bims-cepepe 2023-11-19 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2023–11–19
nine papers selected by
Henry Lamb, Queensland University of Technology

Enhancement of adeno-associated virus serotype 6 transduction into T cells with cell-penetrating peptides.
Impact of Peptide Sequence on Functional siRNA Delivery and Gene Knockdown with Cyclic Amphipathic Peptide Delivery Agents.
Recent advances in delivery of peptide and protein therapeutics to the brain.
Rational Design of TDP-43 Derived α-Helical Peptide Inhibitors: an In-Silico Strategy to Prevent TDP-43 Aggregation in Neurodegenerative Disorders.
Peptide-Mimicking Poly(2-oxazoline)s Possessing Potent Antifungal Activity and BBB Penetrating Property to Treat Invasive Infections and Meningitis.
Peptide aggregation-induced immunogenic cell death in a breast cancer spheroid model.
Protocol for generating embedding-free brain organoids enriched with oligodendrocytes.
Ring Opening of Glycerol Cyclic Phosphates Leads to a Diverse Array of Potentially Prebiotic Phospholipids.
Structural modifications toward improved lead-203/lead-212 peptide-based image-guided alpha-particle radiopharmaceutical therapies for neuroendocrine tumors.

J Gene Med. 2023 Nov 13. e3627

Enhancement of adeno-associated virus serotype 6 transduction into T cells with cell-penetrating peptides.

Pablo D Moço, Shantoshini Dash, Amine A Kamen.

   BACKGROUND: Adeno-associated viruses (AAVs) are gaining interest in the development of cellular immunotherapy. Compared to other viral vectors, AAVs can reduce the risk of insertional oncogenesis. AAV serotype 6 (AAV6) shows the highest efficiency for transducing T cells. Nevertheless, a multiplicity of infection (MOI) of up to one million viral genomes per cell is required to transduce the target cells effectively. Cell-penetrating peptides (CPPs) are short, positively charged peptides that easily translocate the plasma membranes and can facilitate the cellular uptake of a wide variety of cargoes, including small molecules, nucleic acids, drugs, proteins and viral vectors.
METHODS: The present study evaluated five CPPs (Antp, TAT-HA2, LAH4, TAT1 and TAT2) on their effects on enhancing transduction of AAV6 packaging a green fluorescent protein transgene into Jurkat T cell line.
RESULTS: Vector incubation with peptides TAT-HA2 and LAH4 at a final concentration of 0.2 mm resulted in an approximately two-fold increase in transduced cells. At the lowest MOI tested (1.25 × 104 ), using LAH4 resulted in a 10-fold increase in transduction efficiency. The peptide LAH4 increased the uptake of AAV6 viral particles in both Jurkat cells and mouse primary T cells. Regardless of the large size of the AAV6-LAH4 complexes, their internalization does not appear to depend on macropinocytosis.
CONCLUSIONS: Overall, the present study reports an approach to significantly improve the delivery of transgenes into T cells using AAV6 vectors. Notably, the peptides TAT-HA2 and LAH4 contribute to improving the use of AAV6 as a gene delivery vector for the engineering of T cells.

Keywords:  T cells; adeno-associated viral vector; cell-penetrating peptides; transduction

DOI:  https://doi.org/10.1002/jgm.3627
Mol Pharm. 2023 Nov 14.

Impact of Peptide Sequence on Functional siRNA Delivery and Gene Knockdown with Cyclic Amphipathic Peptide Delivery Agents.

Melissa L Jagrosse, Uday K Baliga, Christopher W Jones, Jade J Russell, Claudia I García, Rauf Ahmad Najar, Arshad Rahman, David A Dean, Bradley L Nilsson.

  Short-interfering RNA (siRNA) oligonucleotide therapeutics that modify gene expression by accessing RNA-interference (RNAi) pathways have great promise for the treatment of a range of disorders; however, their application in clinical settings has been limited by significant challenges in cellular delivery. Herein, we report a structure-function study using a series of modified cyclic amphipathic cell-penetrating peptides (CAPs) to determine the impact of peptide sequence on (1) siRNA-binding efficiency, (2) cellular delivery and knockdown efficiency, and (3) the endocytic uptake mechanism. Nine cyclic peptides of the general sequence Ac-C[XZ]4CG-NH2 in which X residues are hydrophobic/aromatic (Phe, Tyr, Trp, or Leu) and Z residues are charged/hydrophilic (Arg, Lys, Ser, or Glu) are assessed along with one acyclic peptide, Ac-(WR)4G-NH2. Cyclization is enforced by intramolecular disulfide bond formation between the flanking Cys residues. Binding analyses indicate that strong cationic character and the presence of aromatic residues that are competent to participate in CH-π interactions lead to CAP sequences that most effectively interact with siRNA. CAP-siRNA binding increases in the following order as a function of CAP hydrophobic/aromatic content: His < Phe < Tyr < Trp. Both cationic charge and disulfide-constrained cyclization of CAPs improve uptake of siRNA in vitro. Net neutral CAPs and an acyclic peptide demonstrate less-efficient siRNA translocation compared to the cyclic, cationic CAPs tested. All CAPs tested facilitated efficient siRNA target gene knockdown of at least 50% (as effective as a lipofectamine control), with the best CAPs enabling >80% knockdown. Significantly, gene knockdown efficiency does not strongly correlate with CAP-siRNA internalization efficiency but moderately correlates with CAP-siRNA-binding affinity. Finally, utilization of small-molecule inhibitors and targeted knockdown of essential endocytic pathway proteins indicate that most CAP-siRNA nanoparticles facilitate siRNA delivery through clathrin- and caveolin-mediated endocytosis. These results provide insight into the design principles for CAPs to facilitate siRNA delivery and the mechanisms by which these peptides translocate siRNA into cells. These studies also demonstrate the nature of the relationships between peptide-siRNA binding, cellular delivery of siRNA cargo, and functional gene knockdown. Strong correlations between these properties are not always observed, which illustrates the complexity in the design of optimal next-generation materials for oligonucleotide delivery.

Keywords:  cell-penetrating peptide; cyclic peptide; drug delivery; siRNA delivery

DOI:  https://doi.org/10.1021/acs.molpharmaceut.3c00455
J Pharmacol Exp Ther. 2023 Nov 17. pii: JPET-MR-2023-001690. [Epub ahead of print]

Recent advances in delivery of peptide and protein therapeutics to the brain.

Sanchit Arora, Tania Bajaj, Jayant Kumar, Manoj Goyal, Arti Singh, Charan Singh.

  The classes of neuropharmaceuticals known as proteins and peptides serve as diagnostic tools and are involved in specific communication in the peripheral and central nervous systems (CNS). However, due to tight junctions resembling epithelial cells found in the blood-brain barrier (BBB) in vivo, they are typically excluded from transport from the blood to the brain. The drugs having molecular weight of less than 400 Dalton are able to cross the BBB via lipid-mediated free diffusion. However, large molecule therapeutics are devoid of these characteristics. As an alternative, these substances may be carried via chimeric peptide drug delivery systems, and assist in transcytosis through BBB with the aid of linker strategies. With their recent developments, several forms of nanoparticles, including PEG-PCL, nanogels, liposomes, NLCs, PLGA-NPs, chitosan, and SLNs, have also been considered for their therapeutic applications. Moreover, the necessity for physiological optimization of current drug delivery methods and their carriers to deliver therapeutic doses of medication into the brain for the treatment of various neurological illnesses has also been emphasized. Therapeutic use of proteins and peptides has no neuroprotective impact in the absence of all these methods. Each tactic, however, has unique drawbacks and considerations. In this review, we discuss different drug delivery methods for therapeutic distribution of pharmaceuticals, primarily neuroproteins and neuropeptides, through endothelial capillaries via blood-brain barrier. Finally, we have also discussed challenges and future perspective of protein and peptide therapeutics delivery to the brain Significance Statement Very few reports on the delivery of therapeutic protein and peptide nanoformulations are available in the literature. Herein, we attempted to discuss these nanoformulations of protein and peptide therapeutics used to treat brain diseases. .

Keywords:  blood-brain barrier; drug delivery; neuropeptides

DOI:  https://doi.org/10.1124/jpet.123.001690
bioRxiv. 2023 Oct 31. pii: 2023.10.26.564235. [Epub ahead of print]

Rational Design of TDP-43 Derived α-Helical Peptide Inhibitors: an In-Silico Strategy to Prevent TDP-43 Aggregation in Neurodegenerative Disorders.

Muthu Raj Salaikumaran, Pallavi P Gopal.

TDP-43, an essential RNA/DNA-binding protein, is central to the pathology of neurodegenerative diseases such as Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Pathological mislocalization and aggregation of TDP-43 disrupts RNA splicing, mRNA stability, and mRNA transport, thereby impairing neuronal function and survival. The formation of amyloid-like TDP-43 filaments is largely facilitated by the destabilization of an α-helical segment within the disordered C-terminal region. In this study, we hypothesized that preventing the destabilization of the α-helical domain could potentially halt the growth of these pathological filaments. To explore this, we utilized a range of in-silico techniques to design and evaluate peptide-based therapeutics. Various pathological TDP-43 amyloid-like filament crystal structures were selected for their potential to inhibit the binding of additional TDP-43 monomers to the growing filaments. Our computational approaches included biophysical and secondary structure property prediction, molecular docking, 3D structure prediction, and molecular dynamics simulations. Through these techniques, we were able to assess the structure, stability, and binding affinity of these peptides in relation to pathological TDP-43 filaments. The results of our in-silico analyses identified a selection of promising peptides, which displayed a stable α-helical structure, exhibited an increased number of intramolecular hydrogen bonds within the helical domain, and demonstrated high binding affinities for pathological TDP-43 amyloid-like filaments. Additionally, molecular dynamics simulations provided further support for the stability of these peptides, as they exhibited lower root mean square deviations in their helical propensity over 100ns. These findings establish α-helical propensity peptides as potential lead molecules for the development of novel therapeutics against TDP-43 aggregation. This structure-based computational approach for rational design of peptide inhibitors opens a new direction in the search for effective interventions for ALS, FTD, and other related neurodegenerative diseases. The peptides identified as the most promising candidates in this study are currently subject to further testing and validation through both in vitro and in vivo experiments.

DOI: https://doi.org/10.1101/2023.10.26.564235
J Am Chem Soc. 2023 Nov 15.

Peptide-Mimicking Poly(2-oxazoline)s Possessing Potent Antifungal Activity and BBB Penetrating Property to Treat Invasive Infections and Meningitis.

Weinan Jiang, Min Zhou, Sheng Chen, Jiayang Xie, Minzhang Chen, Haodong Zhang, Yueming Wu, Xin Chen, Runhui Liu.

Invasive fungal infections, including meningitis, cause a high mortality rate due to few available antifungal drugs and frequently associated side effects and quick emergence of drug-resistant fungi. The restrictive permeability of the blood-brain barrier (BBB) further limits the efficacy of antifungal agents substantially in treating meningitis. Hereby, we design and synthesize guanidinium-functionalized poly(2-oxazoline)s by mimicking cell-penetrating peptides. The optimal polymer, PGMeOx10 bearing a methylene spacer arm, displays potent activities against the drug-resistant fungi and biofilm, negligible toxicity, and insusceptibility to antimicrobial resistance. Moreover, PGMeOx10 can break BBB retractions to exert promising antifungal functions in the brain. PGMeOx10 demonstrates potent in vivo antifungal therapeutic efficacy in mouse models including skin infection, systemic infections, and meningitis. PGMeOx10 effectively rescues infected mice and reduces fungal burden and inflammation in the brain. These results and the excellent biosafety of poly(2-oxazoline)s indicate the effectiveness and potential of our strategy to design promising antifungal agents in treating systemic infections and meningitis.

DOI: https://doi.org/10.1021/jacs.3c09240
bioRxiv. 2023 Nov 02. pii: 2023.10.31.565012. [Epub ahead of print]

Peptide aggregation-induced immunogenic cell death in a breast cancer spheroid model.

Gokhan Gunay, Katelyn N Maier, Seren Hamsici, Filipa Carvalho, Tristan A Timog, Handan Acar.

Utilizing multicellular aggregates (spheroids) for in vitro cancer research offers a physiologically relevant model that closely mirrors the intricate tumor microenvironment, capturing properties of solid tumors such as cell interactions and drug resistance. In this research, we investigated the Peptide-Aggregation Induced Immunogenic Response (PAIIR), an innovative method employing engineered peptides we designed specifically to induce immunogenic cell death (ICD). We contrasted PAIIR-induced ICD with standard ICD and non-ICD inducer chemotherapeutics within the context of three-dimensional breast cancer tumor spheroids. Our findings reveal that PAIIR outperforms traditional chemotherapeutics in its efficacy to stimulate ICD. This is marked by the release of key damage-associated molecular patterns (DAMPs), which bolster the phagocytic clearance of dying cancer cells by dendritic cells (DCs) and, in turn, activate powerful anti-tumor immune responses. Additionally, we observed that PAIIR results in elevated dendritic cell activation and increased antitumor cytokine presence. This study not only showcases the utility of tumor spheroids for efficient high-throughput screening but also emphasizes PAIIR's potential as a formidable immunotherapeutic strategy against breast cancer, setting the stage for deeper exploration and potential clinical implementation.

DOI: https://doi.org/10.1101/2023.10.31.565012
STAR Protoc. 2023 Nov 16. pii: S2666-1667(23)00692-5. [Epub ahead of print]4(4): 102725

Protocol for generating embedding-free brain organoids enriched with oligodendrocytes.

Bahaa Al-Mhanawi, Marta Boira Marti, Sean D Morrison, Pallavi Gupta, Maath Alani, Peter G Noakes, Ernst J Wolvetang, Mohammed R Shaker.

  In response to the scarcity of advanced in vitro models dedicated to human CNS white matter research, we present a protocol to generate neuroectoderm-derived embedding-free human brain organoids enriched with oligodendrocytes. We describe steps for neuroectoderm differentiation, development of neural spheroids, and their transferal to Matrigel. We then detail procedures for the development, maturation, and application of oligodendrocyte-enriched brain organoids. The presence of myelin-producing cells makes these organoids useful for studying human white matter diseases, such as leukodystrophy.

Keywords:  Neuroscience; Organoids; Stem Cells

DOI:  https://doi.org/10.1016/j.xpro.2023.102725
J Am Chem Soc. 2023 Nov 16.

Ring Opening of Glycerol Cyclic Phosphates Leads to a Diverse Array of Potentially Prebiotic Phospholipids.

Maiia Aleksandrova, Fidan Rahmatova, David A Russell, Claudia Bonfio.

Phospholipids are the primary constituents of cell membranes across all domains of life, but how and when phospholipids appeared on early Earth remains unknown. Pressingly, most prebiotic syntheses of complex phospholipids rely upon substrates not yet shown to have been available on early Earth. Here, we describe potentially prebiotic syntheses of a diverse array of complex phospholipids and their building blocks. First, we show that choline could have been produced on early Earth by stepwise N-methylation of ethanolamine. Second, taking a systems chemistry approach, we demonstrate that the intrinsically activated glycerol-2,3-cyclic phosphate undergoes ring opening with combinations of prebiotic amino alcohols to yield complex phospholipid headgroups. Importantly, this pathway selects for the formation of 2-amino alcohol-bearing phospholipid headgroups and enables the accumulation of their natural regioisomers. Finally, we show that the dry-state ring opening of cyclic lysophosphatidic acids leads to a range of self-assembling lysophospholipids. Our results provide new prebiotic routes to key intermediates on the way toward modern phospholipids and illuminate the potential origin and evolution of cell membranes.

DOI: https://doi.org/10.1021/jacs.3c07319
Eur J Nucl Med Mol Imaging. 2023 Nov 13.

Structural modifications toward improved lead-203/lead-212 peptide-based image-guided alpha-particle radiopharmaceutical therapies for neuroendocrine tumors.

Dongyoul Lee, Mengshi Li, Dijie Liu, Nicholas J Baumhover, Edwin A Sagastume, Brenna M Marks, Prerna Rastogi, F Christopher Pigge, Yusuf Menda, Frances L Johnson, Michael K Schultz.

   PURPOSE: The lead-203 (203Pb)/lead-212 (212Pb) elementally identical radionuclide pair has gained significant interest in the field of image-guided targeted alpha-particle therapy for cancer. Emerging evidence suggests that 212Pb-labeled peptide-based radiopharmaceuticals targeting somatostatin receptor subtype 2 (SSTR2) may provide improved effectiveness compared to beta-particle-based therapies for neuroendocrine tumors (NETs). This study aims to improve the performance of SSTR2-targeted radionuclide imaging and therapy through structural modifications to Tyr3-octreotide (TOC)-based radiopharmaceuticals.
METHODS: New SSTR2-targeted peptides were designed and synthesized with the goal of optimizing the incorporation of Pb isotopes through the use of a modified cyclization technique; the introduction of a Pb-specific chelator (PSC); and the insertion of polyethylene glycol (PEG) linkers. The binding affinity of the peptides and the cellular uptake of 203Pb-labeled peptides were evaluated using pancreatic AR42J (SSTR2+) tumor cells and the biodistribution and imaging of the 203Pb-labeled peptides were assessed in an AR42J tumor xenograft mouse model. A lead peptide was identified (i.e., PSC-PEG2-TOC), which was then further evaluated for efficacy in 212Pb therapy studies.
RESULTS: The lead radiopeptide drug conjugate (RPDC) - [203Pb]Pb-PSC-PEG2-TOC - significantly improved the tumor-targeting properties, including receptor binding and tumor accumulation and retention as compared to [203Pb]Pb-DOTA0-Tyr3-octreotide (DOTATOC). Additionally, the modified RPDC exhibited faster renal clearance than the DOTATOC counterpart. These advantageous characteristics of [212Pb]Pb-PSC-PEG2-TOC resulted in a dose-dependent therapeutic effect with minimal signs of toxicity in the AR42J xenograft model. Fractionated administrations of 3.7 MBq [212Pb]Pb-PSC-PEG2-TOC over three doses further improved anti-tumor effectiveness, resulting in 80% survival (70% complete response) over 120 days in the mouse model.
CONCLUSION: Structural modifications to chelator and linker compositions improved tumor targeting and pharmacokinetics (PK) of 203/212Pb peptide-based radiopharmaceuticals for NET theranostics. These findings suggest that PSC-PEG2-TOC is a promising candidate for Pb-based targeted radionuclide therapy for NETs and other types of cancers that express SSTR2.

Keywords:  Alpha-particle therapy; Lead isotopes; PRRT; Peptides; SSTR2; Structural optimization

DOI:  https://doi.org/10.1007/s00259-023-06494-9