Patent Application
20230107186
The present invention relates to cobicistat and its derivatives or prodrugs for use in the prophylaxis and/or treatment of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, severe acute respiratory syndrome coronavirus (SARS-CoV) infection and/or Middle East respiratory syndrome coronavirus (MERS-CoV) infection. The present invention further relates to methods of prevention and/or treatment of SARS-CoV-2 infection.
The announcement of the outbreak of Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) in December 2019 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-2 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-2 can lead to the coronavirus disease 2019 (COVID-19), characterized by severe pneumonia with a high fatality rate, reaching a 40% 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-2 and was the causative agent of the Severe Acute Respiratory Syndrome (SARS).
SARS-CoV-2 belongs to the enveloped positive-sense RNA coronaviruses (Pal et al., 2020), and its genome has a length of 29.9 kb with 12 functional open reading frames (ORFs), along with a set of 9 sub-genomic mRNAs which are carriers of a conserved leader sequence, 9 transcription-regulatory sequences, and 2 terminal untranslated regions (Fehr et al., 2015). The genome encodes a total of 9,860 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 (Pal et al., 2020, Jiang et al., 2020). Additionally, the SARS-CoV-2 genome encodes 16 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). Apart from 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., 2020).
The ongoing pandemic of SARS-CoV-2 poses the challenge of quick development of antiviral therapies. SARS-CoV-2 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. 2020). Currently, there are no widely approved antivirals to treat infection with Coronaviruses. Substantial effort has been devoted to identifying inhibitors of SARS-CoV-2 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. 2019). Large scale clinical trials have identified immune modulating agents (e.g. dexamethasone (Johnson and Vinetz 2020; RECOVERY Collaborative Group, Horby, Lim, et al. 2020)) as potential treatments for Coronavirus disease 2019 (COVID-19). 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. 2020; Beigel et al. 2020; Y. Wang et al. 2020; RECOVERY Collaborative Group, Horby, Mafham, et al. 2020).
Complete inhibition of SARS-CoV-2 replication will likely require combinations of antivirals, in line with previous evidence on other RNA viruses (Pawlotsky et al. 2015; Gulick and Flexner 2019). Candidate inhibitors have been proposed to target several critical steps of SARS-CoV-2 replication, including viral entry, polyprotein cleavage by viral proteases, transcription and viral RNA replication (Guy et al. 2020). SARS-CoV-2 entry is mediated by the spike glycoprotein (S-glycoprotein), which binds through its 51 subunit to the cellular receptor Angiotensin-converting enzyme 2 (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. 2020). The study of candidate inhibitors of SARS-CoV-2 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-2 (Xiu et al. 2020). Interestingly, the intensively studied antimalarials chloroquine and hydroxychloroquine have been suggested to impair SARS-CoV-2 entry in vitro by both decreasing the binding of the RBD to ACE2 and by decreasing endosomal acidification (Liu et al. 2020).
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-2 has focused on research on these viral proteases. Initial drug repurposing efforts focused on inhibitors of the HIV-1 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 (Mandi et al. 2020) and did not offer reproducible clinical benefit (Cao et al. 2020; Chen et al. 2020; E. J. Kim et al. 2020). Larger drug screenings have so far relied on a combination of in-silico and in vitro tools (Jin et al. 2020). In particular, libraries of compounds have been screened through molecular docking and many candidate drugs have shown favorable binding properties to the SARS-CoV-2 proteases when analyzed by molecular dynamics (Razzaghi-Asl et al. 2020). Overall, however, repurposed inhibitors of SARS-CoV-2 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. 2019; Beigel et al. 2020; Y. Wang et al. 2020). Other potential RdRP inhibitors, repurposed from treatment of HCV, HIV-1 and influenza virus have been proposed as well (Jácome et al. 2020; Chien et al. 2020; Jockusch et al. 2020). Among them, Favipiravir and Molnupiravir (MK-4482) 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 2021; Kaptein et al. 2020). 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. 2020). 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. 2020). So far, drug candidates proposed to target viral assembly/budding have not advanced beyond in-silico predictions (Gupta et al. 2020).
A major limitation hampering the development of combined antiviral strategies against SARS-CoV-2 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. 2020; Arribas et al. 2020)]. 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. 2017).
It is an objective of the present invention to provide means for antiviral therapies of SARS.
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 2 (SARS-CoV-2) infection, severe acute respiratory syndrome coronavirus (SARS-CoV) infection and/or Middle East respiratory syndrome coronavirus (MERS-CoV) infection, wherein cobicistat or a derivative or prodrug thereof is used, said derivative or prodrug is ritonavir or desoxy-ritonavir.
According to the present invention this object is solved by a method of prevention and/or treatment of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, severe acute respiratory syndrome coronavirus (SARS-CoV) infection and/or Middle East respiratory syndrome coronavirus (MERS-CoV) infection,
comprising the step of
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. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. For the purpose of the present invention, all references cited herein are incorporated by reference in their entireties.
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 “1 to 20” should be interpreted to include not only the explicitly recited values of 1 to 20, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 1, 2, 3, 4, 5 . . . 17, 18, 19, 20 and sub-ranges such as from 2 to 10, 8 to 15, etc. This same principle applies to ranges reciting only one numerical value, such as “higher than 150 mg 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 coronavirus infection.
As outlined above, the present invention provides cobicistat for use in the prophylaxis and/or treatment of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, severe acute respiratory syndrome coronavirus (SARS-CoV) infection and/or Middle East respiratory syndrome coronavirus (MERS-CoV) infection,
According to the invention, cobicistat or a derivative or prodrug thereof is used. When throughout this application reference is made to “cobicistat” respective compounds which are derivatives and prodrugs are meant to be included under the proviso that said compounds show similar anti-SARS-CoV-2 activity than cobicistat.
A derivative or prodrug of cobicistat is ritonavir or desoxy-ritonavir.
DrugBank ID: DB09065 (https://www.drugbank.ca/drugs/DB09065)
ATC code: V03AX03 (WHO) (https://www.whocc.no/atc_ddd_index/?code=V03 AX03)
Chemical structure: 1,3-thiazol-5-ylmethyl [(2R,5R)-5-{[(2S)-2-({[(2-isopropyl-1,3-thiazol-4-yl)methyl](methyl)carbamoyl}amino)-4-(morpholin-4-yl)butanoyl]amino}-1,6-diphenylhexan-2-yl]carbamate,
with a molecular formula of C40H53N7O5S2 and a molecular weight of 776.0 g/mol (information retrieved from: National Center for Biotechnology Information. PubChem Database. Cobicistat, CID=25151504, https://pubchem.ncbi.nlm.nih.gov/compound/Cobicistat; accessed on Jun. 16, 2020).
Cobicistat, marketed under the trade name of Tybost®, is an approved therapy for treating HIV-1 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 co-administered agents, such as antiretroviral drugs, which are metabolized by CYP3A enzymes (von Hentig et al., 2015).
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-2, as it was only described and utilized as a pharmacological booster of HIV-1 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-1, displaying an EC50 against the HIV-1 protease of more than 30 μM (https://www.selleckchem.com/products/cobicistat-gs-9350.html). Moreover, despite being approved by FDA since 2014, cobicistat was never tested during the MERS-CoV outbreak, for which, as in the case of SARS-CoV-2, there are no effective treatments.
Preferably, cobicistat is provided for use in the prophylaxis and/or treatment of Coronavirus disease 2019 (COVID-19).
Thereby, any stage of COVID-19 is comprised.
Preferably, prophylaxis or prevention comprises pre- and post-exposure prophylaxis to or prevention of SARS-CoV-2 infection.
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.
A preferred substrates of cytochrome P450-3As (CYP3A) and/or P-glycoprotein (P-gp) is plitidepsin.
Plitidepsin (aplidin) is metabolised through cytochrome P450-3A; the cellular target of cobicistat. It inhibits translation elongation factor eEF1A (White et al., 2021).
Preferred anti-inflammatory glucocorticoids are dexamethasone, prednisone, methylprednisolone and hydrocortisone.
Preferred januskinase (JAK) inhibitors are baricitinib, ruxolitinib, and upadacitinib.
A preferred palmitoyl protein thioesterase 1 (PPT1) inhibitor is GNS561.
A preferred monoclonal antibody targeting host inflammation is tocilizumab.
In a preferred embodiment the further drug is remdesivir, i.a. 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.
Route of Administration and Therapeutically Amount
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 a preferred embodiment, cobicistat is administered in a therapeutically amount, which is higher than the dosage used for HIV-1 treatment.
The dosage of cobicistat for HIV-1 treatment is 150 mg per day. Thus, cobicistat is preferably administered in a therapeutically amount higher than 150 mg per day.
In HIV-1 treatment, Cobicistat is administered orally in combination with the HIV-1 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 150 mg/day.
Cobicistat can be administered via systemic delivery, oral, intranasal, via inhalation, intravenous, or any combination thereof.
Preferred routes of administration of cobicistat are:
In one embodiment, cobicistat is administered orally.
The daily dosage for oral administration is preferably in the range from 10 mg to 1,200 mg, more preferably, 300 to 1,000 mg.
In one embodiment, cobicistat is administered intranasally and/or via inhalation.
An inhaled form of cobicistat could overcome its rapid turnover and allow its delivery at micromolar concentrations in the lung.
In one embodiment, the administration is preferably via a dry powder inhaler, and cobicistat is preferably in solid form.
In one embodiment, the administration is preferably via a nebulizer or a soft mist spray dispenser, and cobicistat is preferably resuspended in an aqueous medium.
The amount administered is preferably at micromolar concentrations, such as in the range from about 2 to 30 μM per day, such as 2 to 15 μM per day.
For example, cobicistat is administered intranasally, through inhalation, at an equivalent concentration of 2-15 μM.
Methods of Prevention and/or Treatment
As outlined above, the present invention provides a method of prevention and/or treatment of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, severe acute respiratory syndrome coronavirus (SARS-CoV) infection and/or Middle East respiratory syndrome coronavirus (MERS-CoV) infection.
Said method comprises the step of
As discussed above, said derivative or prodrug is ritonavir or desoxy-ritonavir.
Preferably, the method according to the invention is a method for the prophylaxis and/or treatment of Coronavirus disease 2019 (COVID-19).
Preferably, prophylaxis or prevention comprises pre- and post-exposure prophylaxis to or prevention of SARS-CoV-2 infection.
In a preferred embodiment, cobicistat is administered 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.
A preferred substrate of cytochrome P450-3As (CYP3A) and/or P-glycoprotein (P-gp) is plitidepsin.
Preferred anti-inflammatory glucocorticoids are dexamethasone, prednisone, methylprednisolone and hydrocortisone.
Preferred januskinase (JAK) inhibitors are baricitinib, ruxolitinib and upadacitinib.
A preferred palmitoyl protein thioesterase 1 (PPT1) inhibitor is GNS561.
A preferred monoclonal antibody targeting host inflammation is tocilizumab.
In a preferred embodiment the further drug is remdesivir, i.a. a combination of cobicistat with remdesivir is administered.
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 administered 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 administered 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.
Preferably the administration is oral, intranasal and/or via inhalation.
The therapeutically amount is preferably higher than the dosage used for HIV-1 treatment, i.e. higher than 150 mg per day.
Cobicistat can be administered via systemic delivery, oral, intranasal, via inhalation, intravenous, or any combination thereof.
Preferred routes of administration of cobicistat are:
In one embodiment, the administration of cobicistat is oral, preferably at a daily dosage in the range from 10 mg to 1,200 mg, more preferably, 300 to 1,000 mg.
In one embodiment, the administration of cobicistat is intranasal and/or via inhalation,
wherein, preferably, the administration is via a dry powder inhaler, and cobicistat is preferably in solid form,
or via a nebulizer or a soft mist spray dispenser, and cobicistat is preferably resuspended in an aqueous medium.
Preferably, the amount administered is at micromolar concentrations, such as in the range from about 2 to 30 μM per day, such as 2 to 15 μM per day.
Here, we demonstrate that the FDA-approved CYP3A inhibitor cobicistat, typically used as a booster of HIV-1 protease inhibitors (Sherman et al. 2015), can block SARS-CoV-2 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-1 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-2 replication.
Abstract
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 2 (SARS-CoV-2) 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-2 replication. This was unexpected as cobicistat was specifically developed to be “inert” against the HIV-1 protease and to exert solely a booster effect (Xu et al., 2010). 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., 2010). 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-1 protease inhibitors (Chen et al. 2020). 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-2 infection and as a building block of combination therapies for COVID-19.
Results
To identify potential inhibitors of SARS-CoV-2 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-1 protease inhibitor nelfinavir, which was one of the top scoring compounds in our analysis, was previously shown to decrease SARS-CoV and SARS-CoV-2 replication in vitro (Yamamoto et al. 2004, n.d.) (Table 1). Two additional drugs used for treatment of HIV-1 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., 2010). The latter was a particularly interesting candidate, given its activity as a booster for HIV-1 protease inhibitors (Sherman et al. 2015), 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-2 corroborated a high predicted affinity for the target (
Taken together, these data show that cobicistat has a direct antiviral effect on SARS-CoV-2 replication in vitro.
We next analyzed more thoroughly the antiviral effects of cobicistat using three cell lines of different origin, i.e. Calu-3 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-2 replication. Each cell line was infected using two different multiplicities of infection [(MOI) 0.05 and 0.5] and left untreated or treated with various concentrations of cobicistat 2 h post-infection. In all cell lines, cobicistat showed a dose dependent effect in decreasing viral RNA release in supernatant (
Interestingly, maximum plasma concentrations achievable through standard dosing of cobicistat (150 mg/day as a booster for HIV-1 protease inhibitors) (Deeks 2014) were well below (≈1 μM) most IC50 values obtained in our experiments (
Overall, our data show that non-toxic concentrations of cobicistat can consistently decrease SARS-CoV-2 replication in various cellular infection models. Moreover, these data prove that plasma concentrations obtained through standard HIV-1 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. 2020). Apart from cobicistat, compounds tested included HIV-1 protease inhibitors highlighted by our molecular docking [nelfinavir, tipranavir] or previously administered in clinical trials as SARS-CoV-2 therapeutics [darunavir (Chen et al. 2020), lopinavir (Cao et al. 2020)], as well as two positive controls known to inhibit 3CLpro activity [MG132 and GC376 (Ma et al. 2020)].
While treatment with the known inhibitors of 3CLpro, such as GC376 and MG-132, potently reduced the catalytic activity of the enzyme, cobicistat was surprisingly inactive (
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. 2020). The results showed the reduction of a high molecular weight band (≈250 kDa) when infected cells were incubated with low micromolar concentrations of cobicistat (data not shown). Based on the known molecular weights of SARS-CoV-2 proteins, we postulated that the patterns detected with patient sera corresponded to dimers/trimers of the S-protein (Ou et al. 2020; Algaissi et al. 2020) and to the nucleoprotein (N-protein) (Algaissi et al. 2020) of the virus. To confirm this hypothesis, we performed western blot analysis using monoclonal antibodies against the S and N proteins (
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-2 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-2 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.M.A., Human Medicines Division. Summary on compassionate use Remdesivir Gilead (Siegel et al. 2017)]. We thus examined the in silico predicted affinity of remdesivir for the main members of the CYP3A family (CYP3A4 and 5), as well as for P-gp. The SwissADME server (Daina, Michielin, and Zoete 2017) predicted remdesivir to be both a CYP3A4 and P-gp substrate by using machine learning models with 79% and 88% accuracy, respectively. Similarly, the pkCSM (Pires, Blundell, and Ascher 2015) and CYPreact (Tian et al. 2018) 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-2 infection (
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 (
Overall, our data prove that the combination of cobicistat and remdesivir can suppress viral replication in different cellular models of SARS-CoV-2 infection and show that cobicistat can exert a double activity as direct antiviral agent and as pharmacoenhancer.
Discussion
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-2. 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. 2001; Naggie and Muir 2017). Translating this concept to SARS-CoV-2 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. 2020; Beigel et al. 2020; Y. Wang et al. 2020; RECOVERY Collaborative Group, Horby, Mafham, et al. 2020). Similarly disappointing results were observed in the early stages of HIV-1 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. 2015) could help to overcome these limitations.
While the present study exclusively focused on the combination of cobicistat and remdesivir, more than 30% of all drugs are metabolized by the main cellular targets of cobicistat (i.e. CYP3A4/5) (van Waterschoot et al. 2010). For example, the recently described SARS-CoV-2 inhibitor plitidepsin (White et al. 2021) is mainly metabolized by CYP3A4 in vitro (Brandon et al. 2007). 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-2 patients, such as chloroquine/hydroxychloroquine (K.-A. Kim et al. 2003) and lopinavir (van Waterschoot et al. 2010), 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-2 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-2 replication was higher than that achievable through standard dosages (i.e. 150 mg/day) approved for treatment of HIV-1 infection (Deeks 2014). Therefore, the antiviral effect of cobicistat requires administration of the drug at higher dosages (e.g. 400 mg/day) which result in plasma levels compatible with the antiviral concentrations described in our study (Mathias et al. 2010). These observations can also explain the lack of success of an early trial testing the HIV-1 protease inhibitor darunavir, boosted by a standard cobicistat dose (Chen et al. 2020). 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-2 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. 2020). Of note, previous experience with HIV-1 protease inhibitors suggests that cobicistat might overcome this limitation (Lepist et al. 2012), 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 2020), 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-19, a stage where the efficacy of remdesivir could not be firmly established (Y. Wang et al. 2020).
Overall, our study introduces cobicistat as an agent for inhibiting SARS-CoV-2 replication and for combination therapies aimed at blocking or reversing the onset of COVID-19.
The following examples and drawings illustrate the present invention without, however, limiting the same thereto.
A-C) In-silico docking (A,B) and molecular dynamics (C-E) analysis of the putative mode and energy of binding of cobicistat to SARS-CoV-2 3CLpro.
A) Docking pose showing the ligand interaction of cobicistat to the active site of 3CLpro and the formation of hydrogen bonds to ASN142, GLY143 and GLN189 of 3CLpro.
B) Overlay of crystal structures of SARS-Cov-2 3CLpro showing the amino acids important for the binding of cobicistat to the active site of the enzyme. Residues of the catalytic dyad (Cys145 and His41) of 3CLpro were among the highest contributors to non covalent binding to cobicistat. The source and list of structures used are detailed in Example 1.
C) Schematic representation of time course experiments evaluating in vitro inhibition of SARS-CoV-2 replication by cobicistat.
D,E) Effect of various concentrations of cobicistat, added according to the scheme of (D) on intracellular and supernatant SARS-CoV-2 RNA content in Calu-3 cells. Viral RNA content was measured by qPCR using the 2019-nCoV_N1 primer set (Center of Disease Control). Fold change values in intracellular RNA (D) were calculated by the delta-delta CT method, using the Tata-binding protein (TBP) gene as housekeeper control. Expression levels in supernatant (E) were quantified using an in vitro transcribed standard curve generated as described in Example 1. Data are expressed as mean with SD and were analyzed by two-way ANOVA followed by Dunnet's post-test (N=3 independent experiments). *P<0.05; **P<0.01; ***P<0.001.
A,B) Effect of serial dilutions of cobicistat on SARS-CoV-2 RNA concentration in supernatants (A) and on the viability of infected and uninfected cell lines of lung (Calu-3), gut (T84) and kidney (Vero E6) origin (A,B). Cells were infected with SARS-CoV-2 at two different MOIs (0.05 and 0.5) and left untreated or treated with cobicistat two hours post-infection. Forty-eight hours post-infection supernatants were collected and viral RNA was assayed by qPCR while cellular viability was measured by MTT assay (A) or by crystal violet staining (B). Inhibition of viral replication was calculated as described in Example 1 while viability data were normalized to the uninfected or to the untreated control. Half maximal inhibitory (IC50) concentration values were calculated by nonlinear regression. Each point in panel A represents a mean of 3 independent experiments. Pictures in panel B are derived from infections at MOI 0.5 (Calu-3 and T84 cells) or MOI 0.05 (Vero E6 cells).
C) Comparison between the IC50 and CC50 values of cobicistat determined in vitro and the peak plasma levels detectable in mice and in after administration of a single dose of the drug. Determination of in vitro CC50 values is based on the data shown in (D).
D) Uninfected cell lines of lung (Calu-3), gut (T84) and kidney (Vero E6) origin were left untreated or treated with serial dilutions of cobicistat. Forty-eight hours post-treatment cellular viability was measured by MTT assay. Data, expressed as mean±SD of three independent experiments, were normalized to the untreated control and CC50 values were calculated by nonlinear regression.
A,B). Screening of putative inhibitors of the enzymatic activity of 3CLpro. The activity of 3 CLpro was measured by FRET assay and normalized over the untreated condition (A). Apart from cobicistat, compounds tested included HIV-1 protease inhibitors [nelfinavir, tipranavir] and compounds previously administered in clinical trials as SARS-CoV-2 therapeutics [darunavir, lopinavir], as well as two positive controls known to inhibit 3CLpro activity [MG132 and GC376]. EC50 values were calculated by nonlinear regression (B).
C) Effect of cobicistat on the expression of S- and N-proteins in SARS-CoV-2 infected Vero E6 cells. Cells were infected at 0.5 MOI and left untreated or treated, two hours post-infection, with various concentrations of cobicistat, of the RdRP inhibitor remdesivir, or the 3CLpro inhibitor GC376. Cells were harvested 24 hours post-treatment and subjected to protein extraction and subsequent analysis by Western Blot. Expression of S- and N-proteins, and expression of the housekeeping protein actin-(3, were detected using primary monoclonal antibodies followed by incubation with fluorescent-conjugated secondary antibodies and detection on a LI-COR Odyssey® CLx instrument. Data are representative of three independent experiments.
D,E) Effect of cobicistat on S-protein-mediated syncytia formation. Vero E6 cells were transfected with the SARS-CoV-2 S-protein and left untreated or treated with various concentrations of cobicistat or with sera isolated from convalescent SARS-CoV-2 patients (1:100 dilution). Syncytia formation was examined 24 hours post-transfection by immunofluorescence (IF) staining for DAPI and S-protein (D) and quantified as the number of cells forming syncytia (E). Data were analyzed using the nonparametric Kruskal-Wallis test followed by Dunn's post-test. Horizontal lines represent mean values. **P<0.01; ****P<0.0001. Scale bar=50 μM.
A,B) The relative expression of CYP3A4/5 and P-gp was analyzed by qPCR in uninfected (A) and SARS-CoV-2 infected or mock infected (B) cells. Infections were carried out at MOI 0.5 for 48 hours. Raw data were used to calculate delta CT values (A), by using the TBP gene as housekeeping control. Fold changes, in infected over mock infected cells, were then calculated using the delta-delta CT method. Data in (B) are expressed as mean±SD (N=3).
A-F) Synergistic activity of cobicistat and remdesivir in inhibiting replication and cytopathic effects of SARS-CoV-2 in Vero E6 cells. Cells were infected at 0.5 MOI and left untreated or treated with the drugs at the indicated concentrations two hours-post infection. Forty Eight hours post-treatment: cells were fixed for immunofluorescence (IF) staining (A,B), supernatants were collected for qPCR (C-E) or cellular viability was analyzed (F). For IF detection, cells were stained with sera of SARS-CoV-2 patients and with the J2 antibody, which binds to double stranded RNA (Pape et al. 2020). The percentage of infected cells was determined by automatic acquisition of nine images per well (A), as described in Example 1. Scale bar=100 μM. Viral RNA in supernatants was detected by qPCR using an in vitro transcribed standard curve for absolute quantification (C-E) and data, expressed as mean±SD, were transformed as Logic) to restore normality and analyzed by one-way ANOVA, followed by Holm-Sidak's post-test (C). Cellular viability was measured by MTT assay (F).
Isobologram analysis of synergism (D) (Chou 2010) was performed using the IC90 values for SARS-CoV-2 replication of cobicistat, remdesivir, or their combination, calculated by non-linear regression. Synergism analyses of the inhibition of viral replication (E) or cytopathic effects (F) were performed with the SynergyFinder web-tool using the Zero Interaction Potency (ZIP) model based on inhibition values calculated as described in Example 1.
G) Effect of the combination of cobicistat and remdesivir on SARS-CoV-2 RNA expression in supernatants of a primary human colon organoid. Treatment with cobicistat/remdesivir was performed two hours post-infection and supernatants were collected forty-eight hours post-treatment. Viral RNA was quantified as described for panel (C).
For all panels N=3 independent experiments, except for panel E (N=2 independent experiments) and panel G (N=2 replicates from one colon organoid donor). ***P<0.001; **P<0.01; *P<0.05.
Effect of combined treatment of cobicistat and remdesivir on the viability of SARS-CoV-2 infected Vero E6 cells (A) and on viral replication (B,C) and inhibition of cytopathic effects (D) in T84 cells. Cells were infected at 0.5 MOI and left untreated or treated with the drugs at the indicated concentrations two hours-post infection. Forty Eight hours post-treatment: cells were fixed for crystal violet (A) or immunofluorescence (IF) (B) staining, supernatants were collected for qPCR (C), or cellular viability was analyzed (D). For IF detection, cells were stained with sera of SARS-CoV-2 patients (B). Viral RNA in supernatants was detected by qPCR using an in vitro transcribed standard curve for absolute quantification and data, expressed as mean±SD, were analyzed by non-parametric Friedman test, followed by Dunn's post-test (C). Scale bar=100 μM. Cellular viability was measured by MTT assay and synergism analysis of the inhibition cytopathic effects was performed with the SynergyFinder web-tool using the Zero Interaction Potency (ZIP) model based on inhibition values calculated as described in the Methods section. For panels C,D N=3 independent experiments. *P<0.05.
Effect of combined treatment of cobicistat and remdesivir on the viability of SARS-CoV-2 infected Calu-3 (A) and T84 (B) cells. Cells were infected at 0.5 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-2 inhibitors with desirable Absorption, Distribution, Metabolism, Excretion and Toxicity (ADME-Tox) properties, was performed by structure-based virtual screening (SBVS) of Drugbank V. 5.1.5(72) compounds targeting the three-dimensional structure of SARS-CoV-2 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., 2020), 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 20 Å stored as Glide grid file. Drug screening was performed using the Glide software (Friesner et al., 2004). 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 10% 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 CYP3A4/5. 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 2.5 kcal/mol window. Retained poses for the initial docking phase were set to 5000 poses and only 800 best poses per ligand were selected for energy minimization. Finally, post-docking minimization was carried out for 10 poses per ligand with a 0.5 kcal/mol threshold for rejecting minimized poses.
The following cell lines were used for infection and/or relative quantification of gene expression: Calu-3 (ATCC HTB-55), Caco-2 (ATCC HTB-37), T84 (ATCC CCL-24) and VeroE6 (ATCC CRL-1586). Primary organoids derived from human colon and ileum were seeded in 2D as described in (Stanifer et al. 2020). Culture conditions and susceptibility to SARS-CoV-2 infection have been previously described (Cortese et al. 2020; Stanifer et al. 2020).
Viral stocks used for infections were produced by passaging the BavPatl/2020 SARS-CoV-2 strain in Vero E6 cells and the infectious titer was estimated by plaque assay, as previously described. Infection experiments were conducted using 25,000 or 250,000 cells per well in 96 and 12 well plates, respectively. Cell lines were infected at 0.05 or 0.5 MOI in medium with low FCS content (2%). Colon organoids were infected in a 24-well plate using 60000 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-2 replication: cobicistat (#sc-500831; Santa Cruz Biotechnology), remdesivir (#S78932; Selleckchem Chemicals), tipranavir (#sc-220260; 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-132 (#M8699; Sigma-Aldrich), GC376 (BPS Bioscience), Chloroquine (#C 6628, Sigma Aldrich).
5. RNA Isolation and cDNA Retrotranscription
RNA extraction was performed on cell lysates or supernatants using the NucleoSpin RNA, Mini kit for RNA purification (Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions. The concentration of RNA extracted from cell lysates was measured using a P-class P 300 NanoPhotometer (Implen GmbH, Munich, Germany).
Retrotranscription to cDNA was performed with 500 ng of intracellular RNA or 10 μL of RNA from supernatants, using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., 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-2 N sequence was reverse transcribed from total RNA isolated from cells infected with the SARS-CoV-2 BavPatl stain using Superscript 3 and specific primers (TTAGGCCTGAGTTGAGTCA, SEQ ID NO. 1). The resulting cDNA was amplified and cloned into the pJET1.2 plasmid. Ten μg of plasmid DNA was linearized by Adel 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 2013). In vitro transcripts were purified by phenol-chloroform extraction and resuspended in RNase-free water. RNA integrity was confirmed by agarose gel electrophoresis.
7. qPCR Analysis
Gene and/or viral expression were analyzed by SYBR green qPCR using, for each reaction, 10 μL of SsoFast™ EvaGreen® Supermix (Bio-Rad Laboratories, Hercules, Calif., USA), 500 nM of forward and reverse primer (0.1 μL each from 100 μM stock), 8.8 μL water and 1 μL cDNA. The primers used are listed in Table 2. The qPCR reaction was performed on a CFX96/C1000 Touch qPCR system (Bio-Rad Laboratories, Hercules, Calif., USA) using the following PCR program: polymerase activation/DNA denaturation 98° C. for 3 min, followed by 45 cycles of denaturation at 98° C. for 10 s; annealing/extension at 60° C. for 40 s and a final extension step at the end of the program at 65° C. for 30 s. Gene expression data were normalized using the delta-delta CT method [2(−ΔΔC(T)) method] (Livak and Schmittgen 2001), using the Tata-binding protein (TBP) gene as housekeeper control.
For Western blot experiments 0.5×106 cells were lysed in a buffer (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 0.1% SDS, and 0.5% sodium deoxycholate supplemented with protease and phosphatase inhibitors (Sigma-Aldrich, Saint Louis, Mich., USA). Lysates were boiled at 95° C. for 10 min and sonicated for 5 min using a Bioruptor® Plus sonication device (Diagenode, Liege, Belgium). Protein lysates were then run on a precast NuPAGEBis-Tris 4-12% (Thermo Fisher Scientific, Waltham, Mass., USA) SDS-PAGE at 100-120 V and transferred onto a nitrocellulose membrane (GE Healthcare, Little Chalfont, UK) for 2.5 h at 25 V using a Trans-Blot device for semi-dry transfer (Bio-Rad Laboratories, Hercules, Calif., USA). Membranes were blocked using the LI-COR Intercept (PBS) Blocking Buffer (LI-COR Biosciences, Lincoln, Nebr., USA) for 1 h at RT and incubated overnight at 4° C. with the following primary antibodies in blocking buffer with 0.2% Tween 20: α-β-actin (1:10,000), (Sigma-Aldrich, Saint Louis, Mich., USA), a-SARS-CoV-2 spike protein [(rabbit; 1:1000) ab252690 Abcam], α-SARS-CoV-2 nucleocapsid [(mouse; 1:1000) AB_2827977, Sino Biological)], sera of SARS-CoV-2 positive individuals (1:200). Sera were collected as described in (Pape et al. 2020), 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 0.1% PBS-Tween and incubated for 1 h 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, Nebr., USA). All secondary antibodies were diluted 1:15000 in blocking buffer+0.2% Tween. After three washes with 0.1% PBS-Tween and one wash in PBS, fluorescence signals were acquired using a LI-COR Odyssey® CLx instrument.
Microarray gene expression data for CYP3A4/5 and P-gp in different anatomical tissues or cell lines were retrieved from Homo Sapiens Affymetrix Human Genome U133 Plus 2.0 Array dataset. Data were filtered by applying the criteria “Healthy sample status” and “No experimental treatment”. From the initial list, tissues with sample size<25 were filtered out. The anatomy search tool was used to plot Log 2 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 75)” dataset. The cell line condition filter was used to refine the analysis and include exclusively cell lines susceptible to SARS-CoV-2 infection (i.e. T84, Caco2, Calu-3 and A-549).
Cell viability was evaluated by (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT) assay and by crystal violet staining as previously described (Shytaj et al. 2020; Feoktistova, Geserick, and Leverkus 2016). Briefly, the MTT assay was conducted using the CellTiter 96® Non-Radioactive Cell Proliferation Assay (MTT) (Promega; Madison, Wis., USA). Cells were plated in a 96-well plate at a concentration of 3×106 cells/mL in 100 μl of medium. The MTT solution (15 μl) was added to each well and, after 2-4 h, the reaction was stopped by the addition of 100 μl of 10% SDS. Absorbance values were acquired using an Infinite 200 PRO (Tecan, Männedorf, Switzerland) multimode plate reader at 570 nm wavelength.
For the crystal violet staining, cells were fixed in 6% formaldehyde and incubated with 0.1% crystal violet for 15 mins. Unbound staining was then washed with H2O and cells were imaged using a Nikon Eclipse Ts2-FL microscope.
The activity of 3CLpro was measured by FRET assay (BPS Bioscience, San Diego, Calif., USA) according to the manufacturer's instructions and as previously described (Zhang et al. 2020). Briefly, serial dilutions of test compounds and known 3CLpro were incubated in a 384 well plate with the 3CLpro and its appropriate buffer, containing 0.5 M DTT. Wells without drugs or without 3CLpro were used as positive control of 3CLpro activity and blank control, respectively. After a 30 min incubation, the 3CLpro substrate was added to each well and the plate was stored for 4 hours in the dark. The fluorescence signal was acquired on an Infinite 200 PRO (Tecan, Männedorf, Switzerland) using an excitation wavelength of 360 nm and a detection wavelength of 460 nm. 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 96 well plate and infected with SARS-CoV-2 strain BavPatl/2020 for 24-48 h at MOI 0.5. Cells were rinsed in PBS and fixed with 6% PFA, followed by permeabilization with 0.5% Triton X100 (Sigma) in PBS for 15 minutes. Cells were then subjected to a standard immunofluorescence staining protocol. Briefly, cells were blocked in 2% milk (Roth) in PBS and incubated with primary antibodies in PBS (anti ds-RNA mouse monoclonal J2 antibody (Scicons) 1:2000 and patient serum 1:250). Cells were washed twice in PBS 0.02% tween and incubated with secondary antibody in PBS (1:1000 anti-mouse 568, Goat anti-human IgG-AlexaFluor 488 (Invitrogen, Thermofisher Scientific) for immunoglobulins detection in human serum and goat anti-mouse IgG-AlexaFluor 568 (Invitrogen, Thermofisher Scientific) for dsRNA detection). Nuclei were counterstained with Hoechst 33342 (Thermofisher Scientific, 0.002 μg/ml in PBS) for 5 minutes, washed twice with PBS and stored at +4° C. until imaging.
For syncytia formation assay, Vero E6 cells (0.2×106 cells/well) were seeded on cover slips in a 12 well plate 24 h prior transfection. Cells were transfected using TransIT-2020 or TransIT-LT1 (Mirus) with 0.75 μg of pCDNA3.1(+)-SARS-CoV-2-S and 100 μl Opti-MEM per well. 2 h post transfection, cells were treated with cobicistat (final concentration of 1 μM, 5 μM and 10 μM), serum of patients (1:500 or 1:100) or DMSO (same concentration as in 10 μM cobicistat). 24 h post transfection, cells were washed twice with PBS and fixed in 4% PFA for 20 min at room temperature. After another washing step, cells were permeabilized in 0.5% Triton for 5 min at room temperature, washed and blocked in 3% lipid-free BSA in PBS-0.1% Tween-20 for 1 h at room temperature. After washing, cells were stained with the primary rabbit polyclonal anti-SARS-CoV-2 spike glycoprotein antibody (1:1000, Abcam) for 1 h at room temperature or overnight at 4° C. After washing, cells were incubated with the secondary Alexa Fluor 488 goat anti-rabbit IgG antibody (1:500, Life Technologies) for 1 h at room temperature. After washing, cells were incubated with DAPI (1:1000, Sigma-Aldrich) for 1 min 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 20×/0.75 air objective and a back-illuminated EM-CCD camera (Andor iXon DU-888). JOBS module was used for automatic acquisition of 9 images per well. Images were acquired in 3 channels using the following excitation/emission settings: Ex 377/50, Em 447/60 (Hoechst); Ex 482/35, Em 536/40 (AlexaFluor 488); Ex 562/40, Em 624/40 (AlexaFluor 568). When spinning disc was used the excitation was performed with 405 nm, 488 nm and 561 nm 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 (5 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 >3). 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 relative inhibition values calculated according to the formula: % inhibition=100*(1−(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, Calif., USA). Synergy scores were calculated using the SynergyFinder web-tool (Ianevski et al. 2020) using the Zero Interaction Potency (ZIP) model (Yadav et al. 2015).
The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/055621 | 3/5/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63040758 | Jun 2020 | US |
