Study on the design, synthesis and activity of MDM2/MDMX anti-tumor stapled peptide PROTAC

Study on the design, synthesis and activity of MDM2/MDMX anti-tumor stapled peptide PROTAC | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Study on the design, synthesis and activity of MDM2/MDMX anti-tumor stapled peptide PROTAC Xiufei Liao, Mao Guo, Damin Hu, Chunli Su, Hongli Liao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6970001/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract PROTAC is a drug development technology that uses the Ubiquitin-Proteasome System (UPS) to degrade target proteins, and enhances the degradation ability of target proteins through E3 ubiquitin ligase, which can further enhance the anti-tumor effect of targeted drug molecules. In this study, a series of dual-target MDM2/MDMX stapled peptide PROTAC based on SM3-4 were designed and synthesized, and the stapled peptide PROTAC DSM3-2 and DSM3-5 screened in the study inhibited tumor cell growth in vitro at low µM concentrations. The results showed that the enhancement of stapled peptide activity was positively correlated with the increase of helicity, which provided an effective research basis for the dual-target anti-tumor stapled peptide PROTAC. Molecular docking experiments have shown that the binding peptide DSM3-2 can effectively bind to the target proteins MDM2 and MDMX to exert a dual targeting effect on tumor cells. Physical sciences/Chemistry/Medicinal chemistry/Drug discovery and development Physical sciences/Chemistry/Medicinal chemistry/Structure based drug design Physical sciences/Chemistry/Medicinal chemistry/Target identification Physical sciences/Chemistry/Medicinal chemistry/Target validation Stapled peptides PROTAC Anti-tumor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction PROTAC is a drug development technique that utilizes the UPS for the degradation of target proteins (Fig. 1 ). Structurally, PROTAC consist of three parts: an E3 ubiquitin ligase ligand and a target protein ligand, which are linked together by a specially designed "Linker" structure to form the active form of the triplet "PROTAC" [ 1 – 4 ] . In vivo, the target protein ligand of PROTAC binds to the target protein, and the E3 ubiquitin ligase ligand binds to the E3 ubiquitin ligase in the cell, thereby "pulling the target protein closer" to the E3 ubiquitin ligase through the linker and enabling the UPS system to degrade the target protein [ 5 – 6 ] . A batch of PROTAC bifunctional molecules, the ubiquitin-proteasome system, has been reported to recruit E3 ligases to target proteins, resulting in ubiquitination and protein degradation [ 7 – 8 ] . Based on the crystal structure simulation results, the research group designed and synthesized a series of staple peptides with PMI-M3 as the lead compound, and screened the most active staple peptide SM3-4 [ 9 ] for PROTAC modification. A series of staple peptide PROTACs dual-targeting MDM2/MDMX were synthesized by solid-phase synthesis, and the pure products were purified and lyophilized to determine the anti-tumor cell activity. Compared with the original staple peptide, the PROTAC-modified staple peptide can not only bind to the target protein, but also induce the degradation of the target protein, play a dual inhibitory effect on MDM2/MDMX, and bind the substrate region of E3 ubiquitin ligase in the cell to the ligand of E3 ubiquitin ligase, so that the target protein can be pulled closer to the E3 ubiquitin ligase through Linker, and the target protein can be degraded by the UPS system to further enhance the anti-tumor activity. 2 Experiment Section 2.1 Material: Rink amide Resin (0.3 mmol g -1 loading) was purchased from Tianjin Nankai Hecheng Science & Technology Co. Ltd(Tianjing, China). Fmoc-protected amino acids were purchased from Shanghai GL Biochem Ltd (Shanghai, China). 6-Aminocaoroic Acid(Ahx) were purchased from Shanghai GL Biochem Ltd (Shanghai, China).Phenol, DIC, oxyma TIPS, were obtained from adamas-beta (Shanghai, China). Grubbs Catalyst 1st Generation were purchased from sigma(America). DMF, DCM were purchased from Wohua Chemical Co. Ltd (Shanghai, China). Ethyl ether, NMP, TFA, piperidine and other common reagent were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). The cell U87 MG, U251 was obtained from Shanghai Cellular Institute of Chinese Academy of Sciences (Shanghai, China). 2.2 Method 2.2.1 Synthesis of the stapled peptides Given that this research builds upon the previous double-targeting stapled peptide MDM2/MDMX [9] , the synthesis of the stapled peptide PROTAC and its optimization can be carried out based on the prior research conducted by our group. The detailed synthesis method is presented below (Fig 2). Resin Swelling and Preparation: Rink amide Resin (0.66 g, with a loading capacity of 0.3 mmol g-1) was swollen in DCM (5 mL) for 20 minutes. Subsequently, it was treated with 20% piperidine in DMF (10 mL), followed by washing with DMF (5 times), DCM (5 times), and DMF (5 times) successively. First Amino Acid Coupling: Fmoc-AA-OH (1 mmol), oxyma (1 mmol), DIC (1 mmol), and NMP (6 mL) were combined and added to the resin. The coupling of the first amino acid was completed at 60˚C for 20 minutes. Iterative Deprotection and Coupling: The steps of deprotection, coupling, and washing were repeated. During the 2-hour coupling of S5, two equivalents of the unnatural amino acid were utilized. Fmoc-Ahx-OH (1 mmol), oxyma (1 mmol), DIC (1 mmol), and NMP (6 mL) were mixed and added to the resin, and the coupling was accomplished at 60˚C for 20 minutes. PROTAC Conjugation: PROTAC was linked in a manner similar to that of amino acids. Next, the Ac residue was introduced to the resin using a mixture of 6 mL pyridine and acetic anhydride (v/v = 1:1) at room temperature for 20 minutes. Two RCM reactions, each lasting 2 hours, were conducted in 1, 2-dichloroethane at room temperature using Grubbs’ first-generation catalyst at a concentration of 10 mmol/L. Cleavage and Precipitation: The resin was treated with a reagent composed of 87.5% TFA, 5% H 2 O, 5% phenol, and 2.5% TIPS for 4 hours at room temperature. 2.2.2 Preparative and analytical HPLC and mass spectrometry The stapled peptides PROTAC were purified via RP-HPLC. HPLC analysis indicated that the purity of the purified peptide exceeded 95%. The target peptides were purified with a flow rate of 15 mL/min by means of preparative HPLC (LC-1 SHIMADZU) and a YMC-Pack ODS-AQ column (250 mm×20 mm, I.D.S-10 μm, 12 nm), with monitoring at 214 nm. HR-Q-TOF-MS was carried out using an Agilent 6538 UHD Accurate Mass Q-TOF Mass Spectrometer. 2.2.3 CD spectroscopy The stapled peptides PROTAC could dissolve in PB with a pH value of 7.2 at a concentration range of 10 - 50 mmol/L. CD experiments were carried out using a Jasco J-715 spectropolarimeter at room temperature. A quartz cuvette having a path length of 1 mm was employed for collecting spectra (wavelength: 185 - 255 nm; step resolution: 0.0001 mm; speed: 1.2 µm/h; accumulations: 6; bandwidth: 0.0001 mm). After subtracting the background, the measured spectral data were transformed into a unified scale of molar ellipticity. Then, the curves were normalized using standard parameters, and the helical degree of the polypeptide was calculated. 2.2.4 Cytotoxicity assay Human glioma cell lines U87 MG and U251 were cultured in McCoy's 5A medium. This medium was supplemented with 10% heat-inactivated fetal calf serum (FCS) and 1% penicillin-streptomycin. The cells were maintained under conditions of 37˚C, 5% CO 2 , and complete humidification. A total of 3×10 3 cells per well were seeded into 96-well plates. These cells were then treated with various concentrations of polypeptides for 72 hours. Subsequently, the CCK 8 kit was added, and the absorbance at 450 nm was measured. The percentage of cell survival was computed according to the ratio of the absorbance value of the sample well to that of the reference well at 450 nm. 2.2.5 Molecular docking In order to determine the binding of the book binding peptide with MDM2 and MDMX, the book binding peptide PROTAC DSM3-2 was selected for molecular docking. Suppose the combination modes of DSM3-2 and MDM2 (PDB ID: 4LWV) as well as DSM3-2 and MDMX (PDB ID: 6Q9Y) are established. 3 Results and discussion 3.1 Structural characterization of targeted anti-tumor peptides PROTAC In this study, the stapled peptides PROTAC DSM3-1, DSM3-2, DSM3-3, DSM3-4, DSM3-5 and DSM3-6 of MDM2/MDMX (Table 1) were prepared following the solid-phase synthesis route. The structural characteristics of these splicing peptides were verified by mass spectrometry analysis, and their theoretical molecular weights were consistent with the actual results (Table 2). Due to steric-hinerance effect, even after repeated synthesis, DSM3-3 and DSM3-6 could not be obtained. Table 1 Stapled peptide PROTAC sequence Table 2 Structural characterization results of stapled peptides PROTAC Name Molecular formula Molecular Weight(g/mol) Ion(m/z) DSM3-1 C 154 H 227 N 31 O 36 S 3215.8960 [M+2H] 2+ =1608.8645；[M+3H] 3+ =1072.9131 DSM3-2 C 182 H 280 N 38 O 40 S 2 3727.6260 [M+3H] 3+ =1243.3591；[M+4H] 4+ =932.7721 DSM3-4 C 154 H 227 N 31 O 36 S 3215.8960 [M+2H] 2+ =1608.3724；[M+3H] 3+ =1072.5825 DSM3-5 C 182 H 280 N 38 O 40 S 2 3727.6260 [M+3H] 3+ =1243.3598；[M+4H] 4+ =932.7716 3.2 Circular dichroism characterization This study characterized the secondary structure of the synthetic peptide using circular dichroism spectroscopy and determined its helical degree. As shown in Figure 3, DSM3-1 did not show a significant helical degree; the helical degree of DSM3-2 was 73.56%; the helical degree of DSM3-4 was 6.22%; and the helical degree of DSM3-5 was 96.73%. 3.3 Stapled peptide PROTAC cytotoxicity In this study, tumor cells U87 MG, and U251 were tested for cytotoxicity of the synthesized peptides, and the results of the cell viability of the peptides were tested (Fig.4). The staple peptide IC 50 results were calculated (Table 3). Table 3 IC 50 value of stapled peptide PROTAC Name U87 MG(µM) U251(µM) DSM3-1 ～ ～ DSM3-2 20.27 12.09 DSM3-4 ～ ～ DSM3-5 18.23 10.31 Note: Tumor cells were treated with staple peptides for 2 or 7 days and tested for cytotoxicity using CCK-8 reagent. ～: No obvious activity. DSM3-1 and DSM3-4 showed no significant inhibitory effect on the proliferation of tumor cells U87 MG and U251 in vitro. However, DSM3-2 and DSM3-5 significantly inhibited the growth of tumor cells U87 MG and U251 at a concentration of 16 μM. 3.4 Molecular docking The molecular docking results show that in the interaction between the DSM3-2 and MDM2 pockets, the amino acid residues that interact are concentrated in the 47-69 interval of the protein and the 1-14 interval of the polypeptide (Figure 5A). It is notable that the connection of hydrogen bonds occurs in LYS 47-Thr3, GLY 54-LEU5, TYR 63-TRP 8, and GLU 65-Lys 14. Acceptor-Acceptor interaction occurred in HIS 69-TRP 8 (Figure 5B). In the interaction between DSM3-2 and MDMX pockets (Figure 6A), the connection of hydrogen bonds occurs at Tyr 100-Thr 3, Val 93-Lys 16, and Lys 94-Lys 16, Carbon interaction in Apo 96 - Tyr 7, Pi-Alkyl in VAL 50-Phe 4 (Figure 6B). 4 Conclusion In this study, amino resin was used as the solid-phase support material, DIC-Oxyme was used as the reaction coupling system, the target stapled peptide was obtained by Fmoc solid-phase synthesis, the (S) 2-amino-2-methyl-6-heptanoic acid (S 5 ) amino acid side chain group was synthesized by alkene cross-metathesis reaction, and Ahx was selected as the PROTAC linker to link the selected PROTAC fragment to the staple peptide, and four N-terminal acetylated MDM2/MDMX stapled peptide PROTAC were successfully obtained. Tumor cells U87 MG and U251 were selected for cell viability experiments, and the tumor suppressive activity of peptides DSM3-2 and DSM3-5 was particularly obvious according to the results of peptide cell activity experiments. According to the results of helicity and cell viability, it was found that the activities of DSM3-2 and DSM3-5 with high helicity were significantly higher than those with helicity and decreased to DSM3-1 and DSM3-4, and it was speculated that there was a positive correlation between helicity and cell viability. Molecular docking experiments have shown that the binding peptide DSM3-2 can effectively bind to the target proteins MDM2 and MDMX to exert a dual targeting effect on tumor cells. Declarations Conflict of interest There are no conflicts to declare. Ackonwledgements This work was supported by Fund of the President of Tarim University (TDZKSS202312 and TDZKSS202438), the Sichuan Provincial Central Leading Local Science and Technology Development Fund Project (2023ZYD0062). Author Contribution A. Xiufei Liao wrote the main manuscript and completed the main experiments.B. Damin Hu helped to complete the experiments.C. Mao Guo helped to complete the experiments.D. Chunli Su checked the manuscript.E. Hongli Liao helped to prepare figures 1-6. Data Availability Data is provided within the manuscript or supplementary information files References Li, X. & Song, Y. Proteolysis-targeting chimera (PROTAC) for targeted protein degradation and cancer therapy. J. Hematol. Oncol. 13 (1), 50 (2020). Zeng, S. et al. Proteolysis targeting chimera (PROTAC) in drug discovery paradigm: Recent progress and future challenges. Eur. J. Med. Chem. 210 , 112981 (2021). Jin, J. et al. The peptide PROTAC modality: a novel strategy for targeted protein ubiquitination. Theranostics 10 (22), 10141–10153 (2020). Qi, S. M. et al. PROTAC: An Effective Targeted Protein Degradation Strategy for Cancer Therapy. Front. Pharmacol. 12 , 692574 (2021). Weng, G. et al. PROTAC-DB: an online database of PROTACs. Nucleic Acids Res. 49 (D1), D1381–D1387 (2021). He, S. et al. Aptamer-PROTAC Conjugates (APCs) for Tumor-Specific Targeting in Breast Cancer. Angew Chem. Int. Ed. Engl. 60 (43), 23299–23305 (2021). Hines, J. et al. MDM2-Recruiting PROTAC Offers Superior, Synergistic Antiproliferative Activity via Simultaneous Degradation of BRD4 and Stabilization of p53. Cancer Res. 79 (1), 251–262 (2019). Liao, H. et al. PROTAC peptide induces durable β-catenin degradation and suppresses Wnt-dependent intestinal cancer. Cell. Discov . 6 , 35 (2020). Yang, J. & Liao, X. Study on the design, synthesis, and activity of anti-tumor staple peptides targeting MDM2/MDMX. Front. Chem. 7 , 12 (2024 June). Additional Declarations No competing interests reported. Supplementary Files rawdata2.zip Cite Share Download PDF Status: Published Journal Publication published 25 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 12 Aug, 2025 Reviews received at journal 10 Aug, 2025 Reviews received at journal 08 Aug, 2025 Reviewers agreed at journal 06 Aug, 2025 Reviewers agreed at journal 06 Aug, 2025 Reviewers agreed at journal 06 Aug, 2025 Reviewers invited by journal 06 Aug, 2025 Editor assigned by journal 06 Aug, 2025 Editor invited by journal 06 Aug, 2025 Submission checks completed at journal 31 Jul, 2025 First submitted to journal 31 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6970001","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":498304523,"identity":"5df8a371-e5d5-4519-9f0c-aab3eb59d5bd","order_by":0,"name":"Xiufei Liao","email":"","orcid":"","institution":"Tarim University, Xinjiang Uygur Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Xiufei","middleName":"","lastName":"Liao","suffix":""},{"id":498304524,"identity":"a20eccc6-b9c9-4ba8-9e3f-02bea2ee5457","order_by":1,"name":"Mao Guo","email":"","orcid":"","institution":"Tarim University, Xinjiang Uygur Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Mao","middleName":"","lastName":"Guo","suffix":""},{"id":498304525,"identity":"34ee1719-b989-42f9-a097-7aa54cd8de9a","order_by":2,"name":"Damin Hu","email":"","orcid":"","institution":"Tarim University, Xinjiang Uygur Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Damin","middleName":"","lastName":"Hu","suffix":""},{"id":498304526,"identity":"724be84c-9744-43b7-b8fc-7a9f9acf79f1","order_by":3,"name":"Chunli Su","email":"","orcid":"","institution":"Chengdu Medical College","correspondingAuthor":false,"prefix":"","firstName":"Chunli","middleName":"","lastName":"Su","suffix":""},{"id":498304527,"identity":"19409031-0ac6-4356-ba7e-3334080a18e0","order_by":4,"name":"Hongli Liao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACPmYGBmaGAhseefbGxgcfiNHCBtZikCZj2HO42XAGUVoYwFoO2zDcSG+T5iBKCzvzw8cFBsw8jDMfNkgzMNjJ6TYQdBibsfEMAzYedunEBuMChmRjswMEtTCYSfMY8PAwzk5sSJ7BcCBxG2Et7N+AWiR4GG4ebDjMQ5wWHpAtBjwMNxgbm4nVUmzMY5DAY9iT2Mw4w4AIv/DzH9/4mKfiv708+/HnPz5U2MkR1IIGDEhTPgpGwSgYBaMABwAAOZc1+OJmnwAAAAAASUVORK5CYII=","orcid":"","institution":"Chengdu Medical College","correspondingAuthor":true,"prefix":"","firstName":"Hongli","middleName":"","lastName":"Liao","suffix":""}],"badges":[],"createdAt":"2025-06-25 03:08:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6970001/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6970001/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-18026-6","type":"published","date":"2025-09-25T15:58:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88812291,"identity":"99320caf-322c-495e-ad89-a89eeff3abe3","added_by":"auto","created_at":"2025-08-11 15:43:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":97825,"visible":true,"origin":"","legend":"\u003cp\u003eStabilized α-helical structures of stapled peptides and their advantages\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6970001/v1/1085cd31c49d415fb324a25c.png"},{"id":88812294,"identity":"95bcb2ce-83ca-416c-9f07-1812c5db39b5","added_by":"auto","created_at":"2025-08-11 15:43:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":261556,"visible":true,"origin":"","legend":"\u003cp\u003eStapled peptide PROTAC synthesis route\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6970001/v1/fca1ffa32520858920549e64.png"},{"id":88812292,"identity":"8ead814b-c202-41cb-b509-708b50504f49","added_by":"auto","created_at":"2025-08-11 15:43:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63283,"visible":true,"origin":"","legend":"\u003cp\u003eCircular dichroism and degree of helicity of stapled peptides PROTAC\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6970001/v1/0c1553456eb48889b7e30dd7.png"},{"id":88812713,"identity":"e4b0dd36-ee8e-4b82-aef1-dbaefc2b356f","added_by":"auto","created_at":"2025-08-11 15:51:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":91379,"visible":true,"origin":"","legend":"\u003cp\u003ePeptide cell viability test results. Tumor cells were treated with staple peptides for 2 or 7 days and tested for cytotoxicity using CCK-8 reagent. Results are represented as mean±standard deviation(x̅±SD ), *: p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6970001/v1/70c3a644426663f7c6ecb29a.png"},{"id":88812717,"identity":"57363273-b3bb-42e0-9c45-e0fa205a8977","added_by":"auto","created_at":"2025-08-11 15:51:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":493098,"visible":true,"origin":"","legend":"\u003cp\u003eDSM3-2 was docked into the binding site of MDM2(PDB ID: 4LWV). The key residues of hydrogen bonds and polypeptides are indicated in red font.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6970001/v1/8bb62728b268c7ddf73cc7f3.png"},{"id":88813961,"identity":"d305319a-3148-472c-b47c-4a8c160e2a52","added_by":"auto","created_at":"2025-08-11 15:59:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":605277,"visible":true,"origin":"","legend":"\u003cp\u003eDSM3-2 was docked into the binding site of MDMX(PDB ID: 6Q9Y). The key residues of hydrogen bonds, Carbon and Pi-Alkyl are indicated in red font.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6970001/v1/291d85a2269ec2b0caddb5cd.png"},{"id":92430636,"identity":"45258ef5-fa4d-4ad0-85a3-a8a9c2ce0a58","added_by":"auto","created_at":"2025-09-29 16:07:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2196844,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6970001/v1/e649725f-4ee5-40a8-817d-0d883db4b70d.pdf"},{"id":88813962,"identity":"7ecf2d25-0405-4bc0-8dc0-6bfa06627bfa","added_by":"auto","created_at":"2025-08-11 15:59:28","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":11255305,"visible":true,"origin":"","legend":"","description":"","filename":"rawdata2.zip","url":"https://assets-eu.researchsquare.com/files/rs-6970001/v1/b68c82413d3f90fcb0b2e384.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Study on the design, synthesis and activity of MDM2/MDMX anti-tumor stapled peptide PROTAC","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003ePROTAC is a drug development technique that utilizes the UPS for the degradation of target proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Structurally, PROTAC consist of three parts: an E3 ubiquitin ligase ligand and a target protein ligand, which are linked together by a specially designed \"Linker\" structure to form the active form of the triplet \"PROTAC\" \u003csup\u003e[\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. In vivo, the target protein ligand of PROTAC binds to the target protein, and the E3 ubiquitin ligase ligand binds to the E3 ubiquitin ligase in the cell, thereby \"pulling the target protein closer\" to the E3 ubiquitin ligase through the linker and enabling the UPS system to degrade the target protein \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. A batch of PROTAC bifunctional molecules, the ubiquitin-proteasome system, has been reported to recruit E3 ligases to target proteins, resulting in ubiquitination and protein degradation \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBased on the crystal structure simulation results, the research group designed and synthesized a series of staple peptides with PMI-M3 as the lead compound, and screened the most active staple peptide SM3-4\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e for PROTAC modification. A series of staple peptide PROTACs dual-targeting MDM2/MDMX were synthesized by solid-phase synthesis, and the pure products were purified and lyophilized to determine the anti-tumor cell activity. Compared with the original staple peptide, the PROTAC-modified staple peptide can not only bind to the target protein, but also induce the degradation of the target protein, play a dual inhibitory effect on MDM2/MDMX, and bind the substrate region of E3 ubiquitin ligase in the cell to the ligand of E3 ubiquitin ligase, so that the target protein can be pulled closer to the E3 ubiquitin ligase through Linker, and the target protein can be degraded by the UPS system to further enhance the anti-tumor activity.\u003c/p\u003e"},{"header":"2 Experiment Section","content":"\u003cp\u003e\u003cstrong\u003e2.1 Material:\u003c/strong\u003e Rink amide Resin (0.3 mmol g\u003csup\u003e-1\u003c/sup\u003e loading) was purchased from Tianjin Nankai Hecheng Science \u0026amp; Technology Co. Ltd(Tianjing, China). Fmoc-protected amino acids were purchased from Shanghai GL Biochem Ltd (Shanghai, China). 6-Aminocaoroic Acid(Ahx) were purchased from Shanghai GL Biochem Ltd (Shanghai, China).Phenol, DIC, oxyma TIPS, were obtained from adamas-beta (Shanghai, China). Grubbs Catalyst 1st Generation were purchased from sigma(America). DMF, DCM were purchased from Wohua Chemical Co. Ltd (Shanghai, China). Ethyl ether, NMP, TFA, piperidine and other common reagent were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). The cell U87 MG, U251 was obtained from Shanghai Cellular Institute of Chinese Academy of Sciences (Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1 Synthesis of the stapled peptides\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven that this research builds upon the previous double-targeting stapled peptide MDM2/MDMX\u003csup\u003e[9]\u003c/sup\u003e, the synthesis of the stapled peptide PROTAC and its optimization can be carried out based on the prior research conducted by our group. The detailed synthesis method is presented below (Fig 2).\u003c/p\u003e\n\u003cp\u003eResin Swelling and Preparation: Rink amide Resin (0.66 g, with a loading capacity of 0.3 mmol g-1) was swollen in DCM (5 mL) for 20 minutes. Subsequently, it was treated with 20% piperidine in DMF (10 mL), followed by washing with DMF (5 times), DCM (5 times), and DMF (5 times) successively.\u003c/p\u003e\n\u003cp\u003eFirst Amino Acid Coupling: Fmoc-AA-OH (1 mmol), oxyma (1 mmol), DIC (1 mmol), and NMP (6 mL) were combined and added to the resin. The coupling of the first amino acid was completed at 60˚C for 20 minutes.\u003c/p\u003e\n\u003cp\u003eIterative Deprotection and Coupling: The steps of deprotection, coupling, and washing were repeated. During the 2-hour coupling of S5, two equivalents of the unnatural amino acid were utilized. Fmoc-Ahx-OH (1 mmol), oxyma (1 mmol), DIC (1 mmol), and NMP (6 mL) were mixed and added to the resin, and the coupling was accomplished at 60˚C for 20 minutes.\u003c/p\u003e\n\u003cp\u003ePROTAC Conjugation: PROTAC was linked in a manner similar to that of amino acids. Next, the Ac residue was introduced to the resin using a mixture of 6 mL pyridine and acetic anhydride (v/v = 1:1) at room temperature for 20 minutes. Two RCM reactions, each lasting 2 hours, were conducted in 1, 2-dichloroethane at room temperature using Grubbs\u0026rsquo; first-generation catalyst at a concentration of 10 mmol/L.\u003c/p\u003e\n\u003cp\u003eCleavage and Precipitation: The resin was treated with a reagent composed of 87.5% TFA, 5% H\u003csub\u003e2\u003c/sub\u003eO, 5% phenol, and 2.5% TIPS for 4 hours at room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2 Preparative and analytical HPLC and mass spectrometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe stapled peptides PROTAC were purified via RP-HPLC. HPLC analysis indicated that the purity of the purified peptide exceeded 95%. The target peptides were purified with a flow rate of 15 mL/min by means of preparative HPLC (LC-1 SHIMADZU) and a YMC-Pack ODS-AQ column (250 mm\u0026times;20 mm, I.D.S-10 \u0026mu;m, 12 nm), with monitoring at 214 nm. HR-Q-TOF-MS was carried out using an Agilent 6538 UHD Accurate Mass Q-TOF Mass Spectrometer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.3 CD spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe stapled peptides PROTAC could dissolve in PB with a pH value of 7.2 at a concentration range of 10 - 50 mmol/L. CD experiments were carried out using a Jasco J-715 spectropolarimeter at room temperature. A quartz cuvette having a path length of 1 mm was employed for collecting spectra (wavelength: 185 - 255 nm; step resolution: 0.0001 mm; speed: 1.2 \u0026micro;m/h; accumulations: 6; bandwidth: 0.0001 mm). After subtracting the background, the measured spectral data were transformed into a unified scale of molar ellipticity. Then, the curves were normalized using standard parameters, and the helical degree of the polypeptide was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.4 Cytotoxicity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman glioma cell lines U87 MG and U251 were cultured in McCoy\u0026apos;s 5A medium. This medium was supplemented with 10% heat-inactivated fetal calf serum (FCS) and 1% penicillin-streptomycin. The cells were maintained under conditions of 37˚C, 5% CO\u003csub\u003e2\u003c/sub\u003e, and complete humidification. A total of 3\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well were seeded into 96-well plates. These cells were then treated with various concentrations of polypeptides for 72 hours. Subsequently, the CCK\u003csub\u003e8\u003c/sub\u003e kit was added, and the absorbance at 450 nm was measured. The percentage of cell survival was computed according to the ratio of the absorbance value of the sample well to that of the reference well at 450 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.5 Molecular docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to determine the binding of the book binding peptide with MDM2 and MDMX, the book binding peptide PROTAC DSM3-2 was selected for molecular docking. Suppose the combination modes of DSM3-2 and MDM2 (PDB ID: 4LWV) as well as DSM3-2 and MDMX (PDB ID: 6Q9Y) are established.\u003c/p\u003e"},{"header":"3 Results and discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1 Structural characterization of targeted anti-tumor peptides PROTAC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, the stapled peptides PROTAC DSM3-1, DSM3-2, DSM3-3, DSM3-4, DSM3-5 and DSM3-6 of MDM2/MDMX (Table 1) were prepared following the solid-phase synthesis route. The structural characteristics of these splicing peptides were verified by mass spectrometry analysis, and their theoretical molecular weights were consistent with the actual results (Table 2). Due to steric-hinerance effect, even after repeated synthesis, DSM3-3 and DSM3-6 could not be obtained.\u003c/p\u003e\n\u003cp\u003eTable 1 Stapled peptide PROTAC sequence\u003c/p\u003e\n\u003cdiv\u003e\u003cimg 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\" style=\"width: 382px; height: 202.025px;\" width=\"382\" height=\"202.025\"\u003e\u003c/div\u003e\n\u003cp\u003eTable 2 Structural characterization results of stapled peptides PROTAC\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"622\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eName\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMolecular formula\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMolecular Weight(g/mol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 291px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIon(m/z)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eDSM3-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eC\u003csub\u003e154\u003c/sub\u003eH\u003csub\u003e227\u003c/sub\u003eN\u003csub\u003e31\u003c/sub\u003eO\u003csub\u003e36\u003c/sub\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003e3215.8960\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 291px;\"\u003e\n \u003cp\u003e[M+2H]\u003csup\u003e2+\u003c/sup\u003e=1608.8645；[M+3H]\u003csup\u003e3+\u003c/sup\u003e=1072.9131\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eDSM3-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eC\u003csub\u003e182\u003c/sub\u003eH\u003csub\u003e280\u003c/sub\u003eN\u003csub\u003e38\u003c/sub\u003eO\u003csub\u003e40\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003e3727.6260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 291px;\"\u003e\n \u003cp\u003e[M+3H]\u003csup\u003e3+\u003c/sup\u003e=1243.3591；[M+4H]\u003csup\u003e4+\u003c/sup\u003e=932.7721\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eDSM3-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eC\u003csub\u003e154\u003c/sub\u003eH\u003csub\u003e227\u003c/sub\u003eN\u003csub\u003e31\u003c/sub\u003eO\u003csub\u003e36\u003c/sub\u003eS \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003e3215.8960\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 291px;\"\u003e\n \u003cp\u003e[M+2H]\u003csup\u003e2+\u003c/sup\u003e=1608.3724；[M+3H]\u003csup\u003e3+\u003c/sup\u003e=1072.5825\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eDSM3-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eC\u003csub\u003e182\u003c/sub\u003eH\u003csub\u003e280\u003c/sub\u003eN\u003csub\u003e38\u003c/sub\u003eO\u003csub\u003e40\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003e3727.6260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 291px;\"\u003e\n \u003cp\u003e[M+3H]\u003csup\u003e3+\u003c/sup\u003e=1243.3598；[M+4H]\u003csup\u003e4+\u003c/sup\u003e=932.7716\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Circular dichroism characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study characterized the secondary structure of the synthetic peptide using circular dichroism spectroscopy and determined its helical degree. As shown in Figure 3, DSM3-1 did not show a significant helical degree; the helical degree of DSM3-2 was 73.56%; the helical degree of DSM3-4 was 6.22%; and the helical degree of DSM3-5 was 96.73%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Stapled peptide PROTAC cytotoxicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, tumor cells U87 MG, and U251 were tested for cytotoxicity of the synthesized peptides, and the results of the cell viability of the peptides were tested (Fig.4). The staple peptide IC\u003csub\u003e50\u003c/sub\u003e results were calculated (Table 3).\u003c/p\u003e\n\u003cp\u003eTable 3 IC\u003csub\u003e50\u003c/sub\u003e value of stapled peptide PROTAC\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 19.8864%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eName\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 23.2955%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eU87 MG(\u0026micro;M)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.1288%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eU251(\u0026micro;M)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 21.2121%;\"\u003e\n \u003cp\u003eDSM3-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 23.2955%;\"\u003e\n \u003cp\u003e～\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.1288%;\"\u003e\n \u003cp\u003e～\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 21.2121%;\"\u003e\n \u003cp\u003eDSM3-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 23.2955%;\"\u003e\n \u003cp\u003e20.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.1288%;\"\u003e\n \u003cp\u003e12.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 21.2121%;\"\u003e\n \u003cp\u003eDSM3-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 23.2955%;\"\u003e\n \u003cp\u003e～\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.1288%;\"\u003e\n \u003cp\u003e～\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 21.2121%;\"\u003e\n \u003cp\u003eDSM3-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 23.2955%;\"\u003e\n \u003cp\u003e18.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.1288%;\"\u003e\n \u003cp\u003e10.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: Tumor cells were treated with staple peptides for 2 or 7 days and tested for cytotoxicity using CCK-8 reagent.\u0026nbsp;～: No obvious activity.\u003c/p\u003e\n\u003cp\u003eDSM3-1 and DSM3-4 showed no significant inhibitory effect on the proliferation of tumor cells U87 MG and U251 in vitro. However, DSM3-2 and DSM3-5 significantly inhibited the growth of tumor cells U87 MG and U251 at a concentration of 16 \u0026mu;M.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Molecular docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe molecular docking results show that in the interaction between the DSM3-2 and MDM2 pockets, the amino acid residues that interact are concentrated in the 47-69 interval of the protein and the 1-14 interval of the polypeptide (Figure 5A). It is notable that the connection of hydrogen bonds occurs in LYS 47-Thr3, GLY 54-LEU5, TYR 63-TRP 8, and GLU 65-Lys 14. Acceptor-Acceptor interaction occurred in HIS 69-TRP 8 (Figure 5B).\u003c/p\u003e\n\u003cp\u003eIn the interaction between DSM3-2 and MDMX pockets (Figure 6A), the connection of hydrogen bonds occurs at Tyr 100-Thr 3, Val 93-Lys 16, and Lys 94-Lys 16, Carbon interaction in Apo 96 - Tyr 7, Pi-Alkyl in VAL 50-Phe 4 (Figure 6B).\u003c/p\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eIn this study, amino resin was used as the solid-phase support material, DIC-Oxyme was used as the reaction coupling system, the target stapled peptide was obtained by Fmoc solid-phase synthesis, the (S) 2-amino-2-methyl-6-heptanoic acid (S\u003csub\u003e5\u003c/sub\u003e) amino acid side chain group was synthesized by alkene cross-metathesis reaction, and Ahx was selected as the PROTAC linker to link the selected PROTAC fragment to the staple peptide, and four N-terminal acetylated MDM2/MDMX stapled peptide PROTAC were successfully obtained.\u003c/p\u003e\u003cp\u003eTumor cells U87 MG and U251 were selected for cell viability experiments, and the tumor suppressive activity of peptides DSM3-2 and DSM3-5 was particularly obvious according to the results of peptide cell activity experiments.\u003c/p\u003e\u003cp\u003eAccording to the results of helicity and cell viability, it was found that the activities of DSM3-2 and DSM3-5 with high helicity were significantly higher than those with helicity and decreased to DSM3-1 and DSM3-4, and it was speculated that there was a positive correlation between helicity and cell viability.\u003c/p\u003e\u003cp\u003eMolecular docking experiments have shown that the binding peptide DSM3-2 can effectively bind to the target proteins MDM2 and MDMX to exert a dual targeting effect on tumor cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eAckonwledgements\u003c/h2\u003e\u003cp\u003eThis work was supported by Fund of the President of Tarim University (TDZKSS202312 and TDZKSS202438), the Sichuan Provincial Central Leading Local Science and Technology Development Fund Project (2023ZYD0062).\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA. Xiufei Liao wrote the main manuscript and completed the main experiments.B. Damin Hu helped to complete the experiments.C. Mao Guo helped to complete the experiments.D. Chunli Su checked the manuscript.E. Hongli Liao helped to prepare figures 1-6.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi, X. \u0026amp; Song, Y. Proteolysis-targeting chimera (PROTAC) for targeted protein degradation and cancer therapy. \u003cem\u003eJ. Hematol. Oncol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (1), 50 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZeng, S. et al. Proteolysis targeting chimera (PROTAC) in drug discovery paradigm: Recent progress and future challenges. \u003cem\u003eEur. J. Med. Chem.\u003c/em\u003e \u003cb\u003e210\u003c/b\u003e, 112981 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJin, J. et al. The peptide PROTAC modality: a novel strategy for targeted protein ubiquitination. \u003cem\u003eTheranostics\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e (22), 10141\u0026ndash;10153 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQi, S. M. et al. PROTAC: An Effective Targeted Protein Degradation Strategy for Cancer Therapy. \u003cem\u003eFront. Pharmacol.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 692574 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeng, G. et al. PROTAC-DB: an online database of PROTACs. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e49\u003c/b\u003e (D1), D1381\u0026ndash;D1387 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe, S. et al. Aptamer-PROTAC Conjugates (APCs) for Tumor-Specific Targeting in Breast Cancer. \u003cem\u003eAngew Chem. Int. Ed. Engl.\u003c/em\u003e \u003cb\u003e60\u003c/b\u003e (43), 23299\u0026ndash;23305 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHines, J. et al. MDM2-Recruiting PROTAC Offers Superior, Synergistic Antiproliferative Activity via Simultaneous Degradation of BRD4 and Stabilization of p53. \u003cem\u003eCancer Res.\u003c/em\u003e \u003cb\u003e79\u003c/b\u003e (1), 251\u0026ndash;262 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiao, H. et al. PROTAC peptide induces durable β-catenin degradation and suppresses Wnt-dependent intestinal cancer. \u003cem\u003eCell. Discov\u003c/em\u003e. \u003cb\u003e6\u003c/b\u003e, 35 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang, J. \u0026amp; Liao, X. Study on the design, synthesis, and activity of anti-tumor staple peptides targeting MDM2/MDMX. \u003cem\u003eFront. Chem.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 12 (2024 June).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Stapled peptides, PROTAC, Anti-tumor","lastPublishedDoi":"10.21203/rs.3.rs-6970001/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6970001/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePROTAC is a drug development technology that uses the Ubiquitin-Proteasome System (UPS) to degrade target proteins, and enhances the degradation ability of target proteins through E3 ubiquitin ligase, which can further enhance the anti-tumor effect of targeted drug molecules. In this study, a series of dual-target MDM2/MDMX stapled peptide PROTAC based on SM3-4 were designed and synthesized, and the stapled peptide PROTAC DSM3-2 and DSM3-5 screened in the study inhibited tumor cell growth in vitro at low \u0026micro;M concentrations. The results showed that the enhancement of stapled peptide activity was positively correlated with the increase of helicity, which provided an effective research basis for the dual-target anti-tumor stapled peptide PROTAC. Molecular docking experiments have shown that the binding peptide DSM3-2 can effectively bind to the target proteins MDM2 and MDMX to exert a dual targeting effect on tumor cells.\u003c/p\u003e","manuscriptTitle":"Study on the design, synthesis and activity of MDM2/MDMX anti-tumor stapled peptide PROTAC","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-11 15:43:23","doi":"10.21203/rs.3.rs-6970001/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-12T09:43:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-10T13:47:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-08T07:25:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154594296679719194813078705212894034116","date":"2025-08-06T15:17:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"56396764410746100500336688540047689791","date":"2025-08-06T15:08:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"132556066452707245698932716117192625587","date":"2025-08-06T10:07:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-06T09:54:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-06T09:44:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-06T09:29:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-31T08:06:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-31T08:04:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"97820188-0d17-47fa-8f82-0d80d0ec213f","owner":[],"postedDate":"August 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52928328,"name":"Physical sciences/Chemistry/Medicinal chemistry/Drug discovery and development"},{"id":52928329,"name":"Physical sciences/Chemistry/Medicinal chemistry/Structure based drug design"},{"id":52928330,"name":"Physical sciences/Chemistry/Medicinal chemistry/Target identification"},{"id":52928331,"name":"Physical sciences/Chemistry/Medicinal chemistry/Target validation"}],"tags":[],"updatedAt":"2025-09-29T16:04:01+00:00","versionOfRecord":{"articleIdentity":"rs-6970001","link":"https://doi.org/10.1038/s41598-025-18026-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-09-25 15:58:00","publishedOnDateReadable":"September 25th, 2025"},"versionCreatedAt":"2025-08-11 15:43:23","video":"","vorDoi":"10.1038/s41598-025-18026-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-18026-6","workflowStages":[]},"version":"v1","identity":"rs-6970001","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6970001","identity":"rs-6970001","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}