bims-cepepe Biomed News
on Cell-penetrating peptides
Issue of 2024‒03‒31
twenty-one papers selected by
Henry Lamb, Queensland University of Technology
  1. A Cell-Penetrating Peptide Improves Anti-HER2 Single-Chain Variable Fragment Internalization and Antitumor Activity against HER2-Positive Breast Cancer In Vitro and In Vivo.
  2. A Tag-Free Platform for Synthesis and Screening of Cyclic Peptide Libraries.
  3. Therapeutic stapled peptides: Efficacy and molecular targets.
  4. Understanding the Structural Requirements of Peptide-Protein Interaction and Applications for Peptidomimetic Development.
  5. Disulfide-constrained peptide scaffolds enable a robust peptide-therapeutic discovery platform.
  6. A Lipopeptidomimetic of Transcriptional Activation Domains Selectively Disrupts Med25 Protein-Protein Interactions.
  7. Asparaginyl Endopeptidase-Mediated Peptide Cyclization for Phage Display.
  8. Evaluation of the Cytosolic Uptake of HaloTag Using a pH-Sensitive Dye.
  9. Chemoenzymatic tandem cyclization for the facile synthesis of bicyclic peptides.
  10. The funnel-web spider venom derived single knot peptide Hc3a modulates acid-sensing ion channel 1a desensitisation.
  11. Discovery of macrocyclic CDK2/4/6 inhibitors with improved potency and DMPK properties through a highly efficient macrocyclic drug design platform.
  12. N-terminal modification of an LAH4-derived peptide increases mRNA delivery in the presence of serum.
  13. Accelerating Cellular Uptake with Unnatural Amino Acid for Inhibiting Immunosuppressive Cancer Cells.
  14. Bioinspired Synthesis of Allysine for Late-Stage Functionalization of Peptides.
  15. A dynamic study of VEGF-A siDOX-EVs trafficking through the in-vitro insert co-culture blood-brain barrier model by digital holographic microscopy.
  16. A phage-displayed disulfide constrained peptide discovery platform yields novel human plasma protein binders.
  17. The solute carrier SLC7A1 may act as a protein transporter at the blood-brain barrier.
  18. Aiming the magic bullet: targeted delivery of imaging and therapeutic agents to solid tumors by pHLIP peptides.
  19. Acod1 expression in cancer cells promotes immune evasion through the generation of inhibitory peptides.
  20. Structural analysis of the macrocyclic inhibitor BI-4020 binding to EGFR kinase.
  21. Co-Culture Models: Key Players in In Vitro Neurotoxicity, Neurodegeneration and BBB Modeling Studies.
  1. Molecules. 2024 Mar 11. pii: 1247. [Epub ahead of print]29(6):
    A Cell-Penetrating Peptide Improves Anti-HER2 Single-Chain Variable Fragment Internalization and Antitumor Activity against HER2-Positive Breast Cancer In Vitro and In Vivo.
    Junmin Li, Yanting Zhou, Zhuowei Su, Xue Li, Lei Zhang, Shan Li.
      Cell-penetrating peptides (CPPs) are invaluable tools for delivering various substances into cells by crossing biological membranes. However, the effects of cell-penetrating peptide fusion proteins on the biological activity of antibodies remain to be fully understood. Here, we engineered a recombinant protein, LP-scFv, which combines the single-chain variable region of anti-human epidermal growth factor receptor-2 with a novel and non-oxic cell-penetrating peptide as a leader peptide. The introduction of this leader peptide led to a more than twofold increase in the internalization efficiency of the single-chain antibody, as confirmed using microscopic analysis and flow cytometry. The effects of the single-chain antibodies and LP-scFv on cell viability were evaluated using the MTT assay. Both the single-chain antibodies and LP-scFv reduced the viability of BT474 and NCI-N87 cells in a dose-dependent manner while exhibiting minimal toxicity towards MCF-7 and MCF-10A cells. Further investigation into LP-scFv's mechanism revealed that the induced leader peptide does not alter the MAPK-ERK1/2 and PI3K/AKT pathways of single-chain antibodies. An enhanced antitumor activity was also confirmed in an NCI-N87 tumor xenograft model in mice with a reduction of 45.2% in tumor growth inhibition (vs. 23.1% for scFv) with a 50 mg/kg dose after orthotopic injection administration, which was equivalent to that of trastuzumab (vs. 55.7% for trastuzumab). Overall, these results indicate that LP-scFv exhibits significant permeation activity in HER2-positive cells to enhance the intracellular dose effect on antitumor activity in vitro and in vivo. This research lays the foundation for designing novel antibody-based therapies for cancer.
    Keywords:  HER2-positive breast cancer; antitumor activity; internalization; leader peptide-scFv; scFv
    DOI:  https://doi.org/10.3390/molecules29061247
  2. Angew Chem Int Ed Engl. 2024 Mar 26. e202320045
    A Tag-Free Platform for Synthesis and Screening of Cyclic Peptide Libraries.
    Angele Bruce, Victor Adebomi, Patrick Czabala, Jonathan Palmer, Gaurav Bhardwaj, Monika Raj, William M McFadden, Zachary C Lorson, Ryan L Slack, S G Sarafianos.
      In the realm of high-throughput screening (HTS), macrocyclic peptide libraries traditionally necessitate decoding tags, essential for both library synthesis and identifying hit peptide sequences post-screening. Our innovation introduces a tag-free technology platform for synthesizing cyclic peptide libraries in solution and facilitates screening against biological targets to identify peptide binders through unconventional intramolecular CyClick and DeClick Chemistries (CCDC) discovered within our research. This combination allows for the synthesis of diverse cyclic peptide libraries, the incorporation of various amino acids, and facile linearization and decoding of cyclic peptide binder sequences. Our sensitivity-enhancing derivatization method, utilized in tandem with nano LC-MS/MS, enables the sequencing of peptides even at exceedingly low picomolar concentrations. Employing our technology platform, we've successfully unearthed novel cyclic peptide binders against a monoclonal antibody and the first cyclic peptide binder of HIV CAPSID protein responsible for viral infections as validated by microscale thermalshift assays (TSA), biolayer Interferometry (BLI) and functional assays.
    Keywords:  Tag-Free * Peptide Macrocyclization * CyClick Chemistry * De-Click Chemistry * Affinity Selection
    DOI:  https://doi.org/10.1002/anie.202320045
  3. Pharmacol Res. 2024 Mar 23. pii: S1043-6618(24)00081-1. [Epub ahead of print]203 107137
    Therapeutic stapled peptides: Efficacy and molecular targets.
    Yulei Li, Minghao Wu, Yinxue Fu, Jingwen Xue, Fei Yuan, Tianci Qu, Anastassia N Rissanou, Yilin Wang, Xiang Li, Honggang Hu.
      Peptide stapling, by employing a stable, preformed alpha-helical conformation, results in the production of peptides with improved membrane permeability and enhanced proteolytic stability, compared to the original peptides, and provides an effective solution to accelerate the rapid development of peptide drugs. Various reviews present peptide stapling chemistries, anchoring residues and one- or two-component cyclization, however, therapeutic stapled peptides have not been systematically summarized, especially focusing on various disease-related targets. This review highlights the latest advances in therapeutic peptide drug development facilitated by the application of stapling technology, including different stapling techniques, synthetic accessibility, applicability to biological targets, potential for solving biological problems, as well as the current status of development. Stapled peptides as therapeutic drug candidates have been classified and analysed mainly by receptor- and ligand-based stapled peptide design against various diseases, including cancer, infectious diseases, inflammation, and diabetes. This review is expected to provide a comprehensive reference for the rational design of stapled peptides for different diseases and targets to facilitate the development of therapeutic peptides with enhanced pharmacokinetic and biological properties.
    Keywords:  Staple peptide; Targets; Therapeutic peptide drug; α-helical peptide
    DOI:  https://doi.org/10.1016/j.phrs.2024.107137
  4. Methods Mol Biol. 2024 ;2793 65-82
    Understanding the Structural Requirements of Peptide-Protein Interaction and Applications for Peptidomimetic Development.
    Angy Liseth Davalos Macias, Lilian Costa Alecrim, Fabio C L Almeida, Ricardo Jose Giordano.
      Protein-protein interaction is at the heart of most biological processes, and small peptides that bind to protein binding sites are resourceful tools to explore and understand the structural requirements for these interactions. In that sense, phage display is a well-suited technology to study protein-protein interactions, as it allows for unbiased screening of billions of peptides in search for those that interact with a protein binding domain. Here, we will illustrate how two distinct but complementary approaches, phage display and nuclear magnetic resonance (NMR), can be utilized to unveil structural details of peptide-protein interaction. Finally, knowledge derived from phage mutagenesis and NMR studies can be streamlined for quick peptidomimetic design and synthesis using the retroinversion approach to validate using in vitro and in vivo assays the therapeutic potential of peptides identified by phage display.
    Keywords:  NMRNuclear magnetic resonance (NMR); Nuclear magnetic resonance; Peptide and protein interaction; Peptidomimetic design; Phage display; Phage mutagenesis
    DOI:  https://doi.org/10.1007/978-1-0716-3798-2_5
  5. PLoS One. 2024 ;19(3): e0300135
    Disulfide-constrained peptide scaffolds enable a robust peptide-therapeutic discovery platform.
    Lijuan Zhou, Fei Cai, Yanjie Li, Xinxin Gao, Yuehua Wei, Anna Fedorova, Daniel Kirchhofer, Rami N Hannoush, Yingnan Zhang.
      Peptides present an alternative modality to immunoglobulin domains or small molecules for developing therapeutics to either agonize or antagonize cellular pathways associated with diseases. However, peptides often suffer from poor chemical and physical stability, limiting their therapeutic potential. Disulfide-constrained peptides (DCP) are naturally occurring and possess numerous desirable properties, such as high stability, that qualify them as drug-like scaffolds for peptide therapeutics. DCPs contain loop regions protruding from the core of the molecule that are amenable to peptide engineering via direct evolution by use of phage display technology. In this study, we have established a robust platform for the discovery of peptide therapeutics using various DCPs as scaffolds. We created diverse libraries comprising seven different DCP scaffolds, resulting in an overall diversity of 2 x 1011. The effectiveness of this platform for functional hit discovery has been extensively evaluated, demonstrating a hit rate comparable to that of synthetic antibody libraries. By utilizing chemically synthesized and in vitro folded peptides derived from selections of phage displayed DCP libraries, we have successfully generated functional inhibitors targeting the HtrA1 protease. Through affinity maturation strategies, we have transformed initially weak binders against Notch2 with micromolar Kd values to high-affinity ligands in the nanomolar range. This process highlights a viable hit-to-lead progression. Overall, our platform holds significant potential to greatly enhance the discovery of peptide therapeutics.
    DOI:  https://doi.org/10.1371/journal.pone.0300135
  6. Angew Chem Int Ed Engl. 2024 Mar 25. e202400781
    A Lipopeptidomimetic of Transcriptional Activation Domains Selectively Disrupts Med25 Protein-Protein Interactions.
    Olivia N Pattelli, Estefanía Martínez Valdivia, Matthew S Beyersdorf, Clint S Regan, Mónica Rivas, Katherine A Hebert, Sofia D Merajver, Tomasz Cierpicki, Anna K Mapp.
      Short amphipathic peptides are capable of binding to transcriptional coactivators, often targeting the same binding surfaces as native transcriptional activation domains. However, they do so with modest affinity and generally poor selectivity, limiting their utility as synthetic modulators. Here we show that incorporation of a medium-chain, branched fatty acid to the N-terminus of one such heptameric lipopeptidomimetic (LPPM-8) increases the affinity for the coactivator Med25 >20-fold (Ki >100 μM to 4 μM), rendering it an effective inhibitor of Med25 protein-protein interactions (PPIs). The lipid structure, the peptide sequence, and the C-terminal functionalization of the lipopeptidomimetic each influence the structural propensity of LPPM-8 and its effectiveness as an inhibitor. LPPM-8 engages Med25 through interaction with the H2 face of its Activator Interaction Domain and in doing so stabilizes full-length protein in the cellular proteome. Further, genes regulated by Med25-activator PPIs are inhibited in a cell model of triple-negative breast cancer. Thus, LPPM-8 is a useful tool for studying Med25 and Mediator complex biology and the results indicate that lipopeptidomimetics may be a robust source of inhibitors for activator-coactivator complexes.
    Keywords:  Coactivator; Lipopeptide; Med25; Protein-protein interaction inhibitor; Transcription Factor
    DOI:  https://doi.org/10.1002/anie.202400781
  7. Org Lett. 2024 Mar 26.
    Asparaginyl Endopeptidase-Mediated Peptide Cyclization for Phage Display.
    Xiao-Cui Wan, Yan-Ni Zhang, Hua Zhang, Ying Chen, Zhi-Hui Cui, Wen-Jing Zhu, Ge-Min Fang.
      We report here an enzymatic strategy for asparaginyl endopeptidase-mediated peptide cyclization. Incorporation of chloroacetyl groups into the recognition sequence of OaAEP1 enabled intramolecular cyclization with Cys residues. Combining this strategy and phage display, we identified nanomolar macrocyclic peptide ligands targeting TEAD4. One of the bicyclic peptides binds to TEAD4 with a KD value of 139 nM, 16 times lower than its linear analogue, demonstrating the utility of this platform in discovering high-affinity macrocyclic peptide ligands.
    DOI:  https://doi.org/10.1021/acs.orglett.4c00602
  8. ACS Chem Biol. 2024 Mar 25.
    Evaluation of the Cytosolic Uptake of HaloTag Using a pH-Sensitive Dye.
    JoLynn B Giancola, Jonathan B Grimm, Joomyung V Jun, Yana D Petri, Luke D Lavis, Ronald T Raines.
      The efficient cytosolic delivery of proteins is critical for advancing novel therapeutic strategies. Current delivery methods are severely limited by endosomal entrapment, and detection methods lack sophistication in tracking the fate of delivered protein cargo. HaloTag, a commonly used protein in chemical biology and a challenging delivery target, is an exceptional model system for understanding and exploiting cellular delivery. Here, we employed a combinatorial strategy to direct HaloTag to the cytosol. We established the use of Virginia Orange, a pH-sensitive fluorophore, and Janelia Fluor 585, a similar but pH-agnostic fluorophore, in a fluorogenic assay to ascertain protein localization within human cells. Using this assay, we investigated HaloTag delivery upon modification with cell-penetrating peptides, carboxyl group esterification, and cotreatment with an endosomolytic agent. We found efficacious cytosolic entry with two distinct delivery methods. This study expands the toolkit for detecting the cytosolic access of proteins and highlights that multiple intracellular delivery strategies can be used synergistically to effect cytosolic access. Moreover, HaloTag is poised to serve as a platform for the delivery of varied cargo into human cells.
    DOI:  https://doi.org/10.1021/acschembio.3c00713
  9. Commun Chem. 2024 Mar 28. 7(1): 67
    Chemoenzymatic tandem cyclization for the facile synthesis of bicyclic peptides.
    Masakazu Kobayashi, Naho Onozawa, Kenichi Matsuda, Toshiyuki Wakimoto.
      Bicyclic peptides exhibit improved metabolic stabilities and target specificities when compared to their linear or mono-cyclic counterparts; however, efficient and straightforward synthesis remains challenging due to their intricate architectures. Here, we present a highly selective and operationally simple one-pot chemoenzymatic tandem cyclization approach to synthesize bicyclic peptides with small to medium ring sizes. Penicillin-binding protein-type thioesterases (PBP-type TEs) efficiently cyclized azide/alkyne-containing peptides in a head-to-tail manner. Successive copper (I)-catalyzed azide-alkyne cycloaddition generated bicyclic peptides in one-pot, thus omitting the purification of monocyclic intermediates. This chemoenzymatic strategy enabled the facile synthesis of bicyclic peptides bearing hexa-, octa-, and undecapeptidyl head-to-tail cyclic scaffolds.
    DOI:  https://doi.org/10.1038/s42004-024-01147-w
  10. Biochem Pharmacol. 2024 Mar 27. pii: S0006-2952(24)00158-8. [Epub ahead of print] 116175
    The funnel-web spider venom derived single knot peptide Hc3a modulates acid-sensing ion channel 1a desensitisation.
    Elena Budusan, Colton D Payne, Tye I Gonzalez, Alison Obergrussberger, Nadine Becker, Richard J Clark, K Johan Rosengren, Lachlan D Rash, Ben Cristofori-Armstrong.
      Acid-sensing ion channel 1a (ASIC1a) is a proton-gated channel involved in synaptic transmission, pain signalling, and several ischemia-associated pathological conditions. The spider venom-derived peptides PcTx1 and Hi1a are two of the most potent ASIC1a inhibitors known and have been instrumental in furthering our understanding of the structure, function, and biological roles of ASICs. To date, homologous spider peptides with different pharmacological profiles at ASIC1a have yet to be discovered. Here we report the characterisation of Hc3a, a single inhibitor cystine knot peptide from the Australian funnel-web spider Hadronyche cerberea with sequence similarity to PcTx1. We show that Hc3a has complex pharmacology and binds different ASIC1a conformational states (closed, open, and desensitised) with different affinities, with the most prominent effect on desensitisation. Hc3a slows the desensitisation kinetics of proton-activated ASIC1a currents across multiple application pHs, and when bound directly to ASIC1a in the desensitised conformation promotes current inhibition. The solution structure of Hc3a was solved, and the peptide-channel interaction examined via mutagenesis studies to highlight how small differences in sequence between Hc3a and PcTx1 can lead to peptides with distinct pharmacology. The discovery of Hc3a expands the pharmacological diversity of spider venom peptides targeting ASIC1a and adds to the toolbox of compounds to study the intricacies of ASIC1 gating.
    Keywords:  Acid-sensing ion channel; Funnel-web spider; Hc3a; Inhibitor cystine knot; Peptide
    DOI:  https://doi.org/10.1016/j.bcp.2024.116175
  11. Bioorg Chem. 2024 Mar 15. pii: S0045-2068(24)00190-1. [Epub ahead of print]146 107285
    Discovery of macrocyclic CDK2/4/6 inhibitors with improved potency and DMPK properties through a highly efficient macrocyclic drug design platform.
    Fanye Meng, Jinxin Liu, Zhongying Cao, Jiaojiao Yu, Barbara Steurer, Yilin Yang, Yazhou Wang, Xin Cai, Man Zhang, Feng Ren, Alex Aliper, Xiao Ding, Alex Zhavoronkov.
      Cyclin-dependent kinases (CDKs) are critical cell cycle regulators that are often overexpressed in tumors, making them promising targets for anti-cancer therapies. Despite substantial advancements in optimizing the selectivity and drug-like properties of CDK inhibitors, safety of multi-target inhibitors remains a significant challenge. Macrocyclization is a promising drug discovery strategy to improve the pharmacological properties of existing compounds. Here we report the development of a macrocyclization platform that enabled the highly efficient discovery of a novel, macrocyclic CDK2/4/6 inhibitor from an acyclic precursor (NUV422). Using dihedral angle scan and structure-based, computer-aided drug design to select an optimal ring-closing site and linker length for the macrocycle, we identified compound 8 as a potent new CDK2/4/6 inhibitor with optimized cellular potency and safety profile compared to NUV422. Our platform leverages both experimentally-solved as well as generative chemistry-derived macrocyclic structures and can be deployed to streamline the design of macrocyclic new drugs from acyclic starting compounds, yielding macrocyclic compounds with enhanced potency and improved drug-like properties.
    Keywords:  CDK2/4/6; Cyclin-dependent kinases; Enhanced drug-like properties; Macrocyclization platform
    DOI:  https://doi.org/10.1016/j.bioorg.2024.107285
  12. J Pept Sci. 2024 Mar 25. e3597
    N-terminal modification of an LAH4-derived peptide increases mRNA delivery in the presence of serum.
    Candice Dussouillez, Morane Lointier, Mohammed-Karim Sebane, Sylvie Fournel, Burkhard Bechinger, Antoine Kichler.
      The recently developed mRNA-based coronavirus SARS-CoV-2 vaccines highlighted the great therapeutic potential of the mRNA technology. Although the lipid nanoparticles used for the delivery of the mRNA are very efficient, they showed, in some cases, the induction of side effects as well as the production of antibodies directed against particle components. Thus, the development of alternative delivery systems is of great interest in the pursuit of more effective mRNA treatments. In the present work, we evaluated the mRNA transfection capacities of a series of cationic histidine-rich amphipathic peptides derived from LAH4. We found that while the LAH4-A1 peptide was an efficient carrier for mRNA, its activity was highly serum sensitive. Interestingly, modification of this cell penetrating peptide at the N-terminus with two tyrosines or with salicylic acid allowed to confer serum resistance to the carrier.
    Keywords:  LAH4 peptide family; amphipathic peptide; cell penetrating peptide; gene delivery; mRNA transfection
    DOI:  https://doi.org/10.1002/psc.3597
  13. Chemistry. 2024 Mar 25. e202400691
    Accelerating Cellular Uptake with Unnatural Amino Acid for Inhibiting Immunosuppressive Cancer Cells.
    Meihui Yi, Gabriel Ashton-Rickardt, Weiyi Tan, Zhiyu Liu, Hongjian He, Jer-Tsong Hsieh, Bing Xu.
      Targeting immunosuppressive metastatic cancer cells is a key challenge in therapy. We recently have shown that a rigid-rod aromatic, pBP-NBD, that responds to enzymes and kill immunosuppressive metastatic osteosarcoma (mOS) and castration resistant prostate cancer (CRPC) cells in mimetic bone microenvironment. However, pBP-NBD demonstrated moderate efficacy against CRPC cells. To enhance activity, we incorporated the unnatural amino acid L- or D-4,4'-biphenylalanine (L- or D-BiP) into pBP-NBD, drastically increasing cellular uptake and CRPC inhibition. Specifically, we inserted BiP into pBP-NBD to target mOS (Saos2 and SJSA1) and CRPC (VCaP and PC3) cells with overexpressed phosphatases. Our results show that the D-peptide backbone with an aspartate methyl diester at the C-terminal offers the highest activity against these immunosuppressive mOS and CRPC cells. Importantly, imaging shows that the peptide assemblies almost instantly enter the cells and accumulate primarily within the endoplasmic reticulum of Saos2, SJSA1, and PC3 cells and at the lysosomes of VCaP cells. By using BiP to boost cellular uptake and self-assembly within cancer cells, this work illustrates an unnatural hydrophobic amino acid as a versatile and effective residue to boost endocytosis of synthetic peptides for intracellular self-assembly.
    Keywords:  Accelerated cellular uptake Unnatural amino acids Self-assembly
    DOI:  https://doi.org/10.1002/chem.202400691
  14. Angew Chem Int Ed Engl. 2024 Mar 26. e202403215
    Bioinspired Synthesis of Allysine for Late-Stage Functionalization of Peptides.
    Benjamin Emenike, Sophia Shahin, Monika Raj.
      Inspired by the enzyme lysyl oxidase, which selectively converts the side chain of lysine into allysine, an aldehyde-containing post-translational modification, we report herein the first chemical method for the synthesis of allysine by selective oxidation of dimethyl lysine. This approach is highly chemoselective for dimethyl lysine on proteins. We highlight the utility of this biomimetic approach for generating aldehydes in a variety of pharmaceutically active linear and cyclic peptides at a late stage for their diversification with various affinity and fluorescent tags. Notably, we utilized this approach for generating small-molecule aldehydes from the corresponding tertiary amines. We further demonstrated the potential of this approach in generating cellular models for studying allysine-associated diseases.
    Keywords:  Aldehydes; Chemoselectivity; Dimethyl lysine; Late-stage modification; protein modification
    DOI:  https://doi.org/10.1002/anie.202403215
  15. Front Oncol. 2024 ;14 1292083
    A dynamic study of VEGF-A siDOX-EVs trafficking through the in-vitro insert co-culture blood-brain barrier model by digital holographic microscopy.
    Parisa Shamshiripour, Mehrana Rahnama, Mehdi Nikoobakht, Fahimeh Hajiahmadi, Ali-Reza Moradi, Davoud Ahmadvand.
      Introduction: Modeling the blood-brain barrier has long been a challenge for pharmacological studies. Up to the present, numerous attempts have been devoted to recapitulating the endothelial barrier in vitro to assess drug delivery vehicles' efficiency for brain disorders. In the current work, we presented a new approach for analyzing the morphometric parameters of the cells of an insert co-culture blood-brain barrier model using rat brain astrocytes, rat brain microvascular endothelial cells, and rat brain pericytes. This analytical approach could aid in getting further information on drug trafficking through the blood-brain barrier and its impact on the brain indirectly.Methods: In the current work, we cultured rat brain astrocytes, rat brain microvascular endothelial cells, and rat brain pericytes and then used an insert well to culture the cells in contact with each other to model the blood-brain barrier. Then, the morphometric parameters of the porous membrane of the insert well, as well as each cell type were imaged by digital holographic microscopy before and after cell seeding. At last, we performed folate conjugation on the surface of the EVs we have previously tested for glioma therapy in our previous work called VEGF-A siDOX-EVs and checked how the trafficking of EVs improves after folate conjugation as a clathrin-mediated delivery setup. the trafficking and passage of EVs were assessed by flow cytometry and morphometric analysis of the digital holographic microscopy holograms.
    Results: Our results indicated that EVs successfully entered through the proposed endothelial barrier assessed by flow cytometry analysis and furthermore, folate conjugation significantly improved EV passage through the blood-brain barrier. Moreover, our results indicated that the VEGF-A siDOX-EVs insert cytotoxic impact on the cells of the bottom of the culture plate.
    Conclusion: folate-conjugation on the surface of EVs improves their trafficking through the blood-brain barrier and by using digital holographic microscopy analysis, we could directly assess the morphometric changes of the blood-brain barrier cells for pharmacological purposes as an easy, label-free, and real-time analysis.
    Keywords:  blood tumor barrier; blood-brain barrier; co-culture; drug delivery; glioblastoma
    DOI:  https://doi.org/10.3389/fonc.2024.1292083
  16. PLoS One. 2024 ;19(3): e0299804
    A phage-displayed disulfide constrained peptide discovery platform yields novel human plasma protein binders.
    Xinxin Gao, Harini Kaluarachchi, Yingnan Zhang, Sunhee Hwang, Rami N Hannoush.
      Disulfide constrained peptides (DCPs) show great potential as templates for drug discovery. They are characterized by conserved cysteine residues that form intramolecular disulfide bonds. Taking advantage of phage display technology, we designed and generated twenty-six DCP phage libraries with enriched molecular diversity to enable the discovery of ligands against disease-causing proteins of interest. The libraries were designed based on five DCP scaffolds, namely Momordica charantia 1 (Mch1), gurmarin, Asteropsin-A, antimicrobial peptide-1 (AMP-1), and potato carboxypeptidase inhibitor (CPI). We also report optimized workflows for screening and producing synthetic and recombinant DCPs. Examples of novel DCP binders identified against various protein targets are presented, including human IgG Fc, serum albumin, vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor (PDGF). We identified DCPs against human IgG Fc and serum albumin with sub-micromolar affinity from primary panning campaigns, providing alternative tools for potential half-life extension of peptides and small protein therapeutics. Overall, the molecular diversity of the DCP scaffolds included in the designed libraries, coupled with their distinct biochemical and biophysical properties, enables efficient and robust identification of de novo binders to drug targets of therapeutic relevance.
    DOI:  https://doi.org/10.1371/journal.pone.0299804
  17. Eur J Cell Biol. 2024 Mar 21. pii: S0171-9335(24)00023-2. [Epub ahead of print]103(2): 151406
    The solute carrier SLC7A1 may act as a protein transporter at the blood-brain barrier.
    Magdalena Kurtyka, Frank Wessely, Sarah Bau, Eseoghene Ifie, Liqun He, Nienke M de Wit, Alberte Bay Villekjær Pedersen, Maximilian Keller, Caleb Webber, Helga E de Vries, Olaf Ansorge, Christer Betsholtz, Marijke De Bock, Catarina Chaves, Birger Brodin, Morten S Nielsen, Winfried Neuhaus, Robert D Bell, Tamás Letoha, Axel H Meyer, Germán Leparc, Martin Lenter, Dominique Lesuisse, Zameel M Cader, Stephen T Buckley, Irena Loryan, Claus U Pietrzik.
      Despite extensive research, targeted delivery of substances to the brain still poses a great challenge due to the selectivity of the blood-brain barrier (BBB). Most molecules require either carrier- or receptor-mediated transport systems to reach the central nervous system (CNS). These transport systems form attractive routes for the delivery of therapeutics into the CNS, yet the number of known brain endothelium-enriched receptors allowing the transport of large molecules into the brain is scarce. Therefore, to identify novel BBB targets, we combined transcriptomic analysis of human and murine brain endothelium and performed a complex screening of BBB-enriched genes according to established selection criteria. As a result, we propose the high-affinity cationic amino acid transporter 1 (SLC7A1) as a novel candidate for transport of large molecules across the BBB. Using RNA sequencing and in situ hybridization assays, we demonstrated elevated SLC7A1 gene expression in both human and mouse brain endothelium. Moreover, we confirmed SLC7A1 protein expression in brain vasculature of both young and aged mice. To assess the potential of SLC7A1 as a transporter for larger proteins, we performed internalization and transcytosis studies using a radiolabelled or fluorophore-labelled anti-SLC7A1 antibody. Our results showed that SLC7A1 internalised a SLC7A1-specific antibody in human colorectal carcinoma (HCT116) cells. Moreover, transcytosis studies in both immortalised human brain endothelial (hCMEC/D3) cells and primary mouse brain endothelial cells clearly demonstrated that SLC7A1 effectively transported the SLC7A1-specific antibody from luminal to abluminal side. Therefore, here in this study, we present for the first time the SLC7A1 as a novel candidate for transport of larger molecules across the BBB.
    Keywords:  BBB; CAT-1; SLC7A1; brain drug delivery; brain therapeutics; solute carriers
    DOI:  https://doi.org/10.1016/j.ejcb.2024.151406
  18. Front Pharmacol. 2024 ;15 1355893
    Aiming the magic bullet: targeted delivery of imaging and therapeutic agents to solid tumors by pHLIP peptides.
    Yana K Reshetnyak, Oleg A Andreev, Donald M Engelman.
      The family of pH (Low) Insertion Peptides (pHLIP) comprises a tumor-agnostic technology that uses the low pH (or high acidity) at the surfaces of cells within the tumor microenvironment (TME) as a targeted biomarker. pHLIPs can be used for extracellular and intracellular delivery of a variety of imaging and therapeutic payloads. Unlike therapeutic delivery targeted to specific receptors on the surfaces of particular cells, pHLIP targets cancer, stromal and some immune cells all at once. Since the TME exhibits complex cellular crosstalk interactions, simultaneous targeting and delivery to different cell types leads to a significant synergistic effect for many agents. pHLIPs can also be positioned on the surfaces of various nanoparticles (NPs) for the targeted intracellular delivery of encapsulated payloads. The pHLIP technology is currently advancing in pre-clinical and clinical applications for tumor imaging and treatment.
    Keywords:  cell acidity as a biomarker; immune therapy strategies; pHLIP targeting of cell acidity; targeted delivery to the cytoplasm; targeted drug delivery; targeted nanoparticle delivery; targeting diverse tumor cells; tumor imaging and diagnosis
    DOI:  https://doi.org/10.3389/fphar.2024.1355893
  19. Cell Rep. 2024 Mar 22. pii: S2211-1247(24)00312-7. [Epub ahead of print]43(4): 113984
    Acod1 expression in cancer cells promotes immune evasion through the generation of inhibitory peptides.
    James H Schofield, Joseph Longo, Ryan D Sheldon, Emma Albano, Abigail E Ellis, Mark A Hawk, Sean Murphy, Loan Duong, Sharif Rahmy, Xin Lu, Russell G Jones, Zachary T Schafer.
      Targeting programmed cell death protein 1 (PD-1) is an important component of many immune checkpoint blockade (ICB) therapeutic approaches. However, ICB is not an efficacious strategy in a variety of cancer types, in part due to immunosuppressive metabolites in the tumor microenvironment. Here, we find that αPD-1-resistant cancer cells produce abundant itaconate (ITA) due to enhanced levels of aconitate decarboxylase (Acod1). Acod1 has an important role in the resistance to αPD-1, as decreasing Acod1 levels in αPD-1-resistant cancer cells can sensitize tumors to αPD-1 therapy. Mechanistically, cancer cells with high Acod1 inhibit the proliferation of naive CD8+ T cells through the secretion of inhibitory factors. Surprisingly, inhibition of CD8+ T cell proliferation is not dependent on the secretion of ITA but is instead a consequence of the release of small inhibitory peptides. Our study suggests that strategies to counter the activity of Acod1 in cancer cells may sensitize tumors to ICB therapy.
    Keywords:  Acod1; CP: Cancer; CP: Immunology; TCA cycle; immune evasion; itaconate
    DOI:  https://doi.org/10.1016/j.celrep.2024.113984
  20. ChemMedChem. 2024 Mar 24. e202300343
    Structural analysis of the macrocyclic inhibitor BI-4020 binding to EGFR kinase.
    Tyler S Beyett, Jaimin K Rana, Ilse K Schaeffner, David E Heppner, Michael Eck.
      A novel macrocyclic inhibitor of mutant EGFR (BI-4020) has shown promise in pre-clinical studies of T790M and C797S drug-resistant non-small cell lung cancer. To better understand the molecular basis for BI-4020 selectivity and potency, we have carried out biochemical activity assays and structural analysis with X-ray crystallography. Biochemical potencies agree with previous studies indicating that BI-4020 is uniquely potent against drug-resistant L858R/T790M and L858R/T790M/C797S variants. Structures show that BI-4020 is likely rendered selective due to interactions with the kinase domain hinge region as well as T790M, akin to Osimertinib. Additionally, BI-4020 is also rendered more potent due to its constrained macrocycle geometry as well as additional H-bonds to conserved K745 and T845 residues in both active and inactive conformations. These findings taken together show how this novel macrocyclic inhibitor is both highly potent and selective for mutant EGFR in a reversible mechanism and motivate structure- inspired approaches to developing targeted therapies in medicinal oncology.
    Keywords:  EGFR; Inhibitor; kinase; macrocycle
    DOI:  https://doi.org/10.1002/cmdc.202300343
  21. Biomedicines. 2024 Mar 12. pii: 626. [Epub ahead of print]12(3):
    Co-Culture Models: Key Players in In Vitro Neurotoxicity, Neurodegeneration and BBB Modeling Studies.
    Ana Rita Monteiro, Daniel José Barbosa, Fernando Remião, Renata Silva.
      The biological barriers existing in the human body separate the blood circulation from the interstitial fluid in tissues. The blood-brain barrier (BBB) isolates the central nervous system from the bloodstream, presenting a dual role: the protection of the human brain against potentially toxic/harmful substances coming from the blood, while providing nutrients to the brain and removing metabolites. In terms of architectural features, the presence of junctional proteins (that restrict the paracellular transport) and the existence of efflux transporters at the BBB are the two major in vivo characteristics that increase the difficulty in creating an ideal in vitro model for drug permeability studies and neurotoxicity assessments. The purpose of this work is to provide an up-to-date literature review on the current in vitro models used for BBB studies, focusing on the characteristics, advantages, and disadvantages of both primary cultures and immortalized cell lines. An accurate analysis of the more recent and emerging techniques implemented to optimize the in vitro models is also provided, based on the need of recreating as closely as possible the BBB microenvironment. In fact, the acceptance that the BBB phenotype is much more than endothelial cells in a monolayer has led to the shift from single-cell to multicellular models. Thus, in vitro co-culture models have narrowed the gap between recreating as faithfully as possible the human BBB phenotype. This is relevant for permeability and neurotoxicity assays, and for studies related to neurodegenerative diseases. Several studies with these purposes will be also presented and discussed.
    Keywords:  blood–brain barrier; co-culture models; in vitro; neurodegeneration; neurotoxicity; neurovascular unit
    DOI:  https://doi.org/10.3390/biomedicines12030626
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