Modular Peptide Research Articles

Release of functional dexamethasone by intracellular enzymes: A modular peptide-based strategy for ocular drug delivery

Tissue barriers limit drug delivery in the eye. Therefore, retinal diseases are treated with intravitreal injections. Delivery systems with reduced dosing frequency and/or cellular drug delivery properties are needed. We present here a modular peptide-based delivery system for cell targeted release of dexamethasone in the retinal pigment epithelial cells. The peptide–dexamethasone conjugates consist of cell penetrating peptide, enzyme cleavable linker and dexamethasone that is conjugated with hydrazone bond. The conjugates are chemically stable in the vitreous, internalize into the retinal pigment epithelial cells and release dexamethasone intracellularly by enzymatic action of cathepsin D. In vitro binding assay and molecular docking confirm binding of the released dexamethasone fragment to the human glucocorticoid receptor. In vivo rabbit studies show increased vitreal retention of dexamethasone with a peptide conjugate. Modular peptide conjugates are a promising approach for drug delivery into the retinal cells.

Dynamic peptide-folding mediated biofunctionalization and modulation of hydrogels for 4D bioprinting

Hydrogels are used in a wide range of biomedical applications, including three-dimensional (3D) cell culture, cell therapy and bioprinting. To enable processing using advanced additive fabrication techniques and to mimic the dynamic nature of the extracellular matrix (ECM), the properties of the hydrogels must be possible to tailor and change over time with high precision. The design of hydrogels that are both structurally and functionally dynamic, while providing necessary mechanical support is challenging using conventional synthesis techniques. Here, we show a modular and 3D printable hydrogel system that combines a robust but tunable covalent bioorthogonal cross-linking strategy with specific peptide-folding mediated interactions for dynamic modulation of cross-linking and functionalization. The hyaluronan-based hydrogels were covalently cross-linked by strain-promoted alkyne-azide cycloaddition using multi-arm poly(ethylene glycol). In addition, a de novo designed helix-loop-helix peptide was conjugated to the hyaluronan backbone to enable specific peptide-folding modulation of cross-linking density and kinetics, and hydrogel functionality. An array of complementary peptides with different functionalities was developed and used as a toolbox for supramolecular tuning of cell-hydrogel interactions and for controlling enzyme-mediated biomineralization processes. The modular peptide system enabled dynamic modifications of the properties of 3D printed structures, demonstrating a novel route for design of more sophisticated bioinks for four-dimensional bioprinting.

Cys-Flanked Cationic Peptides For Cell Delivery of the Herpes Simplex Virus Thymidine Kinase Gene for Suicide Gene Therapy of Uterine Leiomyoma

Uterine leiomyoma (UL) is the most common benign tumor in women of reproductive age. Gene therapy using suicidal genes appears to be a promising approach for UL treatment. One of key factors for success of gene therapy is the right choice of genetic construct carrier. A promising group of non-viral carriers for cell delivery of expression vectors is cationic Cys-flanked peptides which form tight complexes with DNA due to electrostatic interactions and the presence of interpeptide disulfide bonds. The paper reports a comparative study of the physico-chemical, toxic, and transfectional properties of the DNA-peptide complexes obtained by matrix polymerization or oxidative polycondensation of Cys-flanked peptides using the chain growth terminator 2-amino ethanethiol. We have demonstrated the therapeutic effect of the delivery of the pPTK-1 plasmid carrying the herpes simplex virus type 1 (HSV-1) thymidine kinase gene into PANC-1, and HEK-293T cell culture as well as into primary UL cells. It has been shown that the carriers obtained by oxidative polycondensation transform primary UL cells more efficiently than those produced by matrix polymerization. Treatment with ganciclovir resulted in the death of up to 40% of UL cells transfected with the pPTK-1 plasmid. The perspectives of use of the polyR6 carrier produced by oxidative polycondensation as a tool for the development of modular peptide carriers for the purposes of UL gene therapy were discussed.

Bacteriocin enterocin CRL35 is a modular peptide that induces non-bilayer states in bacterial model membranes

The mechanism of action of the anti-Listeria peptide enterocin CRL35 was studied with biophysical tools by using lipid mixtures that mimicked Gram-positive plasma membranes. Langmuir monolayers and infrared spectroscopy indicated that the peptide readily interacted with phospholipid assembled in monolayers and bilayers to produce a dual effect, depending on the acyl chains. Indeed, short chain mixtures were disordered by enterocin CRL35, but the gel-phases of membranes composed by longer acyl chains were clearly stabilized by the bacteriocin. Structural and functional studies indicated that non-bilayer states were formed when liposomes were co-incubated with enterocin CRL35, whereas significant permeabilization could be detected when bilayer and non-bilayer states co-existed. Results can be explained by a two-step model in which the N-terminal of the peptide firstly docks enterocin CRL35 on the lipid surface by means of electrostatic interactions; then, C-terminal triggers membrane perturbation by insertion of hydrophobic α-helix.

Synergistic Anti-Angiogenic Effects Using Peptide-Based Combinatorial Delivery of siRNAs Targeting VEGFA, VEGFR1, and Endoglin Genes.

Angiogenesis is a process of new blood vessel formation, which plays a significant role in carcinogenesis and the development of diseases associated with pathological neovascularization. An important role in the regulation of angiogenesis belongs to several key pathways such as VEGF-pathways, TGF-β-pathways, and some others. Introduction of small interfering RNA (siRNA) against genes of pro-angogenic factors is a promising strategy for the therapeutic suppression of angiogenesis. These siRNA molecules need to be specifically delivered into endothelial cells, and non-viral carriers modified with cellular receptor ligands can be proposed as perspective delivery systems for anti-angiogenic therapy purposes. Here we used modular peptide carrier L1, containing a ligand for the CXCR4 receptor, for the delivery of siRNAs targeting expression of VEGFA, VEGFR1 and endoglin genes. Transfection properties of siRNA/L1 polyplexes were studied in CXCR4-positive breast cancer cells MDA-MB-231 and endothelial cells EA.Hy926. We have demonstrated the efficient down-regulation of endothelial cells migration and proliferation by anti-VEGFA, anti-VEGFR1, and anti-endoglin siRNA-induced silencing. It was found that the efficiency of anti-angiogenic treatment can be synergistically improved via the combinatorial delivery of anti-VEGFA and anti-VEGFR1 siRNAs. Thus, this approach can be useful for the development of therapeutic angiogenesis inhibition.

Flexible and modular 3D-printed peptide models.

A flexible and modular peptide modeling set was designed using freely available software tools. The set consists of space-filling models of all 20 naturally occurring amino acid side chains and a modular kit for constructing peptides employing C-alpha carbons and amide bond groups. Connectors that allow free rotation about phi and psi angles on the peptide, together with explicit representation of peptide backbone hydrogen bond donor and acceptors allows for the construction of a wide range of protein secondary structure motifs. The space-filling side chain models highlight the steric, acid-base, and polarity of these groups. These models were printed using relatively affordable commercially available fused filament fabrication printers with minimal postprinting processing. Use of these models in student group activities focused on amino acids and protein secondary structural features is described. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):432-437, 2019.

Front Cover: Targeted Nanoswitchable Inhibitors of Protein–Protein Interactions Involved in Apoptosis (ChemMedChem 1/2019)

The Front Cover shows the concept of a generalized template design strategy to obtain light-regulated peptide inhibitors of helix-mediated protein–protein interactions (PPIs). Using the available information about hot spots at protein–peptide interfaces, we developed modular peptides containing a photoisomerizable crosslinker that provide reversible and reproducible photocontrol of the protein-binding ability and the inhibitory activity of peptides. This effect can be imagined as different components of toy houses (peptide inhibitors) that are reassembled with light to be active or not. This approach has been validated in two important targets (Bcl-xL-Bak and MDM2-p53) and could potentially be applied in a broad range of therapeutically relevant PPIs. More information can be found in the Full Paper by Pau Gorostiza, Ernest Giralt et al. on page 100 in Issue 1, 2019 (DOI: 10.1002/cmdc.201800647).

VEGFA Gene Silencing in CXCR4-Expressing Cells via siRNA Delivery by Means of Targeted Peptide Carrier.

Discovery of small interfering RNA as a tool for specific gene inhibition led to the development of new therapeutic strategy for the treatment of cancers. The efficacious delivery of therapeutic siRNAs into the cells is a crucial step in RNA interference (RNAi) application, but it remains challenging. Non-viral vectors can provide specific cellular uptake, stable siRNA complex formation, and intracellular siRNA release. Recently, we evaluated modular peptide carrier L1 bearing CXCR4 targeting ligand for its ability to condense siRNA and facilitate endosomal escape and VEGFA gene silencing in CXCR4-expressing endothelial and glioblastoma cells. The present chapter showcases the ability of L1 targeted peptide carrier to form complexes with siRNA and provide efficient VEGFA gene knockdown. We showed that siRNA delivery by means of L1 peptide carrier can result in significant decrease of VEGFA gene expression in A172 glioblastoma cells and in EA.hy 926 endothelial cells. Also, delivery of anti-VEGFA siRNA/peptide complexes led to significant inhibition of endothelial cell migration. Our results showed that L1 peptide carrier modified with CXCR4 ligand is a promising tool for targeted siRNA delivery into CXCR4-expressing cancer and endothelial cells.

Peptide binding affinity redistributes preassembled repeat protein fragments.

Designed armadillo repeat proteins (dArmRPs) are modular peptide binders composed of N- and C-terminal capping repeats Y and A and a variable number of internal modules M that each specifically recognize two amino acids of the target peptide. Complementary fragments of dArmRPs obtained by splitting the protein between helices H1 and H2 of an internal module show conditional and specific assembly only in the presence of a target peptide (Michel, E., Plückthun, A., and Zerbe, O. (2018). Peptide-guided assembly of repeat protein fragments. Angew. Chem. Int. Ed. 57, 4576-4579). Here, we investigate dArmRP fragments that already spontaneously assemble with high affinity, e.g. those obtained from splits between entire modules or between helices H2 and H3. We find that the interaction of the peptide with the assembled fragments induces distal conformational rearrangements that suggest an induced fit on a global protein level. A population analysis of an equimolar mixture of an N-terminal and three C-terminal fragments with various affinities for the target peptide revealed predominant assembly of the weakest peptide binder. However, adding a target peptide to this mixture altered the population of the protein complexes such that the combination with the highest affinity for the peptide increased and becomes predominant when adding excess of peptide, highlighting the feasibility of peptide-induced enrichment of best binders from inter-modular fragment mixtures.

Peptide-siRNA Supramolecular Particles for Neural Cell Transfection.

Small interfering ribonucleic acid (siRNA)‐based gene knockdown is an effective tool for gene screening and therapeutics. However, the use of nonviral methods has remained an enormous challenge in neural cells. A strategy is reported to design artificial noncationic modular peptides with amplified affinity for siRNA via supramolecular assembly that shows efficient protein knockdown in neural cells. By solid phase synthesis, a sequence that binds specifically double‐stranded ribonucleic acid (dsRNA) with a self‐assembling peptide for particle formation is integrated. These supramolecular particles can be further functionalized with bioactive sequences without affecting their biophysical properties. The peptide carrier is found to silence efficiently up to 83% of protein expression in primary astroglial and neuronal cell cultures without cytotoxicity. In the case of neurons, a reduction in electrical activity is observed once the presynaptic protein synaptophysin is downregulated by the siRNA–peptide particles. The results demonstrate that the supramolecular particles offer an siRNA delivery platform for efficient nonviral gene screening and discovery of novel therapies for neural cells.

Programmable Construction of Peptide-Based Materials in Living Subjects: From Modular Design and Morphological Control to Theranostics.

Self-assembled nanomaterials show potential high efficiency as theranostics for high-performance bioimaging and disease treatment. However, the superstructures of pre-assembled nanomaterials may change in the complicated physiological conditions, resulting in compromised properties and/or biofunctions. Taking advantage of chemical self-assembly and biomedicine, a new strategy of "in vivo self-assembly" is proposed to in situ construct functional nanomaterials in living subjects to explore new biological effects. Herein, recent advances on peptide-based nanomaterials constructed by the in vivo self-assembly strategy are summarized. Modular peptide building blocks with various functions, such as targeting, self-assembly, tailoring, and biofunctional motifs, are employed for the construction of nanomaterials. Then, self-assembly of these building blocks in living systems to construct various morphologies of nanostructures and corresponding unique biological effects, such as assembly/aggregation-induced retention (AIR), are introduced, followed by their applications in high-performance drug delivery and bioimaging. Finally, an outlook and perspective toward future developments of in vivo self-assembled peptide-based nanomaterials for translational medicine are concluded.

Peptide Self-Assembly: Modular Self-Assembling Peptide Platform with a Tunable Thermoresponsiveness via a Single Amino Acid Substitution (Adv. Funct. Mater. 35/2018)

Peptide Self-Assembly: Modular Self-Assembling Peptide Platform with a Tunable Thermoresponsiveness via a Single Amino Acid Substitution (Adv. Funct. Mater. 35/2018)

Modular peptide-functionalized gold nanorods for effective glioblastoma multicellular tumor spheroid targeting.

Glioblastoma multiforme (GBM) contains a population of tumor initiating stem-like cells, termed cancer stem cells (CSCs). These CSCs, which are resistant to chemo- and radiotherapy, are thought to persist after treatment and drive tumor recurrence. Thus, it is believed that the elimination of CSCs can lead to GBM remission. GBM CSCs express Nestin on their surface, and can be therefore targeted via this protein. Gold nanorods (AuNRs) functionalized with an engineered, modular peptide that recognizes Nestin (NesPEG-AuNRs) were used to target the models of solid tumors originated from human GBM CSC multicellular tumor spheroids (MCTS). In our study, we show that NesPEG-AuNRs have low cytotoxicity, are efficiently taken up by MCTS, and distribute uniformly throughout our tumor models, not only at the periphery as often seen in other nanoparticle systems. NesPEG-AuNR uptake by MCTS appears to be mediated by an energy/caveolae endocytic mechanism. Moreover, plasmon excitation of AuNRs in the near-infrared (NIR) region results in the production of localized heat. Consequently, NesPEG-AuNR cytotoxicity is only observed during NIR-irradiation in MCTS with a high intracellular AuNR content. The intracellular accumulation/diffusion of NesPEG-AuNRs and NIR-irradiation result in photothermally induced GBM CSC apoptosis and MCTS growth inhibition. In summary, these data suggest that the combination of the Nestin recognizing peptide with AuNRs contributes to better tumor accumulation/penetration, and thus in GBM CSC elimination. Moreover, due to the modularity of our peptide design, the Nestin-binding peptide sequence can be exchanged for peptides targeting other surface markers for the treatment of various types of tumors.

Internal Peptide Late‐Stage Diversification: Peptide‐Isosteric Triazoles for Primary and Secondary C(sp3)−H Activation

AbstractSecondary C(sp3)−H arylations were accomplished by palladium catalysis with triazoles as peptide bond isosteres. The unique power of this approach is highlighted by the possibility of achieving secondary C(sp3)−H functionalizations on terminal peptides as well as the unprecedented positional‐selective C(sp3)−H functionalization of internal peptide positions, setting the stage for modular peptide late‐stage diversification.

Internal Peptide Late-Stage Diversification: Peptide-Isosteric Triazoles for Primary and Secondary C(sp3 )-H Activation.

Secondary C(sp3 )-H arylations were accomplished by palladium catalysis with triazoles as peptide bond isosteres. The unique power of this approach is highlighted by the possibility of achieving secondary C(sp3 )-H functionalizations on terminal peptides as well as the unprecedented positional-selective C(sp3 )-H functionalization of internal peptide positions, setting the stage for modular peptide late-stage diversification.

Multifunctional RNA Binding Protein OsTudor-SN in Storage Protein mRNA Transport and Localization

The multifunctional RNA-binding protein Tudor-SN plays multiple roles in transcriptional and posttranscriptional processes due to its modular domain structure, consisting of four tandem Staphylococcus nuclease (SN)-like domains (4SN), followed by a carboxyl-terminal Tudor domain, followed by a fifth partial SN sequence (Tsn). In plants, it confers stress tolerance, is a component of stress granules and P-bodies, and may participate in stabilizing and localizing RNAs to specific subdomains of the cortical-endoplasmic reticulum in developing rice (Oryza sativa) endosperm. Here, we show that, in addition to the intact rice OsTudor-SN protein, the 4SN and Tsn modules exist as independent polypeptides, which collectively may coassemble to form a complex population of homodimer and heteroduplex species. The 4SN and Tsn modules exhibit different roles in RNA binding and as a protein scaffold for stress-associated proteins and RNA-binding proteins. Despite their distinct individual properties, mutations in both the 4SN and Tsn modules mislocalize storage protein mRNAs to the cortical endoplasmic reticulum. These results indicate that the two modular peptide regions of OsTudor-SN confer different cellular properties but cooperate in mRNA localization, a process linking its multiple functions in the nucleus and cytoplasm.

Peptide Logic Circuits Based on Chemoenzymatic Ligation for Programmable Cell Apoptosis

AbstractA novel and versatile peptide‐based bio‐logic system capable of regulating cell function is developed using sortase A (SrtA), a peptide ligation enzyme, as a generic processor. By modular peptide design, we demonstrate that mammalian cells apoptosis can be programmed by peptide‐based logic operations, including binary and combination gates (AND, INHIBIT, OR, and AND‐INHIBIT), and a complex sequential logic circuit (multi‐input keypad lock). Moreover, a proof‐of‐concept peptide regulatory circuit was developed to analyze the expression profile of cell‐secreted protein biomarkers and trigger cancer‐cell‐specific apoptosis.

Peptide Logic Circuits Based on Chemoenzymatic Ligation for Programmable Cell Apoptosis.

A novel and versatile peptide-based bio-logic system capable of regulating cell function is developed using sortase A (SrtA), a peptide ligation enzyme, as a generic processor. By modular peptide design, we demonstrate that mammalian cells apoptosis can be programmed by peptide-based logic operations, including binary and combination gates (AND, INHIBIT, OR, and AND-INHIBIT), and a complex sequential logic circuit (multi-input keypad lock). Moreover, a proof-of-concept peptide regulatory circuit was developed to analyze the expression profile of cell-secreted protein biomarkers and trigger cancer-cell-specific apoptosis.

Curvature of designed armadillo repeat proteins allows modular peptide binding.

Designed armadillo repeat proteins (dArmRPs) were developed to create a modular peptide binding technology where each of the structural repeats binds two residues of the target peptide. An essential prerequisite for such a technology is a dArmRP geometry that matches the peptide bond length. To this end, we determined a large set (n=27) of dArmRP X-ray structures, of which 12 were previously unpublished, to calculate curvature parameters that define their geometry. Our analysis shows that consensus dArmRPs exhibit curvatures close to the optimal range for modular peptide recognition. Binding of peptide ligands can induce a curvature within the desired range, as confirmed by single-molecule FRET experiments in solution. On the other hand, computationally designed ArmRPs, where side chains have been chosen with the intention to optimally fit into a geometrically optimized backbone, turned out to be more divergent in reality, and thus not suitable for continuous peptide binding. Furthermore, we show that the formation of a crystal lattice can induce small but significant deviations from the curvature adopted in solution, which can interfere with the evaluation of repeat protein scaffolds when high accuracy is required. This study corroborates the suitability of consensus dArmRPs as a scaffold for the development of modular peptide binders.

Modification of Poly(propylene fumarate)–Bioglass Composites with Peptide Conjugates to Enhance Bioactivity

Poly(propylene fumarate) (PPF) has been highlighted as one of the most promising materials for bone regeneration. Despite the promising advantages of using polymer scaffolds for biomedical applications, their inherent lack of bioactivity has limited their clinical application. In this study, PPF was successfully functionalized with Bioglass and a novel catechol-bearing peptide bioconjugate containing bioactive short peptide sequences of basic fibroblast growth factor, bone morphogenetic protein 2, and osteogenic growth peptide. The binding affinity was assessed to be around 110 nmol/cm2 with the Bioglass content at 10 wt %. Fluorescence imaging studies show that the catechol-bearing modular peptide binds preferentially to the Bioglass. A 4 week in vitro cell study using human mesenchymal stem cells showed that cell adhesion, spreading, proliferation, and osteogenic differentiation at both gene and protein levels were all improved by the introduction of peptides, demonstrating the potential approach of dually functionalized polymers for bone regeneration.