Screening of an FDA-Approved Compound Library Identifies Four Small-Molecule Inhibitors of Middle East Respiratory Syndrome Coronavirus Replication in Cell Culture
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ABSTRACT
Coronaviruses can cause respiratory and enteric disease in a wide variety of human and animal hosts. The 2003 outbreak of severe acute respiratory syndrome (SARS) first demonstrated the potentially lethal consequences of zoonotic coronavirus infections in humans. In 2012, a similar previously unknown coronavirus emerged, Middle East respiratory syndrome coronavirus (MERS-CoV), thus far causing over 650 laboratory-confirmed infections, with an unexplained steep rise in the number of cases being recorded over recent months. The human MERS fatality rate of ∼30% is alarmingly high, even though many deaths were associated with underlying medical conditions. Registered therapeutics for the treatment of coronavirus infections are not available. Moreover, the pace of drug development and registration for human use is generally incompatible with strategies to combat emerging infectious diseases. Therefore, we have screened a library of 348 FDA-approved drugs for anti-MERS-CoV activity in cell culture. If such compounds proved sufficiently potent, their efficacy might be directly assessed in MERS patients. We identified four compounds (chloroquine, chlorpromazine, loperamide, and lopinavir) inhibiting MERS-CoV replication in the low-micromolar range (50% effective concentrations [EC50s], 3 to 8 μM). Moreover, these compounds also inhibit the replication of SARS coronavirus and human coronavirus 229E. Although their protective activity (alone or in combination) remains to be assessed in animal models, our findings may offer a starting point for treatment of patients infected with zoonotic coronaviruses like MERS-CoV. Although they may not necessarily reduce viral replication to very low levels, a moderate viral load reduction may create a window during which to mount a protective immune response.
INTRODUCTION
In June 2012, a previously unknown coronavirus was isolated from a patient who died from acute pneumonia and renal failure in Saudi Arabia (1, 2). Since then, the virus, now known as the Middle East respiratory syndrome coronavirus (MERS-CoV) (3), has been contracted by hundreds of others in geographically distinct locations in the Middle East, and evidence for limited human-to-human transmission has accumulated (4). Travel-related MERS-CoV infections were reported from a variety of countries in Europe, Africa, Asia, and the United States, causing small local infection clusters in several cases (http://www.who.int/csr/disease/coronavirus_infections/en/). About 200 laboratory-confirmed human MERS cases were registered during the first 2 years of this outbreak, but recently, for reasons that are poorly understood thus far, this number has more than tripled within just 2 months' time (April-May 2014 [5]). This sharp increase in reported infections has enhanced concerns that we might be confronted with a repeat of the 2003 severe acute respiratory syndrome (SARS) episode, concerns aggravated by the fact that the animal reservoir for MERS-CoV remains to be identified with certainty (6–9). Furthermore, at about 30%, the current human case fatality rate is alarmingly high, even though many deaths were associated with underlying medical conditions. MERS-CoV infection in humans can cause clinical symptoms resembling SARS, such as high fever and acute pneumonia, although the two viruses were reported to use different entry receptors, dipeptidyl peptidase 4 (DPP4) (10) and angiotensin-converting enzyme 2 (ACE2) (11), respectively.
Coronaviruses are currently divided across four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus (12). MERS-CoV was identified as a member of lineage C of the genus Betacoronavirus (2), which also includes coronaviruses of bat (13, 14) and hedgehog (6) origin. Following the 2003 SARS epidemic, studies into the complex genome, proteome, and replication cycle of coronaviruses were intensified. Coronaviruses are enveloped viruses with a positive-sense RNA genome of unprecedented length (25 to 32 kb [12, 15, 16]). The crystal structures of a substantial number of viral nonstructural and structural proteins were solved, and targeted drug design was performed for some of those (reviewed in reference 17). Unfortunately, thus far, none of these efforts resulted in antiviral drugs that were advanced beyond the preclinical phase (18). The 2003 SARS-CoV epidemic was controlled within a few months after its onset, and since then, the virus has not reemerged, although close relatives continue to circulate in bat species (14). Consequently, the interest in anticoronavirus drug development has been limited, until the emergence of MERS-CoV. Despite the modest size of this CoV outbreak thus far, the lack of effective methods to prevent or treat coronavirus infections in humans is a serious concern for the control of MERS-CoV or the next zoonotic coronavirus.
Antiviral research in the post-SARS era resulted in the identification of several compounds that may target coronavirus replication directly or modulate the immune response to coronavirus infection. For example, entry inhibitors targeting the coronavirus spike protein were developed (reviewed in reference 19). In addition, several of the replicative enzymes (including both proteases and the helicase) were targeted with small-molecule inhibitors, some of which can inhibit coronavirus infection in cell culture at low-micromolar concentrations (20–26; reviewed in reference 27). Broad-spectrum antiviral agents, like the nucleoside analogue ribavirin and interferon (IFN), were tested for their ability to inhibit SARS-CoV infection and were—to a limited extent—used for the treatment of SARS patients during the outbreak (reviewed in references 28 and 29). In the case of ribavirin, mixed results were reported from studies in different cell lines, animal models, and patients. Also, the merits of treating SARS patients with immunomodulatory corticosteroids have remained a matter of debate (reviewed in references 28–30). For MERS-CoV, partial ribavirin sensitivity was observed in cell culture and in a macaque animal model, but only when using very high doses of the compound in combination with IFN-α2b (31, 32). However, in a small-scale clinical trial, this combination therapy did not benefit critically ill MERS patients (33). Nevertheless, the anticoronavirus effects of type I IFN treatment deserve further evaluation, in particular since MERS-CoV seems to be considerably more sensitive than SARS-CoV (34, 35). Treatment with type I IFNs inhibits SARS-CoV and MERS-CoV replication in cell culture (31, 34–41) and, for example, protected macaques against SARS-CoV (36) or MERS-CoV (32) infection. Based on experiments in cell culture, mycophenolic acid was recently reported to inhibit MERS-CoV infection (41, 42), and we and others showed that low-micromolar concentrations of cyclosporine inhibit coronavirus replication (34, 43–45).
We recently described (34) a high-throughput assay for antiviral compound screening that is based on the pronounced cytopathic effect (CPE) caused by MERS-CoV infection in Vero and Huh7 cells. This assay was now further exploited to screen a library of 348 FDA-approved drugs for their potential to inhibit MERS-CoV replication. Chloroquine, chlorpromazine, loperamide, and lopinavir were found to inhibit MERS-CoV replication in vitro at low-micromolar concentrations. In addition, these molecules appear to be broad-spectrum coronavirus inhibitors, as they blocked the replication of human coronavirus 229E and SARS-CoV with comparable efficacy. Since these compounds have already been approved for clinical use in humans, their anti-MERS-CoV activity merits further investigation, in particular in a small-animal model for MERS-CoV infection, of which a first example has recently been described (46).
MATERIALS AND METHODS
Cell culture and virus infection.
Vero, Vero E6, and Huh7 cells were cultured as described previously (34, 47). Infection of Vero and Huh7 cells with MERS-CoV (strain EMC/2012 [1]) at high or low multiplicity of infection (MOI) and SARS-CoV infection of Vero E6 cells (strain Frankfurt-1 [48]) were done as described before (34). Infection with green fluorescent protein (GFP)-expressing recombinant HCoV-229E (HCoV-229E-GFP [49]) was performed in Dulbecco's modified Eagle medium (DMEM) containing 8% fetal calf serum (FCS), 2 mM l-glutamine (PAA Laboratories), nonessential amino acids (PAA Laboratories), and antibiotics. HCoV-229E-GFP was used to infect monolayers of Huh7 cells at an MOI of 5 as described previously (43). MERS-CoV and SARS-CoV titrations by plaque assay were performed essentially as described before (50). For titrations after high-MOI MERS-CoV infections (MOI of 1), cells were washed twice with phosphate-buffered saline (PBS), and the virus titer at 1 h postinfection (p.i.) was determined to correct for the remainder of the inoculum. All work with live MERS-CoV and SARS-CoV was performed inside biosafety cabinets in biosafety level 3 facilities at Leiden University Medical Center or Erasmus Medical Center, Rotterdam, Netherlands.
Screening of an FDA-approved compound library.
A library of 348 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). Compounds were stored as 10 mM stock solutions in dimethyl sulfoxide (DMSO) at 4°C until use. Compound stocks were diluted to a concentration of 200 or 60 μM in Iscove's modified Dulbecco's medium (Life Technologies) containing 1% FCS (PAA Laboratories) and antibiotics. For MERS-CoV studies, Vero cells were seeded in 96-well plates at a density of 2 × 104 cells per well. After overnight incubation of the cells at 37°C, each well was given 50 μl of compound dilution, which was mixed with 100 μl of Eagle's minimal essential medium (EMEM) containing 2% FCS (EMEM–2% FCS) and 50 μl of MERS-CoV inoculum in EMEM–2% FCS. The MOI used was 0.005, and the final compound concentrations tested were 15 or 50 μM. As a solvent control, a subset of wells was given 0.5% DMSO instead of compound dilution. At 3 days postinfection (dpi), differences in cell viability caused by virus-induced CPE and/or compound-specific side effects were analyzed using the CellTiter 96 AQueous nonradioactive cell proliferation (monotetrazolium salt [MTS]) assay (Promega), as described previously (34). The cytotoxic effects of compound treatment were monitored in parallel plates containing mock-infected cells, which were given regular medium instead of virus inoculum.
Compound validation.
For validation experiments, we separately reordered chlorpromazine (CPZ; S2456; SelleckChem), lopinavir (LPV; ABT-378; SelleckChem), and loperamide (LPM; S2480; SelleckChem), which were dissolved in DMSO, and chloroquine (CQ; C6628; Sigma) which was dissolved in PBS. For all compounds, 20 mM stock solutions were stored at −20°C as aliquots for single use. To verify the antiviral effect of CQ, CPZ, LPM, and LPV on MERS-CoV replication, the assay described above was repeated in 96-well plates using Huh7 cells (104 cells seeded per well on the day before infection), and cell viability was assayed at 2 dpi. Likewise, compounds were tested for their inhibitory effect on SARS-CoV infection at 3 dpi (104 Vero E6 cells seeded per well; MOI, 0.005). For HCoV-229E-GFP infections, 104 Huh7 cells were seeded per well, incubated overnight, and infected at an MOI of 5. Medium containing 0 to 50 μM compound was given 1 h before the start of infection (t = −1), and the compound remained present during infection. HCoV-229E-GFP-infected Huh7 cells were fixed at 24 h p.i., and GFP expression was quantified by fluorometry, as described previously (43).
Statistical analysis.
The 50% effective concentration (EC50) and the compound-specific toxicity (50% cytotoxic concentration [CC50]) were calculated with GraphPad Prism 5 software using the nonlinear regression model. The relative efficacy of a compound in specifically inhibiting viral replication (as opposed to inducing cytopathic side effects) was defined as the selectivity index (SI; calculated as CC50/EC50). Statistical analyses were performed using the results of at least two independent experiments.
RESULTS
Screening for FDA-approved compounds with anti-MERS-CoV activity.
A primary library screen was performed using a set of 348 FDA-approved drugs that were evaluated for their ability to inhibit the replication of MERS-CoV in Vero cells (for a complete list of compounds tested, see Dataset S1 in the supplemental material) according to a recently published method that employs a colorimetric cell viability assay to quantify virus-induced CPE (34).
The primary screen resulted in the identification of 11 hits that showed at least 50% inhibition of virus-induced CPE in the absence of cytotoxicity (which was defined as >75% viability in compound-treated mock-infected cultures). Next, these drugs, as well as the earlier reported coronavirus inhibitor chloroquine (51–55), were tested over a broader concentration range (2 to 62.5 μM; see Fig. S1 in the supplemental material). In this screen, compounds were considered confirmed hits when they inhibited MERS-CoV-induced CPE by >60% at nontoxic concentrations (defined as >75% remaining viability in compound-treated mock-infected cultures). Following this second round of testing, cilnidipine, fluoxetine HCl, ivermectin, manidipine, oxybutynin, pyrimethamine, rifabutin, and rifapentine were not further retained (see Fig. S1 in the supplemental material).
Low-micromolar concentrations of chloroquine, chlorpromazine, loperamide, and lopinavir inhibit MERS-CoV replication.
Four compounds were selected for further validation. Chloroquine (CQ) was found to inhibit MERS-CoV replication in a dose-dependent manner with an EC50 of 3.0 μM (SI, 19.4; Fig. 1A and Table 1). Interestingly, another reported inhibitor of clathrin-mediated endocytosis (56), chlorpromazine (CPZ), was also found to inhibit MERS-CoV-induced CPE (EC50, 4.9 μM; SI, 4.3) with a 12 μM dose achieving complete inhibition (Fig. 1B and Table 1). Loperamide (LPM), an antidiarrheal agent, inhibited MERS-CoV-induced CPE with an EC50 of 4.8 μM (Fig. 1C and Table 1) but proved relatively toxic in Huh7 cells. An SI of 3.2 was calculated, and a maximum of 82% inhibition was observed at 8 μM, a concentration that was not cytotoxic. The fourth hit was the human immunodeficiency virus type 1 (HIV-1) protease inhibitor lopinavir (LPV), which was previously shown to inhibit SARS-CoV main protease activity and SARS-CoV replication in vitro (24). LPV inhibited MERS-CoV-induced CPE with an EC50 of 8.0 μM (SI, 3.1; Fig. 1D and Table 1), and a maximal protective effect (89% inhibition) was observed at a dose of 12 μM. Two other MERS-CoV isolates (MERS-HCoV/KSA/UK/Eng-2/2012 and MERS-HCoV/Qatar/UK/Eng-1/2012) (57) were found to be equally sensitive to CQ, CPZ, and LPM while being somewhat less sensitive to treatment with LPV (data not shown).
FIG 1
TABLE 1
| Compound | MERS-CoV | SARS-CoV | HCoV-229E-GFP | ||||||
|---|---|---|---|---|---|---|---|---|---|
| EC50 (μM) | CC50 (μM) | SI | EC50 (μM) | CC50 (μM) | SI | EC50 (μM) | CC50 (μM) | SI | |
| Chloroquine | 3.0 (± 1.1) | 58.1 (± 1.1) | 19.4 | 4.1 (± 1.0) | >128 | >31 | 3.3 (± 1.2) | >50 | >15 |
| Chlorpromazine | 4.9 (± 1.2) | 21.3 (± 1.0) | 4.3 | 8.8 (± 1.0) | 24.3 (± 1.1) | 2.8 | 2.5 (± 1.0) | 23.5 (± 1.0) | 9.4 |
| Loperamide | 4.8 (± 1.5) | 15.5 (± 1.0) | 3.2 | 5.9 (± 1.1) | 53.8 (± 1.7) | 9.1 | 4.0 (± 1.1) | 25.9 (± 1.0) | 6.0 |
| Lopinavir | 8.0 (± 1.5) | 24.4 (± 1.0) | 3.1 | 17.1 (± 1.0) | >32 | >2 | 6.6 (± 1.1) | 37.6 (± 1.3) | 5.7 |
a
EC50 and CC50 values are means (± SD) from a representative experiment (n = 4) that was repeated at least twice. Antiviral activity was determined in Huh7 cells (for MERS-CoV and HCoV-229E-GFP) or VeroE6 cells (for SARS-CoV). See the text for more details.
CQ, CPZ, LPV, and LPM also inhibit replication of SARS-CoV and HCoV-229E.
To investigate whether the MERS-CoV inhibitors identified above are potential broad-spectrum coronavirus inhibitors, we assessed their activity against two other coronaviruses: the alphacoronavirus HCoV-229E and the lineage B betacoronavirus SARS-CoV (MERS-CoV belongs to lineage C). All four compounds inhibited SARS-CoV-induced CPE in a dose-dependent manner (Fig. 2 and Table 1). For CQ, an EC50 of 4.1 μM was observed (Fig. 2A), which is in line with earlier reports (51, 52). This compound did not affect the metabolism of Vero E6 cells or induce alterations in cell morphology at concentrations of up to 128 μM (CC50, >128 μM; SI, >31). LPM and CPZ blocked SARS-CoV CPE with comparable EC50s (4.8 versus 4.9 μM [Fig. 2B and C]). LPV completely blocked SARS-CoV-induced CPE at 12 μM, with an EC50 of 8.0 μM (Fig. 2D).
FIG 2
Anti-HCoV-229E activity was assessed employing a GFP-expressing recombinant virus, as described previously (43, 49). All four compounds inhibited HCoV-229E-GFP replication at concentrations comparable to those needed to inhibit MERS-CoV and SARS-CoV replication (Fig. 3 and Table 1). The CQ EC50 of 3.3 μM (SI, >15) for HCoV-229E-GFP was in the same range as the previously reported concentration (10 μM) needed to significantly reduce HCoV-229E production in the human cell line L132 (53). Furthermore, CPZ, LPM, and LPV inhibited HCoV-229E-GFP replication with EC50s of 2.5 μM (SI, 9.4), 4.2 μM (SI, 6.0), and 6.6 μM (SI, 5.7), respectively.
FIG 3
Time-of-addition experiments suggest that CQ, CPZ, and LPM inhibit an early step in the replicative cycle whereas LPV inhibits a postentry step.
Both CQ and CPZ are known inhibitors of clathrin-mediated endocytosis and may thus inhibit MERS-CoV infection at a very early stage. To investigate this, both compounds were added to cells 1 h before (t = −1) or after (t = +1) infection (MOI, 1). Viral titers were determined at 24 h p.i. by plaque assay (Fig. 4). Virus production was not affected by CQ treatment when the compound was added at 1 h p.i. However, when added prior to infection, 16 and 32 μM concentrations of CQ induced an ∼1-log and 2-log reduction in virus production, respectively (Fig. 4A). Comparable results were obtained upon CQ treatment of MERS-CoV-infected Huh7 cells (Fig. 4B). The results were less unambiguous for CPZ: addition 1 h prior to infection led to an ∼2-log reduction of virus progeny titers; however, when added at 1 h p.i., a modest effect (0.5- to 1-log reduction) was observed (Fig. 4C and D), suggesting that the compound may also affect MERS-CoV infection at a postentry stage. Treatment with 16 μM LPM in Vero cells reduced virus production by ∼2 log when added prior to infection, while a 1-log reduction was observed when LPM was added at 1 h p.i. (Fig. 4E). Although this suggests a more pronounced effect early in MERS-CoV replication, this difference was not clearly observed when using Huh7 (compare Fig. 4E and F). Treatment with LPV from t of −1 or +1 h p.i. was equally effective in inhibiting MERS-CoV progeny production (2- to 3-log reduction), suggesting that LPV blocks a postentry step in the MERS-CoV replicative cycle (Fig. 4G to H).
FIG 4
DISCUSSION
The ongoing MERS-CoV outbreak has made it painfully clear that our current options for treatment of life-threatening zoonotic coronavirus infections in humans are very limited. At present, no drug is available for the treatment of any of the human or zoonotic coronaviruses (reviewed in reference 58), despite the extensive research efforts triggered by the 2003 SARS outbreak (reviewed in references 26 and 27). The brevity of that epidemic is a major reason why, thus far, none of the prototypic coronavirus inhibitors was advanced beyond the (early) preclinical stage. Like SARS-CoV a decade ago and MERS-CoV at present, future emerging coronaviruses will likely continue to pose a threat to global public health. Therefore, the search for broad-spectrum inhibitors that may reduce the impact of coronavirus infections in humans remains a challenging research priority. Given the time-consuming nature of antiviral drug development and registration, existing therapeutics for other conditions may constitute the only immediate treatment option in the case of emerging infectious diseases. For most of these drugs, ample experience is available with dosing in humans, and their safety and absorption, distribution, metabolism, and excretion (ADME) profiles are well known.
At the time of this study, a MERS-CoV infection model in (small) animals was not available. For initial antiviral testing, we therefore used our cell culture-based screening assay (34) to search for compounds that may inhibit MERS-CoV infection. We identified four FDA-approved compounds (chloroquine, chlorpromazine, loperamide, and lopinavir) that inhibit the in vitro replication of MERS-CoV at low-micromolar concentrations (Fig. 1 and Table 1). While for some of these molecules the SI was limited (<10), for each of them we established at least one concentration at which MERS-CoV replication was inhibited by more than 80% without a detectable reduction of cell viability. The same four drugs were also found to inhibit, with comparable potency, the in vitro replication of two other coronaviruses, i.e., HCoV-229E and SARS-CoV (Fig. 2 and 3 and Table 1).
CQ inhibited MERS-CoV replication with an EC50 of 3.0 μM (Fig. 1A) and blocked infection at an early step (Fig. 4A and B). CQ has a tendency to accumulate in lysosomes, where it sequesters protons and increases the pH. In addition, it interacts with many different proteins and cellular processes, resulting in the modulation of autophagy and the immune response (for a review, see reference 59). CQ has also been reported to inhibit the replication of multiple flaviviruses, influenza viruses, HIV (reviewed in reference 60), Ebola virus (61), and Nipah-Hendra virus (62), as well as several coronaviruses, including SARS-CoV, in cell culture (51–55, 63, 64). Early reports showed that high doses of CQ inhibit an early step of the replication of the coronavirus mouse hepatitis virus (MHV). However, in SARS-CoV-infected BALB/c mice, systemically administered CQ did not result in a significant viral load reduction in the lungs. Intranasal administration of CQ (50 mg/kg of body weight) resulted in a minor reduction of viral titers in the lung (65). When pregnant mice were treated with CQ (at 15 mg/kg), their newborn offspring were protected against a lethal challenge with HCoV-OC43 (54). Likely, the accumulation of CQ in the milk glands, resulting in high drug concentrations in maternal milk, was a major factor in reaching a sufficiently high concentration of the drug in blood plasma. CQ was also shown to inhibit the in vitro replication (EC50, 2 μM) of the feline coronavirus infectious peritonitis virus (FIPV) (55). Treatment of naturally infected cats with CQ resulted in a clinical improvement, which was, however, not attributed to a direct antiviral effect but likely due to the immunomodulatory properties of CQ. These results highlight that, e.g., the drug delivery route, virus strain used, and drug dosage might influence the outcome in animal models. In BALB/c mice, steady-state concentrations of 8 μM in plasma were observed following repeated administration of CQ at 90 mg/kg (61), which is above the EC50 of CQ for inhibition of MERS-CoV-induced CPE in this study. Levels of 9 μM in plasma were observed in humans following CQ treatment with 8 mg/kg/day for three consecutive days (66).
The second FDA-approved drug found to block MERS-CoV infection was CPZ, the first antipsychotic drug developed for treatment of schizophrenia (67). CPZ affects the assembly of clathrin-coated pits at the plasma membrane (56) and has been reported to inhibit the replication of alphaviruses (68), hepatitis C virus (69), and the coronaviruses SARS-CoV (70), infectious bronchitis virus (71), and MHV-2 (72). Our time-of-addition studies, however, suggest that CPZ inhibits MERS-CoV replication at both an early and a postentry stage, implying that an effect on clathrin-mediated endocytosis is unlikely to be the sole antiviral mechanism (Fig. 4C and D). CPZ plasma concentrations in patients treated for psychotic disorders range between 0.3 and 3 μM (73), which is somewhat below the observed EC50s observed here (which range between 2 and 9 μM).
The replication of MERS-CoV in vitro was also inhibited by LPM, an antidiarrheal opioid receptor agonist that reduces intestinal motility (reviewed in reference 74). LPM also inhibits the replication of two other coronaviruses at low-micromolar concentrations (4 to 6 μM). Upon oral or intravenous administration, the molecule rapidly concentrates in the small intestine. Less than 1% of orally taken LPM is absorbed from the gut lumen, and its tendency to concentrate at the site of action is the probable basis for its antidiarrheal effect (75). This property would very much limit systemic use for the treatment of respiratory coronavirus infections, although administration in the form of an aerosol might be explored. In the veterinary field, it would be interesting to test whether the compound has the potential to inhibit enteric coronaviruses, such as the porcine transmissible gastroenteritis coronavirus.
Finally, the HIV-1 protease inhibitor (PI) LPV was shown to inhibit MERS-CoV replication with EC50s of about 8 μM, which is in the range of the LPV concentrations in plasma (8 to 24 μM) that have been observed in AIDS patients (76). LPV was previously shown to block the SARS-CoV main protease (Mpro) (24). This is somehow unexpected, since the retro- and coronavirus proteases belong to different protease families (the aspartic and chymotrypsin-like protease families, respectively). Since MERS-CoV and SARS-CoV are relatively closely related, LPV may also target the Mpro of MERS-CoV. However, several anti-HIV PIs are also known to influence intracellular pathways leading to side effects in patients undergoing highly active antiretroviral therapy, including lipodystrophy and insulin resistance (77). The exact cellular targets of these PIs have not yet been identified, and most likely multiple pathways are involved. It remains to be investigated if the effect of LPV on these intracellular pathways is associated with the anti-CoV activity found here. Interestingly, no selective anti-CoV activity was found for two other HIV PIs in the compound library (atazanavir and ritonavir; see Dataset S1 in the supplemental material). During the SARS outbreak, treatment with LPV, in combination with ritonavir, was explored with some success in nonrandomized clinical trials (for reviews, see references 78 and 79).
The efficacy of the most promising compounds identified in this study, CQ and LPV, should now be evaluated in (small-) animal models for MERS-CoV infection, which are still in development. In a nonhuman primate model (macaques), only mild clinical signs developed, in contrast to the frequently severe clinical outcome in humans (80, 81). Unfortunately, Syrian hamsters (82), BALB/c mice (83), and ferrets (84) were found to resist MERS-CoV infection. A very recent study (46) reported that mice can be rendered susceptible to MERS-CoV infection by prior transduction with a recombinant adenovirus that expresses human DPP4, a documented receptor for MERS-CoV entry (10). Subsequent MERS-CoV infection resulted in severe pneumonia and high MERS-CoV titers in the lungs (46). Despite some practical and conceptual limitations, this model may provide a useful starting point for further evaluation of inhibitors of MERS-CoV infection.
In 2003, the ∼10% mortality rate among SARS patients was one of the major reasons for the worldwide public unrest caused by the emergence of SARS-CoV. Clearly, and despite the recent sharp increase in the number of registered cases (5), the course of the MERS-CoV outbreak has been quite different thus far. Although only about 650 laboratory-confirmed cases have been registered in the 2 years that have passed since the first documented human infections, in particular the ∼30% mortality rate within this group remains a grave concern. In this context, efficacious anticoronavirus drugs, administered alone or in combination, can constitute an important first line of defense. It typically takes over 10 years to develop a newly discovered molecule and obtain approval for clinical use. To the best of our knowledge, there are currently no potent and selective coronavirus inhibitors in (early or advanced) preclinical development. Hence, drugs that have been registered for the treatment of other conditions and that also inhibit MERS-CoV replication might be used (off-label) in an attempt to save the life of MERS patients. A combination of two or more of such drugs may cause a modest reduction in viral load, which might aid to control viral replication, slow down the course of infection, and allow the immune system to mount a protective response. In an accompanying paper by Dyall et al. (85), CQ and CPZ were identified as inhibitors of the MERS-CoV as well. Follow-up studies will include in-depth mechanism of action studies, including resistance development of MERS-CoV against the compounds identified. Furthermore, the efficacy of combinations of two or more of these drugs will be explored, also in combination with interferon. In particular, CQ and LPV may constitute valuable candidates for further testing in animal models or direct off-label use, since the concentrations needed to inhibit viral replication in cell culture are in the range of the concentrations that can be achieved in human plasma.
ACKNOWLEDGMENTS
We thank Ali Tas, Corrine Beugeling, and Dennis Ninaber for excellent technical assistance and Bart Haagmans and Ron Fouchier for helpful discussions. We thank Volker Thiel for providing HCoV-229E-GFP.
The research leading to these results has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 260644-SILVER and was supported in part by the Council for Chemical Sciences (CW) of the Netherlands Organization for Scientific Research (NWO) through TOP grant 700.57.301.
Supplemental Material
REFERENCES
1.
Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, and Fouchier RA. 2012. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N. Engl. J. Med. 367:1814–1820.
2.
van Boheemen S, de Graaf M, Lauber C, Bestebroer TM, Raj VS, Zaki AM, Osterhaus AD, Haagmans BL, Gorbalenya AE, Snijder EJ, and Fouchier RA. 2012. Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. mBio 3:e00473-12.
3.
de Groot RJ, Baker SC, Baric RS, Brown CS, Drosten C, Enjuanes L, Fouchier RA, Galiano M, Gorbalenya AE, Memish ZA, Perlman S, Poon LL, Snijder EJ, Stephens GM, Woo PC, Zaki AM, Zambon M, and Ziebuhr J. 2013. Middle East respiratory syndrome coronavirus (MERS-CoV): announcement of the Coronavirus Study Group. J. Virol. 87:7790–7792.
4.
Cotten M, Watson SJ, Zumla AI, Makhdoom HQ, Palser AL, Ong SH, Al Rabeeah AA, Alhakeem RF, Assiri A, Al-Tawfiq JA, Albarrak A, Barry M, Shibl A, Alrabiah FA, Hajjar S, Balkhy HH, Flemban H, Rambaut A, Kellam P, and Memish ZA. 2014. Spread, circulation, and evolution of the Middle East respiratory syndrome coronavirus. mBio 5(1):e01062-13.
5.
Kupferschmidt K. 2014. Emerging diseases. Soaring MERS cases in Saudi Arabia raise alarms. Science 344:457–458.
6.
Corman VM, Kallies R, Philipps H, Gopner G, Muller MA, Eckerle I, Brunink S, Drosten C, and Drexler JF. 2014. Characterization of a novel betacoronavirus related to Middle East respiratory syndrome coronavirus in European hedgehogs. J. Virol. 88:717–724.
7.
Haagmans BL, Al Dhahiry SH, Reusken CB, Raj VS, Galiano M, Myers R, Godeke GJ, Jonges M, Farag E, Diab A, Ghobashy H, Alhajri F, Al-Thani M, Al-Marri SA, Al Romaihi HE, Al Khal A, Bermingham A, Osterhaus AD, Alhajri MM, and Koopmans MP. 2014. Middle East respiratory syndrome coronavirus in dromedary camels: an outbreak investigation. Lancet Infect. Dis. 14:140–145.
8.
Reusken CB, Haagmans BL, Muller MA, Gutierrez C, Godeke GJ, Meyer B, Muth D, Raj VS, Vries LS, Corman VM, Drexler JF, Smits SL, El Tahir YE, De Sousa R, van Beek J, Nowotny N, van Maanen K, Hidalgo-Hermoso E, Bosch BJ, Rottier P, Osterhaus A, Gortazar-Schmidt C, Drosten C, and Koopmans MP. 2013. Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study. Lancet Infect. Dis. 13:859–866.
9.
Memish ZA, Mishra N, Olival KJ, Fagbo SF, Kapoor V, Epstein JH, Alhakeem R, Durosinloun A, Al Asmari M, Islam A, Kapoor A, Briese T, Daszak P, Al Rabeeah AA, and Lipkin WI. 2013. Middle East respiratory syndrome coronavirus in bats, Saudi Arabia. Emerg. Infect. Dis. 19:1819–1823.
10.
Raj VS, Mou H, Smits SL, Dekkers DH, Muller MA, Dijkman R, Muth D, Demmers JA, Zaki A, Fouchier RA, Thiel V, Drosten C, Rottier PJ, Osterhaus AD, Bosch BJ, and Haagmans BL. 2013. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature 495:251–254.
11.
Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, Berne MA, Somasundaran M, Sullivan JL, Luzuriaga K, Greenough TC, Choe H, and Farzan M. 2003. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426:450–454.
12.
de Groot RJ, Cowley JA, Enjuanes L, Faaberg KS, Perlman S, Rottier PJ, Snijder EJ, Ziebuhr J, and Gorbalenya AE. 2012. Order of Nidovirales, p 785–795. In King A, Adams M, Carstens E, and Lefkowitz EJ (ed), Virus taxonomy, the 9th report of the International Committee on Taxonomy of Viruses. Academic Press, New York, NY.
13.
Drexler JF, Corman VM, and Drosten C. 2013. Ecology, evolution and classification of bat coronaviruses in the aftermath of SARS. Antiviral Res. 101:45–56.
14.
Ge XY, Li JL, Yang XL, Chmura AA, Zhu G, Epstein JH, Mazet JK, Hu B, Zhang W, Peng C, Zhang YJ, Luo CM, Tan B, Wang N, Zhu Y, Crameri G, Zhang SY, Wang LF, Daszak P, and Shi ZL. 2013. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 503:535–538.
15.
Woo PC, Lau SK, Lam CS, Tsang AK, Hui SW, Fan RY, Martelli P, and Yuen KY. 2014. Discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in gammacoronavirus. J. Virol. 88:1318–1331.
16.
Woo PC, Lau SK, Lam CS, Lau CC, Tsang AK, Lau JH, Bai R, Teng JL, Tsang CC, Wang M, Zheng BJ, Chan KH, and Yuen KY. 2012. Discovery of seven novel mammalian and avian coronaviruses in the genus Deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J. Virol. 86:3995–4008.
17.
Tong TR. 2009. Drug targets in severe acute respiratory syndrome (SARS) virus and other coronavirus infections. Infect. Disord. Drug Targets 9:223–245.
18.
Hilgenfeld R and Peiris M. 2013. From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses. Antiviral Res. 100:286–295.
19.
Tong TR. 2009. Therapies for coronaviruses. Part I of II—viral entry inhibitors. Expert Opin. Ther. Pat. 19:357–367.
20.
Yang H, Xie W, Xue X, Yang K, Ma J, Liang W, Zhao Q, Zhou Z, Pei D, Ziebuhr J, Hilgenfeld R, Yuen KY, Wong L, Gao G, Chen S, Chen Z, Ma D, Bartlam M, and Rao Z. 2005. Design of wide-spectrum inhibitors targeting coronavirus main proteases. PLoS Biol. 3:e324.
21.
Ghosh AK, Takayama J, Rao KV, Ratia K, Chaudhuri R, Mulhearn DC, Lee H, Nichols DB, Baliji S, Baker SC, Johnson ME, and Mesecar AD. 2010. Severe acute respiratory syndrome coronavirus papain-like novel protease inhibitors: design, synthesis, protein-ligand X-ray structure and biological evaluation. J. Med. Chem. 53:4968–4979.
22.
Hsu JT, Kuo CJ, Hsieh HP, Wang YC, Huang KK, Lin CP, Huang PF, Chen X, and Liang PH. 2004. Evaluation of metal-conjugated compounds as inhibitors of 3CL protease of SARS-CoV. FEBS Lett. 574:116–120.
23.
Kuo CJ, Liu HG, Lo YK, Seong CM, Lee KI, Jung YS, and Liang PH. 2009. Individual and common inhibitors of coronavirus and picornavirus main proteases. FEBS Lett. 583:549–555.
24.
Wu CY, Jan JT, Ma SH, Kuo CJ, Juan HF, Cheng YS, Hsu HH, Huang HC, Wu D, Brik A, Liang FS, Liu RS, Fang JM, Chen ST, Liang PH, and Wong CH. 2004. Small molecules targeting severe acute respiratory syndrome human coronavirus. Proc. Natl. Acad. Sci. U. S. A. 101:10012–10017.
25.
Ratia K, Pegan S, Takayama J, Sleeman K, Coughlin M, Baliji S, Chaudhuri R, Fu W, Prabhakar BS, Johnson ME, Baker SC, Ghosh AK, and Mesecar AD. 2008. A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication. Proc. Natl. Acad. Sci. U. S. A. 105:16119–16124.
26.
Kilianski A, Mielech AM, Deng X, and Baker SC. 2013. Assessing activity and inhibition of Middle East respiratory syndrome coronavirus papain-like and 3C-like proteases using luciferase-based biosensors. J. Virol. 87:11955–11962.
27.
Graham RL, Donaldson EF, and Baric RS. 2013. A decade after SARS: strategies for controlling emerging coronaviruses. Nat. Rev. Microbiol. 11:836–848.
28.
Wong SS and Yuen KY. 2008. The management of coronavirus infections with particular reference to SARS. J. Antimicrob. Chemother. 62:437–441.
29.
Hui DS. 2013. Severe acute respiratory syndrome (SARS): lessons learnt in Hong Kong. J. Thorac. Dis. 5:S122–S126.
30.
Stockman LJ, Bellamy R, and Garner P. 2006. SARS: systematic review of treatment effects. PLoS Med. 3:e343.
31.
Falzarano D, de Wit E, Martellaro C, Callison J, Munster VJ, and Feldmann H. 2013. Inhibition of novel beta coronavirus replication by a combination of interferon-alpha2b and ribavirin. Sci. Rep. 3:1686.
32.
Falzarano D, de Wit E, Rasmussen AL, Feldmann F, Okumura A, Scott DP, Brining D, Bushmaker T, Martellaro C, Baseler L, Benecke AG, Katze MG, Munster VJ, and Feldmann H. 2013. Treatment with interferon-alpha2b and ribavirin improves outcome in MERS-CoV-infected rhesus macaques. Nat. Med. 19:1313–1317.
33.
Al-Tawfiq JA, Momattin H, Dib J, and Memish ZA. 2014. Ribavirin and interferon therapy in patients infected with the Middle East respiratory syndrome coronavirus: an observational study. Int. J. Infect. Dis. 20:42–46.
34.
de Wilde AH, Raj VS, Oudshoorn D, Bestebroer TM, van Nieuwkoop S, Limpens RW, Posthuma CC, van der Meer Y, Barcena M, Haagmans BL, Snijder EJ, and van den Hoogen BG. 2013. MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-alpha treatment. J. Gen. Virol. 94:1749–1760.
35.
Zielecki F, Weber M, Eickmann M, Spiegelberg L, Zaki AM, Matrosovich M, Becker S, and Weber F. 2013. Human cell tropism and innate immune system interactions of human respiratory coronavirus EMC compared to those of severe acute respiratory syndrome coronavirus. J. Virol. 87:5300–5304.
36.
Haagmans BL, Kuiken T, Martina BE, Fouchier RA, Rimmelzwaan GF, van Amerongen G, van Riel D, de Jong T, Itamura S, Chan KH, Tashiro M, and Osterhaus AD. 2004. Pegylated interferon-alpha protects type 1 pneumocytes against SARS coronavirus infection in macaques. Nat. Med. 10:290–293.
37.
Paragas J, Blatt LM, Hartmann C, Huggins JW, and Endy TP. 2005. Interferon alfacon1 is an inhibitor of SARS-corona virus in cell-based models. Antiviral Res. 66:99–102.
38.
Zheng B, He ML, Wong KL, Lum CT, Poon LL, Peng Y, Guan Y, Lin MC, and Kung HF. 2004. Potent inhibition of SARS-associated coronavirus (SCOV) infection and replication by type I interferons (IFN-alpha/beta) but not by type II interferon (IFN-gamma). J. Interferon Cytokine Res. 24:388–390.
39.
Chan RW, Chan MC, Agnihothram S, Chan LL, Kuok DI, Fong JH, Guan Y, Poon LL, Baric RS, Nicholls JM, and Peiris JS. 2013. Tropism and innate immune responses of the novel human betacoronavirus lineage C virus in human ex vivo respiratory organ cultures. J. Virol. 87:6604–6614.
40.
Kindler E, Jonsdottir HR, Muth D, Hamming OJ, Hartmann R, Rodriguez R, Geffers R, Fouchier RA, Drosten C, Muller MA, Dijkman R, and Thiel V. 2013. Efficient replication of the novel human betacoronavirus EMC on primary human epithelium highlights its zoonotic potential. mBio 4:e00611-12.
41.
Chan JF, Chan KH, Kao RY, To KK, Zheng BJ, Li CP, Li PT, Dai J, Mok FK, Chen H, Hayden FG, and Yuen KY. 2013. Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus. J. Infect. 67:606–616.
42.
Hart BJ, Dyall J, Postnikova E, Zhou H, Kindrachuk J, Johnson RF, Olinger GG Jr, Frieman MB, Holbrook MR, Jahrling PB, and Hensley L. 2014. Interferon-beta and mycophenolic acid are potent inhibitors of Middle East respiratory syndrome coronavirus in cell-based assays. J. Gen. Virol. 95:571–577.
43.
de Wilde AH, Zevenhoven-Dobbe JC, van der Meer Y, Thiel V, Narayanan K, Makino S, Snijder EJ, and van Hemert MJ. 2011. Cyclosporin A inhibits the replication of diverse coronaviruses. J. Gen. Virol. 92:2542–2548.
44.
Pfefferle S, Schopf J, Kogl M, Friedel CC, Muller MA, Carbajo-Lozoya J, Stellberger T, von Dall'armi E, Herzog P, Kallies S, Niemeyer D, Ditt V, Kuri T, Zust R, Pumpor K, Hilgenfeld R, Schwarz F, Zimmer R, Steffen I, Weber F, Thiel V, Herrler G, Thiel HJ, Schwegmann-Wessels C, Pohlmann S, Haas J, Drosten C, and von Brunn A. 2011. The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors. PLoS Pathog. 7:e1002331.
45.
Tanaka Y, Sato Y, Osawa S, Inoue M, Tanaka S, and Sasaki T. 2012. Suppression of feline coronavirus replication in vitro by cyclosporin A. Vet. Res. 43:41.
46.
Zhao J, Li K, Wohlford-Lenane C, Agnihothram SS, Fett C, Zhao J, Gale MJ Jr, Baric RS, Enjuanes L, Gallagher T, McCray PB Jr, and Perlman S. 2014. Rapid generation of a mouse model for Middle East respiratory syndrome. Proc. Natl. Acad. Sci. U. S. A. 111:4970–4975.
47.
Snijder EJ, van der MY, Zevenhoven-Dobbe J, Onderwater JJ, van der MJ, Koerten HK, and Mommaas AM. 2006. Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex. J. Virol. 80:5927–5940.
48.
Drosten C, Gunther S, Preiser W, van der Werf S, Brodt HR, Becker S, Rabenau H, Panning M, Kolesnikova L, Fouchier RA, Berger A, Burguiere AM, Cinatl J, Eickmann M, Escriou N, Grywna K, Kramme S, Manuguerra JC, Muller S, Rickerts V, Sturmer M, Vieth S, Klenk HD, Osterhaus AD, Schmitz H, and Doerr HW. 2003. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med. 348:1967–1976.
49.
Cervantes-Barragan L, Zust R, Maier R, Sierro S, Janda J, Levy F, Speiser D, Romero P, Rohrlich PS, Ludewig B, and Thiel V. 2010. Dendritic cell-specific antigen delivery by coronavirus vaccine vectors induces long-lasting protective antiviral and antitumor immunity. mBio 1:e00171-10.
50.
van den Worm SH, Eriksson KK, Zevenhoven JC, Weber F, Zust R, Kuri T, Dijkman R, Chang G, Siddell SG, Snijder EJ, Thiel V, and Davidson AD. 2012. Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination. PLoS One 7:e32857.
51.
Keyaerts E, Vijgen L, Maes P, Neyts J, and Van Ranst M. 2004. In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine. Biochem. Biophys. Res. Commun. 323:264–268.
52.
Vincent MJ, Bergeron E, Benjannet S, Erickson BR, Rollin PE, Ksiazek TG, Seidah NG, and Nichol ST. 2005. Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virol. J. 2:69.
53.
Kono M, Tatsumi K, Imai AM, Saito K, Kuriyama T, and Shirasawa H. 2008. Inhibition of human coronavirus 229E infection in human epithelial lung cells (L132) by chloroquine: involvement of p38 MAPK and ERK. Antiviral Res. 77:150–152.
54.
Keyaerts E, Li S, Vijgen L, Rysman E, Verbeeck J, Van Ranst M, and Maes P. 2009. Antiviral activity of chloroquine against human coronavirus OC43 infection in newborn mice. Antimicrob. Agents Chemother. 53:3416–3421.
55.
Takano T, Katoh Y, Doki T, and Hohdatsu T. 2013. Effect of chloroquine on feline infectious peritonitis virus infection in vitro and in vivo. Antiviral Res. 99:100–107.
56.
Wang LH, Rothberg KG, and Anderson RG. 1993. Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J. Cell Biol. 123:1107–1117.
57.
Cotten M, Lam TT, Watson SJ, Palser AL, Petrova V, Grant P, Pybus OG, Rambaut A, Guan Y, Pillay D, Kellam P, and Nastouli E. 2013. Full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus. Emerg. Infect. Dis. 19:736B–742B.
58.
Barnard DL and Kumaki Y. 2011. Recent developments in anti-severe acute respiratory syndrome coronavirus chemotherapy. Future Virol. 6:615–631.
59.
Thome R, Lopes SC, Costa FT, and Verinaud L. 2013. Chloroquine: modes of action of an undervalued drug. Immunol. Lett. 153:50–57.
60.
Savarino A, Boelaert JR, Cassone A, Majori G, and Cauda R. 2003. Effects of chloroquine on viral infections: an old drug against today's diseases? Lancet Infect. Dis. 3:722–727.
61.
Madrid PB, Chopra S, Manger ID, Gilfillan L, Keepers TR, Shurtleff AC, Green CE, Iyer LV, Dilks HH, Davey RA, Kolokoltsov AA, Carrion R Jr, Patterson JL, Bavari S, Panchal RG, Warren TK, Wells JB, Moos WH, Burke RL, and Tanga MJ. 2013. A systematic screen of FDA-approved drugs for inhibitors of biological threat agents. PLoS One 8:e60579.
62.
Porotto M, Orefice G, Yokoyama CC, Mungall BA, Realubit R, Sganga ML, Aljofan M, Whitt M, Glickman F, and Moscona A. 2009. Simulating henipavirus multicycle replication in a screening assay leads to identification of a promising candidate for therapy. J. Virol. 83:5148–5155.
63.
Mallucci L. 1966. Effect of chloroquine on lysosomes and on growth of mouse hepatitis virus (MHV-3). Virology 28:355–362.
64.
Krzystyniak K and Dupuy JM. 1984. Entry of mouse hepatitis virus 3 into cells. J. Gen. Virol. 65:227–231.
65.
Barnard DL, Day CW, Bailey K, Heiner M, Montgomery R, Lauridsen L, Chan PK, and Sidwell RW. 2006. Evaluation of immunomodulators, interferons and known in vitro SARS-coV inhibitors for inhibition of SARS-coV replication in BALB/c mice. Antivir. Chem. Chemother. 17:275–284.
66.
Marques MM, Costa MR, Santana Filho FS, Vieira JL, Nascimento MT, Brasil LW, Nogueira F, Silveira H, Reyes-Lecca RC, Monteiro WM, Lacerda MV, and Alecrim MG. 2014. Plasmodium vivax chloroquine resistance and anemia in the Western Brazilian Amazon. Antimicrob. Agents Chemother. 58:342–347.
67.
Miyamoto S, Miyake N, Jarskog LF, Fleischhacker WW, and Lieberman JA. 2012. Pharmacological treatment of schizophrenia: a critical review of the pharmacology and clinical effects of current and future therapeutic agents. Mol. Psychiatry 17:1206–1227.
68.
Pohjala L, Utt A, Varjak M, Lulla A, Merits A, Ahola T, and Tammela P. 2011. Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays. PLoS One 6:e28923.
69.
Blanchard E, Belouzard S, Goueslain L, Wakita T, Dubuisson J, Wychowski C, and Rouille Y. 2006. Hepatitis C virus entry depends on clathrin-mediated endocytosis. J. Virol. 80:6964–6972.
70.
Inoue Y, Tanaka N, Tanaka Y, Inoue S, Morita K, Zhuang M, Hattori T, and Sugamura K. 2007. Clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ACE2 with the cytoplasmic tail deleted. J. Virol. 81:8722–8729.
71.
Chu VC, McElroy LJ, Ferguson AD, Bauman BE, and Whittaker GR. 2006. Avian infectious bronchitis virus enters cells via the endocytic pathway. Adv. Exp. Med. Biol. 581:309–312.
72.
Pu Y and Zhang X. 2008. Mouse hepatitis virus type 2 enters cells through a clathrin-mediated endocytic pathway independent of Eps15. J. Virol. 82:8112–8123.
73.
Rivera-Calimlim L and Hershey L. 1984. Neuroleptic concentrations and clinical response. Annu. Rev. Pharmacol. Toxicol. 24:361–386.
74.
Regnard C, Twycross R, Mihalyo M, and Wilcock A. 2011. Loperamide. J. Pain Symptom Manage. 42:319–323.
75.
Awouters F, Niemegeers CJ, and Janssen PA. 1983. Pharmacology of antidiarrheal drugs. Annu. Rev. Pharmacol. Toxicol. 23:279–301.
76.
Lopez Aspiroz E, Santos Buelga D, Cabrera Figueroa S, Lopez Galera RM, Ribera Pascuet E, Dominguez-Gil Hurle A, and Garcia Sanchez MJ. 2011. Population pharmacokinetics of lopinavir/ritonavir (Kaletra) in HIV-infected patients. Ther. Drug Monit. 33:573–582.
77.
Caron-Debarle M, Boccara F, Lagathu C, Antoine B, Cervera P, Bastard JP, Vigouroux C, and Capeau J. 2010. Adipose tissue as a target of HIV-1 antiretroviral drugs. Potential consequences on metabolic regulations. Curr. Pharm. Des. 16:3352–3360.
78.
Tai DY. 2007. Pharmacologic treatment of SARS: current knowledge and recommendations. Ann. Acad. Med. Singapore 36:438–443.
79.
Cheng VC, Chan JF, To KK, and Yuen KY. 2013. Clinical management and infection control of SARS: lessons learned. Antiviral Res. 100:407–419.
80.
Munster VJ, de Wit E, and Feldmann H. 2013. Pneumonia from human coronavirus in a macaque model. N. Engl. J. Med. 368:1560–1562.
81.
Yao Y, Bao L, Deng W, Xu L, Li F, Lv Q, Yu P, Chen T, Xu Y, Zhu H, Yuan J, Gu S, Wei Q, Chen H, Yuen KY, and Qin C. 2014. An animal model of MERS produced by infection of rhesus macaques with MERS coronavirus. J. Infect. Dis. 209:236–242.
82.
de Wit E, Rasmussen AL, Falzarano D, Bushmaker T, Feldmann F, Brining DL, Fischer ER, Martellaro C, Okumura A, Chang J, Scott D, Benecke AG, Katze MG, Feldmann H, and Munster VJ. 2013. Middle East respiratory syndrome coronavirus (MERS-CoV) causes transient lower respiratory tract infection in rhesus macaques. Proc. Natl. Acad. Sci. U. S. A. 110:16598–16603.
83.
Coleman CM, Matthews KL, Goicochea L, and Frieman MB. 2014. Wild-type and innate immune-deficient mice are not susceptible to the Middle East respiratory syndrome coronavirus. J. Gen. Virol. 95:408–412.
84.
Raj VS, Smits SL, Provacia LB, van den Brand JM, Wiersma L, Ouwendijk WJ, Bestebroer TM, Spronken MI, van Amerongen G, Rottier PJ, Fouchier RA, Bosch BJ, Osterhaus AD, and Haagmans BL. 2014. Adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the Middle East respiratory syndrome coronavirus. J. Virol. 88:1834–1838.
85.
Dyall J, Coleman CM, Hart BJ, Venkataraman T, Holbrook MR, Kindrachuk J, Johnson RF, Olinger GG Jr, Jahrling PB, Laidlaw M, Johansen LM, Lear-Rooney CM, Glass PJ, Hensley LE, and Frieman MB. 2014. Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection. Antimicrob. Agents Chemother. 58:4885–4893.
Information & Contributors
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Published In
Antimicrobial Agents and Chemotherapy
Volume 58 • Number 8 • August 2014
Pages: 4875 - 4884
PubMed: 24841269
Copyright
© 2014 American Society for Microbiology.
History
Received: 10 April 2014
Returned for modification: 2 May 2014
Accepted: 14 May 2014
Published online: 15 July 2014
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2014. Screening of an FDA-Approved Compound Library Identifies Four Small-Molecule Inhibitors of Middle East Respiratory Syndrome Coronavirus Replication in Cell Culture. Antimicrob Agents Chemother 58:.
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