bims-cepepe 2025-06-22 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2025–06–22
fourteen papers selected by
Henry Lamb, Queensland University of Technology

Total Synthesis of N-hydroxy Cyclic Peptide: Talarolide A.
Accurate de novo design of high-affinity protein-binding macrocycles using deep learning.
Hydrophobic And Polarized Aromatic Residues Promote Internalization Of Arg-Rich Cell Penetrating Peptides Through Ionpair-π Interactions.
Synthesis of Bicyclic Peptides Using Cyanopyridine-Aminothiol Click Chemistry.
Antimigratory and Antiproliferative Effects of the Brain-Targeted Peptide Conjugate PepH3-vCPP2319 on Triple Negative Breast Cancer Cell Cultures.
π-Turns in Peptides: A Crystal-State Literature Survey.
Impact of stereochemical replacement on activity and selectivity of membrane-active antibacterial and antifungal cyclic peptides.
Investigating the Site-Specific Impact of Fluorine Substitution on Aromatic Interactions in a Tryptophan Zipper Peptide.
An intracellular release peptide display technology unveils an antimicrobial peptide with low probability for resistance development.
Membrane disruption properties of poly-glycine arginine dipeptide repeats affected by peptide repeats continuity and membrane composition.
Development of Cysteic Acid-Modified FAP Radioligands for Enhanced Renal Clearance: From Preclinical Optimization to First-in-Human Study.
Somatostatin receptor 2 targeting peptide modifications for peptide-drug conjugate treatment of small cell lung cancer.
Effects of toxin peptides PepA1 and PepA2 on human red blood cell membrane model: A molecular dynamics study.
Optimizing the Synthesis of Desotamides and Related Cyclic Antimicrobial Peptides.

Methods Mol Biol. 2025 ;2931 1-12

Total Synthesis of N-hydroxy Cyclic Peptide: Talarolide A.

Waleed M Hussein, Yuxuan Zhu, Angela A Salim, Robert J Capon.

  This chapter reports the successful synthesis of talarolide A (1), a natural product exhibiting a unique hydroxamate H-bond bridge, through a carefully designed solid-phase approach. By systematically selecting the disconnection site, we synthesized a protected linear precursor, which was then sequentially deprotected and cyclized. We discovered that the order of deprotection and cyclization was critical: precyclization of the unprotected peptide facilitated the correct conformational folding essential for achieving the natural product's structure. This approach not only yielded talarolide A but also revealed a noncanonical atropisomer (atrop-talarolide A 5), providing new insights into the role of hydroxamate H-bond bridging in atropisomerism among nonribosomal peptide synthetase (NRPS)-derived cyclic peptides.

Keywords:  Conformer stabilization; Cyclic peptide; Hydroxamate H-bond bridge; Marine-derived fungus; Natural product; Talarolide A; Talaromyces; Total synthesis

DOI:  https://doi.org/10.1007/978-1-0716-4562-8_1
Nat Chem Biol. 2025 Jun 20.

Accurate de novo design of high-affinity protein-binding macrocycles using deep learning.

Stephen A Rettie, David Juergens, Victor Adebomi, Yensi Flores Bueso, Qinqin Zhao, Alexandria N Leveille, Andi Liu, Asim K Bera, Joana A Wilms, Alina Üffing, Alex Kang, Evans Brackenbrough, Mila Lamb, Stacey R Gerben, Analisa Murray, Paul M Levine, Maika Schneider, Vibha Vasireddy, Sergey Ovchinnikov, Oliver H Weiergräber, Dieter Willbold, Joshua A Kritzer, Joseph D Mougous, David Baker, Frank DiMaio, Gaurav Bhardwaj.

Developing macrocyclic binders to therapeutic proteins typically relies on large-scale screening methods that are resource intensive and provide little control over binding mode. Despite progress in protein design, there are currently no robust approaches for de novo design of protein-binding macrocycles. Here we introduce RFpeptides, a denoising diffusion-based pipeline for designing macrocyclic binders against protein targets of interest. We tested 20 or fewer designed macrocycles against each of four diverse proteins and obtained binders with medium to high affinity against all targets. For one of the targets, Rhombotarget A (RbtA), we designed a high-affinity binder (Kd < 10 nM) despite starting from the predicted target structure. X-ray structures for macrocycle-bound myeloid cell leukemia 1, γ-aminobutyric acid type A receptor-associated protein and RbtA complexes match closely with the computational models, with a Cα root-mean-square deviation < 1.5 Å to the design models. RFpeptides provides a framework for rapid and custom design of macrocyclic peptides for diagnostic and therapeutic applications.

DOI: https://doi.org/10.1038/s41589-025-01929-w
Chemistry. 2025 Jun 20. e202501138

Hydrophobic And Polarized Aromatic Residues Promote Internalization Of Arg-Rich Cell Penetrating Peptides Through Ionpair-π Interactions.

Sonia Khemaissa, Antonio Bauzá, Émilie Lesur, Françoise Illien, Sandrine Sagan, Antonio Frontera, Astrid Walrant.

  Cell penetrating peptides (CPPs) are small sequences that can cross cell membranes. Arg and Trp are highly prevalent amino-acids in natural and synthetic efficient CPP sequences. In particular, Trp is essential and cannot be substituted by other hydrophobic or aromatic amino-acids. The aim of the present study is to decipher the role of Trp in synthetic Arg/Trp CPP sequences. To do so, a small peptide library in which this residue was substituted by other natural or non-natural amino-acids was designed. Internalization of these peptides in cells was evaluated and it appeared that combining aromaticity and hydrophobicity in the presence of Arg residues leads to enhanced internalization. The study of the interaction of these peptides with model lipid membranes revealed that the modulation of hydrophobicity promoted insertion in bilayers, but had little impact on the binding affinity. On the other hand, more hydrophobic substitutes of Trp led to more favorable binding enthalpies to heparin. With DFT analysis, we suggest that ion-pair···π interactions between the aromatic ring and the ion pair formed by the positively charges Arg and the negatively charged cell surface groups can be established, and could be at the origin of the unique internalization properties of Trp-containing Arg-rich CPPs.

Keywords:  Cell-penetrating peptides; DFT; Glycosaminoglycans; Tryptophan

DOI:  https://doi.org/10.1002/chem.202501138
Methods Mol Biol. 2025 ;2931 13-25

Synthesis of Bicyclic Peptides Using Cyanopyridine-Aminothiol Click Chemistry.

Sven Ullrich, Christoph Nitsche.

  Constrained peptides are emerging as promising structures in therapeutic development. Offering compatibility with genetically encoded libraries for drug discovery, biocompatible methods to constrain peptides are particularly attractive. While there are many such methods to construct cyclic and stapled peptides, the biocompatible generation of bicyclic peptides is less explored. Addressing this need for biocompatible and selective ways to generate peptide bicycles, we previously developed a strategy based on noncanonical amino acids leveraging the reactivity of cyanopyridine and 1,2-aminothiol. This protocol provides detailed step-by-step instructions for the synthesis of these peptide bicycles and is designed to be accessible even to laboratories with limited synthetic chemistry resources. It outlines the solid-phase peptide synthesis of linear peptide precursors that efficiently form bicyclic structures in aqueous buffer at physiological pH. Utilizing commercially available building blocks, we devised a method to synthesize the noncanonical amino acids that are essential for bicyclization directly on the solid support during peptide synthesis.

Keywords:  Biocompatible chemistry; Constrained peptides; Noncanonical amino acids; Peptide macrocyclization; Solid-phase peptide synthesis

DOI:  https://doi.org/10.1007/978-1-0716-4562-8_2
J Pept Sci. 2025 Aug;31(8): e70035

Antimigratory and Antiproliferative Effects of the Brain-Targeted Peptide Conjugate PepH3-vCPP2319 on Triple Negative Breast Cancer Cell Cultures.

Catarina Gonçalves, Miguel A R B Castanho, Marco Cavaco, Vera Neves.

  Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype affecting mostly younger women with a poor 5-year overall survival. It is characterized by a high metastization rate, particularly to the brain, where the blood-brain barrier (BBB) hinders the pharmaceuticals delivery. New anticancer drugs able to inhibit cell migration are required to effectively prevent the development of metastasis. PepH3-vCPP2319 (AGILKRW(Ahx)WRRRYRRWRRRRRQRRRPRR-amide), consisting of the conjugation of the BBB peptide shuttle (BBBpS) PepH3 (AGILKRW-amide) to the anticancer peptide (ACP) vCPP2319 (WRRRYRRWRRRRRQRRRPRR-amide), was reported to have high anticancer activity (IC50 = 5.0 μM) toward highly aggressive TNBC cells (MDA-MB-231) paired with 2-fold increased accumulation in the brain when compared to unconjugated vCPP2319. Herein, we demonstrate that PepH3-vCPP2319 inhibits cell migration and proliferation in wound healing assays, outperforming the gold standard small chemical inhibitor, iCRT-3. The concentration required to inhibit cell migration is 10-fold lower for PepH3-vCPP2319 (0.5 μM) when compared with iCRT-3 (50 μM). Likewise, PepH3-vCPP2319 at 2.5 μM was more efficient in preventing cell proliferation when compared with 50 μM iCRT-3, with 45% reduction in spheroid diameter. This study sheds light on the antimetastatic potential of PepH3-vCPP2319 through abrogation of cell migration to distant sites, including the brain.

Keywords:  anticancer peptides; cancer metastasis; cancer stem cells; cell migration; triple negative breast cancer

DOI:  https://doi.org/10.1002/psc.70035
J Pept Sci. 2025 Jul;31(7): e70036

π-Turns in Peptides: A Crystal-State Literature Survey.

Barbara Biondi, Fernando Formaggio, Claudio Toniolo, Cristina Peggion, Marco Crisma.

  The results of an analysis on the presence of π-turns, characterized by an i ← i + 5 C=O···H-N intramolecular hydrogen bond, in the X-ray diffraction structures of peptides are discussed. The survey returned a total of 55 π-turn occurrences in linear and cyclic peptides. π-Turns characterized by a helical conformation for residue i + 4, but with a screw sense opposite to that of the three preceding residues, are largely prevailing. They are often found at the C-end of incipient or fully developed α-helices, 310-helices, and mixed α-/310-helices, thus acting as a C-capping motif. However, the structures of two linear peptides and 15 cyclopeptides indicate that these types of π-turns can exist in isolation, without the support of a preceding helix. The frequent presence of additional intramolecular hydrogen bonds internal to the π-turn is also investigated. Cyclopeptides offered examples of two types of π-turns that have no parallel in the structures of proteins. Differently from proteins, π-turns characterized by helical ϕ, ψ sets of the same screw sense for all internal residues are hitherto unreported in the X-ray diffraction structures of peptides. A suggestion for the rational design in peptides/peptidomimetics of a π-turn featuring the screw-sense reversal of residue i + 4 is proposed.

Keywords:  X‐ray diffraction; linear and cyclic peptides; peptide conformation; statistical analysis; π‐turns

DOI:  https://doi.org/10.1002/psc.70036
NPJ Antimicrob Resist. 2025 Jun 17. 3(1): 56

Impact of stereochemical replacement on activity and selectivity of membrane-active antibacterial and antifungal cyclic peptides.

Sandeep Lohan, Anastasia G Konshina, Eman H M Mohammed, Naiera M Helmy, Srishhti K Jha, Rakesh Kumar Tiwari, Innokentiy Maslennikov, Roman G Efremov, Keykavous Parang.

Herein, we report a library of 7-mer macrocyclic peptides designed by systematically replacing one, multiple, or all L-amino acids with their D-isomers in our previously identified hit compounds. Lead peptides, 15c and 16c, showed broad-spectrum activity against bacteria (Gram-positive minimum inhibitory activity (MIC 1.5-6.2 µg/mL and Gram-negative MIC 6.2-25 µg/mL) and fungi (MIC = 3.1-25 µg/mL). Additionally, peptides 15c and 16c showed rapid kill kinetics and biofilm degradation potential against both bacteria and fungi, while resistance development was not observed. The antimicrobial effect of these macrocyclic peptides was attributed to their membranolytic action, which was confirmed by calcein dye leakage assay and scanning electron microscopy analysis. Both peptides, 15c (HC50 = 335 µg/mL) and 16c (HC50 = 310 µg/mL), exhibited significantly lower hemolytic activity compared to their parent peptide p3 (HC50 = 230 µg/mL). At 100 µg/mL, both peptides showed >90% cell viability after 24 h incubation across four normal mammalian cell lines. Both peptides showed plasma stability (t1/2 ≥ 6 h), further supporting their therapeutic potential. Finally, the molecular mechanisms determining the pharmacological properties of a number of typical representatives of each series of synthesized peptides were investigated by NMR spectroscopy and computer simulations. The study revealed specific combinations of structural, dynamic, and hydrophobic parameters of these amphiphilic peptides that allow a reasonable prediction of their hemolytic activity. This Structure-Activity Relationship provides a basis for the rational design of peptides or peptidomimetics with predefined pharmacological profiles.

DOI: https://doi.org/10.1038/s44259-025-00121-3
Chemistry. 2025 Jun 19. e202501263

Investigating the Site-Specific Impact of Fluorine Substitution on Aromatic Interactions in a Tryptophan Zipper Peptide.

David Reiter, Ferhat Mutlu, Dominik Ebert, Marceline Noutio, Joachim Heberle, Mario Schubert, Beate Koksch.

  The stability of pairwise tryptophan (Trp) edge-to-face aromatic interactions has been exploited in the design of small tryptophan zipper (Trpzip) peptides. Herein, we report a systematic study of the regiospecific impact of four constitutional isomers of non-natural fluoro-Trp, regarding their incorporation at either edge- or face-position. Single fluorine substituents withdraw electron density from the indole moiety and introduce a highly electronegative component while the native geometry of Trp is maintained. We employed a library approach based on the sequence of Trpzip2 and assessed peptide structure and stability using CD, FTIR, and NMR spectroscopy. Global hairpin stability was improved or compromised upon site-specific incorporation of a single monofluoro-Trp regioisomer. Fluorine substitution revealed key CH/π interactions within the Trp/Trp packing and holds potential for the future optimization of aromatic interactions involving Trp.

Keywords:  Fluorine; NMR spectroscopy; Pi interactions; Tryptophan; peptides

DOI:  https://doi.org/10.1002/chem.202501263
iScience. 2025 Jun 20. 28(6): 112619

An intracellular release peptide display technology unveils an antimicrobial peptide with low probability for resistance development.

Anna Ebbensgaard, Catrina Olivera, Thomas Bentin, Henrik Franzyk, Godefroid Charbon, Peter E Nielsen, Anders Løbner-Olesen.

  Discovery of bioactive peptides, including those acting to permeabilize and/or kill bacterial cells (antimicrobial peptides) has drawn extensive interest in recent years. However, current technologies for their identification are limited. To address these limitations, the Intracellular Release Peptide Display (IRPD) technology allowing the recombinant "display" of intracellular linear peptides was developed. IRPD uses the protease domain of the capsid protein from the Semliki Forest virus as a scaffold to express and liberate linear peptides intracellularly in Escherichia coli. IRPD is a universal platform that allows screening of millions of peptides and the discovery of bioactive peptides from direct target interactions and independent of the cell envelope barrier. Here, we identified peptides that cause increased bacterial cell envelope permeability and lysis. The most promising candidate, P38, effectively kills Gram-negative pathogens by disrupting the inner membrane without detectable resistance development. Thus, P38 constitutes an interesting hit peptide for further development.

Keywords:  Applied microbiology; Microbiology; Peptides

DOI:  https://doi.org/10.1016/j.isci.2025.112619
J Mol Biol. 2025 Jun 11. pii: S0022-2836(25)00362-6. [Epub ahead of print] 169296

Membrane disruption properties of poly-glycine arginine dipeptide repeats affected by peptide repeats continuity and membrane composition.

Chia-Yi Ho, Yu-Jen Chang, Chih-Wen Yang, Orion Shih, U-Ser Jeng, Ing-Shouh Hwang, Wan-Chen Huang, Yun-Ru Chen.

  C9ORF72 hexanucleotide expansion is the most common genetic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTD). This expansion can be translated into dipeptide repeats (DPRs) through repeat-associated non-ATG (RAN) translation. Arginine-rich DPRs, i.e., poly-glycine arginine (poly-GR) and poly-proline arginine (poly-PR) are considered the most toxic ones among the five types of DPRs. We recently discovered that poly-GR forms helical conformation and is able to penetrate cell membranes, leading to cytotoxicity, but the mechanism remains unclear. Here, we investigated the membrane disruption mechanism of poly-GR related to its sequence and membrane composition. To test this, we stopped its continuously repeated sequence by inserting several proline residues to disrupt its helical structure. We found that the modification reduced its cytotoxicity and membrane disruption capability. Next, we examined the influence of lipid composition on the membrane-disrupting ability of poly-GR using various liposomes. Poly-GR caused higher leakage in the negatively charged liposomes compared to the neutral or positively charged ones. Cholesterol content affected the extent of disruption, while gangliosides had no significant effect. Using small-angle x-ray scattering (SAXS), total internal reflection fluorescence (TIRF) microscopy, and atomic force microscopy (AFM), we observed the behavior of poly-GR on lipid membranes. Finally, we directly treated mouse neuroblastoma to modulate the cholesterol content and found that cholesterol depletion inhibited the internalization of poly-GR into the cells and reduced cytotoxicity. These findings reveal that the conformation of poly-GR and the lipid composition influence its membrane penetration, offering insights into potential therapeutic strategies for C9ORF72-related diseases.

Keywords:  ALS; cholesterol; dipeptide repeats; liposome; membrane; poly-glycine arginine

DOI:  https://doi.org/10.1016/j.jmb.2025.169296
J Med Chem. 2025 Jun 18.

Development of Cysteic Acid-Modified FAP Radioligands for Enhanced Renal Clearance: From Preclinical Optimization to First-in-Human Study.

Simin Peng, Baoyuan Li, Mingming Sun, Jiaqi Yang, Zhikai Cai, Yang Liu, Peipei Tang, Dan Feng, Qianyong Cao, Pengcheng Ran, Kongzhen Hu.

Fibroblast activation protein (FAP)-targeting radioligands hold promise for cancer theranostics. Cyclic peptide-based DOTA-FAP-2286 radioligands have demonstrated high kidney uptake and retention, raising concerns regarding potential nephrotoxicity. Hence, we aimed to design three cysteic acid-modified FAP-targeting cyclic peptide ligands (DOTA-C1/C2/C3-FAP-2286) for reducing renal retention and optimizing pharmacokinetic properties. Competitive binding assays revealed maintained potent affinity for FAP (IC50 < 150 nM). Following systematic preclinical evaluation, [68Ga]Ga-C1-FAP-2286 exhibited optimal biodistribution characteristics, reducing renal uptake by 50% (2.12 ± 0.19% ID/g, P < 0.05), while maintaining tumor accumulation (7.08 ± 0.35 vs 6.26 ± 0.82% ID/g for [68Ga]Ga-FAP-2286), yielding a significantly improved tumor-to-kidney ratio (3.34 ± 0.15 vs 1.59 ± 0.53% ID/g). First-in-human PET/CT imaging in a metastatic gastric cancer patient demonstrated superior diagnostic performance compared to [18F]FDG, with intense uptake in primary lesions (SUVmax = 3.0), including [18F]FDG-negative and metastatic lesions. Thus, [68Ga]Ga-C1-FAP-2286 is a clinically translatable tracer for imaging FAP-expressing malignancies.

DOI: https://doi.org/10.1021/acs.jmedchem.5c01163
Acta Pharmacol Sin. 2025 Jun 18.

Somatostatin receptor 2 targeting peptide modifications for peptide-drug conjugate treatment of small cell lung cancer.

Qing Bo, Meng-Ge Zhang, Fan Yang, Yong Zheng, Ze-Lin Li, Yan-Min Zheng, Fang-Ming Wu, Jun Liang, Li Zhou, Dong-Sheng Li, Yun Wu, Chang-Lin Tian, Pei Lv, Pan Shi.

  Peptide-drug conjugate (PDC) represents a special therapeutic strategy to enhance drug delivery by targeting tumor cell receptors while minimizing off-target effects. Comparing the antibody-drug conjugate (ADC), the targeting peptide constitutes the pivotal component of PDC, especially with easy optimization of peptides to promote their in vivo stability, and with the agonist stimulated GPCR internalization to facilitate drug distribution into tumor cell plasma. Herein, we have optimized a highly stable peptide molecule LanTC targeting somatostatin receptor 2 (SSTR2), through amino acid substitution and disulfide bond modification from an FDA proved peptide drug Lanreotide. The LanTC based PDC was constructed through conjugation of the cytotoxic drug emtansine (DM1). The LanTC-DM1 PDC exhibited high stability and high agonist affinity to SSTR2. Subsequent in vitro and in vivo pharmacological data revealed that LanTC-DM1 PDC exhibited antitumor activity in small cell lung cancers (SCLC) which was known to have over-expressing SSTR2. The LanTC-DM1 PDC with specific targeting and antitumor activity provides a solid basis not only for advancing SSTR2-targeted PDCs as a promising therapy for SCLC, but also for other PDC developments targeting GPCRs in plasma membrane of tumor cells.

Keywords:  Cryo-EM; Peptide-Drug Conjugate (PDC); Somatostatin Receptor 2; anti-tumor efficacy; peptide modification; small cell lung cancer

DOI:  https://doi.org/10.1038/s41401-025-01584-w
Comput Biol Chem. 2025 Jun 14. pii: S1476-9271(25)00212-9. [Epub ahead of print]119 108552

Effects of toxin peptides PepA1 and PepA2 on human red blood cell membrane model: A molecular dynamics study.

Liling Zhao, Li Zhao, Haiyan Li, Zanxia Cao.

  PepA1 and PepA2 are type I toxin peptides with similar amino acid sequences, and both these peptides inhibit Staphylococcus aureus intracellularly and are toxic to human red blood cells (RBCs). PepA2 is 10-fold more cytotoxic than PepA1. The present study investigated the effects of these two peptides on human RBC membrane by performing microsecond-level molecular dynamics simulations using the GROMACS software package. The results indicated that both toxin peptides distorted and destabilized the RBC membrane. At low concentrations, the peptides showed no significant difference in their effects on the cell membrane; however, at high concentrations, PepA2 induced more distortion and destabilization of the cell membrane than PepA1 and enhanced cell permeability, resulting in the diffusion of more water molecules through the membrane. Polar residues in PepA2 and hydrophobic residues in PepA1 mainly formed hydrogen bonds with the water molecules penetrating the membrane. PepA2 formed more alpha-helical structures and was more stable than PepA1, which may explain its strong toxicity. PepA2 also strongly interacted with the RBC membrane as compared to PepA1. The results of the present study could facilitate better understanding of the mechanisms of toxic peptides and assist in designing efficient antibacterial drugs.

Keywords:  Cytotoxicity; Molecular dynamics simulation; PepA1; PepA2; Type I toxin peptides

DOI:  https://doi.org/10.1016/j.compbiolchem.2025.108552
Methods Mol Biol. 2025 ;2931 299-311

Optimizing the Synthesis of Desotamides and Related Cyclic Antimicrobial Peptides.

Archi Bansal, Yuzhang Li, Zyta M Ziora, Prashant Mishra, Peter M Moyle.

  The protocol described in this chapter details the synthesis of Desotamides, a class of cyclic hexapeptide antibiotics from Streptomyces species. These peptides, which often include D-amino acids and non-proteogenic amino acids, are challenging to produce using molecular biology techniques. Their synthesis involves loading the first amino acid onto 2-chlorotrityl chloride resin, followed by fluorenylmethyloxycarbonyl (Fmoc) solid phase synthesis of the linear peptide, cleavage of a protected peptide fragment from the resin using mild acidic conditions, and head-to-tail cyclization in solution. These synthetic processes permit the scalable production and engineering of Desotamides and novel-related antimicrobial species, without the limitations of biological synthesis methods.

Keywords:  Antimicrobials; Peptide cyclization; Solution phase cyclization

DOI:  https://doi.org/10.1007/978-1-0716-4562-8_23