bims-cepepe Biomed News
on Cell-penetrating peptides
Issue of 2025–05–18
fourteen papers selected by
Henry Lamb, Queensland University of Technology
  1. Predicting the structures of cyclic peptides containing unnatural amino acids by HighFold2.
  2. Discovery of Bioactive Peptides Through Peptide Scanning.
  3. Optimization of antimicrobial peptide Smp43 as novel inhibitors of Cancer.
  4. CABS-flex 3.0: an online tool for simulating protein structural flexibility and peptide modeling.
  5. De Novo Design of Highly Stable Binders Targeting Dihydrofolate Reductase in Klebsiella pneumoniae.
  6. Chirality Effects in Peptide-Based Dynamic Combinatorial Chemistry.
  7. PCFT Independent Cellular Uptake of Cyclic Cell Penetrating Peptide Conjugated Folic Acid.
  8. Macrocyclic RGD-peptides with high selectivity for αvβ3 integrin in cancer imaging and therapy.
  9. Chelating [227Th]Th4+ for Peptide Receptor Radionuclide Therapy of Neuroendocrine Tumors.
  10. Design Guidelines to Control Rippled β-Sheets versus Pleated β-Sheets in Mixed-Chirality Peptides.
  11. Selectivity of membrane-active peptides: the role of electrostatics and other membrane biophysical properties.
  12. Macrocyclization: Enhancing Drug-like Properties of Discoidin Domain Receptor Kinase Inhibitors.
  13. Small Organic Carbonic Anhydrase IX Ligands from DNA-Encoded Chemical Libraries for Tumor-Targeted Delivery of Radionuclides.
  14. Self-Assembled Peptide PROTAC Prodrugs Targeting FOXM1 for Cancer Therapy.
  1. Brief Bioinform. 2025 May 01. pii: bbaf202. [Epub ahead of print]26(3):
    Predicting the structures of cyclic peptides containing unnatural amino acids by HighFold2.
    Cheng Zhu, Sen Cao, Tianfeng Shang, Jingjing Guo, An Su, Chengxi Li, Hongliang Duan.
      Cyclic peptides containing unnatural amino acids possess many excellent properties and have become promising candidates in drug discovery. Therefore, accurately predicting the 3D structures of cyclic peptides containing unnatural residues will significantly advance the development of cyclic peptide-based therapeutics. Although deep learning-based structural prediction models have made tremendous progress, these models still cannot predict the structures of cyclic peptides containing unnatural amino acids. To address this gap, we introduce a novel model, HighFold2, built upon the AlphaFold-Multimer framework. HighFold2 first extends the pre-defined rigid groups and their initial atomic coordinates from natural amino acids to unnatural amino acids, thus enabling structural prediction for these residues. Then, it incorporates an additional neural network to characterize the atom-level features of peptides, allowing for multi-scale modeling of peptide molecules while enabling the distinction between various unnatural amino acids. Besides, HighFold2 constructs a relative position encoding matrix for cyclic peptides based on different cyclization constraints. Except for training using spatial structures with unnatural amino acids, HighFold2 also parameterizes the unnatural amino acids to relax the predicted structure by energy minimization for clash elimination. Extensive empirical experiments demonstrate that HighFold2 can accurately predict the 3D structures of cyclic peptide monomers containing unnatural amino acids and their complexes with proteins, with the median RMSD for Cα reaching 1.891 Å. All these results indicate the effectiveness of HighFold2, representing a significant advancement in cyclic peptide-based drug discovery.
    Keywords:  cyclic peptides; multi-scale modeling; structure prediction; unnatural amino acids
    DOI:  https://doi.org/10.1093/bib/bbaf202
  2. J Pept Sci. 2025 Jun;31(6): e70029
    Discovery of Bioactive Peptides Through Peptide Scanning.
    Debora Iaculli, Steven Ballet.
      Therapeutic peptides targeted at various diseases are becoming increasingly relevant for the pharmaceutical industry. Several of these drugs were originally designed by mimicking a segment of a protein of interest. As such, protein mimicry represents a promising strategy both in immunology, for the identification of B- and T-cell epitopes, as well as for the modulation of protein activity, including the disruption of protein-protein interactions (PPIs) and the interference with biological or pathological cellular functions. Several methods have been developed to pinpoint the (binding) epitopes of a protein or the regions responsible for biological activity. One of such strategies is the scanning of the protein or selected domains with synthetic overlapping peptides. As the mechanism of action of a mimetic peptide can be similar to that of the whole protein, this method offers a powerful tool for the investigation of protein function, along with providing a solid basis for the development of therapeutic candidates. This review gives a general overview of different applications of the peptide scanning methodology, describing a comparison of the preparation and use of solid-phase libraries (peptide arrays) with isolated peptide libraries and highlighting their strengths and most common applications.
    Keywords:  bioactive peptides; epitope mapping; peptide libraries; peptide scanning; protein–protein interactions
    DOI:  https://doi.org/10.1002/psc.70029
  3. Bioorg Chem. 2025 May 09. pii: S0045-2068(25)00441-9. [Epub ahead of print]161 108561
    Optimization of antimicrobial peptide Smp43 as novel inhibitors of Cancer.
    Zhou Yu, Dan Han, Kai Zhang, Conghao Gai, Xiaoyun Chai, Qingjie Zhao, Yan Zou, Lei Yin.
      Smp43 is a natural antimicrobial peptide derived from scorpion venom containing 43 amino acids, which inhibits the proliferation of various cancer cells, providing a new surrogate for antitumor drug discovery. However, Smp43 is large (Mw: 4653 Da) and unstable as a linear peptide. We truncated Smp43 to identify the key fragment and found that Smp(1-14) displayed significant antitumor activity. Subsequent optimization of Smp(1-14) led to a series of stapled peptides, among which SSmp6 demonstrated enhanced antitumor activity, stability, and membrane permeability compared to the linear peptides. Moreover, SSmp6 could decrease the phosphorylation of AKT, damage cancer cell membranes, and induce cell apoptosis. The results highlight the potential of SSmp6 as a lead stapled peptide for developing novel antitumor therapies. The study introduces a concept for using antimicrobial peptides to develop anticancer drugs.
    Keywords:  Anticancer; Antimicrobial peptides; Smp43; Stapled peptide
    DOI:  https://doi.org/10.1016/j.bioorg.2025.108561
  4. Nucleic Acids Res. 2025 May 14. pii: gkaf412. [Epub ahead of print]
    CABS-flex 3.0: an online tool for simulating protein structural flexibility and peptide modeling.
    Karol Wróblewski, Mateusz Zalewski, Aleksander Kuriata, Sebastian Kmiecik.
      Simulating protein structure flexibility using classical methods is computationally demanding, especially for large proteins. To address this challenge, we have been developing the CABS-flex method, which enables fast simulations of protein structural flexibility by combining a coarse-grained simulation approach with all-atom detail. Previously available as the CABS-flex 2.0 web server, the method has now undergone a major upgrade with the release of CABS-flex 3.0. Key improvements include the introduction of intuitive flexibility modes that simplify the control of distance restraints and allow users to reflect known or expected dynamic regions; improved all-atom reconstruction for higher-quality model generation; a new feature for de novo peptide structure prediction, supporting both linear and cyclic peptides along with their conformational flexibility; and new tools for result analysis and visualization, facilitating deeper insights into structural flexibility. Additionally, AlphaFold pLDDT-derived restraints can be used as optional input for guiding simulations. The method accepts input as either a PDB/mmCIF structure or a sequence (for peptide modeling). Advanced options allow users to incorporate experimental or computational restraints. The CABS-flex 3.0 web server is available at https://lcbio.pl/cabsflex3. This website is free and open to all users, with no login requirement.
    DOI:  https://doi.org/10.1093/nar/gkaf412
  5. Proteins. 2025 May 15.
    De Novo Design of Highly Stable Binders Targeting Dihydrofolate Reductase in Klebsiella pneumoniae.
    Ihteshamul Haq, Faheem Anwar, Yigang Tong.
      The study aims to design novel therapeutic inhibitors targeting the DHFR protein of Klebsiella pneumoniae. However, challenges like bacterial resistance to peptides and the limitations of computational models in predicting in vivo behavior must be addressed to refine the design process and improve therapeutic efficacy. This study employed deep learning-based bioinformatics techniques to tackle these issues. The study involved retrieving DHFR protein sequences from Klebsiella strains, aligning them to identify conserved regions, and using deep learning models (OmegaFold, ProteinMPNN) to design de novo inhibitors. Cell-penetrating peptide (CPP) motifs were added to enhance delivery, followed by allergenicity and thermal stability assessments. Molecular docking and dynamics simulations evaluated the binding affinity and stability of the inhibitors with DHFR. A conserved 60-residue region was identified, and 60 de novo binders were generated, resulting in 7200 sequences. After allergenicity prediction and stability testing, 10 sequences with melting points near 70°C were shortlisted. Strong binding affinities were observed, especially for complexes 4OR7-1787 and 4OR7-1811, which remained stable in molecular dynamics simulations, indicating their potential as therapeutic agents. This study designed stable de novo peptides with cell-penetrating properties and strong binding affinity to DHFR. Future steps include in vitro validation to assess their effectiveness in inhibiting DHFR, followed by in vivo studies to evaluate their therapeutic potential and stability. These peptides offer a promising strategy against Klebsiella pneumoniae infections, providing potential alternatives to current antibiotics. Experimental validation will be key to assessing their clinical relevance.
    Keywords:  ProteinMPNN; antibiotic resistance; de novo design; deep learning; dihydrofolate reductase
    DOI:  https://doi.org/10.1002/prot.26835
  6. Chemistry. 2025 May 13. e202501298
    Chirality Effects in Peptide-Based Dynamic Combinatorial Chemistry.
    Alice Gable, Emmi Pohjolainen, Gerrit Groenhof, Fabien B L Cougnon.
      Naturally occurring peptides are almost exclusively composed of L-amino acids, and the incorporation of D-amino acids can profoundly alter their ability to fold and self-assemble. Here we explore the effects of chirality on the formation of disulfide dynamic combinatorial libraries generated by short cysteine-rich peptides. Our findings consistently show that heterochiral tripeptides form more diverse dynamic combinatorial libraries than their homochiral counterparts. The most complex library appears to encompass all possible cyclic species up to 19mers. Given that each of these species exists as a mixture of parallel and antiparallel isomers, we estimate this library to contain a total of 2,045 distinct compounds - a remarkable result considering that the library generated by the analogous homochiral peptide predominantly contains two dimers. In certain situations, peptide chirality also affects the relative stability of parallel and antiparallel isomers. Taken together, these results show that small changes in peptide chirality can be dramatically amplified through the formation of cyclic species.
    Keywords:  cysteine-rich peptides * chirality * dynamic combinatorial chemistry * macrocycles * supramolecular chemistry
    DOI:  https://doi.org/10.1002/chem.202501298
  7. Chembiochem. 2025 May 13. e202500242
    PCFT Independent Cellular Uptake of Cyclic Cell Penetrating Peptide Conjugated Folic Acid.
    Vineet Kumar Mishra, Rheal Towner, Juan Carlos Rodriguez-Lecompte, Marya Ahmed.
      Folic acid is an essential component of many metabolic processes, including the synthesis of nucleoproteins, purines, and pyrimidines and is a recommended supplement to lower the incidence of various disorders. Folic acid and folate loaded nanoparticles are extensively evaluated for sustained release and enhanced stability of the molecule, however malfunctioning of Proton Coupled Folate Transporters (PCFT) present on the intestinal cells, and subsequent folate deficiency remain a major issue in this context. This study provides first demonstration where cell-penetrating peptide conjugated folic acid mediate PCFT independent folic acid permeabilization and intracellular bioavailability in vitro in the intestinal cells and macrophages. Cyclic-Transactivating transcriptional activator (cTAT) folic acid conjugates are prepared by solid phase peptide synthesis and are evaluated for the cellular uptake and bioavailability in the presence and absence of PCFT inhibitors. Compared with free folic acid that showed PCFT mediated cellular uptake, cTAT-folic acid conjugates exhibited enhanced cellular uptake at all studied pH and improved intracellular bioavailability of the cargo, as was determined by dihydrofolate reductase (DHFR) assay. Folic acid and cTAT-folic acid conjugates also dampened the production of pro-inflammatory mediators in the presence of toxins in vitro in macrophage cell lines.
    Keywords:  peptide folic acid conjugates, PCFT downregulation, DHFR assay, immunomodulation
    DOI:  https://doi.org/10.1002/cbic.202500242
  8. RSC Med Chem. 2025 May 03.
    Macrocyclic RGD-peptides with high selectivity for αvβ3 integrin in cancer imaging and therapy.
    Xiaozhong Cheng, Chen Li, Haofei Hong, Zhifang Zhou, Zhimeng Wu.
      Integrins, particularly the αvβ3 subtype, are critical receptors involved in cell adhesion, migration, and signaling, playing a significant role in tumor progression and metastasis. Despite extensive research into integrin-targeted therapies, challenges remain in developing ligands that exhibit high selectivity for αvβ3 over other integrin subtypes, such as αvβ5. This study employs a one-pot sortase A-mediated on-resin peptide cleavage and in situ cyclization method to synthesize two generations of macrocyclic RGD-peptide libraries. Systematic screening through surface plasmon resonance and cell-based competition assays identified the lead compound, c-(G5RGDKcLPET), which demonstrated high affinity and selectivity for αvβ3. Additionally, the optimized cyclic peptide was functionalized with a fluorescent dye (Cy5) and the cytotoxic drug monomethyl auristatin E (MMAE), enhancing its potential for cancer imaging and targeted therapy. This work contributes a novel platform for developing integrin-targeted diagnostics and therapeutics, highlighting the importance of macrocyclic peptides in cancer treatment strategies.
    DOI:  https://doi.org/10.1039/d5md00280j
  9. Bioconjug Chem. 2025 May 10.
    Chelating [227Th]Th4+ for Peptide Receptor Radionuclide Therapy of Neuroendocrine Tumors.
    Luke Wharton, Scott W McNeil, Harrison Meeres, Diduo Zhang, Aidan Ingham, Helen Merkens, Maryam Osooly, Cristina Rodríguez-Rodríguez, François Bénard, Hua Yang.
      Targeted alpha therapy (TAT) has shown high promise for the effective treatment of advanced stage cancers. Of the proposed radionuclides for TAT, Thorium-227 represents an interesting candidate given its relatively long half-life, 18.7 days, and the cascade of short-lived, high-potency, alpha-emitting daughter progeny in its decay scheme. However, to date few chelators exist which can effectively and stably bind [227Th]Th4+ at molar activities high enough for TAT. To address this challenge, this study investigated various chelating ligands for coordination of [227Th]Th4+. H4noneunpaX was identified as a promising chelator, demonstrating radiolabeling with [227Th]Th4+ at concentrations of 10-6 M (Am = 272 kBq/nmol). The coordination characteristics of [Th(noneunpaX)] have been investigated through 1H NMR spectroscopy, mass spectrometry, and DFT calculations. In this study, we also investigate for the first time the pairing of Th-227 with a peptide-based bioconjugate and evaluate the in vivo biodistribution characteristics. [227Th]Th-nonenupaX-Ahx-Tyr3-TATE was prepared under mild conditions (ambient temperature, 30 min) and evaluated in NRG mice bearing AR42J xenografts as a model for pancreatic neuroendocrine tumors. The 227Th-labeled radiopeptide showed high uptake in tumors (25.8±6.2 %IA/g at 3 h p.i.) and low uptake in non-targeted organs. Although some release of Th-227 was noted in serum stability studies this was not observed in vivo. This ligand architecture serves as an interesting framework for future optimization, which will involve improvements to the overall stability by enhancing the rigidity of the backbone and assessing other pendent donor groups with a stronger affinity toward [227Th]Th4+. Overall, this study demonstrated for the first time the viability of using peptide-based targeting to effectively deliver Th-227 to tumor sites.
    DOI:  https://doi.org/10.1021/acs.bioconjchem.5c00129
  10. J Am Chem Soc. 2025 May 15.
    Design Guidelines to Control Rippled β-Sheets versus Pleated β-Sheets in Mixed-Chirality Peptides.
    Hyeonju Lee, Amaruka Hazari, Jevgenij A Raskatov, Hyungjun Kim, William A Goddard.
      Decoding how amino acid sequences determine structure facilitates the design of functional proteins, advanced biomaterials, and selective, low-side-effect drugs. The rippled β-sheet, theorized by Pauling and Corey in 1953, has only recently begun to gain experimental support. However, research on rippled β-sheets remains limited, leaving gaps in our understanding of when and how they occur. To understand the relationship between sequences and rippled β-sheet formation propensities, we carried out molecular dynamics (MD) and density functional theory (DFT) simulations to predict the energetics for six systems of forming either a rippled or pleated β-sheets that are ordered either parallel or antiparallel. Notably, among these four possible structures of each system, the structure predicted to have the lowest energy agrees with the single case observed experimentally! To understand why this form is favored, we investigate the local structures of all six systems, with particular attention to the role of hydrogen bonds (H-bonds) in stabilization. In each system, the peptide consistently adopts a motif that allows it to form the maximum number of H-bonds between backbones, even when amidated, and composed of a single-component with mixed chirality or a cyclic peptide. We find that an achiral glycine-glycine bridge acts as a spacer between valine residues, effectively reducing steric hindrance between side chains. Furthermore, we conclude that the structures of cyclic peptides are stabilized by intramolecular H-bonds in an anhydrous environment. Our findings provide deeper insights into how sequences influence β-sheet conformations, enabling us to propose guidelines for the preferred structures of novel peptides.
    DOI:  https://doi.org/10.1021/jacs.4c16425
  11. Biophys Rev. 2025 Apr;17(2): 591-604
    Selectivity of membrane-active peptides: the role of electrostatics and other membrane biophysical properties.
    Iván Felsztyna, Vanesa V Galassi, Natalia Wilke.
      Membrane-active peptides (MAPs) are versatile molecules that interact with lipid bilayers, facilitating processes such as antimicrobial defense, anticancer activity, and membrane translocation. Given that most MAPs are cationic, their selectivity for specific cell membranes has traditionally been attributed to variations in membrane surface charge. However, growing evidence suggests that electrostatics alone cannot fully explain MAPs selectivity. Instead, MAPs activity is also strongly influenced by other membrane biophysical properties, such as lipid packing, phase state, curvature, and the spatial distribution of hydrophobic and charged residues within the peptide sequence. In this review, we summarize the current knowledge on the biophysical determinants of MAPs selectivity. We begin by examining membrane and cell surface electrostatics and their influence on MAPs-membrane interactions, including electrostatically driven peptide conformational changes and lipid recruitment. We then broaden the discussion to include non-electrostatic factors, such as membrane curvature and rheology, which are primarily influenced by sterol or hopanoid content, as well as acyl chain unsaturation and branching. Together, these processes highlight that MAPs selectivity is not governed by any single membrane property but instead emerges from a synergistic interplay of electrostatic, hydrophobic, and topological factors.
    Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-025-01309-7.
    Keywords:  Arginine; Hopanoids; Sterols; Zeta potential of cells; pH-regulated activity
    DOI:  https://doi.org/10.1007/s12551-025-01309-7
  12. ACS Med Chem Lett. 2025 May 08. 16(5): 784-789
    Macrocyclization: Enhancing Drug-like Properties of Discoidin Domain Receptor Kinase Inhibitors.
    Laura Carzaniga, Roberta Mazzucato, Valentina Mileo, Andrea Rizzi, Maura Vallaro, Giuseppe Ermondi, Silvia Cattani, Andrea Secchi, Giulia Caron.
      Macrocyclization, a well-established strategy for developing ligands against challenging drug targets, was employed to design macrocyclic alternatives to a linear discoidin domain receptor (DDR) inhibitor (1) with potential applications in treating fibrotic diseases. This study aimed to enhance the drug-like profile of 1 through innovative design strategies encompassing molecular docking and chameleonicity considerations. These efforts resulted in the synthesis of matched pairs of macrocycles differing in flexibility and linker features. Compound 5a emerged as a promising lead, exhibiting nanomolar-range activity, significantly improved solubility, and excellent permeability. Comprehensive experimental physicochemical characterization further highlighted the modest impact of ionization, the major role played by lipophilicity (but not polarity) in driving permeability of the investigated matched pairs, and the limitations of traditional 2D computational descriptors in predicting macrocycle ADME-related properties.
    DOI:  https://doi.org/10.1021/acsmedchemlett.4c00611
  13. J Am Chem Soc. 2025 May 16.
    Small Organic Carbonic Anhydrase IX Ligands from DNA-Encoded Chemical Libraries for Tumor-Targeted Delivery of Radionuclides.
    Marco Müller, Tony Georgiev, Jacqueline Mock, Dario Neri, Samuele Cazzamalli, Sebastian Oehler.
      Carbonic anhydrase IX (CAIX) is a membrane protein that is highly expressed in clear cell renal cell carcinoma (ccRCC) and in hypoxic tumors. Being virtually absent in most healthy tissues, CAIX became an attractive target for the selective delivery of diagnostic and therapeutic payloads. Here, we report the discovery and characterization of DNA-encoded chemical library (DEL)-derived CAIX ligands for radionuclide-based imaging applications.
    METHODS: DELs were screened against CAIX and CAII to prioritize hits based on their selectivity and enrichment against CAIX. In vitro characterization of hits was performed by fluorescence polarization (FP), surface plasmon resonance (SPR), and flow cytometry. In vivo biodistribution studies of Lutetium-177 and Gallium-68-radiolabeled compounds were performed in SK-RC-52 tumor-bearing mice.
    RESULTS: DEL-based CAIX ligands with different affinities and selectivities could be identified. Selectivity and high affinity toward the target correlated with higher tumor-to-organ ratios and improved tumor retention. The best candidate, named OncoCAIX, reached up to ∼55% injected dose per gram in SK-RC-52 lesions at early time points with very low healthy organ uptake (tumor-to-kidney ratio of >23).
    CONCLUSION: OncoCAIX demonstrated rapid and selective tumor uptake, which is a key feature for the development of radionuclide-based imaging agents for early and late-stage ccRCC and hypoxic tumors.
    DOI:  https://doi.org/10.1021/jacs.5c05198
  14. Mol Pharm. 2025 May 14.
    Self-Assembled Peptide PROTAC Prodrugs Targeting FOXM1 for Cancer Therapy.
    Huajie Zeng, Zhiguo Fang, Yinghua Feng, Tong Su, Weibing Miao, Zihua Wang.
      Proteolysis-targeting chimeras (PROTACs) represent a promising strategy for addressing ″undruggable″ proteins in cancer therapy. However, challenges such as poor bioavailability, limited cellular permeability, and inadequate targeting hinder their effectiveness. Herein, we present a novel PROTAC prodrug, NFTP, designed for FOXM1 degradation, which leverages self-assembled peptides functionalized with an integrin α-6 ligand to enhance tumor targeting and proteolysis in vivo. NFTP effectively penetrates tumor cells, induces FOXM1 degradation, inhibits cancer cell survival and migration, and promotes apoptosis in vitro. In a 4T1 mouse xenograft model, NFTP demonstrated efficient FOXM1-targeted degradation, significant tumor growth inhibition, and low systemic toxicity. This self-assembling FOXM1 PROTAC platform demonstrates enhanced tumor-targeting precision and superior therapeutic performance in vivo, representing a promising paradigm shift in targeted cancer therapy.
    Keywords:  FOXM1; PROTAC; cancer therapy; self-assembled peptide
    DOI:  https://doi.org/10.1021/acs.molpharmaceut.5c00219
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