Enisamium is an inhibitor of the SARS-CoV-2 RNA polymerase and shows improvement of recovery in COVID-19 patients in an interim analysis of a clinical trial

Abstract

Pandemic SARS-CoV-2 causes a mild to severe respiratory disease called Coronavirus Disease 2019 (COVID-19). Control of SARS-CoV-2 spread will depend on vaccine-induced or naturally acquired protective herd immunity. Until then, antiviral strategies are needed to manage COVID-19, but approved antiviral treatments, such as remdesivir, can only be delivered intravenously. Enisamium (laboratory code FAV00A, trade name Amizon®) is an orally active inhibitor of influenza A and B viruses in cell culture and clinically approved in countries of the Commonwealth of Independent States. Here we show that enisamium can inhibit SARS-CoV-2 infections in NHBE and Caco-2 cells. In vitro, the previously identified enisamium metabolite VR17-04 directly inhibits the activity of the SARS-CoV-2 RNA polymerase. Docking and molecular dynamics simulations suggest that VR17-04 prevents GTP and UTP incorporation. To confirm enisamium's antiviral properties, we conducted a double-blind, randomized, placebo-controlled trial in adult, hospitalized COVID-19 patients, which needed medical care either with or without supplementary oxygen. Patients received either enisamium (500 mg per dose) or placebo for 7 days. A pre-planned interim analysis showed in the subgroup of patients needing supplementary oxygen (n = 77) in the enisamium group a mean recovery time of 11.1 days, compared to 13.9 days for the placebo group (log-rank test; p=0.0259). No significant difference was found for all patients (n = 373) or those only needing medical care (n = 296). These results thus suggest that enisamium is an inhibitor of SARS-CoV-2 RNA synthesis and that enisamium treatment shortens the time to recovery for COVID-19 patients needing oxygen.

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

Figure 1.. Enisamium inhibits SARS-CoV-2 infection and replication in vitro.

(A) Chemical structures of 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 in Caco-2 cells by enisamium chloride. (C) Inhibition of SARS-CoV-2 cytopathic effect in Caco-2 cells by enisamium iodide and chloride. (D) Quantification of SARS-CoV-2 RNA genome levels in NHBE cells infected with SARS-CoV-2 after treatment with enisamium iodide. (E) Quantification of HCoV-NL63 N mRNA levels in NHBE cells infected with HCoV-NL63 after treatment with enisamium chloride. (F) Inhibition of the SARS-CoV-2 nsp12/7/8 RNA polymerase complex by VR17–04 on a hairpin template. A mutant containing a double amino acid substitution in the nsp12 active site (SDD=>SAA) was used as negative control. DMSO was used as solvent control. (G) Quantification of SARS-CoV-2 nsp12/7/8 RNA polymerase complex inhibition by enisamium or VR17–04 on a hairpin template. For VR17–04 two nucleotide triphosphate concentrations were used. Polymerase activity was plotted against drug concentration and dose-response curves were fit to the data. Quantification is from n=3 independently prepared reactions using the same nsp12/7/8 protein preparation. Error bars represent standard deviation.

Figure 2.. Enisamium metabolite VR17–04.

(A) Schematic of putative hydrogen bond formation between cytosine and adenine bases with VR17–04. (B) Schematic of the trans and eclipsed conformations of VR17–04. (C) 2D-NOESY and 1H proton (above) 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. (D) Structure of the SARS-CoV-2 nsp12/7/8 complex bound to RNA and remdesivir monophosphate. Rendering based on PDB 7bv2. (E) Model (top) and MD simulation (bottom) of VR17–04 binding to cytosine in nsp12 active site. (F) Model (top) and MD simulation (bottom) of enisamium binding to cytosine in nsp12 active site. (G) Model (top) and MD simulation (bottom) of VR17–04 binding to adenine in nsp12 active site. (H) MD simulation of dihedral angle of VR17–04 or enisamium binding to cytosine in nsp12 active site. (I) MD simulation of dihedral angle of VR17–04 binding to cytosine or adenine in nsp12 active site (J) Effect of VR17–04 on SARS-CoV-2 nsp12/7/8 activity on two different hairpin templates in the presence of GTP (left) or UTP (right). In the presence of wildtype nsp12/7/8 and GTP or UTP, the radiolabelled primer was extended by 1 nt. (K) Effect of VR17–04 on SARS-CoV-2 nsp12/7/8 primer extension activity. A mutant containing a double amino acid substitution in the nsp12 active site (SDD=>SAA) was used as negative control. ATP and GTP were added to the reaction to allow extension of the template by 2 nt.

Figure 3.. Enisamium improves recovery of COVID-19 patients requiring supplementary oxygen.

(A) Schematic of patient recruitment, randomization and treatment. (B) Kaplan-Meier plot of the percentage improvement observed in all COVID-19 patients, (C) RS 5 COVID-19 patients (medical care with no oxygen support), and (D) RS 4 COVID-19 patients (medical care with non-invasive oxygen support).