Tumor-activated prodrug (TAP)-conjugated nanoparticles with cleavable domains for safe doxorubicin delivery.

A major issue in chemotherapy is the lack of specificity of many antitumor drugs, which cause severe side effects and an impaired therapeutic response. Here we report on the design and characterization of model tumor activated prodrug-conjugated polystyrene (PS) nanoparticles (TAP-NPs) for the release of doxorubicin (Dox) triggered by matrix metalloprotease-2 (MMP2) enzyme, which is overexpressed in the extracellular matrix of tumors. In particular, TAP-NPs were produced by attaching Dox to poly(ethylene glycol) (PEG) through two MMP2-cleavable enzymes. The resulting adduct was then tethered to PS NPs. Results showed that Dox release was actually triggered by MMP2 cleavage and was dependent on enzyme concentration, with a plateau around 20 nM. Furthermore, significant cell cytotoxicity was observed towards three cell lines only in the presence of MMP2, but not in cells without enzyme pre-treatment, even after NP internalization by cells. These findings indicate the potential of TAP-NPs as suitable nanocarriers for an on demand, tumor--specific delivery of antitumor drugs after the response to an endogenous stimulus. Further advancements will focus on the translation of this production technology to biodegradable systems for the safe transport of cytotoxic drug to tumor tissues.

Structural insights into and activity analysis of the antimicrobial peptide myxinidin.

The marine environment has been poorly explored in terms of potential new molecules possessing antibacterial activity. Antimicrobial peptides (AMPs) offer a new potential class of pharmaceuticals; however, further optimization is needed if AMPs are to find broad use as antibiotics. We focused our studies on a peptide derived from the epidermal mucus of hagfish (Myxine glutinosa L.), which was previously characterized and showed high antimicrobial activity against human and fish pathogens. In the present work, the activities of myxinidin peptide analogues were analyzed with the aim of widening the original spectrum of action of myxinidin by suitable changes in the peptide primary structure. The analysis of key residues by alanine scanning allowed for the design of novel peptides with increased activity. We identified the amino acids that are of the utmost importance for the observed antimicrobial activities against a set of pathogens comprising both Gram-negative and Gram-positive bacteria. Overall, optimized bactericidal potency was achieved by adding a tryptophan residue at the N terminus and by the simultaneous substitution of residues present in positions 3, 4, and 11 with arginine. These results indicate that the myxinidin analogues emerge as an attractive alternative for treating drug-resistant infectious diseases and provide key insights into a rational design for novel agents against these pathogens.

Structure-activity relations of myxinidin, an antibacterial peptide derived from the epidermal mucus of hagfish.

The structure-activity relations of myxinidin, a peptide derived from epidermal mucus of hagfish, Myxine glutinosa L., were investigated. Analysis of key residues allowed us to design new peptides with increased efficiency. Antimicrobial activity of native and modified peptides demonstrated the key role of uncharged residues in the sequence; the loss of these residues reduces almost entirely myxinidin antimicrobial activity, while insertion of arginine at charged and uncharged position increases antimicrobial activity compared with that of native myxinidin. Particularly, we designed a peptide capable of achieving a high inhibitory effect on bacterial growth. Experiments were conducted using both Gram-negative and Gram-positive bacteria. Nuclear magnetic resonance (NMR) studies showed that myxinidin is able to form an amphipathic α-helical structure at the N terminus and a random coil region at the C terminus.

Chimeric beta-defensin analogs, including the novel 3NI analog, display salt-resistant antimicrobial activity and lack toxicity in human epithelial cell lines.

Human beta-defensins (hBDs) are crucial peptides for the innate immune response and are thus prime candidates as therapeutic agents directed against infective diseases. Based on the properties of wild-type hBD1 and hBD3 and of previously synthesized analogs (1C, 3I, and 3N), we have designed a new analog, 3NI, and investigated its potential as an antimicrobial drug. Specifically, we evaluated the antimicrobial activities of 3NI versus those of hBD1, hBD3, 1C, 3I, and 3N. Our results show that 3NI exerted greater antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis than did hBD1 and hBD3, even with elevated salt concentrations. Moreover, its antiviral activity against herpes simplex virus 1 was greater than that of hBD1 and similar to that of hBD3. Subsequently, we investigated the cytotoxic effects of all peptides in three human epithelial carcinoma cell lines: A549 from lung, CaCo-2 from colon, and Capan-1 from pancreas. None of the analogs significantly reduced cell viability versus wild-type hBD1 and hBD3. They did not induce genotoxicity or cause an increase in the number of apoptotic cells. Using confocal microscopy, we also investigated the localization of the peptides during their incubation with epithelial cells and found that they were distributed on the cell surface, from which they were internalized. Finally, we show that hBD1 and hBD3 are characterized by high resistance to serum degradation. In conclusion, the new analog 3NI seems to be a promising anti-infective agent, particularly given its high salt resistance--a feature that is relevant in diseases such as cystic fibrosis.

Peptide inhibitors against herpes simplex virus infections.

Herpes simplex virus (HSV) is a significant human pathogen causing mucocutaneous lesions primarily in the oral or genital mucosa. Although acyclovir (ACV) and related nucleoside analogs provide successful treatment, HSV remains highly prevalent worldwide and is a major cofactor for the spread of human immunodeficiency virus. Encephalitis, meningitis, and blinding keratitis are among the most severe diseases caused by HSV. ACV resistance poses an important problem for immunocompromised patients and highlights the need for new safe and effective agents; therefore, the development of novel strategies to eradicate HSV is a global public health priority. Despite the continued global epidemic of HSV and extensive research, there have been few major breakthroughs in the treatment or prevention of the virus since the introduction of ACV in the 1980s. A therapeutic strategy at the moment not fully addressed is the use of small peptide molecules. These can be either modeled on viral proteins or derived from antimicrobial peptides. Any peptide that interrupts protein-protein or viral protein-host cell membrane interactions is potentially a novel antiviral drug and may be a useful tool for elucidating the mechanisms of viral entry. This review summarizes current knowledge and strategies in the development of synthetic and natural peptides to inhibit HSV infectivity.

Peptide-lipid interactions: experiments and applications.

The interactions between peptides and lipids are of fundamental importance in the functioning of numerous membrane-mediated cellular processes including antimicrobial peptide action, hormone-receptor interactions, drug bioavailability across the blood-brain barrier and viral fusion processes. Moreover, a major goal of modern biotechnology is obtaining new potent pharmaceutical agents whose biological action is dependent on the binding of peptides to lipid-bilayers. Several issues need to be addressed such as secondary structure, orientation, oligomerization and localization inside the membrane. At the same time, the structural effects which the peptides cause on the lipid bilayer are important for the interactions and need to be elucidated. The structural characterization of membrane active peptides in membranes is a harsh experimental challenge. It is in fact accepted that no single experimental technique can give a complete structural picture of the interaction, but rather a combination of different techniques is necessary.

Corrigendum: Intracellular Delivery: Exploiting Viral Membrane Topic Peptide.

The authors declare that after the publication of the article, it was noticed that two citations were inadvertently omitted. The references have now been included as [74b] and [74c]: [74] (b) Vitiello, M.; Galdiero, M.; Galdiero, M. Inhibition of Viral-Induced Membrane Fusion by Peptides. Protein Pep. Lett., 2009, 16(7), 786-793. (c) Galdiero, S.; Falanga, A.; Vitiello, M.; D'Isanto, M.; Cantisani, M.; Kampanaraki, A.; Benedetti, E.; Browne, H.; Galdiero, M. Peptides containing membrane-interacting motifs inhibit herpes simplex virus type 1 infectivity. Peptides, 2008, 29(9), 1461- 1471. The authors would like to include this reference in the online version of the article to ensure completeness.

Structure and orientation of the gH625-644 membrane interacting region of herpes simplex virus type 1 in a membrane mimetic system.

Glycoprotein H (gH) of the herpes simplex virus type 1 is involved in the complex mechanism of membrane fusion of the viral envelope with host cells. The virus requires four glycoproteins (gB, gD, gH, gL) to execute fusion and the role played by gH remains mysterious. Mutational studies have revealed several regions of gH ectodomain required for fusion and identified the segment from amino acid 625 to 644 as the most fusogenic region. Here, we studied the behavior in a membrane-mimicking DPC micellar environment of a peptide encompassing this region (gH625-644) and determined its NMR solution structure and its orientation within the micelles.

P2 porin and loop L7 from Haemophilus influenzae modulate expression of IL-6 and adhesion molecules in astrocytes.

During neuropathological conditions such as infections and degenerative diseases, astrocytes can be activated by infiltrating immune cells. Activated astrocytes can produce chemokines, cytokines and adhesion molecules. In this study, the production of IL-6 and adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1) and E-selectin by human astroglioma cells stimulated with Gram-negative surface components was investigated. Haemophilus influenzae type b porin P2 and its selected active peptide, loop L7, were found to induce MEK1-MEK2/ mitogen-activated protein kinase phosphorylation in U87-MG cells as demonstrated by ELISA, and up-regulate cellular adhesion molecule and interleukin-6 (IL-6) production as shown by RT-PCR and ELISA. Using two potent and selective inhibitors of MEK activation by Raf-1 (PD-098059) and p38 (SB-203580), it was also demonstrated that both ERK1/2 and p38 pathways play key roles in the production of IL-6 as well as in ICAM-1, VCAM-1 and E-selectin expression by Hib porin.

A peptide derived from herpes simplex virus type 1 glycoprotein H: membrane translocation and applications to the delivery of quantum dots.

Cell membranes are impermeable to most molecules that are not actively imported by living cells, including all macromolecules and even small molecules whose physiochemical properties prevent passive membrane diffusion. However, recently, we have seen the development of increasingly sophisticated methodology for intracellular drug delivery. Cell-penetrating peptides (CPPs), short peptides believed to enter cells by penetrating cell membranes, have attracted great interest in the hope of enhancing gene therapy, vaccine development and drug delivery. Nevertheless, to achieve an efficient intracellular delivery, further strategies to bypass the endocytotic pathway must be investigated. We report on a novel peptide molecule derived from glycoprotein gH of herpes simplex type I virus that is able to traverse the membrane bilayer and to transport a cargo into the cytoplasm with novel properties in comparison with existing CPPs. We use as cargo molecule quantum dots that do not significantly traverse the membrane bilayer on their own. FROM THE CLINICAL EDITOR: Cell-penetrating peptides have recently attracted great interest in optimizing gene therapy, vaccine development and drug delivery. In this study, a peptide derived from glycoprotein gH of herpes simplex I is investigated from this standpoint.

Silver nanoparticles as potential antiviral agents.

Virus infections pose significant global health challenges, especially in view of the fact that the emergence of resistant viral strains and the adverse side effects associated with prolonged use continue to slow down the application of effective antiviral therapies. This makes imperative the need for the development of safe and potent alternatives to conventional antiviral drugs. In the present scenario, nanoscale materials have emerged as novel antiviral agents for the possibilities offered by their unique chemical and physical properties. Silver nanoparticles have mainly been studied for their antimicrobial potential against bacteria, but have also proven to be active against several types of viruses including human imunodeficiency virus, hepatitis B virus, herpes simplex virus, respiratory syncytial virus, and monkey pox virus. The use of metal nanoparticles provides an interesting opportunity for novel antiviral therapies. Since metals may attack a broad range of targets in the virus there is a lower possibility to develop resistance as compared to conventional antivirals. The present review focuses on the development of methods for the production of silver nanoparticles and on their use as antiviral therapeutics against pathogenic viruses.

Proteomic analysis of human U937 cell line activation mediated by Haemophilus influenzae type b P2 porin and its surface-exposed loop 7.

The virulence of Haemophilus influenzae type b (Hib) has been attributed to a variety of potential factors associated with its cell surface, including lipopolysaccharides (LPS) and major outer membrane proteins (OMPs). P2 porin, one of the best-characterized porins in terms of its functional characteristics, is the most abundant OMP in Hib and has also been shown to possess proinflammatory activity. To characterize the role played by bacterial surface components in disease onset and development, the proteomic profiling of human U937 cell line activated by H. influenzae type b P2 porin and its most active surface-exposed loop (L7) was performed by means of two-dimensional electrophoresis and mass spectrometry. The study provided a list of candidate proteins with potential relevance in the host immune and inflammatory response. Most of the differentially expressed proteins are involved in metabolic processes, remodelling of cytoskeleton, stress response and signal transduction pathways. The results constitute the basis for dissecting signal transduction cascades activated by P2 stimulation and gain insights into the molecular events involved in the modulation of pathogen-host cell interactions.

Novel synthetic, salt-resistant analogs of human beta-defensins 1 and 3 endowed with enhanced antimicrobial activity.

Human beta-defensins (hBDs) are antimicrobial peptides of human innate immunity. The antibacterial activities of hBDs 1, 2, and 4 but not the activity of hBD3 are impaired by high salt levels. We have designed and synthesized seven novel hBD analogs, constituted by different domains of hBD1 (which is constitutively expressed in humans) and of hBD3 (which is induced by microorganisms and inflammatory factors in humans), that would maintain and potentially increase the wild-type antimicrobial activities and be salt resistant. We have compared the antibacterial, antiviral, and chemotactic activities of the analogs with those of hBD1 and hBD3. We show that the hBD1 internal region and the hBD3 C-terminal region are critical for antibacterial activity also at high salt concentrations, whereas deletion of the N-terminal region of hBD3 results in an increase in antibacterial activity. All analogs inhibited herpes simplex virus; antiviral activity was enhanced by the hBD1 internal region and the hBD3 C-terminal region. Wild-type and analog peptides were chemotactic for granulocytes and monocytes, irrespective of the salt concentrations. These new peptides may have therapeutic potential.

Viral fusion peptides induce several signal transduction pathway activations that are essential for interleukin-10 and beta-interferon production.

OBJECTIVES: The deciphering of intracellular signaling pathways that are activated by the interaction between viral fusion peptides and cellular membranes are important for the understanding of both viral replication strategies and host defense mechanisms. METHODS: Fusion peptides of several enveloped viruses belonging to different virus families were prepared by standard 9-fluorenylmethoxycarbonyl polyamine solid-phase synthesis and used to stimulate U937 cells in vitro to analyze the phosphorylation patterns of the signaling pathways (PKC, Src, Akt, and MAPK pathways). Immunoprecipitation and Western blotting were carried out by using phosphospecific antibodies. All samples were also assayed for the presence of IL-10 and IFN-beta by ELISA and activation of nuclear factors (AP-1 and NF-kappaB). RESULTS: We have demonstrated that hydrophobic domains of fusion proteins are able to induce several transduction pathways that lead to cytokine (IFN-beta and IL-10) production, an event that appears to be dependent on early activation of AP-1 and NF-kappaB. CONCLUSIONS: The results obtained on the signaling activity of fusion peptides from different viruses enabled us to shed some light on the complex mechanism of viral entry and more precisely we focused on the exact signaling event induced by hydrophobic domains characteristic of fusion peptides interacting with the cell membrane.

Pathophysiological changes of gram-negative bacterial infection can be reproduced by a synthetic peptide mimicking loop L7 sequence of Haemophilus influenzae porin.

Several in vivo models have been used to dissect the molecular mechanisms that contribute to activate the coagulation and fibrinolytic systems by bacteria and bacterial products but many aspects remain poorly understood. In this study we examined the in vivo effect of the synthetic peptide corresponding to loop L7 from Haemophilus influenzae type b (Hib) porin to evaluate its role on the coagulative/fibrinolytic cascade and the circulating markers of endothelial injury. Plasma was obtained from rats injected intravenously with loop L7, Hib porin or a scrambled peptide and tested for fragment 1+2 (F1+2), tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type I (PAI-1) antigen, von Willebrand factor (vWF) and soluble E-selectin (sE-selectin). The coagulative/fibrinolytic cascade was impaired as shown by PAI-1 level increased. Concomitantly, E-selectin, a marker of endothelial injury, was also significantly elevated. In addition either loop L7 or Hib porin injection induced hyperglycaemia and inflammatory cytokine production. The data were correlated with hemodynamic functions. The results indicate that loop L7 plays an essential role in the pathophysiologic events observed during gram-negative infection. These findings may have implications for the development of alternative therapies to counteract excessive inflammatory responses during septic shock.

The identification and characterization of fusogenic domains in herpes virus glycoprotein B molecules.

The molecular mechanism of entry of herpes viruses requires a multicomponent fusion system. Virus entry and cell-cell fusion of Herpes simplex virus (HSV) requires four glycoproteins: gD, gB and gH/gL. The role of gB remained elusive until recently, when the crystal structure of HSV-1 gB became available. Glycoprotein B homologues represent the most highly conserved group of herpes virus glycoproteins; however, despite the high degree of sequence and structural conservation, differences in post-translational processing are observed for different members of this virus family. Whereas gB of HSV is not proteolytically processed after oligomerization, most other gB homologues are cleaved by a cellular protease into subunits that remain linked through disulfide bonds. Proteolytic cleavage is common for activation of many other viral fusion proteins, so it remains difficult to envisage a common role for different herpes virus gB structures in the fusion mechanism. We selected bovine herpes virus type 1 (BoHV-1) and herpes simplex virus type 1 (HSV-1) as representative viruses expressing cleaved and uncleaved gBs, and have screened their amino acid sequences for regions of highly interfacial hydrophobicity. Synthetic peptides corresponding to such regions were tested for their ability to induce the fusion of large unilamellar vesicles and to inhibit herpes virus infection. These results underline that several regions of the gB protein are involved in the mechanism of membrane interaction.

Peptides containing membrane-interacting motifs inhibit herpes simplex virus type 1 infectivity.

Herpes simplex virus (HSV) membrane fusion represents an attractive target for anti-HSV therapy. To investigate the structural basis of HSV membrane fusion and identify new targets for inhibition, we have investigated the different membranotropic domains of HSV-1 gH envelope glycoprotein. We observed that fusion peptides when added exogenously are able to inhibit viral fusion likely by intercalating with viral fusion peptides upon adopting functional structure in membranes. Interestingly, peptides analogous to the predicted HSV-1 gH loop region inhibited viral plaque formation more significantly. Their inhibitory effect appears to be a consequence of their ability to partition into membranes and aggregate within them. Circular dichroism spectra showed that peptides self-associate in aqueous and lipidic solutions, therefore the inhibition of viral entry may occur via peptides association with their counterpart on wild-type gH. The antiviral activity of HSV-1 peptides tested provides an attractive basis for the development of new fusion peptide inhibitors corresponding to regions outside the fusion protein heptad repeat regions.

Biocompatible nanoparticles sensing the matrix metallo-proteinase 2 for the on-demand release of anticancer drugs in 3D tumor spheroids.

The balance between dose-dependent tolerability, effectiveness and toxicity of systemically administered antitumor drugs is extremely delicate. This issue highlights the striking need for targeted release of chemotherapeutic drugs within tumors. In this work, a smart strategy of drug targeting to tumors relying upon biodegradable/biocompatible nanoparticles releasing cytotoxic drugs after sensing physiological variations intrinsic to the very nature of tumor tissues is exploited. Here, the well-known over-expression of matrix metallo-proteinase 2 (MMP2) enzyme in tumors has been chosen as a trigger for the release of a cytotoxic drug. Nanoparticles made up of a biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA)--block--polyethylene glycol (PEG) copolymer (namely PELGA), blended with a tumor-activated prodrug (TAP) composed of a MMP2-sensitive peptide bound to doxorubicin (Dox) and to PLGA chain have been produced. The obtained devices are able to release Dox specifically upon MMP2 cleavage of the TAP. More interestingly, they can sense the differences in the expression levels of endogenous MMP2 protein, thus modulating drug penetration within a three-dimensional (3D) tumor spheroid matrix, accordingly. Therefore, the proposed nanoparticles hold promise as a useful tool for in vivo investigations aimed at an improved therapeutic efficacy of the conjugated drug payload.

Design and activity of a cyclic mini-ß-defensin analog: a novel antimicrobial tool.

We have designed a cyclic 17-amino acid ß-defensin analog featuring a single disulfide bond. This analog, designated "AMC" (ie, antimicrobial cyclic peptide), combines the internal hydrophobic domain of hBD1 and the C-terminal charged region of hBD3. The novel peptide was synthesized and characterized by nuclear magnetic resonance spectroscopy. The antimicrobial activities against gram-positive and gram-negative bacteria as well as against herpes simplex virus type 1 were analyzed. The cytotoxicity and serum stability were assessed. Nuclear magnetic resonance of AMC in aqueous solution suggests that the structure of the hBD1 region, although not identical, is preserved. Like the parent defensins, AMC is not cytotoxic for CaCo-2 cells. Interestingly, AMC retains the antibacterial activity of the parent hBD1 and hBD3 against Pseudomonas aeruginosa, Enterococcus faecalis, and Escherichia coli, and exerts dose-dependent activity against herpes simplex virus type 1. Moreover, while the antibacterial and antiviral activities of the oxidized and reduced forms of the parent defensins are similar, those of AMC are significantly different, and oxidized AMC is also considerably more stable in human serum. Taken together, our data also suggest that this novel peptide may be added to the arsenal of tools available to combat antibiotic-resistant infectious diseases, particularly because of its potential for encapsulation in a nanomedicine vector.

Conformational modifications of gB from herpes simplex virus type 1 analyzed by synthetic peptides.

Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. The crystallized trimeric glycoprotein gB of herpes simplex virus has been described as a postfusion conformation, and several studies prove that like other class III fusion proteins, gB undergoes a pH-dependent switch between the pre- and postfusion conformations. Using several biophysical techniques, we show that peptides corresponding to the long helix of the gB postfusion structure interfere with the membrane fusion event, likely hampering the conformational rearrangements from the pre- to the postfusion structures. Those peptides represent good candidates for further design of peptidomimetic antagonists capable of blocking the fusion process.