Influenza viruses can cause severe respiratory infections in humans, leading to nearly half a million deaths worldwide each year. Improved antiviral drugs are needed to address the threat of development of novel pandemic strains. Current therapeutic interventions target three key proteins in the viral life cycle: neuraminidase, the M2 channel and RNA-dependent-RNA polymerase. Protein-protein interactions between influenza polymerase subunits are potential new targets for drug development. Using a newly developed assay based on AlphaScreen technology, we screened a peptide panel for protein-protein interaction inhibitors to identify a minimal PB1 subunit-derived peptide that retains high inhibition potential and can be further modified. Here, we present an X-ray structure of the resulting decapeptide bound to the C-terminal domain of PA polymerase subunit from pandemic isolate A/California/07/2009 H1N1 at 1.6 Å resolution and discuss its implications for the design of specific, potent influenza polymerase inhibitors.

• MeSH
• antivirové látky farmakologie   MeSH
• interakční proteinové domény a motivy účinky léků   fyziologie   MeSH
• krystalizace MeSH
• lidé MeSH
• RNA-dependentní RNA-polymerasa chemie   metabolismus   MeSH
• vazba proteinů MeSH
• virové proteiny antagonisté a inhibitory   chemie   metabolismus   MeSH
• virus chřipky A, podtyp H1N1 účinky léků   enzymologie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap. An inhibitor of this interaction called pimodivir (VX-787) recently entered the third phase of clinical trial; however, several mutations in PB2 that cause resistance to pimodivir were observed. First major mutation, F404Y, causing resistance was identified during preclinical testing, next the mutation M431I was identified in patients during the second phase of clinical trials. The mutation H357N was identified during testing of IAV strains at Centers for Disease Control and Prevention. We set out to provide a structural and thermodynamic analysis of the interactions between cap-binding domain of PB2 wild-type and PB2 variants bearing these mutations and pimodivir. Here we present four crystal structures of PB2-WT, PB2-F404Y, PB2-M431I and PB2-H357N in complex with pimodivir. We have thermodynamically analysed all PB2 variants and proposed the effect of these mutations on thermodynamic parameters of these interactions and pimodivir resistance development. These data will contribute to understanding the effect of these missense mutations to the resistance development and help to design next generation inhibitors.

• MeSH
• krystalografie rentgenová MeSH
• kvantová teorie MeSH
• molekulární modely MeSH
• mutace genetika   MeSH
• mutantní proteiny metabolismus   MeSH
• podjednotky proteinů antagonisté a inhibitory   chemie   metabolismus   MeSH
• proteinové domény MeSH
• pyridiny chemie   farmakologie   MeSH
• pyrimidiny chemie   farmakologie   MeSH
• pyrroly chemie   farmakologie   MeSH
• RNA-dependentní RNA-polymerasa antagonisté a inhibitory   chemie   metabolismus   MeSH
• termodynamika MeSH
• virová léková rezistence účinky léků   MeSH
• virové proteiny antagonisté a inhibitory   chemie   metabolismus   MeSH
• virus chřipky A účinky léků   enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH

A series of 7-deazaadenine ribonucleosides bearing alkyl, alkenyl, alkynyl, aryl, or hetaryl groups at position 7 as well as their 5'-O-triphosphates and two types of monophosphate prodrugs (phosphoramidates and S-acylthioethanol esters) were prepared and tested for antiviral activity against selected RNA viruses (Dengue, Zika, tick-borne encephalitis, West Nile, and SARS-CoV-2). The modified triphosphates inhibited the viral RNA-dependent RNA polymerases at micromolar concentrations through the incorporation of the modified nucleotide and stopping a further extension of the RNA chain. 7-Deazaadenosine nucleosides bearing ethynyl or small hetaryl groups at position 7 showed (sub)micromolar antiviral activities but significant cytotoxicity, whereas the nucleosides bearing bulkier heterocycles were still active but less toxic. Unexpectedly, the monophosphate prodrugs were similarly or less active than the corresponding nucleosides in the in vitro antiviral assays, although the bis(S-acylthioethanol) prodrug 14h was transported to the Huh7 cells and efficiently released the nucleoside monophosphate.

• MeSH
• antivirové látky farmakologie   MeSH
• COVID-19 farmakoterapie   virologie   MeSH
• fosfáty farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• prekurzory léčiv farmakologie   MeSH
• purinové nukleosidy MeSH
• puriny farmakologie   MeSH
• ribonukleosidy farmakologie   MeSH
• RNA-dependentní RNA-polymerasa metabolismus   MeSH
• RNA-viry účinky léků   MeSH
• SARS-CoV-2 účinky léků   MeSH
• virus dengue účinky léků   MeSH
• virus západního Nilu účinky léků   MeSH
• virus zika účinky léků   MeSH
• viry klíšťové encefalitidy účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The biological effects of flavonoids on mammal cells are diverse, ranging from scavenging free radicals and anti-cancer activity to anti-influenza activity. Despite appreciable effort to understand the anti-influenza activity of flavonoids, there is no clear consensus about their precise mode-of-action at a cellular level. Here, we report the development and validation of a screening assay based on AlphaScreen technology and illustrate its application for determination of the inhibitory potency of a large set of polyols against PA N-terminal domain (PA-Nter) of influenza RNA-dependent RNA polymerase featuring endonuclease activity. The most potent inhibitors we identified were luteolin with an IC50 of 72 ± 2 nM and its 8-C-glucoside orientin with an IC50 of 43 ± 2 nM. Submicromolar inhibitors were also evaluated by an in vitro endonuclease activity assay using single-stranded DNA, and the results were in full agreement with data from the competitive AlphaScreen assay. Using X-ray crystallography, we analyzed structures of the PA-Nter in complex with luteolin at 2.0 Å resolution and quambalarine B at 2.5 Å resolution, which clearly revealed the binding pose of these polyols coordinated to two manganese ions in the endonuclease active site. Using two distinct assays along with the structural work, we have presumably identified and characterized the molecular mode-of-action of flavonoids in influenza-infected cells.

• MeSH
• antivirové látky chemie   metabolismus   MeSH
• endonukleasy antagonisté a inhibitory   chemie   metabolismus   MeSH
• enzymatické testy metody   MeSH
• flavonoidy chemie   metabolismus   MeSH
• inhibitory enzymů chemie   metabolismus   MeSH
• krystalografie rentgenová MeSH
• mikrobiální testy citlivosti MeSH
• molekulární struktura MeSH
• preklinické hodnocení léčiv MeSH
• proteinové domény MeSH
• RNA-dependentní RNA-polymerasa antagonisté a inhibitory   chemie   metabolismus   MeSH
• vazba proteinů MeSH
• virové proteiny antagonisté a inhibitory   chemie   metabolismus   MeSH
• virus chřipky A enzymologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH

Remdesivir was shown to inhibit RNA-dependent RNA-polymerases (RdRp) from distinct viral families such as from Filoviridae (Ebola) and Coronaviridae (SARS-CoV, SARS-CoV-2, MERS). In this study, we tested the ability of remdesivir to inhibit RdRps from the Flaviviridae family. Instead of remdesivir, we used the active species that is produced in cells from remdesivir, the appropriate triphosphate, which could be directly tested in vitro using recombinant flaviviral polymerases. Our results show that remdesivir can efficiently inhibit RdRps from viruses causing severe illnesses such as Yellow fever, West Nile fever, Japanese and Tick-borne encephalitis, Zika and Dengue. Taken together, this study demonstrates that remdesivir or its derivatives have the potential to become a broad-spectrum antiviral agent effective against many RNA viruses.

• MeSH
• adenosintrifosfát analogy a deriváty   chemie   farmakologie   MeSH
• antivirové látky chemie   farmakologie   MeSH
• Betacoronavirus účinky léků   enzymologie   MeSH
• COVID-19 MeSH
• Flavivirus účinky léků   enzymologie   MeSH
• inhibiční koncentrace 50 MeSH
• koronavirové infekce farmakoterapie   virologie   MeSH
• lidé MeSH
• pandemie MeSH
• RNA-dependentní RNA-polymerasa antagonisté a inhibitory   metabolismus   MeSH
• RNA-viry účinky léků   enzymologie   MeSH
• SARS-CoV-2 MeSH
• virová pneumonie farmakoterapie   virologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Tick-borne encephalitis virus (TBEV) is a medically important representative of the Flaviviridae family. The TBEV genome encodes a single polyprotein, which is co/post-translationally cleaved into three structural and seven non-structural proteins. Of the non-structural proteins, NS5, contains an RNA-dependent RNA polymerase (RdRp) domain that is highly conserved and is responsible for the genome replication. Screening for potential antivirals was done using a hybrid receptor and ligand-based pharmacophore search likely targeting the RdRp domain. For the identification of pharmacophores, a mixture of small probe molecules and nucleotide triphosphates were used. The ligand/receptor interaction screenings of structures from the ZINC database resulted in five compounds. Zinc 3677 and 7151 exhibited lower cytotoxicity and were tested for their antiviral effect against TBEV in vitro. Zinc 3677 inhibited TBEV at micromolar concentrations. The results indicate that Zinc 3677 represents a good target for structure-activity optimizations leading potentially to a discovery of effective TBEV antivirals.

• MeSH
• antivirové látky farmakologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• klíšťata virologie   MeSH
• klíšťová encefalitida virologie   MeSH
• lidé MeSH
• replikace viru účinky léků   MeSH
• RNA-dependentní RNA-polymerasa antagonisté a inhibitory   genetika   metabolismus   MeSH
• virové proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• viry klíšťové encefalitidy účinky léků   enzymologie   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

RNA-dependent RNA polymerase 3Dpol is a key enzyme for the replication of picornaviruses. The viral genome is translated into a single polyprotein that is subsequently proteolytically processed into matured products. The 3Dpol enzyme arises from a stable 3CD precursor that has high proteolytic activity but no polymerase activity. Upon cleavage of the precursor the newly established N-terminus of 3Dpol is liberated and inserts itself into a pocket on the surface of the 3Dpol enzyme. The essential residue for this mechanism is the very first glycine that is conserved among almost all picornaviruses. However, kobuviruses and siciniviruses have a serine residue instead. Intrigued by this anomaly we sought to solve the crystal structure of these 3Dpol enzymes. The structures revealed a unique fold of the 3Dpol N-termini but the very first serine residues were inserted into a charged pocket in a similar manner as the glycine residue in other picornaviruses. These structures revealed a common underlying mechanism of 3Dpol activation that lies in activation of the α10 helix containing a key catalytical residue Asp238 that forms a hydrogen bond with the 2' hydroxyl group of the incoming NTP nucleotide.

• MeSH
• HeLa buňky MeSH
• Kobuvirus enzymologie   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• mutageneze cílená MeSH
• Picornaviridae enzymologie   MeSH
• průtoková cytometrie MeSH
• RNA-dependentní RNA-polymerasa chemie   genetika   metabolismus   MeSH
• virové proteiny chemie   genetika   metabolismus   MeSH
• vodíková vazba MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

DNA and RNA guanine-rich oligonucleotides can form non-canonical structures called G-quadruplexes or "G4" that are based on the stacking of G-quartets. The role of DNA and RNA G4 is documented in eukaryotic cells and in pathogens such as viruses. Yet, G4 have been identified only in a few RNA viruses, including the Flaviviridae family. In this study, we analysed the last 157 nucleotides at the 3'end of the HCV (-) strand. This sequence is known to be the minimal sequence required for an efficient RNA replication. Using bioinformatics and biophysics, we identified a highly conserved G4-prone sequence located in the stem-loop IIy' of the negative strand. We also showed that the formation of this G-quadruplex inhibits the in vitro RNA synthesis by the RdRp. Furthermore, Phen-DC3, a specific G-quadruplex binder, is able to inhibit HCV viral replication in cells in conditions where no cytotoxicity was measured. Considering that this domain of the negative RNA strand is well conserved among HCV genotypes, G4 ligands could be of interest for new antiviral therapies.

• MeSH
• buněčné linie MeSH
• G-kvadruplexy * MeSH
• Hepacivirus genetika   fyziologie   MeSH
• konzervovaná sekvence MeSH
• lidé MeSH
• replikace viru MeSH
• RNA virová biosyntéza   chemie   genetika   metabolismus   MeSH
• RNA-dependentní RNA-polymerasa metabolismus   MeSH
• sekvence nukleotidů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Molecular dynamics simulations of complexes between Norwalk virus RNA dependent RNA polymerase and its natural CTP and 2dCTP (both containing the O5'-C5'-C4'-O4' sequence of atoms bridging the triphosphate and sugar moiety) or modified coCTP (C5'-O5'-C4'-O4'), cocCTP (C5'-O5'-C4'-C4'') substrates were produced by means of CUDA programmable graphical processing units and the ACEMD software package. It enabled us to gain microsecond MD trajectories clearly showing that similar nucleoside triphosphates can bind surprisingly differently into the active site of the Norwalk virus RNA dependent RNA polymerase. It corresponds to their different modes of action (CTP-substrate, 2dCTP-poor substrate, coCTP-chain terminator, cocCTP-inhibitor). Moreover, extremely rare events-as repetitive pervasion of Arg182 into a potentially reaction promoting arrangement-were captured.

• MeSH
• cytidintrifosfát analogy a deriváty   metabolismus   MeSH
• infekce viry z čeledi Caliciviridae virologie   MeSH
• lidé MeSH
• Norovirus enzymologie   metabolismus   MeSH
• RNA-dependentní RNA-polymerasa metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• simulace molekulového dockingu MeSH
• substrátová specifita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH