bims-cepepe 2023-11-12 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2023–11–12
thirteen papers selected by
Henry Lamb, Queensland University of Technology

Disulfide Click Reaction for Stapling of S-terminal Peptides.
Integrin Facilitates the Internalization of TAT Peptide Conjugated to RGD Motif in Model Lipid Membranes.
Evaluating the cytotoxicity mechanism of the cell-penetrating peptide TP10 on Jurkat cells.
Mechanism of selective recognition of Lys48-linked polyubiquitin by macrocyclic peptide inhibitors of proteasomal degradation.
Peptide-based approaches to directly target alpha-synuclein in Parkinson's disease.
Structural flexibility of apolipoprotein E-derived arginine-rich peptides improves their cell penetration capability.
A new shuttle for drug delivery across the blood-brain barrier.
A tissue-engineered model of the blood-tumor barrier during metastatic breast cancer.
Targeting of intracellular oncoproteins with peptide-centric CARs.
MicroED as a Powerful Tool for Structure Determination of Macrocyclic Drug Compounds Directly from Their Powder Formulations.
Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease.
Peptide Conjugates of a 2'-O-Methoxyethyl Phosphorothioate Splice-Switching Oligonucleotide Show Increased Entrapment in Endosomes.
Discovery of a Hydroxylamine-Based Brain-Penetrant EGFR Inhibitor for Metastatic Non-Small-Cell Lung Cancer.

Angew Chem Int Ed Engl. 2023 Nov 10. e202314379

Disulfide Click Reaction for Stapling of S-terminal Peptides.

Qing Yu, Leiyang Bai, Xuefeng Jiang.

  A disulfide click strategy was disclosed for stapling to enhance metabolic stability and cellular permeability for therapeutic peptides. A seventeen-membered library of stapling reagents with adjustable lengths and angles was established for rapid double/triple click reactions, bridging S-terminal peptides from 3 to 18 cysteine residues to provide 18- to 48-membered macrocyclic peptides under biocompatible conditions. The constrained peptides exhibited enhanced anti-HCT-116 activity with locked α-helical conformation (IC50 = 6.81 μM vs biological incompetence for acyclic linear peptides), which can be unstapled for rehabilitating the native peptides under the assistance of tris(2-carboxyethyl)phosphine (TCEP). This protocol assembles linear peptides into cyclic peptides controllably to retain the diverse three-dimensional conformations, enabling the cellular uptake followed by release of the disulfides for peptide delivery.

Keywords:  cyclic peptides; disulfide stapling reagents; peptide delivery; reversible stapling; α-helicity

DOI:  https://doi.org/10.1002/anie.202314379
Chembiochem. 2023 Nov 10. e202300642

Integrin Facilitates the Internalization of TAT Peptide Conjugated to RGD Motif in Model Lipid Membranes.

Corina Ciobanasu, Julien Pernier, Christophe Le Clainche.

  In recent years, targeted drug delivery has attracted a great interest for enhanced therapeutic efficiency, with diminished side effects, especially in cancer therapy. Cell penetrating peptides like HIV1-TAT peptide, appear to be the perfect vectors for translocating drugs or other cargoes across the plasma membrane, but their application is limited mostly due to insufficient specificity for intended targets. The tripeptide motif RGD (arginine-glycine-aspartate), found in extracellular matrix proteins has high affinity for integrin receptors overexpressed in cancer and it is involved in different phases of disease progression, including proliferation, invasion and migration. Discovery of new peptides with high binding affinity for disease receptors and permeability of plasma membranes is desirable for both, development of targeted drug delivery systems and early detection and diagnosis. To complement the TAT peptide with specific targeting ability, we conjugated it with an integrin-binding RGD motif. In this paper we describe the permeability abilities and specificity for integrin receptor of RGD-TAT peptides in model membranes. Our findings reveal that RGD-TAT peptide maintains its ability to permeate lipid membranes and exhibits specificity for integrin receptors embedded in giant unilamellar vesicles. This promising outcome suggests that the peptide has significant potential for aplications in targeted drug delivery systems.

Keywords:  RGD peptide; TAT peptide; cell penetrating peptides; fluorescence microscopy; kinetics of peptides internalization

DOI:  https://doi.org/10.1002/cbic.202300642
Biochimie. 2023 Nov 02. pii: S0300-9084(23)00293-6. [Epub ahead of print]

Evaluating the cytotoxicity mechanism of the cell-penetrating peptide TP10 on Jurkat cells.

Kun Ji, Yufan Yao, Yuxuan Gao, Sujie Huang, Ling Ma, Qing Pan, Jun Wu, Wei Zhang, Hongmei Chen, Lei Zhang.

  TP10, a classic cell-penetrating peptide, shows a high degree of similarity to AMPs in structure. Although TP10 has been widely used in drug delivery, the mechanism underlying its cytotoxicity is yet to be elucidated. Herein, we explored the cell-killing mechanism of TP10 against human leukemia Jurkat cells. TP10 induced necrosis in Jurkat cells via rapid disruption of cell membranes, particularly at high concentrations. Although mitochondria in Jurkat cells were damaged by TP10, mitochondria-mediated apoptosis did not occur, possibly due to intracellular ATP depletion. Necroptosis in TP10-treated Jurkat cells became an alternative route of apoptosis. Our results demonstrate that necrosis and necroptosis rather than apoptosis are involved in the cell-killing mechanism of TP10, which contributes to the understanding of its toxicity.

Keywords:  Cell-penetrating peptide; Cytotoxicity; Membrane disruption; Necroptosis; TP10

DOI:  https://doi.org/10.1016/j.biochi.2023.11.001
Nat Commun. 2023 Nov 08. 14(1): 7212

Mechanism of selective recognition of Lys48-linked polyubiquitin by macrocyclic peptide inhibitors of proteasomal degradation.

Betsegaw Lemma, Di Zhang, Ganga B Vamisetti, Bryan G Wentz, Hiroaki Suga, Ashraf Brik, Jacek Lubkowski, David Fushman.

Post-translational modification of proteins with polyubiquitin chains is a critical cellular signaling mechanism in eukaryotes with implications in various cellular states and processes. Unregulated ubiquitin-mediated protein degradation can be detrimental to cellular homeostasis, causing numerous diseases including cancers. Recently, macrocyclic peptides were developed that selectively target long Lysine-48-linked polyubiquitin chains (tetra-ubiquitin) to inhibit ubiquitin-proteasome system, leading to attenuation of tumor growth in vivo. However, structural determinants of the chain length and linkage selectivity by these cyclic peptides remained unclear. Here, we uncover the mechanism underlying cyclic peptide's affinity and binding selectivity by combining X-ray crystallography, solution NMR, and biochemical studies. We found that the peptide engages three consecutive ubiquitins that form a ring around the peptide and determined requirements for preferential selection of a specific trimer moiety in longer polyubiquitin chains. The structural insights gained from this work will guide the development of next-generation cyclic peptides with enhanced anti-cancer activity.

DOI: https://doi.org/10.1038/s41467-023-43025-4
Mol Neurodegener. 2023 Nov 09. 18(1): 80

Peptide-based approaches to directly target alpha-synuclein in Parkinson's disease.

Scott G Allen, Richard M Meade, Lucy L White Stenner, Jody M Mason.

  Peptides and their mimetics are increasingly recognised as drug-like molecules, particularly for intracellular protein-protein interactions too large for inhibition by small molecules, and inaccessible to larger biologics. In the past two decades, evidence associating the misfolding and aggregation of alpha-synuclein strongly implicates this protein in disease onset and progression of Parkinson's disease and related synucleinopathies. The subsequent formation of toxic, intracellular, Lewy body deposits, in which alpha-synuclein is a major component, is a key diagnostic hallmark of the disease. To reach their therapeutic site of action, peptides must both cross the blood-brain barrier and enter dopaminergic neurons to prevent the formation of these intracellular inclusions. In this review, we describe and summarise the current efforts made in the development of peptides and their mimetics to directly engage with alpha-synuclein with the intention of modulating aggregation, and importantly, toxicity. This is a rapidly expanding field with great socioeconomic impact; these molecules harbour significant promise as therapeutics, or as early biomarkers during prodromal disease stages, or both. As these are age-dependent conditions, an increasing global life expectancy means disease prevalence is rising. No current treatments exist to either prevent or slow disease progression. It is therefore crucial that drugs are developed for these conditions before health care and social care capacities become overrun.

Keywords:  Alpha-Synuclein; Parkinson’s Disease; Peptide therapeutics; Peptidomimetics; Synucleinopathies

DOI:  https://doi.org/10.1186/s13024-023-00675-8
Sci Rep. 2023 11 08. 13(1): 19396

Structural flexibility of apolipoprotein E-derived arginine-rich peptides improves their cell penetration capability.

Yuki Takechi-Haraya, Takashi Ohgita, Akiko Usui, Kazuchika Nishitsuji, Kenji Uchimura, Yasuhiro Abe, Ryuji Kawano, Monika I Konaklieva, Mart Reimund, Alan T Remaley, Yoji Sato, Ken-Ichi Izutsu, Hiroyuki Saito.

Amphipathic arginine-rich peptide, A2-17, exhibits moderate perturbation of lipid membranes and the highest cell penetration among its structural isomers. We investigated the direct cell-membrane penetration mechanism of the A2-17 peptide while focusing on structural flexibility. We designed conformationally constrained versions of A2-17, stapled (StpA2-17) and stitched (StchA2-17), whose α-helical conformations were stabilized by chemical crosslinking. Circular dichroism confirmed that StpA2-17 and StchA2-17 had higher α-helix content than A2-17 in aqueous solution. Upon liposome binding, only A2-17 exhibited a coil-to-helix transition. Confocal microscopy revealed that A2-17 had higher cell penetration efficiency than StpA2-17, whereas StchA2-17 remained on the cell membrane without cell penetration. Although the tryptophan fluorescence analysis suggested that A2-17 and its analogs had similar membrane-insertion positions between the interface and hydrophobic core, StchA2-17 exhibited a higher membrane affinity than A2-17 or StpA2-17. Atomic force microscopy demonstrated that A2-17 reduced the mechanical rigidity of liposomes to a greater extent than StpA2-17 and StchA2-17. Finally, electrophysiological analysis showed that A2-17 induced a higher charge influx through transient pores in a planer lipid bilayer than StpA2-17 and StchA2-17. These findings indicate that structural flexibility, which enables diverse conformations of A2-17, leads to a membrane perturbation mode that contributes to cell membrane penetration.

DOI: https://doi.org/10.1038/s41598-023-46754-0
Nat Rev Drug Discov. 2023 Nov 03.

A new shuttle for drug delivery across the blood-brain barrier.

Katie Kingwell.

DOI: https://doi.org/10.1038/d41573-023-00180-z
Fluids Barriers CNS. 2023 Nov 03. 20(1): 80

A tissue-engineered model of the blood-tumor barrier during metastatic breast cancer.

Raleigh M Linville, Joanna Maressa, Zhaobin Guo, Tracy D Chung, Alanna Farrell, Ria Jha, Peter C Searson.

Metastatic brain cancer has poor prognosis due to challenges in both detection and treatment. One contributor to poor prognosis is the blood-brain barrier (BBB), which severely limits the transport of therapeutic agents to intracranial tumors. During the development of brain metastases from primary breast cancer, the BBB is modified and is termed the 'blood-tumor barrier' (BTB). A better understanding of the differences between the BBB and BTB across cancer types and stages may assist in identifying new therapeutic targets. Here, we utilize a tissue-engineered microvessel model with induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial-like cells (iBMECs) and surrounded by human breast metastatic cancer spheroids with brain tropism. We directly compare BBB and BTB in vitro microvessels to unravel both physical and chemical interactions occurring during perivascular cancer growth. We determine the dynamics of vascular co-option by cancer cells, modes of vascular degeneration, and quantify the endothelial barrier to antibody transport. Additionally, using bulk RNA sequencing, ELISA of microvessel perfusates, and related functional assays, we probe early brain endothelial changes in the presence of cancer cells. We find that immune cell adhesion and endothelial turnover are elevated within the metastatic BTB, and that macrophages exert a unique influence on BTB identity. Our model provides a novel three-dimensional system to study mechanisms of cancer-vascular-immune interactions and drug delivery occurring within the BTB.

DOI: https://doi.org/10.1186/s12987-023-00482-9
Nature. 2023 Nov 08.

Targeting of intracellular oncoproteins with peptide-centric CARs.

Mark Yarmarkovich, Quinlen F Marshall, John M Warrington, Rasika Premaratne, Alvin Farrel, David Groff, Wei Li, Moreno di Marco, Erin Runbeck, Hau Truong, Jugmohit S Toor, Sarvind Tripathi, Son Nguyen, Helena Shen, Tiffany Noel, Nicole L Church, Amber Weiner, Nathan Kendsersky, Dan Martinez, Rebecca Weisberg, Molly Christie, Laurence Eisenlohr, Kristopher R Bosse, Dimiter S Dimitrov, Stefan Stevanovic, Nikolaos G Sgourakis, Ben R Kiefel, John M Maris.

The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.

DOI: https://doi.org/10.1038/s41586-023-06706-0
ACS Chem Biol. 2023 Nov 09.

MicroED as a Powerful Tool for Structure Determination of Macrocyclic Drug Compounds Directly from Their Powder Formulations.

Emma Danelius, Guanhong Bu, Lianne H E Wieske, Tamir Gonen.

Macrocycles are important drug leads with many advantages including the ability to target flat and featureless binding sites as well as to act as molecular chameleons and thereby reach intracellular targets. However, due to their complex structures and inherent flexibility, macrocycles are difficult to study structurally, and there are limited structural data available. Herein, we use the cryo-EM method MicroED to determine the novel atomic structures of several macrocycles that have previously resisted structural determination. We show that structures of similar complexity can now be obtained rapidly from nanograms of material and that different conformations of flexible compounds can be derived from the same experiment. These results will have an impact on contemporary drug discovery as well as natural product exploration.

DOI: https://doi.org/10.1021/acschembio.3c00611
J Vis Exp. 2023 Oct 20.

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease.

Camille Goldman, Natalie Suhy, Jessica E Schwarz, Emily Ruth Sartori, Rikki B Rooklin, Braxton R Schuldt, Louise A Mesentier-Louro, Joel W Blanchard.

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer's disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

DOI: https://doi.org/10.3791/65921
ACS Omega. 2023 Oct 31. 8(43): 40463-40481

Peptide Conjugates of a 2'-O-Methoxyethyl Phosphorothioate Splice-Switching Oligonucleotide Show Increased Entrapment in Endosomes.

Alyssa C Hill, J Philipp Becker, Daria Slominski, François Halloy, Christoffer Søndergaard, Jacob Ravn, Jonathan Hall.

Antisense oligonucleotides (ASOs) are short, single-stranded nucleic acid molecules that alter gene expression. However, their transport into appropriate cellular compartments is a limiting factor in their potency. Here, we synthesized splice-switching oligonucleotides (SSOs) previously developed to treat the rare disease erythropoietic protoporphyria. Using chemical ligation-quantitative polymerase chain reaction (CL-qPCR), we quantified the SSOs in cells and subcellular compartments following free uptake. To drive nuclear localization, we covalently conjugated nuclear localization signal (NLS) peptides to a lead 2'-O-methoxyethyl phosphorothioate SSO using thiol-maleimide chemistry. The conjugates and parent SSO displayed similar RNA target-binding affinities. CL-qPCR quantification of the conjugates in cells and subcellular compartments following free uptake revealed one conjugate with better nuclear accumulation relative to the parent SSO. However, compared to the parent SSO, which altered the splicing of the target pre-mRNA, the conjugates were inactive at splice correction under free uptake conditions in vitro. Splice-switching activity could be conferred on the conjugates by delivering them into cells via cationic lipid-mediated transfection or by treating the cells into which the conjugates had been freely taken up with chloroquine, an endosome-disrupting agent. Our results identify the major barrier to the activity of the peptide-oligonucleotide conjugates as endosomal entrapment.

DOI: https://doi.org/10.1021/acsomega.3c05144
J Med Chem. 2023 Nov 07.

Discovery of a Hydroxylamine-Based Brain-Penetrant EGFR Inhibitor for Metastatic Non-Small-Cell Lung Cancer.

Jarvis Hill, Robert M Jones, David Crich.

Metastases to the brain remain a significant problem in lung cancer, as treatment by most small-molecule targeted therapies is severely limited by efflux transporters at the blood-brain barrier (BBB). Here, we report the discovery of a selective, orally bioavailable, epidermal growth factor receptor (EGFR) inhibitor, 9, that exhibits high brain penetration and potent activity in osimertinib-resistant cell lines bearing L858R/C797S and exon19del/C797S EGFR resistance mutations. In vivo, 9 induced tumor regression in an intracranial patient-derived xenograft (PDX) murine model suggesting it as a potential lead for the treatment of localized and metastatic non-small-cell lung cancer (NSCLC) driven by activating mutant bearing EGFR. Overall, we demonstrate that an underrepresented functional group in medicinal chemistry, the trisubstituted hydroxylamine moiety, can be incorporated into a drug scaffold without the toxicity commonly surmised to accompany these units, all while maintaining potent biological activity and without the molecular weight creep common to drug optimization campaigns.

DOI: https://doi.org/10.1021/acs.jmedchem.3c01669