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. 2021 Sep 17;9(9):1254.
doi: 10.3390/biomedicines9091254.

Enisamium Inhibits SARS-CoV-2 RNA Synthesis

Stefano Elli  1 , Denisa Bojkova  2 , Marco Bechtel  2 , Thomas Vial  3 , David Boltz  4 , Miguel Muzzio  4 , Xinjian Peng  4 , Federico Sala  1 , Cesare Cosentino  1 , Andrew Goy  5 , Marco Guerrini  1 , Lutz Müller  6 , Jindrich Cinatl  2 , Victor Margitich  5 , Aartjan J W Te Velthuis  3
Affiliations

Enisamium Inhibits SARS-CoV-2 RNA Synthesis

Stefano Elli et al. Biomedicines. .

Abstract

Pandemic SARS-CoV-2 causes a mild to severe respiratory disease called coronavirus disease 2019 (COVID-19). While control of the SARS-CoV-2 spread partly depends on vaccine-induced or naturally acquired protective herd immunity, antiviral strategies are still needed to manage COVID-19. Enisamium is an inhibitor of influenza A and B viruses in cell culture and clinically approved in countries of the Commonwealth of Independent States. In vitro, enisamium acts through metabolite VR17-04 and inhibits the activity of the influenza A virus RNA polymerase. Here we show that enisamium can inhibit coronavirus infections in NHBE and Caco-2 cells, and the activity of the SARS-CoV-2 RNA polymerase in vitro. Docking and molecular dynamics simulations provide insight into the mechanism of action and indicate that enisamium metabolite VR17-04 prevents GTP and UTP incorporation. Overall, these results suggest that enisamium is an inhibitor of SARS-CoV-2 RNA synthesis in vitro.

Keywords: Amizon; COVID-19; FAV00A; RNA polymerase; SARS-CoV-2; molecular dynamics simulation.

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Conflict of interest statement

V.M. and A.G. are employees of the Farmak Public Joint Stock Company, Kiev, Ukraine. Part of this research was funded by the Farmak Public Joint Stock Company, Kiev, Ukraine. The University of Cambridge was compensated by Farmak for experiments performed by A.J.W.t.V.

Figures

Figure 1
Figure 1
Enisamium inhibits SARS-CoV-2 infection of Caco-2 cells in vitro. (A) Chemical structures of enisamium iodide (FAV00A) and VR17-04. The chemical structure of FAV00B is identical to FAV00A except that chloride ions are present instead of iodide. (B) Inhibition of SARS-CoV-2 N expression by enisamium chloride in Caco-2 cells. (C) Inhibition of SARS-CoV-2 cytopathic effect by enisamium iodide and chloride in Caco-2 cells. (D) Effect of enisamium iodide or (E) enisamium chloride on SARS-CoV-2 infected Caco-2 cells.
Figure 2
Figure 2
Enisamium inhibits HCoV-NL63 infection of NHBE cells and SARS-CoV-2 nsp12/7/8 activity in vitro. (A) Quantification of HCoV-NL63 N mRNA levels in NHBE cells infected with HCoV-NL63 after treatment with enisamium chloride. (B). Inhibition of SARS-CoV-2 nsp12/7/8 RNA polymerase complex activity by ensamium as measured with a mini-genome assay. Quantification is from n = 3 independently prepared reactions using the same nsp12/7/8 protein preparation. Error bars represent standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 3
Figure 3
NMR spectrum of enisamium metabolite VR17-04. (A) Schematic of the trans and eclipsed conformations of VR17-04. Dihedral angles are indicated with φ. (B) 2D-NOESY and 1H proton spectra of VR17-04 acquired at 277 K in water. The NOE correlation between the HN and H5′ proton is highlighted with a dashed line. (C) 2D-NOESY and 1H proton spectra of VR17-04 acquired at 277 K in DMSO. The NOE correlation between the HN and H5′ proton is highlighted as in panel B.
Figure 4
Figure 4
Molecular docking of VR17-04 binding into the nsp12/7/8 complex. (A) Schematic of putative hydrogen bond formation between cytosine and adenine bases with VR17-04. (B) Structure of the SARS-CoV-2 nsp12/7/8 complex bound to RNA and remdesivir monophosphate. Rendering based on PDB 7bv2. (C) Docking of VR17-04 binding to cytosine in nsp12 active site. (D) Docking of enisamium binding to cytosine in nsp12 active site. (E) Docking of VR17-04 binding to adenine in nsp12 active site.
Figure 5
Figure 5
MD simulations of VR17-04 binding to nsp12/7/8. (A) MD simulation snapshot of VR17-04 binding to cytosine in nsp12 active site. Black dotted lines indicate hydrogen bonds. Template is shown in bright orange and nascent strand in dark red. Grey spheres represent magnesium ions. Positive charges on amino acids and base are dark blue and negative charges light red. Nsp12 is colored light blue and shown in a cartoon presentation. (B) MD simulation snapshot of enisamium binding to cytosine in nsp12 active site. Colors as in Figure 5A. (C) MD simulation plot of hydrogen bond distances during VR17-04 binding to cytosine in nsp12 active site (left) and histogram of hydrogen bond distances (right). (D) MD simulation of hydrogen bond distance (C=O---HN-Cyt, blue color) during enisamium binding to cytosine in nsp12 active site. Since no second hydrogen bond can form in the case of enisamium binding to nsp12/7/8, the distance C-H---HN-Cyt is reported in magenta color for comparison. (E) MD simulation plot of the coplanarity angle of VR17-04 or enisamium binding to cytosine in nsp12 active site. (F) MD simulation of hydrogen bond distances during VR17-04 binding to adenine in nsp12 active site. (G) MD simulation of coplanarity angle of VR17-04 binding to cytosine or adenine in nsp12 active site.
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