bims-cepepe Biomed News
on Cell-penetrating peptides
Issue of 2023–10–15
fourteen papers selected by
Henry Lamb, Queensland University of Technology



  1. Cells. 2023 Oct 02. pii: 2395. [Epub ahead of print]12(19):
      Antisense oligonucleotide-based (ASO) therapeutics have emerged as a promising strategy for the treatment of human disorders. Charge-neutral PMOs have promising biological and pharmacological properties for antisense applications. Despite their great potential, the efficient delivery of these therapeutic agents to target cells remains a major obstacle to their widespread use. Cellular uptake of naked PMO is poor. Cell-penetrating peptides (CPPs) appear as a possibility to increase the cellular uptake and intracellular delivery of oligonucleotide-based drugs. Among these, the DG9 peptide has been identified as a versatile CPP with remarkable potential for enhancing the delivery of ASO-based therapeutics due to its unique structural features. Notably, in the context of phosphorodiamidate morpholino oligomers (PMOs), DG9 has shown promise in enhancing delivery while maintaining a favorable toxicity profile. A few studies have highlighted the potential of DG9-conjugated PMOs in DMD (Duchenne Muscular Dystrophy) and SMA (Spinal Muscular Atrophy), displaying significant exon skipping/inclusion and functional improvements in animal models. The article provides an overview of a detailed understanding of the challenges that ASOs face prior to reaching their targets and continued advances in methods to improve their delivery to target sites and cellular uptake, focusing on DG9, which aims to harness ASOs' full potential in precision medicine.
    Keywords:  DG9 peptide; antisense oligonucleotides; cell penetrating peptides; delivery; phosphorodiamidate morpholino oligomers (PMO)
    DOI:  https://doi.org/10.3390/cells12192395
  2. Cells. 2023 Sep 26. pii: 2358. [Epub ahead of print]12(19):
      Cell-penetrating peptides (CPPs) are short peptide sequences that have the ability to cross the cell membrane and deliver cargo. Although it is critical that CPPs accomplish this task with minimal off-target effects, such actions have in many cases not been robustly screened. We presently investigated whether the commonly used CPPs TAT and the polyarginines Arg9 and Arg11 exert off-target effects on cellular Ca2+ homeostasis. In experiments employing myocytes and homogenates from the cardiac left ventricle or soleus muscle, we observed marked inhibition of Ca2+ recycling into the sarcoplasmic reticulum (SR) following incubation with polyarginine CPPs. In both tissues, the rate of SR Ca2+ leak remained unchanged, indicating that protracted Ca2+ removal from the cytosol stemmed from inhibition of the SR Ca2+ ATPase 2 (SERCA2). No such inhibition occurred following treatment with TAT, or in preparations from the SERCA1-expressing extensor digitorum longus muscle. Experiments in HEK cells overexpressing individual SERCA isoforms confirmed that polyarginine incubation specifically inhibited the activity of SERCA2a and 2b, but not SERCA1 or 3. The attenuation of SERCA2 activity was not dependent on the presence of phospholamban, and ELISA-based analyses rather revealed direct interaction between the polyarginines and the actuator domain of the protein. Surface plasmon resonance experiments confirmed strong binding within this region of SERCA2, and slow dissociation between the two species. Based on these observations, we urge caution when employing polyarginine CPPs. Indeed, as SERCA2 is expressed in diverse cell types, the wide-ranging consequences of SERCA2 binding and inhibition should be anticipated in both experimental and therapeutic settings.
    Keywords:  SERCA2; calcium homeostasis; cell-permeating peptides
    DOI:  https://doi.org/10.3390/cells12192358
  3. Molecules. 2023 Oct 09. pii: 6999. [Epub ahead of print]28(19):
      Milk-derived peptides are known to confer anti-inflammatory effects. We hypothesised that milk-derived cell-penetrating peptides might modulate inflammation in useful ways. Using computational techniques, we identified and synthesised peptides from the milk protein Alpha-S1-casein that were predicted to be cell-penetrating using a machine learning predictor. We modified the interpretation of the prediction results to consider the effects of histidine. Peptides were then selected for testing to determine their cell penetrability and anti-inflammatory effects using HeLa cells and J774.2 mouse macrophage cell lines. The selected peptides all showed cell penetrating behaviour, as judged using confocal microscopy of fluorescently labelled peptides. None of the peptides had an effect on either the NF-κB transcription factor or TNFα and IL-1β secretion. Thus, the identified milk-derived sequences have the ability to be internalised into the cell without affecting cell homeostatic mechanisms such as NF-κB activation. These peptides are worthy of further investigation for other potential bioactivities or as a naturally derived carrier to promote the cellular internalisation of other active peptides.
    Keywords:  NF-κB; TNFα; cell-penetrating peptides; macrophages; milk
    DOI:  https://doi.org/10.3390/molecules28196999
  4. J Chem Inf Model. 2023 Oct 12.
      Peptides have recently regained interest as therapeutic candidates, but their development remains confronted with several limitations including low bioavailability. Backbone head-to-tail cyclization, i.e., setting a covalent peptide bond linking the last amino acid with the first one, is one effective strategy of peptide-based drug design to stabilize the conformation of bioactive peptides while preserving peptide properties in terms of low toxicity, binding affinity, target selectivity, and preventing enzymatic degradation. Starting from an active peptide, it usually requires the design of a linker of a few amino acids to make it possible to cyclize the peptide, possibly preserving the conformation of the initial peptide and not affecting its activity. However, very little is known about the sequence-structure relationship requirements of designing linkers for peptide cyclization in a rational manner. Recently, we have shown that large-scale data-mining of available protein structures can lead to the precise identification of protein loop conformations, even from remote structural classes. Here, we transpose this approach to linkers, allowing head-to-tail peptide cyclization. First we show that given a linker sequence and the conformation of the linear peptide, it is possible to accurately predict the cyclized peptide conformation. Second, and more importantly, we show that it seems possible to elaborate on the information inferred from protein structures to propose effective candidate linker sequences constrained by length and amino acid composition, providing the first framework for the rational design of head-to-tail cyclization linkers. Finally, we illustrate this for two peptides using a limited set of amino-acids likely not to interfere with peptide function. For a linear peptide derived from Nrf2, the peptide cyclized starting from the experimental structure showed a 26-fold increase in the binding affinity. For urotensin II, a peptide already cyclized by a disulfide bond that exerts a broad array of biological activities, we were able, starting from models of the structure, to design a head-to-tail cyclized peptide, the first synthesized bicyclic 14-residue long urotensin II analogue, showing a retention of in vitro activity. Although preliminary, our results strongly suggest that such an approach has strong potential for cyclic peptide-based drug design.
    DOI:  https://doi.org/10.1021/acs.jcim.3c00865
  5. Int J Pharm. 2023 Oct 11. pii: S0378-5173(23)00920-1. [Epub ahead of print] 123499
      Our previous studies demonstrated that L-octaarginine grafted onto hyaluronic acid via a tetraglycine spacer significantly enhanced intranasal absorption of protein drugs with a molecular weight (Mw) of 22 kDa or less. The present study focused on its potential as an absorption enhancer for antibody drugs with a larger Mw and the enhancement mechanism. When ranibizumab (48 kDa) alone was intranasally administered in mice, its absolute bioavailability was 0.67% on average. The mean bioavailability elevated to 6.2% under coadministration with tetraglycine-L-octaarginine-linked hyaluronic acid. A similar result was observed under substitution of ranibizumab with certolizumab pegol (91 kDa), although bioavailability itself decreased with the Mw increase, irrespective of coadministration with the hyaluronic acid derivative. Rat experiments also revealed that coadministration with the polysaccharide derivative resulted in significant enhancement of intranasal absorption of trastuzumab (148 kDa). In vitro studies using gene-knocked down cells indicated that syndecan-4-induced macropinocytosis played a crucial role on acceleration of antibody uptake into epithelial cells on the nasal mucosa, irrespective of their Mw. It appeared that neither clathrin heavy chain nor caveolin-1 involved in cellular uptake of antibodies. Tetraglycine-L-octaarginine-linked hyaluronic acid was concluded to be a promising delivery tool that possessed universal absorption-enhancing abilities independent to Mw of biologics.
    Keywords:  antibody; cell-penetrating peptide-linked hyaluronic acid; cell-penetrating peptides; hyaluronic acid; macropinocytosis; mucosal absorption; syndecan-4
    DOI:  https://doi.org/10.1016/j.ijpharm.2023.123499
  6. Chem Sci. 2023 Oct 11. 14(39): 10800-10805
      The disruption of the protein-protein interaction (PPI) between Nrf2 and Keap1 is an attractive strategy to counteract the oxidative stress that characterises a variety of severe diseases. Peptides represent a complementary approach to small molecules for the inhibition of this therapeutically important PPI. However, due to their polar nature and the negative net charge required for binding to Keap1, the peptides reported to date exhibit either mid-micromolar activity or are inactive in cells. Herein, we present a two-component peptide stapling strategy to rapidly access a variety of constrained and functionalised peptides that target the Nrf2/Keap1 PPI. The most promising peptide, P8-H containing a fatty acid tag, binds to Keap1 with nanomolar affinity and is effective at inducing transcription of ARE genes in a human lung epithelial cell line at sub-micromolar concentration. Furthermore, crystallography of the peptide in complex with Keap1 yielded a high resolution X-ray structure, adding to the toolbox of structures available to develop cell-permeable peptidomimetic inhibitors.
    DOI:  https://doi.org/10.1039/d3sc04083f
  7. Arch Microbiol Immunol. 2023 ;7(3): 165-177
      There is increasing evidence that the T-cell protein, Lck, is involved in the pathogenesis of chronic lymphocytic leukemia (CLL) as well as other leukemias and lymphomas. We previously discovered that Lck binds to domain 5 of inositol 1,4,5-trisphosphate receptors (IP3R) to regulate Ca2+ homeostasis. Using bioinformatics, we targeted a region within domain 5 of IP3R-1 predicted to facilitate protein-protein interactions (PPIs). We generated a synthetic 21 amino acid peptide, KKRMDLVLELKNNASKLLLAI, which constitutes a domain 5 sub-domain (D5SD) of IP3R-1 that specifically binds Lck via its SH2 domain. With the addition of an HIV-TAT sequence to enable cell permeability of D5SD peptide, we observed wide-spread, Ca2+-dependent, cell killing of hematological cancer cells when the Lck-IP3R PPI was disrupted by TAT-D5SD. All cell lines and primary cells were sensitive to D5SD peptide, but malignant T-cells were less sensitive compared with B-cell or myeloid malignancies. Mining of RNA-seq data showed that LCK was expressed in primary diffuse large B-cell lymphoma (DLBCL) as well as acute myeloid leukemia (AML). In fact, LCK shows a similar pattern of expression as many well-characterized AML oncogenes and is part of a protein interactome that includes FLT3-ITD, Notch-1, and Kit. Consistent with these findings, our data suggest that the Lck-IP3R PPI may protect malignant hematopoietic cells from death. Importantly, TAT-D5SD showed no cytotoxicity in three different non-hematopoietic cell lines; thus its ability to induce cell death appears specific to hematopoietic cells. Together, these data show that a peptide designed to disrupt the Lck-IP3R PPI has a wide range of pre-clinical activity in leukemia and lymphoma.
    Keywords:  Biochemistry, Proteins; Biological sciences; Cell biology; Cell death; Diseases, Hematological diseases; Drug discovery; Health sciences; Hematological cancer; Leukaemia, Biological sciences; Oncogene proteins; Target identification
    DOI:  https://doi.org/10.26502/ami.936500114
  8. Bioorg Med Chem Lett. 2023 Oct 05. pii: S0960-894X(23)00377-3. [Epub ahead of print] 129499
      A4K14-Citropin 1.1 (GLFAVIKKVASVIKGL-NH2) is a derived antimicrobial peptide (AMP) with a more stable α-helical structure at the C-terminal compared to prototype Citropin 1.1 which was obtained from glandular skin secretions of Australian freetail lizards. In a previous report, A4K14-Citropin 1.1 has been considered as an anti-cancer lead compound. However, linear peptides are difficult to maintain stable secondary structure, resulted in poor pharmacokinetic properties. In this study, we designed and synthesized a series of benzyl-stapled derivatives of A4K14-Citropin 1.1. And their physical and chemical properties, as well as biological activity, were both explored. The result showed that AC-CCSP-2-o and AC-CCSP-3-o exhibited a higher degree of helicity and greater anti-cancer activity compared with the prototype peptide. Besides, there was no significant difference in the hemolytic effect between the stapled peptides and the prototype peptide. AC-CCSP-2-o and AC-CCSP-3-o could serve as promising anti-cancer lead compounds for the novel anti-cancer drug development.
    Keywords:  A4K14-Citropin 1.1; AMP; anti-cancer activity; antimicrobial peptide; thiol-halogen click chemistry
    DOI:  https://doi.org/10.1016/j.bmcl.2023.129499
  9. Chembiochem. 2023 Oct 10. e202300688
      Target-directed dynamic combinatorial chemistry is a very attractive strategy for the discovery of bioactive peptides. However, its application has not yet been demonstrated, presumably due to analytical challenges that arise from the diversity of a peptide library with combinatorial side-chains. We previously reported an efficient method to generate, under biocompatible conditions, large dynamic libraries of cyclic peptides grafted with amino acid's side-chains, by thiol-to-thioester exchanges. In this work, we present analytical tools to easily characterize such libraries by HPLC and mass spectrometry, and in particular to simplify the isomers' distinction requiring sequencing by MS/MS fragmentations. After structural optimization, the cyclic scaffold exhibits a UV-tag, absorbing at 415 nm, and an ornithine residue which favors the regioselective ring-opening and simultaneous MS/MS fragmentation, in the gas-phase, upon CID activation.
    Keywords:  Dynamic Combinatorial Chemistry Thioester exchange Cyclic peptide Mass Spectrometry Lignad Screening
    DOI:  https://doi.org/10.1002/cbic.202300688
  10. Bull Cancer. 2023 Oct 07. pii: S0007-4551(23)00371-5. [Epub ahead of print]
      One of the primary threats in tumor treatment revolves around the limited ability to penetrate tumor sites, leading to reduced therapeutic effectiveness, which remains a critical concern. Recently gaining importance are novel peptides, namely CRGDK/RGPD/EC (iRGD), that possess enhanced tumor-penetrating and inhibitory properties. These peptides specifically target and penetrate tumors by binding to αvβ integrins, namely αvβ3 and αvβ5, as well as NRP-1 receptors. Remarkably abundant on both the vasculature and tumor cell surfaces, these peptides show promising potential for improving tumor treatment outcomes. As a result, iRGD penetrated deep into the tumor tissues with biological products, contrast agents (imaging agents), antitumor drugs, and immune modulators after co-injecting them with peptides or chemically linked to peptides. The synthesis of iRGD peptides is a relatively straightforward process compared to the synthesis of other traditional peptides, and they significantly improved tumor tissue penetration inhibiting tumor metastasis effectively. Recent studies demonstrate the effectiveness of iRGD-driven dual-targeting chemotherapeutics on cancer cells, and the nanocarriers were modified with iRGD, serving as a favorable delivery strategy of payloads for deeper tumor regions. This review aims to provide an overview to emphasize the recent advancements and advantages of iRGD in treating and imaging various cancers.
    Keywords:  Cancer diagnosis; Dual-target mechanisms; Therapy; iRGD; αvβ integrins and neuropilin-1
    DOI:  https://doi.org/10.1016/j.bulcan.2023.08.009
  11. Endocr Oncol. 2023 Jan 01. 3(1): e230010
      Androgen receptor (AR) plays a vital role in the development and progression of prostate cancer from the primary stage to the usually lethal stage known as castration-resistant prostate cancer (CRPC). Constitutively active AR splice variants (AR-Vs) lacking the ligand-binding domain are partially responsible for the abnormal activation of AR and may be involved in resistance to AR-targeting drugs occurring in CRPC. There is increasing consensus on the potential of drugs targeting protein-protein interactions. Our lab has recently identified transmembrane 4 superfamily 3 (TM4SF3) as a critical interacting partner for AR and AR-V7 and mapped the minimal interaction regions. Thus, we hypothesized that these interaction domains can be used to design peptides that can disrupt the AR/TM4SF3 interaction and kill prostate cancer cells. Peptides TA1 and AT1 were designed based on the TM3SF3 or AR interaction domain, respectively. TA1 or AT1 was able to decrease AR/TM4SF3 protein interaction and protein stability. Peptide TA1 reduced the recruitment of AR and TM4SF3 to promoters of androgen-regulated genes and subsequent activation of these AR target genes. Peptides TA1 and AT1 were strongly cytotoxic to prostate cancer cells that express AR and/or AR-V7. Peptide TA1 inhibited the growth and induced apoptosis of both enzalutamide-sensitive and importantly enzalutamide-resistant prostate cancer cells. TA1 also blocked the migration and malignant transformation of prostate cancer cells. Our data clearly demonstrate that using peptides to target the important interaction AR has with TM4SF3 provides a novel method to kill enzalutamide-resistant prostate cancer cells that can potentially lead to new more effective therapy for CRPC.
    Keywords:  AR; AR-V7; TM4SF3; peptides; prostate cancer
    DOI:  https://doi.org/10.1530/EO-23-0010
  12. Biofabrication. 2023 Oct 11.
      Recent developments in digital light processing (DLP) can advance the structural and biochemical complexity of perfusable in vitro models of the blood-brain barrier. Here, we describe a strategy to functionalize complex, DLP-printed vascular models with multiple peptide motifs in a single hydrogel. Different peptides can be clicked into the walls of distinct topologies, or the peptide motifs lining channel walls can differ from those in the bulk of the hydrogel. The flexibility of this approach is used to both characterize the effects of various bioactive domains on endothelial coverage and tight junction formation, in addition to facilitating astrocyte attachment in the hydrogel surrounding the endothelialized vessel to mimic endothelial-astrocyte interaction. Peptides derived from proteins mediating cell-extracellular matrix (ECM) (e.g. RGD and IKVAV) and cell-cell (e.g. HAVDI) adhesions are used to mediate endothelial cell attachment and coverage. HAVDI and IKVAV-lined channels exhibit significantly greater endothelialization and increased ZO-1 localization to cell-cell junctions of endothelial cells, indicative of tight junction formation. RGD is then used in the bulk hydrogel to create an endothelial-astrocyte co-culture model of the blood-brain barrier that exceeds the limitations of previous platforms incapable of complex topology or tunable bioactive domains. This approach yields an adjustable, biofabricated platform to interrogate the effects of cell-matrix interaction on blood-brain barrier mechanobiology.
    Keywords:  blood-brain barrier; cell-matrix interaction; digital light processing; engineered microenvironment
    DOI:  https://doi.org/10.1088/1758-5090/ad0260
  13. PLoS One. 2023 ;18(10): e0292681
      The peptide domain extending from residues 49 to 57 of the HIV-1 Tat protein (TAT) has been widely shown to facilitate cell entry of and blood-brain barrier (BBB) permeability to covalently bound macromolecules; therefore, TAT-linked therapeutic peptides trafficked through peripheral routes have been used to treat brain diseases in preclinical and clinical studies. Although the mechanisms underlying cell entry by similar peptides have been established to be temperature-dependent and cell-type specific and to involve receptor-mediated endocytosis, how these peptides cross the BBB remains unclear. Here, using an in vitro model, we studied the permeability of TAT, which was covalently bound to the fluorescent probe fluorescein isothiocyanate (FITC), and evaluated whether it crossed the "in vitro BBB", a monolayer of brain endothelial cells, and whether the mechanisms were similar to those involved in TAT entry into cells. Our results show that although TAT crossed the monolayer of brain endothelial cells in a temperature-dependent manner, in contrast to the reported mechanism of cell entry, it did not require receptor-mediated endocytosis. Furthermore, we revisited the hypothesis that TAT facilitates brain delivery of covalently bound macromolecules by causing BBB disruption. Our results demonstrated that the dose of TAT commonly used in preclinical and clinical studies did not exert an effect on BBB permeability in vitro or in vivo; however, an extremely high TAT concentration caused BBB disruption in vitro. In conclusion, the BBB permeability to TAT is temperature-dependent, but at treatment-level concentrations, it does not involve receptor-mediated endocytosis or BBB disruption.
    DOI:  https://doi.org/10.1371/journal.pone.0292681
  14. Expert Opin Investig Drugs. 2023 Oct 10. 1-6
       INTRODUCTION: Atherosclerotic cardiovascular disease (ASCVD) is a leading cause of morbidity and mortality worldwide. Lowering LDL-cholesterol, by lifestyle modification or therapeutically, reduces the risk of ASCVD. Proprotein convertase subtilisin/kexin type 9 (PCSK9), a protein which binds to the LDL-receptor and induces degradation, is a clinically validated target to lower LDL-cholesterol. Injectable PCSK9 inhibitor therapies have demonstrated substantial reductions in LDL-cholesterol with associated decreased risk of ASCVD events.
    AREAS COVERED: MK-0616 is an orally bioavailable, renally excreted, macrocyclic peptide inhibitor of PCSK9. The article provides an understanding of the chemistry and development, pharmacokinetic and pharmacodynamic characteristics of MK-0616 and insight into its clinical efficacy and safety. In clinical trials, MK-0616 produced dose-dependent reductions in LDL-cholesterol, non-HDL-cholesterol, and apolipoprotein (apo) B levels. Furthermore, MK-0616 modestly lowered lipoprotein (a) [Lp(a)].
    EXPERT OPINION: MK-0616 is a potent, oral macrocyclic peptide inhibitor of PCSK9 that is not only able to reduce LDL-cholesterol, non-HDL-cholesterol, and apoB, but can also lower Lp(a). Safety and tolerability studies reported to date are promising. MK-0616 may offer advantages over injectable anti-PCSK9 therapies in terms of ease of dosing, patient preference and cost. The results from phase III trials of MK-0616 on cardiovascular outcomes are awaited with interest.
    Keywords:  ApoB; LDL-cholesterol; Lp(a); MK-0616; PCSK9; PCSK9 inhibitor; non-HDL-cholesterol
    DOI:  https://doi.org/10.1080/13543784.2023.2267972