
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4
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Camps-Fajol C, Cavero D, Minguillón J, Surrallés J. Targeting protein-protein interactions in drug discovery: Modulators approved or in clinical trials for cancer treatment. Pharmacol Res 2025; 211:107544. [PMID: 39667542 DOI: 10.1016/j.phrs.2024.107544] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/16/2024] [Revised: 11/27/2024] [Accepted: 12/09/2024] [Indexed: 12/14/2024]
Abstract
Protein-protein interactions (PPIs) form complex cellular networks fundamental to many key biological processes, including signal transduction, cell proliferation and DNA repair. In consequence, their perturbation is often associated with many human diseases. Targeting PPIs offers a promising approach in drug discovery and ongoing advancements in this field hold the potential to provide highly specific therapies for a wide range of complex diseases. Despite the development of PPI modulators is challenging, advances in the genetic, proteomic and computational level have facilitated their discovery and optimization. Focusing on anticancer drugs, in the last years several PPI modulators have entered clinical trials and venetoclax, which targets Bcl-2 family proteins, has been approved for treating different types of leukemia. This review discusses the clinical development status of drugs modulating several PPIs, such as MDM2-4/p53, Hsp90/Hsp90, Hsp90/CDC37, c-Myc/Max, KRAS/SOS1, CCR5/CCL5, CCR2/CCL2 or Smac/XIAP, in cancer drug discovery.
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Affiliation(s)
- Cristina Camps-Fajol
- Unitat Mixta de Recerca en Medicina Genòmica, Universitat Autònoma de Barcelona (UAB)-IR SANT PAU, Barcelona, Spain; Institut de Bioenginyeria de Catalunya (IBEC), Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras, Instituto de Salud Carlos III (CIBERER, ISCIII), Madrid, Spain
| | - Debora Cavero
- Unitat Mixta de Recerca en Medicina Genòmica, Universitat Autònoma de Barcelona (UAB)-IR SANT PAU, Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras, Instituto de Salud Carlos III (CIBERER, ISCIII), Madrid, Spain
| | - Jordi Minguillón
- CIBERER-ISCIII, IdiPAZ-CNIO Translational Research Unit in Pediatric Hemato-Oncology, La Paz University Hospital Research Institute; Spanish National Cancer Center, Madrid, Spain; Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain
| | - Jordi Surrallés
- Unitat Mixta de Recerca en Medicina Genòmica, Universitat Autònoma de Barcelona (UAB)-IR SANT PAU, Barcelona, Spain; Institut de Bioenginyeria de Catalunya (IBEC), Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras, Instituto de Salud Carlos III (CIBERER, ISCIII), Madrid, Spain; Servei de Genètica, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Spain.
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5
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Swenson CS, Mandava G, Thomas DM, Moellering RE. Tackling Undruggable Targets with Designer Peptidomimetics and Synthetic Biologics. Chem Rev 2024; 124:13020-13093. [PMID: 39540650 PMCID: PMC12036645 DOI: 10.1021/acs.chemrev.4c00423] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2024]
Abstract
The development of potent, specific, and pharmacologically viable chemical probes and therapeutics is a central focus of chemical biology and therapeutic development. However, a significant portion of predicted disease-causal proteins have proven resistant to targeting by traditional small molecule and biologic modalities. Many of these so-called "undruggable" targets feature extended, dynamic protein-protein and protein-nucleic acid interfaces that are central to their roles in normal and diseased signaling pathways. Here, we discuss the development of synthetically stabilized peptide and protein mimetics as an ever-expanding and powerful region of chemical space to tackle undruggable targets. These molecules aim to combine the synthetic tunability and pharmacologic properties typically associated with small molecules with the binding footprints, affinities and specificities of biologics. In this review, we discuss the historical and emerging platforms and approaches to design, screen, select and optimize synthetic "designer" peptidomimetics and synthetic biologics. We examine the inspiration and design of different classes of designer peptidomimetics: (i) macrocyclic peptides, (ii) side chain stabilized peptides, (iii) non-natural peptidomimetics, and (iv) synthetic proteomimetics, and notable examples of their application to challenging biomolecules. Finally, we summarize key learnings and remaining challenges for these molecules to become useful chemical probes and therapeutics for historically undruggable targets.
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Affiliation(s)
- Colin S Swenson
- Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, United States
| | - Gunasheil Mandava
- Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, United States
| | - Deborah M Thomas
- Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, United States
| | - Raymond E Moellering
- Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, United States
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6
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Xu L, Fan X, He Y, Xia X, Zhang J. Design, Synthesis, and Biological Evaluation of Lysine-Stapled Peptide Inhibitors of p53-MDM2/MDMX Interactions with Potent Antitumor Activity In Vivo. J Med Chem 2024; 67:17893-17904. [PMID: 39300610 DOI: 10.1021/acs.jmedchem.4c01939] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/22/2024]
Abstract
We introduce novel lysine-stapled peptide inhibitors targeting p53-MDM2/MDMX interactions. Leveraging the model peptides pDI (LTFEHYWAQLTS) and PMI-M3 (LTFLEYWAQLMQ) as starting points, a series of lysine-stapled analogues were designed and synthesized. Through in vitro cell assay screening, two lead compounds, SPDI-48-T1 and SPMI-48-T3, were identified for their excellent antiproliferation activity. Fluorescence polarization assays revealed that both compounds exhibited strong binding affinities against MDM2 and MDMX, ascertained by Kd values within the low micromolar spectrum. Further characterization of SPDI-48-T1 and SPMI-48-T3 demonstrated that SPDI-48-T1 possessed superior cell permeability and serum stability. Notably, SPDI-48-T1 displayed a dose-dependent suppression of tumor growth in an HCT116 xenograft mouse model. Our findings indicate that SPDI-48-T1 holds promise as a lead compound for further development as an anticancer agent by modulating p53-MDM2/MDMX interactions. Additionally, this study also proved that the lysine stapling strategy may serve as a robust approach for generating peptide ligands targeting other protein-protein interactions.
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Affiliation(s)
- Lei Xu
- Chongqing Key Laboratory of Natural Product Synthesis and Drug Research, School of Pharmaceutical Sciences, Chongqing University, Chongqing 401331, People's Republic of China
| | - Xin Fan
- Chongqing Key Laboratory of Natural Product Synthesis and Drug Research, School of Pharmaceutical Sciences, Chongqing University, Chongqing 401331, People's Republic of China
| | - Yi He
- Chongqing Key Laboratory of Natural Product Synthesis and Drug Research, School of Pharmaceutical Sciences, Chongqing University, Chongqing 401331, People's Republic of China
| | - Xuefeng Xia
- Chongqing Key Laboratory of Natural Product Synthesis and Drug Research, School of Pharmaceutical Sciences, Chongqing University, Chongqing 401331, People's Republic of China
| | - Jinqiang Zhang
- Chongqing Key Laboratory of Natural Product Synthesis and Drug Research, School of Pharmaceutical Sciences, Chongqing University, Chongqing 401331, People's Republic of China
- Chongqing University Industrial Technology Research Institute, Chongqing 401329, People's Republic of China
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7
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Yin Q, Hu Y, Dong Z, Lu J, Wang H. Cellular, Structural Basis, and Recent Progress for Targeting Murine Double Minute X (MDMX) in Tumors. J Med Chem 2024; 67:14723-14741. [PMID: 39185935 DOI: 10.1021/acs.jmedchem.4c00913] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/27/2024]
Abstract
Murine double minute X (MDMX) is an oncoprotein that mainly has a negative regulatory effect on the tumor suppressor p53 to induce tumorigenesis. As MDMX is highly expressed in various types of tumor cells, targeting and inhibiting MDMX are becoming a promising strategy for treating cancers. However, the high degree of structural homology between MDMX and its homologous protein murine double minute 2 (MDM2) is a great challenge for the development of MDMX-targeted therapies. This review introduces the structure, distribution, and regulation of the MDMX, summarizes the structural features and structure-activity relationships (SARs) of MDMX ligands, and focuses on the differences between MDMX and MDM2 in these aspects. Our purpose of this work is to propose potential strategies to achieve the specific targeting of MDMX.
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Affiliation(s)
- Qikun Yin
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China
| | - Yuemiao Hu
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China
| | - Zhiwen Dong
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China
| | - Jing Lu
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China
| | - Hongbo Wang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China
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8
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Yang W, Wang J, Zhao L, Chen J. Insights into the Interaction Mechanisms of Peptide and Non-Peptide Inhibitors with MDM2 Using Gaussian-Accelerated Molecular Dynamics Simulations and Deep Learning. Molecules 2024; 29:3377. [PMID: 39064955 PMCID: PMC11279683 DOI: 10.3390/molecules29143377] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2024] [Revised: 07/15/2024] [Accepted: 07/16/2024] [Indexed: 07/28/2024] Open
Abstract
Inhibiting MDM2-p53 interaction is considered an efficient mode of cancer treatment. In our current study, Gaussian-accelerated molecular dynamics (GaMD), deep learning (DL), and binding free energy calculations were combined together to probe the binding mechanism of non-peptide inhibitors K23 and 0Y7 and peptide ones PDI6W and PDI to MDM2. The GaMD trajectory-based DL approach successfully identified significant functional domains, predominantly located at the helixes α2 and α2', as well as the β-strands and loops between α2 and α2'. The post-processing analysis of the GaMD simulations indicated that inhibitor binding highly influences the structural flexibility and collective motions of MDM2. Calculations of molecular mechanics-generalized Born surface area (MM-GBSA) and solvated interaction energy (SIE) not only suggest that the ranking of the calculated binding free energies is in agreement with that of the experimental results, but also verify that van der Walls interactions are the primary forces responsible for inhibitor-MDM2 binding. Our findings also indicate that peptide inhibitors yield more interaction contacts with MDM2 compared to non-peptide inhibitors. Principal component analysis (PCA) and free energy landscape (FEL) analysis indicated that the piperidinone inhibitor 0Y7 shows the most pronounced impact on the free energy profiles of MDM2, with the piperidinone inhibitor demonstrating higher fluctuation amplitudes along primary eigenvectors. The hot spots of MDM2 revealed by residue-based free energy estimation provide target sites for drug design toward MDM2. This study is expected to provide useful theoretical aid for the development of selective inhibitors of MDM2 family members.
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Affiliation(s)
- Wanchun Yang
- School of Science, Shandong Jiaotong University, Jinan 250357, China; (J.W.); (L.Z.)
| | | | | | - Jianzhong Chen
- School of Science, Shandong Jiaotong University, Jinan 250357, China; (J.W.); (L.Z.)
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9
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Chen FJ, Lin W, Chen FE. Non-symmetric stapling of native peptides. Nat Rev Chem 2024; 8:304-318. [PMID: 38575678 DOI: 10.1038/s41570-024-00591-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/23/2024] [Indexed: 04/06/2024]
Abstract
Stapling has emerged as a powerful technique in peptide chemistry. It enables precise control over peptide conformation leading to enhanced properties such as improved stability and enhanced binding affinity. Although symmetric stapling methods have been extensively explored, the field of non-symmetric stapling of native peptides has received less attention, largely as a result of the formidable challenges it poses - in particular the complexities involved in achieving the high chemo-selectivity and site-selectivity required to simultaneously modify distinct proteinogenic residues. Over the past 5 years, there have been significant breakthroughs in addressing these challenges. In this Review, we describe the latest strategies for non-symmetric stapling of native peptides, elucidating the protocols, reaction mechanisms and underlying design principles. We also discuss current challenges and opportunities this field offers for future applications, such as ligand discovery and peptide-based therapeutics.
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Affiliation(s)
- Fa-Jie Chen
- College of Chemistry, Fuzhou University, Fuzhou, P. R. China.
| | - Wanzhen Lin
- College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, P. R. China
| | - Fen-Er Chen
- College of Chemistry, Fuzhou University, Fuzhou, P. R. China.
- Engineering Center of Catalysis and Synthesis for Chiral Molecules, Department of Chemistry, Fudan University, Shanghai, P. R. China.
- Shanghai Engineering Research Center of Industrial Asymmetric Catalysis of Chiral Drugs, Fudan University, Shanghai, P. R. China.
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10
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Li Y, Wu M, Fu Y, Xue J, Yuan F, Qu T, Rissanou AN, Wang Y, Li X, Hu H. Therapeutic stapled peptides: Efficacy and molecular targets. Pharmacol Res 2024; 203:107137. [PMID: 38522761 DOI: 10.1016/j.phrs.2024.107137] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/06/2023] [Revised: 03/06/2024] [Accepted: 03/06/2024] [Indexed: 03/26/2024]
Abstract
Peptide stapling, by employing a stable, preformed alpha-helical conformation, results in the production of peptides with improved membrane permeability and enhanced proteolytic stability, compared to the original peptides, and provides an effective solution to accelerate the rapid development of peptide drugs. Various reviews present peptide stapling chemistries, anchoring residues and one- or two-component cyclization, however, therapeutic stapled peptides have not been systematically summarized, especially focusing on various disease-related targets. This review highlights the latest advances in therapeutic peptide drug development facilitated by the application of stapling technology, including different stapling techniques, synthetic accessibility, applicability to biological targets, potential for solving biological problems, as well as the current status of development. Stapled peptides as therapeutic drug candidates have been classified and analysed mainly by receptor- and ligand-based stapled peptide design against various diseases, including cancer, infectious diseases, inflammation, and diabetes. This review is expected to provide a comprehensive reference for the rational design of stapled peptides for different diseases and targets to facilitate the development of therapeutic peptides with enhanced pharmacokinetic and biological properties.
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Affiliation(s)
- Yulei Li
- School of Pharmaceutical Sciences & Institute of Materia Medica, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong 250117, China.
| | - Minghao Wu
- School of Medicine, Shanghai University, 99 Shangda Road, Shanghai 200444, China
| | - Yinxue Fu
- School of Pharmaceutical Sciences & Institute of Materia Medica, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong 250117, China
| | - Jingwen Xue
- School of Pharmaceutical Sciences & Institute of Materia Medica, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong 250117, China
| | - Fei Yuan
- School of Pharmaceutical Sciences & Institute of Materia Medica, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong 250117, China
| | - Tianci Qu
- School of Pharmaceutical Sciences & Institute of Materia Medica, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong 250117, China
| | - Anastassia N Rissanou
- Theoretical & Physical Chemistry Institute, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, Athens 11635, Greece
| | - Yilin Wang
- Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, 131 Dong'an Road, Shanghai 200032, China
| | - Xiang Li
- School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai, 200433, China.
| | - Honggang Hu
- School of Medicine, Shanghai University, 99 Shangda Road, Shanghai 200444, China.
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11
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Liu J, Yang J, Pan Q, Wang X, Wang X, Chen H, Zheng X, Huang Q. MDM4 was associated with poor prognosis and tumor-immune infiltration of cancers. Eur J Med Res 2024; 29:79. [PMID: 38281029 PMCID: PMC10821240 DOI: 10.1186/s40001-024-01684-z] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2023] [Accepted: 01/17/2024] [Indexed: 01/29/2024] Open
Abstract
MDM4 is one of the MDM protein family and is generally recognized as the key negative regulator of p53. As a cancer-promoting factor, it plays a non-negligible role in tumorigenesis and development. In this article, we analyzed the expression levels of MDM4 in pan-cancer through multiple databases. We also investigated the correlations between MDM4 expression and prognostic value, immune features, genetic mutation, and tumor-related pathways. We found that MDM4 overexpression is often accompanied by adverse clinical features, poor prognosis, oncogenic mutations, tumor-immune infiltration and aberrant activation of oncogenic signaling pathways. We also conducted transcriptomic sequencing to investigate the effect of MDM4 on transcript levels in colon cancer and performed qPCR to verify this. Finally, we carried out some in vitro experiments including colony formation assay, chemoresistance and senescence-associated β-galactosidase activity assay to study the anti-tumor treatment effect of small molecule MDM4 inhibitor, NSC146109. Our research confirmed that MDM4 is a prognostic biomarker and potential therapeutic target for a variety of malignancies.
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Affiliation(s)
- Jie Liu
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China
- Department of Endoscopy, Shengli Clinical Medical College of Fujian Medical University, Fujian Provincial Hospital, Fuzhou, China
- The Graduate School of Fujian Medical University, Fuzhou, China
| | - Jie Yang
- Department of Endoscopy, Shengli Clinical Medical College of Fujian Medical University, Fujian Provincial Hospital, Fuzhou, China
- The Graduate School of Fujian Medical University, Fuzhou, China
| | - Qilong Pan
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China
| | - Xiangyu Wang
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China
- Department of Endoscopy, Shengli Clinical Medical College of Fujian Medical University, Fujian Provincial Hospital, Fuzhou, China
- The Graduate School of Fujian Medical University, Fuzhou, China
| | - Xinyin Wang
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China
| | - Han Chen
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China
| | - Xiaoling Zheng
- Department of Endoscopy, Shengli Clinical Medical College of Fujian Medical University, Fujian Provincial Hospital, Fuzhou, China.
- The Graduate School of Fujian Medical University, Fuzhou, China.
| | - Qingling Huang
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China.
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12
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Fujimura A, Ishida H, Nozaki T, Terada S, Azumaya Y, Ishiguro T, Kamimura YR, Kujirai T, Kurumizaka H, Kono H, Yamatsugu K, Kawashima SA, Kanai M. Designer Adaptor Proteins for Functional Conversion of Peptides to Small-Molecule Ligands toward In-Cell Catalytic Protein Modification. ACS CENTRAL SCIENCE 2023; 9:2115-2128. [PMID: 38033808 PMCID: PMC10683481 DOI: 10.1021/acscentsci.3c00930] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 07/26/2023] [Revised: 09/19/2023] [Accepted: 10/12/2023] [Indexed: 12/02/2023]
Abstract
Peptides are privileged ligands for diverse biomacromolecules, including proteins; however, their utility is often limited due to low membrane permeability and in-cell instability. Here, we report peptide ligand-inserted eDHFR (PLIED) fusion protein as a universal adaptor for targeting proteins of interest (POI) with cell-permeable and stable synthetic functional small molecules (SFSM). PLIED binds to POI through the peptide moiety, properly orienting its eDHFR moiety, which then recruits trimethoprim (TMP)-conjugated SFSM to POI. Using a lysine-acylating BAHA catalyst as SFSM, we demonstrate that POI (MDM2 and chromatin histone) are post-translationally and synthetically acetylated at specific lysine residues. The residue-selectivity is predictable in an atomic resolution from molecular dynamics simulations of the POI/PLIED/TMP-BAHA (MTX was used as a TMP model) ternary complex. This designer adaptor approach universally enables functional conversion of impermeable peptide ligands to permeable small-molecule ligands, thus expanding the in-cell toolbox of chemical biology.
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Affiliation(s)
- Akiko Fujimura
- Graduate
School of Pharmaceutical Sciences, The University
of Tokyo, Tokyo 113-0033, Japan
| | - Hisashi Ishida
- Institute
for Quantum Life Science, National Institutes
for Quantum Science and Technology, Chiba 263-8555, Japan
| | - Tamiko Nozaki
- Graduate
School of Pharmaceutical Sciences, The University
of Tokyo, Tokyo 113-0033, Japan
| | - Shuhei Terada
- Graduate
School of Pharmaceutical Sciences, The University
of Tokyo, Tokyo 113-0033, Japan
| | - Yuto Azumaya
- Graduate
School of Pharmaceutical Sciences, The University
of Tokyo, Tokyo 113-0033, Japan
| | - Tadashi Ishiguro
- Graduate
School of Pharmaceutical Sciences, The University
of Tokyo, Tokyo 113-0033, Japan
| | - Yugo R. Kamimura
- Graduate
School of Pharmaceutical Sciences, The University
of Tokyo, Tokyo 113-0033, Japan
| | - Tomoya Kujirai
- Institute
for Quantitative Biosciences, The University
of Tokyo, Tokyo 113-0032, Japan
| | - Hitoshi Kurumizaka
- Institute
for Quantitative Biosciences, The University
of Tokyo, Tokyo 113-0032, Japan
| | - Hidetoshi Kono
- Institute
for Quantum Life Science, National Institutes
for Quantum Science and Technology, Chiba 263-8555, Japan
| | - Kenzo Yamatsugu
- Graduate
School of Pharmaceutical Sciences, The University
of Tokyo, Tokyo 113-0033, Japan
| | - Shigehiro A. Kawashima
- Graduate
School of Pharmaceutical Sciences, The University
of Tokyo, Tokyo 113-0033, Japan
| | - Motomu Kanai
- Graduate
School of Pharmaceutical Sciences, The University
of Tokyo, Tokyo 113-0033, Japan
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13
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Kasper SH, Otten S, Squadroni B, Orr‐Terry C, Kuang Y, Mussallem L, Ge L, Yan L, Kannan S, Verma CS, Brown CJ, Johannes CW, Lane DP, Chandramohan A, Partridge AW, Roberts LR, Josien H, Therien AG, Hett EC, Howell BJ, Peier A, Ai X, Cassaday J. A high-throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell-based assays. Bioeng Transl Med 2023; 8:e10542. [PMID: 37693049 PMCID: PMC10487316 DOI: 10.1002/btm2.10542] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2023] [Revised: 04/20/2023] [Accepted: 04/24/2023] [Indexed: 09/12/2023] Open
Abstract
Cyclic peptides are poised to target historically difficult to drug intracellular protein-protein interactions, however, their general cell impermeability poses a challenge for characterizing function. Recent advances in microfluidics have enabled permeabilization of the cytoplasmic membrane by physical cell deformation (i.e., mechanoporation), resulting in intracellular delivery of impermeable macromolecules in vector- and electrophoretic-free approaches. However, the number of payloads (e.g., peptides) and/or concentrations delivered via microfluidic mechanoporation is limited by having to pre-mix cells and payloads, a manually intensive process. In this work, we show that cells are momentarily permeable (t 1/2 = 1.1-2.8 min) after microfluidic vortex shedding (μVS) and that lower molecular weight macromolecules can be cytosolically delivered upon immediate exposure after cells are processed/permeabilized. To increase the ability to screen peptides, we built a system, dispensing-microfluidic vortex shedding (DμVS), that integrates a μVS chip with inline microplate-based dispensing. To do so, we synced an electronic pressure regulator, flow sensor, on/off dispense valve, and an x-y motion platform in a software-driven feedback loop. Using this system, we were able to deliver low microliter-scale volumes of transiently mechanoporated cells to hundreds of wells on microtiter plates in just several minutes (e.g., 96-well plate filled in <2.5 min). We validated the delivery of an impermeable peptide directed at MDM2, a negative regulator of the tumor suppressor p53, using a click chemistry- and NanoBRET-based cell permeability assay in 96-well format, with robust delivery across the full plate. Furthermore, we demonstrated that DμVS could be used to identify functional, low micromolar, cellular activity of otherwise cell-inactive MDM2-binding peptides using a p53 reporter cell assay in 96- and 384-well format. Overall, DμVS can be combined with downstream cell assays to investigate intracellular target engagement in a high-throughput manner, both for improving structure-activity relationship efforts and for early proof-of-biology of non-optimized peptide (or potentially other macromolecular) tools.
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Affiliation(s)
| | | | | | | | - Yi Kuang
- Merck & Co., Inc.CambridgeMassachusettsUSA
| | | | - Lan Ge
- Merck & Co., Inc.KenilworthNew JerseyUSA
| | - Lin Yan
- Merck & Co., Inc.KenilworthNew JerseyUSA
| | | | - Chandra S. Verma
- Agency for Science, Technology and Research (A*STAR)SingaporeSingapore
| | | | | | - David P. Lane
- Agency for Science, Technology and Research (A*STAR)SingaporeSingapore
| | | | | | | | | | | | | | | | | | - Xi Ai
- Merck & Co., Inc.KenilworthNew JerseyUSA
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14
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Sola M, Rendon-Angel A, Rojo Martinez V, Sgrignani J, Magrin C, Piovesana E, Cavalli A, Paganetti P, Papin S. Tau protein binds to the P53 E3 ubiquitin ligase MDM2. Sci Rep 2023; 13:10208. [PMID: 37353565 PMCID: PMC10290082 DOI: 10.1038/s41598-023-37046-8] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2023] [Accepted: 06/14/2023] [Indexed: 06/25/2023] Open
Abstract
Tau gene mutations cause a progressive dementia and neurotoxic Tau forms deposited in neurofibrillary tangles are hallmarks of neurodegenerative tauopathies. Loss of non-canonical Tau functions may contribute to disease. In fact, Tau depletion affects the cellular response to DNA damage and tauopathies exhibit the accumulation of DNA lesions. Moreover, Tau modulates P53 activity and cell fate. Considering that MDM2 is the main antagonist of P53, we investigated, using orthogonal assays, if Tau interacts with MDM2. We report the existence in cells and brain of a Tau-MDM2 complex that, in vitro, exhibits reduced P53 ubiquitination activity in a manner sensitive to a Tau mutation. The Tau-MDM2 interaction involves the microtubule-binding domain of Tau and the acidic domain of MDM2, reminiscent of the binding of Tau to negatively charged microtubules. Notably, MDM2 accumulates aberrantly in neurofibrillary tangles. Aging-associated insults may expose a novel loss-of-function of Tau in neurodegeneration and cancer.
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Affiliation(s)
- Martina Sola
- Laboratory for Aging Disorders, Laboratories for Translational Research, Ente Ospedaliero Cantonale, Room 102a, Via Chiesa 5, 6500, Bellinzona, Switzerland
- PhD Program in Neurosciences, Faculty of Biomedical Sciences, Università Della Svizzera Italiana, Lugano, Switzerland
| | - Azucena Rendon-Angel
- Laboratory for Aging Disorders, Laboratories for Translational Research, Ente Ospedaliero Cantonale, Room 102a, Via Chiesa 5, 6500, Bellinzona, Switzerland
| | - Viviana Rojo Martinez
- Laboratory for Aging Disorders, Laboratories for Translational Research, Ente Ospedaliero Cantonale, Room 102a, Via Chiesa 5, 6500, Bellinzona, Switzerland
| | - Jacopo Sgrignani
- Computational Structural Biology, Institute for Research in Biomedicine, Università Della Svizzera Italiana, Bellinzona, Switzerland
| | - Claudia Magrin
- Laboratory for Aging Disorders, Laboratories for Translational Research, Ente Ospedaliero Cantonale, Room 102a, Via Chiesa 5, 6500, Bellinzona, Switzerland
- PhD Program in Neurosciences, Faculty of Biomedical Sciences, Università Della Svizzera Italiana, Lugano, Switzerland
| | - Ester Piovesana
- Laboratory for Aging Disorders, Laboratories for Translational Research, Ente Ospedaliero Cantonale, Room 102a, Via Chiesa 5, 6500, Bellinzona, Switzerland
- PhD Program in Neurosciences, Faculty of Biomedical Sciences, Università Della Svizzera Italiana, Lugano, Switzerland
| | - Andrea Cavalli
- Computational Structural Biology, Institute for Research in Biomedicine, Università Della Svizzera Italiana, Bellinzona, Switzerland
- Swiss Institute of Bioinformatics, Lausanne, Switzerland
| | - Paolo Paganetti
- Laboratory for Aging Disorders, Laboratories for Translational Research, Ente Ospedaliero Cantonale, Room 102a, Via Chiesa 5, 6500, Bellinzona, Switzerland.
- PhD Program in Neurosciences, Faculty of Biomedical Sciences, Università Della Svizzera Italiana, Lugano, Switzerland.
- Neurocentro Della Svizzera Italiana, Ente Ospedaliero Cantonale, Lugano, Switzerland.
| | - Stéphanie Papin
- Laboratory for Aging Disorders, Laboratories for Translational Research, Ente Ospedaliero Cantonale, Room 102a, Via Chiesa 5, 6500, Bellinzona, Switzerland
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15
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Lang L, Frontera A, Perez A, Bauzá A. Computational Study of Driving Forces in ATSP, PDIQ, and P53 Peptide Binding: C═O···C═O Tetrel Bonding Interactions at Work. J Chem Inf Model 2023; 63:3018-3029. [PMID: 37014944 PMCID: PMC10207270 DOI: 10.1021/acs.jcim.3c00024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2023] [Indexed: 04/06/2023]
Abstract
Understanding the molecular interactions that drive peptide folding is crucial to chemistry and biology. In this study, we analyzed the role of CO···CO tetrel bonding (TtB) interactions in the folding mechanism of three different peptides (ATSP, pDIQ, and p53), which exhibit a different propensity to fold in an α helix motif. To achieve this goal, we used both a recently developed Bayesian inference approach (MELDxMD) and Quantum Mechanics (QM) calculations at the RI-MP2/def2-TZVP level of theory. These techniques allowed us to study the folding process and to evaluate the strength of the CO···CO TtBs as well as the synergies between TtBs and hydrogen-bonding (HB) interactions. We believe that the results derived from our study will be helpful for those scientists working in computational biology, peptide chemistry, and structural biology.
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Affiliation(s)
- Lijun Lang
- Chemistry
Department, University of Florida, Gainesville, Florida 32611, United States
| | - Antonio Frontera
- Department
of Chemistry, Universitat de les Illes Balears, Crta. de Valldemossa km 7.5, 07122 Palma, Baleares, Spain
| | - Alberto Perez
- Chemistry
Department, University of Florida, Gainesville, Florida 32611, United States
| | - Antonio Bauzá
- Department
of Chemistry, Universitat de les Illes Balears, Crta. de Valldemossa km 7.5, 07122 Palma, Baleares, Spain
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16
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Rehman AU, Khurshid B, Ali Y, Rasheed S, Wadood A, Ng HL, Chen HF, Wei Z, Luo R, Zhang J. Computational approaches for the design of modulators targeting protein-protein interactions. Expert Opin Drug Discov 2023; 18:315-333. [PMID: 36715303 PMCID: PMC10149343 DOI: 10.1080/17460441.2023.2171396] [Citation(s) in RCA: 23] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2022] [Accepted: 01/18/2023] [Indexed: 01/31/2023]
Abstract
BACKGROUND Protein-protein interactions (PPIs) are intriguing targets for designing novel small-molecule inhibitors. The role of PPIs in various infectious and neurodegenerative disorders makes them potential therapeutic targets . Despite being portrayed as undruggable targets, due to their flat surfaces, disorderedness, and lack of grooves. Recent progresses in computational biology have led researchers to reconsider PPIs in drug discovery. AREAS COVERED In this review, we introduce in-silico methods used to identify PPI interfaces and present an in-depth overview of various computational methodologies that are successfully applied to annotate the PPIs. We also discuss several successful case studies that use computational tools to understand PPIs modulation and their key roles in various physiological processes. EXPERT OPINION Computational methods face challenges due to the inherent flexibility of proteins, which makes them expensive, and result in the use of rigid models. This problem becomes more significant in PPIs due to their flexible and flat interfaces. Computational methods like molecular dynamics (MD) simulation and machine learning can integrate the chemical structure data into biochemical and can be used for target identification and modulation. These computational methodologies have been crucial in understanding the structure of PPIs, designing PPI modulators, discovering new drug targets, and predicting treatment outcomes.
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Affiliation(s)
- Ashfaq Ur Rehman
- Departments of Molecular Biology and Biochemistry, Chemical and Biomolecular Engineering, Materials Science and Engineering, and Biomedical Engineering, Graduate Program in Chemical and Materials Physics, University of California Irvine, Irvine, California, USA
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Medicinal Bioinformatics Center, Shanghai Jiao-Tong University School of Medicine, Shanghai, Zhejiang, China
| | - Beenish Khurshid
- Department of Biochemistry, Abdul Wali Khan University Mardan, Pakistan
| | - Yasir Ali
- National Center for Bioinformatics, Quaid-e-Azam University, Islamabad, Pakistan
| | - Salman Rasheed
- National Center for Bioinformatics, Quaid-e-Azam University, Islamabad, Pakistan
| | - Abdul Wadood
- Department of Biochemistry, Abdul Wali Khan University Mardan, Pakistan
| | - Ho-Leung Ng
- Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA
| | - Hai-Feng Chen
- State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, Department of Bioinformatics and Biostatistics, National Experimental Teaching Center for Life Sciences and Biotechnology, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, Zhejiang, China
| | - Zhiqiang Wei
- Medicinal Chemistry and Bioinformatics Center, Ocean University of China, Qingdao, Shandong, China
| | - Ray Luo
- Departments of Molecular Biology and Biochemistry, Chemical and Biomolecular Engineering, Materials Science and Engineering, and Biomedical Engineering, Graduate Program in Chemical and Materials Physics, University of California Irvine, Irvine, California, USA
| | - Jian Zhang
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Medicinal Bioinformatics Center, Shanghai Jiao-Tong University School of Medicine, Shanghai, Zhejiang, China
- School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan, China
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17
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Chang L, Perez A. Ranking Peptide Binders by Affinity with AlphaFold. Angew Chem Int Ed Engl 2023; 62:e202213362. [PMID: 36542066 DOI: 10.1002/anie.202213362] [Citation(s) in RCA: 35] [Impact Index Per Article: 17.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2022] [Revised: 12/20/2022] [Accepted: 12/21/2022] [Indexed: 12/24/2022]
Abstract
AlphaFold has revolutionized structural biology by predicting highly accurate structures of proteins and their complexes with peptides and other proteins. However, for protein-peptide systems, we are also interested in identifying the highest affinity binder among a set of candidate peptides. We present a novel competitive binding assay using AlphaFold to predict structures of the receptor in the presence of two peptides. For systems in which the individual structures of the peptides are well predicted, the assay captures the higher affinity binder in the bound state, and the other peptide in the unbound form with statistical significance. We test the application on six protein receptors for which we have experimental binding affinities to several peptides. We find that the assay is best suited for identifying medium to strong peptide binders that adopt stable secondary structures upon binding.
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Affiliation(s)
- Liwei Chang
- Department of Chemistry, University of Florida, Gainesville, FL, USA.,Quantum Theory Project, University of Florida, Gainesville, FL, USA
| | - Alberto Perez
- Department of Chemistry, University of Florida, Gainesville, FL, USA.,Quantum Theory Project, University of Florida, Gainesville, FL, USA
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18
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Fenton M, Borcherds W, Chen L, Anbanandam A, Levy R, Chen J, Daughdrill G. The MDMX Acidic Domain Uses Allovalency to Bind Both p53 and MDMX. J Mol Biol 2022; 434:167844. [PMID: 36181774 PMCID: PMC9644833 DOI: 10.1016/j.jmb.2022.167844] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2022] [Revised: 09/05/2022] [Accepted: 09/22/2022] [Indexed: 01/10/2023]
Abstract
Autoinhibition of p53 binding to MDMX requires two short-linear motifs (SLiMs) containing adjacent tryptophan (WW) and tryptophan-phenylalanine (WF) residues. NMR spectroscopy was used to show the WW and WF motifs directly compete for the p53 binding site on MDMX and circular dichroism spectroscopy was used to show the WW motif becomes helical when it is bound to the p53 binding domain (p53BD) of MDMX. Binding studies using isothermal titration calorimetry showed the WW motif is a stronger inhibitor of p53 binding than the WF motif when they are both tethered to p53BD by the natural disordered linker. We also investigated how the WW and WF motifs interact with the DNA binding domain (DBD) of p53. Both motifs bind independently to similar sites on DBD that overlap the DNA binding site. Taken together our work defines a model for complex formation between MDMX and p53 where a pair of disordered SLiMs bind overlapping sites on both proteins.
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Affiliation(s)
- Malissa Fenton
- Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL 33620, United States
| | - Wade Borcherds
- Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL 33620, United States
| | - Lihong Chen
- Molecular Oncology Department, Moffitt Cancer Center, Tampa, FL 33612, United States
| | - Asokan Anbanandam
- Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL 33620, United States
| | - Robin Levy
- Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL 33620, United States
| | - Jiandong Chen
- Molecular Oncology Department, Moffitt Cancer Center, Tampa, FL 33612, United States
| | - Gary Daughdrill
- Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL 33620, United States.
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19
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Sedzro DM, Idris MO, Durojaye OA, Yekeen AA, Fadahunsi AA, Alakanse SO. Identifying Potential p53‐MDM2 Interaction Antagonists: An Integrated Approach of Pharmacophore‐Based Virtual Screening, Interaction Fingerprinting, MD Simulation and DFT Studies. ChemistrySelect 2022. [DOI: 10.1002/slct.202202380] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Affiliation(s)
- Divine Mensah Sedzro
- MOE Key Laboratory of Membraneless Organelle and Cellular Dynamics Hefei National Laboratory for Physical Sciences at the Microscale University of Science and Technology of China Hefei Anhui 230027 China
- School of Life Sciences University of Science and Technology of China Hefei Anhui 230027 China
| | - Mukhtar Oluwaseun Idris
- School of Life Sciences University of Science and Technology of China Hefei Anhui 230027 China
| | - Olanrewaju Ayodeji Durojaye
- MOE Key Laboratory of Membraneless Organelle and Cellular Dynamics Hefei National Laboratory for Physical Sciences at the Microscale University of Science and Technology of China Hefei Anhui 230027 China
- School of Life Sciences University of Science and Technology of China Hefei Anhui 230027 China
- Department of Chemical Sciences Coal City University, Emene Enugu State Nigeria
- ACAII BIOHEALTH LTD, Ikotun Lagos State Nigeria
| | - Abeeb Abiodun Yekeen
- School of Life Sciences University of Science and Technology of China Hefei Anhui 230027 China
| | - Adeola Abraham Fadahunsi
- Graduate School of Biomedical Engineering (GSBSE) University of Maine Orono ME 04469 USA
- Department of Oncology the First Affiliated Hospital of USTC Division of Life Sciences and Medicine University of Science and Technology of China Hefei Anhui 230027 China
- School of Information Science and Technology University of Science and Technology of China Hefei Anhui 230027 China
| | - Suleiman Oluwaseun Alakanse
- School of Life Sciences University of Science and Technology of China Hefei Anhui 230027 China
- Department of Biochemistry Faculty of Life Sciences University of Ilorin Ilorin Kwara State Nigeria
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20
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Chatterjee C, Singh SK. Peptide and protein chemistry approaches to study the tumor suppressor protein p53. Org Biomol Chem 2022; 20:5500-5509. [PMID: 35786742 PMCID: PMC10112546 DOI: 10.1039/d2ob00902a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The tumor suppressor and master gene regulator protein p53 has been the subject of intense investigation for several decades due to its mutation in about half of all human cancers. However, mechanistic studies of p53 in cells are complicated by its many dynamic binding partners and heterogeneous post-translational modifications. The design of therapeutics that rescue p53 functions in cells requires a mechanistic understanding of its protein-protein interactions in specific protein complexes and identifying changes in p53 activity by diverse post-translational modifications. This review highlights the important roles that peptide and protein chemistry have played in biophysical and biochemical studies aimed at elucidating p53 regulation by several key binding partners. The design of various peptide inhibitors that rescue p53 function in cells and new opportunities in targeting p53-protein interactions are discussed. In addition, the review highlights the importance of a protein semisynthesis approach to comprehend the role of site-specific PTMs in p53 regulation.
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Affiliation(s)
- Champak Chatterjee
- Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
| | - Sumeet K Singh
- Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
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21
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Kosugi T, Ohue M. Solubility-Aware Protein Binding Peptide Design Using AlphaFold. Biomedicines 2022; 10:1626. [PMID: 35884931 PMCID: PMC9312799 DOI: 10.3390/biomedicines10071626] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2022] [Revised: 07/02/2022] [Accepted: 07/05/2022] [Indexed: 01/02/2023] Open
Abstract
New protein-protein interactions (PPIs) are identified, but PPIs have different physicochemical properties compared with conventional targets, making it difficult to use small molecules. Peptides offer a new modality to target PPIs, but designing appropriate peptide sequences by computation is challenging. Recently, AlphaFold and RoseTTAFold have made it possible to predict protein structures from amino acid sequences with ultra-high accuracy, enabling de novo protein design. We designed peptides likely to have PPI as the target protein using the "binder hallucination" protocol of AfDesign, a de novo protein design method using AlphaFold. However, the solubility of the peptides tended to be low. Therefore, we designed a solubility loss function using solubility indices for amino acids and developed a solubility-aware AfDesign binder hallucination protocol. The peptide solubility in sequences designed using the new protocol increased with the weight of the solubility loss function; moreover, they captured the characteristics of the solubility indices. Moreover, the new protocol sequences tended to have higher affinity than random or single residue substitution sequences when evaluated by docking binding affinity. Our approach shows that it is possible to design peptide sequences that can bind to the interface of PPI while controlling solubility.
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Affiliation(s)
| | - Masahito Ohue
- Department of Computer Science, School of Computing, Tokyo Institute of Technology, G3-56-4259 Nagatsutacho, Midori-ku, Yokohama City 226-8501, Kanagawa, Japan;
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22
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Hurwitz N, Zaidman D, Wolfson HJ. Pep–Whisperer: Inhibitory peptide design. Proteins 2022; 90:1886-1895. [DOI: 10.1002/prot.26384] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2021] [Revised: 04/07/2022] [Accepted: 04/29/2022] [Indexed: 11/07/2022]
Affiliation(s)
- Naama Hurwitz
- Blavatnik School of Computer Science Tel Aviv University Tel Aviv Israel
| | - Daniel Zaidman
- Department of Organic Chemistry Weizmann Institute of Science Rehovot Israel
| | - Haim J. Wolfson
- Blavatnik School of Computer Science Tel Aviv University Tel Aviv Israel
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23
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Chen T, Sun T, Bian Y, Pei Y, Feng F, Chi H, Li Y, Tang X, Sang S, Du C, Chen Y, Chen Y, Sun H. The Design and Optimization of Monomeric Multitarget Peptides for the Treatment of Multifactorial Diseases. J Med Chem 2022; 65:3685-3705. [DOI: 10.1021/acs.jmedchem.1c01456] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
- Tingkai Chen
- School of Pharmacy, China Pharmaceutical University, Nanjing 211198, People’s Republic of China
| | - Tianyu Sun
- School of Pharmacy, China Pharmaceutical University, Nanjing 211198, People’s Republic of China
| | - Yaoyao Bian
- College of Acupuncture and Massage, College of Regimen and Rehabilitation, Nanjing University of Chinese Medicine, Nanjing 210023, People’s Republic of China
| | - Yuqiong Pei
- School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, People’s Republic of China
| | - Feng Feng
- Food and Pharmaceutical Research Institute, Jiangsu Food and Pharmaceuticals Science College, Huaian 223003, People’s Republic of China
| | - Heng Chi
- Food and Pharmaceutical Research Institute, Jiangsu Food and Pharmaceuticals Science College, Huaian 223003, People’s Republic of China
| | - Yuan Li
- Department of Pharmaceutical Engineering, Jiangsu Food and Pharmaceuticals Science College, Huaian 223005, People’s Republic of China
| | - Xu Tang
- School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, People’s Republic of China
| | - Shenghu Sang
- School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, People’s Republic of China
| | - Chenxi Du
- School of Pharmacy, China Pharmaceutical University, Nanjing 211198, People’s Republic of China
| | - Ying Chen
- School of Pharmacy, China Pharmaceutical University, Nanjing 211198, People’s Republic of China
| | - Yao Chen
- School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, People’s Republic of China
| | - Haopeng Sun
- School of Pharmacy, China Pharmaceutical University, Nanjing 211198, People’s Republic of China
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24
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Sabale PM, Imiołek M, Raia P, Barluenga S, Winssinger N. Suprastapled Peptides: Hybridization-Enhanced Peptide Ligation and Enforced α-Helical Conformation for Affinity Selection of Combinatorial Libraries. J Am Chem Soc 2021; 143:18932-18940. [PMID: 34739233 DOI: 10.1021/jacs.1c07013] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Stapled peptides with an enforced α-helical conformation have been shown to overcome major limitations in the development of short peptides targeting protein-protein interactions (PPIs). While the growing arsenal of methodologies to staple peptides facilitates their preparation, stapling methodologies are not broadly embraced in synthetic library screening. Herein, we report a strategy leveraged on hybridization of short PNA-peptide conjugates wherein nucleobase driven assembly facilitates ligation of peptide fragments and constrains the peptide's conformation into an α-helix. Using native chemical ligation, we show that a mixture of peptide fragments can be combinatorially ligated and used directly in affinity selection against a target of interest. This approach was exemplified with a focused library targeting the p-53/MDM2 interaction. One hundred peptides were obtained in a one-pot ligation reaction, selected by affinity against MDM2 immobilized on beads, and the best binders were identified by mass spectrometry.
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Affiliation(s)
- Pramod M Sabale
- Faculty of Science, NCCR Chemical Biology, University of Geneva, 30 Quai Ernest Ansermet, CH-1205 Geneva, Switzerland
| | - Mateusz Imiołek
- Faculty of Science, NCCR Chemical Biology, University of Geneva, 30 Quai Ernest Ansermet, CH-1205 Geneva, Switzerland
| | - Pierre Raia
- Faculty of Science, NCCR Chemical Biology, University of Geneva, 30 Quai Ernest Ansermet, CH-1205 Geneva, Switzerland
| | - Sofia Barluenga
- Faculty of Science, NCCR Chemical Biology, University of Geneva, 30 Quai Ernest Ansermet, CH-1205 Geneva, Switzerland
| | - Nicolas Winssinger
- Faculty of Science, NCCR Chemical Biology, University of Geneva, 30 Quai Ernest Ansermet, CH-1205 Geneva, Switzerland
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25
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Lian C, Li Y, Hou Z, Zhong W, Tian Y, Yin F, Li Z, Zhou D, Wang R. Proximity-induced amino-yne reaction for selective MDM4 conjugation via propargylated sulfonium. CHINESE CHEM LETT 2021. [DOI: 10.1016/j.cclet.2021.04.015] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
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26
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Li X, Gohain N, Chen S, Li Y, Zhao X, Li B, Tolbert WD, He W, Pazgier M, Hu H, Lu W. Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression. Acta Pharm Sin B 2021; 11:2655-2669. [PMID: 34589387 PMCID: PMC8463443 DOI: 10.1016/j.apsb.2021.06.010] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2021] [Revised: 05/28/2021] [Accepted: 06/03/2021] [Indexed: 12/13/2022] Open
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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Affiliation(s)
- Xiang Li
- School of Pharmacy, Second Military Medical University, Shanghai 200433, China
- Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
| | - Neelakshi Gohain
- Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
| | - Si Chen
- School of Medicine, Shanghai University, Shanghai 200444, China
| | - Yinghua Li
- Institute of Translational Medicine, Shanghai University, Shanghai 200444, China
| | - Xiaoyuan Zhao
- Institute of Translational Medicine, Shanghai University, Shanghai 200444, China
- State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
| | - Bo Li
- State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
| | - William D. Tolbert
- Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
| | - Wangxiao He
- Department of Talent Highland, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China
| | - Marzena Pazgier
- Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
- Corresponding authors. Tel./fax: +86 21 54237607 (Wuyuan Lu), +86 21 66131281 (Honggang Hu), +1 301 295 3291 (Marzena Pazgier).
| | - Honggang Hu
- Institute of Translational Medicine, Shanghai University, Shanghai 200444, China
- Corresponding authors. Tel./fax: +86 21 54237607 (Wuyuan Lu), +86 21 66131281 (Honggang Hu), +1 301 295 3291 (Marzena Pazgier).
| | - Wuyuan Lu
- 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
- Corresponding authors. Tel./fax: +86 21 54237607 (Wuyuan Lu), +86 21 66131281 (Honggang Hu), +1 301 295 3291 (Marzena Pazgier).
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27
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Zhang S, Lou J, Li Y, Zhou F, Yan Z, Lyu X, Zhao Y. Recent Progress and Clinical Development of Inhibitors that Block MDM4/p53 Protein-Protein Interactions. J Med Chem 2021; 64:10621-10640. [PMID: 34286973 DOI: 10.1021/acs.jmedchem.1c00940] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
MDM4 is a homologue of MDM2, serving cooperatively as the negative regulator of tumor suppressor p53. Under the shadow of MDM2 inhibitors, limited efforts had been put into the discovery of MDM4 modulators. Recent studies of the experimental drug ALRN-6924, a dual MDM4 and MDM2 inhibitor, suggest that concurrent inhibition of MDM4 and MDM2 might be beneficial over only MDM2 inhibition. In view of the present research progress, we summarized published inhibitors of MDM4/p53 interactions including both peptide-based compounds and small molecules. Cocrystal structures of ligand/MDM4 complexes have been examined, and their structural features were compiled and compared in order to show the molecular basis required for high MDM4 binding affinities. Representative examples of small-molecule MDM4 inhibitors were discussed, followed by clinical results of ALRN-6924, together, providing a consolidated reference for further development of MDM4 inhibitors, either dual or selective.
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Affiliation(s)
- Shiyan Zhang
- University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China.,State Key Laboratory of Drug Research and Small-Molecule Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
| | - Jianfeng Lou
- University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China.,State Key Laboratory of Drug Research and Small-Molecule Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
| | - Yafang Li
- Nano Science and Technology Institute, University of Science and Technology of China, Suzhou, Jiangsu 215123, China.,State Key Laboratory of Drug Research and Small-Molecule Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
| | - Feilong Zhou
- State Key Laboratory of Drug Research and Small-Molecule Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
| | - Ziqin Yan
- State Key Laboratory of Drug Research and Small-Molecule Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
| | - Xilin Lyu
- State Key Laboratory of Drug Research and Small-Molecule Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
| | - Yujun Zhao
- University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China.,State Key Laboratory of Drug Research and Small-Molecule Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China.,School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
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28
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Perez JJ, Perez RA, Perez A. Computational Modeling as a Tool to Investigate PPI: From Drug Design to Tissue Engineering. Front Mol Biosci 2021; 8:681617. [PMID: 34095231 PMCID: PMC8173110 DOI: 10.3389/fmolb.2021.681617] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2021] [Accepted: 05/05/2021] [Indexed: 12/13/2022] Open
Abstract
Protein-protein interactions (PPIs) mediate a large number of important regulatory pathways. Their modulation represents an important strategy for discovering novel therapeutic agents. However, the features of PPI binding surfaces make the use of structure-based drug discovery methods very challenging. Among the diverse approaches used in the literature to tackle the problem, linear peptides have demonstrated to be a suitable methodology to discover PPI disruptors. Unfortunately, the poor pharmacokinetic properties of linear peptides prevent their direct use as drugs. However, they can be used as models to design enzyme resistant analogs including, cyclic peptides, peptide surrogates or peptidomimetics. Small molecules have a narrower set of targets they can bind to, but the screening technology based on virtual docking is robust and well tested, adding to the computational tools used to disrupt PPI. We review computational approaches used to understand and modulate PPI and highlight applications in a few case studies involved in physiological processes such as cell growth, apoptosis and intercellular communication.
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Affiliation(s)
- Juan J Perez
- Department of Chemical Engineering, Universitat Politecnica de Catalunya, Barcelona, Spain
| | - Roman A Perez
- Bioengineering Institute of Technology, Universitat Internacional de Catalunya, Sant Cugat, Spain
| | - Alberto Perez
- The Quantum Theory Project, Department of Chemistry, University of Florida, Gainesville, FL, United States
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29
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Su A, Tabata Y, Aoki K, Sada A, Ohki R, Nagatoishi S, Tsumoto K, Wang S, Otani Y, Ohwada T. Elaboration of Non-naturally Occurring Helical Tripeptides as p53-MDM2/MDMX Interaction Inhibitors. Chem Pharm Bull (Tokyo) 2021; 69:681-692. [PMID: 33952867 DOI: 10.1248/cpb.c21-00238] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Protein-protein interactions (PPIs) are often mediated by helical, strand and/or coil secondary structures at the interface regions. We previously showed that non-naturally occurring, stable helical trimers of bicyclic β-amino acids (Abh) with all-trans amide bonds can block the p53-MDM2/MDMX α-helix-helix interaction, which plays a role in regulating p53 function. Here, we conducted docking and molecular dynamics calculations to guide the structural optimization of our reported compounds, focusing on modifications of the C-terminal/N-terminal residues. We confirmed that the modified peptides directly bind to MDM2 by means of thermal shift assay, isothermal titration calorimetry, and enzyme-linked immunosorbent assay (ELISA) experiments. Biological activity assay in human osteosarcoma cell line SJSA-1, which has wild-type p53 and amplification of the Mdm2 gene, indicated that these peptides are membrane-permeable p53-MDM2/MDMX interaction antagonists that can rescue p53 function in the cells.
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Affiliation(s)
- Aoze Su
- Graduate School of Pharmaceutical Sciences, The University of Tokyo
| | - Yuko Tabata
- Laboratory of Fundamental Oncology, National Cancer Center Research Institute
| | - Kiyono Aoki
- Laboratory of Fundamental Oncology, National Cancer Center Research Institute
| | - Akane Sada
- Laboratory of Fundamental Oncology, National Cancer Center Research Institute
| | - Rieko Ohki
- Laboratory of Fundamental Oncology, National Cancer Center Research Institute
| | | | - Kouhei Tsumoto
- The Institute of Medical Science, The University of Tokyo.,School of Engineering, The University of Tokyo
| | - Siyuan Wang
- Graduate School of Pharmaceutical Sciences, The University of Tokyo
| | - Yuko Otani
- Graduate School of Pharmaceutical Sciences, The University of Tokyo
| | - Tomohiko Ohwada
- Graduate School of Pharmaceutical Sciences, The University of Tokyo
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30
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Binding Ensembles of p53-MDM2 Peptide Inhibitors by Combining Bayesian Inference and Atomistic Simulations. Molecules 2021; 26:molecules26010198. [PMID: 33401765 PMCID: PMC7795311 DOI: 10.3390/molecules26010198] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2020] [Revised: 12/26/2020] [Accepted: 12/28/2020] [Indexed: 01/21/2023] Open
Abstract
Designing peptide inhibitors of the p53-MDM2 interaction against cancer is of wide interest. Computational modeling and virtual screening are a well established step in the rational design of small molecules. But they face challenges for binding flexible peptide molecules that fold upon binding. We look at the ability of five different peptides, three of which are intrinsically disordered, to bind to MDM2 with a new Bayesian inference approach (MELD × MD). The method is able to capture the folding upon binding mechanism and differentiate binding preferences between the five peptides. Processing the ensembles with statistical mechanics tools depicts the most likely bound conformations and hints at differences in the binding mechanism. Finally, the study shows the importance of capturing two driving forces to binding in this system: the ability of peptides to adopt bound conformations (ΔGconformation) and the interaction between interface residues (ΔGinteraction).
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31
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Caggiano C, Guida E, Todaro F, Bielli P, Mori M, Ghirga F, Quaglio D, Botta B, Moretti F, Grimaldi P, Rossi P, Jannini EA, Barchi M, Dolci S. Sempervirine inhibits RNA polymerase I transcription independently from p53 in tumor cells. Cell Death Discov 2020; 6:111. [PMID: 33298840 PMCID: PMC7595235 DOI: 10.1038/s41420-020-00345-4] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2020] [Accepted: 10/05/2020] [Indexed: 12/18/2022] Open
Abstract
In the search of small molecules that can target MDM2/p53 pathway in testicular germ cell tumors (TGCTs), we identified sempervirine (2,3,4,13-tetrahydro-1H-benz[g]indolo[2,3-a]quinolizin-6-ium), an alkaloid of Gelsemium sempervirens, that has been previously proposed as an inhibitor of MDM2 that targets p53-wildtype (wt) tumor cells. We found that sempervirine not only affects cell growth of p53-wt cancer cells, but it is also active in p53-mutated and p53-null cells by triggering p53-dependent and independent pathways without affecting non-transformed cells. To understand which mechanism/s could be activated both in p53-wt and -null cells, we found that sempervirine induced nucleolar remodeling and nucleolar stress by reducing protein stability of RPA194, the catalytic subunit of RNA polymerase I, that led to rRNA synthesis inhibition and to MDM2 block. As shown for other cancer cell models, MDM2 inhibition by nucleolar stress downregulated E2F1 protein levels both in p53-wt and p53-null TGCT cells with the concomitant upregulation of unphosphorylated pRb. Finally, we show that sempervirine is able to enter the nucleus and accumulates within the nucleolus where it binds rRNA without causing DNA damage. Our results identify semperivirine as a novel rRNA synthesis inhibitor and indicate this drug as a non-genotoxic anticancer small molecule.
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Affiliation(s)
- Cinzia Caggiano
- Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
| | - Eugenia Guida
- Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
| | - Federica Todaro
- Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
| | - Pamela Bielli
- Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
| | - Mattia Mori
- Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy
| | - Francesca Ghirga
- Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Rome, Italy
| | - Deborah Quaglio
- Department of Chemistry and Drug Technology, University of Rome La Sapienza, Rome, Italy
| | - Bruno Botta
- Department of Chemistry and Drug Technology, University of Rome La Sapienza, Rome, Italy
| | - Fabiola Moretti
- Institute of Cell Biology and Neurobiology, National Research Council of Italy (CNR), Rome, Italy
| | - Paola Grimaldi
- Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
| | - Pellegrino Rossi
- Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
| | | | - Marco Barchi
- Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy.
| | - Susanna Dolci
- Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy.
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32
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Sang P, Shi Y, Huang B, Xue S, Odom T, Cai J. Sulfono-γ-AApeptides as Helical Mimetics: Crystal Structures and Applications. Acc Chem Res 2020; 53:2425-2442. [PMID: 32940995 DOI: 10.1021/acs.accounts.0c00482] [Citation(s) in RCA: 41] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Foldamers have defined and predictable structures, improved resistance to proteolytic degradation, enhanced chemical diversity, and are versatile in their mimicry of biological molecules, making them promising candidates in biomedical and material applications. However, as natural macromolecules exhibit endless folding structures and functions, the exploration of the applications of foldamers remains crucial. As such, it is imperative to continue to discover unnatural foldameric architectures with new frameworks and molecular scaffolds. To this end, we recently developed a new class of peptidomimetics termed ″γ-AApeptides", oligomers of γ-substituted-N-acylated-N-aminoethyl amino acids, which are inspired by the chiral peptide nucleic acid backbone. To date γ-AApeptides have been shown to be resistant to proteolytic degradation and possess limitless potential to introduce chemically diverse functional groups, demonstrating promise in biomedical and material sciences. However, the structures of γ-AApeptides were initially unknown, rendering their rational design for the mimicry of a protein helical domain impossible in the beginning, which limited their potential development. To our delight, in the past few years, we have obtained a series of crystal structures of helical sulfono-γ-AApeptides, a subclass of γ-AApeptides. The single-crystal X-ray crystallography indicates that sulfono-γ-AApeptides fold into unprecedented and well-defined helices with unique helical parameters. On the basis of the well-established size, shape, and folding conformation, the design of sulfono-γ-AApeptide-based foldamers opens a new avenue for the development of alternative unnatural peptidomimetics for their potential applications in chemistry, biology, medicine, materials science, and so on.In this Account, we will outline our journey on sulfono-γ-AApeptides and their application as helical mimetics. We will first briefly introduce the design and synthetic strategy of sulfono-γ-AApeptides and then describe the crystal structures of helical sulfono-γ-AApeptides, including left-handed homogeneous sulfono-γ-AApeptides, right-handed 1:1 α/sulfono-γ-AA peptide hybrids, and right-handed 2:1 α/sulfono-γ-AA peptide hybrids. After that, we will illustrate the potential of helical sulfono-γ-AApeptides for biological applications such as the disruption of medicinally relevant protein-protein interactions (PPIs) of BCL9-β-catenin and p53-MDM2/MDMX as well as the mimicry of glucagon-like peptide 1 (GLP-1). In addition, we also exemplify their potential application in material science. We expect that this Account will shed light on the structure-based design and function of helical sulfono-γ-AApeptides, which can provide a new and alternative way to explore and generate novel foldamers with distinctive structural and functional properties.
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Affiliation(s)
- Peng Sang
- Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, Tampa, Florida 33620, United States
| | - Yan Shi
- Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, Tampa, Florida 33620, United States
| | - Bo Huang
- Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, Tampa, Florida 33620, United States
| | - Songyi Xue
- Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, Tampa, Florida 33620, United States
| | - Timothy Odom
- Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, Tampa, Florida 33620, United States
| | - Jianfeng Cai
- Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, Tampa, Florida 33620, United States
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33
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Structure-based designing efficient peptides based on p53 binding site residues to disrupt p53-MDM2/X interaction. Sci Rep 2020; 10:11449. [PMID: 32651397 PMCID: PMC7351717 DOI: 10.1038/s41598-020-67510-8] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2019] [Accepted: 06/09/2020] [Indexed: 12/23/2022] Open
Abstract
MDM2 and MDMX are known as overexpressed oncoproteins in several wild-type p53 cancer cells. The development of potent and dual antagonist peptides for p53-MDM2/X is a continuous challenge. In this study, we intended to investigate the pivotal structural points respecting the development of potent and dual inhibitors of MDM2/X. Correspondingly, MD simulation was performed on the experimentally confirmed peptides, comprising p53, pDI, pDIQ, PMI, and computationally screened mutant pDI and pDIQ. A follow-up secondary structure analysis showed the last three C-terminal residues provide the helicity reservation of peptides bound to MDM2/X. Furthermore, a delicate residue-residue examination displayed Met 11 and Ser12 in the modified peptides contribute significantly to dual inhibition of MDM2/X. Additionally, the peptides_MDM2/X complexes' ΔGbinding extracted by the umbrella sampling method were in agreement with the pattern of their experimental affinity values. It was concluded the screened pDI mutants were considered as suitable anti-MDM2/X peptides, and the data obtained could be exploited as the theoretical structure-based guide for rational peptide design. Taking account of results, the suitable C-terminal residues of p53-based peptides especially Met11, and Ser12, as well as higher umbrella sampling, generated ΔGbinding to MDM2/X would be considered as the positive structural markers of a promising anti-cancer agent.
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34
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35
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Identification of a Structural Determinant for Selective Targeting of HDMX. Structure 2020; 28:847-857.e5. [PMID: 32359398 DOI: 10.1016/j.str.2020.04.011] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2020] [Revised: 03/11/2020] [Accepted: 04/11/2020] [Indexed: 11/21/2022]
Abstract
p53 is a critical tumor-suppressor protein that guards the human genome against mutations by inducing cell-cycle arrest or apoptosis. Cancer cells subvert p53 by deletion, mutation, or overexpression of the negative regulators HDM2 and HDMX. For tumors that retain wild-type p53, its reactivation by pharmacologic targeting of HDM2 and/or HDMX represents a promising strategy, with a series of selective small-molecule HDM2 inhibitors and a dual HDM2/HDMX stapled-peptide inhibitor being evaluated in clinical trials. Because selective HDM2 targeting can cause hematologic toxicity, selective HDMX inhibitors could provide an alternative p53-reactivation strategy, but clinical candidates remain elusive. Here, we applied a mutation-scanning approach to uncover p53-based stapled peptides that are selective for HDMX. Crystal structures of stapled-peptide/HDMX complexes revealed a molecular mechanism for the observed specificity, which was validated by HDMX mutagenesis. Thus, we provide a blueprint for the development of HDMX-selective inhibitors to dissect and target the p53/HDMX interaction.
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36
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Sang P, Shi Y, Lu J, Chen L, Yang L, Borcherds W, Abdulkadir S, Li Q, Daughdrill G, Chen J, Cai J. α-Helix-Mimicking Sulfono-γ-AApeptide Inhibitors for p53-MDM2/MDMX Protein-Protein Interactions. J Med Chem 2020; 63:975-986. [PMID: 31971801 PMCID: PMC7025332 DOI: 10.1021/acs.jmedchem.9b00993] [Citation(s) in RCA: 40] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
The use of peptidomimetic scaffolds is a promising strategy for the inhibition of protein-protein interactions (PPIs). Herein, we demonstrate that sulfono-γ-AApeptides can be rationally designed to mimic the p53 α-helix and inhibit p53-MDM2 PPIs. The best inhibitor, with Kd and IC50 values of 26 nM and 0.891 μM toward MDM2, respectively, is among the most potent unnatural peptidomimetic inhibitors disrupting the p53-MDM2/MDMX interaction. Using fluorescence polarization assays, circular dichroism, nuclear magnetic resonance spectroscopy, and computational simulations, we demonstrate that sulfono-γ-AApeptides adopt helical structures resembling p53 and competitively inhibit the p53-MDM2 interaction by binding to the hydrophobic cleft of MDM2. Intriguingly, the stapled sulfono-γ-AApeptides showed promising cellular activity by enhancing p53 transcriptional activity and inducing expression of MDM2 and p21. Moreover, sulfono-γ-AApeptides exhibited remarkable resistance to proteolysis, augmenting their biological potential. Our results suggest that sulfono-γ-AApeptides are a new class of unnatural helical foldamers that disrupt PPIs.
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Affiliation(s)
- Peng Sang
- Department of Chemistry , University of South Florida , 4202 E. Fowler Avenue , Tampa , Florida 33620 , United States
| | - Yan Shi
- Department of Chemistry , University of South Florida , 4202 E. Fowler Avenue , Tampa , Florida 33620 , United States
| | - Junhao Lu
- Department of Molecular Oncology , H. Lee Moffitt Cancer Center and Research Institute , 12902 Magnolia Drive , Tampa , Florida 33612 , United States
| | - Lihong Chen
- Department of Molecular Oncology , H. Lee Moffitt Cancer Center and Research Institute , 12902 Magnolia Drive , Tampa , Florida 33612 , United States
| | - Leixiang Yang
- Department of Molecular Oncology , H. Lee Moffitt Cancer Center and Research Institute , 12902 Magnolia Drive , Tampa , Florida 33612 , United States
| | - Wade Borcherds
- Department of Cell Biology, Microbiology and Molecular Biology , University of South Florida , Tampa , Florida 33620 , United States
| | - Sami Abdulkadir
- Department of Chemistry , University of South Florida , 4202 E. Fowler Avenue , Tampa , Florida 33620 , United States
| | - Qi Li
- Department of Medical Oncology , Shuguang Hospital, Shanghai University of Traditional Chinese Medicine , Shanghai 201203 , China
| | - Gary Daughdrill
- Department of Cell Biology, Microbiology and Molecular Biology , University of South Florida , Tampa , Florida 33620 , United States
| | - Jiandong Chen
- Department of Molecular Oncology , H. Lee Moffitt Cancer Center and Research Institute , 12902 Magnolia Drive , Tampa , Florida 33612 , United States
| | - Jianfeng Cai
- Department of Chemistry , University of South Florida , 4202 E. Fowler Avenue , Tampa , Florida 33620 , United States
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37
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Rasafar N, Barzegar A, Mehdizadeh Aghdam E. Design and development of high affinity dual anticancer peptide-inhibitors against p53-MDM2/X interaction. Life Sci 2020; 245:117358. [PMID: 32001262 DOI: 10.1016/j.lfs.2020.117358] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2019] [Revised: 01/22/2020] [Accepted: 01/24/2020] [Indexed: 12/16/2022]
Abstract
AIMS Inhibition of P53-MDM2/X interaction is known as an effective cancer therapy strategy. In this regard, pDI peptide was introduced previously with the potential of targeting MDM2. In this research, the large-scale peptide mutation screening was used to achieve the best sequence of pDI with the highest affinity for inhibition activity against MDM2/X. MAIN METHODS Three mutant peptides of pDI as dual inhibitor peptides including single mutations of pDIm/4W, pDIm/11M and double mutations of pDIdm/4W11M were presented with the high affinities to inhibit both MDM2/X. The selected mutants were then evaluated comprehensively to confirm their ability as potent MDM2/X inhibitors, using a theoretical simulation approach. KEY FINDINGS MD simulations analyses confirmed their dual inhibition potential against both MDM2/X interactions with p53 protein. The developed pDIm and mainly pDIdm peptides showed stable conformations over the simulation time with conserved secondary structure and effective interaction with MDM2/X by physical binding such as hydrogen bonding. Besides, umbrella sampling free energy calculation indicated higher binding energy, ΔGbinding, of pDIm-MDM2/X and pDIdm-MDM2/X compared to pDI-MDM2/X. SIGNIFICANCE The optimized and improved mutant pDI, pDIdm, with more effective ΔGbinding values of -30 and -25 kcal/mol to MDMX and MDM2, respectively, is recommended as a promising anticancer agent and suitable candidate for experimental evaluations.
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Affiliation(s)
- Nasim Rasafar
- Research Institute of Bioscience and Biotechnology, University of Tabriz, Tabriz, Iran
| | - Abolfazl Barzegar
- Research Institute of Bioscience and Biotechnology, University of Tabriz, Tabriz, Iran.
| | - Elnaz Mehdizadeh Aghdam
- Molecular Medicine Research Center, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
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38
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Sharma K, Strizhak AV, Fowler E, Xu W, Chappell B, Sore HF, Galloway WRJD, Grayson MN, Lau YH, Itzhaki LS, Spring DR. Functionalized Double Strain-Promoted Stapled Peptides for Inhibiting the p53-MDM2 Interaction. ACS OMEGA 2020; 5:1157-1169. [PMID: 31984273 PMCID: PMC6977200 DOI: 10.1021/acsomega.9b03459] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/17/2019] [Accepted: 12/24/2019] [Indexed: 06/10/2023]
Abstract
The Sondheimer dialkyne reagent has previously been employed in strain-promoted double-click cycloadditions with bis-azide peptides to generate stapled peptide inhibitors of protein-protein interactions. The substituted variants of the Sondheimer dialkyne can be used to generate functionalized stapled peptide inhibitors with improved biological properties; however, this remains a relatively underdeveloped field. Herein, we report the synthesis of new substituted variants of Sondheimer dialkyne and their application in the stapling of p53-based diazido peptides to generate potent stapled peptide-based inhibitors of the oncogenic p53-MDM2 interaction. The functionalized stapled peptide formed from a meta-fluoro-substituted Sondheimer dialkyne was found to be the most potent inhibitor. Furthermore, through experimental studies and density functional theory calculations, we investigated the impact of the substituent on the strain-promoted double-click reactivity of Sondheimer dialkyne.
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Affiliation(s)
- Krishna Sharma
- Department
of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
| | - Alexander V. Strizhak
- Department
of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
| | - Elaine Fowler
- Department
of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
| | - Wenshu Xu
- Department
of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K.
| | - Ben Chappell
- Department
of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
| | - Hannah F. Sore
- Department
of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
| | | | - Matthew N. Grayson
- Department
of Chemistry, University of Bath, Claverton Down, Bath, BA2 7AY, U.K.
| | - Yu Heng Lau
- School
of Chemistry, The University of Sydney, Eastern Avenue, Sydney, New South Wales 2006, Australia
| | - Laura S. Itzhaki
- Department
of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K.
| | - David R. Spring
- Department
of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
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39
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Navaratna T, Atangcho L, Mahajan M, Subramanian V, Case M, Min A, Tresnak D, Thurber GM. Directed Evolution Using Stabilized Bacterial Peptide Display. J Am Chem Soc 2020; 142:1882-1894. [PMID: 31880439 DOI: 10.1021/jacs.9b10716] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Chemically stabilized peptides have attracted intense interest by academics and pharmaceutical companies due to their potential to hit currently "undruggable" targets. However, engineering an optimal sequence, stabilizing linker location, and physicochemical properties is a slow and arduous process. By pairing non-natural amino acid incorporation and cell surface click chemistry in bacteria with high-throughput sorting, we developed a method to quantitatively select high affinity ligands and applied the Stabilized Peptide Evolution by E. coli Display technique to develop disrupters of the therapeutically relevant MDM2-p53 interface. Through in situ stabilization on the bacterial surface, we demonstrate rapid isolation of stabilized peptides with improved affinity and novel structures. Several peptides evolved a second loop including one sequence (Kd = 1.8 nM) containing an i, i+4 disulfide bond. NMR structural determination indicated a bent helix in solution and bound to MDM2. The bicyclic peptide had improved protease stability, and we demonstrated that protease resistance could be measured both on the bacterial surface and in solution, enabling the method to test and/or screen for additional drug-like properties critical for biologically active compounds.
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Affiliation(s)
- Tejas Navaratna
- Department of Chemical Engineering , University of Michigan , Ann Arbor , Michigan 48109 , United States
| | - Lydia Atangcho
- Department of Chemical Engineering , University of Michigan , Ann Arbor , Michigan 48109 , United States
| | - Mukesh Mahajan
- Department of Chemical Engineering , University of Michigan , Ann Arbor , Michigan 48109 , United States
| | | | - Marshall Case
- Department of Chemical Engineering , University of Michigan , Ann Arbor , Michigan 48109 , United States
| | - Andrew Min
- Department of Chemical Engineering , University of Michigan , Ann Arbor , Michigan 48109 , United States
| | - Daniel Tresnak
- Department of Chemical Engineering , University of Michigan , Ann Arbor , Michigan 48109 , United States
| | - Greg M Thurber
- Department of Chemical Engineering , University of Michigan , Ann Arbor , Michigan 48109 , United States.,Department of Biomedical Engineering , University of Michigan , Ann Arbor , Michigan 48109 , United States
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40
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Sharma K, Strizhak AV, Fowler E, Wang X, Xu W, Hatt Jensen C, Wu Y, Sore HF, Lau YH, Hyvönen M, Itzhaki LS, Spring DR. Water-soluble, stable and azide-reactive strained dialkynes for biocompatible double strain-promoted click chemistry. Org Biomol Chem 2020; 17:8014-8018. [PMID: 31418442 DOI: 10.1039/c9ob01745c] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
The Sondheimer dialkyne is extensively used in double strain-promoted azide-alkyne cycloadditions. This reagent suffers with poor water-solubility and rapidly decomposes in aqueous solutions. This intrinsically limits its application in biological systems, and no effective solutions are currently available. Herein, we report the development of novel highly water-soluble, stable, and azide-reactive strained dialkyne reagents. To demonstrate their extensive utility, we applied our novel dialkynes to a double strain-promoted macrocyclisation strategy to generate functionalised p53-based stapled peptides for inhibiting the oncogenic p53-MDM2 interaction. These functionalised stapled peptides bind MDM2 with low nanomolar affinity and show p53 activation in a cellular environment. Overall, our highly soluble, stable and azide-reactive dialkynes offer significant advantages over the currently used Sondheimer dialkyne, and could be utilised for numerous biological applications.
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Affiliation(s)
- Krishna Sharma
- Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK
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41
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Diller DJ, Swanson J, Bayden AS, Brown CJ, Thean D, Lane DP, Partridge AW, Sawyer TK, Audie J. Rigorous Computational and Experimental Investigations on MDM2/MDMX-Targeted Linear and Macrocyclic Peptides. Molecules 2019; 24:E4586. [PMID: 31847417 PMCID: PMC6943714 DOI: 10.3390/molecules24244586] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2019] [Revised: 12/11/2019] [Accepted: 12/12/2019] [Indexed: 12/25/2022] Open
Abstract
There is interest in peptide drug design, especially for targeting intracellular protein-protein interactions. Therefore, the experimental validation of a computational platform for enabling peptide drug design is of interest. Here, we describe our peptide drug design platform (CMDInventus) and demonstrate its use in modeling and predicting the structural and binding aspects of diverse peptides that interact with oncology targets MDM2/MDMX in comparison to both retrospective (pre-prediction) and prospective (post-prediction) data. In the retrospective study, CMDInventus modules (CMDpeptide, CMDboltzmann, CMDescore and CMDyscore) were used to accurately reproduce structural and binding data across multiple MDM2/MDMX data sets. In the prospective study, CMDescore, CMDyscore and CMDboltzmann were used to accurately predict binding affinities for an Ala-scan of the stapled α-helical peptide ATSP-7041. Remarkably, CMDboltzmann was used to accurately predict the results of a novel D-amino acid scan of ATSP-7041. Our investigations rigorously validate CMDInventus and support its utility for enabling peptide drug design.
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Affiliation(s)
- David J. Diller
- CMDBioscience, 5 Park Avenue, New Haven, CT 06511, USA; (D.J.D.); (J.S.); (A.S.B.)
- Venenum BioDesign, LLC, 8 Black Forest Road, Hamilton, NJ 08691, USA
| | - Jon Swanson
- CMDBioscience, 5 Park Avenue, New Haven, CT 06511, USA; (D.J.D.); (J.S.); (A.S.B.)
- ChemModeling, LLC, Suite 101, 500 Huber Park Ct, Weldon Spring, MO 63304, USA
| | - Alexander S. Bayden
- CMDBioscience, 5 Park Avenue, New Haven, CT 06511, USA; (D.J.D.); (J.S.); (A.S.B.)
- Kleo Pharmaceuticals, 25 Science Park, Ste 235, New Haven, CT 06511, USA
| | - Chris J. Brown
- A*STAR, p53 Laboratory, Singapore 138648, Singapore; (C.J.B.); (D.T.); (D.P.L.)
| | - Dawn Thean
- A*STAR, p53 Laboratory, Singapore 138648, Singapore; (C.J.B.); (D.T.); (D.P.L.)
| | - David P. Lane
- A*STAR, p53 Laboratory, Singapore 138648, Singapore; (C.J.B.); (D.T.); (D.P.L.)
| | - Anthony W. Partridge
- MSD International GmbH, Singapore 138665, Singapore;
- Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA
| | - Tomi K. Sawyer
- Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA
| | - Joseph Audie
- CMDBioscience, 5 Park Avenue, New Haven, CT 06511, USA; (D.J.D.); (J.S.); (A.S.B.)
- College of Arts and Sciences, Department of Chemistry, Sacred Heart University, 5151 Park Avenue, Fairfield, CT 06825, USA
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42
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Shi S, Sui K, Liu W, Lei Y, Zhang S, Zhang Q. Revealing binding selectivity of ligands toward murine double minute 2 and murine double minute X based on molecular dynamics simulations and binding free energy calculations. J Biomol Struct Dyn 2019; 38:5081-5094. [PMID: 31755361 DOI: 10.1080/07391102.2019.1695671] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
It is well known that the interactions of p53 with murine double minute 2 and murine double minute X, namely MDM2 and MDMX, have been significant targets of efficient anti-cancer drug design. In this study, molecular dynamics (MD) simulations, principal component (PC) analysis and binding free energy calculations are combined to recognize binding selectivity of three ligands to MDM2 and MDMX. The binding free energies were estimated by using molecular mechanics generalized Born surface area (MM-GBSA) method and the obtained results display that the increase in the binding enthalpy of three ligands to MDM2 relative to MDMX mainly drives the binding selectivity of them toward MDM2 and MDMX. The information obtained from PC analysis shows that the associations of ligands exert important impacts on internal dynamics of MDM2 and MDMX. Meanwhile, the calculations of residue-based free energy decomposition not only identify the hot interaction spots of ligands with MDM2 and MDMX, but also show the residues (L54, M53), (Y67, Y66), (V93, V92), (H96, P95), (I99, I98) and (Y100, Y99) in (MDM2, MDMX) are responsible for most contributions to the binding selectivity of three ligands toward MDM2 and MDMX. It is believed that this work can provide useful information for design of highly selective and dual inhibitors targeting MDM2 and MDMX.Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Shuhua Shi
- School of Science, Shandong Jianzhu University, Jinan, China
| | - Kai Sui
- School of Science, Shandong Jianzhu University, Jinan, China
| | - Weizhe Liu
- School of Science, Shandong Jianzhu University, Jinan, China
| | - Yanzi Lei
- School of Science, Shandong Jianzhu University, Jinan, China
| | - Shaolong Zhang
- College of Physics and Electronics, Shandong Normal University, Jinan, China
| | - Qinggang Zhang
- College of Physics and Electronics, Shandong Normal University, Jinan, China
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43
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Dougherty PG, Wen J, Pan X, Koley A, Ren JG, Sahni A, Basu R, Salim H, Appiah Kubi G, Qian Z, Pei D. Enhancing the Cell Permeability of Stapled Peptides with a Cyclic Cell-Penetrating Peptide. J Med Chem 2019; 62:10098-10107. [PMID: 31657556 DOI: 10.1021/acs.jmedchem.9b00456] [Citation(s) in RCA: 55] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Stapled peptides recapitulate the binding affinity and specificity of α-helices in proteins, resist proteolytic degradation, and may provide a novel modality against challenging drug targets such as protein-protein interactions. However, most of the stapled peptides have limited cell permeability or are impermeable to the cell membrane. We show herein that stapled peptides can be rendered highly cell-permeable by conjugating a cyclic cell-penetrating peptide to their N-terminus, C-terminus, or stapling unit. Application of this strategy to two previously reported membrane-impermeable peptidyl inhibitors against the MDM2/p53 and β-catenin/TCF interactions resulted in the generation of potent proof-of-concept antiproliferative agents against key therapeutic targets.
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Affiliation(s)
- Patrick G Dougherty
- Department of Chemistry and Biochemistry , The Ohio State University , Columbus , Ohio 43210 , United States.,Entrada Therapeutics Inc. , 50 Northern Avenue , Boston , Massachusetts 02210 , United States
| | - Jin Wen
- Department of Chemistry and Biochemistry , The Ohio State University , Columbus , Ohio 43210 , United States
| | - Xiaoyan Pan
- Department of Chemistry and Biochemistry , The Ohio State University , Columbus , Ohio 43210 , United States
| | - Amritendu Koley
- Department of Chemistry and Biochemistry , The Ohio State University , Columbus , Ohio 43210 , United States
| | - Jian-Guo Ren
- Entrada Therapeutics Inc. , 50 Northern Avenue , Boston , Massachusetts 02210 , United States
| | - Ashweta Sahni
- Department of Chemistry and Biochemistry , The Ohio State University , Columbus , Ohio 43210 , United States
| | - Ruchira Basu
- Department of Chemistry and Biochemistry , The Ohio State University , Columbus , Ohio 43210 , United States
| | - Heba Salim
- Department of Chemistry and Biochemistry , The Ohio State University , Columbus , Ohio 43210 , United States
| | - George Appiah Kubi
- Department of Chemistry and Biochemistry , The Ohio State University , Columbus , Ohio 43210 , United States
| | - Ziqing Qian
- Department of Chemistry and Biochemistry , The Ohio State University , Columbus , Ohio 43210 , United States.,Entrada Therapeutics Inc. , 50 Northern Avenue , Boston , Massachusetts 02210 , United States
| | - Dehua Pei
- Department of Chemistry and Biochemistry , The Ohio State University , Columbus , Ohio 43210 , United States
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44
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Carvajal LA, Neriah DB, Senecal A, Benard L, Thiruthuvanathan V, Yatsenko T, Narayanagari SR, Wheat JC, Todorova TI, Mitchell K, Kenworthy C, Guerlavais V, Annis DA, Bartholdy B, Will B, Anampa JD, Mantzaris I, Aivado M, Singer RH, Coleman RA, Verma A, Steidl U. Dual inhibition of MDMX and MDM2 as a therapeutic strategy in leukemia. Sci Transl Med 2019; 10:10/436/eaao3003. [PMID: 29643228 DOI: 10.1126/scitranslmed.aao3003] [Citation(s) in RCA: 187] [Impact Index Per Article: 31.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2017] [Revised: 02/12/2018] [Accepted: 03/23/2018] [Indexed: 12/14/2022]
Abstract
The tumor suppressor p53 is often inactivated via its interaction with endogenous inhibitors mouse double minute 4 homolog (MDM4 or MDMX) or mouse double minute 2 homolog (MDM2), which are frequently overexpressed in patients with acute myeloid leukemia (AML) and other cancers. Pharmacological disruption of both of these interactions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in cancers with wild-type p53. Selective targeting of this pathway has thus far been limited to MDM2-only small-molecule inhibitors, which lack affinity for MDMX. We demonstrate that dual MDMX/MDM2 inhibition with a stapled α-helical peptide (ALRN-6924), which has recently entered phase I clinical testing, produces marked antileukemic effects. ALRN-6924 robustly activates p53-dependent transcription at the single-cell and single-molecule levels and exhibits biochemical and molecular biological on-target activity in leukemia cells in vitro and in vivo. Dual MDMX/MDM2 inhibition by ALRN-6924 inhibits cellular proliferation by inducing cell cycle arrest and apoptosis in cell lines and primary AML patient cells, including leukemic stem cell-enriched populations, and disrupts functional clonogenic and serial replating capacity. Furthermore, ALRN-6924 markedly improves survival in AML xenograft models. Our study provides mechanistic insight to support further testing of ALRN-6924 as a therapeutic approach in AML and other cancers with wild-type p53.
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Affiliation(s)
- Luis A Carvajal
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Daniela Ben Neriah
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Adrien Senecal
- Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Lumie Benard
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | | | - Tatyana Yatsenko
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Swathi-Rao Narayanagari
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.,Ruth L. and David S. Gottesman Institute for Stem Cell Research and Regenerative Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Justin C Wheat
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Tihomira I Todorova
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Kelly Mitchell
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Charles Kenworthy
- Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | | | | | - Boris Bartholdy
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Britta Will
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.,Ruth L. and David S. Gottesman Institute for Stem Cell Research and Regenerative Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.,Division of Hemato-Oncology, Department of Medicine (Oncology), Albert Einstein College of Medicine, Bronx, NY 10461, USA.,Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Jesus D Anampa
- Division of Hemato-Oncology, Department of Medicine (Oncology), Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Ioannis Mantzaris
- Division of Hemato-Oncology, Department of Medicine (Oncology), Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | | | - Robert H Singer
- Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Robert A Coleman
- Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Amit Verma
- Ruth L. and David S. Gottesman Institute for Stem Cell Research and Regenerative Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.,Division of Hemato-Oncology, Department of Medicine (Oncology), Albert Einstein College of Medicine, Bronx, NY 10461, USA.,Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Ulrich Steidl
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. .,Ruth L. and David S. Gottesman Institute for Stem Cell Research and Regenerative Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.,Division of Hemato-Oncology, Department of Medicine (Oncology), Albert Einstein College of Medicine, Bronx, NY 10461, USA.,Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA
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45
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Insights into the binding mechanisms of inhibitors of MDM2 based on molecular dynamics simulations and binding free energy calculations. Chem Phys Lett 2019. [DOI: 10.1016/j.cplett.2019.05.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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46
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Yang Y, Dong Z, Hu H, Peng J, Sheng Y, Tong Y, Yuan S, Li Z, Yang J, Wells T, Qu Y, Farrell NP, Liu Y. The facile and visualizable identification of broad-spectrum inhibitors of MDM2/p53 using co-expressed protein complexes. Analyst 2019; 144:3773-3781. [PMID: 31089613 DOI: 10.1039/c9an00350a] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
MDM2 is a well-known oncoprotein overexpressed in a variety of cancers, and the identification of inhibitors that disrupt the MDM2/p53 interaction is of great interest in anticancer drug development. Here we designed a platform for the facile and visualizable identification of inhibitors of MDM2 using co-expressed protein complexes of MDM2/p53. A hexahistidine-tag on MDM2 allows the binding of the protein complex to the Ni-NTA affinity resin, while the fluorescent protein fused to p53 enables the direct visualization of the interaction of p53 with MDM2. Hence, the inhibition of the MDM2/p53 interaction can be observed with the naked eye. The assay can be set up by directly loading cell lysate to the Ni-NTA affinity resin, and no chemical modification of proteins is needed. In addition to the qualitative analyses, the binding affinity of inhibitors to the MDM2 protein can be quantified by fluorescence titration. The applications of this system have been verified using small molecules and peptide inhibitors. As a proof of concept, we screened a small library using this platform. Interestingly, two types of novel inhibitors of MDM2, including cyclohexyl-triphenylamine derivatives and platinum complexes, were identified and their binding affinities were obtained. Quantitative measurements show that these new types of inhibitors demonstrate a high binding affinity (up to Kd = 51.9 nM) to MDM2.
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Affiliation(s)
- Yang Yang
- CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China.
| | - Zhiqiang Dong
- CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China.
| | - Hongze Hu
- CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China.
| | - Junhui Peng
- CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China.
| | - Yaping Sheng
- CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China.
| | - Yang Tong
- CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China.
| | - Siming Yuan
- CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China.
| | - Zigang Li
- School of Chemical Biology and Biotechnology, Shenzhen Graduate School of Peking University, Shenzhen, 518055, China
| | - Jiaxiang Yang
- Department of Chemistry, Anhui University, Hefei 230601, China
| | - Thomas Wells
- Department of Chemistry, Virginia Commonwealth University, 1001 West Main Street, Richmond, VA 23284-2006, USA
| | - Yun Qu
- Department of Chemistry, Virginia Commonwealth University, 1001 West Main Street, Richmond, VA 23284-2006, USA
| | - Nicholas P Farrell
- Department of Chemistry, Virginia Commonwealth University, 1001 West Main Street, Richmond, VA 23284-2006, USA
| | - Yangzhong Liu
- CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China.
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47
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In-solution enrichment identifies peptide inhibitors of protein-protein interactions. Nat Chem Biol 2019; 15:410-418. [PMID: 30886434 DOI: 10.1038/s41589-019-0245-2] [Citation(s) in RCA: 58] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2017] [Accepted: 02/13/2019] [Indexed: 12/14/2022]
Abstract
The use of competitive inhibitors to disrupt protein-protein interactions (PPIs) holds great promise for the treatment of disease. However, the discovery of high-affinity inhibitors can be a challenge. Here we report a platform for improving the affinity of peptide-based PPI inhibitors using non-canonical amino acids. The platform utilizes size exclusion-based enrichment from pools of synthetic peptides (1.5-4 kDa) and liquid chromatography-tandem mass spectrometry-based peptide sequencing to identify high-affinity binders to protein targets, without the need for 'reporter' or 'encoding' tags. Using this approach-which is inherently selective for high-affinity binders-we realized gains in affinity of up to ~100- or ~30-fold for binders to the oncogenic ubiquitin ligase MDM2 or HIV capsid protein C-terminal domain, which inhibit MDM2-p53 interaction or HIV capsid protein C-terminal domain dimerization, respectively. Subsequent macrocyclization of select MDM2 inhibitors rendered them cell permeable and cytotoxic toward cancer cells, demonstrating the utility of the identified compounds as functional PPI inhibitors.
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48
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Wang X, Magdziarz P, Enriquez E, Zhao W, Quan C, Darabedian N, Momand J, Zhou F. Surface plasmon resonance and cytotoxicity assays of drug efficacies predicted computationally to inhibit p53/MDM2 interaction. Anal Biochem 2019; 569:53-58. [PMID: 30721669 DOI: 10.1016/j.ab.2019.01.012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2018] [Revised: 10/17/2018] [Accepted: 01/31/2019] [Indexed: 01/10/2023]
Abstract
Docking on the p53-binding site of murine double minute 2 (MDM2) by small molecules restores p53's tumor-suppressor function. We previously assessed 3244 FDA-approved drugs via "computational conformer selection" for inhibiting MDM2 and p53 interaction. Here, we developed a surface plasmon resonance method to experimentally confirm the inhibitory effects of the known MDM2 inhibitor, nutlin-3a, and two drug candidates predicted by our computational method. This p53/MDM2 interaction displayed a dosage-dependent weakening when MDM2 is pre-mixed with drug candidates. The inhibition efficiency order is nutlin-3a (IC50 = 97 nM) > bepridil (206 nM) > azelastine (307 nM). Furthermore, we verified their anti-proliferation effects on SJSA-1 (wild-type p53 and overexpressed MDM2), SW480 (mutated p53), and SaOs-2 (deleted p53) cancer cell lines. The inhibitory order towards SJSA-1 cell line is nutlin-3a (IC50 = 0.8 μM) > bepridil (23 μM) > azelastine (25 μM). Our experimental results are in line with the computational prediction, and the higher IC50 values from the cell-based assays are due to the requirement of higher drug concentrations to penetrate cell membranes. The anti-proliferation effects of bepridil and azelastine on the cell lines with mutated and deleted p53 implied some p53-independent anti-proliferation effects.
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Affiliation(s)
- Xiaoying Wang
- College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, 410083, PR China
| | - Patrycja Magdziarz
- Department of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, CA, 90032, USA
| | - Ernest Enriquez
- Department of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, CA, 90032, USA
| | - Wang Zhao
- Department of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, CA, 90032, USA
| | - Chris Quan
- Department of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, CA, 90032, USA
| | - Narek Darabedian
- Department of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, CA, 90032, USA
| | - Jamil Momand
- Department of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, CA, 90032, USA.
| | - Feimeng Zhou
- Department of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, CA, 90032, USA.
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49
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Atangcho L, Navaratna T, Thurber GM. Hitting Undruggable Targets: Viewing Stabilized Peptide Development through the Lens of Quantitative Systems Pharmacology. Trends Biochem Sci 2019; 44:241-257. [PMID: 30563724 PMCID: PMC6661118 DOI: 10.1016/j.tibs.2018.11.008] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2018] [Revised: 08/31/2018] [Accepted: 11/22/2018] [Indexed: 01/10/2023]
Abstract
Stabilized peptide therapeutics have the potential to hit currently undruggable targets, dramatically expanding the druggable genome. However, major obstacles to their development include poor intracellular delivery, rapid degradation, low target affinity, and membrane toxicity. With the emergence of multiple stabilization techniques and screening technologies, the high efficacy of various bioactive peptides has been demonstrated in vitro, albeit with limited success in vivo. We discuss here the chemical and pharmacokinetic barriers to achieving in vivo efficacy, analyze the characteristics of FDA-approved peptide drugs, and propose a developmental tool that considers the molecular properties of stabilized peptides in a comprehensive and quantitative manner to achieve the necessary rates for in vivo delivery to the target, efficacy, and ultimately clinical translation.
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Affiliation(s)
- Lydia Atangcho
- Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA
| | - Tejas Navaratna
- Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA
| | - Greg M Thurber
- Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA.
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50
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Peptidomimetics: A Synthetic Tool for Inhibiting Protein–Protein Interactions in Cancer. Int J Pept Res Ther 2019. [DOI: 10.1007/s10989-019-09831-5] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
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