Search Results (31)

Objectives: The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the current treatment limitations—particularly the emergence of drug resistance and the reduced efficacy of some existing drugs against new variants—highlight the need for novel antiviral strategies with novel action mechanisms. Fusion inhibitors that disrupt six-helix bundle (6-HB) formation during viral entry represent a promising approach. Posaconazole, an antifungal agent, has been identified as a weak fusion inhibitor, but suffers from poor membrane permeability and modest activity. This study aimed to enhance its antiviral potency by conjugating it with cell-penetrating polyarginine peptides and to investigate the mechanism of action. Methods: A series of posaconazole-polyarginine conjugates were synthesized via click chemistry. Antiviral activity was evaluated using pseudotyped SARS-CoV-2 Omicron XDV in HEK293T cells. Mechanisms were investigated by circular dichroism, native PAGE, size-exclusion HPLC, molecular docking, and isothermal titration calorimetry. Metabolic stability was assessed using hepatic microsomes. Results: Posa-R8 exhibited potent antiviral activity comparable to the clinical candidate EK1, with minimal cytotoxicity. Mechanistic studies confirmed that Posa-R8 binds the HR2 region of the spike protein, disrupts 6-HB formation, and inhibits membrane fusion. It also showed strong lipid bilayer affinity and improved phase I metabolic stability over EK1. Conclusions: Polyarginine conjugation enhances the membrane-binding affinity and antiviral efficacy of posaconazole. Posa-R8 represents a promising lead for developing next-generation SARS-CoV-2 fusion inhibitors. Full article

This review examines the feasibility of electrochemical synthesis of poly-L-arginine (PArg) using repetitive cyclic voltammetry in neutral aqueous phosphate-buffered saline. Previous studies on electrochemical deposition of PArg onto different carbonaceous electrode materials are discussed with respect to the already reported mechanistic models. Some controversial interpretations are of interest, predominantly the formation of peptide bonds during the electropolymerisation of L-arginine. Several alternative anodic pathways are considered via the possibilities and limitations of ways of attaching L-arginine molecules to the electrode surface. Furthermore, the role of oxygen-containing surface groups is discussed, as this aspect has been largely overlooked in the context of L-arginine deposition, despite the O-terminating character of the electrode surface and its effect on the reactivity of the nucleophilic guanidine group in L-arginine. Also, the application of extremely high potentials around +2 V vs. Ag/AgCl/3 mol L⁻¹ KCl is considered, as it can lead to the generation of reactive oxygen species that may interfere with or even govern the entire deposition process. Thus, the absence of such considerations may raise doubts about the peptide nature of the electrochemically assisted polymerisation of this basic amino acid. Finally, it seems that the identity of the electrochemically synthesised PArg does not correspond to that of this polymer prepared by conventional methods, such as solid-phase peptide synthesis, solution-phase synthesis, or N-carboxy-anhydride polymerisation, and therefore the whole process remains unproved. Full article

Legumain (LGMN), a lysosomal cysteine protease, is crucial for tumor progression, invasion, and metastasis, making it a promising target for cancer imaging and therapy. This study developed novel ⁶⁷Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-conjugated LGMN-cleavable peptide probes ([⁶⁷Ga]Ga-NOTA-LCPs) composed of polyarginine and polyglutamic acid sequences linked by LGMN-cleavable sites for nuclear medicine imaging of LGMN activity. The probes were synthesized via fluorenylmethoxycarbonyl solid-phase peptide synthesis and radiolabeled in high radiochemical yields. In vitro assays with HCT116 cells showed significantly higher uptake of [⁶⁷Ga]Ga-NOTA-LCPs compared to non-cleavable controls, confirming efficient cleavage and cellular uptake. In vivo studies in tumor-bearing mice revealed rapid renal clearance, low non-specific binding, and favorable tumor-to-blood ratios, particularly for [⁶⁷Ga]Ga-NOTA-LCP-1. These results demonstrate the potential of [⁶⁷Ga]Ga-NOTA-LCPs as effective LGMN-responsive imaging agents, with further optimization needed to improve tumor specificity and reduce off-target accumulation. Full article

Background/Objectives: The role of α-synuclein (α-syn) pathology in Parkinson’s disease (PD) is well established; however, effective therapies remain elusive. Two mechanisms central to PD neurodegeneration are the intracellular aggregation of misfolded α-syn and the uptake of α-syn aggregates into neurons. Cationic arginine-rich peptides (CARPs) are an emerging class of molecule with multiple neuroprotective mechanisms of action, including protein stabilisation. This study characterised both intracellular α-syn aggregation and α-syn uptake in cortical neurons in vitro. Thereafter, this study examined the therapeutic potential of the neuroprotective CARP, R18D (18-mer of D-arginine), to prevent the aforementioned PD pathogenic processes through a cell-free thioflavin-T (ThT) assay and in cortical neurons. Methods: To induce intracellular α-syn aggregation, rat primary cortical neurons were exposed to α-syn seed (0.14 μM) for 2 h to allow uptake of the protein, followed by R18D treatment (0.0625, 0.125, 0.25, 0.5 μM), and a subsequent measurement of α-syn aggregates 48 h later using a homogenous time-resolved fluorescence (HTRF) assay. To assess neuronal uptake, α-syn seeds were covalently labelled with an Alexa-Fluor 488 fluorescent tag, pre-incubated with R18D (0.125, 0.25, 0.5 μM), and then exposed to cortical neurons for 24 h and assessed via confocal microscopy. Results: It was demonstrated that R18D significantly reduced both intracellular α-syn aggregation and α-syn seed uptake in neurons by 37.8% and 77.7%, respectively. Also, R18D reduced the aggregation of α-syn monomers in the cell-free assay. Conclusions: These findings highlight the therapeutic potential of R18D to inhibit key α-syn pathological processes and PD progression. Full article

Background/Objectives: Peptide-based treatments represent an expanding area and require innovative approaches to enhance bioavailability. Combination with cell-penetrating peptides (CPPs) is an attractive strategy to improve non-invasive delivery across nasal epithelial barriers for systemic and direct nose-to-brain transport. We previously developed a modified CPP system termed Glycosaminoglycan-binding Enhanced Transduction (GET) that improves insulin delivery across gastrointestinal epithelium. It contains a membrane docking sequence to promote cellular interactions (P21), a cationic polyarginine domain to stimulate uptake (8R) and an endosomal escaping sequence to maximize availability for onward distribution (LK15). It is synthesized as a single 44-residue peptide (P21-LK15-8R; PLR). Methods: The current research used in vitro assays for a novel exploration of PLR’s ability to improve the transport of two contrasting peptides, insulin (51 residues, net negative charge) and oxytocin (9 residues, weak positive charge) across an RPMI 2650 human nasal epithelial cell barrier cultured at the air–liquid interface. Results: PLR enhanced insulin transcytosis over a 6 h period by 7.8-fold when used at a 2:1 molar ratio of insulin/PLR (p < 0.0001 versus insulin alone). Enhanced oxytocin transcytosis (5-fold) occurred with a 1:10 ratio of oytocin/PLR (p < 0.01). Importantly, these were independent of any impact on transepithelial electrical resistance (TEER) or cell viability (p > 0.05). Conclusions: We advocate the continued evaluation of insulin–PLR and oxytocin–PLR formulations, including longer-term assessments of ciliotoxicity and cytotoxicity in vitro followed by in vivo assessments of systemic and nose-to-brain delivery. Full article

Virus-like particles (VLPs) offer an attractive possibility for the development of vaccines. Recombinant core antigen (HBc) of Hepatitis B virus (HBV) was expressed in different systems, and the E. coli expression system was shown to be effective for the production of HBc VLPs. Here, we used HBc of the HBV genotype G (HBc/G) as a technologically promising VLP carrier for the presentation of spike RBM and nucleocapsid protein-derived peptides of the SARS-CoV-2 Delta variant for subsequent immunological evaluations of obtained fusion proteins. The major immunodominant region (MIR) of the HBc/G protein was modified through the insertion of a receptor binding motif (RBM) from the S protein or B-cell epitope-containing peptide from the N protein. The C-terminus of the two truncated HBc/G proteins was used for the insertion of a group of five cytotoxic T lymphocyte (CTL) epitopes from the N protein. After expression in E. coli, the MIR-derived proteins were found to be insoluble and were recovered through step-wise solubilization with urea, followed by refolding. Despite the lack of correct VLPs, the chimeric proteins induced high levels of antibodies in BALB/c mice. These antibodies specifically recognized either eukaryotically expressed hRBD or bacterially expressed N protein (2–220) of SARS-CoV-2. CTL-epitope-containing proteins were purified as VLPs. The production of cytokines was analyzed through flow cytometry after stimulation of T-cells with target CTL peptides. Only a protein with a deleted polyarginine (PA) domain was able to induce the specific activation of T-cells. At the same time, the T-cell response against the carrier HBc/G protein was detected for both proteins. The neutralization of SARS-CoV-2 pseudotyped murine retrovirus with anti-HBc/G-RBM sera was found to be low. Full article

Cell-penetrating peptides (CPPs) are short peptide sequences that have the ability to cross the cell membrane and deliver cargo. Although it is critical that CPPs accomplish this task with minimal off-target effects, such actions have in many cases not been robustly screened. We presently investigated whether the commonly used CPPs TAT and the polyarginines Arg₉ and Arg₁₁ exert off-target effects on cellular Ca²⁺ homeostasis. In experiments employing myocytes and homogenates from the cardiac left ventricle or soleus muscle, we observed marked inhibition of Ca²⁺ recycling into the sarcoplasmic reticulum (SR) following incubation with polyarginine CPPs. In both tissues, the rate of SR Ca²⁺ leak remained unchanged, indicating that protracted Ca²⁺ removal from the cytosol stemmed from inhibition of the SR Ca²⁺ ATPase 2 (SERCA2). No such inhibition occurred following treatment with TAT, or in preparations from the SERCA1-expressing extensor digitorum longus muscle. Experiments in HEK cells overexpressing individual SERCA isoforms confirmed that polyarginine incubation specifically inhibited the activity of SERCA2a and 2b, but not SERCA1 or 3. The attenuation of SERCA2 activity was not dependent on the presence of phospholamban, and ELISA-based analyses rather revealed direct interaction between the polyarginines and the actuator domain of the protein. Surface plasmon resonance experiments confirmed strong binding within this region of SERCA2, and slow dissociation between the two species. Based on these observations, we urge caution when employing polyarginine CPPs. Indeed, as SERCA2 is expressed in diverse cell types, the wide-ranging consequences of SERCA2 binding and inhibition should be anticipated in both experimental and therapeutic settings. Full article

In Parkinson’s disease (PD), gut inflammation is hypothesised to contribute to α-synuclein aggregation, but gastrointestinal α-synuclein expression is poorly characterised. Cationic arginine-rich peptides (CARPs) are an emerging therapeutic option that exerts various neuroprotective effects and may target the transmission of protein aggregates. This study aimed to investigate endogenous α-synuclein expression in enteroendocrine STC-1 cells and the potential of the CARP, R18D (18-mer of D-arginine), to prevent internalisation of pre-formed α-synuclein fibrils (PFFs) in enteroendocrine cells in vitro. Through confocal microscopy, the immunoreactivity of full-length α-synuclein and the serine-129 phosphorylated form (pS129) was investigated in STC-1 (mouse enteroendocrine) cells. Thereafter, STC-1 cells were exposed to PFFs tagged with Alexa-Fluor 488 (PFF-488) for 2 and 24 h and R18D-FITC for 10 min. After confirming the uptake of both PFFs and R18D-FITC through fluorescent microscopy, STC-1 cells were pre-treated with R18D (5 or 10 μM) for 10 min prior to 2 h of PFF-488 exposure. Immunoreactivity for endogenous α-synuclein and pS129 was evident in STC-1 cells, with prominent pS129 staining along cytoplasmic processes and in perinuclear areas. STC-1 cells internalised PFFs, confirmed through co-localisation of PFF-488 and human-specific α-synuclein immunoreactivity. R18D-FITC entered STC-1 cells within 10 min and pre-treatment of STC-1 cells with R18D interfered with PFF uptake. The endogenous presence of α-synuclein in enteroendocrine cells, coupled with their rapid uptake of PFFs, demonstrates a potential for pathogenic spread of α-synuclein aggregates in the gut. R18D is a novel therapeutic approach to reduce the intercellular transmission of α-synuclein pathology. Full article

The outer membrane of Gram-negative bacteria contains a variety of pore-forming structures collectively referred to as porins. Some of these are voltage dependent, but weakly so, closing at high voltages. Triplin, a novel bacterial pore-former, is a three-pore structure, highly voltage dependent, with a complex gating process. The three pores close sequentially: pore 1 at positive potentials, 2 at negative and 3 at positive. A positive domain containing 14 positive charges (the voltage sensor) translocates through the membrane during the closing process, and the translocation is proposed to take place by the domain entering the pore and thus blocking it, resulting in the closed conformation. This mechanism of pore closure is supported by kinetic measurements that show that in the closing process the voltage sensor travels through most of the transmembrane voltage before reaching the energy barrier. Voltage-dependent blockage of the pores by polyarginine, but not by a 500-fold higher concentrations of polylysine, is consistent with the model of pore closure, with the sensor consisting mainly of arginine residues, and with the presence, in each pore, of a complementary surface that serves as a binding site for the sensor. Full article

Intranasal corticosteroids are effective medications against allergic rhinitis (AR). However, mucociliary clearance promptly eliminates these drugs from the nasal cavity and delays their onset of action. Therefore, a faster, longer-lasting therapeutic effect on the nasal mucosa is required to enhance the efficacy of AR management. Our previous study showed that polyarginine, a cell-penetrating peptide, can deliver cargo to nasal cells; moreover, polyarginine-mediated cell-nonspecific protein transduction into the nasal epithelium exhibited high transfection efficiency with minimal cytotoxicity. In this study, poly-arginine-fused forkhead box P3 (FOXP3) protein, the “master transcriptional regulator” of regulatory T cells (Tregs), was administered into the bilateral nasal cavities of the ovalbumin (OVA)-immunoglobulin E mouse model of AR. The effects of these proteins on AR following OVA administration were investigated using histopathological, nasal symptom, flow cytometry, and cytokine dot blot analyses. Polyarginine-mediated FOXP3 protein transduction induced Treg-like cell generation in the nasal epithelium and allergen tolerance. Overall, this study proposes FOXP3 activation-mediated Treg induction as a novel and potential therapeutic strategy for AR, providing a potential alternative to conventional intranasal drug application for nasal drug delivery. Full article

Safe and effective T cell vaccines are needed for the treatment or prevention of cancers as well as infectious agents where vaccines for neutralizing antibodies have performed poorly. Recent research highlights an important role for tissue-resident memory T cells (T_RM cells) in protective immunity and the role of a subset of dendritic cells that are capable of cross-priming for the induction of T_RM cells. However, efficient vaccine technologies that operate through cross-priming and induce robust CD8+ T cell responses are lacking. We developed a platform technology by genetically engineering the bovine papillomavirus L1 major capsid protein to insert a polyglutamic acid/cysteine motif in place of wild-type amino acids in the HI loop. Virus-like particles (VLPs) are formed by self-assembly in insect cells infected with a recombinant baculovirus. Polyarginine/cysteine-tagged antigens are linked to the VLP by a reversible disulfide bond. The VLP possesses self-adjuvanting properties due to the immunostimulatory activity of papillomavirus VLPs. Polyionic VLP vaccines induce robust CD8+ T cell responses in peripheral blood and tumor tissues. A prostate cancer polyionic VLP vaccine was more efficacious than other vaccines and immunotherapies for the treatment of prostate cancer in a physiologically relevant murine model and successfully treated more advanced diseases than the less efficacious technologies. The immunogenicity of polyionic VLP vaccines is dependent on particle size, reversible linkage of the antigen to the VLP, and an interferon type 1 and Toll-like receptor (TLR)3/7-dependent mechanism. Full article

In recent years, the coupling of structurally and functionally controllable polymers with biologically active peptide materials to obtain polymer-peptide hybrids with excellent properties and biocompatibility has led to important research progress in the field of polymers. In this study, a pH-responsive hyperbranched polymer hPDPA was prepared by combining atom transfer radical polymerization (ATRP) with self-condensation vinyl polymerization (SCVP) using a three-component reaction of Passerini to obtain a monomeric initiator ABMA containing functional groups. The pH-responsive polymer peptide hybrids hPDPA/PArg/HA were obtained by using the molecular recognition of polyarginine (β-CD-PArg) peptide modified with β-cyclodextrin (β-CD) on the hyperbranched polymer, followed by the electrostatic adsorption of hyaluronic acid (HA). The two hybrid materials, h₁PDPA/PArg₁₂/HA and h₂PDPA/PArg₈/HA could self-assemble to form vesicles with narrow dispersion and nanoscale dimensions in phosphate-buffered (PB) at pH = 7.4. The assemblies exhibited low toxicity as drug carriers of β-lapachone (β-lapa), and the synergistic therapy based on ROS and NO generated by β-lapa had significant inhibitory effects on cancer cells. Full article

We report the advantages of glassy carbon electrodes (GCE) modified with multi-walled carbon nanotubes (MWCNTs) non-covalently functionalized with polyarginine (PolyArg) for the adsorption and electrooxidation of different DNAs and the analytical applications of the resulting platform. The presence of the carbon nanostructures, and mainly the charge of the PolyArg that supports them, facilitates the adsorption of calf-thymus and salmon sperm double-stranded DNAs and produces an important decrease in the overvoltages for the oxidation of guanine and adenine residues and a significant enhancement in the associated currents. As a proof-of-concept of possible GCE/MWCNTs-PolyArg biosensing applications, we develop an impedimetric genosensor for the quantification of microRNA-21 at femtomolar levels, using GCE/MWCNTs-PolyArg as a platform for immobilizing the DNA probe, with a detection limit of 3fM, a sensitivity of 1.544 × 10³ Ω M⁻¹, and a successful application in enriched biological fluids. Full article

Purpose: Prolyl 3-hydroxylase family member 4 (P3H4) is a potent prognostic oncogene in bladder cancer (BC), and the inhibition of P3H4 suppresses BC tumor growth. This study aimed to evaluate the efficiency of P3H4 inhibition for BC tumor therapy via tumor-targeting nanoparticles. Methods and results: A linear polyarginine peptide (R9) was synthesized, azide-modified, and then assembled with cyclic pentapeptide cRGDfK. Chlorin e6 (ce6)-conjugated CH3-R9-RGD nanoparticles were prepared for the delivery of siP3H4 into T24 cells in vitro and BC tumors in vivo. Dynamic light scattering analysis identified that the optimum CH3-R9-RGD@siP3H4 molar ratio was 30/1. CH3-R9-RGD@ce6/siP3H4 nanocomposites decreased P3H4 expression and cell proliferation and promoted reactive oxygen species production, apoptosis, and calreticulin exposure in T24 cells in vitro. In vivo experiments showed that CH3-R9-RGD@ce6/siP3H4 nanocomposites caused pathological changes, suppressed BC tumor growth, promoted caspase 3 expression, and enhanced calreticulin exposure in tumor cells. Conclusions: The tumor-targeting CH3-R9-RGD nanocomposites encapsulating siP3H4 and ce6 might be an alternative therapeutic strategy or intravesical instillation chemotherapy for BC. Full article

Peptide-based subunit vaccines include only minimal antigenic determinants, and, therefore, are less likely to induce allergic immune responses and adverse effects compared to traditional vaccines. However, peptides are weakly immunogenic and susceptible to enzymatic degradation when administered on their own. Hence, we designed polyelectrolyte complex (PEC)-based delivery systems to protect peptide antigens from degradation and improve immunogenicity. Lipopeptide (LCP-1) bearing J8 B-cell epitope derived from Group A Streptococcus (GAS) M-protein was selected as the model peptide antigen. In the pilot study, LCP-1 incorporated in alginate/cross-linked polyarginine-J8-based PEC induced high J8-specific IgG antibody titres. The PEC system was then further modified to improve its immune stimulating capability. Of the formulations tested, PEC-4, bearing LCP-1, alginate and cross-linked polylysine, induced the highest antibody titres in BALB/c mice following subcutaneous immunisation. The antibodies produced were more opsonic than those induced by mice immunised with other PECs, and as opsonic as those induced by antigen adjuvanted with powerful complete Freund’s adjuvant. Full article