5-(Perylen-3-ylethynyl)uracil as an antiviral scaffold: Potent suppression of enveloped virus reproduction by 3-methyl derivatives in vitro.

Amphipathic nucleoside and non-nucleoside derivatives of pentacyclic aromatic hydrocarbon perylene are known as potent non-cytotoxic broad-spectrum antivirals. Here we report 3-methyl-5-(perylen-3-ylethynyl)-uracil-1-acetic acid and its amides, a new series of compounds based on a 5-(perylen-3-ylethynyl)-uracil scaffold. The compounds demonstrate pronounced in vitro activity against arthropod-borne viruses, namely tick-borne encephalitis virus (TBEV) and yellow fever virus (YFV), in plaque reduction assays with EC50 values below 1.9 and 1.3 nM, respectively, and Chikungunya virus (CHIKV) in cytopathic effect inhibition test with EC50 values below 3.2 µM. The compounds are active against respiratory viruses as well: severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) in cytopathic effect inhibition test and influenza A virus (IAV) in virus titer reduction experiments are inhibited - EC50 values below 51 nM and 2.2 µM, respectively. The activity stems from the presence of a hydrophobic perylene core, and all of the synthesized compounds exhibit comparable 1O2 generation rates. Nonetheless, activity can vary by orders of magnitude depending on the hydrophilic part of the molecule, suggesting a complex mode of action. A time-of-addition experiment and fluorescent imaging indicate that the compounds inhibit viral fusion in a dose-dependent manner. The localization of the compound in the lipid bilayers and visible damage to the viral envelope suggest the membrane as the primary target. Dramatic reduction of antiviral activity with limited irradiation or under treatment with antioxidants further cements the idea of photoinduced ROS-mediated viral envelope damage being the mode of antiviral action.

Simplistic perylene-related compounds as inhibitors of tick-borne encephalitis virus reproduction.

Rigid amphipathic fusion inhibitors are potent broad-spectrum antivirals based on the perylene scaffold, usually decorated with a hydrophilic group linked via ethynyl or triazole. We have sequentially simplified these structures by removing sugar moiety, then converting uridine to aniline, then moving to perylenylthiophenecarboxylic acids and to perylenylcarboxylic acid. All these polyaromatic compounds, as well as antibiotic heliomycin, still showed pronounced activity against tick-borne encephalitis virus (TBEV) with limited toxicity in porcine embryo kidney (PEK) cell line. 5-(Perylen-3-yl)-2-thiophenecarboxylic acid (5a) showed the highest antiviral activity with 50% effective concentration of approx. 1.6 nM.

4-Chloro-L-kynurenine as fluorescent amino acid in natural peptides.

4-Chloro-L-kynurenine (3-(4-chloroanthraniloyl)-L-alanine, L-4-ClKyn), an amino acid known as a prospective antidepressant, was recently for the first time found in nature in the lipopeptide antibiotic taromycin. Here, we report another instance of its identification in a natural product: 4-chloro-L-kynurenine was isolated from acidic hydrolysis of a new complex peptide antibiotic INA-5812. L-4-ClKyn is a fluorescent compound responsible for the fluorescence of the above antibiotic. Whereas fluorescence of 4-chlorokynurenine was not reported before, we synthesized the racemic compound and studied its emission in various solvents. Next, we prepared conjugates of DL-4-ClKyn with two suitable energy acceptors, BODIPY FL and 3-(phenylethynyl)perylene (PEPe), and studied fluorescence of the derivatives. 4-Chloro-DL-kynurenine emission is not detected in both conjugates, thus evidencing effective energy transfer. However, BODIPY FL emission in the conjugate is substantially reduced, probably due to collisional or photoinduced charge-transfer-mediated quenching. The intrinsic fluorescence of L-4-ClKyn amino acid in antibiotics paves the way for spectral studies of their mode of action.

3'-O-Substituted 5-(perylen-3-ylethynyl)-2'-deoxyuridines as tick-borne encephalitis virus reproduction inhibitors.

A series of analogues of potent antiviral perylene nucleoside dUY11 with methylthiomethyl (MTM), azidomethyl (AZM) and HO-C1-4-alkyl-1,2,3-triazol-1,4-diyl groups at 3'-O-position as well as the two products of copper-free alkyne-azide cycloaddition of the AZM derivative were prepared and evaluated against tick-borne encephalitis virus (TBEV). Four compounds (4, 6, 8a, 8b) showed EC50 ≤ 10 nM, thus appearing the most potent TBEV inhibitors to date. Moreover, these nucleosides have higher lipophilicity (clogP) and increased solubility in aq. DMSO vs. parent compound dUY11.

Antivirals acting on viral envelopes via biophysical mechanisms of action.

Most antivirals target viral proteins and are specific for only one virus, or viral type. Whereas viral proteins are encoded in the plastic viral genome, virion lipids are not and their rearrangements during fusion are conserved among otherwise unrelated enveloped viruses. Antivirals that inhibit these lipid rearrangements could thus pose a high barrier to resistance and have broad-spectrum activity. Fusion occurs through a hemifusion stalk in which only the outer leaflets are fused and thus curved with a smaller radius for the polar heads than for the hydrophobic tails (negative curvature). Outer leaflets enriched in phospholipids with head groups of larger cross sections than their lipid tails ("inverted cone") disfavor negative curvature, inhibiting fusion. The rigid amphipathic fusion inhibitors (RAFIs) are synthetic compounds of inverted cone molecular geometry. They inhibit infectivity of otherwise unrelated enveloped viruses. The leading RAFI, aUY11, has an ethynyl-perylene hydrophobic and an uracil-arabinose polar moiety. aUY11 intercalates in viral envelopes and inhibits virion-to-cell fusion of a broad spectrum of otherwise unrelated enveloped viruses. Previous studies showed that amphipathicity, rigidity, and inverted cone molecular geometry were required. We propose that the inverted cone molecular geometry of the RAFIs increases the energy barrier for the hemifusion stalk, inhibiting fusion. Then, chemically distinct compounds with similar amphipathicity, rigidity, and inverted cone shape would have similar antiviral potencies, regardless of specific chemical groups. Alternatively, the perylene group exposed to visible light may induce viral lipid peroxidation. Then, the perylene group and absorbance at visible spectrum would be required. We now evaluated twenty-five chemically distinct RAFIs. The perylene moiety and absorption at visible spectrum were not required, but a minimum length of the hydrophobic moiety was, 10.3 Å. The arabino moiety could be modified or replaced by other groups. Cytidine was not tolerated. Bilayer intercalation was required but not sufficient. The vast majority of RAFIs had no overt cytotoxicity (CC50 > 20 µM; TI > 250-1200). Carbonyl or butylamide substitutions for arabino, or cytidine replacement for uracil, increased cytotoxicity. Cytotoxicity was mainly determined by the polar moiety and there was no correlation between antiviral and cytostatic activities. The definition of the effects of shape and chemical groups of the RAFIs opens the possibility to the rational design of lipid-acting antivirals active against a broad spectrum of enveloped viruses.

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.

Perylenyltriazoles inhibit reproduction of enveloped viruses.

1-Substituted 4-perylen-2(3)-yl-1,2,3-triazoles, easily accessible by 'click' reaction and combining in one molecule a polyaromatic unit and a nitrogen heterocycle, were found to strongly inhibit the reproduction of enveloped viruses. 5-[4-(Perylen-3-yl)-1,2,3-triazol-1-yl]-uridine and 2-[1-(2-hydroxyethyl)-1,2,3-triazol-4-yl]perylene show EC50 of 0.031 and 0.023 µM, respectively, against tick-borne encephalitis virus (TBEV). Remarkably, the nucleoside unit appears to be not essential for antiviral activity. These results provide deeper understanding of structural basis of activity for this new class of antivirals.