bims-cepepe Biomed News
on Cell-penetrating peptides
Issue of 2025–07–06
twelve papers selected by
Henry Lamb, Queensland University of Technology
  1. Enhanced Intracellular Delivery by Twisted CC-Loop Conformation in Cyclic Cell-Penetrating Peptides.
  2. Development of Cell-Penetrating Peptides and Their Application to DDS.
  3. Proline-Mediated Enhancement in Evolvability of Disulfide-Rich Peptides for Discovering Protein Binders.
  4. Antibody-Free Immunopeptide Nanoconjugates for Brain-Targeted Drug Delivery in Glioblastoma Multiforme.
  5. Mixed-Chirality Prohibitin Peptide: D-(RLARLAR)2 Enhances Stability and In Vivo Effects on Obesity.
  6. CPPsite3: An updated large repository of experimentally validated cell-penetrating peptides.
  7. Addition of Oligoarginine to a Membrane Permeabilizing Peptide M-Lycotoxin Facilitates Intracellular Antibody Infusion from Microcondensate.
  8. Unsaturated Phosphine Oxides for Modular Antibody Rebridging and Single Reagent Peptide-Cyclisation-Bioconjugation.
  9. Fragments of cathelicidins PMAP-36 and BMAP-27 and their D-enantiomers: Effects of all D substitutions on structure, protease resistance and antimicrobial properties.
  10. Fibulin-4 is highly expressed in metastatic breast cancer and can serve as a target of peptide-based imaging probes and experimental therapeutics.
  11. Mechanistic Design of Cell-Penetrating Disruptors for a Phospho-Dependent Interaction.
  12. Amphipathic helical peptide-Nile red probes for fluorescence probing of the lipid packing defects and their surrounding membranes on exosomes.
  1. Biomacromolecules. 2025 Jul 01.
    Enhanced Intracellular Delivery by Twisted CC-Loop Conformation in Cyclic Cell-Penetrating Peptides.
    Qipeng Yan, Xingyue Jiang, Wei Xie, Xia Wu, Dalian Gong, Zenghui Li, Dan Yuan, Junfeng Shi.
      Biological drugs hold great promise for treating various diseases, but their efficacy is often limited by poor cellular uptake. Herein, we introduce cyclic cell-penetrating peptides (CPPs) to enhance the delivery efficiency. Three cyclic peptides with varying ring sizes were designed from a classic amphiphilic CPP via disulfide bond formation. Among them, Y2-13-OX adopted a twisted CC-loop conformation distinct from the typical β-hairpin on negatively charged cell surfaces. This unique structure enhanced membrane penetration, enabling superior delivery compared with linear and other cyclic variants. Y2-13-OX efficiently delivered functional siRNA targeting METTL3, achieving knockdown comparable to that of Lipofectamine 2000. It also transported GFP and plasmids, demonstrating versatility. Computational analysis revealed molecular-level insights into the enhanced interaction between the CC-loop structure and membranes. These findings establish a new CPP conformation that advances therapeutic delivery and opens new avenues for drug transport strategies.
    DOI:  https://doi.org/10.1021/acs.biomac.5c00378
  2. Chem Pharm Bull (Tokyo). 2025 ;73(7): 574-580
    Development of Cell-Penetrating Peptides and Their Application to DDS.
    Makoto Oba.
      Cell-penetrating peptides (CPPs) hold great potential as tools for drug delivery systems (DDSs). Numerous research groups have developed novel CPPs with enhanced functionality and safety. This review highlights recent advancements in CPP research by my research group and our collaborators. We have designed novel CPPs incorporating unnatural amino acids, explored their applications in DDSs, and elucidated their functional mechanisms.
    Keywords:  cell-penetrating peptide; drug delivery system; gene therapy; helical structure; nucleic acid therapeutics; unnatural amino acid
    DOI:  https://doi.org/10.1248/cpb.c25-00272
  3. J Am Chem Soc. 2025 Jul 02.
    Proline-Mediated Enhancement in Evolvability of Disulfide-Rich Peptides for Discovering Protein Binders.
    Hongtan Liu, Lulu Song, Xiaoting Meng, Jinjing Li, Shihui Fan, Huilei Dong, Xiaoran Wang, Maolin Li, Haipeng Yu, Yu-Hsuan Tsai, Yizhen Yin, Chuanliu Wu.
      Disulfide-rich peptides (DRPs), particularly those featuring the inhibitor cystine knot (ICK) motif, represent promising scaffolds for developing next-generation protein modulators and therapeutic agents due to their remarkable stability and specificity. However, their inherent structural integrity and lack of structural plasticity significantly limit their evolvability, creating a fundamental bottleneck in engineering novel functionalities. To address this challenge, we developed a novel proline scanning strategy aimed at enhancing the evolvability of the ICK scaffolds. This strategy leverages the proline-mediated structural decoupling between scaffold and nonscaffold residues in DRPs to promote their evolvability. By strategically incorporating prolines as pre-encoded scaffold residues, we engineered ICK variants with significantly improved foldability and tolerance to sequence variations. This advancement enabled the construction of diverse peptide libraries suitable for screening platforms, including mRNA and phage display. Utilizing this approach, we successfully identified DRPs exhibiting low-nanomolar affinity to clinically important targets, such as TROP2 and 4-1BB. Structural characterization revealed that these evolved DRPs adopted unique three-dimensional structures stabilized by up to four disulfide bonds, demonstrating both high oxidative folding efficiency and enhanced evolvability due to proline incorporation. To evaluate their therapeutic potential, we developed a DRP-based chimeric antigen receptor (CAR) targeting TROP2. The DRP-based CAR T cells exhibited potency comparable to conventional single-chain variable fragment (scFv)-based CAR T cells but with a notably improved safety profile. Overall, our work establishes a robust framework for expanding the functional versatility of DRP scaffolds, facilitating the discovery and development of structurally diverse and functional DRPs for broad applications in therapeutics and drug development.
    DOI:  https://doi.org/10.1021/jacs.5c07075
  4. Bioconjug Chem. 2025 Jul 02.
    Antibody-Free Immunopeptide Nanoconjugates for Brain-Targeted Drug Delivery in Glioblastoma Multiforme.
    Saurabh Sharma, David Lee, Surjendu Maity, Prabhjeet Singh, Jay Chadokiya, Neda Mohaghegh, Alireza Hassani, Hanjun Kim, Ankit Gangarade, Julia Y Ljubimova, Amanda Kirane, Eggehard Holler.
      Glioblastoma Multiforme (GBM) represents a significant clinical challenge among central nervous system tumors, with a dismal mean survival rate of less than 8 months, a statistic that has remained largely unchanged for decades (National Brain Society, 2022). The specialized intricate anatomical features of the brain, notably the blood-brain barrier (BBB), pose significant challenges to effective therapeutic interventions, limiting the potential reach of modern advancements in immunotherapy to impact these types of tumors. This study introduces an innovative, actively targeted immunotherapeutic nanoconjugate (P-12/AP-2/NCs) designed to serve as an immunotherapeutic agent capable of traversing the BBB via LRP-1 receptor-mediated transcytosis. P-12/AP-2/NCs exert their immune-modulating effects by inhibiting the PD-1/PD-L1 axis through a small-sized PD-L1/PD-L2 antagonist peptide, Aurigene NP-12 (P-12). P-12/AP-2/NCs are synthesized from completely biodegradable, functionalized high molecular weight β-poly(l-malic acid) (PMLA) polymer conjugated with P-12 and Angiopep-2 (AP-2) to yield P-12/AP-2/NCs. Evaluating nanoconjugates for BBB permeability and 3D tumor model efficacy using an in vitro BBB-Transwell spheroid-based model demonstrated successful crossing of the BBB and internalization in brain 3D tumor environments. In addition, the nanoconjugate mediated T cells' cytotoxicity on 3D tumor region death in a U87 GBM 3D spheroid model. AP-2/P-12/NCs are selectively inhibited in PD1/PDL1 interaction on T cells and the tumor site, increasing inflammatory cytokine secretion and T cell proliferation. In an in vivo murine brain environment, rhodamine fluorophore-labeled AP-2/P-12/NCs displayed significantly increased accumulation in the brain during 2-6 h time intervals postinjection with a prolonged bioavailability over unconjugated peptides. AP-2/P-12/NCs demonstrated a safety profile at both low and high doses based on major organ histopathology evaluations. Our findings introduce a novel, programmable nanoconjugate platform capable of penetrating the BBB for directed delivery of small peptides and significant immune environment modulation without utilizing antibodies, offering promise for treating challenging brain diseases such as glioblastoma multiforme and beyond.
    DOI:  https://doi.org/10.1021/acs.bioconjchem.5c00168
  5. J Am Chem Soc. 2025 Jul 01.
    Mixed-Chirality Prohibitin Peptide: D-(RLARLAR)2 Enhances Stability and In Vivo Effects on Obesity.
    Lai Yue Chan, Benjamin D Weger, Meltem Weger, Junqiao Du, Nicole Lawrence, Marlon H Cardoso, Samilla B de Rezende, Jéssica A I Muller, Kuok Yap, Brian W C Tse, Kamil A Sokolowski, Robert K P Sullivan, Jennifer Gunter, Octávio L Franco, Frédéric Gachon, David J Craik.
      Obesity stands as a global epidemic and is the primary risk factor for type 2 diabetes, ranking as the fifth leading cause of death worldwide. While lifestyle changes can address body fat accumulation, pharmacotherapies can also assist in sustained weight loss. Here, we report the design of a new generation of prohibitin peptide-based therapeutics engineered to target white adipose tissues. These peptides demonstrate significant reduction of body weight in a high-fat diet-induced obesity mouse model and represent a paradigm shift in approaches to the treatment of obesity by inducing mitochondrial uncoupling. The most potent compound, PTP-r, was prohibitin-TP01 substituted with d-arginine. Overall, the study reveals the promising development of next-generation adipose-targeting prohibitin peptides, capable of curbing adipocyte expansion and body weight, with favorable preclinical safety profiles. These peptides hold immense potential for developing new treatments to address obesity and metabolic syndrome.
    DOI:  https://doi.org/10.1021/jacs.5c05536
  6. Drug Discov Today. 2025 Jun 27. pii: S1359-6446(25)00134-5. [Epub ahead of print] 104421
    CPPsite3: An updated large repository of experimentally validated cell-penetrating peptides.
    Nisha Bajiya, Sohana Najrin, Pankaj Kumar, Shubham Choudhury, Ritu Tomer, Gajendra P S Raghava.
      Over the past two decades, cell-penetrating peptides (CPPs) have emerged as key intracellular drug delivery vehicles. This review provides a systematic overview of advancements in CPP research, encompassing both experimental techniques and computational resources crucial for their discovery. Our primary focus is on CPPsite3, a meticulously curated repository that currently houses 6788 entries, representing 4285 unique CPPs. This updated version builds upon CPPsite and CPPsite2, both of which have been extensively utilized by the scientific community for designing CPPs and developing robust prediction methods. Furthermore, we explore the clinical applications of CPPs, specifically highlighting those currently undergoing clinical use. This comprehensive review aims to be a vital resource for the drug delivery research community.
    Keywords:  CPPsite3 database; cell-penetrating peptides; delivery carriers; therapeutic agents
    DOI:  https://doi.org/10.1016/j.drudis.2025.104421
  7. Bioconjug Chem. 2025 Jul 02.
    Addition of Oligoarginine to a Membrane Permeabilizing Peptide M-Lycotoxin Facilitates Intracellular Antibody Infusion from Microcondensate.
    Junya Michibata, Yoshimasa Kawaguchi, Yusei Furuyama, Yoshihiro Sasaki, Kazunari Akiyoshi, Shiroh Futaki.
      Coacervate-based intracellular delivery of biomacromolecules has attracted our attention due to the feasibility of easy condensation of the biomacromolecules and their controllable release. Our laboratory has developed a unique, coacervate-based delivery system that uses the conjugate of the polysaccharide pullulan with membrane-permeabilizing peptides, including L17E and M-lycotoxin. This system enables immunoglobulin G (IgG) antibodies labeled with the negatively charged fluorophore Alexa Fluor 488 to enter the cytosol directly through the plasma membrane. Cyotosolic IgG distribution is complete within a few minutes after infusion initiation, and infusion can be achieved in serum-containing medium. The purpose of this study was to refine this system to reduce the amount of antibody required while maintaining satisfactory delivery efficiencies. Therefore, pullulan conjugates with M-lytocoxin bearing two to eight arginine residues were designed to enhance the interaction of M-lycotoxin with the cell membrane. The conjugates were able to form microcondensates with Alexa Fluor 488 labeled IgGs. The addition of arginine residues improved the efficiency of cytosolic infusion and successfully reduced the amounts of both antibodies and pullulan-peptide conjugates required for the delivery.
    DOI:  https://doi.org/10.1021/acs.bioconjchem.5c00176
  8. Angew Chem Int Ed Engl. 2025 Jun 30. e202508656
    Unsaturated Phosphine Oxides for Modular Antibody Rebridging and Single Reagent Peptide-Cyclisation-Bioconjugation.
    Christian E Stieger, Alastair McMillan, Mark A R de Geus, Jan Vincent Arafiles, Luise Franz, Christian Hackenberger.
      Achieving modular, selective and homogeneous protein modifications is of utmost importance for the design of next generation biopharmaceuticals; especially in the context of antibody-drug conjugates (ADCs). Here, we introduce unsaturated phosphine oxides as versatile triple-reactive reagents, allowing orthogonal chemoselective bioconjugation schemes. Starting from triethynyl-phosphine oxide, a variety of functionalized diethynyl-triazolyl-phosphine oxides (DTPOs) could be accessed by using CuI-catalysed azide-alkyne cycloaddition (CuAAC). We showcase DTPO-reagents in the fast and selective generation of various highly stable antibody-conjugates via antibody disulfide rebridging. A highlight from this methodology is the synthesis of a DAR 4 ADC following a modular 2-step strategy using bioorthogonal tetrazine-labeling with bicyclo[6.1.0]non-4-yne (BCN) or trans-cyclooctene (TCO) containing payloads. Notably, the DTPO-rebridged ADC exhibited potent cytotoxicity against Her2+ cancer cells. Moreover, we utilize triethynyl-phosphine oxide to obtain ethynyl-ditriazolyl-phosphine oxides (EDPOs) which enable a unique, single-reagent peptide-cyclization-bioconjugation protocol resulting in functional cyclic peptide-protein conjugates. Overall, our work provides versatile and powerful chemoselective modalities for the controlled modification of antibodies, peptide-cyclization and peptide-protein conjugation, expanding the toolkit for chemical biology and therapeutic development.
    Keywords:  Antibody Rebridging * Bioconjugation * Click Chemistry * Antibody-Drug-Conjugates * Peptide Cyclization
    DOI:  https://doi.org/10.1002/anie.202508656
  9. Bioorg Chem. 2025 Jun 30. pii: S0045-2068(25)00595-4. [Epub ahead of print]163 108715
    Fragments of cathelicidins PMAP-36 and BMAP-27 and their D-enantiomers: Effects of all D substitutions on structure, protease resistance and antimicrobial properties.
    Francesca Albini, Barbara Biondi, Adriana Di Stasi, Andrea Schivo, Mario Mardirossian, Marco Scocchi, Cristina Peggion.
      Antimicrobial peptides (AMPs) are natural molecules of great interest in the fight against bacteria and in addressing antibiotic resistance. Their use as antimicrobial drugs is still limited due to their cytotoxicity and poor resistance to proteolysis. PMAP-36 and BMAP-27 are cathelicidins that contribute to innate immunity. Two short sequences, PMAP(12-24) and BMAP(1-18), are the most promising analogues identified but despite their broad antibacterial activity, they undergo rapid proteolytic degradation. In this study, the D and L enantiomers of the two peptides were synthesised, and their structures were elucidated and compared by circular dichroism (CD) spectroscopy and 2D-NMR analysis. Conformational studies revealed that, despite their short sequence, all peptides adopted helical structures in membrane-mimetic environments, with subtle differences between the two AMP groups, PMAP(12-24) being more prone to adopt a mixed α/310-helical conformation (R([Θ]222/[Θ]206 = 0.4-0.5) and BMAP(1-18) being closer to a pure α-helix (R([Θ]222/[Θ]208 = 0.7-0.8). These structural differences between the two peptides were found to influence their antimicrobial activity and mode of membrane permeabilization. Moreover, the D enantiomers of both analogues were resistant to proteolysis. All peptides showed a broad spectrum of antibacterial activity (main MIC range: 1-16 μM). Cytotoxicity studies in fibroblast showed that the peptides were non-cytotoxic at concentrations corresponding to their antibacterial activity. Overall, this study led to the identification of structurally interesting short peptides that could serve as prototypes for the development of effective and practical antibacterial drugs.
    Keywords:  Antimicrobial peptide; BMAP(1–18); BMAP-27; Cathelicidin; D-enantiomer; PMAP-36
    DOI:  https://doi.org/10.1016/j.bioorg.2025.108715
  10. bioRxiv. 2025 Jun 19. pii: 2025.06.18.657655. [Epub ahead of print]
    Fibulin-4 is highly expressed in metastatic breast cancer and can serve as a target of peptide-based imaging probes and experimental therapeutics.
    Alexes C Daquinag, Solmaz AghaAmiri, Stephen M Farmer, Sheng Zhang, Sukhen C Ghosh, Servando Hernandez Vargas, Ashwin K Ramesh, Zhiqiang An, Ali Azhdarinia, Mikhail G Kolonin.
      We have previously reported a cyclic peptide CRAGVGRGC (termed BLMP6) that homes to disseminating tumor cells in mouse cancer models and could be used for metastasis detection and intervention. Here, based on BLMP6 similarity to latent transforming growth factor beta binding protein 4 (LTBP4), we discovered fibulin-4 as a BLMP6 target. We show that BLMP6 mimics the LTBP4 domain binding to fibulin-4 and selectively binds to fibulin-4 in vitro. Fibulin-4 knockout in mouse 4T1 cancer cells abrogated BLMP6 homing to lung metastases. Fibulin-4 expression was found to be increased in invasive and metastatic human breast cancer. AZDye555 fluorophore-labeled BLMP6 was developed as a reagent selectively binding to invasive and metastatic human breast cancer cells in tissue sections and homing to MDA-MB-231 metastases in mice. We show that radiolabeling BLMP6 with 68 Ga can be used for the detection of MDA-MB-231 metastases. We designed a peptide-drug conjugate consisting of monomethyl auristatin E (MMAE) and BLMP6 that preferentially kills aggressive cancer cells. Cytotoxicity of MMAE-BLMP6 against MDA-MB-231 tumors was confirmed in vivo . In an immunocompetent mouse model of B16F10 experimental lung metastases, treatment with MMAE-BLMP6 suppressed metastasis growth and improved survival. There was also a trend for metastasis suppression and survival improvement in the MDA-MB-231 experimental metastasis model. Our results suggest that fibulin-4 and BLMP6 may be further developed for the detection and targeting of metastatic human cancers.
    Statement of significance: This study identifies fibulin-4 as a protein highly expressed in breast cancer metastasis. It evaluates the application of peptide conjugates targeting fibulin-4 in mouse models as non-invasive probes for metastasis detection and cytotoxic drug delivery.
    DOI:  https://doi.org/10.1101/2025.06.18.657655
  11. Res Sq. 2025 Jun 17. pii: rs.3.rs-6862805. [Epub ahead of print]
    Mechanistic Design of Cell-Penetrating Disruptors for a Phospho-Dependent Interaction.
    Vanda Gunning, Matthew Batchelor, Krista K Alexander, Martin Walko, Selena G Burgess, Stephen J Royle, Eileen J Kennedy, Richard Bayliss.
      The complex formed by transforming acidic coiled coil 3 (TACC3) and clathrin heavy chain (CHC) enhances mitotic spindle stability and strength by cross-linking microtubules. The interaction is dependent on phosphorylation of TACC3 at S558 by Aurora-A. Previously, we elucidated the structural basis of the TACC3/CHC interaction, which is driven by hydrophobic residues on both proteins and the formation of an α-helix in TACC3 that docks into the helical repeats of CHC. Here we find that this phosphorylation event plays an unusual role in the protein-protein interaction; rather than direct bond formation, the phosphorylated residue acts by overcoming an inherent electrostatic repulsion between K507 of CHC and basic residues in TACC3. Leveraging this insight, we optimized the sequence using peptide arrays to develop a hydrocarbon-stapled peptide (SP TACC3) that binds CHC with over a hundred-fold higher affinity than the parental TACC3 peptide, effectively disrupting the native interaction. The crystal structure of the SP TACC3/CHC complex reveals the basis for the enhanced interaction and highlights the contribution of additional polar and hydrophobic interactions. SP TACC3 efficiently penetrates cells and displaces TACC3 from the mitotic spindle, causing a delay in mitotic progression in two out of three cancer cell lines. This work showcases the novel application of hydrocarbon-stapled peptides to disrupt the TACC3/CHC protein-protein interaction in a cellular context, highlighting the potential of targeting this interface for future cancer therapies.
    DOI:  https://doi.org/10.21203/rs.3.rs-6862805/v1
  12. Sci Rep. 2025 Jul 03. 15(1): 23790
    Amphipathic helical peptide-Nile red probes for fluorescence probing of the lipid packing defects and their surrounding membranes on exosomes.
    Yusuke Sato, Kazuya Segawa, Tomomi Sakamoto, Arihiro Narita, Kota Matsumoto, Seiichi Nishizawa.
      Amphipathic helical (AH) peptide-based fluorescent probes were explored for analysis of lipid packing defects (LPDs) in the membrane surface of exosomes. Two kinds of AH peptide sequences, derived from the C-terminal sequence of Apolipoprotein A-I (ApoC) and from human α-synuclein (p2-23), were examined, where they differ in the hydrophobic face that can be inserted into LPDs. From the examination of the insertion depth of the AH peptides and the competitive binding using synthetic liposomes as exosome models, we found that ApoC peptide could serve as a binder for deep LPDs whereas p2-23 peptide preferentially recognizes shallow LPDs. These peptides conjugated with an environment-sensitive dye Nile Red (NR) were demonstrated to be useful for assessing both the abundance of target LPDs by the fluorescent enhancement response and the membrane properties surrounding these LPDs by the emission wavelength of the probes, respectively. With these properties, our probes successfully characterized the LPDs of exosomes from three kinds of cancer cells (A549, Hela and MCF7 cells). We showed that exosomal membranes exhibited unique structural properties regarding deep and shallow LPDs and their surrounding membrane polarity. In addition, these properties significantly depended on the donor cells. Our probes would serve as powerful tools for LPD analysis with a view toward a better understanding of exosomal membranes.
    Keywords:  Exosome; Fluorescent probe; Lipid packing defect; Membranes; Polarity
    DOI:  https://doi.org/10.1038/s41598-025-08534-w
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