bims-cepepe 2025-04-13 papers

Chembiochem. 2025 Apr 10. e202500240

A ligase-based two-step approach for the generation of bicyclic peptides containing a benzylphenyl thioether framework.

Gemin Fang, Hua Zhang, Hui-Min Wei, Jun-Hao Xue, Zhe-Min Xia, Feng-Hao Zheng, Xiao-Cui Wan, Li Zhou.

This study describes a ligase-based two-step strategy to prepare a unique type of bicyclic peptide molecules containing a benzyl phenyl thioether arm. Different from the conventional bicyclic peptide construction method, we first utilized peptide ligases (SrtA or OaAEP1) to introduce an arylthiol group into the parent peptides, and then performed bicyclization of the peptides by using tris-(bromomethyl)benzene to generate the desired bicyclic peptides. Since the pKa of aryl thiols is lower than that of alkyl thiols, the bicyclization reaction of the peptides in our system can occur under low concentrations of tris-(bromomethyl)benzene or low pH conditions. The low concentrations of tris-(bromomethyl)benzene have little effect on the phage infectivity, which will help maintain the diversity of phage-displayed cyclic peptides. This study established a biocompatible ligase-mediated two-step strategy for the preparation of bicyclic peptides, which has potential applications in the discovery of bioactive cyclic peptide ligands.

Keywords: peptide ligation * peptide cyclization * bicyclic peptide * enzymatic peptide ligation * ligases

DOI: https://doi.org/10.1002/cbic.202500240

Chembiochem. 2025 Apr 09. e202500032

Efficient synthesis of cleavable CPP-cargo conjugate via low-equivalent of CPP activated by 2,2'-dithiodipyridine.

Pincheng Li, Xiaona Han, Beichen Wang, Yanyan Guo, Yu Wang, Yiming Li.

Cell-penetrating peptides (CPPs) are favored for protein delivery due to their efficiency, rapidity, and low toxicity. However, conjugation of CPPs to proteins often requires significant amounts of CPPs to ensure yields, which also may result in increased proteins dimer formation. Here, we report that the use of low equivalents 2,2'-dithiodipyridine (DPDS)-activated CPP for conjugation cargo allows for high-conversion-rate CPP-cargo conjugates. Using this strategy, we obtained high-conversion-rate conjugates of cyclic deca-arginine peptide (cR10) with ubiquitin (Ub) and UbcH7 using only low equivalents of cR10. Furthermore, we successfully conjugate three CPPs to cargo via DPDS and confirmed its successful cytosolic delivery through fluorescence imaging.

Keywords: 2,2'-dithiodipyridine; CPP; disulfide bonds; protein delivery

DOI: https://doi.org/10.1002/cbic.202500032

J Pept Sci. 2025 May;31(5): e70014

No Evidence for Plasma Membrane Potential-Independent Cell Penetrating Peptide Direct Translocation.

Ali Hallaj, Francisco Tomas Ribeiro, Christian Widmann.

Cell-penetrating peptides (CPPs) are small peptides that can carry bioactive cargoes into cells. CPPs access the cell's cytosol via direct translocation across the plasma membrane. We and others have shown that direct translocation of CPPs occurs through water pores that are formed upon hyperpolarization of the cell's membrane. Direct translocation through water pores can therefore be blocked by depolarizing the plasma membrane. Other direct translocation mechanisms have been proposed that would not rely on membrane hyperpolarization. It has been reported, for example, that in HEK cells, CPP translocation occurs in a plasma membrane potential-independent manner, in contrast to HeLa cells, where CPP access to the cytosol required plasma membrane hyperpolarization. To address these apparent discrepant data, we have tested the requirement of plasma membrane hyperpolarization in a series of cell lines, including HEK and HeLa cells, for CPP direct translocation. Our data, obtained from a wide range of CPP concentrations, show that efficient direct translocation always requires plasma membrane hyperpolarization. We discuss the possible reasons why earlier studies have not evidenced the importance of the plasma membrane potential in the cytosolic uptake of CPPs in some cell lines.

Keywords: Plasma membrane potential; cell‐penetrating peptides; direct translocation

DOI: https://doi.org/10.1002/psc.70014

Nat Commun. 2025 Apr 05. 16(1): 3273

Flexibility-tuning of dual-display DNA-encoded chemical libraries facilitates cyclic peptide ligand discovery.

Dimitar Petrov, Louise Plais, Kristina Schira, Junyu Cai, Michelle Keller, Alice Lessing, Gabriele Bassi, Samuele Cazzamalli, Dario Neri, Andreas Gloger, Jörg Scheuermann.

Cyclic peptides constitute an important drug modality since they offer significant advantages over small molecules and macromolecules. However, access to diverse chemical sets of cyclic peptides is difficult on a large library scale. DNA-encoded Chemical Libraries (DELs) provide a suitable tool to obtain large chemical diversity, but cyclic DELs made by standard DEL implementation cannot efficiently explore their conformational diversity. On the other hand, dual-display Encoded Self-Assembling Chemical (ESAC) Libraries can be used for modulating macrocycle flexibility since the two displayed peptides can be connected in an incremental fashion. In this work, we construct a 56 million dual-display ESAC library using a two-step cyclization strategy. We show that varying the level of conformational restraint is essential for the discovery of specific ligands for the three protein targets thrombin, human alkaline phosphatase and streptavidin.

DOI: https://doi.org/10.1038/s41467-025-58507-w

Nat Commun. 2025 Apr 10. 16(1): 3395

Antibacterial macrocyclic peptides reveal a distinct mode of BamA inhibition.

Outer membrane proteins (OMPs) produced by Gram-negative bacteria contain a cylindrical amphipathic β-sheet ("β-barrel") that functions as a membrane spanning domain. The assembly (folding and membrane insertion) of OMPs is mediated by the heterooligomeric β-barrel assembly machine (BAM). The central BAM subunit (BamA) is an attractive antibacterial target because its structure and cell surface localization are conserved, it catalyzes an essential reaction, and potent bactericidal compounds that inhibit its activity have been described. Here we utilize mRNA display to discover cyclic peptides that bind to Escherichia coli BamA with high affinity. We describe three peptides that arrest the growth of BAM deficient E. coli strains, inhibit OMP assembly in live cells and in vitro, and bind to unique sites within the BamA β-barrel lumen. Remarkably, we find that if the peptides are added to cultures after a slowly assembling OMP mutant binds to BamA, they accelerate its biogenesis. The data strongly suggest that the peptides trap BamA in conformations that block the initiation of OMP assembly but favor a later assembly step. Molecular dynamics simulations provide further evidence that the peptides bind stably to BamA and function by a previously undescribed mechanism.

DOI: https://doi.org/10.1038/s41467-025-58086-w

Chembiochem. 2025 Apr 10. e202500218

First steps towards the design of peptides that influence the intracellular palmitoylation machinery.

Ines Neundorf, Katharina Stillger, Eric Platz-Baudin, Florian Friedland, Melina Ruppel, Coco-Louisa Sticker, Anne Bodenhausen, Eric Noetzel.

Protein S-palmitoylation is a reversible post-translational modification transferring the 16-carbon fatty acid palmitate to cysteines. It plays a critical role in many cellular processes by influencing protein function, localization, stability, and protein-protein interactions and has a significant impact on various physiological and pathological conditions. This emphasizes the need to develop new technologies to study and treat diseases associated with aberrant palmitoylation. To address these challenges, we present cell-permeable peptides containing a DHHC palmitoylation motif aiming to affect intracellular protein S-palmitoylation. A small library of peptides was generated and screened for cellular uptake and cell compatibility. Interestingly, the newly designed peptides internalized to high extent into different cell lines and human breast cell spheroids dependent on their palmitoylation motif. In addition, out of this screen we identified DC-2 as very potent and investigated this peptide in more detail concerning its impact on palmitoylated proteins that are connected to cancer progression. These initial explorations highlighted that DC-2 affected the localization of HRas and altered S-palmitoylation-related signaling cascades of EGFR. Our findings suggest a peptide-driven impact on proteins having palmitoylation sites and highlight cell-permeable DHHC-peptides as a potential tool to be further evolved in the context of palmitoylation and cancer.

Keywords: EGF receptor; Peptides; ZDHHC enzymes; cysteine palmitoylation; protein modification

DOI: https://doi.org/10.1002/cbic.202500218

ACS Omega. 2025 Apr 01. 10(12): 12495-12504

68Ga-Labeled Peptide for Noninvasive Quantifying Tumor Exposure of PD-L1 Therapeutics.

Hui Nie, Lei Peng, Tianhong Yang, Junyu Chen, Chengde Xie, Lingyu Xue, Dake Zhang, Renbo Wu, Xiangsong Zhang, Zhihao Zha.

PURPOSE: Targeting the programmed death protein 1/programmed death-ligand 1 (PD-1/PD-L1) immune checkpoint blockade therapy plays a critical role in cancer therapy. However, not all patients benefit from this approach, with PD-L1 expression levels being a significant contributing factor. Positron emission tomography (PET) imaging of PD-L1 offers a noninvasive, whole-body, and dynamic assessment of its expression. This study aims to develop a novel peptide-based PD-L1 tracer, [68Ga]HF12, to quantitatively evaluate PD-L1 expression in tumors, thereby offering clinical guidance.
METHODS: HF12 was successfully synthesized and radiolabeled with 68Ga to yield [68Ga]HF12. In vitro binding assays confirmed the specific binding affinity of HF12 for PD-L1 using CHO-hPD-L1 and CHO cell lines. Subsequent in vivo positron emission tomography (PET) imaging and biodistribution studies assessed [68Ga]HF12 for monitoring PD-L1 expression levels in tumor-bearing mice, including those subjected to immunotherapy. Furthermore, PD-L1 expression in tumor tissues was evaluated by using autoradiography, Western blotting, and immunohistochemical (IHC) analysis.
RESULTS: The synthesis of [68Ga]HF12 was successfully achieved with a radiochemical purity and yield exceeding 95%. Cellular uptake studies indicated that [68Ga]HF12 demonstrated both high specificity and significant uptake in PD-L1-positive CHO-hPD-L1 cells. Micro-PET imaging and biodistribution studies revealed that [68Ga]HF12 was preferentially accumulated in CHO-hPD-L1 tumors compared to PD-L1-negative CHO tumors. Treatment with Atezolizumab resulted in a significant reduction in [68Ga]HF12 uptake in CHO-hPD-L1 tumors relative to pretreatment levels, whereas no significant changes were observed in the phosphate-buffered saline (PBS) control group. Subsequent biodistribution studies, along with Western blotting and immunohistochemical analyses, confirmed that PD-L1 expression levels in tumors were reduced following immunotherapy, consistent with the results obtained from PET imaging.
CONCLUSIONS: [68Ga]HF12 was successfully synthesized as a radiotracer for noninvasive quantitative PET imaging of PD-L1 expression levels. This radiotracer exhibited the potential to quantify PD-L1 expression across various tumors, thereby facilitating the prediction of patient response to anti-PD-1 and anti-PD-L1 immunotherapies and monitoring therapeutic efficacy.

DOI: https://doi.org/10.1021/acsomega.4c11396