bims-cepepe Biomed News
on Cell-penetrating peptides
Issue of 2024–09–01
23 papers selected by
Henry Lamb, Queensland University of Technology
  1. Mixed Stereochemistry Macrocycle Acts as a Helix-Stabilizing Peptide N-Cap.
  2. Proximity-driven site-specific cyclization of phage-displayed peptides.
  3. 177Lu-labeling of nuclear localization sequence (NLS)-grafted HER2-receptor affine peptide.
  4. The prospect of substrate-based kinase inhibitors to improve target selectivity and overcome drug resistance.
  5. Hybrid Diffusion Model for Stable, Affinity-Driven, Receptor-Aware Peptide Generation.
  6. Cell Penetrating Peptide Enhances the Aphidicidal Activity of Spider Venom-Derived Neurotoxin.
  7. Discovery of AMPs from random peptides via deep learning-based model and biological activity validation.
  8. Melittin can permeabilize membranes via large transient pores.
  9. Amine-bearing hydrocarbon cross-links: Tailoring helix stability, hydrophilicity, and synthetic adaptability in peptides.
  10. Picking the tyrosine-lock: chemical synthesis of the tyrosyl-DNA phosphodiesterase I inhibitor recifin A and analogues.
  11. Multi-omics analysis delineates resistance mechanisms associated with BRAF inhibition in melanoma cells.
  12. Bioproduction Platform to Generate Functionalized Disulfide-Constrained Peptide Analogues.
  13. Activity of Membrane-Permeabilizing Lpt Peptides.
  14. Selective Activation of Peptide-Thioester Precursors for Templated Native Chemical Ligations.
  15. Detection of Hepatocellular Carcinoma in an Orthotopic Patient-Derived Xenograft with an Epithelial Cell Adhesion Molecule-Specific Peptide.
  16. A Novel Workflow for In Silico Prediction of Bioactive Peptides: An Exploration of Solanum lycopersicum By-Products.
  17. A lysosome-targeted fluorescent probe for thiol detection in drug analysis and multiple biological systems.
  18. FRAP analysis of peptide diffusion in extracellular matrix mimetic hydrogels as an in vitro model for subcutaneous injection.
  19. A versatile fluorinated azamacrocyclic chelator enabling 18F PET or 19F MRI: a first step towards new multimodal and smart contrast agents.
  20. Metabolic Plasticity of Glioblastoma Cells in Response to DHODH Inhibitor BAY2402234 Treatment.
  21. Inhibition of enhanced green fluorescent protein cytosolic delivery mediated by modified cell-penetrating peptide due to high overexpression of Caveolin-1.
  22. Dual Antimicrobial and Anticancer Activity of Membrane-Active Peptide BP52.
  23. Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids.
  1. J Am Chem Soc. 2024 Aug 25.
    Mixed Stereochemistry Macrocycle Acts as a Helix-Stabilizing Peptide N-Cap.
    Fabian Hink, Julen Aduriz-Arrizabalaga, Xabier Lopez, Hiroaki Suga, David De Sancho, Joseph M Rogers.
      Interactions between proteins and α-helical peptides have been the focus of drug discovery campaigns. However, the large interfaces formed between multiple turns of an α-helix and a binding protein represent a significant challenge to inhibitor discovery. Modified peptides featuring helix-stabilizing macrocycles have shown promise as inhibitors of these interactions. Here, we tested the ability of N-terminal to side-chain thioether-cyclized peptides to inhibit the α-helix binding protein Mcl-1, by screening a trillion-scale library. The enriched peptides were lariats featuring a small, four-amino-acid N-terminal macrocycle followed by a short linear sequence that resembled the natural α-helical Mcl-1 ligands. These "Heliats" (helical lariats) bound Mcl-1 with tens of nM affinity, and inhibited the interaction between Mcl-1 and a natural peptide ligand. Macrocyclization was found to stabilize α-helical structures and significantly contribute to affinity and potency. Yet, the 2nd and 3rd positions within the macrocycle were permissible to sequence variation, so that a minimal macrocyclic motif, of an N-acetylated d-phenylalanine at the 1st position thioether connected to a cysteine at the 4th, could be grafted into a range of peptides and stabilize helical conformations. We found that d-stereochemistry is more helix-stabilizing than l- at the 1st position in the motif, as the d-amino acid can utilize polyproline II torsional angles that allow for more optimal intrachain hydrogen bonding. This mixed stereochemistry macrocyclic N-cap is synthetically accessible, requiring only minor modifications to standard solid-phase peptide synthesis, and its compatibility with peptide screening can provide ready access to helix-focused peptide libraries for de novo inhibitor discovery.
    DOI:  https://doi.org/10.1021/jacs.4c05378
  2. Nat Commun. 2024 Aug 24. 15(1): 7308
    Proximity-driven site-specific cyclization of phage-displayed peptides.
    Libby Brown, Aldrin V Vidal, Ana Laura Dias, Tiago Rodrigues, Anna Sigurdardottir, Toby Journeaux, Siobhan O'Brien, Thomas V Murray, Peter Ravn, Monika Papworth, Gonçalo J L Bernardes.
      Cyclization provides a general strategy for improving the proteolytic stability, cell membrane permeability and target binding affinity of peptides. Insertion of a stable, non-reducible linker into a disulphide bond is a commonly used approach for cyclizing phage-displayed peptides. However, among the vast collection of cysteine reactive linkers available, few provide the selectivity required to target specific cysteine residues within the peptide in the phage display system, whilst sparing those on the phage capsid. Here, we report the development of a cyclopropenone-based proximity-driven chemical linker that can efficiently cyclize synthetic peptides and peptides fused to a phage-coat protein, and cyclize phage-displayed peptides in a site-specific manner, with no disruption to phage infectivity. Our cyclization strategy enables the construction of stable, highly diverse phage display libraries. These libraries can be used for the selection of high-affinity cyclic peptide binders, as exemplified through model selections on streptavidin and the therapeutic target αvβ3.
    DOI:  https://doi.org/10.1038/s41467-024-51610-4
  3. Bioorg Med Chem. 2024 Aug 21. pii: S0968-0896(24)00297-9. [Epub ahead of print]112 117883
    177Lu-labeling of nuclear localization sequence (NLS)-grafted HER2-receptor affine peptide.
    Sushree Arpitabala Yadav, V Kusum Vats, Rohit Sharma, Nitish Chauhan, Mahesh Subramanian, Amit Das, Drishty Satpati.
      Tagging of cell permeable nuclear localization sequence (NLS) with receptor targeting peptide vectors is an attractive strategy for selectively targeted translocation of therapeutic cargoes. The present study aimed at grafting nuclear localization sequence (NLS) onto breast cancer targeting rL-A9 peptide. Molecular docking analysis revealed higher binding affinity of the peptide, DOTA-NLS-rL-A9 (-26.1 kJ/mol) towards HER2 receptor in comparison to DOTA-rL-A9 peptide (-22.2 kJ/mol). Confocal microscopy data suggested significantly enhanced cellular internalization of NLS-tagged peptide. The engineered HER2-selective, DOTA-NLS-rL-A9 peptide scaffold was radiolabeled with Lu-177 for intracellular delivery of the theranostic radionuclide into tumor cells. [177Lu]Lu-DOTA-NLS-rL-A9 exhibited significantly enhanced binding affinity (4.58 ± 1.77 nM) towards human breast carcinoma SKBR3 cells and cellular internalization (85 % at 24 h) compared to its original analog, [177Lu]Lu-DOTA-rL-A9. In vivo biodistribution studies showed consistent retention of [177Lu]Lu-DOTA-NLS-rL-A9 in the tumor with negligible washout of radioactivity (∼4.1 % ID/g at 48 h). Prolonged tumor activity with rapid off-target tissue clearance resulted in significantly high tumor-to-background ratios. The radiopeptide, [177Lu]Lu-DOTA-NLS-rL-A9 thus, being precisely confined into HER2-expressing tumor cells and exhibiting favourable pharmacokinetic features is an efficient candidate for further screening.
    Keywords:  A9 peptide; HER2; Internalization; Lu-177; NLS
    DOI:  https://doi.org/10.1016/j.bmc.2024.117883
  4. Chem Sci. 2024 Aug 22. 15(33): 13130-13147
    The prospect of substrate-based kinase inhibitors to improve target selectivity and overcome drug resistance.
    Biswajit Biswas, Yen-Hua Huang, David J Craik, Conan K Wang.
      Human kinases are recognized as one of the most important drug targets associated with cancer. There are >80 FDA-approved kinase inhibitors to date, most of which work by inhibiting ATP binding to the kinase. However, the frequent development of single-point mutations within the kinase domain has made overcoming drug resistance a major challenge in drug discovery today. Targeting the substrate site of kinases can offer a more selective and resistance-resilient solution compared to ATP inhibition but has traditionally been challenging. However, emerging technologies for the discovery of drug leads using recombinant display and stabilization of lead compounds have increased interest in targeting the substrate site of kinases. This review discusses recent advances in the substrate-based inhibition of protein kinases and the potential of such approaches for overcoming the emergence of resistance.
    DOI:  https://doi.org/10.1039/d4sc01088d
  5. J Chem Inf Model. 2024 Aug 28.
    Hybrid Diffusion Model for Stable, Affinity-Driven, Receptor-Aware Peptide Generation.
    Vishva Saravanan R, Soham Choudhuri, Bhaswar Ghosh.
      The convergence of biotechnology and artificial intelligence has the potential to transform drug development, especially in the field of therapeutic peptide design. Peptides are short chains of amino acids with diverse therapeutic applications that offer several advantages over small molecular drugs, such as targeted therapy and minimal side effects. However, limited oral bioavailability and enzymatic degradation have limited their effectiveness. With advances in deep learning techniques, innovative approaches to peptide design have become possible. In this work, we demonstrate HYDRA, a hybrid deep learning approach that leverages the distribution modeling capabilities of a diffusion model and combines it with a binding affinity maximization algorithm that can be used for de novo design of peptide binders for various target receptors. As an application, we have used our approach to design therapeutic peptides targeting proteins expressed by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) genes. The ability of HYDRA to generate peptides conditioned on the target receptor's binding sites makes it a promising approach for developing effective therapies for malaria and other diseases.
    DOI:  https://doi.org/10.1021/acs.jcim.4c01020
  6. Toxins (Basel). 2024 Aug 14. pii: 358. [Epub ahead of print]16(8):
    Cell Penetrating Peptide Enhances the Aphidicidal Activity of Spider Venom-Derived Neurotoxin.
    Wenxian Wu, Abid Ali, Jinbo Shen, Maozhi Ren, Yi Cai, Limei He.
      HxTx-Hv1h, a neurotoxic peptide derived from spider venom, has been developed for use in commercial biopesticide formulations. Cell Penetrating Peptides (CPPs) are short peptides that facilitate the translocation of various biomolecules across cellular membranes. Here, we evaluated the aphidicidal efficacy of a conjugated peptide, HxTx-Hv1h/CPP-1838, created by fusing HxTx-Hv1h with CPP-1838. Additionally, we aimed to establish a robust recombinant expression system for HxTx-Hv1h/CPP-1838. We successfully achieved the secretory production of HxTx-Hv1h, its fusion with Galanthus nivalis agglutinin (GNA) forming HxTx-Hv1h/GNA and HxTx-Hv1h/CPP-1838 in yeast. Purified HxTx-Hv1h exhibited contact toxicity against Megoura crassicauda, with a 48 h median lethal concentration (LC50) of 860.5 μg/mL. Fusion with GNA or CPP-1838 significantly enhanced its aphidicidal potency, reducing the LC50 to 683.5 μg/mL and 465.2 μg/mL, respectively. The aphidicidal efficacy was further improved with the addition of surfactant, decreasing the LC50 of HxTx-Hv1h/CPP-1838 to 66.7 μg/mL-over four times lower compared to HxTx-Hv1h alone. Furthermore, we engineered HxTx-Hv1h/CPP-1838 multi-copy expression vectors utilizing the BglBrick assembly method and achieved high-level recombinant production in laboratory-scale fermentation. This study is the first to document a CPP fusion strategy that enhances the transdermal aphidicidal activity of a natural toxin like HxTx-Hv1h and opens up the possibility of exploring the recombinant production of HxTx-Hv1h/CPP-1838 for potential applications.
    Keywords:  aphicide; cell penetrating peptide; fusion protein; insecticidal neurotoxin; multi-copy expression
    DOI:  https://doi.org/10.3390/toxins16080358
  7. Eur J Med Chem. 2024 Aug 26. pii: S0223-5234(24)00678-0. [Epub ahead of print]277 116797
    Discovery of AMPs from random peptides via deep learning-based model and biological activity validation.
    Jun Du, Changyan Yang, Yabo Deng, Hai Guo, Mengyun Gu, Danna Chen, Xia Liu, Jinqi Huang, Wenjin Yan, Jian Liu.
      The ample peptide field is the best source for discovering clinically available novel antimicrobial peptides (AMPs) to address emerging drug resistance. However, discovering novel AMPs is complex and expensive, representing a major challenge. Recent advances in artificial intelligence (AI) have significantly improved the efficiency of identifying antimicrobial peptides from large libraries, whereas using random peptides as negative data increases the difficulty of discovering antimicrobial peptides from random peptides using discriminative models. In this study, we constructed three multi-discriminator models using deep learning and successfully screened twelve AMPs from a library of 30,000 random peptides. three candidate peptides (P2, P11, and P12) were screened by antimicrobial experiments, and further experiments showed that they not only possessed excellent antimicrobial activity but also had extremely low hemolytic activity. Mechanistic studies showed that these peptides exerted their bactericidal effects through membrane disruption, thus reducing the possibility of bacterial resistance. Notably, peptide 12 (P12) showed significant efficacy in a mouse model of Staphylococcus aureus wound infection with low toxicity to major organs at the highest tested dose (400 mg/kg). These results suggest deep learning-based multi-discriminator models can identify AMPs from random peptides with potential clinical applications.
    Keywords:  Antimicrobial peptides; Artificial intelligence (AI); Deep learning; Drug-resistant bacteria; Wound infection modeling
    DOI:  https://doi.org/10.1016/j.ejmech.2024.116797
  8. Nat Commun. 2024 Aug 23. 15(1): 7281
    Melittin can permeabilize membranes via large transient pores.
    Jakob P Ulmschneider, Martin B Ulmschneider.
      Membrane active peptides are known to porate lipid bilayers, but their exact permeabilization mechanism and the structure of the nanoaggregates they form in membranes have often been difficult to determine experimentally. For many sequences at lower peptide concentrations, transient leakage is observed in experiments, suggesting the existence of transient pores. For two well-know peptides, alamethicin and melittin, we show here that molecular mechanics simulations i) can directly distinguish equilibrium poration and non-equilibrium transient leakage processes, and ii) can be used to observe the detailed pore structures and mechanism of permeabilization in both cases. Our results are in very high agreement with numerous experimental evidence for these two peptides. This suggests that molecular simulations can capture key membrane poration phenomena directly and in the future may develop to be a useful tool that can assist experimental peptide design.
    DOI:  https://doi.org/10.1038/s41467-024-51691-1
  9. Bioorg Med Chem. 2024 Aug 22. pii: S0968-0896(24)00307-9. [Epub ahead of print]112 117893
    Amine-bearing hydrocarbon cross-links: Tailoring helix stability, hydrophilicity, and synthetic adaptability in peptides.
    Duc V H Tran, Thanh K Pham, Young-Woo Kim.
      This study comprehensively explored the helix-stabilizing effects of amine-bearing hydrocarbon cross-links (ABXs), revealing their context-dependent nature influenced by various structural parameters. Notably, we identified a 9-atom ABX as a robust helix stabilizer, showcasing versatile synthetic adaptability while preserving peptide water solubility. Future investigations are imperative to fully exploit this system's potential and enrich our chemical toolkit for designing innovative peptide-based biomolecules.
    Keywords:  Amine cross-link; Peptide drugs; Peptide stapling; Ring-closing metathesis; α-Helix
    DOI:  https://doi.org/10.1016/j.bmc.2024.117893
  10. Chem Sci. 2024 Aug 22. 15(33): 13227-13233
    Picking the tyrosine-lock: chemical synthesis of the tyrosyl-DNA phosphodiesterase I inhibitor recifin A and analogues.
    Taylor B Smallwood, Lauren R H Krumpe, Colton D Payne, Victoria G Klein, Barry R O'Keefe, Richard J Clark, Christina I Schroeder, K Johan Rosengren.
      The peptide recifin A is the inaugural member of the structurally intriguing new fold referred to as a tyrosine-lock. Its central four stranded β-sheet is stabilized by a unique arrangement in which three disulfide bonds and their interconnecting backbone form a ring that wraps around one of the strands, resulting in a Tyr side chain being buried in the molecular core. Here we aimed to establish a synthetic route to this complex class of natural products. Full length recifin A was successfully generated through native chemical ligation chemistry joining two 21 amino acid residue fragments. Surprisingly, reduced linear recifin A readily adopts the correct, topologically-complex fold via random oxidation of the cysteines, suggesting it is highly energetically favored. Utilizing our synthetic strategy, we generated five recifin A analogues to investigate the structural role of the central Tyr residue and provide the first insights into the structure activity relationship of recifin A towards its cancer target tyrosyl-DNA phosphodiesterase I.
    DOI:  https://doi.org/10.1039/d4sc01976h
  11. Exp Cell Res. 2024 Aug 24. pii: S0014-4827(24)00306-9. [Epub ahead of print]442(1): 114215
    Multi-omics analysis delineates resistance mechanisms associated with BRAF inhibition in melanoma cells.
    Keshava K Datta, Hitesh Kore, Harsha Gowda.
      Mutant BRAF is a critical oncogenic driver in melanoma, making it an attractive therapeutic target. However, the success of targeted therapy using BRAF inhibitors vemurafenib and dabrafenib has been limited due to development of resistance, restricting their clinical efficacy. A prior knowledge of resistance mechanisms to BRAFi or any cancer drug can lead to development of drugs that overcome resistance thus improving clinical outcomes. In vitro cellular models are powerful systems that can be utilized to mimic and study resistance mechanisms. In this study, we employed a multi-omics approach to characterize a panel of BRAF mutant melanoma cell lines to develop and systematically characterize BRAFi persister and resistant cells using exome sequencing, proteomics and phosphoproteomics. Our datasets revealed frequently observed intrinsic and acquired, genetic and non-genetic mechanisms of BRAFi resistance that have been studied in patients who developed resistance. In addition, we identified proteins that can be potentially targeted to overcome BRAFi resistance. Overall, we demonstrate that in vitro systems can be utilized not only to predict resistance mechanisms but also to identify putative therapeutic targets.
    Keywords:  Exome sequencing; Kinase inhibitors; Phosphoproteomics; Proteomics; Resistance
    DOI:  https://doi.org/10.1016/j.yexcr.2024.114215
  12. ACS Bio Med Chem Au. 2024 Aug 21. 4(4): 190-203
    Bioproduction Platform to Generate Functionalized Disulfide-Constrained Peptide Analogues.
    Sunhee Hwang, Aaron T Balana, Bryan Martin, Michael Clarkson, Paola Di Lello, Hao Wu, Yanjie Li, Jakob Fuhrmann, Yavuz Dagdas, Patrick Holder, Christina I Schroeder, Stephen E Miller, Xinxin Gao.
      Disulfide-constrained peptides (DCPs) have gained increased attention as a drug modality due to their exceptional stability and combined advantages of large biologics and small molecules. Chemical synthesis, although widely used to produce DCPs, is associated with high cost, both economically and environmentally. To reduce the dependence on solid phase peptide synthesis and the negative environmental footprint associated with it, we present a highly versatile, low-cost, and environmentally friendly bioproduction platform to generate DCPs and their conjugates as well as chemically modified or isotope-labeled DCPs. Using the DCP against the E3 ubiquitin ligase Zinc and Ring Finger 3, MK1-3.6.10, as a model peptide, we have demonstrated the use of bacterial expression, combined with Ser ligation or transglutaminase-mediated XTEN ligation, to produce multivalent MK1-3.6.10 and MK1-3.6.10 with N-terminal functional groups. We have also developed a bioproduction method for the site-specific incorporation of unnatural amino acids into recombinant DCPs by the amber codon suppression system. Lastly, we produced 15N/13C-labeled MK1-3.6.10 with high yield and assessed the performance of a semiautomated resonance assignment workflow that could be used to accelerate binding studies and structural characterization of DCPs. This study provides a proof of concept to generate functionalized DCPs using bioproduction, providing a potential solution to alleviate the reliance on hazardous chemicals, reduce the cost, and expedite the timeline for DCP discovery.
    DOI:  https://doi.org/10.1021/acsbiomedchemau.4c00026
  13. Biomolecules. 2024 Aug 13. pii: 994. [Epub ahead of print]14(8):
    Activity of Membrane-Permeabilizing Lpt Peptides.
    Stefano Maggi, Giulia Mori, Luigi Maglie, Dario Carnuccio, Danila Delfino, Emanuele Della Monica, Claudio Rivetti, Claudia Folli.
      Herein, we investigated the toxicity and membrane-permeabilizing capabilities of Lpt and Lpt-like peptides, belonging to type I toxin-antitoxin systems carried by plasmid DNA of Lacticaseibacillus strains. These 29 amino acid peptides are predicted to form α-helical structures with a conserved central hydrophobic sequence and differently charged hydrophilic termini. Like Lpt, the expression of Lpt-like in E. coli induced growth arrest, nucleoid condensation, and cell membrane damage, suggesting membrane interaction as the mode of action. The membrane permeabilization activity of both peptides was evaluated by using liposome leakage assays, dynamic light scattering, and CD spectroscopy. Lpt and Lpt-like showed liposome leakage activity, which did not lead to liposome disruption but depended on peptide concentration. Lpt was generally more effective than Lpt-like, probably due to different physical chemical properties. Leakage was significantly reduced in larger liposomes and increased with negatively charged PCPS liposomes, indicating that electrostatic interactions and membrane curvature influence peptide activity. Contrary to most membrane-active peptides, Lpt an Lpt-like progressively lost their α-helical structure upon interaction with liposomes. Our data are inconsistent with the formation of membrane-spanning peptide pores but support a mechanism relying on the transient failure of the membrane permeability barrier possibly through the formation of "lipid pores".
    Keywords:  Lacticaseibacillus; liposome leakage; membrane active peptides; membrane permeabilization; nucleoid condensation; toxin–antitoxin systems; type I toxins
    DOI:  https://doi.org/10.3390/biom14080994
  14. Angew Chem Int Ed Engl. 2024 Aug 28. e202413644
    Selective Activation of Peptide-Thioester Precursors for Templated Native Chemical Ligations.
    Paul Spaltenstein, Riley J Giesler, Samuel R Scherer, Patrick W Erickson, Michael S Kay.
      Chemical protein synthesis enables access to proteins that would otherwise be difficult or impossible to obtain with traditional means such as recombinant expression. Chemoselective ligations provide the ability to join peptide segments prepared by solid-phase peptide synthesis. While native chemical ligation (NCL) is widely used, it is limited by the need for C-terminal thioesters with suitable reaction kinetics, properly placed native Cys or thiolated derivatives, and peptide segment solubility at low mM concentrations. Moreover, repetitive purifications to isolate ligated products are often yield-sapping, hampering efficiency and progress. In this work, we demonstrate the use of Controlled Activation of Peptides for Templated NCL (CAPTN). This traceless multi-segment templated NCL approach permits the one-pot synthesis of proteins by harnessing selective thioester activation and orthogonal conjugation chemistries to favor formation of full-length ligated product while minimizing side reactions. Importantly, CAPTN provides kinetic enhancements allowing ligations at sterically hindered junctions and low peptide concentrations. Additionally, this one-pot approach removes the need for intermediate purification. We report the synthesis of two E.coli ribosomal subunits S16 and S17 enabled by the chemical tools described herein. We anticipate that CAPTN will expedite the synthesis of valuable proteins and expand on templated approaches for chemical protein synthesis.
    Keywords:  chemical protein synthesis; native chemical ligation; peptide conjugation; peptide-thioester precursor; templated peptide ligation
    DOI:  https://doi.org/10.1002/anie.202413644
  15. Cancers (Basel). 2024 Aug 10. pii: 2818. [Epub ahead of print]16(16):
    Detection of Hepatocellular Carcinoma in an Orthotopic Patient-Derived Xenograft with an Epithelial Cell Adhesion Molecule-Specific Peptide.
    Xiaoli Wu, Shuo Feng, Tse-Shao Chang, Ruoliu Zhang, Sangeeta Jaiswal, Eun-Young K Choi, Yuting Duan, Hui Jiang, Thomas D Wang.
      Hepatocellular carcinoma (HCC) has emerged as a major contributor to the worldwide cancer burden. Improved methods are needed for early cancer detection and image-guided surgery. Peptides have small dimensions that can overcome delivery challenges to achieve high tumor concentrations and deep penetration. We used phage display methods to biopan against the extra-cellular domain of the purified EpCAM protein, and used IRDye800 as a near-infrared (NIR) fluorophore. The 12-mer sequence HPDMFTRTHSHN was identified, and specific binding to EpCAM was validated with HCC cells in vitro. A binding affinity of kd = 67 nM and onset of k = 0.136 min-1 (7.35 min) were determined. Serum stability was measured with a half-life of T1/2 = 2.6 h. NIR fluorescence images showed peak uptake in vivo by human HCC patient-derived xenograft (PDX) tumors at 1.5 h post-injection. Also, the peptide was able to bind to foci of local and distant metastases in liver and lung. Peptide biodistribution showed high uptake in tumor versus other organs. No signs of acute toxicity were detected during animal necropsy. Immunofluorescence staining of human liver showed specific binding to HCC compared with cirrhosis, adenoma, and normal specimens.
    Keywords:  EpCAM; fluorescence; hepatocellular carcinoma; imaging; near infrared; peptide
    DOI:  https://doi.org/10.3390/cancers16162818
  16. Biomolecules. 2024 Jul 31. pii: 930. [Epub ahead of print]14(8):
    A Novel Workflow for In Silico Prediction of Bioactive Peptides: An Exploration of Solanum lycopersicum By-Products.
    Francesco Morena, Chiara Cencini, Eleonora Calzoni, Sabata Martino, Carla Emiliani.
      Resource-intensive processes currently hamper the discovery of bioactive peptides (BAPs) from food by-products. To streamline this process, in silico approaches present a promising alternative. This study presents a novel computational workflow to predict peptide release, bioactivity, and bioavailability, significantly accelerating BAP discovery. The computational flowchart has been designed to identify and optimize critical enzymes involved in protein hydrolysis but also incorporates multi-enzyme screening. This feature is crucial for identifying the most effective enzyme combinations that yield the highest abundance of BAPs across different bioactive classes (anticancer, antidiabetic, antihypertensive, anti-inflammatory, and antimicrobial). Our process can be modulated to extract diverse BAP types efficiently from the same source. Here, we show the potentiality of our method for the identification of diverse types of BAPs from by-products generated from Solanum lycopersicum, the widely cultivated tomato plant, whose industrial processing generates a huge amount of waste, especially tomato peel. In particular, we optimized tomato by-products for bioactive peptide production by selecting cultivars like Line27859 and integrating large-scale gene expression. By integrating these advanced methods, we can maximize the value of by-products, contributing to a more circular and eco-friendly production process while advancing the development of valuable bioactive compounds.
    Keywords:  bioactive peptides; food-derived peptides; functional foods; health-promoting peptides; in silico prediction; nutritional genomics; plant-derived peptides; tomato bioactive compounds
    DOI:  https://doi.org/10.3390/biom14080930
  17. Anal Bioanal Chem. 2024 Aug 28.
    A lysosome-targeted fluorescent probe for thiol detection in drug analysis and multiple biological systems.
    Yitong Liu, Juan Song, Yan Li, Peng Hou, Haijun Wang, Jiaming Wang, Chuan He, Song Chen.
      Biothiols, characterized by their unique sulfhydryl (-SH) groups, possess excellent antioxidant properties, effectively neutralizing the damage to cellular structures caused by reactive oxygen species (ROS) in living organisms. Additionally, lysosomes play a crucial role in decomposing damaged biomolecules through the action of their internal enzymes, regulating the cellular redox state, and mitigating oxidative stress. To facilitate rapid monitoring of intracellular biothiols, particularly within lysosomes, we constructed a lysosome-targeted biothiol fluorescent probe, PHL-DNP, in this study. PHL-DNP exhibited excellent photophysical properties in an aqueous test system, including strong fluorescence enhancement response, excellent selectivity, and low detection limits (Cys 16.5 nM, Hcy 16.8 nM, GSH 21.3 nM, Cap 26.6 nM). These attributes enabled easy and efficient qualification of Cys on test strips and accurate determination of the effective content of captopril tablets. Notably, PHL-DNP demonstrated low cytotoxicity and precise lysosomal targeting. Through bioimaging, PHL-DNP not only monitored changes in biothiol levels under oxidative stress but also assessed biothiols in complex biological systems such as live HeLa cells, zebrafish, tumor tissue sections, and radish roots. This provides a promising tool for quantitative analysis of biothiols, disease marker detection, and drug testing.
    Keywords:  Bioimaging; Biothiols; Captopril content determination; Lysosomal targeting; Pyrazoline derivatives
    DOI:  https://doi.org/10.1007/s00216-024-05495-3
  18. Int J Pharm. 2024 Aug 22. pii: S0378-5173(24)00862-7. [Epub ahead of print]664 124628
    FRAP analysis of peptide diffusion in extracellular matrix mimetic hydrogels as an in vitro model for subcutaneous injection.
    Julia Parlow, Agnes Rodler, Johan Gråsjö, Helen Sjögren, Per Hansson.
      Subcutaneous (SC) injection is a common route of administration for drug compounds with poor oral bioavailability. However, bioavailability is often variable and incomplete, and there is as yet no standard accepted medium for simulation of the human SC environment. In this work we evaluate a FRAP based method for quantitative determination of local self-diffusion coefficients within extracellular matrix (ECM) mimetic hydrogels, potentially useful as in vitro models for drug transport in the ECM after SC injection. Gels were made consisting of either agarose, cross-linked collagen (COL) and hyaluronic acid (HA) or cross-linked HA. The diffusivities of uncharged FITC-dextran (FD4), the highly charged poly-lysine (PLK20) and poly-glutamic acid (PLE20) as well as the GLP-1 analogue exenatide were determined within the gels using FRAP. The diffusion coefficients in uncharged agarose gels were in the range of free diffusion in PBS. The diffusivity of cationic PLK20 in gels containing anionic HA was substantially decreased due to strong electrostatic interactions. Peptide aggregation could be observed as immobile fractions in experiments with exenatide. We conclude that the FRAP method provides useful information of peptides' interactions and transport properties in hydrogel networks, giving insight into the mechanisms affecting absorption of drug compounds after subcutaneous injection.
    Keywords:  Diffusion; Extracellular matrix; FRAP; Hydrogel; In vitro; Peptide; Subcutaneous
    DOI:  https://doi.org/10.1016/j.ijpharm.2024.124628
  19. Chem Sci. 2024 Aug 22. 15(33): 13550-13557
    A versatile fluorinated azamacrocyclic chelator enabling 18F PET or 19F MRI: a first step towards new multimodal and smart contrast agents.
    Charline Sire, Vincent Meneyrol, Nathalie Saffon-Merceron, Enzo Terreno, Francesca Garello, Lorenzo Tei, Emmanuelle Jestin, Raphaël Tripier, Thibault Troadec.
      Macrocyclic chelators play a central role in medical imaging and nuclear medicine owing to their unparalleled metal cation coordination abilities. Their functionalization by fluorinated groups is an attractive design, to combine their properties with those of 18F for Positron Emission Tomography (PET) or natural 19F for Magnetic Resonance Imaging (MRI), and access potential theranostic or smart medical imaging probes. For the first time, a compact fluorinated macrocyclic architecture has been synthesized, based on a cyclen chelator bearing additional pyridine coordinating units and simple methyltrifluoroborate prosthetic groups. This ligand and its corresponding model Zn(ii) complex were investigated to evaluate the 18F-PET or 19F MRI abilities provided by this novel molecular structure. The chelator and the complex were obtained via a simple and high-yielding synthetic route, present excellent solvolytic stability of the trifluoroborate groups at various pH, and provide facile late-stage 18F-radiolabeling (up to 68% radiochemical yield with high activity) as well as a satisfying detection limit for 19F MRI imaging (low mM range).
    DOI:  https://doi.org/10.1039/d4sc02871f
  20. Metabolites. 2024 Jul 27. pii: 413. [Epub ahead of print]14(8):
    Metabolic Plasticity of Glioblastoma Cells in Response to DHODH Inhibitor BAY2402234 Treatment.
    Ayenachew Bezawork-Geleta, Diane Moujalled, David P De Souza, Vinod K Narayana, James Dimou, Rodney Luwor, Matthew J Watt.
      Glioblastoma (IDH-wildtype) represents a formidable challenge in oncology, lacking effective chemotherapeutic or biological interventions. The metabolic reprogramming of cancer cells is a hallmark of tumor progression and drug resistance, yet the role of metabolic reprogramming in glioblastoma during drug treatment remains poorly understood. The dihydroorotate dehydrogenase (DHODH) inhibitor BAY2402234 is a blood-brain barrier penetrant drug showing efficiency in in vivo models of many brain cancers. In this study, we investigated the effect of BAY2402234 in regulating the metabolic phenotype of EGFRWT and EGFRvIII patient-derived glioblastoma cell lines. Our findings reveal the selective cytotoxicity of BAY2402234 toward EGFRWT glioblastoma subtypes with minimal effect on EGFRvIII patient cells. At sublethal doses, BAY2402234 induces triglyceride synthesis at the expense of membrane lipid synthesis and fatty acid oxidation in EGFRWT glioblastoma cells, while these effects are not observed in EGFRvIII glioblastoma cells. Furthermore, BAY2402234 reduced the abundance of signaling lipid species in EGFRWT glioblastoma. This study elucidates genetic mutation-specific metabolic plasticity and efficacy in glioblastoma cells in response to drug treatment, offering insights into therapeutic avenues for precision medicine approaches.
    Keywords:  brain cancer; lipid droplets; lipid metabolism
    DOI:  https://doi.org/10.3390/metabo14080413
  21. Biochem Biophys Res Commun. 2024 Aug 22. pii: S0006-291X(24)01122-7. [Epub ahead of print]733 150586
    Inhibition of enhanced green fluorescent protein cytosolic delivery mediated by modified cell-penetrating peptide due to high overexpression of Caveolin-1.
    Akiko Okuda, Keito Sugai, Shujiro Okuda.
      The modified cell-penetrating peptide Pas2r12 can deliver antibodies (IgG, 150 kDa) and enhanced green fluorescent protein (EGFP1, 27 kDa) into the cytosol through caveolae-dependent endocytosis. In this study, we determined the effect of Caveolin-1 overexpression on the cytosolic delivery of EGFP by Pas2r12. Three types of Caveolin-1 overexpressing strains were isolated, including Cav1L (low), Cav1M (medium), and Cav1H (high), using HEK293 as the parent cell line. We found that the number of caveolae on the surface of the Caveolin-1-overexpressing strains was similar to that of HEK293. We examined the cytosolic delivery rate of EGFP by Pas2r12. In the Cav1L and Cav1M cells, there was little change compared with HEK293; however, in Cav1H, the rate was significantly decreased. Moreover, the amount of EGFP uptake into the cells (total intracellular EGFP) showed an increasing trend in Cav1H compared with HEK293. These results indicate that in Cav1H, the amount of EGFP uptake into the cells increases, whereas the cytosolic delivery rate of EGFP decreases. This suggests that high overexpression of Caveolin-1 inhibits the transition of EGFP from endosomes to the cytosol.
    Keywords:  Caveolae; Caveolin-1; Cell-penetrating peptide; Drug delivery system; Endocytosis; Enhanced green fluorescent protein
    DOI:  https://doi.org/10.1016/j.bbrc.2024.150586
  22. Protein J. 2024 Aug 27.
    Dual Antimicrobial and Anticancer Activity of Membrane-Active Peptide BP52.
    Hai Bui Thi Phuong, Hoa Doan Ngan, Hue Pham Thi, Binh Nguyen Thi Thanh, Tien T Dang, Thao N T Ho, Tung Truong Thanh, Minh Nguyen Hong, Huy Luong Xuan.
      The linear undecapeptide BP52 was previously reported to have antibacterial activity against phytopathogenic bacteria species. Due to the structural similarities to naturally occurring cationic helical antimicrobial peptides, it was speculated that this peptide could potentially target microbial pathogens and cancer cells found in mammals. Consequently, this study aims to further investigate the structural and biological properties of this peptide. Our findings indicate that BP52 exhibits strong antimicrobial and anticancer activity while displaying relatively low levels of hemolytic activity. Hence, this study suggests that BP52 could be a potential lead compound for drug discovery against infectious diseases and cancer. Besides, new insights into the relationships between the structure and the multifunctional properties of antimicrobial peptides were also explored.
    Keywords:  Alternative antibiotics; Amphipathic helix; Anticancer peptides; Antimicrobial peptides; Structure-activity relationships
    DOI:  https://doi.org/10.1007/s10930-024-10231-y
  23. J Vis Exp. 2024 Aug 09.
    Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids.
    Angela C Debruyne, Gabriele Ferrari, Hang Zhou, Nore Van Loon, Nina Heymans, Irina A Okkelman, Ruslan I Dmitriev.
      Multicellular tumor spheroids are a popular 3D tissue microaggregate model for reproducing tumor microenvironment, testing and optimizing drug therapies and using bio- and nanosensors in a 3D context. Their ease of production, predictable size, growth, and observed nutrient and metabolite gradients are important to recapitulate the 3D niche-like cell microenvironment. However, spheroid heterogeneity and variability of their production methods can influence overall cell metabolism, viability, and drug response. This makes it difficult to choose the most appropriate methodology, considering the requirements in size, variability, needs of biofabrication, and use as in vitro 3D tissue models in stem and cancer cell biology. In particular, spheroid production can influence their compatibility with quantitative live microscopies, such as optical metabolic imaging, fluorescence lifetime imaging microscopy (FLIM), monitoring of spheroid hypoxia with nanosensors, or viability. Here, a number of conventional spheroid formation protocols are presented, highlighting their compatibility with the live widefield, confocal, and two-photon microscopies. The follow-up imaging to analysis pipeline with multiplexed autofluorescence FLIM and, using various types of cancer and stem cell spheroids, is also presented.
    DOI:  https://doi.org/10.3791/66845
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