bims-cepepe 2024-06-16 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2024–06–16
eight papers selected by
Henry Lamb, Queensland University of Technology

The coming of age of cyclic peptide drugs: an update on discovery technologies.
Discovery of a new class of cell-penetrating peptides by novel phage display platform.
Charge-switchable cell-penetrating peptides for rerouting nanoparticles to glioblastoma treatment.
Progresses in the establishment, evaluation, and application of in vitro blood-brain barrier models.
Establishment of a Four-Cell In Vitro Blood-Brain Barrier Model With Human Primary Brain Cells.
Developing a dormancy-associated ECM signature in TNBC that is linked to immunosuppressive tumor microenvironment and selective sensitivity to MAPK inhibitors.
Discovery and biological evaluation of biaryl acetamide derivatives as selective and in vivo active sphingosine kinase-2 inhibitors.
Synthesis of 64Cu-, 55Co-, and 68Ga-Labeled Radiopharmaceuticals Targeting Neurotensin Receptor-1 for Theranostics: Adjusting In Vivo Distribution Using Multiamine Macrocycles.

Expert Opin Drug Discov. 2024 Jun 14. 1-13

The coming of age of cyclic peptide drugs: an update on discovery technologies.

Sophia You, Glen McIntyre, Toby Passioura.

   INTRODUCTION: Cyclic peptides are an established class of pharmaceuticals, with the ability to bind to a broader range of protein targets than traditional small molecules while also being capable of oral availability and cell penetration. Historically, cyclic peptide drugs have been discovered almost exclusively through natural product mining approaches; however, the last two decades have seen the development of display screening approaches capable of rapidly identifying de novo (i.e. not natural product derived) cyclic peptide ligands to targets of interest.
AREAS COVERED: In this review, the authors describe the current clinical landscape for cyclic peptide pharmaceuticals. This article focuses on the discovery approaches that have led to the development of different classes of molecules and how the development of newer technologies, particularly phage and mRNA display, has broadened the clinical applicability of such molecules.
EXPERT OPINION: The field of de novo cyclic peptide drug discovery is reaching maturity, with the first drugs identified through display screening approaches reaching the market in recent years. Many more are in clinical trials; however, significant technical challenges remain. Technological improvements will be required over the coming years to facilitate the identification of membrane permeable cyclic peptides capable of oral availability and targeting intracellular proteins.

Keywords:  Macrocycle; display screening; drug discovery; peptide

DOI:  https://doi.org/10.1080/17460441.2024.2367024
Sci Rep. 2024 06 11. 14(1): 13437

Discovery of a new class of cell-penetrating peptides by novel phage display platform.

Jinsha Liu, John Heddleston, Douglas Raymond Perkins, Jack Jia Hua Chen, Ahmadreza Ghanbarpour, Bill William Smith, Rebecca Miles, Eitaro Aihara, Sepideh Afshar.

  The primary hurdles for small interference RNA (siRNA) in clinical use are targeted and cytosolic delivery. To overcome both challenges, we have established a novel platform based on phage display, called NNJA. In this approach, a lysosomal cathepsin substrate is engineered within the flexible loops of PIII, that is displaying a unique random sequence at its N-terminus. NNJA library selection targeting cell-expressed targets should yield specific peptides localized in the cytoplasm. That is because phage internalization and subsequent localization to lysosome, upon peptide binding to the cell expressed target, will result in cleavage of PIII, rendering phage non-infective. Such phage will be eliminated from the selected pool and only peptide-phage that escapes lysosomes will advance to the next round. Proof of concept studies with the NNJA library demonstrated cytosolic localization of selected peptide-phage and peptide-siRNA, confirmed through confocal microscopy. More importantly, conjugation of siHPRT to monomeric or multimeric NNJA peptides resulted in significant reduction in HPRT mRNA in various cell types without significant cytotoxicity. Sequence similarity and clustering analysis from NGS dataset provide insights into sequence composition facilitating cell penetration. NNJA platform offers a highly efficient peptide discovery engine for targeted delivery of oligonucleotides to cytosol.

Keywords:  Cell-penetrating peptide (CPP); Cytoplasmic delivery; NNJA peptides; Novel phage display platform

DOI:  https://doi.org/10.1038/s41598-024-64405-w
Colloids Surf B Biointerfaces. 2024 May 28. pii: S0927-7765(24)00242-X. [Epub ahead of print]241 113983

Charge-switchable cell-penetrating peptides for rerouting nanoparticles to glioblastoma treatment.

Maria Mendes, Sandra Nunes, Tânia Cova, Francisco Branco, Michael Dyrks, Beate Koksch, Nuno Vale, João Sousa, Alberto Pais, Carla Vitorino.

  Glioblastoma (GB) is one of the most lethal types of neoplasms with unique anatomic, physiologic, and pathologic features that usually persist after exposure to standard therapeutic modalities. It is biologically aggressive, and the existence of the blood-brain barrier (BBB) limits the efficacy of standard therapies. In this work, we hypothesize the potential of surface-functionalized ultra-small nanostructured lipid carriers (usNLCs) with charge-switchable cell-penetrating peptides (CPPs) to overcome this biological barrier and improve targeted delivery to brain tumor tissues. The big question is: what is the potential of CPPs in directing nanoparticles toward brain tumor tissue? To answer this question, the usNLCs were functionalized with distinct biomolecules [five CPPs, c(RGDfK) and transferrin, Tf] through electrostatic interaction and its ability as a targeting approach to BBB (HBMEC) and glioma cells (U87 cells) evaluated in terms of physicochemical properties, cellular uptake, permeability in a 2D-BBB model, and tumor growth inhibition. Monte Carlo simulations elucidated CPP adsorption patterns. The permeability studies revealed that targeted usNLCs, especially usNLCsTf and usNLCsCPP4, exhibited an increased permeability coefficient compared to the non-targeted usNLCs. Functionalized usNLCs evidenced enhanced uptake in BBB cells, with smaller CPPs showing higher internalization (CPP1 and CPP2). Similarly, functionalized usNLCs exhibited more significant cytotoxicity in glioma cells, with specific CPPs promoting favorable internalization. Analysis of the endocytic pathway indicated that usNLCsCPPs were mainly internalized by direct translocation and caveolae-mediated endocytosis. Optimal usNLCs with dual targeting capabilities to both BBB and GB cells provide a promising therapeutic strategy for GB.

Keywords:  Blood-brain barrier; Cell-penetrating peptides; Glioblastoma; Tumor-targeting peptides; Ultra-small nanostructured lipid carriers

DOI:  https://doi.org/10.1016/j.colsurfb.2024.113983
J Neurosci Res. 2024 Jun;102(6): e25359

Progresses in the establishment, evaluation, and application of in vitro blood-brain barrier models.

Panfeng Yin, Xianchun Wang.

  The blood-brain barrier (BBB) is a barrier between the circulatory system and the central nervous system (CNS), contributing to CNS protection and maintaining the brain homeostasis. Establishment of in vitro BBB models that are closer to the microenvironment of the human brain is helpful for evaluating the potential and efficiency of a drug penetrating BBB and thus the clinical application value of the drug. The in vitro BBB models not only provide great convenience for screening new drugs that can access to CNS but also help people to have a deeper study on the mechanism of substances entering and leaving the brain, which makes people have greater opportunities in the treatment of CNS diseases. Up to now, although much effort has been paid to the researches on the in vitro BBB models and many progresses have been achieved, no unified method has been described for establishing a BBB model and there is much work to do and many challenges to be faced with in the future. This review summarizes the research progresses in the establishment, evaluation, and application of in vitro BBB models.

Keywords:  application; blood–brain barrier; establishment; evaluation; in vitro model; progress

DOI:  https://doi.org/10.1002/jnr.25359
Curr Protoc. 2024 Jun;4(6): e1067

Establishment of a Four-Cell In Vitro Blood-Brain Barrier Model With Human Primary Brain Cells.

Johid R Malik, Ukamaka O Modebelu, Courtney V Fletcher, Anthony T Podany, Kimberly K Scarsi, Siddappa N Byrareddy, Robbyn K Anand, Shilpa Buch, Susmia Sil, Jennifer Le, John S Bradley, Ashley N Brown, Debapriya Sutar, Sean N Avedissian.

  The blood-brain barrier (BBB) constitutes a crucial protective anatomical layer with a microenvironment that tightly controls material transit. Constructing an in vitro BBB model to replicate in vivo features requires the sequential layering of constituent cell types. Maintaining heightened integrity in the observed tight junctions during both the establishment and post-experiment phases is crucial to the success of these models. We have developed an in vitro BBB model that replicates the cellular composition and spatial orientation of in vivo BBB observed in humans. The experiment includes comprehensive procedures and steps aimed at enhancing the integration of the four-cell model. Departing from conventional in vitro BBB models, our methodology eliminates the necessity for pre-coated plates to facilitate cell adhesion, thereby improving cell visualization throughout the procedure. An in-house coating strategy and a simple yet effective approach significantly reduce costs and provides superior imaging of cells and corresponding tight junction protein expression. Also, our BBB model includes all four primary cell types that are structural parts of the human BBB. With its innovative and user-friendly features, our in-house optimized in vitro four-cell-based BBB model showcases novel methodology and provides a promising experimental platform for drug screening processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Coating and culture system Basic Protocol 2: Cell seeding and Transwell insert handling Basic Protocol 3: Assessment of model functionality.

Keywords:  blood‐brain barrier; central nervous system; human primary cells; in vitro model

DOI:  https://doi.org/10.1002/cpz1.1067
Heliyon. 2024 Jun 15. 10(11): e32106

Developing a dormancy-associated ECM signature in TNBC that is linked to immunosuppressive tumor microenvironment and selective sensitivity to MAPK inhibitors.

Yang Dong, Jin Bai, Jianjun Zhou.

   Aims: Cellular dormancy is a state of quiescence subpopulation of tumor cells, characterized by low differentiation and lack of mitotic activity. They could evade chemotherapy and targeted therapy, leading to drug resistance and disease recurrence. Recent studies have shown a correlation between dormant cancer cells and unique extracellular matrix (ECM) composition, which is critical in regulating cell behavior. However, their interacting roles in TNBC patients remains to be characterized.
Main methods: Dormant cancer cells in MDA-MB-231 cell line with highest PKH26 dye-retaining were FACS-sorted and gene expression was then analyzed. Dormant associated ECM (DA-ECM) signature was characterized by pathway analysis. Unsupervised hierarchical clustering was used to define distinct ECM features for TNBC patients. ECM-specific tumor biology was defined by integration of bulk RNA-seq with single-cell RNA-seq data, analysis of ligand-receptor interactions and enriched biological pathways, and in silico drug screening. We validated the sensitivity of dormant cancer cells to MAPK inhibitors by flow cytometry in vitro.
Key findings: We observed that dormant TNBC cells preferentially expressed ∼10 % DA-ECM genes. The DA-ECM High subtype defined by unsupervised hierarchical clustering analysis was associated with immunosuppressive tumor microenvironment. Moreover, ligand-receptor interaction and pathway analysis revealed that the DA-ECM High subtype may likely help maintain tumor cell dormancy through MAPK, Hedgehog and Notch signaling pathways. Finally, in silico drug screening against the DA-ECM signature and in vitro assay showed dormant cancer cells were relatively sensitive to the MAPK pathway inhibitors, which may represent a potential therapeutic strategy for treating TNBC.
Significance: Collectively, our research revealed that dormancy-associated ECM characterized tumor cells possess significant ECM remodeling capacity, and treatment strategies towards these cells could improve TNBC patient outcome.

Keywords:  Cellular dormancy; Chemoresistance; Extracellular matrix; MAPK signaling pathway; Triple negative breast cancer

DOI:  https://doi.org/10.1016/j.heliyon.2024.e32106
Eur J Med Chem. 2024 Jun 08. pii: S0223-5234(24)00457-4. [Epub ahead of print]275 116577

Discovery and biological evaluation of biaryl acetamide derivatives as selective and in vivo active sphingosine kinase-2 inhibitors.

Yanan Li, Gang Li, Yiming Wang, Longfei Li, Yali Song, Fei Cao, Kan Yang.

  Sphingosine kinase 2 (SphK2) has emerged as a promising target for cancer therapy due to its critical role in tumor growth. However, the lack of potent and selective inhibitors has hindered its clinical application. Herein, we report the design and synthesis of a series of novel SphK2 inhibitors, culminating in the identification of compound 12q as a highly selective and potent inhibitor of SphK2. Molecular dynamics simulations suggest that the incorporation of larger substitution groups facilitates a more effective occupation of the binding site, thereby stabilizing the complex. Compared to the widely used inhibitor ABC294640, compound 12q exhibits superior anti-proliferative activity against various cancer cells, inducing G2 phase arrest and apoptosis in liver cancer cells HepG2. Notably, 12q inhibited migration and colony formation in HepG2 and altered intracellular sphingolipid content. Moreover, intraperitoneal administration of 12q in mice resulted in decreased levels of S1P. 12q provides a valuable tool compound for exploring the therapeutic potential of targeting SphK2 in cancer.

Keywords:  Anti-tumor; Inhibitors; Sphingolipid; Sphingosine 1-Phosphate; Sphingosine kinase 2

DOI:  https://doi.org/10.1016/j.ejmech.2024.116577
J Nucl Med. 2024 Jun 13. pii: jnumed.124.267469. [Epub ahead of print]

Synthesis of 64Cu-, 55Co-, and 68Ga-Labeled Radiopharmaceuticals Targeting Neurotensin Receptor-1 for Theranostics: Adjusting In Vivo Distribution Using Multiamine Macrocycles.

German O Fonseca Cabrera, Xinrui Ma, Wilson Lin, Tao Zhang, Weiling Zhao, Liqin Pan, Xiaomei Li, Todd E Barnhart, Eduardo Aluicio-Sarduy, Huaifu Deng, Xuedan Wu, Kadalipura P Rakesh, Zibo Li, Jonathan W Engle, Zhanhong Wu.

  The development of theranostic radiotracers relies on their binding to specific molecular markers of a particular disease and the use of corresponding radiopharmaceutical pairs thereafter. This study reports the use of multiamine macrocyclic moieties (MAs), as linkers or chelators, in tracers targeting the neurotensin receptor-1 (NTSR-1). The goal is to achieve elevated tumor uptake, minimal background interference, and prolonged tumor retention in NTSR-1-positive tumors. Methods: We synthesized a series of neurotensin antagonists bearing MA linkers and metal chelators. The MA unit is hypothesized to establish a strong interaction with the cell membrane, and the addition of a second chelator may enhance water solubility, consequently reducing liver uptake. Small-animal PET/CT imaging of [64Cu]Cu-DOTA-SR-3MA, [64Cu]Cu-NT-CB-NOTA, [68Ga]Ga-NT-CB-NOTA, [64Cu]Cu-NT-CB-DOTA, and [64Cu]Cu-NT-Sarcage was acquired at 1, 4, 24, and 48 h after injection using H1299 tumor models. [55Co]Co-NT-CB-NOTA was also tested in HT29 (high NTSR-1 expression) and Caco2 (low NTSR-1 expression) colorectal adenocarcinoma tumor models. Saturation binding assay and internalization of [55Co]Co-NT-CB-NOTA were used to test tracer specificity and internalization in HT29 cells. Results: In vivo PET imaging with [64Cu]Cu-NT-CB-NOTA, [68Ga]Ga-NT-CB-NOTA, and [55Co]Co-NT-CB-NOTA revealed high tumor uptake, high tumor-to-background contrast, and sustained tumor retention (≤48 h after injection) in NTSR-1-positive tumors. Tumor uptake of [64Cu]Cu-NT-CB-NOTA remained at 76.9% at 48 h after injection compared with uptake 1 h after injection in H1299 tumor models, and [55Co]Co-NT-CB-NOTA was retained at 60.2% at 24 h compared with uptake 1 h after injection in HT29 tumor models. [64Cu]Cu-NT-Sarcage also showed high tumor uptake with low background and high tumor retention 48 h after injection Conclusion: Tumor uptake and pharmacokinetic properties of NTSR-1-targeting radiopharmaceuticals were greatly improved when attached with different nitrogen-containing macrocyclic moieties. The study results suggest that NT-CB-NOTA labeled with either 64Cu/67Cu, 55Co/58mCo, or 68Ga (effect of 177Lu in tumor to be determined in future studies) and NT-Sarcage labeled with 64Cu/67Cu or 55Co/58mCo may be excellent diagnostic and therapeutic radiopharmaceuticals targeting NTSR-1-positive cancers. Also, the introduction of MA units to other ligands is warranted in future studies to test the generality of this approach.

Keywords:  55Co; 64Cu; 68Ga; PET; neurotensin receptor; prostate cancer; radiotheranostic agent

DOI:  https://doi.org/10.2967/jnumed.124.267469