bims-cepepe Biomed News
on Cell-penetrating peptides
Issue of 2024‒01‒21
nine papers selected by
Henry Lamb, Queensland University of Technology
  1. Comparative transport analysis of cell penetrating peptides and Lysosomal sequences for selective tropism towards RPE cells.
  2. Solid-phase peptide synthesis in 384-well plates.
  3. IP3RPEP6, a novel peptide inhibitor of IP3 receptor channels that does not affect connexin-43 hemichannels.
  4. Introduction of a fatty acid chain modification to prolong circulatory half-life of a radioligand towards glucose-dependent insulinotropic polypeptide receptor.
  5. The Role of Attractive Non-Covalent Interactions in Peptide Macrocyclization.
  6. Fluorescent peptide for detecting factor XIIIa activity and fibrin in whole blood clots forming under flow.
  7. Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom.
  8. Discovery of the First-in-Class RORγ Covalent Inhibitors for Treatment of Castration-Resistant Prostate Cancer.
  9. Drug resistance in glioblastoma: from chemo- to immunotherapy.
  1. Res Sq. 2023 Dec 27. pii: rs.3.rs-3651531. [Epub ahead of print]
    Comparative transport analysis of cell penetrating peptides and Lysosomal sequences for selective tropism towards RPE cells.
    Kris Grohn, Kyle Parella, Ellie Lumen, Hanna Colegrove, Victor Bjork, Alana Franceski, Aaron Wolfe, Kelsey Moody.
      Cell penetrating peptides are typically nonspecific, targeting multiple cell types without discrimination. However, subsets of Cell penetrating peptides (CPP) have been found, which show a 'homing' capacity or increased likelihood of internalizing into specific cell types and subcellular locations. Therapeutics intended to be delivered to tissues with a high degree of cellular diversity, such as the intraocular space, would benefit from delivery using CPP that can discriminate across multiple cell types. Lysosomal storage diseases in the retinal pigment epithelium (RPE) can impair cargo clearance, leading to RPE atrophy and blindness. Characterizing CPP for their capacity to effectively deliver cargo to the lysosomes of different cell types may expand treatment options for lysosomal storage disorders. We developed a combinatorial library of CPP and lysosomal sorting signals, applied to ARPE19 and B3 corneal lens cells, for the purpose of determining cell line specificity and internal targeting. Several candidate classes of CPP were found to have as much as 4 times the internalization efficiency in ARPE19 compared to B3. Follow-up cargo transport studies were also performed, which demonstrate effective internalization and lysosomal targeting in ARPE19 cells.
    DOI:  https://doi.org/10.21203/rs.3.rs-3651531/v1
  2. J Pept Sci. 2024 Jan 14. e3555
    Solid-phase peptide synthesis in 384-well plates.
    Mischa Schüttel, Edward Will, Gontran Sangouard, Anne Zarda, Sevan Habeshian, Alexander L Nielsen, Christian Heinis.
      Newer solid-phase peptide synthesis and release strategies enable the production of short peptides with high purity, allowing direct screening for desired bioactivity without prior chromatographic purification. However, the maximum number of peptides that can currently be synthesized per microplate reactor is 96, allowing the parallel synthesis of 384 peptides in modern devices that have space for 4 microplate reactors. To synthesize larger numbers of peptides, we modified a commercially available peptide synthesizer to enable the production of peptides in 384-well plates, which allows the synthesis of 1,536 peptides in one run (4 × 384 peptides). We report new hardware components and customized software that allowed for the synthesis of 1,536 short peptides in good quantity (average > 0.5 μmol), at high concentration (average > 10 mM), and decent purity without purification (average > 80%). The high-throughput peptide synthesis, which we developed with peptide drug development in mind, may be widely used for peptide library synthesis and screening, antibody epitope scanning, epitope mimetic development, or protease/kinase substrate screening.
    Keywords:  384-well plate; SPPS; combinatorial chemistry; peptide library; peptide synthesis
    DOI:  https://doi.org/10.1002/psc.3555
  3. Acta Physiol (Oxf). 2024 Jan 19. e14086
    IP3RPEP6, a novel peptide inhibitor of IP3 receptor channels that does not affect connexin-43 hemichannels.
    Siyu Tao, Paco Hulpiau, Larry E Wagner, Katja Witschas, David I Yule, Geert Bultynck, Luc Leybaert.
      AIM: Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are intracellular Ca2+ -release channels with crucial roles in cell function. Current IP3 R inhibitors suffer from off-target effects and poor selectivity towards the three distinct IP3 R subtypes. We developed a novel peptide inhibitor of IP3 Rs and determined its effect on connexin-43 (Cx43) hemichannels, which are co-activated by IP3 R stimulation.METHODS: IP3RPEP6 was developed by in silico molecular docking studies and characterized by on-nucleus patch-clamp experiments of IP3 R2 channels and carbachol-induced IP3 -mediated Ca2+ responses in IP3 R1, 2 or 3 expressing cells, triple IP3 R KO cells and astrocytes. Cx43 hemichannels were studied by patch-clamp and ATP-release approaches, and by inhibition with Gap19 peptide. IP3RPEP6 interactions with IP3 Rs were verified by co-immunoprecipitation and affinity pull-down assays.
    RESULTS: IP3RPEP6 concentration-dependently reduced the open probability of IP3 R2 channels and competitively inhibited IP3 Rs in an IC50 order of IP3 R2 (~3.9 μM) < IP3 R3 (~4.3 μM) < IP3 R1 (~9.0 μM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co-immunoprecipitated with IP3 R2 but not with IP3 R1; interaction with IP3 R3 varied between cell types. The IC50 of IP3RPEP6 inhibition of carbachol-induced Ca2+ responses decreased with increasing cellular Cx43 expression. Moreover, Gap19-inhibition of Cx43 hemichannels significantly reduced the amplitude of the IP3 -Ca2+ responses and strongly increased the EC50 of these responses. Finally, we identified palmitoyl-8G-IP3RPEP6 as a membrane-permeable IP3RPEP6 version allowing extracellular application of the IP3 R-inhibiting peptide.
    CONCLUSION: IP3RPEP6 inhibits IP3 R2/R3 at concentrations that have limited effects on IP3 R1. IP3 R activation triggers hemichannel opening, which strongly affects the amplitude and concentration-dependence of IP3 -triggered Ca2+ responses.
    Keywords:  Ca2+ signaling; IP3; astrocytes; connexin-43; hemichannel; inositol 1,4,5-trisphosphate; peptide inhibitor
    DOI:  https://doi.org/10.1111/apha.14086
  4. Nucl Med Biol. 2024 Jan 10. pii: S0969-8051(24)00002-7. [Epub ahead of print]128-129 108876
    Introduction of a fatty acid chain modification to prolong circulatory half-life of a radioligand towards glucose-dependent insulinotropic polypeptide receptor.
    Amina Khalil, Sona Hakhverdyan, Pierre Cheung, Martin Bossart, Michael Wagner, Olof Eriksson, Irina Velikyan.
      BACKGROUND: The beneficial role of glucose-dependent insulinotropic polypeptide receptor (GIPR) in weight control and maintaining glucose levels has led to the development of several multi-agonistic peptide drug candidates, targeting GIPR and glucagon like peptide 1 receptor (GLP1R) and/or the glucagon receptor (GCGR). The in vivo quantification of target occupancy by these drugs would accelerate the development of new drug candidates. The aim of this study was to evaluate a novel peptide (GIP1234), based on previously reported ligand DOTA-GIP-C803, modified with a fatty acid moiety to prolong its blood circulation. It would allow higher target tissue exposure and consequently improved peptide uptake as well as in vivo PET imaging and quantification of GIPR occupancy by novel drugs of interest.METHOD: A 40 amino acid residue peptide (GIP1234) was synthesized based on DOTA-GIP-C803, in turn based on the sequences of endogenous GIP and Exendin-4 with specific amino acid modifications to obtain GIPR selectivity. A palmitoyl fatty acid chain was furthermore added at Lys14 via a glutamic acid linker to prolong its blood circulation time by the interaction with albumin. GIP1234 was conjugated with a DOTA chelator at the C-terminal cysteine residue to achieve 68Ga radiolabeling. The resulting PET probe, [68Ga]Ga-DOTA-GIP1234 was evaluated for receptor binding specificity and selectivity using HEK293 cells transfected with human GIPR, GLP1R, or GCGR. Blocking experiments with tirzepatide (2 μM) were conducted using huGIPR HEK293 cells to investigate binding specificity. Ex vivo and in vivo organ distribution of [68Ga]Ga-DOTA-GIP1234 was studied in rats and a pig in comparison to [68Ga]Ga-DOTA-C803-GIP. Binding of [68Ga]Ga-DOTA-GIP1234 to albumin was assessed in situ using polyacrylamide gel electrophoresis (PAGE). The stability was tested in formulation buffer and rat blood plasma.
    RESULTS: [68Ga]Ga-DOTA-GIP1234 was synthesized with non-decay corrected radiochemical yield of 88 ± 3.7 % and radiochemical purity of 97.8 ± 0.8 %. The molar activity for the radiotracer was 8.1 ± 1.1 MBq/nmol. [68Ga]Ga-DOTA-GIP1234 was stable and maintained affinity to huGIPR HEK293 cells (dissociation constant (Kd) = 40 ± 12.5 nM). The binding of [68Ga]Ga-DOTA-GIP1234 to huGCGR and huGLP1R cells was insignificant. Pre-incubation of huGIPR HEK293 cell sections with tirzepatide resulted in the decrease of [68Ga]Ga-DOTA-GIP1234 binding by close to 90 %. [68Ga]Ga-DOTA-GIP1234 displayed slow blood clearance in pigs with SUV = 3.5 after 60 min. Blood retention of the tracer in rat was 2-fold higher than that of [68Ga]Ga-DOTA-C803-GIP. [68Ga]Ga-DOTA-GIP1234 also demonstrated strong liver uptake in both pig and rat combined with decreased renal excretion. The concentration dependent binding of [68Ga]Ga-DOTA-GIP1234 to albumin was confirmed in situ by PAGE.
    CONCLUSION: [68Ga]Ga-DOTA-GIP1234 demonstrated nanomolar affinity and selectivity for huGIPR in vitro. Addition of a fatty acid moiety prolonged blood circulation time and tissue exposure in both rat and pig in vivo. However, the liver uptake was also increased which may make PET imaging of abdominal tissues such as pancreas challenging. The investigation of the influence of fatty acid moiety on the biological performance of the peptide ligand paved the way for further rational design of GIPR ligand analogues with improved characteristics.
    Keywords:  Glucose-dependent insulinotropic polypeptide receptor; Incretins; PET imaging; Peptide radioligands
    DOI:  https://doi.org/10.1016/j.nucmedbio.2024.108876
  5. J Org Chem. 2024 Jan 13.
    The Role of Attractive Non-Covalent Interactions in Peptide Macrocyclization.
    Diego B Diaz, Rozhin Rowshanpour, George J Saunders, Travis Dudding, Andrei K Yudin.
      The efficiency of macrocyclization reactions relies on the appropriate conformational preorganization of a linear precursor, ensuring that reactive ends are in spatial proximity prior to ring closure. Traditional peptide cyclization approaches that reduce the extent of terminal ion pairing often disfavor cyclization-conducive conformations and can lead to undesired cyclodimerization or oligomerization side reactions, particularly when they are performed without high dilution. To address this challenge, synthetic strategies that leverage attractive noncovalent interactions, such as zwitterionic attraction between chain termini during macrocyclization, offer a potential solution by reducing the entropic penalty associated with linear peptides adopting precyclization conformations. In this study, we investigate the role of (N-isocyanoimino)triphenylphosphorane (Pinc) in facilitating the cyclization of linear peptides into conformationally rigid macrocycles. The observed moderate diastereoselectivity is consistent with the preferential Si-facial addition of Pinc, where the isocyanide adds to the E-iminium ion on the same face as the l-proline amide group. The resulting peptide chain reveals that the activated phosphonium ylide of Pinc brings the reactive ends close together, promoting cyclization by enclosing the carboxylate within the interior of the pentapeptide and preventing the formation of byproducts. For shorter peptides with modified peptide backbones, the cyclization mechanism and outcome are redirected, as nucleophilic motifs such as thiazole and imidazole can covalently trap nitrilium intermediates. The isolation of the intermediate in the unproductive macrocyclization pathway, along with nuclear magnetic resonance and density functional theory studies, provides insights into heterocycle-dependent selectivity. The Pinc-driven macrocyclization process has generated diverse collections of cyclic molecules, and our models offer a comprehensive understanding of observed trends, facilitating the development of other heterocycle-forming macrocyclization reactions.
    DOI:  https://doi.org/10.1021/acs.joc.3c02084
  6. Res Pract Thromb Haemost. 2024 Jan;8(1): 102291
    Fluorescent peptide for detecting factor XIIIa activity and fibrin in whole blood clots forming under flow.
    Yue Liu, Jennifer Crossen, Timothy J Stalker, Scott L Diamond.
      Background: During clotting, thrombin generates fibrin monomers and activates plasma-derived transglutaminase factor (F) XIIIa; collagen and thrombin-activated platelets offer thrombin-independent cellular FXIIIa (cFXIIIa) for clotting. Detecting fibrin on collagen and tissue factor surfaces in whole blood clotting typically uses complex reagents like fluorescent fibrinogen or antifibrin antibody.Objectives: We want to test whether the peptide using the α2- antiplasmin crosslinking mechanism by FXIIIa is a useful tool in both monitoring FXIIIa activity, and visualize and monitor fibrin formation, deposition, and extent of crosslinking within fibrin structures in whole blood clots formed under flow.
    Methods: We tested a fluorescent peptide derived from α2-antiplasmin sequence (Ac-GNQEQVSPLTLLKWC-fluorescein) to monitor the location of transglutaminase activity and fibrin during whole blood clotting under microfluidic flow (wall shear rate, 100 s-1).
    Results: The peptide rapidly colocated with accumulating fibrin due to transglutaminase activity, confirmed by Phe-Pro-Arg-chloromethylketone inhibiting fibrin and peptide labeling. The FXIIIa inhibitor T101 had no effect on fibrin generation but ablated the labeling of fibrin by the peptide. Similarly, Gly-Pro-Arg-Pro abated fibrin formation and thus strongly attenuated the peptide signal. At arterial wall shear rate (1000 s-1), less fibrin was formed, and consequently, less peptide labeling of fibrin was detected compared with venous conditions. The addition of tissue plasminogen activator caused a reduction of both fibrin and peptide signals. Also, the peptide strongly colocalized with fibrin (but not platelets) in clots from laser-injured mouse cremaster arterioles. For clotting under flow, FXIIIa activity was most likely plasma-derived since a RhoA inhibitor did not block α2-antiplasmin fragment cross-linking to fibrin.
    Conclusion: Under flow, the majority of FXIIIa-dependent fibrin labeling with peptide during clotting was distal of thrombin activity. The synthetic peptide provided a strong and sustained labeling of fibrin as it formed under flow.
    Keywords:  alpha-2-antiplasmin; blood platelets; fibrin; thrombosis
    DOI:  https://doi.org/10.1016/j.rpth.2023.102291
  7. Cell Rep Phys Sci. 2023 Jul 19. pii: 101459. [Epub ahead of print]4(7):
    Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom.
    Andreia Boaro, Lucía Ageitos, Marcelo Der Torossian Torres, Esther Broset Blasco, Sebahat Oztekin, Cesar de la Fuente-Nunez.
      Antimicrobial peptides (AMPs) derived from natural toxins and venoms offer a promising alternative source of antibiotics. Here, through structure-function-guided design, we convert two natural AMPs derived from the venom of the solitary eumenine wasp Eumenes micado into α-helical AMPs with reduced toxicity that kill Gram-negative bacteria in vitro and in a preclinical mouse model. To identify the sequence determinants conferring antimicrobial activity, an alanine scan screen and strategic single lysine substitutions are made to the amino acid sequence of these natural peptides. These efforts yield a total of 34 synthetic derivatives, including alanine substituted and lysine-substituted sequences with stabilized α-helical structures and increased net positive charge. The resulting lead synthetic peptides kill the Gram-negative pathogens Escherichia coli and Pseudomonas aeruginosa (PAO1 and PA14) by rapidly permeabilizing both their outer and cytoplasmic membranes, exhibit anti-infective efficacy in a mouse model by reducing bacterial loads by up to three orders of magnitude, and do not readily select for bacterial resistance.
    DOI:  https://doi.org/10.1016/j.xcrp.2023.101459
  8. J Med Chem. 2024 Jan 16.
    Discovery of the First-in-Class RORγ Covalent Inhibitors for Treatment of Castration-Resistant Prostate Cancer.
    Wei Fang, Jianwei Zheng, Lin Deng, Yana An, Deqin Rong, Jianwei Wei, Xiao-Feng Xiong, Junjian Wang, Yuanxiang Wang.
      Nuclear receptor receptor-related orphan receptor γ (RORγ) is a ligand-dependent transcription factor and has been established as a key player in castration-resistant prostate cancers (CRPC) by driving androgen receptor (AR) overexpression, representing a potential therapeutical target for advanced prostate cancers. Here, we report the identification of the first-in-class RORγ covalent inhibitor 29 via the structure-based drug design approach following structure-activity relationship (SAR) exploration. Mass spectrometry assay validated its covalent inhibition mechanism. Compound 29 significantly inhibited RORγ transcriptional activity and remarkably suppressed the expression levels of AR and AR-targeted genes. Compound 29 also exhibited much superior activity in inhibiting the proliferation and colony formation and inducing apoptosis of the CRPC cell lines relative to the positive control 2 and noncovalent control 33. Importantly, it markedly suppressed the tumor growth in a 22Rv1 mouse tumor xenograft model with good safety. These results clearly demonstrate that 29 is a highly potent and selective RORγ covalent inhibitor.
    DOI:  https://doi.org/10.1021/acs.jmedchem.3c02063
  9. Cancer Drug Resist. 2023 ;6(4): 688-708
    Drug resistance in glioblastoma: from chemo- to immunotherapy.
    Sachin Sharma, Oksana Chepurna, Tao Sun.
      As the most common and aggressive type of primary brain tumor in adults, glioblastoma is estimated to end over 10,000 lives each year in the United States alone. Stand treatment for glioblastoma, including surgery followed by radiotherapy and chemotherapy (i.e., Temozolomide), has been largely unchanged since early 2000. Cancer immunotherapy has significantly shifted the paradigm of cancer management in the past decade with various degrees of success in treating many hematopoietic cancers and some solid tumors, such as melanoma and non-small cell lung cancer (NSCLC). However, little progress has been made in the field of neuro-oncology, especially in the application of immunotherapy to glioblastoma treatment. In this review, we attempted to summarize the common drug resistance mechanisms in glioblastoma from Temozolomide to immunotherapy. Our intent is not to repeat the well-known difficulty in the area of neuro-oncology, such as the blood-brain barrier, but to provide some fresh insights into the molecular mechanisms responsible for resistance by summarizing some of the most recent literature. Through this review, we also hope to share some new ideas for improving the immunotherapy outcome of glioblastoma treatment.
    Keywords:  Glioblastoma; drug resistance; immunosuppression; immunotherapy; tumor microenvironment
    DOI:  https://doi.org/10.20517/cdr.2023.82
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