Alexey V. Ustinov

Rigid amphipathic fusion inhibitors, small molecule antiviral compounds against enveloped viruses

Antiviral drugs targeting viral proteins often result in prompt selection for resistance. Moreover, the number of viral targets is limited. Novel antiviral targets are therefore needed. The unique characteristics of fusion between virion envelopes and cell membranes may provide such targets. Like all fusing bilayers, viral envelopes locally adopt hourglass-shaped stalks during the initial stages of fusion, a process that requires local negative membrane curvature. Unlike cellular vesicles, however, viral envelopes do not redistribute lipids between leaflets, can only use the energy released by virion proteins, and fuse to the extracellular leaflets of cell membranes. Enrichment in phospholipids with hydrophilic heads larger than their hydrophobic tails in the convex outer leaflet of vesicles favors positive curvature, therefore increasing the activation energy barrier for fusion. Such phospholipids can increase the activation barrier beyond the energy provided by virion proteins, thereby inhibiting viral fusion. However, phospholipids are not pharmacologically useful. We show here that a family of synthetic rigid amphiphiles of shape similar to such phospholipids, RAFIs (rigid amphipathic fusion inhibitors), inhibit the infectivity of several otherwise unrelated enveloped viruses, including hepatitis C and HSV-1 and -2 (lowest apparent IC50 48 nM), with no cytotoxic or cytostatic effects (selectivity index > 3,000) by inhibiting the increased negative curvature required for the initial stages of fusion.

5-(Perylen-3-yl)Ethynyl-arabino-Uridine (aUY11), an Arabino-Based Rigid Amphipathic Fusion Inhibitor, Targets Virion Envelope Lipids To Inhibit Fusion of Influenza Virus, Hepatitis C Virus, and Other Enveloped Viruses

ABSTRACT Entry of enveloped viruses requires fusion of viral and cellular membranes. Fusion requires the formation of an intermediate stalk structure, in which only the outer leaflets are fused. The stalk structure, in turn, requires the lipid bilayer of the envelope to bend into negative curvature. This process is inhibited by enrichment in the outer leaflet of lipids with larger polar headgroups, which favor positive curvature. Accordingly, phospholipids with such shape inhibit viral fusion. We previously identified a compound, 5-(perylen-3-yl)ethynyl-2′-deoxy-uridine (dUY11), with overall shape and amphipathicity similar to those of these phospholipids. dUY11 inhibited the formation of the negative curvature necessary for stalk formation and the fusion of a model enveloped virus, vesicular stomatitis virus (VSV). We proposed that dUY11 acted by biophysical mechanisms as a result of its shape and amphipathicity. To test this model, we have now characterized the mechanisms against influenza virus and HCV of 5-(perylen-3-yl)ethynyl-arabino-uridine (aUY11), which has shape and amphipathicity similar to those of dUY11 but contains an arabino-nucleoside. aUY11 interacted with envelope lipids to inhibit the infectivity of influenza virus, hepatitis C virus (HCV), herpes simplex virus 1 and 2 (HSV-1/2), and other enveloped viruses. It specifically inhibited the fusion of influenza virus, HCV, VSV, and even protein-free liposomes to cells. Furthermore, aUY11 inhibited the formation of negative curvature in model lipid bilayers. In summary, the arabino-derived aUY11 and the deoxy-derived dUY11 act by the same antiviral mechanisms against several enveloped but otherwise unrelated viruses. Therefore, chemically unrelated compounds of appropriate shape and amphipathicity target virion envelope lipids to inhibit formation of the negative curvature required for fusion, inhibiting infectivity by biophysical, not biochemical, mechanisms.

Modification of nucleic acids using [3 + 2]-dipolar cycloaddition of azides and alkynes

The use of azide and alkyne cycloaddition reaction in the synthesis of conjugates of nucleic acids and oligodeoxyribonucleotides is reviewed. Data on the chemical and enzymatic methods for introducing azides and alkynes into DNA are summarized.

On magnetic flux dynamics in 1D arrays of underdamped Josephson junctions

Abstract We have measured the current-voltage characteristics of parallel biased arrays of small Nb/NbO x /Pb Josephson junctions connected by superconducting loops. Depending on the temperature of the sample, the application of a magnetic field perpendicular to the planes of the loops gives rise to linear branches and flux-flow steps in the current-voltage characteristics of the arrays. For increasing temperatures the linear branches evolve in resonant flux-flow like singularities. Good agreement between the experimental data and the direct numerical simulations based on the discrete Josephson transmission line model is found.

Rigid amphipathic nucleosides suppress reproduction of the tick-borne encephalitis virus

Rigid amphipathic fusion inhibitors (RAFIs), 5-arylethynyl uracil nucleosides with bulky aryl groups, appeared to have considerable activity against tick-borne encephalitis virus (TBEV) in cell culture. The rigid ethynyl linker and perylene residue are essential structural prerequisites for high activity, giving EC50 values of 18 and 24 nM for 5-(perylen-3-yl)ethynyl-arabino-uridine and 5-(perylen-3-yl)ethynyl-2′-deoxy-uridine, respectively, upon simultaneous mixing of the compounds, cells and virus.

A nascent proteome study combining click chemistry with 2DE.

To investigate the dynamic cellular response to a condition change, selective labeling of the nascent proteome is necessary. Here, we report a method combining click chemistry protein labeling with 2D DIGE. To test the relevance of the method, we compared nascent proteomes of actively growing bacterial cells with that of cells exposed to protein synthesis inhibitor, erythromycin. Cells were incubated with methionine analog, homopropargyl glycin, and their nascent proteome was selectively labeled with monosulfonated neutral Cy3 and Cy5 azides specially synthesized for this purpose. Following fluorescent labeling, the protein samples were mixed and subjected to standard 2D DIGE separation. The method allowed us to reveal a dramatic reduction of newly synthesized proteins upon erythromycin treatment, while the total proteome was not significantly affected. Additionally, several proteins, whose synthesis was resistant to erythromycin, were identified.

Perylene Diimide–Oligonucleotide Conjugates Constructed by Click Chemistry

Oligonucleotide conjugates were prepared by copper-catalyzed cycloaddition of acetylene-modified oligonucleotides to diazido derivative of perylene 3,4,9,10-tetracarboxylic acid diimide.

Fine Tuning of Pyrene Excimer Fluorescence in Molecular Beacons by Alteration of the Monomer Structure

Oligonucleotide probes labeled with pyrene pairs that form excimers have a number of applications in hybridization analysis of nucleic acids. A long excited state lifetime, large Stokes shift, and chemical stability make pyrene excimer an attractive fluorescent label. Here we report synthesis of chiral phosphoramidite building blocks based on (R)-4-amino-2,2-dimethylbutane-1,3-diol, easily available from an inexpensive d-(-)-pantolactone. 1-Pyreneacetamide, 1-pyrenecarboxamide, and DABCYL derivatives have been used in preparation of molecular beacon (MB) probes labeled with one or two pyrenes/quenchers. We observed significant difference in the excimer emission maxima (475-510 nm; Stokes shifts 125-160 nm or 7520-8960 cm-1) and excimer/monomer ratio (from 0.5 to 5.9) in fluorescence spectra depending on the structure and position of monomers in the pyrene pair. The pyrene excimer formed by two rigid 1-pyrenecarboxamide residues showed the brightest emission. This is consistent with molecular dynamics data on excimer stability. Increase of the excimer fluorescence for MBs after hybridization with DNA was up to 24-fold.

Seebeck effect of weak links in a high-Tc superconductor

The authors have observed experimentally the analog of the superfluid helium fountain effect in a polycrystalline YBaCuO film. In a superconductor a temperature gradient induces the counterflow of superconducting electrons and quasiparticles. The supercurrent creates a phase difference at the intra- and intergrain weak links. In the presence of a temperature gradient parallel to an electric current they have detected an asymmetry of the current-voltage characteristic. Reversing the direction of the temperature gradient also reverses the sign of the current asymmetry. They estimate the thermoelectric coefficient for the quasiparticles in the YBaCuO sample.

Improved Large-scale Synthesis of 5-(Perylen-3-ylethynyl)-arabino-uridine (aUY11), the Broad-Spectrum Antiviral

Rigid amphipathic fusion inhibitors (RAFIs), 5-arylethynyl-uracil nucleosides with bulky aryl groups, constitute a class of compounds active against enveloped viruses. Derivatives of perylene, a pentacyclic aromatic hydrocarbon, are the most potent among RAFI nucleosides with IC50 values ranging from 0.2 mM to 0.005 mM. 1–4 5-(Perylen-3-ylethynyl)-arabino-uridine (aUY11, 1) inhibits replication of enveloped viruses (HSV-1, influenza virus, HCV, etc.) without cytotoxic and even cytostatic activities in cell cultures. The only reported synthetic procedure was complicated with a lack of availability of a key precursor, 3-ethynylperylene, and with high polarity and reduced solubility of the key intermediate, a 30,50-O-silyl protected peryleneethynyl nucleoside derivative. Therefore the synthesis was performed in less than 200 mg scale with 21% yield in the last two steps. Studies of antiviral effects in animal models, however, require multigram quantities of this compound, thus more efficient methods of preparation are needed.