Ribavirin shows antiviral activity against SARS-CoV-2 and downregulates the activity of TMPRSS2 and the expression of ACE2 In Vitro

Ribavirin is a guanosine analog and has a broad-spectrum antiviral activity against RNA viruses. Based on this, we aimed to show the anti-SARS-CoV-2 activity of this drug molecule via in vitro, in silico and molecular techniques. Ribavirin showed antiviral activity in Vero E6 cells following SARS-CoV-2 infection. In silico analysis suggested that Ribarivin has a broad-spectrum impact on Vero E6 cells. According to the detailed molecular techniques, Ribavirin was shown to decrease TMPRSS2 expression both at mRNA and protein level 48 hours after treatment. The suppressive effect of Ribavirin in ACE2 protein expression was shown to be dependent on cell types. Finally, proteolytic activity assays showed that Ribavirin also showed an inhibitory effect on TMPRSS2 enzyme. As a conclusion, Ribavirin is a potential antiviral drug for the treatment against SARS-CoV-2, and it interferes with the effect of TMPRSS2 and ACE2 expression.


Introduction
The coronavirus diseases 2019 (COVID-19) pandemic represents the hardest global public health threat since the influenza outbreak in 1918 and has rapidly resulted in significant health and economic burden worldwide [1]. Pre-clinical and clinical trials for the treatment of COVID- 19 have been ongoing in various centers across the World. Currently, there are 2271 clinical trials which involves therapeutic or preventative interventions registered at www.clinicaltrials.gov. Among these trials, different therapeutic approaches have been studies including antiviral, antimalarial, immunomodulator and cell/plasma based therapies [1].
However, results of pre-clinical and clinical studies may vary between each other and there is still no definite therapeutic strategy.
Drug repurposing is a method preferred by researchers to introduce new therapeutics, reduce the time and financial burden of developing molecules or drugs [2]. Ribavirin, one of the FDAapproved antiviral drugs used for the last four decades against RNA viruses, could be a potential candidate to be used for drug repurposing against COVID-19. Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) with broad-spectrum antiviral properties, first synthesized in 1972 [3], has been used mostly to treat hepatitis C virus (HCV) infections in humans. Although ribavirin is used almost exclusively in hepatitis C virus infections in clinical practice, it has an antiviral effect against different viruses [4]. Furthermore, ribavirin has suitable formulations for inhalation, or intravenous administration and oral use. Due to this feature, it has been also preferred in the treatment of different viral infections. It is well-known that ribavirin molecule has different antiviral mechanisms of action against different viruses [5]. In one these mechanisms, ribavirin monophosphate can potently inhibit guanosine derivatives (eg guanosine triphosphate [GTP]) and reduce nucleotide residues resulting in antiviral effect, especially in studies with respiratory syncytial virus (RSV), yellow fever virus and paramyxovirus [6][7][8][9].
Secondly, the main intracellular metabolite of ribavirin is ribavirin triphosphate (RTP) which can act through polymerase inhibition. RTP can bind competitively with molecules such as adenosine triphosphate (ATP) or GTP and inducing the inhibition of polymerases. This mechanism of action has been observed in studies conducted with influenza virus, reovirus and vesicular stomatitis virus [4,10,11]. As a final mechanism of action; ribavirin molecule can bind with translation initiation factor 4E (eIF4E), preventing the initiation of translation, or interacting with the enzymes responsible for RNA cap synthesis, preventing the initiation of translation. This mechanism has been reported via in silico molecular docking analysis for the SARS-CoV-2 virus and Lassa fever [12]. Ribavirin is also used in Crimean-Congo haemorrhagic fever, coronavirus, South American haemorrhagic fevers, hantavirus and adenovirus infections [4]. As stated above, ribavirin exerts antiviral effects in different viruses via exerting different mechanisms. Both its ease of use and its ability to be used against different viruses; suggest that ribavirin is a promising potent and broad-spectrum antiviral drug. For this reason, in this study, the antiviral effect of ribavirin against SARS-CoV-2 has been studies in detail via in silico, in vitro and molecular biology analysis.

Cells and viruses
The  30minutes. The supernatants were collected separately from each well. They were centrifugated at low-speed (3000 rpm, 10 min at 4°C) to remove the debris. The cleared supernantants were replaced to a new 96 well plate. Fluorescence intensity was measured using fluorescence spectrometer at 380 nm (excitation) and 460 (emission).

Statistically Analysis
Statistically analysis was performed to evaluate significancy for each data. MTT, Flow cytometry, western blot and proteolytic activity assays were performed in three biological replicates, and qRT-PCR assays were performed in six biological replicates. The median values of the control groups and drug-treated groups were compared with paired two-tailed T test.
Analysis of variance was conducted on the replicate values of experiment groups. P values <0.05 was accepted as statistically significant. * indicates that p<0.05. The data was analyzed using Graph Pad Prism 8.01. In order to determine the antiviral activity of ribavirin, SARS-CoV-2-Ank1 infected Vero E6 cells were treated with drug molecules across a concentration range (100 µM -5 pM). Cells at which were only infected with SARS-CoV-2-Ank1 showed full CPE at day 4. Therefore, the rest of the samples were analyzed at this time point. According to Figure 1B, it was determined that the lowest 4 µM Ribavirin concentration stopped the infectivity of SARS-CoV-2-Ank1, at the level of 100% under in vitro conditions. It was also observed that the viral infectivity was stopped at a level of 50% when drug was used at a concentration of 800 nM.

Ribarivin has a broad-spectrum impact on Vero E6 cells as evident by in silico analysis
According

Ribavirin decreases TMPRSS2 expression in Vero E6 cells
Since in silico docking analysis showed that Ribavirin has a broad-spectrum impact on Vero E6, we performed further molecular analysis to delineate the effect of the drug on viral lifecycle.  Figure 3A). On the contrary, any significant change was not observed at 48th hours in Caco 2 cells ( Figure 3A).  Figure 3B). These data suggested that Ribavirin treatment significantly reduces TMPRSS2 expression both at protein and gene level at the end of the 48 hours-treatment.

The suppressive effect of Ribavirin in ACE2 protein expression changes depending on the cell type
In addition to evaluation of protein level of TMPRSS2, to elucidate the mechanism of Ribavirin in the regulation of ACE2 expression at protein level we performed western blotting assay. Our

Ribavirin has an inhibitory effect in proteolytic activity of TMPRSS2 enzyme
A well-known mechanism is that SARS-CoV-2 entries a host cell using its spike protein appearing on the surface of the virus binds ACE2 receptor on the host cell surface. After this binding, the virus primes two cut sites and causes a conformational change which allows the fusion of viral and host membranes. Besides ACE2, TMPRSS2 has an extracellular protease domain that cleaves the spike protein to begin membrane fusion. Due to this reason the inhibition of TMPRSS2 protease activity might be a valuable and important sign to analyze the anti-viral effect of drug at molecular level [14]. In vitro measurement of a fluorescence resonance energy transfer of a synthetic protease substrate (Boc-Gln-Ala-Arg-AMC, ENZO Life Sciences) is a very useful tool to examine the TMPRSS2-activated matriptase proteolytic activities [15]. The substrate protein was added into Vero-E6 and Caco-2 cell cultures which were treated with 5, 10, and 25 µM Ribavirin for 48 hours. In accordance with the measurement of soluble forms of proteins released from cells, we detected proteolytic activity of TMPRSS2 in the supernatant of cell culture. It was observed that Ribavirin treatment significantly reduces TMRPRSS2 activity in a dose dependent manner both in Vero-E6 and Caco-2 ( Figure 5) cells comparing to the control. This data indicates that Ribavirin has an inhibitory role in the proteolytic activity of TMPRSS2 enzyme.

Discussion
Ribavirin is a guanosine analog and has a broad-spectrum antiviral activity against RNA interacts with angiotensin converting enzyme 2 (ACE2) receptor located on host organism membrane for viral cell entry [16,17]. Transmembrane protease serine 2 (TMPRSS2) enzyme located on host cell membrane cleaves S protein and reveals S2 domain of S protein which leading to fusion of the viral membrane with the host cell membrane to release viral +ssRNA genome into a host cell cytoplasm [18,19]. Chloroquine phosphate and hydroxychloroquine suggested to inhibit ACE2 [20]. Camostat mesylate and Nafamostat mesylate suggested for the inhibition of TMPRSS2 priming [21]. Therefore, TMPRSS2 is one of the potential targets for anti-SARS-CoV-2 drug development. In this study, we have shown that Ribavirin treatment of Vero E6 cells decreased the expression of TMPRSS2 which could explain the mechanism of action against SARS-CoV-2. The mechanism linking ribavirin to TMPRSS2 expression is beyond the scope of the present study, however we have identified a few possible mechanisms.
A possible explanation for the observed decrease in TMPRSS2 expression is that ribavirinmediated GTP depletion hinders the signaling cascades that activate the androgen receptor (AR), a potent activator of TMPRSS2 expression [22,23]. Ribavirin monophosphate (RMP)mediated inhibition of inosine monophosphate dehydrogenase (IMPDH) is a prominent antiviral mechanism occurring even at relatively low concentrations of ribavirin [22,23]. IMPDH is responsible for the production of GMP (a GTP precursor) from inosine monophosphate (IMP). RMP competes with IMP for binding to IMPDH, thereby reducing GMP and GTP synthesis [24]. Competitive inhibition of IMPDH by RMP depletes local GTP, causing downstream imbalance of nucleotides that interferes with mRNA capping, ribosomal function, and G-protein signaling [22,24]. Because AR is activated by the MAPK pathway involving the G-protein Ras [25], one could conjecture that depletion of GTP could indirectly dampen AR activity and by extension TMPRSS2 expression. Ribavirin-mediated depression of Ras-signaling has not been demonstrated experimentally, but the theoretical connection is worth future exploration.
Ribavirin may also inhibit the expression of TMPRSS2 by modulating the formation of inhibitory G-quadruplex structures at the TMPRSS2 promoter. In a G-quadruplex, G-rich sequences form tetrads around K+ ions and stack to form the G-quadruplex structure [26,27].
Formation of G-quadruplex structures in the promoter region can influence gene expression, and some cancer therapeutics have been explored for stabilizing these structures to silence proto-oncogenes [28]. A recent study identified a guanosine rich sequence upstream of the TMPRSS2 promoter, capable of forming a G-quadruplex structure [29]. Stabilization of Gquadruplex in the TMPRSS2 promoter inhibited TMPRSS2 expression and IAV replication [29]. G-quadruplexes can be unwound by helicase enzymes to relieve the suppression of downstream genes. Ribavirin triphosphate inhibits the activity of helicases by competitive inhibition of ATP-hydrolysis [30]. We propose that ribavirin-mediated G-quadruplex stabilization could potentially explain the observed decrease in TMPRSS2 expression.  Cells (at 4 different wells) were monitored for CPE using an inverted light microscope.
Percentage of cell survival was plotted.