Molecular mechanism of rhinovirus escape from the Pyrazolo[3,4-d]pyrimidine capsid-binding inhibitor OBR-5-340 via mutations distant from the binding pocket: Derivatives that brake resistance.

Rhinoviruses (RVs) cause the common cold. Attempts at discovering small molecule inhibitors have mainly concentrated on compounds supplanting the medium chain fatty acids residing in the sixty icosahedral symmetry-related hydrophobic pockets of the viral capsid of the Rhinovirus-A and -B species. High-affinity binding to these pockets stabilizes the capsid against structural changes necessary for the release of the ss(+) RNA genome into the cytosol of the host cell. However, single-point mutations may abolish this binding. RV-B5 is one of several RVs that are naturally resistant against the well-established antiviral agent pleconaril. However, RV-B5 is strongly inhibited by the pyrazolopyrimidine OBR-5-340. Here, we report on isolation and characterization of RV-B5 mutants escaping OBR-5-340 inhibition and show that substitution of amino acid residues not only within the binding pocket but also remote from the binding pocket hamper inhibition. Molecular dynamics network analysis revealed that strong inhibition occurs when an ensemble of several sequence stretches of the capsid proteins enveloping OBR-5-340 move together with OBR-5-340. Mutations abrogating this dynamic, regardless of whether being localized within the binding pocket or distant from it result in escape from inhibition. Pyrazolo [3,4-d]pyrimidine derivatives overcoming OBR-5-340 escape of various RV-B5 mutants were identified. Our work contributes to the understanding of the properties of capsid-binding inhibitors necessary for potent and broad-spectrum inhibition of RVs.

A Study of the Activity of Adamantyl Amines against Mutant Influenza A M2 Channels Identified a Polycyclic Cage Amine Triple Blocker, Explored by Molecular Dynamics Simulations and Solid-State NMR.

We compared the anti-influenza potencies of 57 adamantyl amines and analogs against influenza A virus with serine-31 M2 proton channel, usually termed as WT M2 channel, which is amantadine sensitive. We also tested a subset of these compounds against viruses with the amantadine-resistant L26F, V27A, A30T, G34E M2 mutant channels. Four compounds inhibited WT M2 virus in vitro with mid-nanomolar potency, with 27 compounds showing sub-micromolar to low micromolar potency. Several compounds inhibited L26F M2 virus in vitro with sub-micromolar to low micromolar potency, but only three compounds blocked L26F M2-mediated proton current as determined by electrophysiology (EP). One compound was found to be a triple blocker of WT, L26F, V27A M2 channels by EP assays, but did not inhibit V27A M2 virus in vitro, and one compound inhibited WT, L26F, V27A M2 in vitro without blocking V27A M2 channel. One compound blocked only L26F M2 channel by EP, but did not inhibit virus replication. The triple blocker compound is as long as rimantadine, but could bind and block V27A M2 channel due to its larger girth as revealed by molecular dynamics simulations, while MAS NMR informed on the interaction of the compound with M2(18-60) WT or L26F or V27A.

1H NMR-Based Biochemometric Analysis of Morus alba Extracts toward a Multipotent Herbal Anti-Infective.

Mulberry Diels-Alder-type adducts (MDAAs) derived from the white mulberry tree were discovered recently as dual inhibitors of influenza viruses and pneumococci. For the development of a natural product based remedy for respiratory infections, the aim was to (i) identify the most prolific natural source of MDAAs, (ii) develop a protocol to maximize the content of MDAAs in Morus alba extracts, (iii) unravel constituents with the highest anti-infective potential within multicomponent mixtures, and (iv) select and characterize a hit extract as a candidate for further studies. Validated quantitative UPLC-PDA analysis of seven MDAAs (1-7) revealed the root bark as the best starting material and pressurized liquid extraction (PLE) as the optimum technique for extraction. Extracts enriched in MDAAs of a total content above 20% exerted a potent dual anti-influenza virus and antipneumococcal activity. For a detailed analysis of the most bioactive chemical features and molecules within the extracts, 1H NMR-based heterocovariance analysis (HetCA) was used. According to the multivariate statistical analysis procedure conducted, MDAAs exclusively accounted for the in vitro anti-influenza viral effect. The anti-infective profile of one hit extract (MA60) investigated showed a good tolerance by lung cells (A549, Calu-3) and pronounced in vitro activities against influenza viruses, S. pneumoniae, S. aureus, and inflammation.

Insights into the direct anti-influenza virus mode of action of Rhodiola rosea.

BACKGROUND: The anti-influenza A virus activities and contents of previously isolated most active flavonoids (rhodiosin and tricin) from a standardized hydro-ethanolic R. rosea root and rhizome extract (SHR-5®), did not fully explain the efficacy of SHR-5®. Moreover, the mode of antiviral action of SHR-5® is unknown. PURPOSE: To determine the anti-influenza viral principle of SHR-5® we evaluated i) the combined anti-influenza virus effect of rhodiosin and tricin, ii) the impact of its tannin-enriched fraction (TE), iii) its antiviral spectrum and mode of action, and iv) its propensity for resistance development in vitro. METHODS: The combined anti-influenza virus effect of rhodiosin and tricin and the impact of TE were investigated with cytopathic effect (CPE)-inhibition assays in MDCK cells. A tannin-depleted fraction (TD) and TE were prepared by polyamide column chromatography and dereplicated by LC-MS. Plaque-reduction assays provided insights into the anti-influenza virus profile, the mode of action, and the propensity for resistance development of SHR-5®. RESULTS: Our results i) did not reveal synergistic anti-influenza A virus effects of rhodiosin and tricin, but ii) proved a strong impact of TE mainly composed of prodelphinidin gallate oligomers. iii) TE inhibited the plaque-production of influenza virus A(H1N1)pdm09, A(H3N2), and B (Victoria and Yamagata) isolates (including isolates resistant to neuraminidase and/or M2 ion channel inhibitors) with 50% inhibitory concentration values between 0.12 - 0.53 µg/ml similar to SHR-5®. Mechanistic studies proved a virucidal activity, inhibition of viral adsorption, viral neuraminidase activity, and virus spread by SHR-5® and TE. iv) No resistance development was observed in vitro. CONCLUSION: For the first time a comprehensive analysis of the anti-influenza virus profile of a hydro-ethanolic R. rosea extract (SHR-5®) was assessed in vitro. The results demonstrating broad-spectrum multiple direct anti-influenza virus activities, and a lack of resistance development to SHR-5® together with its known augmentation of host defense, support its potential role as an adaptogen against influenza virus infection.

High-performance Countercurrent Chromatography to Access Rhodiola rosea Influenza Virus Inhibiting Constituents.

In a cytopathic effect inhibition assay, a standardized Rhodiola rosea root and rhizome extract, also known as roseroot extract (SHR-5), exerted distinct anti-influenza A virus activity against HK/68 (H3N2) (IC50 of 2.8 µg/mL) without being cytotoxic. For fast and efficient isolation and identification of the extract's bioactive constituents, a high-performance countercurrent chromatographic separation method was developed. It resulted in a three-stage gradient elution program using a mobile phase solvent system composed of ethyl acetate/n-butanol/water (1 : 4 : 5 → 2 : 3 : 5 → 3 : 2 : 5) in the reversed-phase mode. The elaborated high-performance countercurrent chromatographic method allowed for fractionation of the complex roseroot extract in a single chromatographic step in a way that only one additional orthogonal isolation/purification step per fraction yielded 12 isolated constituents. They cover a broad polarity range and belong to different structural classes, namely, the phenylethanoid tyrosol and its glucoside salidroside, the cinnamyl alcohol glycosides rosavin, rosarin, and rosin as well as gallic acid, the cyanogenic glucoside lotaustralin, the monoterpene glucosides rosiridin and kenposide A, and the flavonoids tricin, tricin-5-O-ß-D-glucopyranoside, and rhodiosin. The most promising anti-influenza activities were determined for rhodiosin, tricin, and tricin-5-O-ß-D-glucopyranoside with IC50 values of 7.9, 13, and 15 µM, respectively. The herein established high-performance countercurrent chromatographic protocol enables fast and scalable access to major as well as minor roseroot constituents. This is of particular relevance for extract standardization, quality control, and further in-depth pharmacological investigations of the metabolites of this popular traditional herbal remedy.

Comparative in vitro analysis of inhibition of rhinovirus and influenza virus replication by mucoactive secretolytic agents and plant extracts.

BACKGROUND: Rhinoviruses and influenza viruses cause millions of acute respiratory infections annually. Symptoms of mild acute respiratory infections are commonly treated with over-the-counter products like ambroxol, bromhexine, and N-acetyl cysteine, as well as of thyme and pelargonium extracts today. Because the direct antiviral activity of these over-the-counter products has not been studied in a systematic way, the current study aimed to compare their inhibitory effect against rhinovirus and influenza virus replication in an in vitro setting. METHODS: The cytotoxicity of ambroxol, bromhexine, and N-acetyl cysteine, as well as of thyme and pelargonium extracts was analyzed in Madin Darby canine kidney (MDCK) and HeLa Ohio cells. The antiviral effect of these over-the-counter products was compared by analyzing the dose-dependent inhibition (i) of rhinovirus A2- and B14-induced cytopathic effect in HeLa Ohio cells and (ii) of influenza virus A/Hong Kong/68 (subtype H3N2)- and A/Jena/8178/09 (subtype H1N1, pandemic)-induced cytopathic effect in MDCK cells at non-cytotoxic concentrations. To get insights into the mechanism of action of pelargonium extract against influenza virus, we performed time-of-addition assays as well as hemagglutination and neuraminidase inhibition assays. RESULTS: N-acetyl cysteine, thyme and pelargonium extract showed no or only marginal cytotoxicity in MDCK and HeLa Ohio cells in the tested concentration range. The 50% cytotoxic concentration of ambroxol and bromhexine was 51.85 and 61.24 µM, respectively. No anti-rhinoviral activity was detected at non-cytotoxic concentrations in this in vitro study setting. Ambroxol, bromhexine, and N-acetyl cysteine inhibited the influenza virus-induced cytopathic effect in MDCK cells no or less than 50%. In contrast, a dose-dependent anti-influenza virus activity of thyme and pelargonium extracts was demonstrated. The time-of addition assays revealed an inhibition of early and late steps of influenza virus replication by pelargonium extract whereas zanamivir acted on late steps only. The proven block of viral neuraminidase activity might explain the inhibition of influenza virus replication when added after viral adsorption. CONCLUSION: The study results indicate a distinct inhibition of influenza A virus replication by thyme and pelargonium extract which might contribute to the beneficial effects of these plant extracts on acute respiratory infections symptoms.

Genetic polymorphism of thymidine kinase (TK) and DNA polymerase (pol) of clinical varicella-zoster virus (VZV) isolates collected over three decades.

BACKGROUND: Genotypic resistance testing of varicella-zoster virus (VZV) strains to antivirals is of high relevance in immunocompromised patients with VZV reactivations unresponsive to therapy. However, the knowledge on mutations associated with natural gene polymorphism or resistance is limited. OBJECTIVES: To examine the genotype of the thymidine kinase (TK) and DNA polymerase (pol) of unselected clinical VZV isolates collected between 1984 and 2014 and to verify the phenotype related to novel amino acid (aa) substitutions. STUDY DESIGN: The TK and DNA pol genes of 169 VZV isolates were analyzed by amplification and sequencing. Sequences were compared to that of the reference strain Dumas. The phenotype to acyclovir and other antivirals was examined in isolates with novel aa substitutions using modified plaque reduction assay. RESULTS: In the TK of four strains, four different aa substitutions were detected, apart from the known change S288L that was present in all strains compared to Dumas. All four substitutions have hitherto not been described in the literature and were phenotypically classified as natural gene polymorphisms although two out of them (S51L, K186R) were localized in conserved gene centers. The DNA pol of 34 isolates exhibited 19 different substitutions, 14 out of them were novel, and two (R753K, V777I) were within conserved gene regions. Again, these changes were characterized as natural gene polymorphisms. CONCLUSIONS: Non-synonymous mutations in VZV TK or DNA pol conferring natural gene polymorphism are rare events. Nevertheless, the phenotypic characterization of 18 novel polymorphisms can help to provide a better identification of resistance mutations.

Minimization of a eukaryotic mini-intein.

Inteins are internal protein splicing elements that can autocatalytically self-excise from their host protein and ligate the protein flanks (exteins) with a peptide bond. Large inteins comprise independent protein splicing and endonuclease domains whereas mini-inteins lack the central endonuclease domain. To identify mini-intein domains that are essential for protein splicing, deletions were introduced at different sites of the 157-aa PRP8 mini-intein of Penicillium chrysogenum. The removal of eight and six amino acids at two different sites resulted in a functional eukaryotic mini-intein of only 143 aa.