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Displaying 10 papers, 8 pages, start at 1, 27 Hits
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Interferons-Interferons

With regards to MERS, in vitro and in vivo preclinical studies have indicated that IFN-α2b alone or in combination with ribavirin, may have a therapeutic effect if given early in disease [78, 79] . In clinical trials, however, IFN-α2b (given in combination with other treatments) did not lead to a significant benefit to patients (see section 2.1.2). IFN-β1a (EC 50 = 1.37 IU/ml) was superior in activity against MERS-CoV infection in vitro compared to IFN-α2a, IFN-α2b, and IFN-γ; these IFNs had EC 50 values of 160.8, 21.4, and 56.5 IU/ml, respectively [80] . IFN-β1b is currently under evaluation for MERS-CoV in a randomized clinical trial (in combination with lopinavir/ritonavir) [32] . Investigating the IFN-β subtypes (1a and 1b) in combination with other antivirals may be worthwhile as potential synergistic combinations could reduce the effective drug dosage and IFN-associated adverse effects.
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. The Middle East Respiratory Syndrome Coronavirus

The first strategy is to test existing broad-spectrum anti-virals 18 . Interferons, ribavirin, and cyclophilin inhibitors used to treat coronavirus pneumonia fall into this category. The advantages of these therapies are that their metabolic characteristics, dosages used, potential efficacy and side effects are clear as they have been approved for treating viral infections. But the disadvantage is that these therapies are too "broad-spectrum" and cannot kill coronaviruses in a targeted manner, and their side effects should not be underestimated. The second strategy is to use existing molecular databases to screen for molecules that may have therapeutic effect on coronavirus 19, 20 . High-throughput screening makes this strategy possible, and new functions of many drug molecules can be found through this strategy, for example, the discovery of anti-HIV infection drug lopinavir/ritonavir. The third strategy is directly based on the genomic information and pathological characteristics of different coronaviruses to develop new targeted drugs from scratch.
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Abstract

Three anti-HIV drugs, ritonavir, lopinavir and darunavir, might have therapeutic effect on coronavirus disease 2019 . In this study, the structure models of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteases, coronavirus endopeptidase C30 (CEP_C30) and papain like viral protease (PLVP), were built by homology modeling. Ritonavir, lopinavir and darunavir were then docked to the models, respectively, followed by energy minimization of the protease-drug complexes. In the simulations, ritonavir can bind to CEP_C30 most suitably, and induce significant conformation changes of CEP_C30; lopinavir can also bind to CEP_C30 suitably, and induce significant conformation changes of CEP_C30; darunavir can bind to PLVP suitably with slight conformation changes of PLVP. It is suggested that the therapeutic effect of ritonavir and lopinavir on COVID-19 may be mainly due to their inhibitory effect on CEP_C30, while ritonavir may have stronger efficacy; the inhibitory effect of darunavir on SARS-CoV-2 and its potential therapeutic effect may be mainly due to its inhibitory effect on PLVP.

Introduction

The trial fifth edition of diagnosis and treatment guideline of COVID-19 issued by National Health Commission of the People's Republic of China (http://www.nhc.gov.cn/) recommends to use Kaletra for treatment. Kaletra, an anti-HIV drug which is composed of two protease inhibitors, ritonavir (CAS#: 155213-67-5) and lopinavir (CAS#: 192725-17-0), might have therapeutic effect on coronavirus diseases like SARS and MERS [7] [8] [9] [10] . However, whether it can inhibit SARS-CoV-2 or treat COVID-19 lacks clinical evidences and randomized clinical trials, and the safety of its use in COVID-19 patients is unclear. Otherwise, Lanjuan Li, infectious disease scientist, academician of Chinese Academy of Engineering, recommended darunavir (CAS#: 206361-99-1), also an HIV protease inhibitor, as a treatment for COVID-19. Although the inhibitory effect of darunavir on SARS-CoV-2 has been verified in vitro, its therapeutic effect on COVID-19 is still unknown. At the same time, the mechanism of how these drugs inhibit SARS-CoV-2 is also unknown.
As coronaviruses, including SARS-CoV-2, synthesize polyproteins followed by hydrolyzed to produce their structure and function proteins [11] [12] [13] , it is suggested that ritonavir, lopinavir and darunavir may block the multiplication cycle of SARS-CoV-2 by inhibiting its proteases. To preliminarily understand the inhibitory effects of the drugs, in this study, molecular models of the proteases were built by homology modeling, followed by docking the drugs to the proteases, respectively. In addition, the dynamic interactions between the drugs and the protease were also simulated to evaluate the binding effects of the drugs on the proteases.

Molecular Docking of the Drugs

The optimal docking site of CEP_C30 is a pocket between its two subunits. Through docking drugs to this site, 100 poses were found when docking ritonavir, with the Libdock score of the optimal pose 192.346; 88 poses were found when docking lopinavir, with the Libdock score of the optimal pose 147.123; and 49 poses were found when docking darunavir, with the Libdock score of the optimal pose 149.404. The optimal docking site of PLVP is a pocket in one side of the protein. Through docking drugs to this site, 4 poses were found when docking ritonavir, with the Libdock score of the optimal pose 164.153; 3 poses were found when docking lopinavir, with the Libdock score of the optimal pose 107.137; and 8 poses were found when docking darunavir, with the Libdock score of the optimal pose 139.543. The interactions between the proteases and drugs at the optimal poses are shown in Figure 2 , and the structures of the docked protease-drug complexes at the optimal pose before energy minimization are available in Supplemental Files S3 to S8.

The Binding Effects of the Drugs

For ritonavir and lopinavir, these two drugs seem more suitable to bind to CEP_C30 rather than PLVP. Through energy minimization, it is suggested that after docking ritonavir or lopinavir, CEP_C30 can be induced to change its conformation significantly to bind the drug tightly. Moreover, ritonavir is more suitable than lopinavir to bind to CEP_C30. As shown in Figure 2 and 3, there are more interactions between CEP_C30 and ritonavir, and the binding between them is tighter. On the contrary, neither ritonavir nor lopinavir seems suitable to bind to PLVP, since the bindings become less tight after energy minimization.

The Possible Inhibitory Mechanisms of the Drugs

Since the catalytic mechanisms of CEP_C30 and PLVP domains are both unknown, it is unable to determine whether ritonavir, lopinavir and darunavir are competitive or non-competitive inhibitors, although these drugs are all peptide analogues and seem to be competitive.
In conclusion, the therapeutic effect of ritonavir and lopinavir on COVID-19 and other coronavirus disease may be mainly due to their inhibitory effect on CEP_C30, while ritonavir may have stronger efficacy; the inhibitory effect of darunavir on SARS-CoV-2 and its potential therapeutic effect may be mainly due to its inhibitory effect on PLVP. However, there are still limitations in this study. As the catalytic mechanisms of CEP_C30 and PLVP are still unknown, in further studies, it should still be focused on to figure out these mechanisms and how ritonavir, lopinavir and darunavir block these procedures. The safety and actual effects of these drugs on human body should also be carried out and verified by randomized clinical control studies further. author/funder. All rights reserved. No reuse allowed without permission.
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Abstract

Background: It had been more than 5 years since the first case of Middle East Respiratory Syndrome coronavirus infection (MERS-CoV) was recorded, but no specific treatment has been investigated in randomized clinical trials. Results from in vitro and animal studies suggest that a combination of lopinavir/ritonavir and interferon-β1b (IFN-β1b) may be effective against MERS-CoV. The aim of this study is to investigate the efficacy of treatment with a combination of lopinavir/ritonavir and recombinant IFN-β1b provided with standard supportive care, compared to treatment with placebo provided with standard supportive care in patients with laboratory-confirmed MERS requiring hospital admission.

Background

Based on in vitro data, the combination of lopinavir and ritonavir has been considered as a candidate therapy for MERS. Lopinavir and ritonavir are antiretroviral protease inhibitors used in combination for the treatment of human immunodeficiency virus (HIV) infection and have limited side effects [20] . The combination of lopinavir/ritonavir (Kaletra®, Abbott Laboratories, Chicago, IL, USA) has also been used for the treatment of SARS. In one study, the combination of lopinavir/ritonavir used in 41 patients with SARS was associated with significantly fewer adverse clinical outcomes (acute respiratory distress syndrome or death) 21 days after the onset of symptoms compare to ribavirin alone used in 111 historical controls (2.4% versus 28.8%, p = 0.001) [21] . However, the historical nature of the control comparison does not allow for a valid estimate of efficacy. In a high-throughput screening for antiviral compounds, lopinavir inhibited the replication of MERS-CoV at levels below those that occur in the circulation after a single oral dose of lopinavir/ritonavir (400 mg lopinavir with 100 mg ritonavir), suggesting that the drug can achieve therapeutic levels in vivo [16, 22] . The effects of lopinavir/ritonavir, IFN-β1b and mycophenolate mofetil (MMF), all of which have shown viral inhibitory effects in vitro, have been tested in common marmosets with severe MERS-CoV infection [23] . The animals treated with lopinavir/ritonavir or IFN-β1b had improved clinical, radiological, pathological and viral-load outcomes compared with untreated animals. By contrast, treatment with MMF resulted in severe or fatal disease, with higher mean viral loads than in untreated animals. Untreated animals and MMF-treated animals had a mortality of 67% by 36 h compared to 0-33% among animals treated with lopinavir/ritonavir or IFN-β1b [23] .

Study population

Exclusion criteria at eligibility assessment 1. Suicidal ideation based on history (contraindication to IFN-β1b) 2. Known allergy or hypersensitivity reaction to lopinavir/ritonavir or to recombinant IFN-β1b, including, but not limited to, toxic epidermal necrolysis, Stevens-Johnson syndrome, erythema multiforme, urticaria or angioedema 3. Elevated alanine aminotransferase (ALT) more than five-fold the upper limit in the hospital's laboratory 4. Use of medications that are contraindicated with lopinavir/ritonavir and that cannot be replaced or stopped during the study period, such as CYP3A inhibitors (see Table 1 ) 5. Pregnancyeligible and consenting female participants of childbearing age will be tested for pregnancy before enrollment in the study 6. Known HIV infection, because of concerns about the development of resistance to lopinavir/ritonavir if used without combination with other anti-HIV drugs, or 7. Patient likely to be transferred to a non-participating hospital within 72 h.

Safety measures Drug interactions

A clinical pharmacist and the treating physician will evaluate each patient at the time of enrollment and daily throughout the study period until day 14 to identify any interactions between the investigated treatments and other drugs, and they will take action accordingly. Table 1 summarizes common medications that may interact with lopinavir/ritonavir and a suggested action plan.

Liver function tests

Elevated liver enzymes are common in MERS infection due to the liver involvement in the disease. Based on analysis of our historical data, liver enzymes were not more elevated in patients treated with ribavirin and IFN-α. Based on HIV literature, aminotransferase elevation levels (more than five times the upper limit of normal) occur in 3 to 10% of patients taking lopinavir/ ritonavir-containing antiretroviral regimens [33] . These laboratory abnormalities are usually asymptomatic and self-limited and often resolve even without discontinuation of the drug [33]. Clinically apparent hepatotoxicity due to lopinavir/ritonavir occurs, but is rare, with symptoms or jaundice appear usually after 1 to 8 weeks of starting therapy [33]. The hepatotoxicity is usually selflimited; although fatal cases have been reported [33] . The treating team should discontinue or minimize concomitant use of any medication that has hepatotoxic potential.
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Searching for evidence

We performed a search of the literature relating to MERS-CoV and SARS-CoV treatment guidelines published after 2002. We searched the literature from the past 20 years for details of the doses and adverse effects of antiviral drugs including interferon, ribavirin, and lopinavir/ritonavir. Searches were performed on PubMed (www.pubmed.gov) using combinations of the following search terms: Middle East respiratory syndrome, severe acute respiratory syndrome, coronavirus, treatment, therapy, and antiviral. Because the number of original articles on MERS-CoV treatment is small, we reviewed all articles including case reports.

Key question 3. Which antiviral regimens can be used in South Korea?

• A combination regimen of type 1 interferon + ribavirin + lopinavir/ritonavir is recommended for antiviral therapy (BIII). • The use of ribavirin alone is not recommended for antiviral therapy, whereas combined administration with type 1 interferon is recommended (AIII). • In cases in which it is difficult to use ribavirin for antiviral therapy, a combination regimen of type 1 interferon + lopinavir/ritonavir should be considered first (AIII). • The dose of ribavirin for MERS-CoV treatment has not been standardized, but the drug can be used at the same doses as in previous clinical trials or the treatment of other respiratory viruses. Dose adjustment may be required in patients showing signs of a decline in renal function (AIII) ( Table 2 ).
There are currently no antiviral drugs with a clearly proven clinical effect in the treatment of MERS-CoV infection. Antiviral studies reported to date have mostly been laboratory studies, and so the actual clinical data for the use of antivirals are limited. There are data from animal experiments and a small amount of clinical data for type 1 interferon, ribavirin, and lopinavir/ritonavir. Type 1 interferons include interferon-α2a, -α2b, and -β1a. In an animal experiment in rhesus macaques, a combination regimen of interferon-α2b and ribavirin showed clinical improvements and reduced severity [15] . There have been clinical case reports of patients who improved after combination therapy with interferon-α2b and ribavirin [16] . However, room remains for debate due to the lack of sufficient clinical studies on combination therapy using type 1 interferon and ribavirin. In one retrospective comparative analysis of cases treated with ribavirin + interferon-α2a or interferon-β1a, neither regimen was effective [6] . In another retrospective clinical study of the effects of a combination regimen of interferon-α2a and ribavirin, 14 of 20 patients (70%) who received this treatment survived beyond 14 days, whereas only seven of 24 patients (29%) in the non-treatment group survived, seemingly demonstrating an effect of the combination regimen (P = 0.004). However, the interpreta- [5] . Both studies have limited ability to provide statistical proof due to the small number of patients. However, since there was an overall trend for improvement in the type 1 interferon + ribavirin combination therapy group, until the lack of an effect has been shown conclusively, combination therapy is recommended. Evidence for the use of lopinavir/ritonavir as an antiviral for MERS-CoV infection is based on its efficacy in the treatment of SARS-CoV [17, 18] . In an animal experiment of common marmosets, the administration of lopinavir/ritonavir alone significantly reduced the numbers of MERS-CoV colonies in the lungs compared to the non-treatment group, and this effect was identical to that of interferon-β1b [19] . Indeed, one report showed that patients administered lopinavir/ritonavir at the same time as type 1 interferon + ribavirin combination therapy showed improved viremia after 2 days [20] . Hence, if possible, the use of lopinavir/ritonavir in addition to type 1 interferon + ribavirin combination therapy is recommended. As for type 1 interferons, in vitro studies showed a stronger effect of interferon-β than interferon-α. Moreover, of these options, the median effective inhibitory concentration (EC 50 ) to maximum serum concentration ratio of interferon-β1b was lower than those of interferon-α2a, interferon-α2b, and interferon-β1a [21, 22] . However, because clinical studies are lacking, no particular type 1 interferon can yet be concluded to be superior to the others [6] . In one in vitro study, ribavirin and interferon-α2b separately inhibited MERS-CoV proliferation [23] . However, MERS-CoV proliferation was not inhibited at the typical concentrations of ribavirin used clinically, and inhibition was only confirmed at concentrations that show toxicity in humans [21] . Therefore, monotherapy with ribavirin at typical doses is expected to show a reduced clinical effect. Nevertheless, when interferon-α2b and ribavirin are administered in combination, they showed a synergistic effect and a reduced dose of ribavirin was required [23] . Therefore, its combined administration with interferon is recommended.

Key question 5. Should antiviral drugs be used by pregnant women?

Pregnant women are conventionally considered a high-risk group for the progression to severe disease or death, and a case was reported of stillbirth in the second trimester of pregnancy for a woman infected with MERS-CoV [27] . Of the antiviral drugs recommended, ribavirin is in Category X for safety in pregnant women, while lopinavir/ritonavir and type 1 interferon are in Category C. Given the lack of clinical studies on antiviral treatment in pregnant women, it is difficult to recommend these drugs. Considering the physiological adaptations to pregnancy in pregnant women, conservative treatment should be provided [28] . When treating pregnant women infected with human immunodeficiency virus (HIV), the preferred protease inhibitor is lopinavir/ritonavir [29] . Among type 1 interferons, there is evidence supporting the safe use of interferon-β1a, which is used to treat multiple sclerosis, in pregnant women. Although one report showed that the incidence of spontaneous abortion increased in pregnant women who used interferon-β1a, there was no statistically significant difference with the incidence in control individuals [30, 31] . Therefore, the use of antiviral drugs can be considered after a comparison of risks and benefits of the drugs. Possible antiviral treatment would be combination therapy with interferon-β1a and lopinavir/tironavir, but there is no case report of this being used in pregnant women with MERS. Any decision to use antiviral drugs requires the consideration of ethical issues and a consultation with an obstetric specialist.
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Effectiveness of some anti-HIV/AIDS drugs for 2019-nCoV

Lopinavir is an antiretroviral medication used to inhibit HIV/AIDS viral protease. It is often used as a fixed-dose combination with another protease inhibitor, ritonavir, sold under the name Kaletra or Aluvia. Ritonavir, sold under the trade name Norvir, is another antiretroviral medication. Its combination with Lopinavir is known as highly active antiretroviral therapy (HAART). Although there is no tractable clinical evidence, Kaletra or Aluvia has been proposed as a potential anticoronavirus drug for 2019-nCoV. The possibility of repurposing some HIV drugs for SARS-CoV treatment has also studied in the literature. 16 It is important to evaluate their binding affinities, which are obtained with two ligand-based methods (i.e., LS-BP and 2DFP) and two 3D models (3DALL and 3DMT). To carry out 3D model predictions, we dock them to the 2019-nCoV protease inhibition site. The resulting complexes are optimized with molecular dynamics and then evaluated by 3DALL and 3DMT. Table 1 shows the low sequence identity between HIV viral protease and 2019-nCoV protease, which might suggest the limited potential for repurposing Aluvia and Norvir for 2019-nCoV treatment. For Lopinavir, our LS-BP and 2DFP predicted the binding affinities of -5.66 kcal/mol and -5.54 kcal/mol, respectively. For Ritonavir, similar low binding affinities of -5.14 kcal/mol and -4.96 kcal/mol were predicted by our LS-BP and 2DFP, respectively. However, our 3D model 3DALL predicted better binding affinities, i.e., -7.78 kcal/mol and -8.44 kcal/mol for Lopinavir and Ritonavir, respectively. The other 3D model, 3DMT, also predicted moderately high binding affinities of -8.13 kcal/mol and -8.07 kcal/mol for Lopinavir and Ritonavir, respectively. Considering the fact that the small training set for LS-BP and 2DFP models is very small, the results predicted by 3D models are more reliable. Figures 20 and 21 indicate that these drugs have reasonable dock poses with 2019-nCoV protease. Therefore, HIV drugs Kaletra (or Aluvia) and Norvir might indeed have a moderate effect in the treatment of 2019-nCoV. However, Many new compounds generated by our GNC appear to have better druggable properties than these HIV inhibitors do. epidemic. Although we know quite a little about 2019-nCoV, it is fortunate that the sequence identity of the 2019-nCoV protease and that of severe acute respiratory syndrome virus (SARS-CoV) is as high as 96.1%. In this work, we show that the protease inhibitor binding sites of 2019-nCoV and SARS-CoV are almost identical, which provides a foundation for us to hypothesize that all potential anti-SARS-CoV chemotherapies are also effective anti-2019-CoV molecules. Additionally, we employed a recently developed generative network complex (GNC) to seek potential protease inhibitors for effective treatment of pneumonia caused by 2019-nCoV. Two datasets 13 . CC-BY-NC 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.01.30.927889 doi: bioRxiv preprint are utilized in this work. One is a SARS-CoV protease inhibitor dataset, which is constructed by collecting 115 SRAS-CoV inhibitors from open database ChEMBL. The other dataset is a binding affinity training set mainly containing the PDBbind refined set. Our GNC model predicts over 8000 potential anti-2019-nCoV drugs which are evaluated by a latent space binding predictor (LS-BP) and a 2D fingerprint predictor (2DFP). Promising drug candidates are further evaluated by two 3D deep learning models trained with all the training sets together, including the dataset for coronaviral protease (3DALL), and the 3D deep learning multitask model trained with the dataset for coronaviral protease as a separated task (3DMT). Furthermore, we choose 15 potential anti-2019-nCoV drugs to analyze partition coefficient (logP), solubility (logS), and synthetic accessibility score (SAscore) according to binding affinity ranking computed by the 3DALL model. The reasonable logP, logS, and SAscore show that our top 15 anti-2019-nCoV drug candidates are potentially effective for inhibiting 2019-nCoV. Finally, the effectiveness of some anti-HIV/AIDS drugs for treating 2019-nCoV is analyzed. Although HIV drugs Kaletra (or Aluvia) and Norvir might indeed have a moderate effect in the treatment of 2019-nCoV, the analysis of these anti-HIV/AIDS drugs together with our top 15 anti-2019-nCoV molecules shows that the new compounds generated by our GNC appear to have better druggable properties than these HIV inhibitors do.
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Replication and Translation Inhibitors: Targeting NS proteins

Viral proteases have been shown to serve as good inhibitory targets. For instance, protease inhibition was shown to be a successful strategy in treating HIV infection [151] . The HIV-1 protease cleaves the translated polypeptide chain into smaller functional proteins, thereby allowing the virus particle to mature [152] . By inhibiting the protease, the immature virus particles would not be able to transform into the mature virion, hence obstructing the viral replication. Several HIV-1 protease inhibitors were discovered and used clinically, such as saquinavir, ritonavir, indinavir, nelfinavir, amprenavir (and its prodrug, fosamprenavir), lopinavir, atazanavir, and darunavir [153] . Similarly, the NS5, NS3 and NS2B (co-factor) proteins were known to play major roles in enzymatic activities for DENV infection, thus making them ideal antiviral targets [154, 155] . After DENV infection, translation of the viral genome will give rise to a polyprotein containing three structural and seven non-structural proteins. The polyprotein will be cleaved into individual proteins during virus maturation by the host proteases (signalase and furin) on the luminal side of the endoplasmic reticulum, as well as by the viral serine protease (NS2B-NS3 protease) on the cytoplasmic side to ensure the success of viral replication and maturation [49, 154, 156] . DENV NS3 contains a trypsin-like protease and it requires the NS2B cofactor to be active to cleave the DENV polyprotein at the Ser-His-Asp catalytic triad [157] [158] [159] [160] .
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Combining Host-Virus and Drug-Gene Interactions Reveals Novel Activities of Broad-Spectrum Antiviral Agents Against Hepatitis C Virus and Human Metapneumovirus

We selected 28 investigational/experimental/approved antivirals compounds (30) which had DGIdb annotated targets that are part of the hvPPI. We added 12 agents as controls (Table S1 and Figure S6) . We tested 40 broad-spectrumantivirals against GFP-expressing human metapneumovirus (HMPV) NL/1/00 strain (78) . Seven different concentrations of the compounds were added to HMPV or mock-infected cells. HMPV-induced GFP expression and cell viability was measured and after 48 h to determine compound efficiency and toxicity. After the initial screening, we identified five compounds, which lowered GFP-expression without detectable cytotoxicity (with SI > 3). We repeated the experiment with these compounds. The experiments revealed novel activity of azacytidine, lopinavir, nitazoxanide, itraconazole, and oritavancin against HMPV ( Figure S7A and Table 1, Table S2 ). Similarly, we examined toxicity and antiviral activity of broad-spectrum-antivirals against GFP-expressing HCV in Huh-7.5 cells using previously described procedures (49) . Our test identified azithromycin, cidofovir, oritavancin, dibucaine, gefitinib, minocycline, and pirlindole mesylate as novel anti-HCV agents with SI > 3 ( Figure S7B and Table 1, Table S2 ). In summary, our metaanalysis approach of the hvPPI could provide novel and faster approaches for the re-purposing of existing drugs as antiviral agents.

DISCUSSION

This unique dataset can be used for further detailed interrogation of the mechanisms behind viral evasion. This could serve as a starting point for identifying novel host targets and generating hypothesis in the context of viral evasion and development of pan-viral therapeutic intervention strategies. The methods described here also provide unique ways of dissecting the orthogonal datasets. Various analyses from this study have highlighted the existence on pan-viral evasion points that could be utilized for the development of host-directed antiviral therapies. It is also intriguing to see that there is commonality and specificity at the level of sub cellular localization of the viral targets. Our analyses have underlined some salient features in the context of IAV, HPV, DENV, and HCV. Further detailed analysis in this context along with protein sequence features, such as Short Linear Motifs [SLiMs; (79) ] would provide novel insights as well as deeper understanding of how small sequence features are involved in the hijacking of the host machinery. Integration of such data with known drug-gene interactions provides a clear estimate of the druggable proportion in the hvPPI. Our meta-analysis approach of the hvPPI could provide novel avenues of re-purposing existing drugs for antiviral targeting strategies. Our meta-analysis approach of the hvPPI could provide novel avenues of re-purposing existing drugs for antiviral targeting strategies. To prove the concept, we tested 40 BSAs against HMPV, HCV, Sindbis virus (SINV), cytomegalovirus (CMV), and hepatitis B virus (HBV). Importantly, 28 BSAs have DGIdb annotated targets that are part of the hvPPI, whereas 12 were used as controls. These safein-man drugs have already been used as investigational agents or experimental drugs in different virus infections (Table S2) . We demonstrated novel antiviral effects of azacytidine, itraconazole, lopinavir, nitazoxanide, and oritavancin against HMPV, as well as cidofovir, dibucaine, azithromycin, gefitinib, minocycline, oritavancin, and pirlindole against HCV.
FLUAV, RVFV, HIV-1, and HIV-2 agent. Itraconazole is an antifungal medication. It is also used as experimental anti-HEV-B, HRV-B, HRV-A, Par-A3, and SAFV agent. Nitazoxanide is a broad-spectrum antiparasitic drug, which is also investigational agent against FLUAV and HCV and experimental anti-CHIKV, RSV, HBV, HIV-1, VACV, RV, JEV, MERS-CoV, NoV, RuV, and ZIKV agent. Lopinavir is an FDA-approved antiretroviral of the protease inhibitor class. It is also investigational anti-MERS-CoV and experimental anti-ZIKV agent (Table S2 ). In addition to inhibition of viral proteases (Table S2) , Lopinavir was reported to induce host RNaseL production in infected and non-infected cells (80) . RNaseL is endoribonuclease that is a part of interferon (IFN) antiviral response, which is the most critical node of virus-host interactions. Although, the antiviral mechanisms of action of other compounds are still unknown, these agents could inhibit steps of viral infections, which precede reporter protein expression from viral RNA. In summary, our results indicate that existing BSAs could be re-purposed to other viral infections. To further expand a spectrum of their activities, these BSAs could be tested against other viruses. Re-purposing these and other safe-inman antiviral therapeutics could save resources and time needed for development of novel drugs to quickly address unmet medical needs, because safety profiles of these agents in humans are available. Effective treatment with broad-spectrum-antivirals may shortly become available, pending the results of further pre-clinical studies and clinical trials. This, in turn, means that some broad-spectrum-antivirals could be used for rapid management of new or emerging drug-resistant strains, as well as for first-line treatment or for prophylaxis of acute virus infections or for viral co-infections. The most effective and tolerable compounds could expand the available therapeutics for the treatment of viral diseases, improving preparedness and the protection of the general population from viral epidemics and pandemics.