Effective in Vivo Targeting of Influenza Virus through a Cell-Penetrating/Fusion Inhibitor Tandem Peptide Anchored to the Plasma Membrane.

The impact of influenza virus infection is felt each year on a global scale when approximately 5-10% of adults and 20-30% of children globally are infected. While vaccination is the primary strategy for influenza prevention, there are a number of likely scenarios for which vaccination is inadequate, making the development of effective antiviral agents of utmost importance. Anti-influenza treatments with innovative mechanisms of action are critical in the face of emerging viral resistance to the existing drugs. These new antiviral agents are urgently needed to address future epidemic (or pandemic) influenza and are critical for the immune-compromised cohort who cannot be vaccinated. We have previously shown that lipid tagged peptides derived from the C-terminal region of influenza hemagglutinin (HA) were effective influenza fusion inhibitors. In this study, we modified the influenza fusion inhibitors by adding a cell penetrating peptide sequence to promote intracellular targeting. These fusion-inhibiting peptides self-assemble into ∼15-30 nm nanoparticles (NPs), target relevant infectious tissues in vivo, and reduce viral infectivity upon interaction with the cell membrane. Overall, our data show that the CPP and the lipid moiety are both required for efficient biodistribution, fusion inhibition, and efficacy in vivo.

In Vivo Efficacy of Measles Virus Fusion Protein-Derived Peptides Is Modulated by the Properties of Self-Assembly and Membrane Residence.

Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F-derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances biodistribution and the half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides. IMPORTANCE: Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. We show here that specific biophysical properties regulate the in vivo efficacy of MV F-derived peptides.

A focus on glucose-mediated drug delivery to the central nervous system.

Drug delivery to the central nervous system (CNS) is a timely and challenging issue: 95 percent; of the pharmacological drugs cannot be delivered to the brain. This is mainly due to the blood-brain barrier (BBB), a highly selective boundary that hampers the passage of most compounds into the CNS. To overcome this problem, several approaches exist to deliver a therapeutic drug to the brain that takes into account not only the chemical properties of the drug but also the type of transport used at the BBB. One of those strategies is the glucose-mediated drug delivery which will be the focus of the present review. Glucose-mediated drug delivery requires the attachment of glycosyl moieties to a drug and the use of endogenous glucose transporters as a way to circumvent the blood-brain barrier. Glycosylated drugs display improved cell penetrability, enhanced biodistribution, stability and low toxicity. Examples such as glycosylation of ibuprofen and different opioids result in an enhanced central effect and will be discussed.

In vitro blood-brain barrier models--latest advances and therapeutic applications in a chronological perspective.

The first generation of in vitro models providing successful isolation of viable brain endothelial cells from different species, which could be maintained in cell culture, have emerged around thirty years ago. However, the time consuming and the difficulty of working with primary culture cells led to the development of simpler models employing cell lines with blood-brain barrier properties. The creation, in late nineties, of a transgenic mouse harboring the temperature sensitive simian virus 40 large T-antigen as a source of conditionally immortalized brain endothelial cell lines circumvented the problems of in vitro transfection of tumour inducing gene in primary cells. These different ways to obtain cultures of brain endothelial cells have profited from the discovery of different cellular factors that allow the growth of differentiated cells on plastic filters. Although cell preparations and culture conditions of brain endothelial cells are based on the same principle, there are two main models for studying the blood-brain barrier: the static and the more recently described dynamic model. Dynamic models were created in order to replicate the physiological in vivo environment of the blood-brain barrier. The large pool of in vitro models is being enlarged since each laboratory improves its model adding small differences adapted to the research interests. The great impact of blood-brain barrier studies in the development of therapies related to the central nervous system supports the interests of this review about in vitro models.

Hepatitis C virus core protein binding to lipid membranes: the role of domains 1 and 2.

We have analysed and identified different membrane-active regions of the Hepatitis C virus (HCV) core protein by observing the effect of 18-mer core-derived peptide libraries from two HCV strains on the integrity of different membrane model systems. In addition, we have studied the secondary structure of specific membrane-interacting peptides from the HCV core protein, both in aqueous solution and in the presence of model membrane systems. Our results show that the HCV core protein region comprising the C-terminus of domain 1 and the N-terminus of domain 2 seems to be the most active in membrane interaction, although a role in protein-protein interaction cannot be excluded. Significantly, the secondary structure of nearly all the assayed peptides changes in the presence of model membranes. These sequences most probably play a relevant part in the biological action of HCV in lipid interaction. Furthermore, these membranotropic regions could be envisaged as new possible targets, as inhibition of its interaction with the membrane could potentially lead to new vaccine strategies.

Does aliphatic chain length influence carbocyanines' orientation in supported lipid multilayers?

UV-Vis linear dichroism was used to study the orientation of 3,3'-dihexyloxacarbocyanine (DiOC6(3)) and 3,3'-dihexadecyloxacarbocyanine (DiOC16(3)) in supported multilayers of dipalmitoylphosphatidylcholine (DPPC) and dilauroylphosphatidylcholine (DLPC). Orientational probability density functions were similar for the two carbocyanines in both lipids. Multimodal distributions were found in all cases. The main peak is at 9 degrees-11 degrees relative to the bilayer normal axis, except for DiOC16(3) in DLPC multilayers (main peaks at 13 degrees and 90 degrees). Quenching studies revealed that the two carbocyanines are localized at the interface region of the membrane regardless of the lipid matrix they are inserted in. Combining these data with linear dichroism results lead to the conclusion that both the aliphatic chain length of carbocyanines and the lipid phase have little influence in the structural organization of these probes in lipidic bilayers. The partition constants of DiOC6 (3), K(p), were determined from fluorescence anisotropy measurements; the values obtained were K(p) (DPPC) = (2.39 +/- 0.05) x 10(3) and K(p) (DLPC) = (5.01 +/- 1.15) x 10(3).

Joint determination by Brownian dynamics and fluorescence quenching of the in-depth location profile of biomolecules in membranes.

The in-depth molar distribution function of fluorophores is revealed by a new methodology for fluorescence quenching data analysis in membranes. Brownian dynamics simulation was used to study the in-depth location profile of quenchers. A Lorentzian profile was reached. Since the Stern-Volmer equation is valid at every depth in the membrane for low quencher concentrations, the molar distribution of the fluorophore (also regarded as a Lorentzian) can be achieved. The average location and the broadness of the fluorophore distribution can be calculated. The importance of the knowledge of the location width is demonstrated and discussed, since this parameter reveals important conclusions on structural features of the interaction of membranes with probes and biomolecules (e.g., conformational freedom in proteins), as well as photophysical properties (e.g., differential fluorophore quantum yields). Subsequent use of this methodology by the reader does not, necessarily, involve the performance of simulations and is not limited to the use of Lorentzian function distributions.