Conjugation Of Cell-penetrating Peptides Research Articles

Cell-Penetrating and Enzyme-Responsive Peptides for Targeted Cancer Therapy: Role of Arginine Residue Length on Cell Penetration and In Vivo Systemic Toxicity.

For the improved delivery of cancer therapeutics and imaging agents, the conjugation of cell-penetrating peptides (CPPs) increases the cellular uptake and water solubility of agents. Among the various CPPs, arginine-rich peptides have been the most widely used. Combining CPPs with enzyme-responsive peptides presents an innovative strategy to target specific intracellular enzymes in cancer cells and when combined with the appropriate click chemistry can enhance theranostic drug delivery through the formation of intracellular self-assembled nanostructures. However, one drawback of CPPs is their high positive charge which can cause nonspecific binding, leading to off-target accumulation and potential toxicity. Hence, balancing cell-specific penetration, toxicity, and biocompatibility is essential for future clinical efficacy. We synthesized six cancer-specific, legumain-responsive RnAANCK peptides containing one to six arginine residues, with legumain being an asparaginyl endopeptidase that is overexpressed in aggressive prostate tumors. When conjugated to Alexa Fluor 488, R1-R6AANCK peptides exhibited a concentration- and time-dependent cell penetration in prostate cancer cells, which was higher for peptides with higher R values, reaching a plateau after approximately 120 min. Highly aggressive DU145 prostate tumor cells, but not less aggressive LNCaP cells, self-assembled nanoparticles in the cytosol after the cleavage of the legumain-specific peptide. The in vivo biocompatibility was assessed in mice after the intravenous injection of R1-R6AANCK peptides, with concentrations ranging from 0.0125 to 0.4 mmol/kg. The higher arginine content in R4-6 peptides showed blood and urine indicators for the impairment of bone marrow, liver, and kidney function in a dose-dependent manner, with instant hemolysis and morbidity in extreme cases. These findings underscore the importance of designing peptides with the optimal arginine residue length for a proper balance of cell-specific penetration, toxicity, and in vivo biocompatibility.

Enhancing the Antimicrobial Properties of Peptides through Cell-Penetrating Peptide Conjugation: A Comprehensive Assessment

Combining antimicrobial peptides (AMPs) with cell-penetrating peptides (CPPs) has shown promise in boosting antimicrobial potency, especially against Gram-negative bacteria. We examined the CPP-AMP interaction with distinct bacterial types based on cell wall differences. Our investigation focused on AMPs incorporating penetratin CPP and dihybrid peptides containing both cell-penetrating TAT protein fragments from the human immunodeficiency virus and Antennapedia peptide (Antp). Assessment of the peptides TAT-AMP, AMP-Antp, and TAT-AMP-Antp revealed their potential against Gram-positive strains (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), and Bacillus cereus). Peptides TAT-AMP and AMP-Antp using an amyloidogenic AMP from S1 ribosomal protein Thermus thermophilus, at concentrations ranging from 3 to 12 μM, exhibited enhanced antimicrobial activity against B. cereus. TAT-AMP and TAT-AMP-Antp, using an amyloidogenic AMP from the S1 ribosomal protein Pseudomonas aeruginosa, at a concentration of 12 µM, demonstrated potent antimicrobial activity against S. aureus and MRSA. Notably, the TAT-AMP, at a concentration of 12 µM, effectively inhibited Escherichia coli (E. coli) growth and displayed antimicrobial effects similar to gentamicin after 15 h of incubation. Peptide characteristics determined antimicrobial activity against diverse strains. The study highlights the intricate relationship between peptide properties and antimicrobial potential. Mechanisms of AMP action are closely tied to bacterial cell wall attributes. Peptides with the TAT fragment exhibited enhanced antimicrobial activity against S. aureus, MRSA, and P. aeruginosa. Peptides containing only the Antp fragment displayed lower activity. None of the investigated peptides demonstrated cytotoxic or cytostatic effects on either BT-474 cells or human skin fibroblasts. In conclusion, CPP-AMPs offer promise against various bacterial strains, offering insights for targeted antimicrobial development.

Intracellular bacterial eradication using a novel peptide in vitro.

To determine the effect of a novel antimicrobial peptide (AMP; OP145) and cell-penetrating peptide (Octa-arginine/R8) conjugate on the killing of intracellular Enterococcus faecalis, compared to OP145 and an antibiotic combination recommended for regenerative endodontic procedures. The biocompatible concentrations of OP145 and OP145-R8 were determined by assessing their cytotoxicity against human macrophages and red blood cells. Spatiotemporal internalization of the peptides into macrophages was investigated qualitatively and quantitatively by confocal laser scanning microscopy and flow cytometry respectively. Killing of extracellular and intracellular E. faecalis OG1RF by the peptides was determined by counting the colony-forming units (CFU). Intracellular antibacterial activity of the peptides was compared to a double antibiotic combination. Confocal microscopy was used to confirm the intracellular bacterial eradication. Significant differences between the different test groups were analysed using one-way analysis of variance. p < .05 was considered to be statistically significant. Peptides at a concentration of 7.5 μmol/L were chosen for subsequent experiments based on the results of the alamarBlue™ cell viability assay and haemolytic assay. OP145-R8 selectively internalized into lysosomal compartments and the cytosol of macrophages. Conjugation with R8 improved the internalization of OP145 into macrophages in a temporal manner (70.53% at 1 h to 77.13% at 2 h), while no temporal increase was observed for OP145 alone (60.53% at 1 h with no increase at 2 h). OP145-R8 demonstrated significantly greater extracellular and intracellular antibacterial activity compared to OP145 at all investigated time-points and concentrations (p < .05). OP145-R8 at 7.5 μmol/L eradicated intracellular E. faecalis after 2 h (3.5 log reduction compared to the control; p < .05), while the antibiotics could not reduce more than 0.5 log CFU compared to the control (p > .05). Confocal microscopy showed complete absence of E. faecalis within the OP145-R8 treated macrophages. The results of this study demonstrated that the conjugation of an AMP OP145 to a cell-penetrating peptide R8 eradicated extracellular and intracellular E. faecalis OG1RF without toxic effects on the host cells.

Enhancing the Effectiveness of Oligonucleotide Therapeutics Using Cell-Penetrating Peptide Conjugation, Chemical Modification, and Carrier-Based Delivery Strategies.

Oligonucleotide-based therapies are a promising approach for treating a wide range of hard-to-treat diseases, particularly genetic and rare diseases. These therapies involve the use of short synthetic sequences of DNA or RNA that can modulate gene expression or inhibit proteins through various mechanisms. Despite the potential of these therapies, a significant barrier to their widespread use is the difficulty in ensuring their uptake by target cells/tissues. Strategies to overcome this challenge include cell-penetrating peptide conjugation, chemical modification, nanoparticle formulation, and the use of endogenous vesicles, spherical nucleic acids, and smart material-based delivery vehicles. This article provides an overview of these strategies and their potential for the efficient delivery of oligonucleotide drugs, as well as the safety and toxicity considerations, regulatory requirements, and challenges in translating these therapies from the laboratory to the clinic.

Strategies to improve the physicochemical properties of peptide-based drugs.

Peptides are a rapid-growing class of therapeutics with unique and desirable physicochemical properties. Due to disadvantages such as low membrane permeability and susceptibility to proteolytic degradation, peptide-based drugs have limited bioavailability, a short half-life, and rapid in vivo elimination. Various strategies can be applied to improve the physicochemical properties of peptide-based drugs to overcome limitations such as limited tissue residence time, metabolic instability, and low permeability. Applied strategies including backbone modifications, side chain modifications, conjugation with polymers, modification of peptide termini, fusion to albumin, conjugation with the Fc portion of antibodies, cyclization,stapled peptides, pseudopeptides, cell-penetrating peptide conjugates, conjugation with lipids, and encapsulation in nanocarriers are discussed.

Peptide-Platinum(IV) Conjugation Minimizes the Negative Impact of Current Anticancer Chemotherapy on Nonmalignant Cells.

The relative success of platinum (Pt)-based chemotherapy comes at the cost of severe adverse side effects and is associated with a high risk of pro-oncogenic activation in the tumor microenvironment. Here, we report the synthesis of C-POC, a novel Pt(IV) cell-penetrating peptide conjugate showing a reduced impact against nonmalignant cells. In vitro and in vivo evaluation using patient-derived tumor organoids and laser ablation inductively coupled plasma mass spectrometry indicates that C-POC maintains robust anticancer efficacy while displaying diminished accumulation in healthy organs and reduced adverse toxicity compared to the standard Pt-based therapy. Likewise, C-POC uptake is significantly lowered in the noncancerous cells populating the tumor microenvironment. This results in the downregulation of versican, a biomarker of metastatic spreading and chemoresistance that we found upregulated in patients treated with standard Pt-based therapy. Altogether, our findings underscore the importance of considering the off-target impact of anticancer treatment on normal cells to improve drug development and patient care.

Application of Cell Penetrating Peptides as a Promising Drug Carrier to Combat Viral Infections.

Novel effective drugs or therapeutic vaccines have been already developed to eradicate viral infections. Some non-viral carriers have been used for effective drug delivery to a target cell or tissue. Among them, cell penetrating peptides (CPPs) attracted a special interest to enhance drug delivery into the cells with low toxicity. They were also applied to transfer peptide/protein-based and nucleic acids-based therapeutic vaccines against viral infections. CPPs-conjugated drugs or vaccines were investigated in several viral infections including poliovirus, Ebola, coronavirus, herpes simplex virus, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, Japanese encephalitis virus, and influenza A virus. Some studies showed that the uptake of CPPs or CPPs-conjugated drugs can be performed through both non-endocytic and endocytic pathways. Despite high potential of CPPs for cargo delivery, there are some serious drawbacks such as non-tissue-specificity, instability, and suboptimal pharmacokinetics features that limit their clinical applications. At present, some solutions are utilized to improve the CPPs properties such as conjugation of CPPs with targeting moieties, the use of fusogenic lipids, generation of the proton sponge effect, etc. Up to now, no CPP or composition containing CPPs has been approved by the Food and Drug Administration (FDA) due to the lack of sufficient in vivo studies on stability, immunological assays, toxicity, and endosomal escape of CPPs. In this review, we briefly describe the properties, uptake mechanisms, advantages and disadvantages, and improvement of intracellular delivery, and bioavailability of cell penetrating peptides. Moreover, we focus on their application as an effective drug carrier to combat viral infections.

A Pt(IV)-conjugated brain penetrant macrocyclic peptide shows pre-clinical efficacy in glioblastoma.

Glioblastoma (GBM) is the most aggressive primary malignant brain tumor, with a median survival of approximately 15months. Treatment is limited by the blood-brain barrier (BBB) which restricts the passage of most drugs to the brain. We previously reported the design and synthesis of a BBB-penetrant macrocyclic cell-penetrating peptide conjugate (M13) covalently linked at the axial position of a Pt(IV) cisplatin prodrug. Here we show the Pt(IV)-M13 conjugate releases active cisplatin upon intracellular reduction and effects potent in vitro GBM cell killing. Pt(IV)-M13 significantly increased platinum uptake in an in vitro BBB spheroid model and intravenous administration of Pt(IV)-M13 in GBM tumor-bearing mice led to higher platinum levels in brain tissue and intratumorally compared with cisplatin. Pt(IV)-M13 administration was tolerated in naïve nude mice at higher dosage regimes than cisplatin and significantly extended survival above controls in a murine GBM xenograft model (median survival 33days for Pt(IV)-M13 vs 24days for Pt(IV) prodrug, 22.5days for cisplatin and 22days for control). Increased numbers of γH2AX nuclear foci, biomarkers of DNA damage, were observed in tumors of Pt(IV)-M13-treated mice, consistent with elevated platinum levels. The present work provides the first demonstration that systemic injection of a Pt(IV) complex conjugated to a brain-penetrant macrocyclic peptide can lead to increased platinum levels in the brain and extend survival in mouse GBM models, supporting further development of this approach and the utility of brain-penetrating macrocyclic peptide conjugates for delivering non-BBB penetrant drugs to the central nervous system.

Peptide-Conjugated PMOs for the Treatment of Myotonic Dystrophy.

Antisense oligonucleotides (ASOs) have shown great therapeutic potential in the treatment of many neuromuscular diseases including myotonic dystrophy 1 (DM1). However, systemically delivered ASOs display poor biodistribution and display limited penetration into skeletal muscle. The conjugation of cell-penetrating peptides (CPPs) to phosphorodiamidate morpholino oligonucleotides (PMOs), a class of ASOs with a modified backbone, can be used to enhance ASO skeletal muscle penetration. Peptide-PMOs (P-PMOs) have been shown to be highly effective in correcting the DM1 skeletal muscle phenotype in both murine and cellular models of DM1 and at a molecular and functional level. Here we describe the synthesis and conjugation of P-PMOs and methods for analyzing their biodistribution and toxicity in the HSA-LR DM1 mouse model and their efficacy both in vitro and in vivo using FISH and RT-PCR splicing analysis.

Cell penetrating peptide: A potent delivery system in vaccine development.

One of the main obstacles to most medication administrations (such as the vaccine constructs) is the cellular membrane's inadequate permeability, which reduces their efficiency. Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) are well-known as potent biological nanocarriers to overcome this natural barrier, and to deliver membrane-impermeable substances into cells. The physicochemical properties of CPPs, the attached cargo, concentration, and cell type substantially influence the internalization mechanism. Although the exact mechanism of cellular uptake and the following processing of CPPs are still uncertain; but however, they can facilitate intracellular transfer through both endocytic and non-endocytic pathways. Improved endosomal escape efficiency, selective cell targeting, and improved uptake, processing, and presentation of antigen by antigen-presenting cells (APCs) have been reported by CPPs. Different in vitro and in vivo investigations using CPP conjugates show their potential as therapeutic agents in various medical areas such as infectious and non-infectious disorders. Effective treatments for a variety of diseases may be provided by vaccines that can cooperatively stimulate T cell-mediated immunity (T helper cell activity or cytotoxic T cell function), and immunologic memory. Delivery of antigen epitopes to APCs, and generation of a potent immune response is essential for an efficacious vaccine that can be facilitated by CPPs. The current review describes the delivery of numerous vaccine components by various CPPs and their immunostimulatory properties.

Unleashing cell-penetrating peptide applications for immunotherapy

With the advent of cancer immunotherapy, immunomodulation has emerged as an important strategy for the treatment of various diseases. We review recent advances in clinical trials of cell-penetrating peptide (CPP) applications for immunotherapy and also discuss their challenges and opportunities for preclinical studies in various immune diseases. CPP conjugation to antigenic peptides or proteins can enable efficient antigen uptake and cross-presentation by antigen-presenting cells (APCs), which induce both humoral and cytotoxic responses. In addition, CPP-coupled immune modulators can enhance antitumor immunity or anti-inflammatory effects to regulate allergies and autoimmunity. Given their huge advantages in overcoming delivery barriers, CPP-based strategies for immunomodulation could extend drug optimization and advance immunotherapy in various human diseases.

Scaffold size-dependent effect on the enhanced uptake of antibiotics and other compounds by Escherichia coli and Pseudomonas aeruginosa

The outer membrane of Gram-negative bacteria functions as an impermeable barrier to foreign compounds. Thus, modulating membrane transport can contribute to improving susceptibility to antibiotics and efficiency of bioproduction reactions. In this study, the cellular uptake of hydrophobic and large-scaffold antibiotics and other compounds in Gram-negative bacteria was investigated by modulating the homolog expression of bamB encoding an outer membrane lipoprotein and tolC encoding an outer membrane efflux protein via gene deletion and gene silencing. The potential of deletion mutants for biotechnological applications, such as drug screening and bioproduction, was also demonstrated. Instead of being subjected to gene deletion, wild-type bacterial cells were treated with cell-penetrating peptide conjugates of a peptide nucleic acid (CPP-PNA) against bamB and tolC homologs as antisense agents. Results revealed that the single deletion of bamB and tolC in Escherichia coli increased the uptake of large- and small-scaffold hydrophobic compounds, respectively. A bamB-and-tolC double deletion mutant had a higher uptake efficiency for certain antibiotics and other compounds with high hydrophobicity than each single deletion mutant. The CPP-PNA treated E. coli and Pseudomonas aeruginosa cells showed high sensitivity to various antibiotics. Therefore, these gene deletion and silencing approaches can be utilized in therapeutic and biotechnological fields.

Enhanced nitric oxide production by macrophages treated with a cell-penetrating peptide conjugate

The SPRY domain-containing SOCS box protein-2 (SPSB2) plays a critical role in the degradation of inducible nitric oxide synthase (iNOS) in macrophages. In this study, we have conjugated a peptide inhibitor of the iNOS-SPSB2 interaction with a cell-penetrating peptide (CPP) for delivery into macrophages, and confirmed its binding to SPSB2. We have assessed the uptake of a fluorophore-tagged analogue by RAW 264.7 and immortalised bone marrow derived macrophage (iBMDM) cell lines, and shown that the CPP-peptide conjugate enhanced NO production. The findings of this study will be useful in further refinement of CPP-peptide conjugates as leads in the development of new antibiotics that target the host innate immune response.

Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides

Membrane fusion triggered by Ca2+ is orchestrated by a conserved set of proteins to mediate synaptic neurotransmitter release, mucin secretion and other regulated exocytic processes1–4. For neurotransmitter release, the Ca2+ sensitivity is introduced by interactions between the Ca2+ sensor synaptotagmin and the SNARE complex5, and sequence conservation and functional studies suggest that this mechanism is also conserved for mucin secretion6. Disruption of Ca2+-triggered membrane fusion by a pharmacological agent would have therapeutic value for mucus hypersecretion as it is the major cause of airway obstruction in the pathophysiology of respiratory viral infection, asthma, chronic obstructive pulmonary disease and cystic fibrosis7–11. Here we designed a hydrocarbon-stapled peptide that specifically disrupts Ca2+-triggered membrane fusion by interfering with the so-called primary interface between the neuronal SNARE complex and the Ca2+-binding C2B domain of synaptotagmin-1. In reconstituted systems with these neuronal synaptic proteins or with their airway homologues syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, along with Munc13-2 and Munc18-2, the stapled peptide strongly suppressed Ca2+-triggered fusion at physiological Ca2+ concentrations. Conjugation of cell-penetrating peptides to the stapled peptide resulted in efficient delivery into cultured human airway epithelial cells and mouse airway epithelium, where it markedly and specifically reduced stimulated mucin secretion in both systems, and substantially attenuated mucus occlusion of mouse airways. Taken together, peptides that disrupt Ca2+-triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways.

Anticancer and Antimicrobial Evaluations on Alternative Reading Frame (ARF) Peptides and their Derivatives.

Alternative reading frame (ARF) protein up-regulates the intracellular level of a tumour suppressor protein, p53, by blocking MDM2 mediated p53 ubiquitination. The two homologous forms of ARF proteins are p19ARF in mice and p14ARF in humans. In our study, p19ARF-derived peptide ARF (26-44) and its cell-penetrating peptide conjugate Tat-ARF (26-44), p14ARF-derived peptide ARF (1-22), and its NrLS conjugate ARF (1-22)-NrLS were designed, and their anticancer properties were investigated. Our objective is to study the anticancer and antimicrobial properties of ARF-derived peptides and their cell-penetrating and NrLS conjugates. Peptides synthesized using solid-phase peptide synthesis (SPPS) were purified using RPHPLC and characterized using Bruker MALDI-TOF mass spectrometry. Cytotoxicity was evaluated on HeLa and BE(2)-C cells by cell viability IC50 determination. Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. Morphological studies were carried out using SEM and TEM techniques, live/dead staining, ROS and Hoest staining. Peptides Tat-ARF (1-22) and ARF (1-22)-NrLS exhibited potent cytotoxic effects, comparable to the known standard cisplatin. Cellular morphological studies showed signs of apoptosis which were confirmed by reactive oxygen species (ROS) generation and Hoechst nuclear staining. ARF peptides showed potent antimicrobial activities at low micromolar concentrations without haemolysis. Tat modification improved the activity of ARF (26-44) by 9 folds against HeLa and 5 folds against BE(2)-C cells. NrLS modification of ARF (1-22) imparted 12 fold potency against HeLa and 2-fold potency against BE(2)-C cells. This study helps to further understand the effect of these peptides on MDM2 proteins and their role in the apoptosis signalling pathway.

Cell-penetrating peptides in protein mimicry and cancer therapeutics.

Extensive research has been undertaken in the pursuit of anticancer therapeutics. Many anticancer drugs require specificity of delivery to cancer cells, whilst sparing healthy tissue. Cell-penetrating peptides (CPPs), now well established as facilitators of intracellular delivery, have in recent years advanced to incorporate target specificity and thus possess great potential for the targeted delivery of anticancer cargoes. Though none have yet been approved for clinical use, this novel technology has already entered clinical trials. In this review we present CPPs, discuss their classification, mechanisms of cargo internalization and highlight strategies for conjugation to anticancer moieties including their incorporation into therapeutic proteins. As the mainstay of this review, strategies to build specificity into tumor targeting CPP constructs through exploitation of the tumor microenvironment and the use of tumor homing peptides are discussed, whilst acknowledging the extensive contribution made by CPP constructs to target specific protein-protein interactions integral to intracellular signaling pathways associated with tumor cell survival and progression. Finally, antibody/antigen CPP conjugates and their potential roles in cancer immunotherapy and diagnostics are considered. In summary, this review aims to harness the potential of CPP-aided drug delivery for future cancer therapies and diagnostics whilst highlighting some of the most recent achievements in selective delivery of anticancer drugs, including cytostatic drugs, to a range of tumor cells both in vitro and in vivo.

SynB3 Conjugated QBP1 Passes Blood-Brain Barrier Models and Inhibits PolyQ Protein Aggregation.

Polyglutamine diseases are degenerative diseases in the central nervous system caused by CAG trinucleotide repeat expansion which encodes polyglutamine tracts, leading to the misfolding of pathological proteins. Small peptides can be designed to prevent polyglutamine diseases by inhibiting the polyglutamine protein aggregation, for example, polyglutamine binding peptide 1(QBP1). However, the transportation capability of polyglutamine binding peptide 1 across the blood-brain barrier is less efficient. We hypothesized whether its therapeutic effect could be improved by increasing the rate of membrane penetration. The objective of the study was to explore whether polyglutamine binding peptide 1 conjugated cell-penetrating peptides could pass through the blood-brain barrier and inhibit the aggregation of polyglutamine proteins. In order to investigate the toxic effects, we constructed a novel stable inducible PC12 cells to express Huntington protein that either has 11 glutamine repeats or 63 glutamine repeats to mimic wild type and polyglutamine expand Huntington protein, respectively. Both SynB3 and TAT conjugated polyglutamine binding peptide 1 was synthesized, respectively. We tested their capabilities to pass through a Trans-well system and subsequently studied the counteractive effects on polyglutamine protein aggregation. The conjugation of cell-penetrating peptides to SynB3 and TAT enhanced the transportation of polyglutamine binding peptide 1 across the mono-cell layer and ameliorated polyglutamine-- expanded Huntington protein aggregation; moreover, SynB3 showed better delivery efficiency than TAT. Interestingly, it has been observed that polyglutamine binding peptide 1 specifically inhibited polyglutamine-expanded protein aggregation rather than affected other amyloidosis proteins, for example, β-Amyloid. Our study indicated that SynB3 could be an effective carrier for polyglutamine binding peptide 1 distribution through the blood-brain barrier model and ameliorate the formation of polyglutamine inclusions; thus SynB3 conjugated polyglutamine binding peptide 1 could be considered as a therapeutic candidate for polyglutamine diseases.

The Effect of Conjugation with Octaarginine, a Cell-Penetrating Peptide on Antifungal Activity of Imidazoacridinone Derivative.

Acridine cell-penetrating peptide conjugates are an extremely important family of compounds in antitumor chemotherapy. These conjugates are not so widely analysed in antimicrobial therapy, although bioactive peptides could be used as nanocarriers to smuggle antimicrobial compounds. An octaarginine conjugate of an imidazoacridinone derivative (Compound 1-R8) synthetized by us exhibited high antifungal activity against reference and fluconazole-resistant clinical strains (MICs ≤ 4 μg mL−1). Our results clearly demonstrate the qualitative difference in accumulation of the mother compound and Compound 1-R8 conjugate into fungal cells. Only the latter was transported and accumulated effectively. Microscopic and flow cytometry analysis provide some evidence that the killing activity of Compound 1-R8 may be associated with a change in the permeability of the fungal cell membrane. The conjugate exhibited low cytotoxicity against human embryonic kidney (HEK-293) and human liver (HEPG2) cancer cell lines. Nevertheless, the selectivity index value of the conjugate for human pathogenic strains remained favourable and no hemolytic activity was observed. The inhibitory effect of the analysed compound on yeast topoisomerase II activity suggested its molecular target. In summary, conjugation with R8 effectively increased imidazoacridinone derivative ability to enter the fungal cell and achieve a concentration inside the cell that resulted in a high antifungal effect.

Nanoparticular Inhibitors of Flavivirus Proteases from Zika, West Nile and Dengue Virus Are Cell-Permeable Antivirals.

Viral proteases have been established as drug targets in several viral diseases including human immunodeficiency virus and hepatitis C virus infections due to the essential role of these enzymes in virus replication. In contrast, no antiviral therapy is available to date against flaviviral infections including those by Zika virus (ZIKV), West Nile virus (WNV), or dengue virus (DENV). Numerous potent inhibitors of flaviviral proteases have been reported; however, a huge gap remains between the in vitro and intracellular activities, possibly due to low cellular uptake of the charged compounds. Here, we present an alternative, nanoparticular approach to antivirals. Conjugation of peptidomimetic inhibitors and cell-penetrating peptides to dextran yielded chemically defined nanoparticles that were potent inhibitors of flaviviral proteases. Peptide-dextran conjugates inhibited viral replication and infection in cells at nontoxic, low micromolar or even nanomolar concentrations. Thus, nanoparticular antivirals might be alternative starting points for the development of broad-spectrum antiflaviviral drugs.

Evaluating Efficacy of Peptide-Delivered Oligonucleotides Using the Severe Taiwanese SMA Mouse Model.

Oligonucleotides (ONs) are therapeutic macromolecules with great potential for the treatment of neurological conditions, including spinal muscular atrophy (SMA), a neurodegenerative disease. However, the neurovascular unit severely limits their distribution to the neural parenchyma of the brain and the spinal cord. Cell-penetrating peptides (CPPs) can be conjugated to oligonucleotides to increase their delivery across biological barriers. In this chapter, we describe the synthesis and conjugation of CPPs to oligonucleotides, and the use of a severe SMA mouse model to test in vivo the efficacy of CPP-delivered oligonucleotides, using ELISA, western blot, and TaqMan™ RT-qPCR assays.