A comparative analysis of remdesivir and other repurposed antivirals against SARS‐CoV‐2
2020; Springer Nature; Volume: 13; Issue: 1 Linguagem: Inglês
10.15252/emmm.202013105
ISSN1757-4684
AutoresAlexander Simonis, Sebastian J. Theobald, Gerd Fätkenheuer, Jan Rybniker, Jakob J Malin,
Tópico(s)SARS-CoV-2 detection and testing
ResumoReview3 November 2020Open Access A comparative analysis of remdesivir and other repurposed antivirals against SARS-CoV-2 Alexander Simonis Alexander Simonis Department I of Internal Medicine, Division of Infectious Diseases, University of Cologne, Cologne, Germany Faculty of Medicine, Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Sebastian J Theobald Sebastian J Theobald Department I of Internal Medicine, Division of Infectious Diseases, University of Cologne, Cologne, Germany Faculty of Medicine, Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Gerd Fätkenheuer Gerd Fätkenheuer Department I of Internal Medicine, Division of Infectious Diseases, University of Cologne, Cologne, Germany Search for more papers by this author Jan Rybniker Jan Rybniker Department I of Internal Medicine, Division of Infectious Diseases, University of Cologne, Cologne, Germany Faculty of Medicine, Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany German Center for Infection Research (DZIF), Partner Site Bonn-Cologne, Cologne, Germany Search for more papers by this author Jakob J Malin Corresponding Author Jakob J Malin [email protected] orcid.org/0000-0002-2989-0436 Department I of Internal Medicine, Division of Infectious Diseases, University of Cologne, Cologne, Germany Faculty of Medicine, Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Alexander Simonis Alexander Simonis Department I of Internal Medicine, Division of Infectious Diseases, University of Cologne, Cologne, Germany Faculty of Medicine, Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Sebastian J Theobald Sebastian J Theobald Department I of Internal Medicine, Division of Infectious Diseases, University of Cologne, Cologne, Germany Faculty of Medicine, Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Gerd Fätkenheuer Gerd Fätkenheuer Department I of Internal Medicine, Division of Infectious Diseases, University of Cologne, Cologne, Germany Search for more papers by this author Jan Rybniker Jan Rybniker Department I of Internal Medicine, Division of Infectious Diseases, University of Cologne, Cologne, Germany Faculty of Medicine, Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany German Center for Infection Research (DZIF), Partner Site Bonn-Cologne, Cologne, Germany Search for more papers by this author Jakob J Malin Corresponding Author Jakob J Malin [email protected] orcid.org/0000-0002-2989-0436 Department I of Internal Medicine, Division of Infectious Diseases, University of Cologne, Cologne, Germany Faculty of Medicine, Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Author Information Alexander Simonis1,2, Sebastian J Theobald1,2, Gerd Fätkenheuer1, Jan Rybniker1,2,3 and Jakob J Malin *,1,2 1Department I of Internal Medicine, Division of Infectious Diseases, University of Cologne, Cologne, Germany 2Faculty of Medicine, Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany 3German Center for Infection Research (DZIF), Partner Site Bonn-Cologne, Cologne, Germany *Corresponding author. Tel: +49 221 478 38374; E-mail: [email protected] EMBO Mol Med (2021)13:e13105https://doi.org/10.15252/emmm.202013105 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The ongoing SARS-CoV-2 pandemic stresses the need for effective antiviral drugs that can quickly be applied in order to reduce morbidity, mortality, and ideally viral transmission. By repurposing of broadly active antiviral drugs and compounds that are known to inhibit viral replication of related viruses, several advances could be made in the development of treatment strategies against COVID-19. The nucleoside analog remdesivir, which is known for its potent in vitro activity against Ebolavirus and other RNA viruses, was recently shown to reduce the time to recovery in patients with severe COVID-19. It is to date the only approved antiviral for treating COVID-19. Here, we provide a mechanism and evidence-based comparative review of remdesivir and other repurposed drugs with proven in vitro activity against SARS-CoV-2. Glossary Antiviral drugs Drugs that directly interfere with the ability of a virus to replicate in vivo or in cell-based models. Most antiviral drugs interfere with the host cell-dependent life cycle of the virus. Thus, mode of action of most antivirals is the inhibition of the viral entry into the host cell, blockage of viral proteases, or inhibition of viral RNA replicase. Bioavailability Used to describe the fraction of a drug or its active metabolite that reaches the systemic circulation and organ tissue after administration. Cell-based assay The term cell-based assay is commonly used to refer to any assay, where living cells are used as model to study physiologic or pathophysiologic processes under various conditions (e.g., exposure to an antiviral agent). Due to their cost efficiency and high standardization/reproducibility, cell-based assays are essential tools in preclinical drug discovery. COVID-19 (coronavirus disease 2019) The infectious disease caused by SARS-CoV-2 in humans. Coronaviruses (CoV) Coronaviruses are a group of RNA viruses that cause diseases in mammals and birds. Coronavirus-associated diseases in humans include severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease 2019 (COVID-19). In addition, there are endemic human CoVs that cause mild respiratory infections. Drug repositioning or repurposing A term that describes a drug discovery strategy based on the identification of new therapeutic approaches by using already known substances that may be at a preclinical or clinical development stage. This strategy offers a time- and cost-saving method to develop therapeutics against newly emerged or neglected diseases. Half-maximal effective concentration (EC50) The concentration of a substance which is required to obtain 50% of its maximal effect. It is used to determine potency of a drug. For some analyses (for example antibacterial activity), the 50% inhibitory concentration (IC50) is used in analogy. Besides the half-maximal concentration, the 90% maximal effective concentration (EC90) can be determined. MERS-CoV Middle East respiratory syndrome-related coronavirus causes the Middle East respiratory syndrome (MERS) in humans which is associated with severe respiratory symptoms and high mortality. The first confirmed case of MERS was reported in 2012. Nucleoside/nucleotide analogs Nucleosides are endogenous compounds composed of a nucleobase and a five-carbon sugar (ribose or 2'-deoxyribose), while nucleotides contain one more phosphate group. Nucleosides/nucleotides are essential for the synthesis of DNA and RNA but are also involved in other cellular processes like signaling and metabolism. Nucleoside/nucleotide analogs are synthetic, chemically modified nucleosides/nucleotides that are able to mimic their physiological counterparts. Assembly of nucleoside/nucleotide analogs into the RNA/DNA leads to premature termination of the strand synthesis and inhibition of, e.g., viral replication. Pseudovirions/pseudotyped particles Pseudovirions are synthetic viral particles with modified genomes and/or envelope proteins in order to facilitate specific investigations. The particles usually lack genes essential for pathogenicity and cannot replicate. This is an advantage for experiments on otherwise highly pathogenic viruses like SARS-CoV-2. Pseudotyping is the combination of viral particles with foreign viral envelope proteins. Pseudotyping can be used to study the function of viral envelope proteins and mechanisms of viral entry. SARS-CoV The severe acute respiratory syndrome (SARS) coronavirus was first described in 2003. It causes a respiratory disease that accompanies a high rate of complications and mortality. After the epidemic outbreak in Asia in 2002-2003, sporadic cases have been observed in several countries until 2004. SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2, initially described as 2019-nCoV, causes respiratory infections that can progress to viral pneumonia in COVID-19. It emerged in December 2019 in Wuhan, China, and rapidly developed to a pandemic which is still ongoing. Introduction Coronaviruses (CoV) are known to cause respiratory tract infections in humans and animals. Since the emergence and subsequent characterization of the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 (Drosten et al, 2003; Ksiazek et al, 2003; Peiris et al, 2003) and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 (Corman et al, 2012), coronaviruses have increasingly been recognized as potential source of epidemic diseases. Both pathogens seem to cause zoonotic infections that originate from viral reservoirs in bats (Guan et al, 2003; Li et al, 2005; Mohd et al, 2016). In 2020, a novel coronavirus (SARS-CoV-2) emerged in China (Zhu et al, 2020) and spread globally in a very short period of time. The rapid geographical extension of SARS-CoV-2 in comparison to previous outbreaks with SARS-CoV and MERS-CoV may be caused by an increased infectivity of the pathogen (Sigrist et al, 2020; Wrapp et al, 2020). As of September 22, the ongoing coronavirus disease 2019 (COVID-19) pandemic caused over 31 million detected SARS-CoV-2 infections and more than 950,000 deaths (Johns Hopkins University, 2020). The dramatic global implications of this pandemic stressed the urgent need for therapeutic agents that can quickly be applied in the clinic without a long-lasting preclinical development phase. Several therapeutic strategies were therefore investigated by repurposing of known antimicrobial or immunomodulatory substances that might be beneficial for patients with COVID-19. These agents can roughly be divided into compounds with a direct antiviral effect that impairs viral replication and host-directed drugs that may support recovery from COVID-19 by attenuating an excessive host immune response. In this article, we focus on repurposed drugs against COVID-19 with proven antiviral effects against SARS-CoV-2 in cell-based studies. The most advanced developed antiviral of this type is the nucleoside analog remdesivir that was previously unsuccessfully tested against Ebolavirus disease in clinical trials (Mulangu et al, 2019). Based on recent clinical and preclinical data on its efficacy against COVID-19, remdesivir received emergency use authorizations (EMA) in the United States and Japan and was recently approved by the European Medicines Agency (EMA) for the treatment of adult patients with severe COVID-19 that require supplemental oxygen. Although approval of this drug is a very encouraging signal, its clinical efficacy seems to be relatively modest based on available evidence (Beigel et al, 2020; Goldman et al, 2020; Grein et al, 2020; Wang et al, 2020c). We will review preclinical and clinical outcomes of repurposed antivirals and their molecular mechanism of action (MOA) to provide a comparative analysis of remdesivir with the ultimate aim to support a rational appraisal of its efficacy. SARS-CoV-2 life cycle The viral life cycle of SARS-CoV-2 provides several attractive molecular targets for viral inhibition that can be exploited by repurposed antiviral drugs. Like all Coronaviriade, this β-coronavirus, is an enveloped, positive-sense, single-stranded RNA virus. It is composed of a core structure where the viral RNA is encapsulated by the nucleocapsid (N) protein and the envelope, a lipid bilayer in which the spike (S), membrane (M), and envelope (E) protein are anchored (de Haan & Rottier, 2005). Upon viral transmission, mostly via droplet transmission, the life cycle of SARS-CoV-2 is initiated by the attachment of the virion to the host cell by the spike glycoprotein (S-protein) and its receptor. Several studies could show that entry, as shown for SARS-CoV before, depends on binding of the receptor-binding domain (RBD) (subunit S1) of the S-protein to the human angiotensin converting enzyme receptor 2 (ACE2; Hoffmann et al, 2020a; Walls et al, 2020). Notably, the RBD of SARS-CoV-2 shows a 10- to 20-fold higher affinity to ACE2 than SARS-CoV, which may explain its increased transmissibility (Wrapp et al, 2020). Furthermore, single-cell RNA-sequencing data revealed a high expression level of the ACE2 receptor in human nasal epithelial cells, which may also enhance the efficiency of SARS-CoV-2 transmission (Sungnak et al, 2020). After initial binding of the S1 subunit to ACE2, entry into the host cell required proteolytic cleavage of the S-protein at the S1/S2 and S2' site, which leads to fusion of the viral and cellular membrane mediated by the S2 subunit. Proteolytic cleavage of the S-protein is induced by the membranous serine protease TMPRSS2 of the host cell (Hoffmann et al, 2020a). Interestingly, a new furin cleavage site at the S1/S2 boundary could be found in SARS-CoV-2. The exact role of this site in pathogenesis is controversially discussed (Walls et al, 2020; Xia et al, 2020a). Cleavage of S-protein exposes the S2 subunit which contains an internal fusion peptide and two hydrophobic (heptad) repeat regions (HR1 and HR2). HR1 and HR2 self-assemble into a stable helical bundle that brings viral and cellular membranes in close proximity for fusion. Several bundles can form a fusion pore and finally release the viral genome into the cytoplasm (Bosch et al, 2003; Xia et al, 2020b). Moreover, several studies could show that virus entry is not only ensued by direct fusion with the plasma membrane, but rather by endosomal/lysosomal uptake and intra-lysosomal activation of the spike protein by cathepsin L followed by membrane fusion and intracellular release of genomic RNA (Wang et al, 2008; Burkard et al, 2014; Ou et al, 2020). After release of viral RNA into the cytosol viral replication is initiated by the translation of the replicase gene encoded by two large ORFs (rep1a and rep1b), which express the two polyproteins pp1a and pp1ab. The polyproteins contain several non-structural proteins (nsp) (pp1a = nsp 1–11; pp1ab = 1–16) also including a RNA-dependent RNA polymerase (RdRp) domain (nsp12) and proteases that cleave the polyproteins (initiated by the enzyme's own autolytic cleavage from pp1a and pp1ab) (Anand et al, 2003; Pertusati et al, 2012). Most of the nsp forms the replicase–transcriptase complex (RTC): The RTC replicates the genomic RNA and sub-genomic RNA, which encodes the structural proteins and other accessory proteins. While the nucleocapsid (N) protein remains in the cytosol and forms complexes with the genomic RNA, the viral structure proteins M, E, and S are translated, inserted into the membrane of the rough endoplasmatic reticulum (ER) and subsequently transported to the ER-to-Golgi intermediate compartment (ERGIC) (Fehr & Perlman, 2015). Here, the genomic RNA–nucleocapsid complexes get enveloped by the virion precursors, are transported to the cell surface in vesicles, and are released by exocytosis. An overview of the life cycle of SARS-CoV-2 including targets that might be exploited for inhibition of viral replication is illustrated in Figure 1. Based on its MOA, repurposed drugs with anti-SARS-CoV-2 activity can be divided into substances that prevent viral entry into host cells (1–2) and inhibit viral proteases (3) and inhibitors of viral replicase (4). Other compounds elicit multiple effects, or its specific MOA in SARS-CoV-2 is unknown. Figure 1. Life cycle of SARS-CoV-2 and antiviral drug targets Attachment of SARS-CoV-2 to its host cell is mediated by binding of the viral spike protein to the ACE2 receptor. After proteolytic cleavage of the S1 domain by the membrane-anchored serine protease TMPRSS2, fusion of the viral and host cell membrane is initiated by the exposed S2 subunit. Alternatively, SARS-CoV-2 can invade the host cell upon endosomal uptake and activation of the spike protein by cathepsin L. Released viral RNA is translated by ribosomes of the host cell. Polyproteins pp1a/pp1ab are cleaved mainly by the viral main protease (3C-like proteinase). Released non-structural proteins form the replicase–transcriptase complex, which initiates the viral RNA synthesis machinery. Viral structure proteins and genomic RNA form new particles, which are released by exocytosis. The replication cycle of SARS-CoV-2 can be inhibited at various stadiums: viral entry (1-2); protease inhibition (3), and RNA replication (4). Download figure Download PowerPoint Prevention of viral entry into the host cell Viral entry is initiated by the S2 subunit, which requires prior S-protein priming by proteolytic cleavage of the S1 subunit. As shown for other coronaviruses, viral entry in cell lines depends on the serine protease TMPRSS2 and the endosomal cysteine proteases cathepsin B and L (Kawase et al, 2012; Hoffmann et al, 2020a). However, several studies indicate that cell entry is driven preferentially via the cell surface or early endosomes by TMPRSS2 and that proteolytic cleavage of the S-protein by TMPRSS2 is crucial for infection of the host (Shirato et al, 2018; Iwata-Yoshikawa et al, 2019). Thus, inhibition of the TMPRSS2 and/or cathepsin B and L seems a promising target to prevent virus entry. Camostat/Nafamostat TMPRSS2 is a cell membrane-anchored serine protease and belongs to the family of type II transmembrane serine proteases. These proteases share a common catalytic mechanism involving a triad of three amino acids, serine, aspartate, and histidine present in highly conserved sequence motifs (Antalis et al, 2011). Serine proteases underlie a strict regulation by endogenous inhibitors (e.g., α1/α2-antitrypsin, and antithrombin III) and need a prior activation leading to hemostasis under physiological conditions. Thus, imbalance can cause several pathophysiological processes like thrombosis (Rau et al, 2007). However, the exact physiological functions of TMPRSS2 are still unknown. Synthetic protease inhibitors like camostat mesilate or nafamostat mesilate have been clinically tested in patients with acute or chronic pancreatitis, which is pathophysiologically related to an inappropriate activation of digestive enzymes inside the pancreas, including the serine protease trypsin (Chang et al, 2009; Ramsey et al, 2019). Due to their capability to inhibit TMPRSS2, serine protease inhibitors have been tested for their antiviral effects on SARS-CoV-2 and other coronaviruses. Camostat partially blocked the entry of vesicular stomatitis virus (VSV) pseudotyped particles harboring the SARS-CoV-2 spike protein (pseudovirions) into the human epithelial colorectal adenocarcinoma cell line Caco-2, Vero-TMPRSS2+ cells, and human airway epithelial (HAE). A complete inhibition of viral entry could only be reached when camostat was used in combination with E-64d, an inhibitor of cathepsin B/L, suggesting that SARS-CoV-2 can exploit both pathways for entry into the host cell (Hoffmann et al, 2020a). However, TMPRSS2 is essential for viral transmission and pathogenesis while CatB/L activity is dispensable so that inhibition of TMPRSS2 displays a rational antiviral strategy (Iwata-Yoshikawa et al, 2019). Wang et al demonstrated inhibition of SARS-CoV-2 by nafamostat with a 50% effective inhibitory concentration (EC50) of 22.50 μM in Vero E6 cells (Wang et al, 2020a). A comparative assessment of the serine protease inhibitors gabexate mesilate, camostat mesilate, and nafamostat mesilate and their ability to inhibit viral entry was done by Hoffmann et al Efficiency of entry inhibition was determined 16 h post-inoculation by using Calu-3 cells infected with SARS-CoV-2-pseudovirions. Nafamostat demonstrated an almost 15-fold higher efficiency (EC50 5 nM) compared with camostat (87 nM), both superior to gabexate (EC50 1.2 M). Nafamostat also showed to inhibit SARS-CoV-2 infection of lung-derived human Calu-3 cells in vitro even at a low dose of 100 nM (Hoffmann et al, 2020b). Although antiviral efficacy of TMPRSS2 inhibitors seems to be inferior to other strategies (table 1), entry inhibitors may be developed that are beneficial in COVID-19 when given alone or in combination with other antivirals. Three randomized controlled trials (RCT) are currently listed that evaluate nafamostat in patients with COVID-19 (NCT04418128, NCT04352400, NCT04473053), but currently no clinical data can be reported. Table 1. In vitro efficacy and drug targets of repurposed investigational compounds with proven anti-SARS-CoV-2 activity Antiviral target Investigational drug Isolate EC50 in Vero E6 cells (µM) References CPE RT–PCR VY Viral entry Nafamostat Wuhan/WIV04/2019 ND 22.50 ND Wang et al (2020) Umifenovir (Arbidol) Wuhan/WIV04/2019 ND 4.11 ND Wang et al (2020b) France/IDF0571/2020 ND 3.54 ND Pizzorno et al (2020) Viral protease Lopinavir Hong Kong/VM20001061/2020 25a 26.10a 26.62a Choy et al (2020) France/IDF0571/2020 ND 5.25 ND Pizzorno et al (2020) RNA synthesis Favipiravir Wuhan/WIV04/2019 ND 61.88 ND Wang et al (2020) Hong Kong/VM20001061/2020 > 100 > 100 > 100 Choy et al (2020) France/IDF0571/2020 ND > 100 ND Pizzorno et al (2020) Penciclovir Wuhan/WIV04/2019 ND 95.96 ND Wang et al (2020) Remdesivir Wuhan/WIV04/2019 ND 0.77 ND Wang et al (2020) Australia/VIC01/2020 4.9 ND ND Ogando et al (2020) Hong Kong/VM20001061/2020 25a 26.9a 23.15a Choy et al (2020) France/IDF0571/2020 ND 0.99 ND Pizzorno et al (2020) Ribavirin Wuhan/WIV04/2019 ND 109.50 ND Wang et al (2020) Hong Kong/VM20001061/2020 500a > 500 > 500 Choy et al (2020) Miscellaneous Berberine France/IDF0571/2020 ND 10.58 ND Pizzorno et al (2020) Chloroquine Wuhan/WIV04/2019 ND 1.13 ND Wang et al (2020) France/IDF0571/2020 ND 1.38 ND Pizzorno et al (2020) Wuhan/WIV04/2019 ND 2.71-7.36b ND Liu et al (2020) Wuhan/IVDC-HB-01/2019 ND 5.47 ND Yao et al (2020) Hydroxychloroquine Wuhan/WIV04/2019 ND 4.06-12.96b ND Liu et al (2020) Wuhan/IVDC-HB-01/2019 ND 0.72 ND Yao et al (2020) France/lDF0372/2020 ND 2.2-4.4c ND Maisonnasse et al (2020) Cyclosporine A France/IDF0571/2020 ND 3.05 ND Pizzorno et al (2020) Emetine Hong Kong/VM20001061/2020 1.56a 0.50a 0.46a Choy et al (2020) Homoharringtonine Hong Kong/VM20001061/2020 3.13a 2.14a 2.55a Choy et al (2020) Nitazoxanide Wuhan/WIV04/2019 ND 2.12 ND Wang et al (2020) EC50, 50% effective concentrations. Assay types: CPE, cytopathologic effects; RT–PCR, real-time polymerase chain reaction; VY, virus yield assay. a Calculation of EC50 based on viral loads fitted to log10 scale. b Tested in different MOI (0.01, 0.02, 0.8). Umifenovir Umifenovir is a broad-spectrum antiviral approved in Russia and China for the prophylaxis and treatment of human influenza A and B infections (Boriskin et al, 2008). Its antiviral mechanism of action is thought to be related to an impaired virus-mediated membrane fusion that is essential for viral entry. Umifenovir seems to modify the physicochemical properties of the host cell membrane by influencing the negatively charged phospholipids (Villalaín, 2010). Furthermore, it has been shown that umifenovir interacts with hemagglutinin (HA) of the influenza virus by preventing the pH-induced transition of HA into its functional state (Leneva et al, 2009). In a recent study, the efficacy of six currently available and licensed anti-influenza drugs (umifenovir, baloxavir, laninamivir, oseltamivir, peramivir, and zanamivir) were tested against SARS-CoV-2 in Vero E6 cells. Among tested drugs, only umifenovir inhibited SARS-CoV-2 replication efficiently with an EC50 of 4.11 μM (Wang et al, 2020b). These results could be reproduced by another in vitro study with an EC50 of 3.5 μM (Pizzorno et al, 2020). Although umifenovir demonstrated anti-SARS-CoV-2 activity in vitro, a therapeutic role of umifenovir in COVID-19 is uncertain and results of qualitative clinical trials are lacking. Retrospective analyses currently indicate no significant impact on clinical outcomes (Huang et al, 2020). Blockage of viral proteases A crucial step in SARS-CoV-2 replication is the proteolytic cleavage and release of functional polypeptides from the polyproteins pp1a/pp1ab by viral proteases. Subsequently, released non-structural proteins form the replicase–transcriptase complex, which initiates the viral RNA synthesis machinery. Translated viral structure proteins and replicated genomic RNA originate new infectious virus particles, which are released from the infected host cell. In coronaviruses, the main protease (Mpro) also known as 3C-like protease (3CLpro) cleaves the polyprotein at conserved sites between Leu-Gln and Ser-Ala-Gly. This well-characterized enzyme represents an ideal antiviral target as its function is critical for viral replication (Anand et al, 2003; Zhang et al, 2020b). Due to its intrinsic proteolytic activity and the absence of homologous enzymes in humans, toxicity of specific inhibitors is expected to be limited. Of known protease inhibitors that were repurposed for SARS-CoV-2, the combination of lopinavir and ritonavir has been in focus of interest as other protease inhibitors (e.g., darunavir) showed no in vitro activity at applicable concentrations (De Meyer et al, 2020). Lopinavir/ritonavir Lopinavir/ritonavir is used as combination regimen in the treatment of infections with human immune deficiency virus 1 (HIV-1). Both lopinavir and ritonavir are inhibitors of HIV-1 protease, an enzyme that cleaves the HIV polyproteins Gag and Gag-Pol by bond hydrolysis. Since ritonavir also acts as inhibitor of cytochrome P450-3A4 (CYP3A4), an enzyme that normally metabolizes protease inhibitors, ritonavir is added to enhance the bioavailability of lopinavir (Sham et al, 1998). Lopinavir has been tested in vitro against SARS-CoV, MERS-CoV, and human coronavirus 229E (de Wilde et al, 2014). Here, the mean EC50 of lopinavir ranged from 6.6 µM (± 1.1) µM (HCoV-229E) and 8.0 µM (± 1.5) MERS-CoV to 17.1 µM (± 1.0) (SARS-CoV). Recent analysis demonstrated that lopinavir is also active against SARS-CoV-2 with an EC50 of 5.25–26.1 µM (Choy et al, 2020; Pizzorno et al, 2020) while ritonavir alone was not effective (Choy et al, 2020). In vivo efficacy of lopinavir/ritonavir has been assessed in mice and common marmosets for MERS-CoV with ambiguous results: In a study published in 2015, lopinavir/ritonavir-treated marmosets had improved clinical findings and reduced viral loads associated with a better outcome. Animals were treated with 2 mg/kg/day of lopinavir plus 3 mg/kg/day of ritonavir given orally once daily at 6, 30, and 54 h post-infection (Chan et al, 2015). However, treatment of infected mice with lopinavir/ritonavir (160/40 mg + interferon beta) improved pulmonary function but did not reduce virus replication or occurrence of severe lung damage (Sheahan et al, 2020). Clinical effects in patients with severe COVID-19 were evaluated in a randomized controlled clinical trial including 199 patients. Patients were randomized in a 1:1 ratio to receive either lopinavir/ritonavir (standard dose of 400/100 mg) for 14 days or the standard care. The primary end point of the study was clinical improvement or discharge from the hospital. Unfortunately, treatment did not improve clinical symptoms and mortality, or decreased viral loads in pharyngeal swabs (Cao et al, 2020). The disappointing clinical results might be related to sub-therapeutic levels for inhibition of SARS-COV-2 because application of 400/100 mg of lopinavir/ritonavir twice daily was shown to yield median serum concentrations of 7.2 mg/l (11.5 µM) in patients with HIV (van der Lugt et al, 2009), which is significantly lower than the observed EC50 in the in vitro studies. However, summarizing the relatively low efficacy against SARS-CoV-2 in vitro in comparison with other repurposed drugs and available in vivo data it is unlikely that lopinavir/ritonavir will play a significant therapeutic role in COVID-19. Besides lopinavir and ritonavir, other protease inhibitors with activity against SARS-CoV and MERS-CoV were identified that might be repurposed to target SARS-CoV-2 (Anand et al, 2003; He et al, 2020a). Inhibition of viral RNA replicase Once functional, non-structural proteins are released by proteolytic cleavage of the polyproteins, the replicase–transcriptase complex, which catalyzes the synthesis of the viral RNA, can be formed. Synthesis is initiated by binding of the RdRp at or near the 3' end of the RNA strand. Subsequently, the complementary RNA strand is generated in the elongation phase by repetitive nucleotidyl transfer reactions. Several drugs are able to interfere with the RNA synthesis machinery. Mainly, nucleoside/nucleotide analogs have been repurposed and tested against SARS-CoV-2. These drugs disrupt viral replication by competing with endogenous nucleosides during the elongation phase. After their insertion nucleoside
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