Patent Description:
The announcement of the outbreak of Severe Acute Respiratory Syndrome Coronavirus type <NUM> (SARS-CoV-<NUM>) in December <NUM> was followed by quick and pandemic spread of the infection, leading to a medical, economic and social crisis. One of the most challenging health emergencies of the past hundred years, the SARS-CoV-<NUM> pandemic is highlighting the danger posed by RNA viruses, also in countries where they were absent or considered eradicated. The pathogenic effects of SARS CoV-<NUM> can lead to the coronavirus disease <NUM> (COVID-<NUM>), characterized by severe pneumonia with a high fatality rate, reaching a <NUM>% among hospitalized old patients. Other coronaviruses associated with severe disease and high mortality are MERS-CoV, which can lead to the Middle East Respiratory Syndrome (MERS) and SARS-CoV, which bears a close genetic similarity with SARS-CoV-<NUM> and was the causative agent of the Severe Acute Respiratory Syndrome (SARS).

SARS-CoV-<NUM> belongs to the enveloped positive-sense RNA coronaviruses (Pal et al. , <NUM>), and its genome has a length of <NUM> kb with <NUM> functional open reading frames (ORFs), along with a set of <NUM> sub-genomic mRNAs which are carriers of a conserved leader sequence, <NUM> transcription-regulatory sequences, and <NUM> terminal untranslated regions (Fehr et al. The genome encodes a total of <NUM>,<NUM> amino acids and, in particular, four main structural proteins: the spike (S)-glycoprotein, the small envelope/E glycoprotein, the membrane/M glycoprotein and the nucleocapsid/N protein (<NPL>). Additionally, the SARS-CoV-<NUM> genome encodes <NUM> non-structural proteins (NSPs), which encompass the two viral cysteine proteases, i.e. NSP3/papain-like protease and NSP5/3C-like protease (3CLpro, also known as main protease). Apartfrom the proteases, the viral NSPs encompass other key viral enzymes such as NSP12 /RNA-dependent RNA polymerase (RdRP) and NSP13/helicase, which are essential for the transcription and replication of the virus (Pal et al.

The ongoing pandemic of SARS-CoV-<NUM> poses the challenge of quick development of antiviral therapies. SARS-CoV-<NUM> is an enveloped, positive sense, RNA virus of the Coronaviridae family, which includes other human-infecting pathogens such as SARS-CoV and MERS-CoV (V'kovski et al. Currently, there are no widely approved antivirals to treat infection with Coronaviruses. Substantial effort has been devoted to identifying inhibitors of SARS-CoV-<NUM> replication through repurposing of compounds approved for treating other clinical indications. Repositioned drugs offer the advantage of a well-known safety profile and the possibility of faster clinical testing, which is essential during a sudden epidemic outbreak (Pushpakom et al. Large scale clinical trials have identified immune modulating agents (e.g. dexamethasone (Johnson and Vinetz <NUM>; <NPL>)) as potential treatments for Coronavirus disease <NUM> (COVID-<NUM>). However, direct acting antiviral agents have shown limited clinical benefits so far. In particular, a set of antiviral drugs initially identified as effective in vitro (remdesivir, chloroquine/hydroxychloroquine) has been unable to reproducibly decrease mortality in placebo-controlled trials (M. Wang et al. <NUM>; Beigel et al. Wang et al. <NUM>; <NPL>).

Complete inhibition of SARS-CoV-<NUM> replication will likely require combinations of antivirals, in line with previous evidence on other RNA viruses (Pawlotsky et al. <NUM>; Gulick and Flexner <NUM>). Candidate inhibitors have been proposed to target several critical steps of SARS-CoV-<NUM> replication, including viral entry, polyprotein cleavage by viral proteases, transcription and viral RNA replication (Guy et al. SARS-CoV-<NUM> entry is mediated by the spike glycoprotein (S-glycoprotein), which binds through its S1 subunit to the cellular receptor Angiotensinconverting enzyme <NUM> (ACE2). Upon binding, the viral entry requires a proteolytic activation of the S2 subunit leading to the fusion of the viral envelope with the host cell membrane (Hoffmann et al. The study of candidate inhibitors of SARS-CoV-<NUM> entry has mainly focused on monoclonal antibodies and small molecules to target the association of the receptor binding domain (RBD) of the S-glycoprotein to ACE-<NUM> (Xiu et al. Interestingly, the intensively studied antimalarials chloroquine and hydroxychloroquine have been suggested to impair SARS-CoV-<NUM> entry in vitro by both decreasing the binding of the RBD to ACE2 and by decreasing endosomal acidification (Liu et al.

Upon viral membrane fusion, the viral RNA is released to the cytosol and translated into two large polyproteins that are cleaved into non-structural proteins (nsp) by two viral proteases, the main protease (3CLpro) and the papain-like protease (PLpro). A large body of work to identify antivirals against SARS-CoV-<NUM> has focused on research on these viral proteases. Initial drug repurposing efforts focused on inhibitors of the HIV-<NUM> protease, such as lopinavir and darunavir, alone or in combination with pharmacological boosters. These inhibitors, however, proved poorly effective in inhibiting 3CLpro activity in vitro (Mahdi et al. <NUM>) and did not offer reproducible clinical benefit (Cao et al. <NUM>; Chen et al. Larger drug screenings have so far relied on a combination of in-silico and in vitro tools (Jin et al. In particular, libraries of compounds have been screened through molecular docking and many candidate drugs have shown favorable binding properties to the SARS-CoV-<NUM> proteases when analyzed by molecular dynamics (Razzaghi-Asl et al. Overall, however, repurposed inhibitors of SARS-CoV-<NUM> proteases have generally shown half-maximal inhibitory concentration (IC50) values that were incompatible with dosages achievable in vivo.

The nsps generated by polyprotein cleavage by the viral proteases support the transcription and replication of the viral genome, which is catalyzed by the activity of the RNA-dependent RNA polymerase (RdRP). Owing to its crucial role and high evolutionary conservation, this viral enzyme represents a very attractive therapeutic target, which has so far been exploited by repurposing the anti-Ebola virus drug remdesivir (Mulangu et al. <NUM>; Beigel et al. Wang et al. Other potential RdRP inhibitors, repurposed from treatment of HCV, HIV-<NUM> and influenza virus have been proposed as well (Jácome et al. <NUM>; Chien et al. <NUM>; Jockusch et al. Among them, Favipiravir and Molnupiravir (MK-<NUM>) have shown in vivo therapeutic potential by decreasing viral burden and transmission in hamster and ferret models of the infection, respectively (Cox, Wolf, and Plemper <NUM>; Kaptein et al. Viral transcripts generated by the RdRP are used for assembly of new virions by budding into the lumen of the ER-Golgi intermediate compartment (ERGIC) (Klein et al. The assembly is driven by the structural proteins M and E which are responsible for the incorporation of the N protein forming ribonucleoprotein complexes containing the viral genome. After the budding is completed, viruses are released from the cell either by exocytosis or through lysosomal organelle trafficking (V'kovski et al. So far, drug candidates proposed to target viral assembly/budding have not advanced beyond in-silico predictions (Gupta et al.

De Meyer S, et al. (<NPL>) discloses a dose of <NUM> cobicistat for use in the treatment of SARS-CoV-<NUM> in combination with <NUM> darunavir. De Meyer S, et al. does not disclose that cobicistat taken orally alone at a dose of <NUM> to <NUM>,<NUM> daily can be for use in the prophylaxis and/or treatment of severe acute respiratory syndrome coronavirus type <NUM> (SARS-CoV-<NUM>) infection, severe acute respiratory syndrome coronavirus (SARS-CoV) infection and/or Middle East respiratory syndrome coronavirus (MERS-CoV) infection.

A major limitation hampering the development of combined antiviral strategies against SARS-CoV-<NUM> is the lack of data on drug interactions. Initial guidelines have cautioned against the combined use of potentially effective compounds, such as remdesivir and chloroquine/hydroxychloroquine, on the basis of the possible interference of the latter with remdesivir metabolism through the efflux pump P-glycoprotein (P-gp) [(Gilead. Summary on compassionate use) (Leegwater et al. <NUM>; Arribas et al. On the other hand, extensive first pass metabolism by the liver is known to limit bioavailability of remdesivir forcing its intravenous administration, limiting both its scalability and, likely, antiviral efficacy (Siegel et al.

It is an objective of the present invention to provide means for antiviral therapies of SARS.

The scope of the invention is defined by the appended set of claims.

According to the present invention this object is solved by providing cobicistat for use in the prophylaxis and/or treatment of severe acute respiratory syndrome coronavirus type <NUM> (SARS-CoV-<NUM>) infection, severe acute respiratory syndrome coronavirus (SARS-CoV) infection and/or Middle East respiratory syndrome coronavirus (MERS-CoV) infection,wherein cobicistat is administered orally at a daily dosage of <NUM> to <NUM>,<NUM>.

Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary.

Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of "<NUM> to <NUM>" should be interpreted to include not only the explicitly recited values of <NUM> to <NUM>, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. <NUM>, <NUM>, <NUM>, <NUM> and sub-ranges such as from <NUM> to <NUM>, <NUM> to <NUM>. This same principle applies to ranges reciting only one numerical value, such as "higher than <NUM> per day". Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

As outlined above, the present invention provides cobicistat for use in the prophylaxis and/or treatment of severe acute respiratory syndrome coronavirus type <NUM> (SARS-CoV-<NUM>) infection, severe acute respiratory syndrome coronavirus (SARS-CoV) infection and/or Middle East respiratory syndrome coronavirus (MERS-CoV) infection, wherein cobicistat is administered orally at a daily dosage of <NUM> to <NUM>,<NUM>.

Cobicistat, marketed under the trade name of Tybost®, is an approved therapy for treating HIV-<NUM> infection. As of yet, cobicistat has not been used as a direct antretroviral, but rather exerts its effect as a pharmacokinetic enhancer (booster) for other antiretrovirals, such as the integrase inhibitor elvitegravir and some protease inhibitors (e.g. darunavir).

At a molecular level, cobicistat can selectively inhibit cytochrome P450 3A isoforms (CYP3A) and block P-glycoprotein efflux transporters, thus increasing the systemic exposure of coadministered agents, such as antiretroviral drugs, which are metabolized by CYP3A enzymes (von Hentig et al.

Despite the existence of clinical guidelines and observations conducted by experts in the field, so far nobody hypothesized that cobicistat could be a direct-acting antiviral agent against SARS-CoV-<NUM>, as it was only described and utilized as a pharmacological booster of HIV-<NUM> protease inhibitor. This lack of attention was likely due to the fact that it was known that Cobicistat is devoid of any direct antiviral activity against HIV-<NUM>, displaying an EC50 against the HIV-<NUM> protease of more than <NUM> (https://www. selleckchem. com/products/cobicistat-gs-<NUM>. Moreover, despite being approved by FDA since <NUM>, cobicistat was never tested during the MERS-CoV outbreak, for which, as in the case of SARS-CoV-<NUM>, there are no effective treatments.

Preferably, cobicistat is provided for use in the prophylaxis and/or treatment of Coronavirus disease <NUM> (COVID-<NUM>).

Thereby, any stage of COVID-<NUM> is comprised.

In a preferred embodiment, cobicistat is used in combination with one or more further drug.

Preferably, the one or more further drug is selected from.

Preferred antiviral agents are remdesivir, chloroquine or hydroxychloroquine, molnupiravir or favipiravir.

Preferred HIV protease inhibitors are tipranavir, nelfinavir, lopinavir and atazanavir.

Plitidepsin is a substrate of cytochrome P450-3As (CYP3A) and/or P-glycoprotein (P-gp).

Plitidepsin (aplidin) is metabolised through cytochrome P450-3A; the cellular target of cobicistat. It inhibits translation elongation factor eEF1A (White et al.

Preferred anti-inflammatory glucocorticoids are dexamethasone, prednisone, methylprednisolone and hydrocortisone.

Preferred januskinase (JAK) inhibitors are baricitinib, ruxolitinib, and upadacitinib.

A preferred palmitoyl protein thioesterase <NUM> (PPT1) inhibitor is GNS561.

A preferred monoclonal antibody targeting host inflammation is tocilizumab.

In a preferred embodiment the further drug is remdesivir, i. a combination of cobicistat with remdesivir is preferred.

In one embodiment, the one or more further drug is remdesivir, tipranavir, chloroquine, hydroxychloroquine, molnupiravir, favipiravir, nelfinavir, lopinavir, atazanavir, plitidepsin, dexamethasone, baricitinib and/or GNS561.

In a preferred embodiment cobicistat is used in combination with remdesivir in further combination with one or more further drug, preferably with an HIV protease inhibitor, more preferably tipranavir, nelfinavir, lopinavir or atazanavir.

In one embodiment, cobicistat is used in combination with chloroquine in further combination with one or more further drug, preferably with an HIV protease inhibitor, more preferably tipranavir, nelfinavir, lopinavir or atazanavir.

A "therapeutically amount" or "therapeutically effective amount", both of which terms are used herein interchangeably, of cobicistat according to the present invention is the amount which results in the desired therapeutic result.

In HIV-<NUM> treatment, Cobicistat is administered orally in combination with the HIV-<NUM> protease inhibitors atazanavir (trade name of the combination: Evotaz®) or darunavir (trade names of the combination: Prezcobix® in the US and Rezolsta® in the EU) or with a combination of several antiretrovirals (trade names Stribild®, Genvoya®, Symtuza®). In all these fixed-dose combinations Cobicistat is administered in a tablet at <NUM>/day.

Here, we demonstrate that the FDA-approved CYP3A inhibitor cobicistat, typically used as a booster of HIV-<NUM> protease inhibitors (Sherman et al. <NUM>), can block SARS-CoV-<NUM> replication in vitro in cell lines of lung and gut origin. While cobicistat was identified through in-silico screening of 3CLpro inhibitors, our data point towards an effect on the S-protein, which in the presence of cobicistat showed decreased ability to form syncytia in cells overexpressing the S-protein. The antiviral concentrations of cobicistat, while well tolerated in vitro, are clearly above those used for HIV-<NUM> treatment, but compatible with plasma levels previously reached at higher doses in mice as well as in humans. In combination with remdesivir, cobicistat exhibits a synergistic effect in rescuing cell viability and abrogating viral replication in both cell lines and in a primary colon organoid. Overall, our data show that cobicistat has a dual activity both as antiviral drug and as pharmacoenhancer, thus potentially providing a basis for combined therapies aimed at complete suppression SARS-CoV-<NUM> replication.

Combinations of direct-acting antivirals are needed to minimize drug-resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the Severe Acute Respiratory Syndrome Corona Virus <NUM> (SARS-CoV-<NUM>) and testing of a number of drug regimens has led to conflicting results. Here we show for the first time that cobicistat, which is an-FDA approved drug-booster that blocks the activity of the drug metabolizing proteins Cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), can have antiviral activity and inhibit SARS-CoV-<NUM> replication. This was unexpected as cobicistat was specifically developed to be "inert" against the HIV-<NUM> protease and to exert solely a booster effect (Xu et al. Our cell-to-cell membrane fusion assays indicated that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. Incubation with low micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. These concentrations of cobicistat were previously deemed unnecessary as the inhibitory activity of the drug on CYP3A requires only low nanomolar concentrations (Xu et al. Indeed, clinical trials testing drug regimens including cobicistat had only considered standard dosing of cobicistat and had not postulated any antiviral effect of this drug and were aimed solely at testing the antiviral activity of HIV-<NUM> protease inhibitors (Chen et al. When cobicistat was used in combination with the putative CYP3A target and nucleoside analog remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. The cobicistat/remdesivir combination was able to potently abate viral replication to levels comparable to mock-infected cells leading to an almost complete rescue of infected cell viability. These data highlight cobicistat as a therapeutic for treating SARS-CoV-<NUM> infection and as a building block of combination therapies for COVID-<NUM>.

To identify potential inhibitors of SARS-CoV-<NUM> replication we performed a structure-based virtual screening of the Drugbank library of compounds approved for clinical use. Candidate drugs were ranked based on their docking score to the substrate-binding site of 3CLpro, i.e. the site essential for the proteolytic function. Our results highlighted seventeen top candidate inhibitors, including compounds used to treat parasitic as well as viral infections. Among the latter, the HIV-<NUM> protease inhibitor nelfinavir, which was one of the top scoring compounds in our analysis, was previously shown to decrease SARS-CoV and SARS-CoV-<NUM> replication in vitro (Yamamoto et al. ) (Table <NUM>). Two additional drugs used for treatment of HIV-<NUM> displayed top docking scores, i.e. the protease inhibitor tipranavir and, unexpectedly, the CYP3A inhibitor cobicistat, which was previously designed as a molecule devoid of antiviral activity (Xu et al. The latter was a particularly interesting candidate, given its activity as a booster for HIV-<NUM> protease inhibitors (Sherman et al. <NUM>), which renders it a promising candidate for combination therapies. Additional in-silico investigation of the binding poses and stability of cobicistat to the 3CLpro of SARS-CoV-<NUM> corroborated a high predicted affinity for the target (<FIG> and <FIG>). These in-silico results prompted us to test the effect of cobicistat on SARS-CoV-<NUM> replication in vitro. For this purpose, we conducted a time course analysis of the effect of different concentrations of cobicistat on intracellular viral RNA replication and release of virus into the culture supernatant of Calu-<NUM> cells (<FIG>). Analysis of virus RNA amounts by qPCR showed a dose dependent inhibitory effect of low micromolar concentrations of cobicistat (<FIG>). This effect was visible in both supernatants and cellular extracts, and was reproducible when samples were assayed with two different sets of primers [i.e. N1 and N2 primer sets recommended by the Center of Disease Control (<FIG> with the N1 primer set; data for N2 primer set not shown)]. Of note, pre-incubation or treatment upon infection with cobicistat did not increase the antiviral effects as compared to adding the drug two or four hours post-infection, potentially suggesting an effect on late stages of the viral life cycle.

Taken together, these data show that cobicistat has a direct antiviral effect on SARS-CoV-<NUM> replication in vitro.

We next analyzed more thoroughly the antiviral effects of cobicistat using three cell lines of different origin, i.e. Calu-<NUM> cells (human lung), Vero E6 cells (african green monkey kidney) and T84 cells (human gut), to reflect various known or putative tissue compartments of SARS-CoV-<NUM> replication. Each cell line was infected using two different multiplicities of infection [(MOI) <NUM> and <NUM>] and left untreated or treated with various concentrations of cobicistat <NUM> post-infection. In all cell lines, cobicistat showed a dose dependent effect in decreasing viral RNA release in supernatant (<FIG>). In line with this, the higher concentrations of cobicistat tested (<NUM>-<NUM>) were able to partially rescue viability of infected cells, as shown by both MTT and crystal violet assay (<FIG> and <FIG>), while being well tolerated by uninfected cells (<FIG> and <FIG>). Overall, the range of IC50 concentrations of cobicistat (<NUM>-<NUM>) was dependent on the MOI of the infection and on the cell type, but always far below the half cytotoxic concentration (CC50) range of the drug on the same cell types (<NUM>-<NUM>). We then compared our in vitro results with previously known pharmacokinetic properties of cobicistat in humans and mice, namely the peak plasma levels detectable in mice (Pharmacology Review of Cobicistat - Application number: <NUM>- <NUM>) and in humans (Mathias et al. <NUM>; Kakuda et al.

Interestingly, maximum plasma concentrations achievable through standard dosing of cobicistat (<NUM>/day as a booster for HIV-<NUM> protease inhibitors) (Deeks <NUM>) were well below (≈<NUM>) most IC50 values obtained in our experiments (<FIG>). In line with this, clinical testing of the HIV-<NUM> protease darunavir, boosted by standard concentrations of cobicistat, did not yield clinical benefit to SARS-CoV-<NUM> infected patients (Chen et al. On the other hand, plasma levels achievable through a higher dosage of cobicistat, that was tested in tolerability studies of this drug (<NUM>/day) (Mathias et al. <NUM>), were above IC50 values calculated when cells were infected using a <NUM> MOI (<FIG>). Moreover, plasma levels achievable in mice through a higher cobicistat dosage shown to be safe in this animal model (<NUM>/Kg) were clearly above all IC50 values calculated in our experiments, while remaining below the CC50 concentrations (<FIG>).

Overall, our data show that non-toxic concentrations of cobicistat can consistently decrease SARS-CoV-<NUM> replication in various cellular infection models. Moreover, these data prove that plasma concentrations obtained through standard HIV-<NUM> dosing of cobicistat are below those required to highlight the antiviral effect of cobicistat.

To characterize the mechanism of the antiviral effects of cobicistat, we analyzed the catalytic activity of 3CLpro using a previously described FRET assay (Zhang et al. Apart from cobicistat, compounds tested included HIV-<NUM> protease inhibitors highlighted by our molecular docking [nelfinavir, tipranavir] or previously administered in clinical trials as SARS-CoV-<NUM> therapeutics [darunavir (Chen et al. <NUM>), lopinavir (Cao et al. <NUM>)], as well as two positive controls known to inhibit 3CLpro activity [MG132 and GC376 (Ma et al.

While treatment with the known inhibitors of 3CLpro, such as GC376 and MG-<NUM>, potently reduced the catalytic activity of the enzyme, cobicistat was surprisingly inactive (<FIG>). Among the top scoring compounds in our docking analysis (Table <NUM>), only tipranavir proved able to partially inhibit 3CLpro activity, although at relatively high concentrations (EC50 <NUM>; <FIG>).

In light of the lack of effect of cobicistat on 3CLpro, we proceeded to analyze the possible impact of cobicistat on other key viral proteins. To reduce the bias of the analysis, while retaining a representative model of the infection, we performed western blot analysis of Vero E6 cell lysates using previously validated patient sera to detect viral proteins (Pape et al. The results showed the reduction of a high molecular weight band (≈<NUM> kDa) when infected cells were incubated with low micromolar concentrations of cobicistat (data not shown). Based on the known molecular weights of SARS-CoV-<NUM> proteins, we postulated that the patterns detected with patient sera corresponded to dimers/trimers of the S-protein (Ou et al. <NUM>; Algaissi et al. <NUM>) and to the nucleoprotein (N-protein) (Algaissi et al. <NUM>) of the virus. To confirm this hypothesis, we performed western blot analysis using monoclonal antibodies against the S and N proteins (<FIG>). The results confirmed the observation that S-protein levels are decreased by cobicistat (<FIG>). Moreover, the data indicated a high inhibition at the level of the S2 subunit (≈<NUM> KDa) of the S-protein, i.e. the subunit responsible for the fusion to the host cell and subsequent viral entry (<FIG>). To isolate the possible effect of cobicistat on S-protein-mediated fusion, we used a cellular assay measuring syncytia formation in Vero E6 cells transfected with the S-protein. The results showed decreased syncytia formation when cells were incubated with cobicistat or when sera from SARS-CoV-<NUM> patients were used as controls to block S-protein fusion (<FIG> and <FIG>). Of note, both western blot analysis and the syncytia assay indicated an effect of cobicistat in the <NUM>-<NUM> range, which corresponds to the range of most IC50 values calculated on the basis of viral RNA levels in supernatants (<FIG>, <FIG>).

Overall, these data show that the antiviral effect of cobicistat is not mediated by inhibition of 3CLpro activity, but is rather exerted, at least partially, through impairment of S-protein-mediated fusion.

We then tested the potential of cobicistat to exert a double activity as direct inhibitor of SARS-CoV-<NUM> replication and as pharmacoenhancer of other antivirals. To this aim, we evaluated remdesivir as a candidate compound to synergize with cobicistat. The choice of remdesivir was motivated by its known activity as an inhibitor of SARS-CoV-<NUM> RdRP, as well as by its postulated susceptibility to extensive first pass liver metabolism, potentially mediated by the cellular targets of cobicistat CYP3A and P-gp [E. , Human Medicines Division. Summary on compassionate use Remdesivir Gilead (Siegel et al. We thus examined the in silico predicted affinity of remdesivir for the main members of the CYP3A family (CYP3A4 and <NUM>), as well as for P-gp. The SwissADME server (Daina, Michielin, and Zoete <NUM>) predicted remdesivir to be both a CYP3A4 and P-gp substrate by using machine learning models with <NUM>% and <NUM>% accuracy, respectively. Similarly, the pkCSM (Pires, Blundell, and Ascher <NUM>) and CYPreact (Tian et al. <NUM>) servers also predicted remdesivir to be both a P-gp and CYP3A4 substrate, but not an inhibitor. Finally, remdesivir displayed high docking scores to the active sites of CYP3A4, CYP3A5 and P-gp (data not shown), which were comparable to those of ritonavir and cobicistat, i.e. known inhibitors with well characterized binding (data not shown).

To identify the most suitable in vitro model for testing the combination of remdesivir and cobicistat, we first examined the relative expression levels of CYP3A4, CYP3A5 and P-gp in different human tissues and cell lines susceptible to SARS-CoV-<NUM> infection (<FIG>). Both transcriptomic analysis (data not shown) and qPCR analysis highlighted liver, gut and kidney as major compartments of CYP3A4/<NUM> and P-gp expression (<FIG>), as previously described (von Richter et al. <NUM>; Wessler et al. On the other hand, primary lung tissues were characterized by lower CYP3A4/<NUM> and P-gp expression, while the cell line Calu-<NUM> showed intermediate characteristics, with low CYP3A4/<NUM> and high P-gp expression, in line with upregulation of the latter marker in cancer cells (Bradley and Ling <NUM>). Of note, SARS-CoV-<NUM> infection was associated with altered expression of these genes. Overall, infected cells and primary gut organoids showed a trend towards upregulation of P-gp, CYP3A4 and CYP3A5 expression (<FIG>). Of note, cell lines of gut origin and Vero E6 cells displayed a peculiar trend showing opposite expression patterns of CYP3A4 and CYP3A5 upon infection (<FIG>). Given their divergent response to the infection, we decided to use both Vero E6 and T84 cells as models for testing cobicistat and remdesivir, to obtain data on the efficacy of the drug combination and on its possible reliance on increased expression of either CYP3A4 or CYP3A5.

While treatment with remdesivir-only displayed antiviral activity at previously described levels (data not shown), the combined use of cobicistat and remdesivir was able to significantly enhance the effect of each drug alone, in both cell lines (<FIG>, and <FIG>). Surprisingly, this synergistic effect was most visible when cobicistat was administered at low micromolar concentrations, thus proving that the synergism is not merely driven by a booster effect of cobicistat, but also by its direct antiviral activity which had never before been postulated by people skilled in the art. In particular, the drug combination was able to almost completely abrogate viral infection/replication, as measured by IF (<FIG>, <FIG>; <FIG>), and qPCR (<FIG>; <FIG>), analysis. In line with this potent antiviral activity, the cobicistat/remdesivir combination also displayed a synergistic effect in inhibiting the cytopathic effects of SARS-CoV-<NUM>, thus restoring viability of infected cells to levels comparable to mock-infected controls (<FIG>; <FIG>,<FIG>,<FIG>). We then tested the effect of the drug combination on a primary human colon organoid (<FIG>), which is susceptible to SARS-CoV-<NUM> infection, as previously described (Stanifer et al. Also in this case, the addition of cobicistat enhanced the antiviral effect of remdesivir. Finally, we tested the ability of a combination of cobicistat and chloroquine in rescuing cytopathic effects of SARS-CoV-<NUM> infection in two cell lines (<FIG>). While the effects of this combination were lower than those observed when treating with cobicistat and remdesivir, the use of chloroquine with cobicistat was synergistic in one of the two cell lines considered (i.e. Calu-<NUM> cells) (<FIG>).

Overall, our data prove that the combination of cobicistat and remdesivir can suppress viral replication in different cellular models of SARS-CoV-<NUM> infection and show that cobicistat can exert a double activity as direct antiviral agent and as pharmacoenhancer.

The data herein presented demonstrate the antiviral activity of the FDA-approved drug cobicistat and support its role for combined antiviral therapies against SARS-CoV-<NUM>. The use of drug combinations targeting different steps of the viral life cycle is a well-established paradigm for treating RNA-virus infections (Bartlett et al. <NUM>; Naggie and Muir <NUM>). Translating this concept to SARS-CoV-<NUM> drug development has, however, proven challenging due to the paucity of effective drug candidates available. In particular, compounds showing promise in initial studies, have failed to reproducibly decrease the mortality and morbidity of the infection (M. Wang et al. <NUM>; Beigel et al. Wang et al. <NUM>; RECOVERY Collaborative Group, Horby, Mafham, et al. Similarly disappointing results were observed in the early stages of HIV-<NUM> drug discovery, and might be partially explained by the inability of candidate antivirals to reach in vivo concentrations sufficient to completely block viral replication. The use of pharmacoenhancers such as cobicistat (Sherman et al. <NUM>) could help to overcome these limitations.

While the present study exclusively focused on the combination of cobicistat and remdesivir, more than <NUM>% of all drugs are metabolized by the main cellular targets of cobicistat (i.e. CYP3A4/<NUM>) (van Waterschoot et al. For example, the recently described SARS-CoV-<NUM> inhibitor plitidepsin (White et al. <NUM>) is mainly metabolized by CYP3A4 in vitro (Brandon et al. Therefore, it is conceivable that a synergistic effect similar to that described for remdesivir can be obtained by coupling cobicistat to other antiviral agents. In particular, other compounds tested in clinical trials of SARS-CoV-<NUM> patients, such as chloroquine/hydroxychloroquine (K. <NUM>) and lopinavir (van Waterschoot et al. <NUM>), are well known substrates of CYP3A. The booster effect of cobicistat would be further complemented by the own antiviral activity of this drug, which was proven herein in vitro on several models of SARS-CoV-<NUM> infection. In line with this, we observed the strongest synergistic effect with remdesivir, when cobicistat was used at concentrations above its IC50 levels, suggesting that the hitherto unknown antiviral effects of cobicistat contribute to the observed synergism. Of note, the concentration range in which cobicistat could inhibit SARS-CoV-<NUM> replication was higher than that achievable through standard dosages (i.e. <NUM>/day) approved for treatment of HIV-<NUM> infection (Deeks <NUM>). Therefore, the antiviral effect of cobicistat requires administration of the drug at higher dosages (e.g. <NUM>/day) which result in plasma levels compatible with the antiviral concentrations described in our study (Mathias et al. These observations can also explain the lack of success of an early trial testing the HIV-<NUM> protease inhibitor darunavir, boosted by a standard cobicistat dose (Chen et al. It is important to note that the authors of said study never considered the addition of cobicistat to have any antiviral potential and concluded from the study the lack of antiviral activity of darunavir, without hypothesizing the possibility to increase the dosage of cobicistat.

Another possible limitation of candidate antivirals for SARS-CoV-<NUM> treatment is the inability to reach specific tissue reservoirs of the infection. Remdesivir is case in point, due to its quick metabolization and poor intestinal absorption (Hu et al. Of note, previous experience with HIV-<NUM> protease inhibitors suggests that cobicistat might overcome this limitation (Lepist et al. <NUM>), in line with the synergistic effect that we observed when treating primary colon organoid and T84 colon adenocarcinoma cells with the combination of cobicistat and remdesivir. Intriguingly, the tissue penetration and activity of cobicistat in the main sites of CYP3A expression (i.e. gut and liver) can be relevant also for the route of administration of remdesivir. Currently, remdesivir requires intravenous administration due to its extensive first pass metabolism (Jorgensen, Kebriaei, and Dresser <NUM>), but its coupling with cobicistat can improve its absorption, perhaps allowing oral formulation of the drug. Increasing the scalability of remdesivir might per se improve its therapeutic potential, as an early treatment of the infection might prevent hospitalization and development of severe COVID-<NUM>, a stage where the efficacy of remdesivir could not be firmly established (Y. Wang et al.

Overall, our study introduces cobicistat as an agent for inhibiting SARS-CoV-<NUM> replication and for combination therapies aimed at blocking or reversing the onset of COVID-<NUM>.

The following examples and drawings illustrate the present invention without, however, limiting the same thereto. The invention is defined solely by the claims.

Effect of combined treatment of cobicistat and remdesivir on the viability of SARS-CoV-<NUM> infected Calu-<NUM> (A) and T84 (B) cells. Cells were infected at <NUM> MOI and left untreated or treated with the drugs at the indicated concentrations two hours-post infection. Forty Eight hours post-treatment cellular viability was analyzed by MTT assay. Synergism analysis of the inhibition cytopathic effects was performed with the SynergyFinder web-tool using the Zero Interaction Potency (ZIP) model.

Identification of potentially active SARS-CoV-<NUM> inhibitors with desirable Absorption, Distribution, Metabolism, Excretion and Toxicity (ADME-Tox) properties, was performed by structure-based virtual screening (SBVS) of Drugbank V. <NUM>(<NUM>) compounds targeting the three-dimensional structure of SARS-CoV-<NUM>3CLpro. The analysis was focused on the substrate-binding site, which is located between domain I and II of 3CLpro. The binding site was identified using the publicly available 3D crystal structure [Protein Data Bank (PDB) ID: 6W63]. Structures of the previously described non-covalent protease inhibitor X77 (Andrianov et al. , <NUM>), natively co-crystallized with 3CLpro were used as a reference for the identification of binding-site coordinates and dimensions for the virtual screening workflow, as well as for the docking validation of positions generated from the screening.

Protein structure analysis and preparation for docking were performed using the Schrödinger protein preparation wizard (Schrödinger Inc). Missing hydrogen atoms were added, bond orders were corrected and unknown atom types were assigned. Protein side-chain amides were fixed using program default parameters and missing protein side chains were filled-in using the prime tool. All non-amino acid residues, including water molecules, were removed. Further, unrelated ligand molecules were removed and active ligand structures were extracted and isolated in separate files. Finally, the minimization of protein strain energy was achieved through restrained minimization options with default parameters. The centroids of extracted ligands were then used to identify the binding site with coordinates and dimensions extended for <NUM>Å stored as Glide grid file. Drug screening was performed using the Glide software (Friesner et al. High throughput virtual screening (HTVS) was performed with the fastest search configurations. After post-docking minimization, the top-scoring tenth percentile of the output docked structures were subjected to the standard precision docking stage (SP). Then, active ligand structures were extracted and isolated in separate files. Finally, the top <NUM>% scoring compounds were selected and retained only if their good scoring states were confirmed by Extra precision docking.

Remdesivir docking to CYP3A4, CYP3A5 and P-gp structures was performed to assess its capacity as a substrate/inhibitor for these proteins. CYP3A4, CYP3A5 and P-gp structures were collected from Protein Data Bank (PDB), IDs: 5VC0, 5VEU and 6QEE, respectively, and were subjected to the same preparation steps described above. Native inhibitors were used for identification of binding sites; the centroid of the known inhibitor Zosuquidar was used to identify the drug binding pocket of the P-gp protein structure. Further, co-crystallized Ritonavir was used for identification of the drug binding pocket in both CYP3A4/<NUM>. Receptor grids were generated for protein structures, for both CYP3A4 and CYP3A5. The heme iron of the Protoporphyrin ring was added as metal coordination constraint, allowing metal-ligand interaction in the subsequent docking steps. Docking was performed using flexible ligand conformer sampling allowing ring sampling with a <NUM> kcal/mol window. Retained poses for the initial docking phase were set to <NUM> poses and only <NUM> best poses per ligand were selected for energy minimization. Finally, post-docking minimization was carried out for <NUM> poses per ligand with a <NUM> kcal/mol threshold for rejecting minimized poses.

The following cell lines were used for infection and/or relative quantification of gene expression: Calu-<NUM> (ATCC HTB-<NUM>), Caco-<NUM> (ATCC HTB-<NUM>), T84 (ATCC CCL-<NUM>) and VeroE6 (ATCC CRL-<NUM>). Primary organoids derived from human colon and ileum were seeded in 2D as described in (Stanifer et al. Culture conditions and susceptibility to SARS-CoV-<NUM> infection have been previously described (Cortese et al. <NUM>; Stanifer et al.

Viral stocks used for infections were produced by passaging the BavPat1/<NUM> SARS-CoV-<NUM> strain in Vero E6 cells and the infectious titer was estimated by plaque assay, as previously described. Infection experiments were conducted using <NUM>,<NUM> or <NUM>,<NUM> cells per well in <NUM> and <NUM> well plates, respectively. Cell lines were infected at <NUM> or <NUM> MOI in medium with low FCS content (<NUM>%). Colon organoids were infected in a <NUM>-well plate using <NUM> plaque forming units (PFU) per well. Two hours post-infection cells were washed twice in PBS and resuspended in complete medium.

The following compounds were tested to determine their effects on 3CLpro activity, cytotoxicity or inhibition of SARS-CoV-<NUM> replication: cobicistat (#sc-<NUM>; Santa Cruz Biotechnology), remdesivir (#S78932; Selleckchem Chemicals), tipranavir (#sc-<NUM>; Santa Cruz Biotechnology), nelfinavir mesylate hydrate (#PZ0013, Sigma-Aldrich), darunavir, lopinavir (both obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID), MG-<NUM> (#M8699; Sigma-Aldrich), GC376 (BPS Bioscience), Chloroquine (#C <NUM>, Sigma Aldrich).

RNA extraction was performed on cell lysates or supernatants using the NucleoSpin RNA, Mini kit for RNA purification (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions. The concentration of RNA extracted from cell lysates was measured using a P-class P <NUM> NanoPhotometer (Implen GmbH, Munich, Germany).

Retrotranscription to cDNA was performed with 500ng of intracellular RNA or 10µL of RNA from supernatants, using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions.

For the preparation of a viral RNA standard to use in qPCR for quantification of viral copies in supernatants, SARS-CoV-<NUM> N sequence was reverse transcribed from total RNA isolated from cells infected with the SARS-CoV-<NUM> BavPat1 stain using Superscript <NUM> and specific primers (TTAGGCCTGAGTTGAGTCA, SEQ ID NO. The resulting cDNA was amplified and cloned into the pJET1. <NUM> plasmid. Ten µg of plasmid DNA was linearized by AdeI restriction enzyme digestion and DNA was purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany). For in vitro transcription T7 RNA polymerase was used as previously described (Fischl and Bartenschlager <NUM>). In vitro transcripts were purified by phenolchloroform extraction and resuspended in RNase-free water. RNA integrity was confirmed by agarose gel electrophoresis.

Gene and/or viral expression were analyzed by SYBR green qPCR using, for each reaction, 10µL of SsoFast™ EvaGreen® Supermix (Bio-Rad Laboratories, Hercules, CA, USA), <NUM> of forward and reverse primer (<NUM>. 1µL each from <NUM> stock), <NUM>. 8µL water and 1µL cDNA. The primers used are listed in Table <NUM>. The qPCR reaction was performed on a CFX96/C1000 Touch qPCR system (Bio-Rad Laboratories, Hercules, CA, USA) using the following PCR program: polymerase activation/DNA denaturation <NUM> for <NUM>, followed by <NUM> cycles of denaturation at <NUM> for <NUM>; annealing/extension at <NUM> for <NUM> and a final extension step at the end of the program at <NUM> for <NUM>. Gene expression data were normalized using the delta-delta CT method [<NUM>(-ΔΔ C(T)) method] (Livak and Schmittgen <NUM>), using the Tata-binding protein (TBP) gene as housekeeper control.

For Western blot experiments <NUM> × <NUM><NUM> cells were lysed in a buffer (<NUM> Tris-HCl, pH <NUM>, <NUM> EDTA, <NUM> NaCl, <NUM>% Nonidet P-<NUM>, <NUM>% SDS, and <NUM>% sodium deoxycholate supplemented with protease and phosphatase inhibitors (Sigma-Aldrich, Saint Louis, MI, USA). Lysates were boiled at <NUM> for <NUM> and sonicated for <NUM> using a Bioruptor® Plus sonication device (Diagenode, Liège, Belgium). Protein lysates were then run on a precast NuPAGE Bis-Tris <NUM>-<NUM>% (Thermo Fisher Scientific, Waltham, MA, USA) SDS-PAGE at <NUM>-<NUM> V and transferred onto a nitrocellulose membrane (GE Healthcare, Little Chalfont, UK) for <NUM> at <NUM> V using a Trans-Blot device for semi-dry transfer (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked using the LI-COR Intercept (PBS) Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA) for <NUM> at RT and incubated overnight at <NUM> with the following primary antibodies in blocking buffer with <NUM>% Tween <NUM>: α-β-actin (<NUM>:<NUM>,<NUM>), (Sigma-Aldrich, Saint Louis, MI, USA), α-SARS-CoV-<NUM> spike protein [(rabbit; <NUM>:<NUM>) ab252690 Abcam], α-SARS-CoV-<NUM> nucleocapsid [(mouse; <NUM>:<NUM>) AB_2827977, Sino Biological)], sera of SARS-CoV-<NUM> positive individuals (<NUM>:<NUM>). Sera were collected as described in (Pape et al. <NUM>), following signing of informed consent by the donors, as well as ethical approval by Heidelberg University Hospital. After primary antibody incubation, membranes were washed three times with <NUM>% PBS-Tween and incubated for <NUM> with the following fluorescence-conjugated secondary antibodies: IRDye® 800CW Goat anti-Human IgG, IRDye® 800CW anti rabbit, IRDye® 700CW anti mouse (LI-COR Biosciences, Lincoln, NE, USA). All secondary antibodies were diluted <NUM>:<NUM> in blocking buffer + <NUM>% Tween. After three washes with <NUM>% PBS-Tween and one wash in PBS, fluorescence signals were acquired using a LI-COR Odyssey® CLx instrument.

Microarray gene expression data for CYP3A4/<NUM> and P-gp in different anatomical tissues or cell lines were retrieved from Homo Sapiens Affymetrix Human Genome U133 Plus <NUM> Array dataset. Data were filtered by applying the criteria "Healthy sample status" and "No experimental treatment". From the initial list, tissues with sample size < <NUM> were filtered out. The anatomy search tool was used to plot Log2 expression ratios of the tested genes. Gene expression data in cell lines were retrieved, apart from the aforementioned microarray dataset, from the RNAseq "mRNA Gene Level Homo sapiens (ref: Ensembl <NUM>)" dataset. The cell line condition filter was used to refine the analysis and include exclusively cell lines susceptible to SARS-CoV-<NUM> infection (i.e. T84, Caco2, Calu-<NUM> and A-<NUM>).

Cell viability was evaluated by (<NUM>- [<NUM>,<NUM>-dimethylthiazol-<NUM>-yl]-<NUM>,<NUM> diphenyl tetrazolium bromide) (MTT) assay and by crystal violet staining as previously described (Shytaj et al. <NUM>; Feoktistova, Geserick, and Leverkus <NUM>). Briefly, the MTT assay was conducted using the CellTiter <NUM>® Non-Radioactive Cell Proliferation Assay (MTT) (Promega; Madison, WI, USA). Cells were plated in a <NUM>-well plate at a concentration of <NUM> × <NUM><NUM> cells/mL in <NUM>µl of medium. The MTT solution (<NUM>µl) was added to each well and, after <NUM>-<NUM>, the reaction was stopped by the addition of <NUM>µl of <NUM>% SDS. Absorbance values were acquired using an Infinite <NUM> PRO (Tecan, Männedorf, Switzerland) multimode plate reader at <NUM> wavelength.

For the crystal violet staining, cells were fixed in <NUM>% formaldehyde and incubated with <NUM>% crystal violet for <NUM> mins. Unbound staining was then washed with H<NUM>O and cells were imaged using a Nikon Eclipse Ts2-FL microscope.

The activity of 3CLpro was measured by FRET assay (BPS Bioscience, San Diego, CA, USA) according to the manufacturer's instructions and as previously described (Zhang et al. Briefly, serial dilutions of test compounds and known 3CLpro were incubated in a <NUM> well plate with the 3CLpro and its appropriate buffer, containing <NUM> DTT. Wells without drugs or without 3CLpro were used as positive control of 3CLpro activity and blank control, respectively. After a <NUM> incubation, the 3CLpro substrate was added to each well and the plate was stored for <NUM> hours in the dark. The fluorescence signal was acquired on an Infinite <NUM> PRO (Tecan, Männedorf, Switzerland) using an excitation wavelength of <NUM> and a detection wavelength of <NUM>. All Three separate experiments were conducted, with each experiment performed in duplicate. Relative 3CLpro was expressed as percentage of the positive control after subtraction of the blank.

Cells were seeded on iBIDI glass bottom <NUM> well plate and infected with SARS-CoV-<NUM> strain BavPat1/<NUM> for <NUM>-<NUM> at MOI <NUM>. Cells were rinsed in PBS and fixed with <NUM>% PFA, followed by permeabilization with <NUM>% Triton X100 (Sigma) in PBS for <NUM> minutes. Cells were then subjected to a standard immunofluorescence staining protocol. Briefly, cells were blocked in <NUM>% milk (Roth) in PBS and incubated with primary antibodies in PBS (anti ds-RNA mouse monoclonal J2 antibody (Scicons) <NUM>:<NUM> and patient serum <NUM>:<NUM>). Cells were washed twice in PBS <NUM>% tween and incubated with secondary antibody in PBS (<NUM>: <NUM> anti-mouse <NUM>, Goat anti-human IgG-AlexaFluor <NUM> (Invitrogen, Thermofisher Scientific) for immunoglobulins detection in human serum and goat anti-mouse IgG-AlexaFluor <NUM> (Invitrogen, Thermofisher Scientific) for dsRNA detection). Nuclei were counterstained with Hoechst <NUM> (Thermofisher Scientific, <NUM>. 002µg/ml in PBS) for <NUM> minutes, washed twice with PBS and stored at +<NUM> until imaging.

For syncytia formation assay, Vero E6 cells (<NUM> x <NUM><NUM> cells/well) were seeded on cover slips in a <NUM> well plate <NUM> prior transfection. Cells were transfected using TransIT-<NUM> or TransIT-LT1 (Mirus) with <NUM>µg of pCDNA3. <NUM>(+)-SARS-CoV-<NUM>-S and <NUM>µl Opti-MEM per well. <NUM> post transfection, cells were treated with cobicistat (final concentration of <NUM>, <NUM> and <NUM>), serum of patients (<NUM>:<NUM> or <NUM>:<NUM>) or DMSO (same concentration as in <NUM> cobicistat). <NUM> post transfection, cells were washed twice with PBS and fixed in <NUM> % PFA for <NUM> at room temperature. After another washing step, cells were permeabilized in <NUM> % Triton for <NUM> at room temperature, washed and blocked in <NUM>% lipid-free BSA in PBS-<NUM>% Tween-<NUM> for <NUM> at room temperature. After washing, cells were stained with the primary rabbit polyclonal anti-SARS-CoV-<NUM> spike glycoprotein antibody (<NUM>:<NUM>, Abcam) for <NUM> at room temperature or overnight at <NUM>. After washing, cells were incubated with the secondary Alexa Fluor <NUM> goat anti-rabbit IgG antibody (<NUM>:<NUM>, Life Technologies) for <NUM> at room temperature. After washing, cells were incubated with DAPI (<NUM>:<NUM>, Sigma-Aldrich) for <NUM> followed by washing with PBS and deionized water. Images were acquired with Nikon Eclipse Ts2-FL Inverted Microscope. Syncytia with three or more nuclei surrounded by the antibody staining were used for the quantification. The edges of the antibody staining were overdrawn with the polygon selection tool in ImageJ.

Cells were imaged using motorized Nikon Ti2 widefield microscope or with Nikon/Andor (CSU W1) spinning disc using a Plan Apo lambda 20x/<NUM> air objective and a back-illuminated EM-CCD camera (Andor iXon DU-<NUM>). JOBS module was used for automatic acquisition of <NUM> images per well. Images were acquired in <NUM> channels using the following excitation/emission settings: Ex <NUM>/<NUM>, Em <NUM>/<NUM> (Hoechst); Ex <NUM>/<NUM>, Em <NUM>/<NUM> (AlexaFluor <NUM>); Ex <NUM>/<NUM>, Em <NUM>/<NUM> (AlexaFluor <NUM>). When spinning disc was used the excitation was performed with <NUM>, <NUM> and <NUM> lasers.

Quantification of infected cells (expressed as percentage of total cells imaged per well) was performed using a custom-made macro in ImageJ. After camera offset subtraction and local background subtraction using the rolling ball algorithm, nuclei were segmented using automated local thresholding based on the Niblack method. Region of interest (represented by the ring (<NUM> pixel wide) around the nucleus) was determined for each individual cell. Median signal intensity was measured in the region of interest in Alexa488 (serum) and Alexa568 (dsRNA) channels. Threshold for calling infected cells was manually determined for each individual experiment using the data from mock transfected cells. The same image analysis procedure and threshold was used for all wells within one experiment.

Data normality assumptions were tested by D'Agostino & Pearson normality test (for > <NUM>). Multiple group comparisons were conducted by non-parametric Kruksal Wallis test, followed by Dunn's post-test, or by Two-Way ANOVA followed by Dunnet's post-test. Half maximal inhibitory (IC50) and cytotoxic (CC50) concentrations of the compounds tested were estimated by nonlinear regression using using relative inhibition values calculated according to the formula: % inhibition = <NUM> * (<NUM> - (X - mock infected)/(infected untreated - mock infected)), where X is each given treatment condition. Data analysis was conducted using GraphPad Prism v6 (GraphPad Software, San Diego, CA, USA). Synergy scores were calculated using the SynergyFinder web-tool (Ianevski et al. <NUM>) using the Zero Interaction Potency (ZIP) model (Yadav et al.

Claim 1:
Cobicistat for use in the prophylaxis and/or treatment of a severe acute respiratory syndrome coronavirus type <NUM> (SARS-CoV-<NUM>) infection, a severe acute respiratory syndrome coronavirus (SARS-CoV) infection, and/or a Middle East respiratory syndrome coronavirus (MERS-CoV) infection,
wherein cobicistat is administered orally at a daily dosage of <NUM> to <NUM>,<NUM>.