bims-cepepe 2025-07-13 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2025–07–13
thirteen papers selected by
Henry Lamb, Queensland University of Technology

Cyclotides as novel plant-derived scaffolds for orally active cyclic peptide therapeutics.
Global Protein-Ligand Binding Affinity Profiling via Photocatalytic Labeling.
Peptide cyclization via late-stage functionalization of tryptophan by sulfonium.
A Computational Workflow for Structure-Guided Design of Potent and Selective Kinase Peptide Substrates.
The impact of peptide surface density on PD-L1 receptor inhibition as a therapeutic strategy for triple negative breast cancer.
Prediction of protein-protein interaction sites between Russell's viper PLA2 and the γ-phospholipase inhibitor PIP from the amino acid frequency distribution of a bio-panned peptide set.
EGFRp4 Peptides: A Novel Strategy for Epidermal Growth Factor Receptor (EGFR) Inhibitor.
Investigation of an OATP1B inhibitory effect by a cyclic peptide using the endogenous biomarker coproporphyrin-I in monkeys.
Tri-arabinosylation facilitates the bioactivity of CLE3 peptide in Arabidopsis.
Cysteine pattern barcoding-based dataset filtration enhances the machine learning-assisted interpretation of Conus venom peptide therapeutics.
A 3D In Vitro Blood-Brain Barrier Model to Study Pathogen and Drug Effects on the BBB.
Macrocyclization of backbone N-methylated peptides by a prolyl oligopeptidase with a distinctive substrate recognition mechanism.
Structure-guided design and development of cyclic peptide allosteric activators of Polycomb Repressive Complex 2.

Mol Cells. 2025 Jul 04. pii: S1016-8478(25)00076-7. [Epub ahead of print] 100252

Cyclotides as novel plant-derived scaffolds for orally active cyclic peptide therapeutics.

Youbong Hyun.

  Cyclotides are a group of plant-derived small peptides, characterized by a head-to-tail cyclic backbone and three conserved cysteine knots. The unique structure endows cyclotides with exceptional chemical stability and intrinsic cell-penetrating capacities, allowing them to serve as orally active agents for host defense. These properties also position cyclotides as ideal scaffolds for the design of novel edible drugs. This review provides a comprehensive overview of the discovery, distribution, and evolutionary divergence of cyclotides in flowering plants, while also discussing their classification and applications in structure-based engineering for various purposes. Additionally, recent advancements in the biological and pharmaceutical activities of both naturally occurring and synthetic 'designer' cyclotides are summarized. The complexities of cyclotide biosynthetic pathway have posed challenges for their synthesis in non-native organisms. Thus, the article concludes by exploring the latest strategies for large-scale cyclotide production in plants, highlighting Viola species as rich sources for future cyclotide engineering.

Keywords:  Cyclic peptides; Cyclotide; Orally active drug delivery; Viola

DOI:  https://doi.org/10.1016/j.mocell.2025.100252
bioRxiv. 2025 Jul 06. pii: 2025.07.04.662444. [Epub ahead of print]

Global Protein-Ligand Binding Affinity Profiling via Photocatalytic Labeling.

Charles David Warren, Noah Yardeny, Siyang Peng, Colin Spencer Burdette, Jacob B Geri.

Protein-ligand binding, selectivity, and affinity dictate the effects of drugs and endogenous molecules in cells. Currently, potential protein-ligand interactions are identified by qualitative interpretation of proteomic, transcriptomic, or genomic data, then binding affinities of hits are measured using purified proteins or engineered reporter systems to validate and quantify the strength of individual interactions. Few methods enable simultaneous target identification and biophysical affinity measurement, and these either apply to specific enzyme classes or proteins with ligand dependent shifts in stability. Here we describe a general platform, termed Affinity Map, which leverages competitive binding analysis, high fidelity photocatalytic labeling, and high throughput proteomics for global quantitative binding affinity profiling. We show that this method is applicable to major classes of ligands, including small molecules, linear peptides, cyclic peptides, and proteins, and can measure affinities between unmodified ligands and proteins in cell lysates, organ extracts, and live cell surfaces.

DOI: https://doi.org/10.1101/2025.07.04.662444
Org Biomol Chem. 2025 Jul 07.

Peptide cyclization via late-stage functionalization of tryptophan by sulfonium.

Yusong Han, Jia Chen, Shan Feng, Mingxuan Wu.

Cyclic peptides exhibit important biological activities and are widely found in natural products and peptide-based drugs. Therefore, the development of synthesis methods for cyclic peptides is essential. In recent years, tryptophan-mediated cyclic peptides have emerged as bioactive molecules, but current methods require unique unnatural amino acids and transition metals as catalysts. Our group recently reported a tryptophan site-selective crosslinking in the protein binding pocket by sulfonium peptide via single-electron transfer under UV irradiation. Herein, we expanded the reaction from intermolecular to intramolecular crosslinking to achieve peptide cyclization. This method enables the preparation of tryptophan-mediated cyclic peptides from peptides via two steps: transformation of Met or Cys to dimethylsulfonium followed by crosslinking under UV irradiation with tryptophan. Therefore, this method enables synthesis using only natural amino acids, without any special catalysts. We also investigated the regioselectivity of the indole ring and found that C(6) was favoured, followed by C(4) and C(7). Next, we investigated crosslinking regioselectivity in the reader CBX1 protein binding pocket and found that C(7) was favoured, which was quite distinct from peptide cyclization. The collected data indicated that indole regioselectivity is determined by local interactions between the indole and sulfonium. Overall, this study demonstrated a feasible method for peptide cyclization using sulfonium and tryptophan. Although yield and regioselectivity are not great at the moment, we believe that this study will pave the way for future improvements based on mechanistic insights.

DOI: https://doi.org/10.1039/d5ob00776c
bioRxiv. 2025 Jul 05. pii: 2025.07.04.663216. [Epub ahead of print]

A Computational Workflow for Structure-Guided Design of Potent and Selective Kinase Peptide Substrates.

Abeeb A Yekeen, Cynthia J Meyer, Melissa McCoy, Bruce Posner, Kenneth D Westover.

Kinases are pivotal cell signaling regulators and prominent drug targets. Short peptide substrates are widely used in kinase activity assays essential for investigating kinase biology and drug discovery. However, designing substrates with high activity and specificity remains challenging. Here, we present Subtimizer (substrate optimizer), a streamlined computational pipeline for structure-guided kinase peptide substrate design using AlphaFold-Multimer for structure modeling, ProteinMPNN for sequence design, and AlphaFold2-based interface evaluation. Applied to five kinases, four showed substantially improved activity (up to 350%) with designed peptides. Kinetic analyses revealed >2-fold reductions in Michaelis constant (Km), indicating improved enzyme-substrate affinity. Two designed peptides exhibited >5-fold improvement in selectivity. This study demonstrates AI-driven structure-guided protein design as an effective approach for developing potent and selective kinase substrates, facilitating assay development for drug discovery and functional investigation of the kinome.

DOI: https://doi.org/10.1101/2025.07.04.663216
J Control Release. 2025 Jul 07. pii: S0168-3659(25)00640-6. [Epub ahead of print] 114019

The impact of peptide surface density on PD-L1 receptor inhibition as a therapeutic strategy for triple negative breast cancer.

Naira Keshishian, Rudolf G Abdelmessih, Debra T Auguste.

  Immune checkpoint inhibitors (ICIs) disrupt receptor signaling, allowing the immune system to recognize and eliminate tumor cells across various cancers. ICI therapies have success in some cancers; however, most cancer patients do not show a therapeutic response. Antibodies, the current standard for ICIs, exhibit limited tumor penetration and immune-related side effects prompting the need for alternative strategies. Peptides are a promising alternative with the potential to overcome these limitations; however, their moderate binding affinity necessitates delivery platforms that enhance their therapeutic potential. Coupling peptides to nanoscale drug delivery systems such as liposomes has enhanced peptide binding affinity, selectivity and in vivo effectiveness. In this study, programmed cell death ligand 1 (PD-L1) antagonist peptides were conjugated to liposomes at varying surface densities to investigate the impact of density on tumor progression and immune cell infiltration. In vitro studies revealed that increasing the peptide density from 9000 peptides/μm2 (1 mol%) to 53,000 peptides/μm2 (3 mol%) enhanced binding extent, affinity, and rate. In contrast, in vivo studies showed that higher-density formulations had reduced tumor accumulation and no therapeutic effect, while the 24,000 peptides/μm2 (1.5 mol%) formulation significantly inhibited tumor growth and promoted CD8+ T cell infiltration. These findings highlight the importance of optimizing ligand density for effective peptide-based ICI therapies.

Keywords:  Immunotherapy; PD-L1; Peptide conjugated liposomes; Peptide density; TNBC; Targeted drug delivery

DOI:  https://doi.org/10.1016/j.jconrel.2025.114019
Integr Biol (Camb). 2025 Jan 08. pii: zyaf012. [Epub ahead of print]17

Prediction of protein-protein interaction sites between Russell's viper PLA2 and the γ-phospholipase inhibitor PIP from the amino acid frequency distribution of a bio-panned peptide set.

Karthika Rajan, Margaret Reick, Aswathy Alangode, Martin Reick.

  We screened a random peptide phage display library using Russell's viper venom phospholipase A2 (RV-PLA2) as bait. Sequence information from the resulting set of bio-panned heptapeptides was analyzed and mined to determine likely sites of interaction between two subunits of RV-PLA2 homo dimers and between RV-PLA2 and the γPLA2 inhibitor PIP from Malayopython reticulatus. This was accomplished in part by sequence alignment of the affinity-selected peptides with the sequences of RV-PLA2 and PIP. Because similarity scores calculated from sequence alignments proved inadequate to determine interaction interfaces accurately for RV-PLA2 dimers, we explored the use of amino acid frequency-based interactions scores (SFI/SFIN) for a more accurate prediction of protein-protein interaction sites. Heptamers with elevated SFI(N) scores were compared to interfaces of interaction observed in crystal structures of RV-PLA2 homodimers and to sites of interaction predicted by protein-protein docking between structures of RV-PLA2 and model of PIP. Segments with a high density of protein-protein contacts coincided with heptamer sequences exhibiting SFI and/or SFIN scores significantly above average, in both RV-PLA2 homodimers and in RV-PLA2 γPLI heteromeric structures. Elevated SFI and SFIN scores were associated with peptide function since the heptamers with some of the highest SFI and SFI(N) scores, LPGLPLS, GLPLSLQ and SLQNGLY constitute the known PLA2 inhibitor P-PB.I (LPGLPLSLQNGLY) while KLGRVDI, and WDGVYIR, constitute PIP-17 (LGRVDIHVWDGVYIRGR), IC50 for hsPLA2: 5.3 μM. A graph showing the alignment of maxima between SFI scores and average solvent accessibility (per heptamer) suggests that solvent accessibility is a major driver of both protein-protein interaction and phage selection. Insights We show by computational methods that in sets of small phage-displayed peptides of the same length selected for binding to the same target protein, amino acids contributing to binding at a particular position occur at higher frequencies than in random peptides. This position-specific selection of particular amino acids can be detected in the position-specific amino acid frequency distribution of that set of selected peptides. Therefore, when this position-specific amino acid frequency is mapped back onto a particular amino acid sequence of the same length, the sum of these frequencies can function as a measure of enrichment of selected amino acids.

Keywords:  Russell’s viper; models; peptide library; phage; phospholipase A2; protein–protein interaction; sequence alignment; snake venom; structural

DOI:  https://doi.org/10.1093/intbio/zyaf012
ACS Omega. 2025 Jul 01. 10(25): 26848-26856

EGFRp4 Peptides: A Novel Strategy for Epidermal Growth Factor Receptor (EGFR) Inhibitor.

Nattanan Jiwacharoenchai, Duangnapa Kiriwan, Shu-Yu Chang, Chang Ching Weng, Yaw-Kuen Li, Lueacha Tabtimmai, Kiattawee Choowongkomon.

The epidermal growth factor receptor (EGFR) plays a pivotal role in cell signaling pathways, and its malfunction is implicated in cancers such as lung, breast, and gastric cancer. Numerous studies have explored EGFR-specific peptides using various methods, excluding ribosome display peptide libraries. In this study, EGFRp1 and EGFRp4 peptides (11-mer) were discovered through ribosome display selection against the extracellular domain of EGFR (EGFR ECD or sEGFR). Notably, EGFRp4 exhibited superior binding affinity within the nanomolar range compared with other reported EGFR-specific peptides. It also demonstrated a positive cooperativity model with EGFR ECD across three experimental approaches: fluorescence polarization, surface plasmon resonance, and ligand tracer. Molecular docking substantiated this model, indicating that EGFRp4 prefers binding to active sEGFR conformations rather than inactive conformations. Furthermore, conjugating EGFRp4 onto NP1 using a GFLG linker notably enhanced the NP1 cyclic peptide performance in inhibiting EGFR-expressing cell lines. This conjugate demonstrated efficacy in inducing apoptosis and suppressing EGFR phosphorylation, comparable to that of Erlotinib. These findings underscore the potential of EGFRp4 as a peptide conjugate for EGFR-targeted applications.

DOI: https://doi.org/10.1021/acsomega.5c01684
Drug Metab Pharmacokinet. 2025 Jun 03. pii: S1347-4367(25)00447-1. [Epub ahead of print]63 101497

Investigation of an OATP1B inhibitory effect by a cyclic peptide using the endogenous biomarker coproporphyrin-I in monkeys.

Hiromi Sawada, Yasuto Kido, Makoto Iwasaki, Kimiko Nishida, Kei Mayumi, Ryosuke Watari.

  Peptide drugs are expected to be a new modality that will replace traditional small molecule drugs. As the number of approved peptide drugs increases, they are being co-administered with various drugs, but there is a limited number of reports on their drug-drug interaction (DDI) in both in vitro and in vivo (clinical) studies. In this study, we investigated the transporter inhibitory potential of Compound A, a macrocyclic peptide (3.5 kDa) for the treatment of pain. We found that Compound A exhibited a strong inhibitory effect on the organic anion transporting polypeptide (OATP) 1B in an in vitro study. To assess the in vivo OATP1B inhibitory potential, Compound A was intravenously or subcutaneously administered to monkeys, and the plasma concentration of coproporphyrin-I (CP-I), an endogenous biomarker of OATP1B, was determined. Compound A markedly increased the CP-I concentration in monkeys. A semi-mechanistic pharmacokinetic model analysis using the CP-I concentration revealed that Compound A is a highly potent in vivo OATP1B inhibitor (in vivo Ki, OATP1B: 59.9 ng/mL as total plasma concentration). Our findings suggest that even peptides with a large molecular weight can cause DDI. These results offer valuable information for the further development of DDI guidelines for peptides.

Keywords:  Biomarker; Coproporphyrin-I; Drug-drug interaction; Monkey; Organic anion transporting polypeptide 1B; Peptide; Pharmacokinetics; Transporter

DOI:  https://doi.org/10.1016/j.dmpk.2025.101497
Plant Biotechnol (Tokyo). 2025 Jun 25. 42(2): 163-166

Tri-arabinosylation facilitates the bioactivity of CLE3 peptide in Arabidopsis.

Satoru Nakagami, Taiki Kajiwara, Hajime Hibino, Taku Yoshiya, Masayoshi Mochizuki, Shugo Tsuda, Toshihiro Yamamoto, Shinichiro Sawa.

  Post-translational modification is critical for the bioactivity of small secreted-signaling peptides. The shoot apical meristem (SAM) activity that defines SAM size is controlled by the CLAVATA3 (CLV3) peptide ligand, which belongs to the CLV3/EMBRYO SURROUNDING REGIONRELATED (CLE) family, and its cognate receptor CLV1. The mature CLV3 peptide is post-translationally modified with tri-arabinose, increasing the binding affinity with CLV1. However, the mature form of most CLE peptides is unknown. Here we apply the synthetic CLE3 peptide with tri-arabinose to clv3 mutant to determine whether the CLE3 peptide can reduce the SAM size. We show that tri-arabinosylated CLE3 peptide exhibits stronger bioactivity in the SAM in a CLV1/BAM1-dependent manner. Our data emphasizes the importance of post-translational modification on peptide signaling, helping to characterize bona fide mature peptides.

Keywords:  Arabidopsis; CLE peptide; post-translational modification; shoot apical meristem

DOI:  https://doi.org/10.5511/plantbiotechnology.25.0120b
PLoS One. 2025 ;20(7): e0327578

Cysteine pattern barcoding-based dataset filtration enhances the machine learning-assisted interpretation of Conus venom peptide therapeutics.

Rimsha Bibi, Noshaba Qasmi, Sajid Rashid.

Crude cone snail venom is a rich source of bioactive compounds with significant therapeutic potential. In this study, we conducted a comprehensive analysis of 5,985 cone snail peptides across 82 Conus species to identify unique cysteine (Cys) patterns and associated frameworks. The classification of these Cys patterns, based on conserved framework combinations, enabled the generation of species-level pattern barcodes. These barcodes were then evaluated to assess the species correlations of individual sequences. By analyzing 151 known Conus peptide PDB files, we computed Cys disulfide linkages to assess overall stability profiles. Incorporating barcode data allowed us to filter the dataset and prepare it for machine learning (ML) processing. Random Forest (RF) modeling, a supervised learning technique, was used to predict the therapeutic potential of venom peptides. Feature extraction was based on known venom-derived approved peptide-based drugs. The dataset was split into a 70:30 train-test ratio. A total of 6,430 peptides (5,985 from cone snails and 445 from other venomous species) were used to evaluate model prediction capability. The proposed model achieved ideal accuracy (90.48%) in peptide therapeutic classification. Subsequent model outputs underwent further structural and binding pattern analysis against known targets, revealing significant similarities between the binding patterns of approved and novel peptides. The model's performance could be further enhanced by incorporating additional datasets and optimizing feature selection, potentially broadening its applicability to larger peptide datasets. Overall, this study underscores the potential of ML in advancing pharmacological research on diverse venom peptides.

DOI: https://doi.org/10.1371/journal.pone.0327578
Methods Mol Biol. 2025 ;2950 61-69

A 3D In Vitro Blood-Brain Barrier Model to Study Pathogen and Drug Effects on the BBB.

Alizé Proust, Robert J Wilkinson.

  In vitro blood-brain barrier models play a pivotal role in neuroscience research to study the intricate structure and function of the blood-brain barrier, facilitating drug development, enhancing disease understanding, and advancing treatment strategies for neurological disorders. Here, we present a triple contact coculture model with endothelial cells, astrocytes, and pericytes and offer a physiologically relevant BBB.

Keywords:  Astrocytes; Blood-brain barrier; Contact model; Endothelial cells; Pericytes

DOI:  https://doi.org/10.1007/978-1-0716-4674-8_5
Chem Sci. 2025 Jul 02.

Macrocyclization of backbone N-methylated peptides by a prolyl oligopeptidase with a distinctive substrate recognition mechanism.

Emmanuel Matabaro, Haigang Song, Lukas Sonderegger, Fabio Gherlone, Andrew M Giltrap, Sam Liver, Alvar D Gossert, Markus Künzler, James H Naismith.

Macrocyclization and multiple backbone N-methylations can significantly improve the pharmacological properties of peptides. Since chemical synthesis of such compounds is often challenging, enzyme-based production platforms are an interesting option. Here, we characterized OphP, a serine peptidase involved in the cyclization of omphalotins, a group of ribosomally produced dodecapeptides with multiple backbone N-methylations. OphP displays robust peptidase and macrocyclase activity towards multiply α-N-methylated peptides of various lengths and composition derived from the omphalotin precursor protein OphMA. In addition, OphP processes, with lower efficiency, peptides unrelated to OphMA, containing a MeGly, MeAla or Pro residue at the P1 site. Structural analysis reveals that OphP adopts a canonical prolyl oligopeptidase fold but, unlike other enzymes of this enzyme family, recognizes its substrates by their hydrophobic and multiply backbone N-methylated core rather than by the follower peptide. The activity of OphP could be harnessed for the enzymatic production of therapeutic peptides.

DOI: https://doi.org/10.1039/d5sc03723a
bioRxiv. 2025 Jun 26. pii: 2025.06.26.661818. [Epub ahead of print]

Structure-guided design and development of cyclic peptide allosteric activators of Polycomb Repressive Complex 2.

Yasuaki Tokodai, Mark Matyas, Azumi Ishigamori, Erin O'Connor, Angel Zhang, Sora Yamazaki, Tsunehide Hiraki, Noah Lohar, Liqi Yao, Ryan Stoner, Luis E Paez-Beltran, Kayoko Kanamitsu, Noriko Nakaya, Satoshi Ichikawa, Justin Brumbaugh, Vignesh Kasinath, Fumika Yakushiji.

Dysregulation of the histone methyltransferase Polycomb repressive complex 2 (PRC2) results in aberrant silencing of tumor suppressors and activation of oncogenes. Targeting PRC2 with compounds holds significant potential for both basic research and therapeutic applications. Here, we leveraged extensive structural studies of PRC2 to design a cyclic peptide that robustly activates PRC2. Structure-activity relationship studies guided the functional optimization of this cyclic peptide, yielding a Phenylalanine-type (Phe-type) cyclic peptide with approximately eight-fold activation compared to that of the poised state of PRC2. A 3.3Å cryo-electron microscopy structure of the PRC2-peptide complex, combined with biochemical analyses, revealed a shift in the H3K27 methylation from mono-(me1) and dimethylation (me2) to trimethylation (me3). Finally, we demonstrated that the cyclic peptide exhibits improved mouse plasma stability and can also be readily taken up by cells which results in a shift of the H3K27 methylation landscape to trimethylation, similar to the observed effects in vitro. These findings support the utility of such molecules for probing PRC2 activation and targeting dysregulated H3K27 methylation in cancer.

DOI: https://doi.org/10.1101/2025.06.26.661818