Vladimir A. Korshun

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.

Highly Fluorescent Conjugated Pyrenes in Nucleic Acid Probes: (Phenylethynyl)pyrenecarbonyl‐Functionalized Locked Nucleic Acids

In recent years, fluorescently labeled oligonucleotides have become a widely used tool in diagnostics, DNA sequencing, and nanotechnology. The recently developed (phenylethynyl)pyrenes are attractive dyes for nucleic acid labeling, with the advantages of long-wave emission relative to the parent pyrene, high fluorescence quantum yields, and the ability to form excimers. Herein, the synthesis of six (phenylethynyl)pyrene-functionalized locked nucleic acid (LNA) monomers M(1)-M(6) and their incorporation into DNA oligomers is described. Multilabeled duplexes display higher thermal stabilities than singly modified analogues. An increase in the number of phenylethynyl substituents attached to the pyrene results in decreased binding affinity towards complementary DNA and RNA and remarkable bathochromic shifts of absorption/emission maxima relative to the parent pyrene fluorochrome. This bathochromic shift leads to the bright fluorescence colors of the probes, which differ drastically from the blue emission of unsubstituted pyrene. The formation of intra- and interstrand excimers was observed for duplexes that have monomers M(1)-M(6) in both complementary strands and in numerous single-stranded probes. If more phenylethynyl groups are inserted, the detected excimer signals become more intense. In addition, (phenylethynyl)pyrenecarbonyl-LNA monomers M(4), M(5), and M(6) proved highly useful for the detection of single mismatches in DNA/RNA targets.

1‐, 2‐, and 4‐Ethynylpyrenes in the Structure of Twisted Intercalating Nucleic Acids: Structure, Thermal Stability, and Fluorescence Relationship

A postsynthetic, on-column Sonogashira reaction was applied on DNA molecules modified by 2- or 4-iodophenylmethylglycerol in the middle of the sequence, to give the corresponding ortho- and para-twisted intercalating nucleic acids (TINA) with 1-, 2-, and 4-ethynylpyrene residues. The convenient synthesis of 2- and 4-ethynylpyrenes started from the hydrogenolysis of pyrene that has had the sulfur removed and separation of 4,5,9,10-tetrahydropyrene and 1,2,3,6,7,8-hexahydropyrene, which were later converted to the final compounds by successive Friedel-Crafts acetylation, aromatization by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and a Vilsmeier-Haack-Arnold transformation followed by a Bodendorf fragmentation. Significant alterations in thermal stability of parallel triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in homopyrimidine TINAs. Thus, for para-TINAs the bulge insertion of an intercalator led to high thermal stability of Hoogsteen-type parallel triplexes and duplexes, whereas Watson-Crick-type duplexes were destabilized. In the case of ortho-TINA, both Hoogsteen and Watson-Crick-type complexes were stabilized. Alterations in the thermal stability were highly influenced by the ethynylpyrene isomers used. This also led to TINAs with different changes in fluorescence spectra depending on the secondary structures formed. Stokes shift of approximately 100 nm was detected for pyren-2-ylethynylphenyl derivatives, whereas values for 1- and 4-ethynylpyrenylphenyl conjugates were 10 and 40 nm, respectively. In contrast with para-TINAs, insertion of two ortho-TINAs opposite each other in the duplex as a pseudo-pair resulted in formation of an excimer band at 505 nm for both 1- and 4-ethynylpyrene analogues, which was also accompanied with higher thermal stability.

Fluorescent 5-alkynyl-2'-deoxyuridines: high emission efficiency of a conjugated perylene nucleoside in a DNA duplex.

Four fluorescent 5‐alkynyl‐2′‐deoxyuridines were studied in DNA oligonucleotides and their duplexes. The fluorescence response to hybridization differs dramatically for nucleosides containing a perylene fluorochrome either conjugated or not conjugated to the nucleobase. The conjugated nucleoside, 5‐(perylen‐3‐ylethynyl)‐2′‐deoxyuridine, shows enhanced long‐wavelength emission in the DNA duplex, in contrast to the blue fluorescence of perylene on a flexible linker (in 5‐[(perylen‐3‐yl)methoxyprop‐1‐ynyl]‐2′‐deoxyuridine), which is quenched upon duplex formation.

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.

New Pyrene Derivatives for Fluorescent Labeling of Oligonucleotides

Abstract A series of pyrene-containing reagents have been synthesized and used for the fluorescent labeling of oligonucleotides.

[Synthesis and fluorescent properties of 5-(1-pyrenylethynyl)-2'- deoxyuridine-containing oligodeoxynucleotides].

Novel reagents for the fluorescent labeling of oligo- and polynucleotides have been prepared: 5-(1-pyrenylethynyl)-2′-deoxyuridine 3′-phosphoramidite and a solid support carrying this nucleoside. Oligo-nucleotides containing one or several modified units have been synthesized, and the fluorescence of these probes has been shown to change upon hybridization with the complementary sequence.

Incorporation of a pyrene nucleoside analogue into synthetic oligodeoxynucleotides using a nucleoside-like synthon

Abstract A novel phosphoramidite 4 based on the hydroxyprolinol 1 backbone has been synthesized and used to chemically prepare DNA fragments bearing a pyrene-containing nucleoside analogue.

New Fluorescent Nucleoside Derivatives -5-Alkynylated 2-Deoxyuridines

Abstract Four fluorescent nucleosides, 5-(4-pyrenylethynyl)-, 5-(l-pyrenylbutadiynyl)-, 5-(3-perylenylethynyl)-, and 5-[4-(2-benzoxazolyl)phenylethynyl]-2′-deoxyuridines, were synthesized.