Preparation and characterization of inactivated tick-borne encephalitis virus samples for single-particle imaging at the European XFEL.

X-ray imaging of virus particles at the European XFEL could eventually allow their complete structures to be solved, potentially approaching the resolution of other structural virology methods. To achieve this ambitious goal with today's technologies, about 1 ml of purified virus suspension containing at least 1012 particles per millilitre is required. Such large amounts of concentrated suspension have never before been obtained for enveloped viruses. Tick-borne encephalitis virus (TBEV) represents an attractive model system for the development of enveloped virus purification and concentration protocols, given the availability of large amounts of inactivated virus material provided by vaccine-manufacturing facilities. Here, the development of a TBEV vaccine purification and concentration scheme is presented combined with a quality-control protocol that allows substantial amounts of highly concentrated non-aggregated suspension to be obtained. Preliminary single-particle imaging experiments were performed for this sample at the European XFEL, showing distinct diffraction patterns.

Elongation of N6-benzyladenosine scaffold via Pd-catalyzed C-C bond formation leads to derivatives with antiflaviviral activity.

Decoration of nucleoside analogues with lipophilic groups often leads to compounds with improved antiviral activity. For example, N6-benzyladenosine derivatives containing elongated lipophilic substituents in the benzyl core efficiently inhibit reproduction of tick-borne encephalitis virus (TBEV), while N6-benzyladenosine itself potently inhibits reproduction of human enterovirus A71 (EV-A71). We have extended a series of N6-benzyladenosine analogues using effective synthetic methods of CC bond formation based on Pd-catalyzed cross-coupling reactions (Sonogashira and Suzuki) in order to study the influence of bulky lipophilic substituents in the N6 position of adenosine on the antiviral activity against flaviviruses, such as TBEV, yellow fever virus (YFV) and West Nile virus (WNV), as well as a panel of enteroviruses including EV-A71, Echovirus 30 (E30), and poliovirus type 2 (PV2). Reproduction of tested flaviviruses appeared to be inhibited by the micromolar concentrations of the compounds, while cytotoxicity in most cases was beyond the detection limit. Time-of-addition studies demonstrated that the hit compounds inhibited the stage of viral RNA synthesis, but not the stages of the viral entry or protein translation. As a result, several new promising antiflaviviral leads have been identified. On the other hand, none of the synthesized compounds inhibited enterovirus reproduction, indicating a possibility of involvement of flavivirus-specific pathways in their mechanism of action.

3-[N,N-Bis(sulfonyl)amino]isoxazolines with Spiro-Annulated or 1,2-Annulated Cyclooctane Rings Inhibit Reproduction of Tick-Borne Encephalitis, Yellow Fever, and West Nile Viruses.

Spirocyclic compounds containing heterocyclic moieties represent promising 3D scaffolds for modern drug design. In the search for novel anti-flaviviral agents, we have obtained a series of 3-[N,N-bis(sulfonyl)amino]isoxazolines containing spiro-annulated cyclooctane rings and assessed their antiviral activity against tick-borne encephalitis (TBEV), yellow fever (YFV), and West Nile (WNV) viruses. The structural analogs of spirocyclic compounds with a single sulfonyl group or 1,2-annulated cyclooctane ring were also investigated. Almost all the studied 3-[N,N-bis(sulfonyl)amino]isoxazolines revealed antiviral activity against TBEV and WNV. The most active against TBEV was spiro-isoxazoline derivative containing p-nitrophenyl groups in the sulfonyl part (EC50 2.0 ± 0.5 µM), while the highest potency against WNV was found for the compounds with lipophilic substituents in sulfonyl moiety, naphtyl being the most favorable one (EC50 1.3 ± 0.5 µM). In summary, two novel scaffolds of anti-flaviviral agents based on N,N-bis(sulfonyl)amino]isoxazoline were proposed, and the compounds of this type demonstrated activity against TBEV and WNV.

Tetrahydroquinazoline N-oxide derivatives inhibit reproduction of tick-borne and mosquito-borne flaviviruses.

Tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), and West Nile virus (WNV) are flaviviruses causing emerging arthropod-borne infections of a great public health concern. Clinically approved drugs are not available to complement or replace the existing vaccines, which do not provide sufficient coverage. Thus, the discovery and characterization of new antiflaviviral chemotypes would advance studies in this field. In this study, a series of tetrahydroquinazoline N-oxides was synthesized, and the antiviral activity of the compounds was assessed against TBEV, YFV, and WNV using the plaque reduction assay along with the cytotoxicity to the corresponding cell lines (porcine embryo kidney and Vero). Most of the studied compounds were active against TBEV (EC50 2 to 33 µM) and WNV (EC50 0.15 to 34 µM) and a few also demonstrated inhibitory activity against YFV (EC50 0.18 to 41 µM). To investigate the potential mechanism of action of the synthesized compounds, time-of-addition (TOA) experiments and virus yield reduction assays were performed for TBEV. The TOA studies suggested that the antiviral activity of the compounds should affect the early stages of the viral replication cycle after cell entry. Compounds with tetrahydroquinazoline N-oxide scaffold show a broad spectrum of activity against flaviviruses and represent a promising chemotype for antiviral drug discovery.

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.

Getting to Know the Neighbours with GTM: The Case of Antiviral Compounds.

Recent outbreaks of dangerous viral infections, such as Ebola virus disease, Zika fever, etc., are forcing the search for new antiviral compounds. Preferably, such compounds should possess broad-spectrum antiviral activity, as the development of drugs for the treatment of dozens of viral infections lacking specific treatment would require significant resources. Antiviral activity data present in public resources are very sparse and further investigation of structure-activity relationships is necessary. One of the strategies could be the investigation of chemical space around known active compounds and assessment of activity against closely related viruses in order to fill in the antiviral activity matrix. Here we present an investigation of antiviral activity using universal maps built with generative topographic mapping (GTM) algorithm. The GTM-based maps were used to find commercially available compounds in close proximity to already known compounds with anti-flaviviral and anti-enteroviral activities. Selected compounds were then assessed in cell-based assays against tick-borne encephalitis virus (TBEV) and a panel of enteroviruses. This approach allowed us to identify 23 new compounds showing anti-TBEV activity with EC50 values in micromolar and submicromolar range.

Antiviral activity spectrum of phenoxazine nucleoside derivatives.

The phenoxazine scaffold is widely used to stabilize nucleic acid duplexes, as a part of fluorescent probes for the study of nucleic acid structure, recognition, and metabolism, etc. Here we present the synthesis of phenoxazine-based nucleoside derivatives and their antiviral activity against a panel of structurally diverse viruses: enveloped DNA herpesviruses varicella zoster virus (VZV) and human cytomegalovirus, enveloped RNA tick-borne encephalitis virus (TBEV), and non-enveloped RNA enteroviruses. Studied compounds were effective against DNA and RNA viruses reproduction in cell culture. 3-(2'-Deoxy-ß-D-ribofuranosyl)-1,3-diaza-2-oxophenoxazine proved to be a potent inhibitor of VZV replication with superior activity against wild type than thymidine kinase deficient strains (EC50 0.06 and 10 µM, respectively). This compound did not show cytotoxicity on all the studied cell lines. Several compounds showed promising activity against TBEV (EC50 0.35-0.91 µM), but the activity was accompanied by pronounced cytotoxicity. These compounds may be considered as a good starting point for further structure optimization as antiherpesviral or antiflaviviral compounds.

Ramified derivatives of 5-(perylen-3-ylethynyl)uracil-1-acetic acid and their antiviral properties.

The propargylamide of N3-Pom-protected 5-(perylen-3-ylethynyl)uracil acetic acid, a universal precursor, was used in a CuAAC click reaction for the synthesis of several derivatives, including three ramified molecules with high activities against tick-borne encephalitis virus (TBEV). Pentaerythritol-based polyazides were used for the assembly of molecules containing 2⋯4 antiviral 5-(perylen-3-ylethynyl)uracil scaffolds, the first examples of polyvalent perylene antivirals. Cluster compounds showed enhanced absorbance, however, their fluorescence was reduced due to self-quenching. Due to the solubility issues, Pom group removal succeeded only for compounds with one peryleneethynyluracil unit. Four compounds, including one ramified cluster 9f, showed remarkable 1⋯3 nM EC50 values against TBEV in cell culture.