bims-cepepe 2025-08-17 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2025–08–17
eleven papers selected by
Henry Lamb, Queensland University of Technology

Translocation of penetratin-like peptides involving calcium-dependent interactions between glycosaminoglycans and phosphocholine headgroups of the membrane lipid bilayer.
Target sequence-conditioned design of peptide binders using masked language modeling.
Decoding Protein-Peptide Interactions Using a Large, Target-Agnostic Yeast Surface Display Library.
High-throughput investigation of cyclin docking interactions reveals the complexity of motif binding determinants.
Targeted degradation of cell surface proteins through endocytosis triggered by cell-penetrating peptide-small molecule conjugates.
Engineering Peptide Modulators for T-Cell Migration by Structural Scaffold Matching.
Alchemical Simulation-Aided De Novo Design of Membrane-Active Antimicrobial Hepta-Peptides.
Peptide-mimetics derived from leucyl-tRNA synthetase are potential agents for the therapy of mt-tRNA related diseases.
Impacts of anti-drug antibodies on pharmacokinetic and pharmacodynamic actions of cell-permeable middle molecule peptide drug.
Targeting Cholesterol-Dependent Piezo1 Activation Impairs Amoeboid Migration in Melanoma Cells.
Discovery of Cell-Permeable Macrocyclic Cyclin A/B RxL Inhibitors that Demonstrate Anti-tumor Activity in E2F-Driven Cancers.

RSC Chem Biol. 2025 Aug 02.

Translocation of penetratin-like peptides involving calcium-dependent interactions between glycosaminoglycans and phosphocholine headgroups of the membrane lipid bilayer.

Bingwei He, Sonia Khemaissa, Sébastien Cardon, Rodrigue Marquant, Françoise Illien, Delphine Ravault, Fabienne Burlina, Emmanuelle Sachon, Astrid Walrant, Sandrine Sagan.

Cell-penetrating peptides (CPPs) can internalize ubiquitously in cells. To explore the specific targeting issue of CPPs, we used glycosaminoglycan (GAG)-binding peptides previously identified in Otx2 and En2 homeoproteins (HPs). The Otx2 sequence preferentially recognizes highly sulfated chondroitin (CS) and the En2 one, heparan sulfates (HS) GAGs. The two HPs internalize in specific cells thanks to their GAG-targeting sequence. We studied the capacity of chimeric peptides containing a GAG-targeting and a penetratin-like sequences to enter into various cell lines known to express different levels and types of GAGs. Since GAGs are found at the vicinity the membrane lipid bilayer, we also analyzed the putative binary and ternary interactions between heparin (HI), (4S,6S)-CS (CS-E), zwitterionic phosphocholine (PC) model membranes and those chimeric peptides. Altogether, our results demonstrate the existence of Ca2+-dependent interactions between GAGs and PC lipid bilayers, the major phospholipid headgroup found in animal cell plasma membrane. In addition, the interaction of CS-E (but not HI), with PC favors the binding of the chimeric CS-E-recognition motif-penetratin-like peptide and its subsequent crossing of the lipid membrane to access directly to the cytosol of cells. Altogether, this study brings further understanding of translocation mechanism of CPPs, which requires specific GAGs at the cell-surface. It also shed light on the role of GAGs in the cell transfer specificity and paracrine activity of HPs.

DOI: https://doi.org/10.1039/d5cb00099h
Nat Biotechnol. 2025 Aug 13.

Target sequence-conditioned design of peptide binders using masked language modeling.

Leo Tianlai Chen, Zachary Quinn, Madeleine Dumas, Christina Peng, Lauren Hong, Moises Lopez-Gonzalez, Alexander Mestre, Rio Watson, Sophia Vincoff, Lin Zhao, Jianli Wu, Audrey Stavrand, Mayumi Schaepers-Cheu, Tian Zi Wang, Divya Srijay, Connor Monticello, Pranay Vure, Rishab Pulugurta, Sarah Pertsemlidis, Kseniia Kholina, Shrey Goel, Matthew P DeLisa, Jen-Tsan Ashley Chi, Ray Truant, Hector C Aguilar, Pranam Chatterjee.

The computational design of protein-based binders presents unique opportunities to access 'undruggable' targets, but effective binder design often relies on stable three-dimensional structures or structure-influenced latent spaces. Here we introduce PepMLM, a target sequence-conditioned designer of de novo linear peptide binders. Using a masking strategy that positions cognate peptide sequences at the C terminus of target protein sequences, PepMLM finetunes the ESM-2 protein language model to fully reconstruct the binder region, achieving low perplexities matching or improving upon validated peptide-protein sequence pairs. After successful in silico benchmarking with AlphaFold-based docking, we experimentally validate the efficacy of PepMLM through both binding and degradation assays. PepMLM-derived peptides demonstrate sequence-specific binding to cancer and reproductive targets, including NCAM1 and AMHR2, and enable targeted degradation of proteins across diverse disease contexts, from Huntington's disease to live viral infections. Altogether, PepMLM enables the design of candidate binders to any target protein, without requiring structural input, facilitating broad applications in therapeutic development.

DOI: https://doi.org/10.1038/s41587-025-02761-2
ACS Chem Biol. 2025 Aug 13.

Decoding Protein-Peptide Interactions Using a Large, Target-Agnostic Yeast Surface Display Library.

Joseph D Hurley, Irina Shlosman, Megha Lakshminarayan, Ziyuan Zhao, Hong Yue, Radosław P Nowak, Eric S Fischer, Andrew C Kruse.

Protein-peptide interactions underlie key biological processes and are commonly utilized in biomedical research and therapeutic discovery. It is often desirable to identify peptide sequence properties that confer high-affinity binding to a target protein. However, common approaches to such characterization are typically low throughput and sample only regions of sequence space near an initial hit. To overcome these challenges, we built a yeast surface display library representing ∼6.1 × 109 unique peptides. We then performed screens against diverse protein targets, including two antibodies, an E3 ubiquitin ligase, and an essential membrane-bound bacterial enzyme. In each case, we observed motifs that appear to drive peptide binding, and we identified multiple novel, high-affinity clones. These results highlight the library's utility as a robust and versatile tool for discovering peptide ligands and for characterizing protein-peptide binding interactions more generally. To enable further studies, we will make the library freely available upon request.

DOI: https://doi.org/10.1021/acschembio.5c00265
Nat Commun. 2025 Aug 15. 16(1): 7622

High-throughput investigation of cyclin docking interactions reveals the complexity of motif binding determinants.

Mihkel Örd, Matthew J Winters, Mythili S Subbanna, Natàlia de Martín Garrido, Victoria I Cushing, Johanna Kliche, Caroline Benz, Ylva Ivarsson, Basil J Greber, Peter M Pryciak, Norman E Davey.

Many regulatory protein-protein interactions depend on Short Linear Motifs (SLiMs). In the cell cycle, cyclin-CDKs recognize SLiMs to control substrate recruitment and phosphorylation timing. Here, we measure the relative binding strength of ~100,000 peptides to 11 human cyclins from five families (D, E, A, B, and F). Using a quantitative intracellular binding assay and large-scale tiled peptide screening, we identify multiple non-canonical binders unveiling a broader repertoire of cyclin docking motif types. Cryo-electron microscopy and saturation mutagenesis studies reveal distinct binding modes and sequence features governing motif recognition, binding strength, and cyclin preference. Docking motifs vary from highly selective to pan-cyclin, thereby fine-tuning the timing of CDK phosphorylation during cell cycle. Overall, these findings provide insights into the rules encoding specificity and affinity of SLiM-mediated interactions and offer a framework for understanding motif-driven protein networks across the proteome.

DOI: https://doi.org/10.1038/s41467-025-62765-z
Nat Commun. 2025 Aug 14. 16(1): 7575

Targeted degradation of cell surface proteins through endocytosis triggered by cell-penetrating peptide-small molecule conjugates.

Wanyi He, Congli Chen, Jiwei Zheng, Yanyan Li, Huaihuai Shi, Yimin Zhou, Meiqing Li, Ping Gong, Ke Liu, Ximing Shao, Xiaojun Yao, Hongchang Li, Liang Chen, Lijing Fang.

Targeted degradation of membrane-associated proteins, which constitute a crucial class of drug targets implicated in diverse disease pathologies, has garnered considerable attention in chemical biology and drug discovery recently. Taking advantage of the endosomal entrapment of cell-penetrating peptides (CPPs) in delivering bioactive macromolecules, we successfully construct a CPP-based platform for specific degradation of cell surface proteins by conjugation of target protein-binding small molecules (SMs) with different CPPs, resulting in the formation of CPP-mediated lysosome-targeting chimeras (CPPTACs). Through the endo-lysosomal pathway, CPPTACs exhibit a remarkable ability to degrade clinically significant plasma membrane proteins, including PD-L1, CAIX, and CB2R. In contrast to LYTACs and similar technologies, CPPTACs drive the degradation of targets in a manner independent of specific lysosome-shuttling receptors, thus providing a widely applicable strategy for plasma membrane protein degradation, regardless of the cell types. Additionally, simpler structural design and broader therapeutic window for CPPTACs are expected since CPPs-mediated endocytosis and lysosomal degradation do not necessitate the three-component binding model typically required by other heterobifunctional degraders. Overall, consisting of small molecules and biocompatible cell-penetrating peptides, CPPTACs developed in this study represent a simple, adaptable, and effective approach for selectively degrading cell surface proteins in various cellular contexts with potential for application in both biological research and therapeutic interventions.

DOI: https://doi.org/10.1038/s41467-025-62776-w
J Med Chem. 2025 Aug 12.

Engineering Peptide Modulators for T-Cell Migration by Structural Scaffold Matching.

Jasmin Gattringer, Simon Hasinger, Agnes Weidmann, Katarzyna Walczewska-Szewc, Kirtikumar B Jadhav, Tobias Zrzavy, Anja Steinmaurer, Paulien Baeten, Monika Perisic, Wilson Cochrane, Markus Muttenthaler, Bieke Broux, Dagmar Gotthardt, K Johan Rosengren, Christian W Gruber, Roland Hellinger.

Lymphocyte migration plays a crucial role in the progression of autoimmune and inflammatory diseases, and the inhibition of autoreactive immune cells is an attractive therapeutic strategy. Pepitem is an endogenous modulator of lymphocyte migration. In this study, we implemented a structural scaffold matching approach to engineer of stabilized pepitem-based probes. Prioritizing the native helix-loop-helix structure of pepitem, protein structure databases were mined to identify the structurally closest peptide scaffold. Leveraging this strategy, we developed VhTI-pep 2, inhibiting CD3+ T-lymphocyte migration in vitro with a comparable potency (EC50 = 10.6 ± 16.5 nM) to pepitem (EC50 = 6.0 ± 6.4 nM). Its potency was further extended to T-cell subsets derived from multiple sclerosis patients and highly disease-driving memory and Th1 cell populations. Our approach will guide the design of stabilized peptide probes and future therapeutics, overcoming the challenges associated with flexible and linear peptides.

DOI: https://doi.org/10.1021/acs.jmedchem.5c00677
J Med Chem. 2025 Aug 11.

Alchemical Simulation-Aided De Novo Design of Membrane-Active Antimicrobial Hepta-Peptides.

Suvankar Ghosh, Tanumoy Sarkar, Maitery Yadav, Sunanda Chatterjee, Priyadarshi Satpati.

Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics. This study uses alchemical free energy simulations to design ultrashort, cationic, broad-spectrum membrane-active peptides. Previously, we identified broad-spectrum peptide P4 (LKWLKKL-NH2, charge +4) with moderate activity (10-50 μM) but it was ineffective against Methicillin-resistant Staphylococcus aureus (MRSA). Here, we designed eight analogues of P4 by substituting side chains at the third and fourth positions while retaining the overall charge. Alchemical simulations ranked the peptides by assessing the changes in the POPE/POPG bilayer affinity (ΔΔG) resulting from P4 mutation(s). Testing against Acinetobacter baumannii, MRSA, and Candida albicans showed a strong correlation between in silico ranking and experimental potency. Key findings include the following: (1) substituting L4 with W4 (P4L4W) improved activity by 2- to 5-fold against all the strains; (2) W3 was crucial; (3) peptides were membranolytic; and (4) alanine substitutions compromised activity. This comprehensive approach links energetics, molecular interactions, and activity, accelerating the identification of therapeutic membrane-active peptides.

DOI: https://doi.org/10.1021/acs.jmedchem.5c01410
Front Pharmacol. 2025 ;16 1607343

Peptide-mimetics derived from leucyl-tRNA synthetase are potential agents for the therapy of mt-tRNA related diseases.

Annalinda Pisano, Sara Belloli, Maria Gemma Pignataro, Paolo Rainone, Silvia Valtorta, Angela Coliva, Valeria de Turris, Luciana Mosca, Patrizio Di Micco, Rosa Maria Moresco, Giulia d'Amati, Veronica Morea.

   Introduction: Mitochondrial diseases caused by point mutations in mitochondrial tRNA (mt-tRNA) genes, including MELAS and MERRF syndromes, represent a significant unmet clinical need, due to the lack of effective treatments. We previously identified peptide molecules derived from human leucyl-tRNA synthetase, whose features make them attractive leads for the development of therapeutic agents against mt-tRNA point mutations-related diseases. Indeed, we demonstrated that, upon exogenous administration, these peptides penetrate human cell and mitochondrial membranes; stabilize mitochondrial tRNA structures; and rescue severe mitochondrial defects in cells bearing the point mutations m.3243A>G and m.8344A>G, responsible for MELAS and MERRF syndromes, respectively.
Results: To progress towards therapeutic applications, in this work we designed three peptide-mimetic derivatives (PMTs). These are composed entirely of D-amino acids and potentially endowed with enhanced stability in human plasma and resistance to enzymatic degradation. We show that, like the parent peptide, the PMTs have mitochondrial localization and improve cell viability and oxygen consumption in human cybrid cell lines bearing the aforementioned point mutations. Additionally, as anticipated, the PMTs had significantly higher plasma stability than the parent peptide. The most promising PMT was radiolabelled with Cu-64 and used in in vivo biodistribution and tolerability studies. Importantly, i. v. administered PMT reached all body districts, including heart, muscle and even brain, thus revealing an intrinsic ability to cross the blood-brain barrier. Finally, PMT was safe in adult wild-type mice at dosages up to 10 mg/kg.
Discussion: These findings represent a significant step towards the implementation of therapeutic strategies for mttRNA-related mitochondrial diseases.

Keywords:  Cu-64 radioisotope; MELAS and MERRF cybrids; PET; biodistribution; peptide-mimetic molecules; plasma stability; rescuing effect; tolerability

DOI:  https://doi.org/10.3389/fphar.2025.1607343
J Pharmacol Exp Ther. 2025 Jul 18. pii: S0022-3565(25)39876-9. [Epub ahead of print]392(9): 103663

Impacts of anti-drug antibodies on pharmacokinetic and pharmacodynamic actions of cell-permeable middle molecule peptide drug.

Mengxuan Gao, Ryota Saito, Hiroo Watanabe, Tomofumi Shimojo, Taichi Akahoshi, Shino Kuramoto, Tatsuhiko Tachibana.

  The administration of peptide drugs can induce the generation of anti-drug antibodies (ADAs), potentially leading to unwanted effects such as reduced drug efficacy, which is often seen with therapeutic monoclonal antibodies. However, the influence of ADAs on the pharmacokinetic and pharmacodynamic properties of cell-permeable middle molecules remains unclear. This study investigated ADA impacts using AP2151, a middle molecule cyclic peptide with high cell membrane permeability. ADAs were generated by immunizing rabbits with an AP2151 analog conjugated to keyhole limpet hemocyanin. A dialysis experiment was then performed to determine the binding affinity between AP2151 and the purified ADAs. Using in-house experimental data, we established a SimBiology model to simulate the pharmacokinetic profiles of AP2151 in varying ADA concentrations. The simulation results suggested that higher ADA levels (up to several micromolar) would more strongly increase the total plasma concentrations of AP2151 without apparently changing the free concentrations. Thus, according to the free drug theory, the pharmacological effect was predicted to remain constant despite the presence of ADAs. These predictions were subsequently confirmed by an in vivo study, in which mice were administered with AP2151 following a preliminary infusion with or without ADAs. Our findings suggest that in the case of cell-permeable middle molecules, ADAs raise total drug concentrations but have little impact on the drug efficacy. SIGNIFICANCE STATEMENT: We conducted both model-based simulation and in vivo experiments to examine anti-drug antibody impacts on a cell-permeable middle molecule drug. As predicted by the model and confirmed by mouse studies, the presence of anti-drug antibodies increases the total plasma concentration of the drug without compromising its pharmacological effects, highlighting the significance of model analysis and may contribute to the understanding of middle molecule compounds.

Keywords:  Anti-drug antibody; Cyclic peptide; Free drug; Middle molecule; Modeling and simulation; RAS inhibitor

DOI:  https://doi.org/10.1016/j.jpet.2025.103663
bioRxiv. 2025 Jul 17. pii: 2025.07.11.664494. [Epub ahead of print]

Targeting Cholesterol-Dependent Piezo1 Activation Impairs Amoeboid Migration in Melanoma Cells.

Sylvia Kuang, Alleah Abrenica, Neelakshi Kar, Jeremy S Logue.

Bleb-based migration enables cancer cells to navigate the heterogeneous tumor microenvironment. Here, we report a phenotypic screen identifying drugs that inhibit bleb formation, a driver of amoeboid migration. Statins, including Fluvastatin and Pitavastatin, suppress amoeboid migration of melanoma cells in confined environments by reducing intracellular cholesterol. This disrupts plasma membrane tension sensing by Piezo1, lowering intracellular Ca 2 + levels. Both cholesterol supplementation and Piezo1 activation rescue migration in confined environments, confirming their functional link. Notably, high cholesterol biosynthesis enzyme levels correlate with reduced patient survival in melanoma. These findings reveal that cholesterol is essential for confinement sensing through Piezo1, identifying cholesterol biosynthesis or uptake as rational therapeutic targets against metastasis.
Significance Statement: This study builds on a phenotypic drug screen that identified statins as inhibitors of bleb-based migration, a key mode of cancer cell movement through confined spaces. We show that statins reduce membrane cholesterol, disrupting the function of the mechanosensitive channel Piezo1 and impairing melanoma cell migration. Restoring cholesterol or activating Piezo1 rescues this effect, revealing a functional link between cholesterol and confinement sensing. Our findings highlight cholesterol biosynthesis as essential for invasive cell behavior and identify it as a therapeutic vulnerability. Importantly, elevated cholesterol pathway activity correlates with reduced survival in melanoma patients, underscoring the clinical relevance of targeting this pathway to limit metastasis.

DOI: https://doi.org/10.1101/2025.07.11.664494
J Med Chem. 2025 Aug 14.

Discovery of Cell-Permeable Macrocyclic Cyclin A/B RxL Inhibitors that Demonstrate Anti-tumor Activity in E2F-Driven Cancers.

Andrew T Bockus, Siegfried S F Leung, Breena Fraga-Walton, Miguel P Baldomero, Luis Hernandez, Nathan J Dupper, Justin A Shapiro, Bryan M Lent, David C Spellmeyer, Megan K DeMart, Joshua Luna, Dalena Hoang, Manesh Chand, Yuliana Gritsenko, Cayla McEwen, Mahesh Ramaseshan, Catherine E Gleason, Frances Hamkins-Indik, Miles W Membreno, Jie Zheng, Ranya Odeh, Meisam Nosrati, Daphne He, Ramesh Bambal, Peadar Cremin, Jinshu Fang, Bernard Levin, Evelyn W Wang, Marie Evangelista, David Earp, Constantine Kreatsoulas, Rajinder Singh, Pablo D Garcia, James B Aggen.

The cyclin-dependent kinase (CDK)/retinoblastoma protein (RB)/early region 2 binding factor (E2F) axis forms the core transcriptional machinery driving cell cycle progression. Alterations in RB1 or other pathway members occur in many cancers, resulting in heightened oncogenic E2F activity. The activity of E2F is regulated by RxL-mediated binding to the hydrophobic patch (HP) of Cyclin A; blocking this interaction results in the hyperactivation of E2F and synthetic lethality in E2F-driven tumors. While mechanistically differentiated and potentially more selective than blocking CDK activity (e.g., CDK2 or CDK4 inhibitors), the Cyclin A/E2F RxL interaction was deemed undruggable. Utilizing structure-based design, we have discovered a family of cell-permeable macrocyclic Cyclin A/B RxL inhibitors that show potent and selective activity against RB1/E2F-dysregulated cancer cell lines. Lead compound 34 demonstrated proof-of-concept efficacy via intraperiotoneal (IP) administration in mouse cell line-derived xenograft (CDX) tumor models.

DOI: https://doi.org/10.1021/acs.jmedchem.5c00253