Cell-penetrating Capabilities Research Articles

De Novo Designed Cell-Penetrating Peptide Self-Assembly Featuring Distinctive Tertiary Structure.

Recent attention has focused on the de novo design of proteins, paralleling advancements in biopharmaceuticals. Achieving protein designs with both structure and function poses a significant challenge, particularly considering the importance of quaternary structures, such as oligomers, in protein function. The cell penetration properties of peptides are of particular interest as they involve the penetration of large molecules into cells. We previously suggested a link between the oligomerization propensity of amphipathic peptides and their cell penetration abilities, yet concrete evidence at cellular-relevant concentrations was lacking due to oligomers' instability. In this study, we sought to characterize oligomerization states using various techniques, including X-ray crystallography, acceptor photobleaching Förster resonance energy transfer (FRET), native mass spectrometry (MS), and differential scanning calorimetry (DSC), while exploring the function related to oligomer status. X-ray crystallography revealed the atomic structures of oligomers formed by LK-3, a bis-disulfide bridged dimer with amino acid sequence LKKLCLKLKKLCKLAG, and its derivatives, highlighting the formation of hexamers, specifically the trimer of dimers, which exhibited a stable hydrophobic core. FRET experiments showed that LK-3 oligomer formation was associated with cell penetration. Native MS confirmed higher-order oligomers of LK-3, while an intriguing finding was the enhanced cell-penetrating capability of a 1:1 mixture of l/d-peptide dimers compared to pure enantiomers. DSC analysis supported the notion that this enantiomeric mixture promotes the formation of functional oligomers, crucial for cell penetration. In conclusion, our study provides direct evidence that amphipathic peptide LK-3 forms oligomers at low nanomolar concentrations, underscoring their significance in cell penetration behavior.

Abstract 6061: Development of target cell-penetrating scFv conjugate degrader for the selective inhibition of mutant, resistant KRAS cancer

Abstract The activation of mutations in the RAS gene, which is frequently observed in human cancers, induces downstream critical effectors, contributing to tumorigenesis. While attempts to directly target the GTP-bound, activated RAS have shown some success in certain cancer treatments, resistance remains a challenge. Our objective is to develop a cell-penetrating single-chain fragment variable (scFv) degrader that targets mutant KRAS for degradation. The scFv, binding specifically to antigens, can mitigate the side effects of non-specific binding seen with chemical drugs. However, due to the limited cell-penetrating capability of scFv, a delivery carrier is often necessary. In this study, a cancer targeting, and cell-penetrating synthetic peptide (CPP) was developed as a drug delivery carrier and added to the sequence of scFv. To this conjugate, a von Hippel-Lindau (VHL) expressing sequence was added to enable the scFv binding to mutant KRAS, enabling its proteasomal degradation. These conjugates were engineered as a plasmid DNA to be expressed as a protein conjugate in CHO cells. The purity of the scFv-VHL-CPP degrader was confirmed by SDS PAGE. In SPR results, the degrader not only exhibited high binding ability to mutant KRAS but also influenced the proliferation of KRAS mutant cells in cell viability tests. Mutant GTP-bound KRAS was reduced by the KRAS degrader in KRAS mutant cancer cells. The in vivo study further confirmed the degradation of the KRAS, as reflected by the significantly reduced tumor growth. In conclusion, a degrader protein binding to mutant KRAS that consists of cell penetrating peptide, scFv, and VHL is expected to become a new protein anticancer drug especially effective against mutant, resistant KRAS-mediated cancer cells. Especially, the design of the target cell penetrating peptide-scFv-VHL would be served as a platform for the target protein degrader development. Citation Format: Gookjin Yoon, Beom Soo Jo, Dong Woo Lee, Jinwook Yang, Min-Ho Park, Sanghui Seok, Jue Yeon Lee, Chong Pyoung Chung, Yoon Jeong Park. Development of target cell-penetrating scFv conjugate degrader for the selective inhibition of mutant, resistant KRAS cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6061.

Therapeutic Peptides, Proteins and their Nanostructures for Drug Delivery and Precision Medicine.

Peptide and protein nanostructures with tunable structural features, multifunctionality, biocompatibility and biomolecular recognition capacity enable development of efficient targeted drug delivery tools for precision medicine applications. In this review article, we present various techniques employed for the synthesis and self-assembly of peptides and proteins into nanostructures. We discuss design strategies utilized to enhance their stability, drug-loading capacity, and controlled release properties, in addition to the mechanisms by which peptide nanostructures interact with target cells, including receptor-mediated endocytosis and cell-penetrating capabilities. We also explore the potential of peptide and protein nanostructures for precision medicine, focusing on applications in personalized therapies and disease-specific targeting for diagnostics and therapeutics in diseases such as cancer.

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

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.

Discovery of ICOS-Targeted Small Molecules Using Pharmacophore-Based Screening.

There are currently no small molecules clinically approved as immune checkpoint modulators. Besides possessing oral bioavailability, cell-penetrating capabilities and enhanced tumor penetration compared to monoclonal antibodies (mAbs), small molecules are amenable to pharmacokinetic optimization, which allows adopting flexible dosage regimens that may avoid immune-related adverse events associated with mAbs. The interaction of inducible co-stimulator (ICOS) with its ligand (ICOS-L) plays key roles in T-cell differentiation and activation of T-cell to B-cell functions. This study represents the development and validation of a virtual screening strategy to identify small molecules that bind a novel druggable binding pocket in human ICOS. We used a lipophilic canyon in the apo-structure of ICOS and the ICOS/ICOS-L interface individually as templates for molecular dynamics simulation to generate 3D pharmacophores subsequently used for virtual screening campaigns. Our strategy was successful finding a first-in-class small molecule ICOS binder (5P, KD value=108.08±26.76 μM) and validating biophysical screening platforms for ICOS-targeted small molecules. We anticipate that future structural optimization of 5P will result in the discovery of high affinity chemical ligands for ICOS.

DNA-Based Nanoprobes for Simultaneous Detection of Telomerase and Correlated Biomolecules.

Telomerase (TE), a ribonucleoprotein reverse transcriptase, is enzymatically activated in most tumor cells and is responsible for promoting tumor progression and malignancy by enabling replicative immortality of cancer cells. TE has become an important hallmark for cancer diagnosis and a potential therapy target. Therefore, accurate in situ detection of TE activity, especially the simultaneous imaging of TE activity and its correlated biomolecules, is essential to medical diagnostics and therapeutics. DNA-based nanoprobes, with their effective cell penetration capability and programmability, are the most advantageous for detection of intracellular TE activity. This concept article introduces the recent strategies for in situ sensing and imaging of TE activity, with a focus on simultaneous detection of TE and related biomolecules, and provides challenges and perspectives for the development of new strategies for such correlated imaging.

The role of cell-penetrating peptides in potential anti-cancer therapy.

Due to the complex physiological structure, microenvironment and multiple physiological barriers, traditional anti‐cancer drugs are severely restricted from reaching the tumour site. Cell‐penetrating peptides (CPPs) are typically made up of 5–30 amino acids, and can be utilised as molecular transporters to facilitate the passage of therapeutic drugs across physiological barriers. Up to now, CPPs have widely been used in many anti‐cancer treatment strategies, serving as an excellent potential choice for oncology treatment. However, their drawbacks, such as the lack of cell specificity, short duration of action, poor stability in vivo, compatibility problems (i.e. immunogenicity), poor therapeutic efficacy and formation of unwanted metabolites, have limited their further application in cancer treatment. The cellular uptake mechanisms of CPPs involve mainly endocytosis and direct penetration, but still remain highly controversial in academia. The CPPs‐based drug delivery strategy could be improved by clever design or chemical modifications to develop the next‐generation CPPs with enhanced cell penetration capability, stability and selectivity. In addition, some recent advances in targeted cell penetration that involve CPPs provide some new ideas to optimise CPPs.

Novel tetrameric cell penetrating antimicrobial peptoids effective against mycobacteria and drug-resistant Staphylococcus aureus.

Antimicrobial peptides (AMPs) are short, cationic, amphipathic molecules that have gained tremendous popularity as alternatives to traditional antibiotics due to their lower propensity to develop bacterial resistance. However, the clinical developability of AMPs remains impeded due to shortcomings such as proteolytic instability and poor penetration leading to low bioavailability. To improve the access of AMPs to cells and subsequent bacteria killing, we evaluated the cell-penetrating and antimicrobial properties of three novel libraries of synthetic peptoids using Minimum Inhibitory Concentration, killing efficacy and membrane permeabilization assays against mycobacteria and Staphylococcus aureus. In addition, we investigated cell selectivity using mammalian cells to assess peptoid toxicity. We showed that short tetrameric Rhodamine B-labeled peptoids composed of a balance of aromatic and lipophilic residues have potent selective antimicrobial activity against a range of microorganisms. The most potent candidates were active against drug-resistant S. aureus isolates as well as mycobacterial strains, with cell penetrating capabilities reported in HeLa and RAW 264.7 macrophage cells. These data suggest that peptoids with novel dual functionalities may potentially be an interesting class of therapeutics and/or molecular delivery agents for anti-infective purposes.

Artificial Evolutionary Optimization Process to Improve the Functionality of Cell Penetrating Peptides.

Crossing cellular membranes is a versatile molecular property that allows for a wide variety of peptides with cell penetrating capabilities. This broadness complicates identification of candidates suited best for a specific application. To facilitate the screening of this enormous molecular space in a supervised manner we here present a method to "breed" the desired molecules by applying the rules of Darwinian evolution. With this mate-and-check protocol, which combines an in silico evolution step with an in vitro performance test, cell penetrating peptides that are optimized for a specific task can be achieved in a few rounds of breeding. The procedure is simple and straightforward on the synthetic site but requires robust, highly reproducible and close-to-reality biological assays to yield realistic functional output. With this technology even top-performing peptides can be further improved and functionally adjusted.

Near-Infrared Light-Triggered Lysosome-Targetable Carbon Dots for Photothermal Therapy of Cancer.

Photothermal therapy (PTT) has inherent advantages in the treatment of hypoxic tumors due to its optically controlled selectivity on tumor ablation and oxygen-independent nature. The subcellular organelle-targeting capability and photothermal conversion efficiency (PCE) at near-infrared (NIR) wavelength are the key parameters in the assessment of the photothermal agent (PTA). Here, we report that carbon dots (CDs) prepared by the hydrothermal treatment of coronene derivatives show a high PCE of 54.7% at 808 nm, which can be attributed to the narrow band gap and the presence of amounts of continuous energy bands on CDs. Moreover, the vibrations in the layered graphite structures of the CDs also increase the rate of nonradiative transition and thus enhance the PCE. Furthermore, the CDs also possess excellent photostability, biocompatibility, and cell penetration capability and could mainly accumulate in the lysosomes. These experiment results have proved that the CDs are suitable as an efficient NIR light-triggered PTA for efficient PTT against cancer.

Revealing the importance of carrier-cargo association in delivery of insulin and lipidated insulin

Delivery of therapeutic peptides upon oral administration is highly desired and investigations report that the cell-penetrating peptide (CPP) penetratin and its analogues shuffle and penetramax show potential as carriers to enhance insulin delivery. Exploring this, the specific aim of the present study was to understand the impact that their complexation with a lipidated or non-lipidated therapeutic cargo would have on the delivery, to evaluate the effect of differences in membrane interactions in vitro and in vivo, as well as to deduce the mode of action leading to enhanced delivery. Fundamental biophysical aspects were studied by a range of orthogonal methods. Transepithelial permeation of therapeutic peptide was evaluated using the Caco-2 cell culture model supplemented with epithelial integrity measurements, real-time assessment of the carrier peptide effects on cell viability and on mode of action. Pharmacokinetic and pharmacodynamic (PK/PD) parameters were evaluated following intestinal administration to rats and tissue effects were investigated by histology. The biophysical studies revealed complexation of insulin with shuffle and penetramax, but not with penetratin. This corresponded to enhanced transepithelial permeation of insulin, but not of lipidated insulin, when in physical mixture with shuffle or penetramax. The addition of shuffle and penetramax was associated with a lowering of Caco-2 cell monolayer integrity and viability, where the lowering of cell viability was immediate, but reversible. Insulin delivery in rats was enhanced by shuffle and penetramax and accompanied by a 10–20-fold decrease in blood glucose with immediate effect on the intestinal mucosa. In conclusion, shuffle and penetramax, but not penetratin, demonstrated to be potential candidates as carriers for transmucosal delivery of insulin upon oral administration, and their effect depended on association with both cargo and cell membrane. Interestingly, the present study provides novel mechanistic insight that peptide carrier-induced cargo permeation points towards enhancement via the paracellular route in the tight epithelium. This is different from the anticipated belief being that it is the cell-penetrating capability that facilitate transepithelial cargo permeation via a transcellular route.

Amphipathic design dictates self-assembly, cytotoxicity and cell uptake of arginine-rich surfactant-like peptides.

Amphiphilicity is the most critical parameter in the self-assembly of surfactant-like peptides (SLPs), regulating the way by which hydrophobic attraction holds peptides together. Its effects go beyond supramolecular assembly and may also trigger different cell responses of bioactive peptide-based nanostructures. Herein, we investigate the self-assembly and cellular effects of nanostructures based on isomeric SLPs composed by arginine (R) and phenylalanine (F). Two amphipathic designs were studied: a diblock construct F4R4 and its bolaamphiphile analog R2F4R2. A strong sequence-dependent polymorphism emerges with appearance of globules and vesicle-like assemblies, or flat nanotapes and cylindrical micelles. The diblock construct possesses good cell penetrating capabilities and effectiveness to kill SK-MEL-28 melanoma tumor cells, in contrast to reduced intracellular uptake and low cytotoxicity exhibited by the bolaamphiphilic form. Our findings demonstrate that amphipathic design is a relevant variable for self-assembling SLPs to modulate different cellular responses and may assist in optimizing the production of nanostructures based on arginine-enriched sequences in cell penetrating and antimicrobial peptides.

Rational Design and In Vitro Evaluation of Novel Peptides Binding to Neuroligin-1 for Synaptic Targeting.

Neuroligin-1 (NL1) is a postsynaptic cell adhesion protein that plays a crucial role in synapsis and signaling between neurons. Due to its clustered distribution in synaptic clefts, NL1 appears as a novel potential site for synaptic targeting purposes. In this work, in silico protein topography analysis was employed to identify two prospective binding sites on the NL1 dimer surface in the 2:2 synaptic adhesion complex with β-neurexin (PDB code 3B3Q ). Receptor-based rational design, cell-penetrating capability prediction, molecular docking, molecular dynamics simulations, and binding free energy calculations were used to identify five heptapeptides candidates with favorable predicted profiles as non cell-penetrating NL1-binding agents. Preliminary in vitro colocalization assays with NL1-transfected HEK 293 cells confirmed that peptides remain in the extracellular space without inducing detectable changes in cell morphology. The highest NL1-colocatization capability was attained by the peptide ADEAIVA, which appears as a promising candidate for the future development of specific NL1-targeting systems as part of synapse-directed therapies against central nervous system diseases.

Peptides derived from the C-terminal domain of HIV-1 Viral Protein R in lipid bilayers: Structure, membrane positioning and gene delivery

Viral protein R (Vpr) is a small accessory protein of 96 amino acids that is present in Human and simian immunodeficiency viruses. Among the very different properties that Vpr possesses we can find cell penetrating capabilities. Based on this and on its capacity to interact with nucleic acids we previously investigated the DNA transfection properties of Vpr and subfragments thereof. We found that fragments of the C-terminal helical domain of Vpr are able to deliver efficiently plasmid DNA into different cell lines. As the amphipathic helix may play a role in the interactions with membranes, we investigated whether insertion of a proline residue in the α-helix modifies the transfection properties of Vpr. Unexpectedly, we found that the resulting Vpr55-82 Pro70 peptide was even more efficient than wild type Vpr55-82 in the gene delivery assays. Using circular dichroism, light scattering and solid-state NMR techniques, we characterized the secondary structure and interactions of Vpr and several mutants with model membranes. A model is proposed where the proline shifts the dissociation equilibrium of the peptide-cargo complex and thereby its endosomal release.

Interchain doubly-bridged α-helical peptides for the development of protein binders

Constrained peptide scaffolds that are tolerant to extensive sequence manipulation and amenable to bioactive peptide design are of great value to the development of novel protein binders and peptide therapeutics. In this work, we reported strategies for the design and synthesis of a kind of novel interchain doubly-bridged α-helical peptides, involving mutual stabilization of two peptide α-helices linked by two interchain bisthioether crosslinkers. By taking a MDM2-binding peptide with an α-helical tendency as a model, we demonstrated that α-helical dimers with significantly improved structural and proteolytic stability and nanomolar binding affinity to the target protein can be obtained. By modulating the surface charges on the dimeric peptides, we also obtained a dimeric peptide with enhanced cell-penetrating capability, which can efficiently penetrate into cancer cells and inhibit the intracellular MDM2-p53 interactions to promote cell apoptosis. Considering that many proteins take a surface α-helical segment as the binding motif to mediate their interactions with other proteins, we believe that our interchain doubly-bridged α-helical peptides would provide a promising scaffold for the development of novel high-affinity protein binders.

Breeding Cell Penetrating Peptides: Optimization of Cellular Uptake by a Function-Driven Evolutionary Process.

In nature, building block-based biopolymers can adapt to functional and environmental demands by recombination and mutation of the monomer sequence. We present here an analogous, artificial evolutionary optimization process which we have applied to improve the functionality of cell-penetrating peptide molecules. The "evolution" consisted of repeated rounds of in silico peptide sequence alterations using a genetic algorithm followed by in vitro peptide synthesis, experimental analysis, and ranking according to their "fitness" (i.e., their ability to carry the cargo carboxyfluorescein into cultured cells). The genetic algorithm-based optimization method was customized and adapted from former successful applications in the lab to realize an early convergence and a minimum number of in vitro and in silico processing steps by configured settings derived from empirical in silico simulation. We started out with 20 "lead peptides" which we had previously identified as top performers regarding their ability to enter cultured cells. Ten breeding rounds comprising 240 peptides each yielded a peptide population of which the top 10 candidates displayed a 6-fold (median values) increase in its cell-penetration capability compared with the top 10 lead peptides, and two consensus sequences emerged which represent local fitness optima. In addition, the cell-penetrating potential could be proven independently of the carboxyfluorescein cargo in an alternative setting. Our results demonstrate that we have established a powerful optimization technology that can be used to further improve peptides with known functionality and adapt them to specific applications.

The Remarkable Cationic Peptides: A Boon to Pharmaceutical Sciences?

In this opinion article, the authors discuss a number of interesting, beneficial properties of naturally occurring and synthetic cationic antimicrobial peptides (AMPs) with the prospective aim of bringing these compounds into therapeutic use to avoid antibiotic resistance and utilize their numerous properties. The structural diversity and the conformational freedom of these compounds adversely affects their mechanistic elucidation. Our molecular level mechanistic exploration of these peptides has shown their ion carriage properties and systematically explains their antibiotic activity through disruption of bacterial cell homeostasis and inhibition of 14-α demethylase enzyme. We have also shown self-assemblage in AMPs in different nanoparticulate and tubular forms. Some AMPs possess cell penetration capability and their co-administration with drug enhances antibacterial activity through a non-disruptive mechanism. The anti-HIV activity of AMPs has been explained based on their non-covalent, non-base-pair base-pair type interactions with HIV viral ssRNA template. Design of peptidomimetic compounds with enhanced druggability based on our mechanistic explorations will definitely lead to better non-toxic drugs with antibacterial, anti-HIV activity and may contribute towards development of efficient drug delivery systems.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Design and tuning of a cell-penetrating albumin derivative as a versatile nanovehicle for intracellular drug delivery

Human serum albumin (HSA) is a superior carrier for delivering extracellular drugs. However, the development of a cell-penetrating HSA remains a great challenge due to its low membrane permeability. We report herein on the design of a series of palmitoyl-poly-arginine peptides (CPPs) and an evaluation of their cell-penetrating effects after forming a complex with HSA for use in intracellular drug delivery. The palmitoyl CPPs forms a stable complex with HSA by anchoring itself to the high affinity palmitate binding sites of HSA. Among the CPPs evaluated, a cyclic polypeptide composed of D-dodecaarginines, palmitoyl-cyclic-(D-Arg)12 was the most effective for facilitating the cellular uptake of HSA by HeLa cells. Such a superior cell-penetrating capability is primarily mediated by macropinocytosis. The effect of the CPP on pharmacological activity was examined using three drugs loaded in HSA via three different methods: a) an HSA-paclitaxel complex, b) an HSA-doxorubicin covalent conjugate and c) an HSA-thioredoxin fusion protein. The results showed that cell-penetrating efficiency was increased with a corresponding and significant enhancement in pharmacological activity. In conclusion, palmitoyl-cyclic-(D-Arg)12/HSA is a versatile cell-penetrating drug delivery system with great potential for use as a nano-carrier for a wide diversity of pharmaceutical applications.

Cross-Linking Induced Self-Organization of Polymers into Degradable Assemblies.

Covalently stabilized polymer assemblies are normally fabricated from the self-assembly of polymer chains followed by a cross-linking reaction. In this report, we show that a cross-linking-induced self-assembly approach, in which boronate cross-linking sites are formed by the condensation reaction between boronic and catechol groups, can organize polymer networks into uniform assemblies. Self-assembly of these boronate cross-linked polymer networks adopts two different driving forces in water and methanol solutions. Hydrophobic aggregation of polymer networks in water solution affords spherical assemblies, while B-N dative bond formed between boronate and imine functionalities in methanol solution organizes the polymer networks into bundle-like assemblies. We not only demonstrate the intrinsic stimuli-responsive degradability of these cross-linked assemblies but also show that their degradation can cause a controllable release of guest molecules. Moreover, bundle-like assemblies with rough surface and exposed boronate functionalities exhibit dramatically higher cell penetration capability than the spherical assemblies with smooth surface and embedded boronate functionalities.

Functional manganese dioxide nanosheet for targeted photodynamic therapy and bioimaging in vitro and in vivo

Photodynamic therapy (PDT) has been widely studied as a promising non-invasive therapeutic strategy for the treatment of cancer. However, the poor solubility of photosensitizer (PS) in aqueous solution and inefficient cell-penetrating capability have limited the target-specific PDT. Herein, we develop a novel targeted photodynamic therapeutic and bioimaging system based on folic acid (FA)-conjugated MnO2 (FA-MnO2) nanosheet as a new carrier of PS, zinc phthalocyanine (ZnPc). ZnPc loaded FA-MnO2 nanosheet (FA-MnO2/ZnPc) complex is successfully formed by electrostatic interaction and coordination. We find that FA-MnO2/ZnPc complex exhibits excellent targeted delivery of ZnPc into folate receptor positive cancer cells and the ZnPc is released out from the complex via endogenous glutathione (GSH) stimulus, facilitating simultaneous bioimaging and targeted PDT by singlet oxygen (SO) generation upon light irradiation, showing high efficacy with only one tenth of conventional PS dosage in vitro and in vivo.