Poster 48: Flavivirus Fusion Inhibitors: Identification and Putative Mechanism of ActionEvgenia V. Dueva1, 2, 3, Dmitry I. Osolodkin1, 2, 3, Liubov I. Kozlovskaya3, Victor V. Dotsenko4, Vladimir M. Pentkovski2, Vladimir A. Palyulin1, 2, Galina G. Karganova1, 3, Nikolay S. Zefirov1, 2
|1Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia|
2iScalare Laboratory, Moscow Institute of Physics and Technology, Dolgoprudny, Russia
3FSBI Chumakov Institute of Poliomyelitis and Viral Encephalitides RAMS, Moscow, Russia
4ChemEx Laboratory, Vladimir Dal' East Ukrainian National University, Lugansk, Ukraine
|Tick-borne encephalitis virus (TBEV) belongs to the genus Flavivirus, family Flaviviridae. It is transferred by tick bite, affects central nervous system and causes annually up to 10,000 cases of infections in Europe and Asia. Related viruses, such as Powassan virus (POWV) and Omsk hemorrhagic fever virus, cause more or less severe neurological diseases, and are also responsible for haemorrhagic fever. However, no clinically approved drugs are currently available against these diseases. Thus, development of anti-flaviviral therapeutics is still unsolved, but high priority task.|
Entry of viral particles into the host cell represents the first point available for chemotherapeutics intervention. The fusion of viral and host cell membranes is mediated by flavivirus envelope proteins E, which undergoes rearrangement from dimer to trimer via large-scale pH-dependent conformational changes in monomeric unit. In the crystal structure of dengue virus E protein ectodomain dimer the hydrophobic pocket occupied by a detergent molecule n-octyl-beta-D-glucoside (b-OG) was identified . It was shown that small molecules can bind in this pocket and thus prevent fusion process . This data allowed us to perform homology modelling of E protein ectodomain for TBEV and POWV.
In this work homology model of 'closed' conformation (corresponding to the resting state) of Powassan virus E protein have been constructed using the crystal structures of dengue virus serotype 2 and tick-borne encephalitis virus E proteins as templates (PDB IDs 1OAN and 1SVB, respectively). To obtain 'open' POWV and TBEV E protein models required for docking (corresponding to fusion-inactive state), the structure of dengue E protein in a complex with b-OG (PDB ID 1OKE) was utilized along with 1SVB. The docking-based virtual screening of chemical databases revealed several putative hit compounds against both POWV and TBEV.
Plaque reduction assay of the selected compounds showed that several of them are active against the studied viruses with IC50 values in the micromolar range.
For complexes of two enantiomers of the most active compound with POWV E protein ectodomain dimers as well as for free protein states thirty nanoseconds of molecular dynamics simulations (MD) in implicit water were performed to reveal the mechanistic basis of the fusion inhibition. Clear differences were observed between the MD trajectories of E proteins in the apo form and in complex with the inhibitors, highlighting structural features important for the fusion inhibition. It was also noted that behaviour of ligand-protein complexes varies with different enantiomers. The study of POWV E is of special interest despite the rarity of this virus. Due to transmission by both ticks and mosquitoes, POWV E molecular dynamics makes possible the analysis of host non-specificity determinants.
While histidine residues have been proposed to serve as pH-sensors that trigger conformational rearrangements of surface proteins in fusion process , we also performed simulation of molecular dynamics of E protein dimer models with histidine residues in protonated state to evaluate this hypothesis.
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