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VIRUS ENTRY INHIBITORS\nBinding of spike protein (S) to its receptor represents the host's first confrontation with the virus and its life cycle, thus providing prophylactic intervention opportunities. The genome of SARS‐CoV‐2 has been recently determined to have an 80% identity to that of SARS‐CoV‐1 and 96% identity to the bat‐CoV RaTG13. 26 The SARS‐CoV‐2 S protein shows nucleotide sequence identities of 75% or less to all other previously described CoVs. However, again, the new SARS‐CoV‐2 S protein shares a 93.1% identity to the S protein of RaTG13. SARS‐CoV‐2 spike (S) recognizes, with its receptor‐binding domain (RBD), the cellular ACE2 receptor with high affinity (K d, 14.7 nM) 27 as judged by surface plasmin resonance spectrometry; and intervention at the RBD‐ACE2 interface can potentially disrupt infection efficiency. It was observed that the RBDs of the SARS‐CoV‐2‐ACE2 and SARS‐CoV‐1‐ACE2 complexes are quite similar (Figure 3). 29\nFigure 3 Comparison of the SARS‐CoV‐2 S and SARS‐CoV‐1 S structures: ribbon diagrams of the (A) SARS‐CoV‐2 S and (D) SARS‐CoV‐1 S [PDB 6NB6] ectodomain cryo‐EM structures. S1 subunits of (B) SARS‐CoV‐2 S and (E) SARS‐CoV‐1 S. S2 subunits of (C) SARS‐CoV‐2 S and (F) SARS‐CoV‐1 S. 28 cryo‐EM, cryogenic electron microscopy; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2 [Color figure can be viewed at wileyonlinelibrary.com] As mentioned previously, Zhang et al. 30 determined the full‐length genome sequence of SARS‐CoV‐2 and revealed that the virus is remarkably similar (89.1% sequence identity) to a group of SARS‐like CoVs. Simultaneously, Shi et al. 26 reported that SARS‐CoV‐2 shares 96% sequence identity at a whole‐genome level with a bat coronavirus—and importantly, like SARS‐CoV‐1, SARS‐CoV‐2 utilizes ACE2 receptor for viral entry. Recently, Yan et al. solved the cryo‐EM structure of full‐length human ACE2 bound to the RBD of SARS‐CoV‐2, providing important structural information for therapeutic intervention strategies. 29\nThe sequence identity of the spike protein between SARS‐CoV‐1 (1273 aa) and SARS‐CoV‐2 (1253 aa) is 76%. The spike protein has two regions, S1 and S2. The S1 region of the SARS‐CoV‐1 has a RBD that forms high‐affinity interactions with ACE2. The prevailing understanding is that SARS‐CoV‐2 employs this RBD to enter its human host cell as well. Aligning the two different RBDs revealed a sequence identity of 73.5%. However, many nonconserved mutations that interact directly with ACE2 are located in the two structural regions. 31 And both crystal and cryo‐EM structures of the SARS‐CoV‐1 spike‐ACE2 complex have shown that merely residues of regions 1 and 2 form hydrogen bonds and hydrophobic interactions with ACE2. The mutations in these two regions of SARS‐CoV‐2 will, therefore, likely reduce the number of those interactions. 32\nStudies also have shown that the RdRP, and the Mpro are highly conserved between SARS‐CoV‐2 and SARS‐CoV‐1. 33 , 34 Therefore, it is widely accepted that SARS‐CoV‐2 behaves similarly to SARS‐CoV‐1 with regard to viral entry and replication. Since the general genomic layout and replication kinetics are so conserved among MERS, SARS‐1, and SARS‐2 CoVs, investigating inhibitors of common structures is a logical step.\nThe inhibitory strength against viral enzyme was expressed as IC50, which is the concentration of the inhibitor needed to inhibit half of the enzyme activity in the tested condition. The K i value is reflective of ligand‐binding affinity to the enzyme. The inhibitory activity for cell‐based bioassays is expressed as EC50, which is a half maximal effective concentration required to induce the biological response. The warhead group means a “reactive group” of the inhibitors that can form both covalent and noncovalent interactions with amino acids in the active site of the enzyme.\n\n2.1 Targeting the RBD\nStructural investigations of the RBD‐ACE2 complex provided information about essential residues for viral entry. Hsiang et al. 35 reported a number of peptides that significantly blocked the interaction of the S protein with ACE2 with IC50 values as low as 1.88 nM. Michael et al. found charged residues between positions 22 and 57 crucial for SARS‐CoV‐1 viral entry. Based on this, they designed peptides P4 (IC50, 50 µM) and P5 (IC50, 6.0 µM) with significant inhibitory activity against SARS‐CoV‐1. The antiviral activity was further improved when they introduced the glycine binding linkage of peptide P4 (residues 22–47) with an ACE2‐derived peptide (residues 351–357) against a SARS‐CoV‐1 pseudovirus with an IC50 of 100 nM and devoid of cytotoxicity up to 200 µM. 36 It is worth highlighting that a similar strategy could work for the new SARS‐CoV‐2. The recently solved cryo‐EM structure of SARS‐CoV‐2 in complex with human ACE2 can provide a structural rationale for the peptide design. 29\nFor viral entry, MERS‐CoV uses its spike protein (S) to interact with the host‐receptor DPP4, 37 , 38 , 39 also known as adenosine deaminase‐complexing protein‐2 or CD26. 37 MERS‐CoV was also the first virus reported to use this particular path. 35 , 37 DPP4 is a type II transmembrane glycoprotein, that forms homodimers on the cell surface, and it is involved in the cleavage of dipeptides. 37 , 40 In humans, DPP4 is predominantly found on the bronchial epithelial and alveolar cells in the lower lungs. 40 , 41\nMERS‐4 and MERS‐27 are monoclonal antibodies targeting the RBD of MERS‐CoV S that were discovered in a nonimmune yeast‐display scFv library screening. The more active MERS‐4 potently blocked the infection of DPP4‐expressing Huh‐7 cells with pseudotyped MERS‐CoV (IC50, 0.056 μg/mL). It also prevented MERS‐CoV‐induced cytopathogenic effects in MERS‐infected Vero E6 cells (IC50, 0.5 μg/mL). 42\nA heptad repeat (HR) is a repeating structural pattern of seven amino acids. A crucial membrane fusion framework of SARS‐CoV is the 6‐helix‐bundle (6‐HB) that is formed by HR1 and HR2 of the viral S protein. Enfuvirtide (T‐20) is an FDA approved HR2 peptide and the first HIV fusion inhibitor. It has opened up new avenues toward identifying and developing peptides as viral entry inhibitors. Such molecules represent a promising strategy against enveloped viruses with class 1 fusion proteins such as Nipah virus, Hendra virus, Ebola virus, and other paramyxoviruses, simian immunodeficiency virus, feline immunodeficiency virus, and respiratory syncytial virus. 43 , 44 , 45 , 46 The HR regions of SARS‐CoV‐1 and SARS‐CoV‐2 S protein share a high degree of conservation, and such fusion inhibitors have potential applications in preventing SARS‐CoV‐2 entry.\nSmall molecule entry inhibitors, on the other hand, are reported to target the RBD. Compared to peptides, proteins, and biologics, small molecules have several advantages due to lower production costs, improved pharmacokinetics, stability, and dosage accuracy. Sarafianos et al. identified the oxazole‐carboxamide derivative SSAA09E2 (1; Figure 4) as an entry inhibitor against SARS‐CoV‐1 by screening a chemical library composed of 3000 compounds. 47 This inhibitor directly blocks ACE2 recognition by interfering with the RBD with an EC50 value of 3.1 µM and a 50% cytotoxic concentration (CC50) value of greater than 100 µM, not affecting ACE2 expression levels. 48\nFigure 4 Inhibitors targeting the receptor‐binding domain Xu et al. 49 identified two small molecules, TGG (2; Figure 4) and luteolin (3; Figure 4), that can bind avidly to the SARS‐CoV‐1 S2 protein and inhibit its entry into Vero E6 cells (EC50: 4.5 µM, 10.6 µM; respectively). Compounds 2 and 3 showed cytotoxicity (CC50) of 1.08 and 0.155 mM, and the selectivity index (SI) values of 2 and 3 were 240.0 and 14.62, respectively. Further studies regarding acute toxicity revealed that the 50% lethal doses of 2 and 3 were ~456 and 232 mg/kg, respectively. These results indicate that these small molecules could be used at relatively high concentrations in mice. 49 Quercetin (4; Figure 4), an analog of 3, also showed antiviral activity against SARS‐CoV‐1, with an EC50 value of 83.4 µM and a CC50 value of 3.32 mM. 50\nNgai et al. reported ADS‐J1 (5; Figure 4) as a potential SARS‐CoV‐1 viral entry inhibitor with an EC50 of 3.89 µM. Molecular docking studies predicted that 5 can bind into a deep pocket of the SARS‐CoV‐1 S HR region and block viral entry into host cells. 51 Imatinib (6; Figure 4), an Abelson kinase inhibitor, could inhibit CoV S protein‐induced fusion with an EC50 value of 10 µM and showed no cytotoxic effects in Vero cells up to 100 µM concentration. 52 , 53\n\n2.2 Inhibitors targeting the cellular receptor\nThe genetic code of SARS‐CoV‐2 shares noticeable similarities with SARS‐CoV‐1, which caused the SARS epidemic in 2002. 26 , 54 More importantly, both viruses have identical mechanisms of infection. SARS‐CoV‐1 uses the host's ACE2 as a portal to infect cells, which has high expression in the vascular endothelium 55 and the lung, particularly in type 2 alveolar epithelial cells. 56 SARS‐CoV‐2 shares 76% of its spike (S) protein with SARS‐CoV‐1. Despite a few amino acid differences in its RBD compared to the SARS‐CoV‐1 S protein, the SARS‐CoV‐2 S protein binds to ACE2 with even greater affinity 27 offering an explanation for its greater virulence and preference for the lung.\nACE, a highly glycosylated type I integral membrane protein, is an essential component of the renin‐angiotensin (Ang) system, which controls blood pressure homeostasis. Both ACE1 and ACE2 cleave Ang peptides. However, they differ markedly: ACE1 cuts and converts the inactive decapeptide Ang I into the octapeptide Ang II by removing the dipeptide His‐Leu. This Ang II induces vaso‐ and bronchoconstriction, increased vascular permeability, inflammation, and fibrosis, thus promoting acute respiratory distress syndrome (ARDS) and lung failure in patients infected with SARS‐CoV‐1 or SARS‐CoV‐2 57 (Figure 5). Therefore, ACE‐inhibitors (ACEis) and angiotensin II receptor blockers (ARBs) could block the disease‐propagating effect of Ang II. 58 , 59 , 60\nFigure 5 The roles of ACE1 and 2 in the renin‐angiotensin system. A, Chemical structures of angiotensin‐related peptides and B, Schematic diagram of roles of ACE1 and 2 in renin‐angiotensin system. ACE, angiotensin‐converting enzyme [Color figure can be viewed at wileyonlinelibrary.com] ACE2, on the other hand, is a zinc‐containing metalloenzyme, and shares merely 42% of its amino acid sequence with ACE1. 61 It cleaves only one amino acid residue (Leu or Phe) from Ang I and Ang II, respectively, generating Ang (1–9) and Ang (1–7) (a vasodilator) (Figure 5). Thus, ACE2 has been considered a potential therapeutic target for cardiovascular diseases.\nVirtual screening combined with a molecular docking approach targeting the ACE2 catalytic site with around 140 000 compounds led to the identification of inhibitor N‐(2‐aminoethyl)‐1 aziridine‐ethanamine (7; Figure 6) with an IC50 value of 57 µM and a K i of 459 µM. However, no information about the cytotoxicity of this compound is available so far. 62\nFigure 6 Inhibitors for SARS‐CoV‐1 and 2 targeting ACE2. ACE, angiotensin‐converting enzyme; SARS‐CoV, severe acute respiratory syndrome coronavirus Chloroquine (8; Figure 6) is a relatively safe, cheap, and effective medication for the treatment of malaria and amebiasis. Savarino et al. 63 reported its antiviral effects. At a molecular level, it increases late endosomal and lysosomal pH, resulting in impaired liberation of virions from endosomes or lysosomes. The virus is therefore unable to release its genetic material into the cell and replicate. 64 , 65 Furthermore, they hypothesize that chloroquine might block the production of proinflammatory cytokines (such as IL‐6), thereby blocking the pathway that subsequently leads to ARDS. 63\nChloroquine is reasonably active in vitro against SARS‐CoV‐1, MERS‐CoV, and SARS‐CoV‐2. It was found to inhibit SARS‐CoV‐2 with an EC50 value of 5.47 µM in vitro. 66 Antiviral activity against SARS‐CoV‐1 was reported with an IC50 of 8.8 μM in Vero cells, but it is unclear how this translates into activity in respiratory epithelial cells and in vivo. 67 , 68 Mechanistic studies of chloroquine for SARS‐CoV‐1 infection revealed that it could also weaken the interaction between the RBD of SARS‐CoV‐1 and ACE2 by interfering with terminal glycosylation of ACE2, thereby reducing its affinity to SARS‐CoV‐1 S. 69\nDuring the SARS‐CoV‐2 pandemic, chloroquine has been recommended by Chinese, South Korean, and Italian health authorities for the experimental treatment of COVID‐19, 70 , 71 despite contraindications for patients with heart disease or diabetes. 72 However, health experts and agencies like the US FDA and European Medicines Agency warned against broad uncontrolled use after reports of misuse of low‐quality versions of chloroquine phosphate intended for fish.\nHydroxychloroquine (9; Figure 6) is being studied as an experimental treatment for COVID‐19. 73 However, the benefits of treatment with this drug are unclear. 74\nHydroxychloroquine was found to inhibit SARS‐CoV‐2 with an EC50 value of 0.74 µM in vitro. 66 Some studies imply synergistic effects of hydroxychloroquine and azithromycin. Azithromycin is active in vitro against Zika and Ebola virus 75 , 76 and can be used to guard against life‐threatening bacterial superinfections when administered to patients suffering from viral infections. 77 A small study that compared hydroxychloroquine monotherapy and combination treatment with azithromycin found a significant advantage of the combination. While evaluating the efficacy of therapeutic intervention with hydroxychloroquine as monotherapy and its impact in combination with azithromycin, the number of patients testing negative in polymerase chain reaction (PCR) tests was substantially different in the two groups with 100% of patients cured (6 days post inclusion) in the combination arm of the study versus 57% in the monotherapy group. At the same time, 12% of patients in the control group receiving only standard care were cured. 78 , 79\nThe WHO declared on 18 March that chloroquine and its derivative hydroxychloroquine will be among the four medicines studied in the solidarity clinical trial 80 for the treatment of COVID‐19. In April 2020, the US National Institutes of Health (NIH) also commenced a study with the drug for treating COVID‐19 patients. 81\nThe recent clinical trial involving 96 032 patients with COVID‐19 concluded that it was unable to confirm a benefit of hydroxychloroquine or chloroquine, when used alone or in combination with a macrolide such as azithromycin (or clarithromycin). 82 The study actually reported decreased survival rates for patients treated with each of these drug regimens. Additionally, patients had an increased risk of developing ventricular arrhythmia under treatment. However, still more evidence is needed to adequately assess the drugs' risks or benefits for the treatment or prevention of COVID‐19 (it is important to note that chloroquine and hydroxychloroquine are still considered safe treatment options in certain autoimmune diseases and malaria). Besides, the WHO announced the premature pause of its clinical trials using hydroxychloroquine as a safety precaution on 24 May 2020.\nOn a different note, it was found that ACE2 undergoes proteolytic shedding; releasing an enzymatic ectodomain during viral entry. 83 A disintegrin and metalloproteinase (ADAM), also known as TNF‐α converting enzyme (TACE), assisted the shedding regulation of ACE2. Inhibition of this enzyme led to reduced shedding of ACE2. GW280264X (10; Figure 6) was found to be a specific inhibitor of ADAM‐induced shedding of ACE2 at 1 nM. 84 Two TACE inhibitors, TAPI‐0 (11) and TAPI‐2 (12; Figure 6), reduced ACE2 shedding, with IC50 values of 100 and 200 nM, respectively. 83\nMLN‐4760 (13; Figure 6) inhibited the catalytic activity of ACE2 with an IC50 of around 440 pM. 85 This is the most potent and selective small‐molecule inhibitor against soluble human ACE2 described to date, thus making it a very promising candidate for SARS‐CoV‐2 interference. It binds to the active site zinc and emulates the transition state peptide. However, no antiviral data for this compound is available at this time.\nThe interference of a virus‐host cell fusion, which is mediated by the viral S protein to its receptor ACE2 on host cells, may be a viable prevention strategy. Umifenovir (14; brand name Arbidol), a broad spectrum antiviral drug used against influenza, prevents viral entry by inhibiting virus‐host cell fusion. 86 It is currently being investigated in a clinical trial for the treatment of SARS‐CoV‐2. 87 , 88\nDo ACEis or ARBs amplify SARS‐CoV‐2 pathogenicity and aggravate the clinical course of COVID‐19? After ACE2 was recognized as the SARS‐CoV‐2 receptor, 14 , 29 speculations emerged about potentially negative consequences of ACEi or ARB therapy in COVID‐19 patients. This theory caused confusion in the public and alarmed patients taking these medicines. One report said that the expression of ACE2 was increased in patients with heart disease compared to healthy individuals. It was also insisted that ACE2 expression could be increased by taking ACEis and ARBs, 89 although there is no supporting report of this happening in the lungs.\nIn another report, it was suggested that patients suffering from high blood pressure receiving “ACE2‐increasing drugs” have a higher risk for severe COVID‐19, since ACEis and ARBs could elevate levels of ACE2. 90\nA joint declaration by the presidents of the HFSA/ACC/AHA on 17 March 2020, 91 followed by a similar statement of the European Medicines Agency, 92 clarified that there was no scientific basis for stopping ACEi or ARB therapy. 93 , 94 , 95 This was in accordance with the editors of the New England Journal of Medicine. 96\nIn case of SARS‐CoV, the experimental data showed that such medications may be beneficial rather than damaging, which led to a new therapeutic approach for lung diseases. 97\n\n2.3 Proteolytic processing inhibitors\nCoVs enter the host cells via both clathrin (endosomal) and nonclathrin pathways (nonendosomal); however, both pathways are dependent upon receptor binding. 98 , 99\nThe clathrin‐mediated pathway involves the binding of CoV S protein to the host receptor followed by the internalization of vesicles that maturate to late endosomes. Acidification of the endosome promotes the H+‐dependent activation of cellular cathepsin L proteinase in late endosomes and lysosomes, which cleaves and activates the S protein, thus initiating viral fusion. Recent research shows that in addition to ACE2 SARS‐CoV‐2 can also use the host cell receptor CD147 to gain access into host cells. 100\nMembrane fusion is also the crucial step for the CoV life cycle in the nonclathrin/endosomal route, in which host proteases such as cathepsin L, TMPRSS2, and TMPRSS11D (airway trypsin‐like protease) cut the S protein at the S1/S2 cleavage site to activate the S protein for membrane fusion. 101 Interference with this process by targeting these proteases could become an attractive strategy for combating CoV infections. A recent study confirms the role of TMPRSS2 for the viral life cycle in SARS‐CoV‐2‐infected VeroE6 cells. 5 Furin (a serine endoprotease) activates MERS‐CoV to initiate the nonclathrin mediated membrane fusion event. 102\nThe neurotransmitter receptor blockers chlorpromazine (15), promethazine (16), and fluphenazine (17; Figure 7), were reported to inhibit MERS‐CoV and SARS‐CoV‐1 most probably by impeding S protein‐induced fusion. 103 Chlorpromazine, a clathrin‐mediated viral entry inhibitor, was already described to inhibit human CoV‐229E, hepatitis C virus, infectious bronchitis virus, as well as mouse hepatitis virus‐2 (MHV2). 104 , 105 , 106 , 107 , 108\nFigure 7 Neurotransmitter inhibitors targeting clathrin/nonclathrin pathways Matsuyama et al. identified the commercially available serine protease inhibitor camostat (18; Figure 8) to be a SARS‐CoV‐1 inhibitor, blocking TMPRSS2 activity at 10 µM. However, at a higher concentration (100 µM), inhibition of viral entry via SARS‐CoV‐1 S protein‐mediated cell fusion never exceeded 65% (inhibition efficiency), indicating that 35% of entry events take place via the endosomal cathepsin pathway. Interestingly, treatment with a combination of EST (a cathepsin inhibitor) and 18 resulted in remarkably blocked infection (\u003e95%) activity of pseudotyped viruses. 109\nFigure 8 Inhibitors targeting TMPRSS2. TMPRSS, transmembrane serine protease A similar approach has been investigated to prevent viral entry of SARS‐CoV‐2. Pöhlmann et al. reported the attainment of full inhibition efficiency with a combination of both 18 and E‐64d (a cathepsin inhibitor). Both studies indicate that SARS‐CoV‐1 and 2 enter cells in a similar manner showing the potential of 18 as a candidate for further development. 15\nRecently, K11777 (19; Figure 8), a cysteine protease inhibitor, was shown in tissue cultures to inhibit SARS‐CoV‐1 and MERS‐CoV replication in the subnanomolar range. 110 , 111 Future tissue culture and animal model studies should be conducted to clarify, whether its antiviral activity is mediated by targeting TMPRSS2.\nTeicoplanin is a glycopeptide antibiotic used to prevent infections with Gram‐positive bacteria like methicillin‐resistant Staphylococcus aureus and Enterococcus faecalis. It was found that teicoplanin inhibits the entry of SARS‐CoV‐1, MERS‐CoV, and Ebola virus by specifically targeting cathepsin L. 112 This knowledge has also been used to block the entry of new SARS‐CoV‐2 pseudoviruses with an IC50 value of 1.66 µM. Therefore, teicoplanin could be considered a potential candidate for the treatment of COVID‐19. 113\n\n2.4 Small‐molecules as cathepsin L inhibitors\nHuman cathepsin L is a cysteine endopeptidase and plays a key role for infection efficiency by activation of the S protein into a fusogenic state to escape the late endosomes. Targeting this protease with small molecules could interfere with virus‐cell entry and therefore be a possible intervention strategy for CoV infection. 114 Bates et al. identified MDL28170 (20; Figure 9) as an antiviral compound that specifically inhibited cathepsin‐L‐mediated substrate cleavage, with an IC50 value of 2.5 nM and EC50 value in the range of 100 nM. However, despite its potent inhibitory activity, no cytotoxicity data for 20 is currently available. 115\nFigure 9 Cathepsin L inhibitors with antiviral activity Diamond et al. reported CID 16725315 (21) and CID 23631927 (22; Figure 9) as viral entry inhibitors of SARS‐CoV in a cathepsin L inhibition assay. Compound 21 could block cathepsin L with an IC50 value of 6.9 nM, while 22 showed slightly weaker potency (IC50, 56 nM). Compound 22 was also found to inhibit Ebola virus infection (EC50, 193 nM) of human embryonic kidney 293T cells. This compound did not show any sign of toxicity to human aortic endothelial cells up to 100 µM. This data offers a new promising point for the treatment of SARS and Ebola virus infections. 116\nScreening of ~14 000 compounds in a cell‐based assay resulted in the identification of SSAA09E1 (23; Figure 9) as inhibitor of cathepsin L proteinase, with an IC50 value of 5.33 µM. In a pseudotype‐based assay in 293T cells, the EC50 value of 23 was around 6.4 µM, and no cytotoxicity was detected below 100 µM. 48\nPhenotypic screening approaches led to the identification of several viral entry inhibitors. This approach has the advantage of finding cellular‐active compounds, providing information on drug solubility and cell uptake. 117 On the other hand, it is limited in terms of capacity compared to in silico target‐based screening. Hsiang et al. identified emodin (24; Figure 9), the active component from Polygonum multiflorum and Rheum officinale, could block the interaction of S protein with ACE2, with an IC50 value of 10 µM and an EC50 value of 200 µM in an S protein‐pseudotyped retrovirus assay using Vero E6 cells. However, the mechanism of action of this compound still needs to be determined. 118 Sarafianos et al. 48 found that SSAA09E3 (25), a benzamide derivative of 24, could prevent virus‐cell membrane fusion in pseudotype‐based and antiviral‐based assays, with an EC50 value of 9.7 µM, but a CC50 value of 20 µM indicates additional unknown cellular targets.\nVE607 (26) was identified among 50 240 structurally diverse small molecules to specifically inhibit SARS‐CoV‐1 entry into cells using a phenotype‐based screening. Its EC50 value was reported at 3.0 µM and it inhibited SARS‐CoV‐1 plaque formation with an EC50 of 1.6 µM. 119 Cathepsin inhibitor E‐64‐D (27) blocked MERS‐CoV and SARS‐CoV‐1 infection as well. 120 , 121"}

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The genome of SARS‐CoV‐2 has been recently determined to have an 80% identity to that of SARS‐CoV‐1 and 96% identity to the bat‐CoV RaTG13. 26 The SARS‐CoV‐2 S protein shows nucleotide sequence identities of 75% or less to all other previously described CoVs. However, again, the new SARS‐CoV‐2 S protein shares a 93.1% identity to the S protein of RaTG13. SARS‐CoV‐2 spike (S) recognizes, with its receptor‐binding domain (RBD), the cellular ACE2 receptor with high affinity (K d, 14.7 nM) 27 as judged by surface plasmin resonance spectrometry; and intervention at the RBD‐ACE2 interface can potentially disrupt infection efficiency. It was observed that the RBDs of the SARS‐CoV‐2‐ACE2 and SARS‐CoV‐1‐ACE2 complexes are quite similar (Figure 3). 29\nFigure 3 Comparison of the SARS‐CoV‐2 S and SARS‐CoV‐1 S structures: ribbon diagrams of the (A) SARS‐CoV‐2 S and (D) SARS‐CoV‐1 S [PDB 6NB6] ectodomain cryo‐EM structures. S1 subunits of (B) SARS‐CoV‐2 S and (E) SARS‐CoV‐1 S. S2 subunits of (C) SARS‐CoV‐2 S and (F) SARS‐CoV‐1 S. 28 cryo‐EM, cryogenic electron microscopy; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2 [Color figure can be viewed at wileyonlinelibrary.com] As mentioned previously, Zhang et al. 30 determined the full‐length genome sequence of SARS‐CoV‐2 and revealed that the virus is remarkably similar (89.1% sequence identity) to a group of SARS‐like CoVs. Simultaneously, Shi et al. 26 reported that SARS‐CoV‐2 shares 96% sequence identity at a whole‐genome level with a bat coronavirus—and importantly, like SARS‐CoV‐1, SARS‐CoV‐2 utilizes ACE2 receptor for viral entry. Recently, Yan et al. solved the cryo‐EM structure of full‐length human ACE2 bound to the RBD of SARS‐CoV‐2, providing important structural information for therapeutic intervention strategies. 29\nThe sequence identity of the spike protein between SARS‐CoV‐1 (1273 aa) and SARS‐CoV‐2 (1253 aa) is 76%. The spike protein has two regions, S1 and S2. The S1 region of the SARS‐CoV‐1 has a RBD that forms high‐affinity interactions with ACE2. The prevailing understanding is that SARS‐CoV‐2 employs this RBD to enter its human host cell as well. Aligning the two different RBDs revealed a sequence identity of 73.5%. However, many nonconserved mutations that interact directly with ACE2 are located in the two structural regions. 31 And both crystal and cryo‐EM structures of the SARS‐CoV‐1 spike‐ACE2 complex have shown that merely residues of regions 1 and 2 form hydrogen bonds and hydrophobic interactions with ACE2. The mutations in these two regions of SARS‐CoV‐2 will, therefore, likely reduce the number of those interactions. 32\nStudies also have shown that the RdRP, and the Mpro are highly conserved between SARS‐CoV‐2 and SARS‐CoV‐1. 33 , 34 Therefore, it is widely accepted that SARS‐CoV‐2 behaves similarly to SARS‐CoV‐1 with regard to viral entry and replication. Since the general genomic layout and replication kinetics are so conserved among MERS, SARS‐1, and SARS‐2 CoVs, investigating inhibitors of common structures is a logical step.\nThe inhibitory strength against viral enzyme was expressed as IC50, which is the concentration of the inhibitor needed to inhibit half of the enzyme activity in the tested condition. The K i value is reflective of ligand‐binding affinity to the enzyme. The inhibitory activity for cell‐based bioassays is expressed as EC50, which is a half maximal effective concentration required to induce the biological response. The warhead group means a “reactive group” of the inhibitors that can form both covalent and noncovalent interactions with amino acids in the active site of the enzyme.\n\n2.1 Targeting the RBD\nStructural investigations of the RBD‐ACE2 complex provided information about essential residues for viral entry. Hsiang et al. 35 reported a number of peptides that significantly blocked the interaction of the S protein with ACE2 with IC50 values as low as 1.88 nM. Michael et al. found charged residues between positions 22 and 57 crucial for SARS‐CoV‐1 viral entry. Based on this, they designed peptides P4 (IC50, 50 µM) and P5 (IC50, 6.0 µM) with significant inhibitory activity against SARS‐CoV‐1. The antiviral activity was further improved when they introduced the glycine binding linkage of peptide P4 (residues 22–47) with an ACE2‐derived peptide (residues 351–357) against a SARS‐CoV‐1 pseudovirus with an IC50 of 100 nM and devoid of cytotoxicity up to 200 µM. 36 It is worth highlighting that a similar strategy could work for the new SARS‐CoV‐2. The recently solved cryo‐EM structure of SARS‐CoV‐2 in complex with human ACE2 can provide a structural rationale for the peptide design. 29\nFor viral entry, MERS‐CoV uses its spike protein (S) to interact with the host‐receptor DPP4, 37 , 38 , 39 also known as adenosine deaminase‐complexing protein‐2 or CD26. 37 MERS‐CoV was also the first virus reported to use this particular path. 35 , 37 DPP4 is a type II transmembrane glycoprotein, that forms homodimers on the cell surface, and it is involved in the cleavage of dipeptides. 37 , 40 In humans, DPP4 is predominantly found on the bronchial epithelial and alveolar cells in the lower lungs. 40 , 41\nMERS‐4 and MERS‐27 are monoclonal antibodies targeting the RBD of MERS‐CoV S that were discovered in a nonimmune yeast‐display scFv library screening. The more active MERS‐4 potently blocked the infection of DPP4‐expressing Huh‐7 cells with pseudotyped MERS‐CoV (IC50, 0.056 μg/mL). It also prevented MERS‐CoV‐induced cytopathogenic effects in MERS‐infected Vero E6 cells (IC50, 0.5 μg/mL). 42\nA heptad repeat (HR) is a repeating structural pattern of seven amino acids. A crucial membrane fusion framework of SARS‐CoV is the 6‐helix‐bundle (6‐HB) that is formed by HR1 and HR2 of the viral S protein. Enfuvirtide (T‐20) is an FDA approved HR2 peptide and the first HIV fusion inhibitor. It has opened up new avenues toward identifying and developing peptides as viral entry inhibitors. Such molecules represent a promising strategy against enveloped viruses with class 1 fusion proteins such as Nipah virus, Hendra virus, Ebola virus, and other paramyxoviruses, simian immunodeficiency virus, feline immunodeficiency virus, and respiratory syncytial virus. 43 , 44 , 45 , 46 The HR regions of SARS‐CoV‐1 and SARS‐CoV‐2 S protein share a high degree of conservation, and such fusion inhibitors have potential applications in preventing SARS‐CoV‐2 entry.\nSmall molecule entry inhibitors, on the other hand, are reported to target the RBD. Compared to peptides, proteins, and biologics, small molecules have several advantages due to lower production costs, improved pharmacokinetics, stability, and dosage accuracy. Sarafianos et al. identified the oxazole‐carboxamide derivative SSAA09E2 (1; Figure 4) as an entry inhibitor against SARS‐CoV‐1 by screening a chemical library composed of 3000 compounds. 47 This inhibitor directly blocks ACE2 recognition by interfering with the RBD with an EC50 value of 3.1 µM and a 50% cytotoxic concentration (CC50) value of greater than 100 µM, not affecting ACE2 expression levels. 48\nFigure 4 Inhibitors targeting the receptor‐binding domain Xu et al. 49 identified two small molecules, TGG (2; Figure 4) and luteolin (3; Figure 4), that can bind avidly to the SARS‐CoV‐1 S2 protein and inhibit its entry into Vero E6 cells (EC50: 4.5 µM, 10.6 µM; respectively). Compounds 2 and 3 showed cytotoxicity (CC50) of 1.08 and 0.155 mM, and the selectivity index (SI) values of 2 and 3 were 240.0 and 14.62, respectively. Further studies regarding acute toxicity revealed that the 50% lethal doses of 2 and 3 were ~456 and 232 mg/kg, respectively. These results indicate that these small molecules could be used at relatively high concentrations in mice. 49 Quercetin (4; Figure 4), an analog of 3, also showed antiviral activity against SARS‐CoV‐1, with an EC50 value of 83.4 µM and a CC50 value of 3.32 mM. 50\nNgai et al. reported ADS‐J1 (5; Figure 4) as a potential SARS‐CoV‐1 viral entry inhibitor with an EC50 of 3.89 µM. Molecular docking studies predicted that 5 can bind into a deep pocket of the SARS‐CoV‐1 S HR region and block viral entry into host cells. 51 Imatinib (6; Figure 4), an Abelson kinase inhibitor, could inhibit CoV S protein‐induced fusion with an EC50 value of 10 µM and showed no cytotoxic effects in Vero cells up to 100 µM concentration. 52 , 53\n\n2.2 Inhibitors targeting the cellular receptor\nThe genetic code of SARS‐CoV‐2 shares noticeable similarities with SARS‐CoV‐1, which caused the SARS epidemic in 2002. 26 , 54 More importantly, both viruses have identical mechanisms of infection. SARS‐CoV‐1 uses the host's ACE2 as a portal to infect cells, which has high expression in the vascular endothelium 55 and the lung, particularly in type 2 alveolar epithelial cells. 56 SARS‐CoV‐2 shares 76% of its spike (S) protein with SARS‐CoV‐1. Despite a few amino acid differences in its RBD compared to the SARS‐CoV‐1 S protein, the SARS‐CoV‐2 S protein binds to ACE2 with even greater affinity 27 offering an explanation for its greater virulence and preference for the lung.\nACE, a highly glycosylated type I integral membrane protein, is an essential component of the renin‐angiotensin (Ang) system, which controls blood pressure homeostasis. Both ACE1 and ACE2 cleave Ang peptides. However, they differ markedly: ACE1 cuts and converts the inactive decapeptide Ang I into the octapeptide Ang II by removing the dipeptide His‐Leu. This Ang II induces vaso‐ and bronchoconstriction, increased vascular permeability, inflammation, and fibrosis, thus promoting acute respiratory distress syndrome (ARDS) and lung failure in patients infected with SARS‐CoV‐1 or SARS‐CoV‐2 57 (Figure 5). Therefore, ACE‐inhibitors (ACEis) and angiotensin II receptor blockers (ARBs) could block the disease‐propagating effect of Ang II. 58 , 59 , 60\nFigure 5 The roles of ACE1 and 2 in the renin‐angiotensin system. A, Chemical structures of angiotensin‐related peptides and B, Schematic diagram of roles of ACE1 and 2 in renin‐angiotensin system. ACE, angiotensin‐converting enzyme [Color figure can be viewed at wileyonlinelibrary.com] ACE2, on the other hand, is a zinc‐containing metalloenzyme, and shares merely 42% of its amino acid sequence with ACE1. 61 It cleaves only one amino acid residue (Leu or Phe) from Ang I and Ang II, respectively, generating Ang (1–9) and Ang (1–7) (a vasodilator) (Figure 5). Thus, ACE2 has been considered a potential therapeutic target for cardiovascular diseases.\nVirtual screening combined with a molecular docking approach targeting the ACE2 catalytic site with around 140 000 compounds led to the identification of inhibitor N‐(2‐aminoethyl)‐1 aziridine‐ethanamine (7; Figure 6) with an IC50 value of 57 µM and a K i of 459 µM. However, no information about the cytotoxicity of this compound is available so far. 62\nFigure 6 Inhibitors for SARS‐CoV‐1 and 2 targeting ACE2. ACE, angiotensin‐converting enzyme; SARS‐CoV, severe acute respiratory syndrome coronavirus Chloroquine (8; Figure 6) is a relatively safe, cheap, and effective medication for the treatment of malaria and amebiasis. Savarino et al. 63 reported its antiviral effects. At a molecular level, it increases late endosomal and lysosomal pH, resulting in impaired liberation of virions from endosomes or lysosomes. The virus is therefore unable to release its genetic material into the cell and replicate. 64 , 65 Furthermore, they hypothesize that chloroquine might block the production of proinflammatory cytokines (such as IL‐6), thereby blocking the pathway that subsequently leads to ARDS. 63\nChloroquine is reasonably active in vitro against SARS‐CoV‐1, MERS‐CoV, and SARS‐CoV‐2. It was found to inhibit SARS‐CoV‐2 with an EC50 value of 5.47 µM in vitro. 66 Antiviral activity against SARS‐CoV‐1 was reported with an IC50 of 8.8 μM in Vero cells, but it is unclear how this translates into activity in respiratory epithelial cells and in vivo. 67 , 68 Mechanistic studies of chloroquine for SARS‐CoV‐1 infection revealed that it could also weaken the interaction between the RBD of SARS‐CoV‐1 and ACE2 by interfering with terminal glycosylation of ACE2, thereby reducing its affinity to SARS‐CoV‐1 S. 69\nDuring the SARS‐CoV‐2 pandemic, chloroquine has been recommended by Chinese, South Korean, and Italian health authorities for the experimental treatment of COVID‐19, 70 , 71 despite contraindications for patients with heart disease or diabetes. 72 However, health experts and agencies like the US FDA and European Medicines Agency warned against broad uncontrolled use after reports of misuse of low‐quality versions of chloroquine phosphate intended for fish.\nHydroxychloroquine (9; Figure 6) is being studied as an experimental treatment for COVID‐19. 73 However, the benefits of treatment with this drug are unclear. 74\nHydroxychloroquine was found to inhibit SARS‐CoV‐2 with an EC50 value of 0.74 µM in vitro. 66 Some studies imply synergistic effects of hydroxychloroquine and azithromycin. Azithromycin is active in vitro against Zika and Ebola virus 75 , 76 and can be used to guard against life‐threatening bacterial superinfections when administered to patients suffering from viral infections. 77 A small study that compared hydroxychloroquine monotherapy and combination treatment with azithromycin found a significant advantage of the combination. While evaluating the efficacy of therapeutic intervention with hydroxychloroquine as monotherapy and its impact in combination with azithromycin, the number of patients testing negative in polymerase chain reaction (PCR) tests was substantially different in the two groups with 100% of patients cured (6 days post inclusion) in the combination arm of the study versus 57% in the monotherapy group. At the same time, 12% of patients in the control group receiving only standard care were cured. 78 , 79\nThe WHO declared on 18 March that chloroquine and its derivative hydroxychloroquine will be among the four medicines studied in the solidarity clinical trial 80 for the treatment of COVID‐19. In April 2020, the US National Institutes of Health (NIH) also commenced a study with the drug for treating COVID‐19 patients. 81\nThe recent clinical trial involving 96 032 patients with COVID‐19 concluded that it was unable to confirm a benefit of hydroxychloroquine or chloroquine, when used alone or in combination with a macrolide such as azithromycin (or clarithromycin). 82 The study actually reported decreased survival rates for patients treated with each of these drug regimens. Additionally, patients had an increased risk of developing ventricular arrhythmia under treatment. However, still more evidence is needed to adequately assess the drugs' risks or benefits for the treatment or prevention of COVID‐19 (it is important to note that chloroquine and hydroxychloroquine are still considered safe treatment options in certain autoimmune diseases and malaria). Besides, the WHO announced the premature pause of its clinical trials using hydroxychloroquine as a safety precaution on 24 May 2020.\nOn a different note, it was found that ACE2 undergoes proteolytic shedding; releasing an enzymatic ectodomain during viral entry. 83 A disintegrin and metalloproteinase (ADAM), also known as TNF‐α converting enzyme (TACE), assisted the shedding regulation of ACE2. Inhibition of this enzyme led to reduced shedding of ACE2. GW280264X (10; Figure 6) was found to be a specific inhibitor of ADAM‐induced shedding of ACE2 at 1 nM. 84 Two TACE inhibitors, TAPI‐0 (11) and TAPI‐2 (12; Figure 6), reduced ACE2 shedding, with IC50 values of 100 and 200 nM, respectively. 83\nMLN‐4760 (13; Figure 6) inhibited the catalytic activity of ACE2 with an IC50 of around 440 pM. 85 This is the most potent and selective small‐molecule inhibitor against soluble human ACE2 described to date, thus making it a very promising candidate for SARS‐CoV‐2 interference. It binds to the active site zinc and emulates the transition state peptide. However, no antiviral data for this compound is available at this time.\nThe interference of a virus‐host cell fusion, which is mediated by the viral S protein to its receptor ACE2 on host cells, may be a viable prevention strategy. Umifenovir (14; brand name Arbidol), a broad spectrum antiviral drug used against influenza, prevents viral entry by inhibiting virus‐host cell fusion. 86 It is currently being investigated in a clinical trial for the treatment of SARS‐CoV‐2. 87 , 88\nDo ACEis or ARBs amplify SARS‐CoV‐2 pathogenicity and aggravate the clinical course of COVID‐19? After ACE2 was recognized as the SARS‐CoV‐2 receptor, 14 , 29 speculations emerged about potentially negative consequences of ACEi or ARB therapy in COVID‐19 patients. This theory caused confusion in the public and alarmed patients taking these medicines. One report said that the expression of ACE2 was increased in patients with heart disease compared to healthy individuals. It was also insisted that ACE2 expression could be increased by taking ACEis and ARBs, 89 although there is no supporting report of this happening in the lungs.\nIn another report, it was suggested that patients suffering from high blood pressure receiving “ACE2‐increasing drugs” have a higher risk for severe COVID‐19, since ACEis and ARBs could elevate levels of ACE2. 90\nA joint declaration by the presidents of the HFSA/ACC/AHA on 17 March 2020, 91 followed by a similar statement of the European Medicines Agency, 92 clarified that there was no scientific basis for stopping ACEi or ARB therapy. 93 , 94 , 95 This was in accordance with the editors of the New England Journal of Medicine. 96\nIn case of SARS‐CoV, the experimental data showed that such medications may be beneficial rather than damaging, which led to a new therapeutic approach for lung diseases. 97\n\n2.3 Proteolytic processing inhibitors\nCoVs enter the host cells via both clathrin (endosomal) and nonclathrin pathways (nonendosomal); however, both pathways are dependent upon receptor binding. 98 , 99\nThe clathrin‐mediated pathway involves the binding of CoV S protein to the host receptor followed by the internalization of vesicles that maturate to late endosomes. Acidification of the endosome promotes the H+‐dependent activation of cellular cathepsin L proteinase in late endosomes and lysosomes, which cleaves and activates the S protein, thus initiating viral fusion. Recent research shows that in addition to ACE2 SARS‐CoV‐2 can also use the host cell receptor CD147 to gain access into host cells. 100\nMembrane fusion is also the crucial step for the CoV life cycle in the nonclathrin/endosomal route, in which host proteases such as cathepsin L, TMPRSS2, and TMPRSS11D (airway trypsin‐like protease) cut the S protein at the S1/S2 cleavage site to activate the S protein for membrane fusion. 101 Interference with this process by targeting these proteases could become an attractive strategy for combating CoV infections. A recent study confirms the role of TMPRSS2 for the viral life cycle in SARS‐CoV‐2‐infected VeroE6 cells. 5 Furin (a serine endoprotease) activates MERS‐CoV to initiate the nonclathrin mediated membrane fusion event. 102\nThe neurotransmitter receptor blockers chlorpromazine (15), promethazine (16), and fluphenazine (17; Figure 7), were reported to inhibit MERS‐CoV and SARS‐CoV‐1 most probably by impeding S protein‐induced fusion. 103 Chlorpromazine, a clathrin‐mediated viral entry inhibitor, was already described to inhibit human CoV‐229E, hepatitis C virus, infectious bronchitis virus, as well as mouse hepatitis virus‐2 (MHV2). 104 , 105 , 106 , 107 , 108\nFigure 7 Neurotransmitter inhibitors targeting clathrin/nonclathrin pathways Matsuyama et al. identified the commercially available serine protease inhibitor camostat (18; Figure 8) to be a SARS‐CoV‐1 inhibitor, blocking TMPRSS2 activity at 10 µM. However, at a higher concentration (100 µM), inhibition of viral entry via SARS‐CoV‐1 S protein‐mediated cell fusion never exceeded 65% (inhibition efficiency), indicating that 35% of entry events take place via the endosomal cathepsin pathway. Interestingly, treatment with a combination of EST (a cathepsin inhibitor) and 18 resulted in remarkably blocked infection (\u003e95%) activity of pseudotyped viruses. 109\nFigure 8 Inhibitors targeting TMPRSS2. TMPRSS, transmembrane serine protease A similar approach has been investigated to prevent viral entry of SARS‐CoV‐2. Pöhlmann et al. reported the attainment of full inhibition efficiency with a combination of both 18 and E‐64d (a cathepsin inhibitor). Both studies indicate that SARS‐CoV‐1 and 2 enter cells in a similar manner showing the potential of 18 as a candidate for further development. 15\nRecently, K11777 (19; Figure 8), a cysteine protease inhibitor, was shown in tissue cultures to inhibit SARS‐CoV‐1 and MERS‐CoV replication in the subnanomolar range. 110 , 111 Future tissue culture and animal model studies should be conducted to clarify, whether its antiviral activity is mediated by targeting TMPRSS2.\nTeicoplanin is a glycopeptide antibiotic used to prevent infections with Gram‐positive bacteria like methicillin‐resistant Staphylococcus aureus and Enterococcus faecalis. It was found that teicoplanin inhibits the entry of SARS‐CoV‐1, MERS‐CoV, and Ebola virus by specifically targeting cathepsin L. 112 This knowledge has also been used to block the entry of new SARS‐CoV‐2 pseudoviruses with an IC50 value of 1.66 µM. Therefore, teicoplanin could be considered a potential candidate for the treatment of COVID‐19. 113\n\n2.4 Small‐molecules as cathepsin L inhibitors\nHuman cathepsin L is a cysteine endopeptidase and plays a key role for infection efficiency by activation of the S protein into a fusogenic state to escape the late endosomes. Targeting this protease with small molecules could interfere with virus‐cell entry and therefore be a possible intervention strategy for CoV infection. 114 Bates et al. identified MDL28170 (20; Figure 9) as an antiviral compound that specifically inhibited cathepsin‐L‐mediated substrate cleavage, with an IC50 value of 2.5 nM and EC50 value in the range of 100 nM. However, despite its potent inhibitory activity, no cytotoxicity data for 20 is currently available. 115\nFigure 9 Cathepsin L inhibitors with antiviral activity Diamond et al. reported CID 16725315 (21) and CID 23631927 (22; Figure 9) as viral entry inhibitors of SARS‐CoV in a cathepsin L inhibition assay. Compound 21 could block cathepsin L with an IC50 value of 6.9 nM, while 22 showed slightly weaker potency (IC50, 56 nM). Compound 22 was also found to inhibit Ebola virus infection (EC50, 193 nM) of human embryonic kidney 293T cells. This compound did not show any sign of toxicity to human aortic endothelial cells up to 100 µM. This data offers a new promising point for the treatment of SARS and Ebola virus infections. 116\nScreening of ~14 000 compounds in a cell‐based assay resulted in the identification of SSAA09E1 (23; Figure 9) as inhibitor of cathepsin L proteinase, with an IC50 value of 5.33 µM. In a pseudotype‐based assay in 293T cells, the EC50 value of 23 was around 6.4 µM, and no cytotoxicity was detected below 100 µM. 48\nPhenotypic screening approaches led to the identification of several viral entry inhibitors. This approach has the advantage of finding cellular‐active compounds, providing information on drug solubility and cell uptake. 117 On the other hand, it is limited in terms of capacity compared to in silico target‐based screening. Hsiang et al. identified emodin (24; Figure 9), the active component from Polygonum multiflorum and Rheum officinale, could block the interaction of S protein with ACE2, with an IC50 value of 10 µM and an EC50 value of 200 µM in an S protein‐pseudotyped retrovirus assay using Vero E6 cells. However, the mechanism of action of this compound still needs to be determined. 118 Sarafianos et al. 48 found that SSAA09E3 (25), a benzamide derivative of 24, could prevent virus‐cell membrane fusion in pseudotype‐based and antiviral‐based assays, with an EC50 value of 9.7 µM, but a CC50 value of 20 µM indicates additional unknown cellular targets.\nVE607 (26) was identified among 50 240 structurally diverse small molecules to specifically inhibit SARS‐CoV‐1 entry into cells using a phenotype‐based screening. Its EC50 value was reported at 3.0 µM and it inhibited SARS‐CoV‐1 plaque formation with an EC50 of 1.6 µM. 119 Cathepsin inhibitor E‐64‐D (27) blocked MERS‐CoV and SARS‐CoV‐1 infection as well. 120 , 121"}

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VIRUS ENTRY INHIBITORS\nBinding of spike protein (S) to its receptor represents the host's first confrontation with the virus and its life cycle, thus providing prophylactic intervention opportunities. The genome of SARS‐CoV‐2 has been recently determined to have an 80% identity to that of SARS‐CoV‐1 and 96% identity to the bat‐CoV RaTG13. 26 The SARS‐CoV‐2 S protein shows nucleotide sequence identities of 75% or less to all other previously described CoVs. However, again, the new SARS‐CoV‐2 S protein shares a 93.1% identity to the S protein of RaTG13. SARS‐CoV‐2 spike (S) recognizes, with its receptor‐binding domain (RBD), the cellular ACE2 receptor with high affinity (K d, 14.7 nM) 27 as judged by surface plasmin resonance spectrometry; and intervention at the RBD‐ACE2 interface can potentially disrupt infection efficiency. It was observed that the RBDs of the SARS‐CoV‐2‐ACE2 and SARS‐CoV‐1‐ACE2 complexes are quite similar (Figure 3). 29\nFigure 3 Comparison of the SARS‐CoV‐2 S and SARS‐CoV‐1 S structures: ribbon diagrams of the (A) SARS‐CoV‐2 S and (D) SARS‐CoV‐1 S [PDB 6NB6] ectodomain cryo‐EM structures. S1 subunits of (B) SARS‐CoV‐2 S and (E) SARS‐CoV‐1 S. S2 subunits of (C) SARS‐CoV‐2 S and (F) SARS‐CoV‐1 S. 28 cryo‐EM, cryogenic electron microscopy; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2 [Color figure can be viewed at wileyonlinelibrary.com] As mentioned previously, Zhang et al. 30 determined the full‐length genome sequence of SARS‐CoV‐2 and revealed that the virus is remarkably similar (89.1% sequence identity) to a group of SARS‐like CoVs. Simultaneously, Shi et al. 26 reported that SARS‐CoV‐2 shares 96% sequence identity at a whole‐genome level with a bat coronavirus—and importantly, like SARS‐CoV‐1, SARS‐CoV‐2 utilizes ACE2 receptor for viral entry. Recently, Yan et al. solved the cryo‐EM structure of full‐length human ACE2 bound to the RBD of SARS‐CoV‐2, providing important structural information for therapeutic intervention strategies. 29\nThe sequence identity of the spike protein between SARS‐CoV‐1 (1273 aa) and SARS‐CoV‐2 (1253 aa) is 76%. The spike protein has two regions, S1 and S2. The S1 region of the SARS‐CoV‐1 has a RBD that forms high‐affinity interactions with ACE2. The prevailing understanding is that SARS‐CoV‐2 employs this RBD to enter its human host cell as well. Aligning the two different RBDs revealed a sequence identity of 73.5%. However, many nonconserved mutations that interact directly with ACE2 are located in the two structural regions. 31 And both crystal and cryo‐EM structures of the SARS‐CoV‐1 spike‐ACE2 complex have shown that merely residues of regions 1 and 2 form hydrogen bonds and hydrophobic interactions with ACE2. The mutations in these two regions of SARS‐CoV‐2 will, therefore, likely reduce the number of those interactions. 32\nStudies also have shown that the RdRP, and the Mpro are highly conserved between SARS‐CoV‐2 and SARS‐CoV‐1. 33 , 34 Therefore, it is widely accepted that SARS‐CoV‐2 behaves similarly to SARS‐CoV‐1 with regard to viral entry and replication. Since the general genomic layout and replication kinetics are so conserved among MERS, SARS‐1, and SARS‐2 CoVs, investigating inhibitors of common structures is a logical step.\nThe inhibitory strength against viral enzyme was expressed as IC50, which is the concentration of the inhibitor needed to inhibit half of the enzyme activity in the tested condition. The K i value is reflective of ligand‐binding affinity to the enzyme. The inhibitory activity for cell‐based bioassays is expressed as EC50, which is a half maximal effective concentration required to induce the biological response. The warhead group means a “reactive group” of the inhibitors that can form both covalent and noncovalent interactions with amino acids in the active site of the enzyme.\n\n2.1 Targeting the RBD\nStructural investigations of the RBD‐ACE2 complex provided information about essential residues for viral entry. Hsiang et al. 35 reported a number of peptides that significantly blocked the interaction of the S protein with ACE2 with IC50 values as low as 1.88 nM. Michael et al. found charged residues between positions 22 and 57 crucial for SARS‐CoV‐1 viral entry. Based on this, they designed peptides P4 (IC50, 50 µM) and P5 (IC50, 6.0 µM) with significant inhibitory activity against SARS‐CoV‐1. The antiviral activity was further improved when they introduced the glycine binding linkage of peptide P4 (residues 22–47) with an ACE2‐derived peptide (residues 351–357) against a SARS‐CoV‐1 pseudovirus with an IC50 of 100 nM and devoid of cytotoxicity up to 200 µM. 36 It is worth highlighting that a similar strategy could work for the new SARS‐CoV‐2. The recently solved cryo‐EM structure of SARS‐CoV‐2 in complex with human ACE2 can provide a structural rationale for the peptide design. 29\nFor viral entry, MERS‐CoV uses its spike protein (S) to interact with the host‐receptor DPP4, 37 , 38 , 39 also known as adenosine deaminase‐complexing protein‐2 or CD26. 37 MERS‐CoV was also the first virus reported to use this particular path. 35 , 37 DPP4 is a type II transmembrane glycoprotein, that forms homodimers on the cell surface, and it is involved in the cleavage of dipeptides. 37 , 40 In humans, DPP4 is predominantly found on the bronchial epithelial and alveolar cells in the lower lungs. 40 , 41\nMERS‐4 and MERS‐27 are monoclonal antibodies targeting the RBD of MERS‐CoV S that were discovered in a nonimmune yeast‐display scFv library screening. The more active MERS‐4 potently blocked the infection of DPP4‐expressing Huh‐7 cells with pseudotyped MERS‐CoV (IC50, 0.056 μg/mL). It also prevented MERS‐CoV‐induced cytopathogenic effects in MERS‐infected Vero E6 cells (IC50, 0.5 μg/mL). 42\nA heptad repeat (HR) is a repeating structural pattern of seven amino acids. A crucial membrane fusion framework of SARS‐CoV is the 6‐helix‐bundle (6‐HB) that is formed by HR1 and HR2 of the viral S protein. Enfuvirtide (T‐20) is an FDA approved HR2 peptide and the first HIV fusion inhibitor. It has opened up new avenues toward identifying and developing peptides as viral entry inhibitors. Such molecules represent a promising strategy against enveloped viruses with class 1 fusion proteins such as Nipah virus, Hendra virus, Ebola virus, and other paramyxoviruses, simian immunodeficiency virus, feline immunodeficiency virus, and respiratory syncytial virus. 43 , 44 , 45 , 46 The HR regions of SARS‐CoV‐1 and SARS‐CoV‐2 S protein share a high degree of conservation, and such fusion inhibitors have potential applications in preventing SARS‐CoV‐2 entry.\nSmall molecule entry inhibitors, on the other hand, are reported to target the RBD. Compared to peptides, proteins, and biologics, small molecules have several advantages due to lower production costs, improved pharmacokinetics, stability, and dosage accuracy. Sarafianos et al. identified the oxazole‐carboxamide derivative SSAA09E2 (1; Figure 4) as an entry inhibitor against SARS‐CoV‐1 by screening a chemical library composed of 3000 compounds. 47 This inhibitor directly blocks ACE2 recognition by interfering with the RBD with an EC50 value of 3.1 µM and a 50% cytotoxic concentration (CC50) value of greater than 100 µM, not affecting ACE2 expression levels. 48\nFigure 4 Inhibitors targeting the receptor‐binding domain Xu et al. 49 identified two small molecules, TGG (2; Figure 4) and luteolin (3; Figure 4), that can bind avidly to the SARS‐CoV‐1 S2 protein and inhibit its entry into Vero E6 cells (EC50: 4.5 µM, 10.6 µM; respectively). Compounds 2 and 3 showed cytotoxicity (CC50) of 1.08 and 0.155 mM, and the selectivity index (SI) values of 2 and 3 were 240.0 and 14.62, respectively. Further studies regarding acute toxicity revealed that the 50% lethal doses of 2 and 3 were ~456 and 232 mg/kg, respectively. These results indicate that these small molecules could be used at relatively high concentrations in mice. 49 Quercetin (4; Figure 4), an analog of 3, also showed antiviral activity against SARS‐CoV‐1, with an EC50 value of 83.4 µM and a CC50 value of 3.32 mM. 50\nNgai et al. reported ADS‐J1 (5; Figure 4) as a potential SARS‐CoV‐1 viral entry inhibitor with an EC50 of 3.89 µM. Molecular docking studies predicted that 5 can bind into a deep pocket of the SARS‐CoV‐1 S HR region and block viral entry into host cells. 51 Imatinib (6; Figure 4), an Abelson kinase inhibitor, could inhibit CoV S protein‐induced fusion with an EC50 value of 10 µM and showed no cytotoxic effects in Vero cells up to 100 µM concentration. 52 , 53\n\n2.2 Inhibitors targeting the cellular receptor\nThe genetic code of SARS‐CoV‐2 shares noticeable similarities with SARS‐CoV‐1, which caused the SARS epidemic in 2002. 26 , 54 More importantly, both viruses have identical mechanisms of infection. SARS‐CoV‐1 uses the host's ACE2 as a portal to infect cells, which has high expression in the vascular endothelium 55 and the lung, particularly in type 2 alveolar epithelial cells. 56 SARS‐CoV‐2 shares 76% of its spike (S) protein with SARS‐CoV‐1. Despite a few amino acid differences in its RBD compared to the SARS‐CoV‐1 S protein, the SARS‐CoV‐2 S protein binds to ACE2 with even greater affinity 27 offering an explanation for its greater virulence and preference for the lung.\nACE, a highly glycosylated type I integral membrane protein, is an essential component of the renin‐angiotensin (Ang) system, which controls blood pressure homeostasis. Both ACE1 and ACE2 cleave Ang peptides. However, they differ markedly: ACE1 cuts and converts the inactive decapeptide Ang I into the octapeptide Ang II by removing the dipeptide His‐Leu. This Ang II induces vaso‐ and bronchoconstriction, increased vascular permeability, inflammation, and fibrosis, thus promoting acute respiratory distress syndrome (ARDS) and lung failure in patients infected with SARS‐CoV‐1 or SARS‐CoV‐2 57 (Figure 5). Therefore, ACE‐inhibitors (ACEis) and angiotensin II receptor blockers (ARBs) could block the disease‐propagating effect of Ang II. 58 , 59 , 60\nFigure 5 The roles of ACE1 and 2 in the renin‐angiotensin system. A, Chemical structures of angiotensin‐related peptides and B, Schematic diagram of roles of ACE1 and 2 in renin‐angiotensin system. ACE, angiotensin‐converting enzyme [Color figure can be viewed at wileyonlinelibrary.com] ACE2, on the other hand, is a zinc‐containing metalloenzyme, and shares merely 42% of its amino acid sequence with ACE1. 61 It cleaves only one amino acid residue (Leu or Phe) from Ang I and Ang II, respectively, generating Ang (1–9) and Ang (1–7) (a vasodilator) (Figure 5). Thus, ACE2 has been considered a potential therapeutic target for cardiovascular diseases.\nVirtual screening combined with a molecular docking approach targeting the ACE2 catalytic site with around 140 000 compounds led to the identification of inhibitor N‐(2‐aminoethyl)‐1 aziridine‐ethanamine (7; Figure 6) with an IC50 value of 57 µM and a K i of 459 µM. However, no information about the cytotoxicity of this compound is available so far. 62\nFigure 6 Inhibitors for SARS‐CoV‐1 and 2 targeting ACE2. ACE, angiotensin‐converting enzyme; SARS‐CoV, severe acute respiratory syndrome coronavirus Chloroquine (8; Figure 6) is a relatively safe, cheap, and effective medication for the treatment of malaria and amebiasis. Savarino et al. 63 reported its antiviral effects. At a molecular level, it increases late endosomal and lysosomal pH, resulting in impaired liberation of virions from endosomes or lysosomes. The virus is therefore unable to release its genetic material into the cell and replicate. 64 , 65 Furthermore, they hypothesize that chloroquine might block the production of proinflammatory cytokines (such as IL‐6), thereby blocking the pathway that subsequently leads to ARDS. 63\nChloroquine is reasonably active in vitro against SARS‐CoV‐1, MERS‐CoV, and SARS‐CoV‐2. It was found to inhibit SARS‐CoV‐2 with an EC50 value of 5.47 µM in vitro. 66 Antiviral activity against SARS‐CoV‐1 was reported with an IC50 of 8.8 μM in Vero cells, but it is unclear how this translates into activity in respiratory epithelial cells and in vivo. 67 , 68 Mechanistic studies of chloroquine for SARS‐CoV‐1 infection revealed that it could also weaken the interaction between the RBD of SARS‐CoV‐1 and ACE2 by interfering with terminal glycosylation of ACE2, thereby reducing its affinity to SARS‐CoV‐1 S. 69\nDuring the SARS‐CoV‐2 pandemic, chloroquine has been recommended by Chinese, South Korean, and Italian health authorities for the experimental treatment of COVID‐19, 70 , 71 despite contraindications for patients with heart disease or diabetes. 72 However, health experts and agencies like the US FDA and European Medicines Agency warned against broad uncontrolled use after reports of misuse of low‐quality versions of chloroquine phosphate intended for fish.\nHydroxychloroquine (9; Figure 6) is being studied as an experimental treatment for COVID‐19. 73 However, the benefits of treatment with this drug are unclear. 74\nHydroxychloroquine was found to inhibit SARS‐CoV‐2 with an EC50 value of 0.74 µM in vitro. 66 Some studies imply synergistic effects of hydroxychloroquine and azithromycin. Azithromycin is active in vitro against Zika and Ebola virus 75 , 76 and can be used to guard against life‐threatening bacterial superinfections when administered to patients suffering from viral infections. 77 A small study that compared hydroxychloroquine monotherapy and combination treatment with azithromycin found a significant advantage of the combination. While evaluating the efficacy of therapeutic intervention with hydroxychloroquine as monotherapy and its impact in combination with azithromycin, the number of patients testing negative in polymerase chain reaction (PCR) tests was substantially different in the two groups with 100% of patients cured (6 days post inclusion) in the combination arm of the study versus 57% in the monotherapy group. At the same time, 12% of patients in the control group receiving only standard care were cured. 78 , 79\nThe WHO declared on 18 March that chloroquine and its derivative hydroxychloroquine will be among the four medicines studied in the solidarity clinical trial 80 for the treatment of COVID‐19. In April 2020, the US National Institutes of Health (NIH) also commenced a study with the drug for treating COVID‐19 patients. 81\nThe recent clinical trial involving 96 032 patients with COVID‐19 concluded that it was unable to confirm a benefit of hydroxychloroquine or chloroquine, when used alone or in combination with a macrolide such as azithromycin (or clarithromycin). 82 The study actually reported decreased survival rates for patients treated with each of these drug regimens. Additionally, patients had an increased risk of developing ventricular arrhythmia under treatment. However, still more evidence is needed to adequately assess the drugs' risks or benefits for the treatment or prevention of COVID‐19 (it is important to note that chloroquine and hydroxychloroquine are still considered safe treatment options in certain autoimmune diseases and malaria). Besides, the WHO announced the premature pause of its clinical trials using hydroxychloroquine as a safety precaution on 24 May 2020.\nOn a different note, it was found that ACE2 undergoes proteolytic shedding; releasing an enzymatic ectodomain during viral entry. 83 A disintegrin and metalloproteinase (ADAM), also known as TNF‐α converting enzyme (TACE), assisted the shedding regulation of ACE2. Inhibition of this enzyme led to reduced shedding of ACE2. GW280264X (10; Figure 6) was found to be a specific inhibitor of ADAM‐induced shedding of ACE2 at 1 nM. 84 Two TACE inhibitors, TAPI‐0 (11) and TAPI‐2 (12; Figure 6), reduced ACE2 shedding, with IC50 values of 100 and 200 nM, respectively. 83\nMLN‐4760 (13; Figure 6) inhibited the catalytic activity of ACE2 with an IC50 of around 440 pM. 85 This is the most potent and selective small‐molecule inhibitor against soluble human ACE2 described to date, thus making it a very promising candidate for SARS‐CoV‐2 interference. It binds to the active site zinc and emulates the transition state peptide. However, no antiviral data for this compound is available at this time.\nThe interference of a virus‐host cell fusion, which is mediated by the viral S protein to its receptor ACE2 on host cells, may be a viable prevention strategy. Umifenovir (14; brand name Arbidol), a broad spectrum antiviral drug used against influenza, prevents viral entry by inhibiting virus‐host cell fusion. 86 It is currently being investigated in a clinical trial for the treatment of SARS‐CoV‐2. 87 , 88\nDo ACEis or ARBs amplify SARS‐CoV‐2 pathogenicity and aggravate the clinical course of COVID‐19? After ACE2 was recognized as the SARS‐CoV‐2 receptor, 14 , 29 speculations emerged about potentially negative consequences of ACEi or ARB therapy in COVID‐19 patients. This theory caused confusion in the public and alarmed patients taking these medicines. One report said that the expression of ACE2 was increased in patients with heart disease compared to healthy individuals. It was also insisted that ACE2 expression could be increased by taking ACEis and ARBs, 89 although there is no supporting report of this happening in the lungs.\nIn another report, it was suggested that patients suffering from high blood pressure receiving “ACE2‐increasing drugs” have a higher risk for severe COVID‐19, since ACEis and ARBs could elevate levels of ACE2. 90\nA joint declaration by the presidents of the HFSA/ACC/AHA on 17 March 2020, 91 followed by a similar statement of the European Medicines Agency, 92 clarified that there was no scientific basis for stopping ACEi or ARB therapy. 93 , 94 , 95 This was in accordance with the editors of the New England Journal of Medicine. 96\nIn case of SARS‐CoV, the experimental data showed that such medications may be beneficial rather than damaging, which led to a new therapeutic approach for lung diseases. 97\n\n2.3 Proteolytic processing inhibitors\nCoVs enter the host cells via both clathrin (endosomal) and nonclathrin pathways (nonendosomal); however, both pathways are dependent upon receptor binding. 98 , 99\nThe clathrin‐mediated pathway involves the binding of CoV S protein to the host receptor followed by the internalization of vesicles that maturate to late endosomes. Acidification of the endosome promotes the H+‐dependent activation of cellular cathepsin L proteinase in late endosomes and lysosomes, which cleaves and activates the S protein, thus initiating viral fusion. Recent research shows that in addition to ACE2 SARS‐CoV‐2 can also use the host cell receptor CD147 to gain access into host cells. 100\nMembrane fusion is also the crucial step for the CoV life cycle in the nonclathrin/endosomal route, in which host proteases such as cathepsin L, TMPRSS2, and TMPRSS11D (airway trypsin‐like protease) cut the S protein at the S1/S2 cleavage site to activate the S protein for membrane fusion. 101 Interference with this process by targeting these proteases could become an attractive strategy for combating CoV infections. A recent study confirms the role of TMPRSS2 for the viral life cycle in SARS‐CoV‐2‐infected VeroE6 cells. 5 Furin (a serine endoprotease) activates MERS‐CoV to initiate the nonclathrin mediated membrane fusion event. 102\nThe neurotransmitter receptor blockers chlorpromazine (15), promethazine (16), and fluphenazine (17; Figure 7), were reported to inhibit MERS‐CoV and SARS‐CoV‐1 most probably by impeding S protein‐induced fusion. 103 Chlorpromazine, a clathrin‐mediated viral entry inhibitor, was already described to inhibit human CoV‐229E, hepatitis C virus, infectious bronchitis virus, as well as mouse hepatitis virus‐2 (MHV2). 104 , 105 , 106 , 107 , 108\nFigure 7 Neurotransmitter inhibitors targeting clathrin/nonclathrin pathways Matsuyama et al. identified the commercially available serine protease inhibitor camostat (18; Figure 8) to be a SARS‐CoV‐1 inhibitor, blocking TMPRSS2 activity at 10 µM. However, at a higher concentration (100 µM), inhibition of viral entry via SARS‐CoV‐1 S protein‐mediated cell fusion never exceeded 65% (inhibition efficiency), indicating that 35% of entry events take place via the endosomal cathepsin pathway. Interestingly, treatment with a combination of EST (a cathepsin inhibitor) and 18 resulted in remarkably blocked infection (\u003e95%) activity of pseudotyped viruses. 109\nFigure 8 Inhibitors targeting TMPRSS2. TMPRSS, transmembrane serine protease A similar approach has been investigated to prevent viral entry of SARS‐CoV‐2. Pöhlmann et al. reported the attainment of full inhibition efficiency with a combination of both 18 and E‐64d (a cathepsin inhibitor). Both studies indicate that SARS‐CoV‐1 and 2 enter cells in a similar manner showing the potential of 18 as a candidate for further development. 15\nRecently, K11777 (19; Figure 8), a cysteine protease inhibitor, was shown in tissue cultures to inhibit SARS‐CoV‐1 and MERS‐CoV replication in the subnanomolar range. 110 , 111 Future tissue culture and animal model studies should be conducted to clarify, whether its antiviral activity is mediated by targeting TMPRSS2.\nTeicoplanin is a glycopeptide antibiotic used to prevent infections with Gram‐positive bacteria like methicillin‐resistant Staphylococcus aureus and Enterococcus faecalis. It was found that teicoplanin inhibits the entry of SARS‐CoV‐1, MERS‐CoV, and Ebola virus by specifically targeting cathepsin L. 112 This knowledge has also been used to block the entry of new SARS‐CoV‐2 pseudoviruses with an IC50 value of 1.66 µM. Therefore, teicoplanin could be considered a potential candidate for the treatment of COVID‐19. 113\n\n2.4 Small‐molecules as cathepsin L inhibitors\nHuman cathepsin L is a cysteine endopeptidase and plays a key role for infection efficiency by activation of the S protein into a fusogenic state to escape the late endosomes. Targeting this protease with small molecules could interfere with virus‐cell entry and therefore be a possible intervention strategy for CoV infection. 114 Bates et al. identified MDL28170 (20; Figure 9) as an antiviral compound that specifically inhibited cathepsin‐L‐mediated substrate cleavage, with an IC50 value of 2.5 nM and EC50 value in the range of 100 nM. However, despite its potent inhibitory activity, no cytotoxicity data for 20 is currently available. 115\nFigure 9 Cathepsin L inhibitors with antiviral activity Diamond et al. reported CID 16725315 (21) and CID 23631927 (22; Figure 9) as viral entry inhibitors of SARS‐CoV in a cathepsin L inhibition assay. Compound 21 could block cathepsin L with an IC50 value of 6.9 nM, while 22 showed slightly weaker potency (IC50, 56 nM). Compound 22 was also found to inhibit Ebola virus infection (EC50, 193 nM) of human embryonic kidney 293T cells. This compound did not show any sign of toxicity to human aortic endothelial cells up to 100 µM. This data offers a new promising point for the treatment of SARS and Ebola virus infections. 116\nScreening of ~14 000 compounds in a cell‐based assay resulted in the identification of SSAA09E1 (23; Figure 9) as inhibitor of cathepsin L proteinase, with an IC50 value of 5.33 µM. In a pseudotype‐based assay in 293T cells, the EC50 value of 23 was around 6.4 µM, and no cytotoxicity was detected below 100 µM. 48\nPhenotypic screening approaches led to the identification of several viral entry inhibitors. This approach has the advantage of finding cellular‐active compounds, providing information on drug solubility and cell uptake. 117 On the other hand, it is limited in terms of capacity compared to in silico target‐based screening. Hsiang et al. identified emodin (24; Figure 9), the active component from Polygonum multiflorum and Rheum officinale, could block the interaction of S protein with ACE2, with an IC50 value of 10 µM and an EC50 value of 200 µM in an S protein‐pseudotyped retrovirus assay using Vero E6 cells. However, the mechanism of action of this compound still needs to be determined. 118 Sarafianos et al. 48 found that SSAA09E3 (25), a benzamide derivative of 24, could prevent virus‐cell membrane fusion in pseudotype‐based and antiviral‐based assays, with an EC50 value of 9.7 µM, but a CC50 value of 20 µM indicates additional unknown cellular targets.\nVE607 (26) was identified among 50 240 structurally diverse small molecules to specifically inhibit SARS‐CoV‐1 entry into cells using a phenotype‐based screening. Its EC50 value was reported at 3.0 µM and it inhibited SARS‐CoV‐1 plaque formation with an EC50 of 1.6 µM. 119 Cathepsin inhibitor E‐64‐D (27) blocked MERS‐CoV and SARS‐CoV‐1 infection as well. 120 , 121"}

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VIRUS ENTRY INHIBITORS\nBinding of spike protein (S) to its receptor represents the host's first confrontation with the virus and its life cycle, thus providing prophylactic intervention opportunities. The genome of SARS‐CoV‐2 has been recently determined to have an 80% identity to that of SARS‐CoV‐1 and 96% identity to the bat‐CoV RaTG13. 26 The SARS‐CoV‐2 S protein shows nucleotide sequence identities of 75% or less to all other previously described CoVs. However, again, the new SARS‐CoV‐2 S protein shares a 93.1% identity to the S protein of RaTG13. SARS‐CoV‐2 spike (S) recognizes, with its receptor‐binding domain (RBD), the cellular ACE2 receptor with high affinity (K d, 14.7 nM) 27 as judged by surface plasmin resonance spectrometry; and intervention at the RBD‐ACE2 interface can potentially disrupt infection efficiency. It was observed that the RBDs of the SARS‐CoV‐2‐ACE2 and SARS‐CoV‐1‐ACE2 complexes are quite similar (Figure 3). 29\nFigure 3 Comparison of the SARS‐CoV‐2 S and SARS‐CoV‐1 S structures: ribbon diagrams of the (A) SARS‐CoV‐2 S and (D) SARS‐CoV‐1 S [PDB 6NB6] ectodomain cryo‐EM structures. S1 subunits of (B) SARS‐CoV‐2 S and (E) SARS‐CoV‐1 S. S2 subunits of (C) SARS‐CoV‐2 S and (F) SARS‐CoV‐1 S. 28 cryo‐EM, cryogenic electron microscopy; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2 [Color figure can be viewed at wileyonlinelibrary.com] As mentioned previously, Zhang et al. 30 determined the full‐length genome sequence of SARS‐CoV‐2 and revealed that the virus is remarkably similar (89.1% sequence identity) to a group of SARS‐like CoVs. Simultaneously, Shi et al. 26 reported that SARS‐CoV‐2 shares 96% sequence identity at a whole‐genome level with a bat coronavirus—and importantly, like SARS‐CoV‐1, SARS‐CoV‐2 utilizes ACE2 receptor for viral entry. Recently, Yan et al. solved the cryo‐EM structure of full‐length human ACE2 bound to the RBD of SARS‐CoV‐2, providing important structural information for therapeutic intervention strategies. 29\nThe sequence identity of the spike protein between SARS‐CoV‐1 (1273 aa) and SARS‐CoV‐2 (1253 aa) is 76%. The spike protein has two regions, S1 and S2. The S1 region of the SARS‐CoV‐1 has a RBD that forms high‐affinity interactions with ACE2. The prevailing understanding is that SARS‐CoV‐2 employs this RBD to enter its human host cell as well. Aligning the two different RBDs revealed a sequence identity of 73.5%. However, many nonconserved mutations that interact directly with ACE2 are located in the two structural regions. 31 And both crystal and cryo‐EM structures of the SARS‐CoV‐1 spike‐ACE2 complex have shown that merely residues of regions 1 and 2 form hydrogen bonds and hydrophobic interactions with ACE2. The mutations in these two regions of SARS‐CoV‐2 will, therefore, likely reduce the number of those interactions. 32\nStudies also have shown that the RdRP, and the Mpro are highly conserved between SARS‐CoV‐2 and SARS‐CoV‐1. 33 , 34 Therefore, it is widely accepted that SARS‐CoV‐2 behaves similarly to SARS‐CoV‐1 with regard to viral entry and replication. Since the general genomic layout and replication kinetics are so conserved among MERS, SARS‐1, and SARS‐2 CoVs, investigating inhibitors of common structures is a logical step.\nThe inhibitory strength against viral enzyme was expressed as IC50, which is the concentration of the inhibitor needed to inhibit half of the enzyme activity in the tested condition. The K i value is reflective of ligand‐binding affinity to the enzyme. The inhibitory activity for cell‐based bioassays is expressed as EC50, which is a half maximal effective concentration required to induce the biological response. The warhead group means a “reactive group” of the inhibitors that can form both covalent and noncovalent interactions with amino acids in the active site of the enzyme.\n\n2.1 Targeting the RBD\nStructural investigations of the RBD‐ACE2 complex provided information about essential residues for viral entry. Hsiang et al. 35 reported a number of peptides that significantly blocked the interaction of the S protein with ACE2 with IC50 values as low as 1.88 nM. Michael et al. found charged residues between positions 22 and 57 crucial for SARS‐CoV‐1 viral entry. Based on this, they designed peptides P4 (IC50, 50 µM) and P5 (IC50, 6.0 µM) with significant inhibitory activity against SARS‐CoV‐1. The antiviral activity was further improved when they introduced the glycine binding linkage of peptide P4 (residues 22–47) with an ACE2‐derived peptide (residues 351–357) against a SARS‐CoV‐1 pseudovirus with an IC50 of 100 nM and devoid of cytotoxicity up to 200 µM. 36 It is worth highlighting that a similar strategy could work for the new SARS‐CoV‐2. The recently solved cryo‐EM structure of SARS‐CoV‐2 in complex with human ACE2 can provide a structural rationale for the peptide design. 29\nFor viral entry, MERS‐CoV uses its spike protein (S) to interact with the host‐receptor DPP4, 37 , 38 , 39 also known as adenosine deaminase‐complexing protein‐2 or CD26. 37 MERS‐CoV was also the first virus reported to use this particular path. 35 , 37 DPP4 is a type II transmembrane glycoprotein, that forms homodimers on the cell surface, and it is involved in the cleavage of dipeptides. 37 , 40 In humans, DPP4 is predominantly found on the bronchial epithelial and alveolar cells in the lower lungs. 40 , 41\nMERS‐4 and MERS‐27 are monoclonal antibodies targeting the RBD of MERS‐CoV S that were discovered in a nonimmune yeast‐display scFv library screening. The more active MERS‐4 potently blocked the infection of DPP4‐expressing Huh‐7 cells with pseudotyped MERS‐CoV (IC50, 0.056 μg/mL). It also prevented MERS‐CoV‐induced cytopathogenic effects in MERS‐infected Vero E6 cells (IC50, 0.5 μg/mL). 42\nA heptad repeat (HR) is a repeating structural pattern of seven amino acids. A crucial membrane fusion framework of SARS‐CoV is the 6‐helix‐bundle (6‐HB) that is formed by HR1 and HR2 of the viral S protein. Enfuvirtide (T‐20) is an FDA approved HR2 peptide and the first HIV fusion inhibitor. It has opened up new avenues toward identifying and developing peptides as viral entry inhibitors. Such molecules represent a promising strategy against enveloped viruses with class 1 fusion proteins such as Nipah virus, Hendra virus, Ebola virus, and other paramyxoviruses, simian immunodeficiency virus, feline immunodeficiency virus, and respiratory syncytial virus. 43 , 44 , 45 , 46 The HR regions of SARS‐CoV‐1 and SARS‐CoV‐2 S protein share a high degree of conservation, and such fusion inhibitors have potential applications in preventing SARS‐CoV‐2 entry.\nSmall molecule entry inhibitors, on the other hand, are reported to target the RBD. Compared to peptides, proteins, and biologics, small molecules have several advantages due to lower production costs, improved pharmacokinetics, stability, and dosage accuracy. Sarafianos et al. identified the oxazole‐carboxamide derivative SSAA09E2 (1; Figure 4) as an entry inhibitor against SARS‐CoV‐1 by screening a chemical library composed of 3000 compounds. 47 This inhibitor directly blocks ACE2 recognition by interfering with the RBD with an EC50 value of 3.1 µM and a 50% cytotoxic concentration (CC50) value of greater than 100 µM, not affecting ACE2 expression levels. 48\nFigure 4 Inhibitors targeting the receptor‐binding domain Xu et al. 49 identified two small molecules, TGG (2; Figure 4) and luteolin (3; Figure 4), that can bind avidly to the SARS‐CoV‐1 S2 protein and inhibit its entry into Vero E6 cells (EC50: 4.5 µM, 10.6 µM; respectively). Compounds 2 and 3 showed cytotoxicity (CC50) of 1.08 and 0.155 mM, and the selectivity index (SI) values of 2 and 3 were 240.0 and 14.62, respectively. Further studies regarding acute toxicity revealed that the 50% lethal doses of 2 and 3 were ~456 and 232 mg/kg, respectively. These results indicate that these small molecules could be used at relatively high concentrations in mice. 49 Quercetin (4; Figure 4), an analog of 3, also showed antiviral activity against SARS‐CoV‐1, with an EC50 value of 83.4 µM and a CC50 value of 3.32 mM. 50\nNgai et al. reported ADS‐J1 (5; Figure 4) as a potential SARS‐CoV‐1 viral entry inhibitor with an EC50 of 3.89 µM. Molecular docking studies predicted that 5 can bind into a deep pocket of the SARS‐CoV‐1 S HR region and block viral entry into host cells. 51 Imatinib (6; Figure 4), an Abelson kinase inhibitor, could inhibit CoV S protein‐induced fusion with an EC50 value of 10 µM and showed no cytotoxic effects in Vero cells up to 100 µM concentration. 52 , 53\n\n2.2 Inhibitors targeting the cellular receptor\nThe genetic code of SARS‐CoV‐2 shares noticeable similarities with SARS‐CoV‐1, which caused the SARS epidemic in 2002. 26 , 54 More importantly, both viruses have identical mechanisms of infection. SARS‐CoV‐1 uses the host's ACE2 as a portal to infect cells, which has high expression in the vascular endothelium 55 and the lung, particularly in type 2 alveolar epithelial cells. 56 SARS‐CoV‐2 shares 76% of its spike (S) protein with SARS‐CoV‐1. Despite a few amino acid differences in its RBD compared to the SARS‐CoV‐1 S protein, the SARS‐CoV‐2 S protein binds to ACE2 with even greater affinity 27 offering an explanation for its greater virulence and preference for the lung.\nACE, a highly glycosylated type I integral membrane protein, is an essential component of the renin‐angiotensin (Ang) system, which controls blood pressure homeostasis. Both ACE1 and ACE2 cleave Ang peptides. However, they differ markedly: ACE1 cuts and converts the inactive decapeptide Ang I into the octapeptide Ang II by removing the dipeptide His‐Leu. This Ang II induces vaso‐ and bronchoconstriction, increased vascular permeability, inflammation, and fibrosis, thus promoting acute respiratory distress syndrome (ARDS) and lung failure in patients infected with SARS‐CoV‐1 or SARS‐CoV‐2 57 (Figure 5). Therefore, ACE‐inhibitors (ACEis) and angiotensin II receptor blockers (ARBs) could block the disease‐propagating effect of Ang II. 58 , 59 , 60\nFigure 5 The roles of ACE1 and 2 in the renin‐angiotensin system. A, Chemical structures of angiotensin‐related peptides and B, Schematic diagram of roles of ACE1 and 2 in renin‐angiotensin system. ACE, angiotensin‐converting enzyme [Color figure can be viewed at wileyonlinelibrary.com] ACE2, on the other hand, is a zinc‐containing metalloenzyme, and shares merely 42% of its amino acid sequence with ACE1. 61 It cleaves only one amino acid residue (Leu or Phe) from Ang I and Ang II, respectively, generating Ang (1–9) and Ang (1–7) (a vasodilator) (Figure 5). Thus, ACE2 has been considered a potential therapeutic target for cardiovascular diseases.\nVirtual screening combined with a molecular docking approach targeting the ACE2 catalytic site with around 140 000 compounds led to the identification of inhibitor N‐(2‐aminoethyl)‐1 aziridine‐ethanamine (7; Figure 6) with an IC50 value of 57 µM and a K i of 459 µM. However, no information about the cytotoxicity of this compound is available so far. 62\nFigure 6 Inhibitors for SARS‐CoV‐1 and 2 targeting ACE2. ACE, angiotensin‐converting enzyme; SARS‐CoV, severe acute respiratory syndrome coronavirus Chloroquine (8; Figure 6) is a relatively safe, cheap, and effective medication for the treatment of malaria and amebiasis. Savarino et al. 63 reported its antiviral effects. At a molecular level, it increases late endosomal and lysosomal pH, resulting in impaired liberation of virions from endosomes or lysosomes. The virus is therefore unable to release its genetic material into the cell and replicate. 64 , 65 Furthermore, they hypothesize that chloroquine might block the production of proinflammatory cytokines (such as IL‐6), thereby blocking the pathway that subsequently leads to ARDS. 63\nChloroquine is reasonably active in vitro against SARS‐CoV‐1, MERS‐CoV, and SARS‐CoV‐2. It was found to inhibit SARS‐CoV‐2 with an EC50 value of 5.47 µM in vitro. 66 Antiviral activity against SARS‐CoV‐1 was reported with an IC50 of 8.8 μM in Vero cells, but it is unclear how this translates into activity in respiratory epithelial cells and in vivo. 67 , 68 Mechanistic studies of chloroquine for SARS‐CoV‐1 infection revealed that it could also weaken the interaction between the RBD of SARS‐CoV‐1 and ACE2 by interfering with terminal glycosylation of ACE2, thereby reducing its affinity to SARS‐CoV‐1 S. 69\nDuring the SARS‐CoV‐2 pandemic, chloroquine has been recommended by Chinese, South Korean, and Italian health authorities for the experimental treatment of COVID‐19, 70 , 71 despite contraindications for patients with heart disease or diabetes. 72 However, health experts and agencies like the US FDA and European Medicines Agency warned against broad uncontrolled use after reports of misuse of low‐quality versions of chloroquine phosphate intended for fish.\nHydroxychloroquine (9; Figure 6) is being studied as an experimental treatment for COVID‐19. 73 However, the benefits of treatment with this drug are unclear. 74\nHydroxychloroquine was found to inhibit SARS‐CoV‐2 with an EC50 value of 0.74 µM in vitro. 66 Some studies imply synergistic effects of hydroxychloroquine and azithromycin. Azithromycin is active in vitro against Zika and Ebola virus 75 , 76 and can be used to guard against life‐threatening bacterial superinfections when administered to patients suffering from viral infections. 77 A small study that compared hydroxychloroquine monotherapy and combination treatment with azithromycin found a significant advantage of the combination. While evaluating the efficacy of therapeutic intervention with hydroxychloroquine as monotherapy and its impact in combination with azithromycin, the number of patients testing negative in polymerase chain reaction (PCR) tests was substantially different in the two groups with 100% of patients cured (6 days post inclusion) in the combination arm of the study versus 57% in the monotherapy group. At the same time, 12% of patients in the control group receiving only standard care were cured. 78 , 79\nThe WHO declared on 18 March that chloroquine and its derivative hydroxychloroquine will be among the four medicines studied in the solidarity clinical trial 80 for the treatment of COVID‐19. In April 2020, the US National Institutes of Health (NIH) also commenced a study with the drug for treating COVID‐19 patients. 81\nThe recent clinical trial involving 96 032 patients with COVID‐19 concluded that it was unable to confirm a benefit of hydroxychloroquine or chloroquine, when used alone or in combination with a macrolide such as azithromycin (or clarithromycin). 82 The study actually reported decreased survival rates for patients treated with each of these drug regimens. Additionally, patients had an increased risk of developing ventricular arrhythmia under treatment. However, still more evidence is needed to adequately assess the drugs' risks or benefits for the treatment or prevention of COVID‐19 (it is important to note that chloroquine and hydroxychloroquine are still considered safe treatment options in certain autoimmune diseases and malaria). Besides, the WHO announced the premature pause of its clinical trials using hydroxychloroquine as a safety precaution on 24 May 2020.\nOn a different note, it was found that ACE2 undergoes proteolytic shedding; releasing an enzymatic ectodomain during viral entry. 83 A disintegrin and metalloproteinase (ADAM), also known as TNF‐α converting enzyme (TACE), assisted the shedding regulation of ACE2. Inhibition of this enzyme led to reduced shedding of ACE2. GW280264X (10; Figure 6) was found to be a specific inhibitor of ADAM‐induced shedding of ACE2 at 1 nM. 84 Two TACE inhibitors, TAPI‐0 (11) and TAPI‐2 (12; Figure 6), reduced ACE2 shedding, with IC50 values of 100 and 200 nM, respectively. 83\nMLN‐4760 (13; Figure 6) inhibited the catalytic activity of ACE2 with an IC50 of around 440 pM. 85 This is the most potent and selective small‐molecule inhibitor against soluble human ACE2 described to date, thus making it a very promising candidate for SARS‐CoV‐2 interference. It binds to the active site zinc and emulates the transition state peptide. However, no antiviral data for this compound is available at this time.\nThe interference of a virus‐host cell fusion, which is mediated by the viral S protein to its receptor ACE2 on host cells, may be a viable prevention strategy. Umifenovir (14; brand name Arbidol), a broad spectrum antiviral drug used against influenza, prevents viral entry by inhibiting virus‐host cell fusion. 86 It is currently being investigated in a clinical trial for the treatment of SARS‐CoV‐2. 87 , 88\nDo ACEis or ARBs amplify SARS‐CoV‐2 pathogenicity and aggravate the clinical course of COVID‐19? After ACE2 was recognized as the SARS‐CoV‐2 receptor, 14 , 29 speculations emerged about potentially negative consequences of ACEi or ARB therapy in COVID‐19 patients. This theory caused confusion in the public and alarmed patients taking these medicines. One report said that the expression of ACE2 was increased in patients with heart disease compared to healthy individuals. It was also insisted that ACE2 expression could be increased by taking ACEis and ARBs, 89 although there is no supporting report of this happening in the lungs.\nIn another report, it was suggested that patients suffering from high blood pressure receiving “ACE2‐increasing drugs” have a higher risk for severe COVID‐19, since ACEis and ARBs could elevate levels of ACE2. 90\nA joint declaration by the presidents of the HFSA/ACC/AHA on 17 March 2020, 91 followed by a similar statement of the European Medicines Agency, 92 clarified that there was no scientific basis for stopping ACEi or ARB therapy. 93 , 94 , 95 This was in accordance with the editors of the New England Journal of Medicine. 96\nIn case of SARS‐CoV, the experimental data showed that such medications may be beneficial rather than damaging, which led to a new therapeutic approach for lung diseases. 97\n\n2.3 Proteolytic processing inhibitors\nCoVs enter the host cells via both clathrin (endosomal) and nonclathrin pathways (nonendosomal); however, both pathways are dependent upon receptor binding. 98 , 99\nThe clathrin‐mediated pathway involves the binding of CoV S protein to the host receptor followed by the internalization of vesicles that maturate to late endosomes. Acidification of the endosome promotes the H+‐dependent activation of cellular cathepsin L proteinase in late endosomes and lysosomes, which cleaves and activates the S protein, thus initiating viral fusion. Recent research shows that in addition to ACE2 SARS‐CoV‐2 can also use the host cell receptor CD147 to gain access into host cells. 100\nMembrane fusion is also the crucial step for the CoV life cycle in the nonclathrin/endosomal route, in which host proteases such as cathepsin L, TMPRSS2, and TMPRSS11D (airway trypsin‐like protease) cut the S protein at the S1/S2 cleavage site to activate the S protein for membrane fusion. 101 Interference with this process by targeting these proteases could become an attractive strategy for combating CoV infections. A recent study confirms the role of TMPRSS2 for the viral life cycle in SARS‐CoV‐2‐infected VeroE6 cells. 5 Furin (a serine endoprotease) activates MERS‐CoV to initiate the nonclathrin mediated membrane fusion event. 102\nThe neurotransmitter receptor blockers chlorpromazine (15), promethazine (16), and fluphenazine (17; Figure 7), were reported to inhibit MERS‐CoV and SARS‐CoV‐1 most probably by impeding S protein‐induced fusion. 103 Chlorpromazine, a clathrin‐mediated viral entry inhibitor, was already described to inhibit human CoV‐229E, hepatitis C virus, infectious bronchitis virus, as well as mouse hepatitis virus‐2 (MHV2). 104 , 105 , 106 , 107 , 108\nFigure 7 Neurotransmitter inhibitors targeting clathrin/nonclathrin pathways Matsuyama et al. identified the commercially available serine protease inhibitor camostat (18; Figure 8) to be a SARS‐CoV‐1 inhibitor, blocking TMPRSS2 activity at 10 µM. However, at a higher concentration (100 µM), inhibition of viral entry via SARS‐CoV‐1 S protein‐mediated cell fusion never exceeded 65% (inhibition efficiency), indicating that 35% of entry events take place via the endosomal cathepsin pathway. Interestingly, treatment with a combination of EST (a cathepsin inhibitor) and 18 resulted in remarkably blocked infection (\u003e95%) activity of pseudotyped viruses. 109\nFigure 8 Inhibitors targeting TMPRSS2. TMPRSS, transmembrane serine protease A similar approach has been investigated to prevent viral entry of SARS‐CoV‐2. Pöhlmann et al. reported the attainment of full inhibition efficiency with a combination of both 18 and E‐64d (a cathepsin inhibitor). Both studies indicate that SARS‐CoV‐1 and 2 enter cells in a similar manner showing the potential of 18 as a candidate for further development. 15\nRecently, K11777 (19; Figure 8), a cysteine protease inhibitor, was shown in tissue cultures to inhibit SARS‐CoV‐1 and MERS‐CoV replication in the subnanomolar range. 110 , 111 Future tissue culture and animal model studies should be conducted to clarify, whether its antiviral activity is mediated by targeting TMPRSS2.\nTeicoplanin is a glycopeptide antibiotic used to prevent infections with Gram‐positive bacteria like methicillin‐resistant Staphylococcus aureus and Enterococcus faecalis. It was found that teicoplanin inhibits the entry of SARS‐CoV‐1, MERS‐CoV, and Ebola virus by specifically targeting cathepsin L. 112 This knowledge has also been used to block the entry of new SARS‐CoV‐2 pseudoviruses with an IC50 value of 1.66 µM. Therefore, teicoplanin could be considered a potential candidate for the treatment of COVID‐19. 113\n\n2.4 Small‐molecules as cathepsin L inhibitors\nHuman cathepsin L is a cysteine endopeptidase and plays a key role for infection efficiency by activation of the S protein into a fusogenic state to escape the late endosomes. Targeting this protease with small molecules could interfere with virus‐cell entry and therefore be a possible intervention strategy for CoV infection. 114 Bates et al. identified MDL28170 (20; Figure 9) as an antiviral compound that specifically inhibited cathepsin‐L‐mediated substrate cleavage, with an IC50 value of 2.5 nM and EC50 value in the range of 100 nM. However, despite its potent inhibitory activity, no cytotoxicity data for 20 is currently available. 115\nFigure 9 Cathepsin L inhibitors with antiviral activity Diamond et al. reported CID 16725315 (21) and CID 23631927 (22; Figure 9) as viral entry inhibitors of SARS‐CoV in a cathepsin L inhibition assay. Compound 21 could block cathepsin L with an IC50 value of 6.9 nM, while 22 showed slightly weaker potency (IC50, 56 nM). Compound 22 was also found to inhibit Ebola virus infection (EC50, 193 nM) of human embryonic kidney 293T cells. This compound did not show any sign of toxicity to human aortic endothelial cells up to 100 µM. This data offers a new promising point for the treatment of SARS and Ebola virus infections. 116\nScreening of ~14 000 compounds in a cell‐based assay resulted in the identification of SSAA09E1 (23; Figure 9) as inhibitor of cathepsin L proteinase, with an IC50 value of 5.33 µM. In a pseudotype‐based assay in 293T cells, the EC50 value of 23 was around 6.4 µM, and no cytotoxicity was detected below 100 µM. 48\nPhenotypic screening approaches led to the identification of several viral entry inhibitors. This approach has the advantage of finding cellular‐active compounds, providing information on drug solubility and cell uptake. 117 On the other hand, it is limited in terms of capacity compared to in silico target‐based screening. Hsiang et al. identified emodin (24; Figure 9), the active component from Polygonum multiflorum and Rheum officinale, could block the interaction of S protein with ACE2, with an IC50 value of 10 µM and an EC50 value of 200 µM in an S protein‐pseudotyped retrovirus assay using Vero E6 cells. However, the mechanism of action of this compound still needs to be determined. 118 Sarafianos et al. 48 found that SSAA09E3 (25), a benzamide derivative of 24, could prevent virus‐cell membrane fusion in pseudotype‐based and antiviral‐based assays, with an EC50 value of 9.7 µM, but a CC50 value of 20 µM indicates additional unknown cellular targets.\nVE607 (26) was identified among 50 240 structurally diverse small molecules to specifically inhibit SARS‐CoV‐1 entry into cells using a phenotype‐based screening. Its EC50 value was reported at 3.0 µM and it inhibited SARS‐CoV‐1 plaque formation with an EC50 of 1.6 µM. 119 Cathepsin inhibitor E‐64‐D (27) blocked MERS‐CoV and SARS‐CoV‐1 infection as well. 120 , 121"}

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VIRUS ENTRY INHIBITORS\nBinding of spike protein (S) to its receptor represents the host's first confrontation with the virus and its life cycle, thus providing prophylactic intervention opportunities. The genome of SARS‐CoV‐2 has been recently determined to have an 80% identity to that of SARS‐CoV‐1 and 96% identity to the bat‐CoV RaTG13. 26 The SARS‐CoV‐2 S protein shows nucleotide sequence identities of 75% or less to all other previously described CoVs. However, again, the new SARS‐CoV‐2 S protein shares a 93.1% identity to the S protein of RaTG13. SARS‐CoV‐2 spike (S) recognizes, with its receptor‐binding domain (RBD), the cellular ACE2 receptor with high affinity (K d, 14.7 nM) 27 as judged by surface plasmin resonance spectrometry; and intervention at the RBD‐ACE2 interface can potentially disrupt infection efficiency. It was observed that the RBDs of the SARS‐CoV‐2‐ACE2 and SARS‐CoV‐1‐ACE2 complexes are quite similar (Figure 3). 29\nFigure 3 Comparison of the SARS‐CoV‐2 S and SARS‐CoV‐1 S structures: ribbon diagrams of the (A) SARS‐CoV‐2 S and (D) SARS‐CoV‐1 S [PDB 6NB6] ectodomain cryo‐EM structures. S1 subunits of (B) SARS‐CoV‐2 S and (E) SARS‐CoV‐1 S. S2 subunits of (C) SARS‐CoV‐2 S and (F) SARS‐CoV‐1 S. 28 cryo‐EM, cryogenic electron microscopy; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2 [Color figure can be viewed at wileyonlinelibrary.com] As mentioned previously, Zhang et al. 30 determined the full‐length genome sequence of SARS‐CoV‐2 and revealed that the virus is remarkably similar (89.1% sequence identity) to a group of SARS‐like CoVs. Simultaneously, Shi et al. 26 reported that SARS‐CoV‐2 shares 96% sequence identity at a whole‐genome level with a bat coronavirus—and importantly, like SARS‐CoV‐1, SARS‐CoV‐2 utilizes ACE2 receptor for viral entry. Recently, Yan et al. solved the cryo‐EM structure of full‐length human ACE2 bound to the RBD of SARS‐CoV‐2, providing important structural information for therapeutic intervention strategies. 29\nThe sequence identity of the spike protein between SARS‐CoV‐1 (1273 aa) and SARS‐CoV‐2 (1253 aa) is 76%. The spike protein has two regions, S1 and S2. The S1 region of the SARS‐CoV‐1 has a RBD that forms high‐affinity interactions with ACE2. The prevailing understanding is that SARS‐CoV‐2 employs this RBD to enter its human host cell as well. Aligning the two different RBDs revealed a sequence identity of 73.5%. However, many nonconserved mutations that interact directly with ACE2 are located in the two structural regions. 31 And both crystal and cryo‐EM structures of the SARS‐CoV‐1 spike‐ACE2 complex have shown that merely residues of regions 1 and 2 form hydrogen bonds and hydrophobic interactions with ACE2. The mutations in these two regions of SARS‐CoV‐2 will, therefore, likely reduce the number of those interactions. 32\nStudies also have shown that the RdRP, and the Mpro are highly conserved between SARS‐CoV‐2 and SARS‐CoV‐1. 33 , 34 Therefore, it is widely accepted that SARS‐CoV‐2 behaves similarly to SARS‐CoV‐1 with regard to viral entry and replication. Since the general genomic layout and replication kinetics are so conserved among MERS, SARS‐1, and SARS‐2 CoVs, investigating inhibitors of common structures is a logical step.\nThe inhibitory strength against viral enzyme was expressed as IC50, which is the concentration of the inhibitor needed to inhibit half of the enzyme activity in the tested condition. The K i value is reflective of ligand‐binding affinity to the enzyme. The inhibitory activity for cell‐based bioassays is expressed as EC50, which is a half maximal effective concentration required to induce the biological response. The warhead group means a “reactive group” of the inhibitors that can form both covalent and noncovalent interactions with amino acids in the active site of the enzyme.\n\n2.1 Targeting the RBD\nStructural investigations of the RBD‐ACE2 complex provided information about essential residues for viral entry. Hsiang et al. 35 reported a number of peptides that significantly blocked the interaction of the S protein with ACE2 with IC50 values as low as 1.88 nM. Michael et al. found charged residues between positions 22 and 57 crucial for SARS‐CoV‐1 viral entry. Based on this, they designed peptides P4 (IC50, 50 µM) and P5 (IC50, 6.0 µM) with significant inhibitory activity against SARS‐CoV‐1. The antiviral activity was further improved when they introduced the glycine binding linkage of peptide P4 (residues 22–47) with an ACE2‐derived peptide (residues 351–357) against a SARS‐CoV‐1 pseudovirus with an IC50 of 100 nM and devoid of cytotoxicity up to 200 µM. 36 It is worth highlighting that a similar strategy could work for the new SARS‐CoV‐2. The recently solved cryo‐EM structure of SARS‐CoV‐2 in complex with human ACE2 can provide a structural rationale for the peptide design. 29\nFor viral entry, MERS‐CoV uses its spike protein (S) to interact with the host‐receptor DPP4, 37 , 38 , 39 also known as adenosine deaminase‐complexing protein‐2 or CD26. 37 MERS‐CoV was also the first virus reported to use this particular path. 35 , 37 DPP4 is a type II transmembrane glycoprotein, that forms homodimers on the cell surface, and it is involved in the cleavage of dipeptides. 37 , 40 In humans, DPP4 is predominantly found on the bronchial epithelial and alveolar cells in the lower lungs. 40 , 41\nMERS‐4 and MERS‐27 are monoclonal antibodies targeting the RBD of MERS‐CoV S that were discovered in a nonimmune yeast‐display scFv library screening. The more active MERS‐4 potently blocked the infection of DPP4‐expressing Huh‐7 cells with pseudotyped MERS‐CoV (IC50, 0.056 μg/mL). It also prevented MERS‐CoV‐induced cytopathogenic effects in MERS‐infected Vero E6 cells (IC50, 0.5 μg/mL). 42\nA heptad repeat (HR) is a repeating structural pattern of seven amino acids. A crucial membrane fusion framework of SARS‐CoV is the 6‐helix‐bundle (6‐HB) that is formed by HR1 and HR2 of the viral S protein. Enfuvirtide (T‐20) is an FDA approved HR2 peptide and the first HIV fusion inhibitor. It has opened up new avenues toward identifying and developing peptides as viral entry inhibitors. Such molecules represent a promising strategy against enveloped viruses with class 1 fusion proteins such as Nipah virus, Hendra virus, Ebola virus, and other paramyxoviruses, simian immunodeficiency virus, feline immunodeficiency virus, and respiratory syncytial virus. 43 , 44 , 45 , 46 The HR regions of SARS‐CoV‐1 and SARS‐CoV‐2 S protein share a high degree of conservation, and such fusion inhibitors have potential applications in preventing SARS‐CoV‐2 entry.\nSmall molecule entry inhibitors, on the other hand, are reported to target the RBD. Compared to peptides, proteins, and biologics, small molecules have several advantages due to lower production costs, improved pharmacokinetics, stability, and dosage accuracy. Sarafianos et al. identified the oxazole‐carboxamide derivative SSAA09E2 (1; Figure 4) as an entry inhibitor against SARS‐CoV‐1 by screening a chemical library composed of 3000 compounds. 47 This inhibitor directly blocks ACE2 recognition by interfering with the RBD with an EC50 value of 3.1 µM and a 50% cytotoxic concentration (CC50) value of greater than 100 µM, not affecting ACE2 expression levels. 48\nFigure 4 Inhibitors targeting the receptor‐binding domain Xu et al. 49 identified two small molecules, TGG (2; Figure 4) and luteolin (3; Figure 4), that can bind avidly to the SARS‐CoV‐1 S2 protein and inhibit its entry into Vero E6 cells (EC50: 4.5 µM, 10.6 µM; respectively). Compounds 2 and 3 showed cytotoxicity (CC50) of 1.08 and 0.155 mM, and the selectivity index (SI) values of 2 and 3 were 240.0 and 14.62, respectively. Further studies regarding acute toxicity revealed that the 50% lethal doses of 2 and 3 were ~456 and 232 mg/kg, respectively. These results indicate that these small molecules could be used at relatively high concentrations in mice. 49 Quercetin (4; Figure 4), an analog of 3, also showed antiviral activity against SARS‐CoV‐1, with an EC50 value of 83.4 µM and a CC50 value of 3.32 mM. 50\nNgai et al. reported ADS‐J1 (5; Figure 4) as a potential SARS‐CoV‐1 viral entry inhibitor with an EC50 of 3.89 µM. Molecular docking studies predicted that 5 can bind into a deep pocket of the SARS‐CoV‐1 S HR region and block viral entry into host cells. 51 Imatinib (6; Figure 4), an Abelson kinase inhibitor, could inhibit CoV S protein‐induced fusion with an EC50 value of 10 µM and showed no cytotoxic effects in Vero cells up to 100 µM concentration. 52 , 53\n\n2.2 Inhibitors targeting the cellular receptor\nThe genetic code of SARS‐CoV‐2 shares noticeable similarities with SARS‐CoV‐1, which caused the SARS epidemic in 2002. 26 , 54 More importantly, both viruses have identical mechanisms of infection. SARS‐CoV‐1 uses the host's ACE2 as a portal to infect cells, which has high expression in the vascular endothelium 55 and the lung, particularly in type 2 alveolar epithelial cells. 56 SARS‐CoV‐2 shares 76% of its spike (S) protein with SARS‐CoV‐1. Despite a few amino acid differences in its RBD compared to the SARS‐CoV‐1 S protein, the SARS‐CoV‐2 S protein binds to ACE2 with even greater affinity 27 offering an explanation for its greater virulence and preference for the lung.\nACE, a highly glycosylated type I integral membrane protein, is an essential component of the renin‐angiotensin (Ang) system, which controls blood pressure homeostasis. Both ACE1 and ACE2 cleave Ang peptides. However, they differ markedly: ACE1 cuts and converts the inactive decapeptide Ang I into the octapeptide Ang II by removing the dipeptide His‐Leu. This Ang II induces vaso‐ and bronchoconstriction, increased vascular permeability, inflammation, and fibrosis, thus promoting acute respiratory distress syndrome (ARDS) and lung failure in patients infected with SARS‐CoV‐1 or SARS‐CoV‐2 57 (Figure 5). Therefore, ACE‐inhibitors (ACEis) and angiotensin II receptor blockers (ARBs) could block the disease‐propagating effect of Ang II. 58 , 59 , 60\nFigure 5 The roles of ACE1 and 2 in the renin‐angiotensin system. A, Chemical structures of angiotensin‐related peptides and B, Schematic diagram of roles of ACE1 and 2 in renin‐angiotensin system. ACE, angiotensin‐converting enzyme [Color figure can be viewed at wileyonlinelibrary.com] ACE2, on the other hand, is a zinc‐containing metalloenzyme, and shares merely 42% of its amino acid sequence with ACE1. 61 It cleaves only one amino acid residue (Leu or Phe) from Ang I and Ang II, respectively, generating Ang (1–9) and Ang (1–7) (a vasodilator) (Figure 5). Thus, ACE2 has been considered a potential therapeutic target for cardiovascular diseases.\nVirtual screening combined with a molecular docking approach targeting the ACE2 catalytic site with around 140 000 compounds led to the identification of inhibitor N‐(2‐aminoethyl)‐1 aziridine‐ethanamine (7; Figure 6) with an IC50 value of 57 µM and a K i of 459 µM. However, no information about the cytotoxicity of this compound is available so far. 62\nFigure 6 Inhibitors for SARS‐CoV‐1 and 2 targeting ACE2. ACE, angiotensin‐converting enzyme; SARS‐CoV, severe acute respiratory syndrome coronavirus Chloroquine (8; Figure 6) is a relatively safe, cheap, and effective medication for the treatment of malaria and amebiasis. Savarino et al. 63 reported its antiviral effects. At a molecular level, it increases late endosomal and lysosomal pH, resulting in impaired liberation of virions from endosomes or lysosomes. The virus is therefore unable to release its genetic material into the cell and replicate. 64 , 65 Furthermore, they hypothesize that chloroquine might block the production of proinflammatory cytokines (such as IL‐6), thereby blocking the pathway that subsequently leads to ARDS. 63\nChloroquine is reasonably active in vitro against SARS‐CoV‐1, MERS‐CoV, and SARS‐CoV‐2. It was found to inhibit SARS‐CoV‐2 with an EC50 value of 5.47 µM in vitro. 66 Antiviral activity against SARS‐CoV‐1 was reported with an IC50 of 8.8 μM in Vero cells, but it is unclear how this translates into activity in respiratory epithelial cells and in vivo. 67 , 68 Mechanistic studies of chloroquine for SARS‐CoV‐1 infection revealed that it could also weaken the interaction between the RBD of SARS‐CoV‐1 and ACE2 by interfering with terminal glycosylation of ACE2, thereby reducing its affinity to SARS‐CoV‐1 S. 69\nDuring the SARS‐CoV‐2 pandemic, chloroquine has been recommended by Chinese, South Korean, and Italian health authorities for the experimental treatment of COVID‐19, 70 , 71 despite contraindications for patients with heart disease or diabetes. 72 However, health experts and agencies like the US FDA and European Medicines Agency warned against broad uncontrolled use after reports of misuse of low‐quality versions of chloroquine phosphate intended for fish.\nHydroxychloroquine (9; Figure 6) is being studied as an experimental treatment for COVID‐19. 73 However, the benefits of treatment with this drug are unclear. 74\nHydroxychloroquine was found to inhibit SARS‐CoV‐2 with an EC50 value of 0.74 µM in vitro. 66 Some studies imply synergistic effects of hydroxychloroquine and azithromycin. Azithromycin is active in vitro against Zika and Ebola virus 75 , 76 and can be used to guard against life‐threatening bacterial superinfections when administered to patients suffering from viral infections. 77 A small study that compared hydroxychloroquine monotherapy and combination treatment with azithromycin found a significant advantage of the combination. While evaluating the efficacy of therapeutic intervention with hydroxychloroquine as monotherapy and its impact in combination with azithromycin, the number of patients testing negative in polymerase chain reaction (PCR) tests was substantially different in the two groups with 100% of patients cured (6 days post inclusion) in the combination arm of the study versus 57% in the monotherapy group. At the same time, 12% of patients in the control group receiving only standard care were cured. 78 , 79\nThe WHO declared on 18 March that chloroquine and its derivative hydroxychloroquine will be among the four medicines studied in the solidarity clinical trial 80 for the treatment of COVID‐19. In April 2020, the US National Institutes of Health (NIH) also commenced a study with the drug for treating COVID‐19 patients. 81\nThe recent clinical trial involving 96 032 patients with COVID‐19 concluded that it was unable to confirm a benefit of hydroxychloroquine or chloroquine, when used alone or in combination with a macrolide such as azithromycin (or clarithromycin). 82 The study actually reported decreased survival rates for patients treated with each of these drug regimens. Additionally, patients had an increased risk of developing ventricular arrhythmia under treatment. However, still more evidence is needed to adequately assess the drugs' risks or benefits for the treatment or prevention of COVID‐19 (it is important to note that chloroquine and hydroxychloroquine are still considered safe treatment options in certain autoimmune diseases and malaria). Besides, the WHO announced the premature pause of its clinical trials using hydroxychloroquine as a safety precaution on 24 May 2020.\nOn a different note, it was found that ACE2 undergoes proteolytic shedding; releasing an enzymatic ectodomain during viral entry. 83 A disintegrin and metalloproteinase (ADAM), also known as TNF‐α converting enzyme (TACE), assisted the shedding regulation of ACE2. Inhibition of this enzyme led to reduced shedding of ACE2. GW280264X (10; Figure 6) was found to be a specific inhibitor of ADAM‐induced shedding of ACE2 at 1 nM. 84 Two TACE inhibitors, TAPI‐0 (11) and TAPI‐2 (12; Figure 6), reduced ACE2 shedding, with IC50 values of 100 and 200 nM, respectively. 83\nMLN‐4760 (13; Figure 6) inhibited the catalytic activity of ACE2 with an IC50 of around 440 pM. 85 This is the most potent and selective small‐molecule inhibitor against soluble human ACE2 described to date, thus making it a very promising candidate for SARS‐CoV‐2 interference. It binds to the active site zinc and emulates the transition state peptide. However, no antiviral data for this compound is available at this time.\nThe interference of a virus‐host cell fusion, which is mediated by the viral S protein to its receptor ACE2 on host cells, may be a viable prevention strategy. Umifenovir (14; brand name Arbidol), a broad spectrum antiviral drug used against influenza, prevents viral entry by inhibiting virus‐host cell fusion. 86 It is currently being investigated in a clinical trial for the treatment of SARS‐CoV‐2. 87 , 88\nDo ACEis or ARBs amplify SARS‐CoV‐2 pathogenicity and aggravate the clinical course of COVID‐19? After ACE2 was recognized as the SARS‐CoV‐2 receptor, 14 , 29 speculations emerged about potentially negative consequences of ACEi or ARB therapy in COVID‐19 patients. This theory caused confusion in the public and alarmed patients taking these medicines. One report said that the expression of ACE2 was increased in patients with heart disease compared to healthy individuals. It was also insisted that ACE2 expression could be increased by taking ACEis and ARBs, 89 although there is no supporting report of this happening in the lungs.\nIn another report, it was suggested that patients suffering from high blood pressure receiving “ACE2‐increasing drugs” have a higher risk for severe COVID‐19, since ACEis and ARBs could elevate levels of ACE2. 90\nA joint declaration by the presidents of the HFSA/ACC/AHA on 17 March 2020, 91 followed by a similar statement of the European Medicines Agency, 92 clarified that there was no scientific basis for stopping ACEi or ARB therapy. 93 , 94 , 95 This was in accordance with the editors of the New England Journal of Medicine. 96\nIn case of SARS‐CoV, the experimental data showed that such medications may be beneficial rather than damaging, which led to a new therapeutic approach for lung diseases. 97\n\n2.3 Proteolytic processing inhibitors\nCoVs enter the host cells via both clathrin (endosomal) and nonclathrin pathways (nonendosomal); however, both pathways are dependent upon receptor binding. 98 , 99\nThe clathrin‐mediated pathway involves the binding of CoV S protein to the host receptor followed by the internalization of vesicles that maturate to late endosomes. Acidification of the endosome promotes the H+‐dependent activation of cellular cathepsin L proteinase in late endosomes and lysosomes, which cleaves and activates the S protein, thus initiating viral fusion. Recent research shows that in addition to ACE2 SARS‐CoV‐2 can also use the host cell receptor CD147 to gain access into host cells. 100\nMembrane fusion is also the crucial step for the CoV life cycle in the nonclathrin/endosomal route, in which host proteases such as cathepsin L, TMPRSS2, and TMPRSS11D (airway trypsin‐like protease) cut the S protein at the S1/S2 cleavage site to activate the S protein for membrane fusion. 101 Interference with this process by targeting these proteases could become an attractive strategy for combating CoV infections. A recent study confirms the role of TMPRSS2 for the viral life cycle in SARS‐CoV‐2‐infected VeroE6 cells. 5 Furin (a serine endoprotease) activates MERS‐CoV to initiate the nonclathrin mediated membrane fusion event. 102\nThe neurotransmitter receptor blockers chlorpromazine (15), promethazine (16), and fluphenazine (17; Figure 7), were reported to inhibit MERS‐CoV and SARS‐CoV‐1 most probably by impeding S protein‐induced fusion. 103 Chlorpromazine, a clathrin‐mediated viral entry inhibitor, was already described to inhibit human CoV‐229E, hepatitis C virus, infectious bronchitis virus, as well as mouse hepatitis virus‐2 (MHV2). 104 , 105 , 106 , 107 , 108\nFigure 7 Neurotransmitter inhibitors targeting clathrin/nonclathrin pathways Matsuyama et al. identified the commercially available serine protease inhibitor camostat (18; Figure 8) to be a SARS‐CoV‐1 inhibitor, blocking TMPRSS2 activity at 10 µM. However, at a higher concentration (100 µM), inhibition of viral entry via SARS‐CoV‐1 S protein‐mediated cell fusion never exceeded 65% (inhibition efficiency), indicating that 35% of entry events take place via the endosomal cathepsin pathway. Interestingly, treatment with a combination of EST (a cathepsin inhibitor) and 18 resulted in remarkably blocked infection (\u003e95%) activity of pseudotyped viruses. 109\nFigure 8 Inhibitors targeting TMPRSS2. TMPRSS, transmembrane serine protease A similar approach has been investigated to prevent viral entry of SARS‐CoV‐2. Pöhlmann et al. reported the attainment of full inhibition efficiency with a combination of both 18 and E‐64d (a cathepsin inhibitor). Both studies indicate that SARS‐CoV‐1 and 2 enter cells in a similar manner showing the potential of 18 as a candidate for further development. 15\nRecently, K11777 (19; Figure 8), a cysteine protease inhibitor, was shown in tissue cultures to inhibit SARS‐CoV‐1 and MERS‐CoV replication in the subnanomolar range. 110 , 111 Future tissue culture and animal model studies should be conducted to clarify, whether its antiviral activity is mediated by targeting TMPRSS2.\nTeicoplanin is a glycopeptide antibiotic used to prevent infections with Gram‐positive bacteria like methicillin‐resistant Staphylococcus aureus and Enterococcus faecalis. It was found that teicoplanin inhibits the entry of SARS‐CoV‐1, MERS‐CoV, and Ebola virus by specifically targeting cathepsin L. 112 This knowledge has also been used to block the entry of new SARS‐CoV‐2 pseudoviruses with an IC50 value of 1.66 µM. Therefore, teicoplanin could be considered a potential candidate for the treatment of COVID‐19. 113\n\n2.4 Small‐molecules as cathepsin L inhibitors\nHuman cathepsin L is a cysteine endopeptidase and plays a key role for infection efficiency by activation of the S protein into a fusogenic state to escape the late endosomes. Targeting this protease with small molecules could interfere with virus‐cell entry and therefore be a possible intervention strategy for CoV infection. 114 Bates et al. identified MDL28170 (20; Figure 9) as an antiviral compound that specifically inhibited cathepsin‐L‐mediated substrate cleavage, with an IC50 value of 2.5 nM and EC50 value in the range of 100 nM. However, despite its potent inhibitory activity, no cytotoxicity data for 20 is currently available. 115\nFigure 9 Cathepsin L inhibitors with antiviral activity Diamond et al. reported CID 16725315 (21) and CID 23631927 (22; Figure 9) as viral entry inhibitors of SARS‐CoV in a cathepsin L inhibition assay. Compound 21 could block cathepsin L with an IC50 value of 6.9 nM, while 22 showed slightly weaker potency (IC50, 56 nM). Compound 22 was also found to inhibit Ebola virus infection (EC50, 193 nM) of human embryonic kidney 293T cells. This compound did not show any sign of toxicity to human aortic endothelial cells up to 100 µM. This data offers a new promising point for the treatment of SARS and Ebola virus infections. 116\nScreening of ~14 000 compounds in a cell‐based assay resulted in the identification of SSAA09E1 (23; Figure 9) as inhibitor of cathepsin L proteinase, with an IC50 value of 5.33 µM. In a pseudotype‐based assay in 293T cells, the EC50 value of 23 was around 6.4 µM, and no cytotoxicity was detected below 100 µM. 48\nPhenotypic screening approaches led to the identification of several viral entry inhibitors. This approach has the advantage of finding cellular‐active compounds, providing information on drug solubility and cell uptake. 117 On the other hand, it is limited in terms of capacity compared to in silico target‐based screening. Hsiang et al. identified emodin (24; Figure 9), the active component from Polygonum multiflorum and Rheum officinale, could block the interaction of S protein with ACE2, with an IC50 value of 10 µM and an EC50 value of 200 µM in an S protein‐pseudotyped retrovirus assay using Vero E6 cells. However, the mechanism of action of this compound still needs to be determined. 118 Sarafianos et al. 48 found that SSAA09E3 (25), a benzamide derivative of 24, could prevent virus‐cell membrane fusion in pseudotype‐based and antiviral‐based assays, with an EC50 value of 9.7 µM, but a CC50 value of 20 µM indicates additional unknown cellular targets.\nVE607 (26) was identified among 50 240 structurally diverse small molecules to specifically inhibit SARS‐CoV‐1 entry into cells using a phenotype‐based screening. Its EC50 value was reported at 3.0 µM and it inhibited SARS‐CoV‐1 plaque formation with an EC50 of 1.6 µM. 119 Cathepsin inhibitor E‐64‐D (27) blocked MERS‐CoV and SARS‐CoV‐1 infection as well. 120 , 121"}

{"project":"LitCovid-PD-GO-BP","denotations":[{"id":"T13","span":{"begin":2886,"end":2890},"obj":"http://purl.obolibrary.org/obo/GO_0003968"},{"id":"T14","span":{"begin":5888,"end":5903},"obj":"http://purl.obolibrary.org/obo/GO_0061025"},{"id":"T15","span":{"begin":8385,"end":8412},"obj":"http://purl.obolibrary.org/obo/GO_0046718"},{"id":"T16","span":{"begin":8391,"end":8406},"obj":"http://purl.obolibrary.org/obo/GO_0044409"},{"id":"T17","span":{"begin":8453,"end":8469},"obj":"http://purl.obolibrary.org/obo/GO_0033673"},{"id":"T18","span":{"begin":9321,"end":9330},"obj":"http://purl.obolibrary.org/obo/GO_0016032"},{"id":"T19","span":{"begin":9321,"end":9330},"obj":"http://purl.obolibrary.org/obo/GO_0009405"},{"id":"T20","span":{"begin":9501,"end":9527},"obj":"http://purl.obolibrary.org/obo/GO_0008217"},{"id":"T21","span":{"begin":9516,"end":9527},"obj":"http://purl.obolibrary.org/obo/GO_0042592"},{"id":"T22","span":{"begin":9801,"end":9813},"obj":"http://purl.obolibrary.org/obo/GO_0006954"},{"id":"T23","span":{"begin":12415,"end":12437},"obj":"http://purl.obolibrary.org/obo/GO_0033578"},{"id":"T24","span":{"begin":12424,"end":12437},"obj":"http://purl.obolibrary.org/obo/GO_0070085"},{"id":"T25","span":{"begin":13490,"end":13506},"obj":"http://purl.obolibrary.org/obo/GO_0016032"},{"id":"T26","span":{"begin":15616,"end":15626},"obj":"http://purl.obolibrary.org/obo/GO_0065007"},{"id":"T27","span":{"begin":16400,"end":16411},"obj":"http://purl.obolibrary.org/obo/GO_0140253"},{"id":"T28","span":{"begin":16400,"end":16411},"obj":"http://purl.obolibrary.org/obo/GO_0045026"},{"id":"T29","span":{"begin":16400,"end":16411},"obj":"http://purl.obolibrary.org/obo/GO_0000768"},{"id":"T30","span":{"begin":16400,"end":16411},"obj":"http://purl.obolibrary.org/obo/GO_0000747"},{"id":"T31","span":{"begin":16666,"end":16677},"obj":"http://purl.obolibrary.org/obo/GO_0140253"},{"id":"T32","span":{"begin":16666,"end":16677},"obj":"http://purl.obolibrary.org/obo/GO_0045026"},{"id":"T33","span":{"begin":16666,"end":16677},"obj":"http://purl.obolibrary.org/obo/GO_0000768"},{"id":"T34","span":{"begin":16666,"end":16677},"obj":"http://purl.obolibrary.org/obo/GO_0000747"},{"id":"T35","span":{"begin":18506,"end":18519},"obj":"http://purl.obolibrary.org/obo/GO_0045851"},{"id":"T36","span":{"begin":18597,"end":18607},"obj":"http://purl.obolibrary.org/obo/GO_0004175"},{"id":"T37","span":{"begin":18850,"end":18865},"obj":"http://purl.obolibrary.org/obo/GO_0061025"},{"id":"T38","span":{"begin":19124,"end":19139},"obj":"http://purl.obolibrary.org/obo/GO_0061025"},{"id":"T39","span":{"begin":19324,"end":19340},"obj":"http://purl.obolibrary.org/obo/GO_0019058"},{"id":"T40","span":{"begin":19467,"end":19482},"obj":"http://purl.obolibrary.org/obo/GO_0061025"},{"id":"T41","span":{"begin":20297,"end":20308},"obj":"http://purl.obolibrary.org/obo/GO_0140253"},{"id":"T42","span":{"begin":20297,"end":20308},"obj":"http://purl.obolibrary.org/obo/GO_0045026"},{"id":"T43","span":{"begin":20297,"end":20308},"obj":"http://purl.obolibrary.org/obo/GO_0000768"},{"id":"T44","span":{"begin":20297,"end":20308},"obj":"http://purl.obolibrary.org/obo/GO_0000747"},{"id":"T45","span":{"begin":23354,"end":23364},"obj":"http://purl.obolibrary.org/obo/GO_0004175"},{"id":"T46","span":{"begin":23743,"end":23749},"obj":"http://purl.obolibrary.org/obo/GO_0098739"},{"id":"T47","span":{"begin":23743,"end":23749},"obj":"http://purl.obolibrary.org/obo/GO_0098657"},{"id":"T48","span":{"begin":24336,"end":24351},"obj":"http://purl.obolibrary.org/obo/GO_0061025"},{"id":"T49","span":{"begin":24739,"end":24748},"obj":"http://purl.obolibrary.org/obo/GO_0009058"}],"text":"2 VIRUS ENTRY INHIBITORS\nBinding of spike protein (S) to its receptor represents the host's first confrontation with the virus and its life cycle, thus providing prophylactic intervention opportunities. The genome of SARS‐CoV‐2 has been recently determined to have an 80% identity to that of SARS‐CoV‐1 and 96% identity to the bat‐CoV RaTG13. 26 The SARS‐CoV‐2 S protein shows nucleotide sequence identities of 75% or less to all other previously described CoVs. However, again, the new SARS‐CoV‐2 S protein shares a 93.1% identity to the S protein of RaTG13. SARS‐CoV‐2 spike (S) recognizes, with its receptor‐binding domain (RBD), the cellular ACE2 receptor with high affinity (K d, 14.7 nM) 27 as judged by surface plasmin resonance spectrometry; and intervention at the RBD‐ACE2 interface can potentially disrupt infection efficiency. It was observed that the RBDs of the SARS‐CoV‐2‐ACE2 and SARS‐CoV‐1‐ACE2 complexes are quite similar (Figure 3). 29\nFigure 3 Comparison of the SARS‐CoV‐2 S and SARS‐CoV‐1 S structures: ribbon diagrams of the (A) SARS‐CoV‐2 S and (D) SARS‐CoV‐1 S [PDB 6NB6] ectodomain cryo‐EM structures. S1 subunits of (B) SARS‐CoV‐2 S and (E) SARS‐CoV‐1 S. S2 subunits of (C) SARS‐CoV‐2 S and (F) SARS‐CoV‐1 S. 28 cryo‐EM, cryogenic electron microscopy; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2 [Color figure can be viewed at wileyonlinelibrary.com] As mentioned previously, Zhang et al. 30 determined the full‐length genome sequence of SARS‐CoV‐2 and revealed that the virus is remarkably similar (89.1% sequence identity) to a group of SARS‐like CoVs. Simultaneously, Shi et al. 26 reported that SARS‐CoV‐2 shares 96% sequence identity at a whole‐genome level with a bat coronavirus—and importantly, like SARS‐CoV‐1, SARS‐CoV‐2 utilizes ACE2 receptor for viral entry. Recently, Yan et al. solved the cryo‐EM structure of full‐length human ACE2 bound to the RBD of SARS‐CoV‐2, providing important structural information for therapeutic intervention strategies. 29\nThe sequence identity of the spike protein between SARS‐CoV‐1 (1273 aa) and SARS‐CoV‐2 (1253 aa) is 76%. The spike protein has two regions, S1 and S2. The S1 region of the SARS‐CoV‐1 has a RBD that forms high‐affinity interactions with ACE2. The prevailing understanding is that SARS‐CoV‐2 employs this RBD to enter its human host cell as well. Aligning the two different RBDs revealed a sequence identity of 73.5%. However, many nonconserved mutations that interact directly with ACE2 are located in the two structural regions. 31 And both crystal and cryo‐EM structures of the SARS‐CoV‐1 spike‐ACE2 complex have shown that merely residues of regions 1 and 2 form hydrogen bonds and hydrophobic interactions with ACE2. The mutations in these two regions of SARS‐CoV‐2 will, therefore, likely reduce the number of those interactions. 32\nStudies also have shown that the RdRP, and the Mpro are highly conserved between SARS‐CoV‐2 and SARS‐CoV‐1. 33 , 34 Therefore, it is widely accepted that SARS‐CoV‐2 behaves similarly to SARS‐CoV‐1 with regard to viral entry and replication. Since the general genomic layout and replication kinetics are so conserved among MERS, SARS‐1, and SARS‐2 CoVs, investigating inhibitors of common structures is a logical step.\nThe inhibitory strength against viral enzyme was expressed as IC50, which is the concentration of the inhibitor needed to inhibit half of the enzyme activity in the tested condition. The K i value is reflective of ligand‐binding affinity to the enzyme. The inhibitory activity for cell‐based bioassays is expressed as EC50, which is a half maximal effective concentration required to induce the biological response. The warhead group means a “reactive group” of the inhibitors that can form both covalent and noncovalent interactions with amino acids in the active site of the enzyme.\n\n2.1 Targeting the RBD\nStructural investigations of the RBD‐ACE2 complex provided information about essential residues for viral entry. Hsiang et al. 35 reported a number of peptides that significantly blocked the interaction of the S protein with ACE2 with IC50 values as low as 1.88 nM. Michael et al. found charged residues between positions 22 and 57 crucial for SARS‐CoV‐1 viral entry. Based on this, they designed peptides P4 (IC50, 50 µM) and P5 (IC50, 6.0 µM) with significant inhibitory activity against SARS‐CoV‐1. The antiviral activity was further improved when they introduced the glycine binding linkage of peptide P4 (residues 22–47) with an ACE2‐derived peptide (residues 351–357) against a SARS‐CoV‐1 pseudovirus with an IC50 of 100 nM and devoid of cytotoxicity up to 200 µM. 36 It is worth highlighting that a similar strategy could work for the new SARS‐CoV‐2. The recently solved cryo‐EM structure of SARS‐CoV‐2 in complex with human ACE2 can provide a structural rationale for the peptide design. 29\nFor viral entry, MERS‐CoV uses its spike protein (S) to interact with the host‐receptor DPP4, 37 , 38 , 39 also known as adenosine deaminase‐complexing protein‐2 or CD26. 37 MERS‐CoV was also the first virus reported to use this particular path. 35 , 37 DPP4 is a type II transmembrane glycoprotein, that forms homodimers on the cell surface, and it is involved in the cleavage of dipeptides. 37 , 40 In humans, DPP4 is predominantly found on the bronchial epithelial and alveolar cells in the lower lungs. 40 , 41\nMERS‐4 and MERS‐27 are monoclonal antibodies targeting the RBD of MERS‐CoV S that were discovered in a nonimmune yeast‐display scFv library screening. The more active MERS‐4 potently blocked the infection of DPP4‐expressing Huh‐7 cells with pseudotyped MERS‐CoV (IC50, 0.056 μg/mL). It also prevented MERS‐CoV‐induced cytopathogenic effects in MERS‐infected Vero E6 cells (IC50, 0.5 μg/mL). 42\nA heptad repeat (HR) is a repeating structural pattern of seven amino acids. A crucial membrane fusion framework of SARS‐CoV is the 6‐helix‐bundle (6‐HB) that is formed by HR1 and HR2 of the viral S protein. Enfuvirtide (T‐20) is an FDA approved HR2 peptide and the first HIV fusion inhibitor. It has opened up new avenues toward identifying and developing peptides as viral entry inhibitors. Such molecules represent a promising strategy against enveloped viruses with class 1 fusion proteins such as Nipah virus, Hendra virus, Ebola virus, and other paramyxoviruses, simian immunodeficiency virus, feline immunodeficiency virus, and respiratory syncytial virus. 43 , 44 , 45 , 46 The HR regions of SARS‐CoV‐1 and SARS‐CoV‐2 S protein share a high degree of conservation, and such fusion inhibitors have potential applications in preventing SARS‐CoV‐2 entry.\nSmall molecule entry inhibitors, on the other hand, are reported to target the RBD. Compared to peptides, proteins, and biologics, small molecules have several advantages due to lower production costs, improved pharmacokinetics, stability, and dosage accuracy. Sarafianos et al. identified the oxazole‐carboxamide derivative SSAA09E2 (1; Figure 4) as an entry inhibitor against SARS‐CoV‐1 by screening a chemical library composed of 3000 compounds. 47 This inhibitor directly blocks ACE2 recognition by interfering with the RBD with an EC50 value of 3.1 µM and a 50% cytotoxic concentration (CC50) value of greater than 100 µM, not affecting ACE2 expression levels. 48\nFigure 4 Inhibitors targeting the receptor‐binding domain Xu et al. 49 identified two small molecules, TGG (2; Figure 4) and luteolin (3; Figure 4), that can bind avidly to the SARS‐CoV‐1 S2 protein and inhibit its entry into Vero E6 cells (EC50: 4.5 µM, 10.6 µM; respectively). Compounds 2 and 3 showed cytotoxicity (CC50) of 1.08 and 0.155 mM, and the selectivity index (SI) values of 2 and 3 were 240.0 and 14.62, respectively. Further studies regarding acute toxicity revealed that the 50% lethal doses of 2 and 3 were ~456 and 232 mg/kg, respectively. These results indicate that these small molecules could be used at relatively high concentrations in mice. 49 Quercetin (4; Figure 4), an analog of 3, also showed antiviral activity against SARS‐CoV‐1, with an EC50 value of 83.4 µM and a CC50 value of 3.32 mM. 50\nNgai et al. reported ADS‐J1 (5; Figure 4) as a potential SARS‐CoV‐1 viral entry inhibitor with an EC50 of 3.89 µM. Molecular docking studies predicted that 5 can bind into a deep pocket of the SARS‐CoV‐1 S HR region and block viral entry into host cells. 51 Imatinib (6; Figure 4), an Abelson kinase inhibitor, could inhibit CoV S protein‐induced fusion with an EC50 value of 10 µM and showed no cytotoxic effects in Vero cells up to 100 µM concentration. 52 , 53\n\n2.2 Inhibitors targeting the cellular receptor\nThe genetic code of SARS‐CoV‐2 shares noticeable similarities with SARS‐CoV‐1, which caused the SARS epidemic in 2002. 26 , 54 More importantly, both viruses have identical mechanisms of infection. SARS‐CoV‐1 uses the host's ACE2 as a portal to infect cells, which has high expression in the vascular endothelium 55 and the lung, particularly in type 2 alveolar epithelial cells. 56 SARS‐CoV‐2 shares 76% of its spike (S) protein with SARS‐CoV‐1. Despite a few amino acid differences in its RBD compared to the SARS‐CoV‐1 S protein, the SARS‐CoV‐2 S protein binds to ACE2 with even greater affinity 27 offering an explanation for its greater virulence and preference for the lung.\nACE, a highly glycosylated type I integral membrane protein, is an essential component of the renin‐angiotensin (Ang) system, which controls blood pressure homeostasis. Both ACE1 and ACE2 cleave Ang peptides. However, they differ markedly: ACE1 cuts and converts the inactive decapeptide Ang I into the octapeptide Ang II by removing the dipeptide His‐Leu. This Ang II induces vaso‐ and bronchoconstriction, increased vascular permeability, inflammation, and fibrosis, thus promoting acute respiratory distress syndrome (ARDS) and lung failure in patients infected with SARS‐CoV‐1 or SARS‐CoV‐2 57 (Figure 5). Therefore, ACE‐inhibitors (ACEis) and angiotensin II receptor blockers (ARBs) could block the disease‐propagating effect of Ang II. 58 , 59 , 60\nFigure 5 The roles of ACE1 and 2 in the renin‐angiotensin system. A, Chemical structures of angiotensin‐related peptides and B, Schematic diagram of roles of ACE1 and 2 in renin‐angiotensin system. ACE, angiotensin‐converting enzyme [Color figure can be viewed at wileyonlinelibrary.com] ACE2, on the other hand, is a zinc‐containing metalloenzyme, and shares merely 42% of its amino acid sequence with ACE1. 61 It cleaves only one amino acid residue (Leu or Phe) from Ang I and Ang II, respectively, generating Ang (1–9) and Ang (1–7) (a vasodilator) (Figure 5). Thus, ACE2 has been considered a potential therapeutic target for cardiovascular diseases.\nVirtual screening combined with a molecular docking approach targeting the ACE2 catalytic site with around 140 000 compounds led to the identification of inhibitor N‐(2‐aminoethyl)‐1 aziridine‐ethanamine (7; Figure 6) with an IC50 value of 57 µM and a K i of 459 µM. However, no information about the cytotoxicity of this compound is available so far. 62\nFigure 6 Inhibitors for SARS‐CoV‐1 and 2 targeting ACE2. ACE, angiotensin‐converting enzyme; SARS‐CoV, severe acute respiratory syndrome coronavirus Chloroquine (8; Figure 6) is a relatively safe, cheap, and effective medication for the treatment of malaria and amebiasis. Savarino et al. 63 reported its antiviral effects. At a molecular level, it increases late endosomal and lysosomal pH, resulting in impaired liberation of virions from endosomes or lysosomes. The virus is therefore unable to release its genetic material into the cell and replicate. 64 , 65 Furthermore, they hypothesize that chloroquine might block the production of proinflammatory cytokines (such as IL‐6), thereby blocking the pathway that subsequently leads to ARDS. 63\nChloroquine is reasonably active in vitro against SARS‐CoV‐1, MERS‐CoV, and SARS‐CoV‐2. It was found to inhibit SARS‐CoV‐2 with an EC50 value of 5.47 µM in vitro. 66 Antiviral activity against SARS‐CoV‐1 was reported with an IC50 of 8.8 μM in Vero cells, but it is unclear how this translates into activity in respiratory epithelial cells and in vivo. 67 , 68 Mechanistic studies of chloroquine for SARS‐CoV‐1 infection revealed that it could also weaken the interaction between the RBD of SARS‐CoV‐1 and ACE2 by interfering with terminal glycosylation of ACE2, thereby reducing its affinity to SARS‐CoV‐1 S. 69\nDuring the SARS‐CoV‐2 pandemic, chloroquine has been recommended by Chinese, South Korean, and Italian health authorities for the experimental treatment of COVID‐19, 70 , 71 despite contraindications for patients with heart disease or diabetes. 72 However, health experts and agencies like the US FDA and European Medicines Agency warned against broad uncontrolled use after reports of misuse of low‐quality versions of chloroquine phosphate intended for fish.\nHydroxychloroquine (9; Figure 6) is being studied as an experimental treatment for COVID‐19. 73 However, the benefits of treatment with this drug are unclear. 74\nHydroxychloroquine was found to inhibit SARS‐CoV‐2 with an EC50 value of 0.74 µM in vitro. 66 Some studies imply synergistic effects of hydroxychloroquine and azithromycin. Azithromycin is active in vitro against Zika and Ebola virus 75 , 76 and can be used to guard against life‐threatening bacterial superinfections when administered to patients suffering from viral infections. 77 A small study that compared hydroxychloroquine monotherapy and combination treatment with azithromycin found a significant advantage of the combination. While evaluating the efficacy of therapeutic intervention with hydroxychloroquine as monotherapy and its impact in combination with azithromycin, the number of patients testing negative in polymerase chain reaction (PCR) tests was substantially different in the two groups with 100% of patients cured (6 days post inclusion) in the combination arm of the study versus 57% in the monotherapy group. At the same time, 12% of patients in the control group receiving only standard care were cured. 78 , 79\nThe WHO declared on 18 March that chloroquine and its derivative hydroxychloroquine will be among the four medicines studied in the solidarity clinical trial 80 for the treatment of COVID‐19. In April 2020, the US National Institutes of Health (NIH) also commenced a study with the drug for treating COVID‐19 patients. 81\nThe recent clinical trial involving 96 032 patients with COVID‐19 concluded that it was unable to confirm a benefit of hydroxychloroquine or chloroquine, when used alone or in combination with a macrolide such as azithromycin (or clarithromycin). 82 The study actually reported decreased survival rates for patients treated with each of these drug regimens. Additionally, patients had an increased risk of developing ventricular arrhythmia under treatment. However, still more evidence is needed to adequately assess the drugs' risks or benefits for the treatment or prevention of COVID‐19 (it is important to note that chloroquine and hydroxychloroquine are still considered safe treatment options in certain autoimmune diseases and malaria). Besides, the WHO announced the premature pause of its clinical trials using hydroxychloroquine as a safety precaution on 24 May 2020.\nOn a different note, it was found that ACE2 undergoes proteolytic shedding; releasing an enzymatic ectodomain during viral entry. 83 A disintegrin and metalloproteinase (ADAM), also known as TNF‐α converting enzyme (TACE), assisted the shedding regulation of ACE2. Inhibition of this enzyme led to reduced shedding of ACE2. GW280264X (10; Figure 6) was found to be a specific inhibitor of ADAM‐induced shedding of ACE2 at 1 nM. 84 Two TACE inhibitors, TAPI‐0 (11) and TAPI‐2 (12; Figure 6), reduced ACE2 shedding, with IC50 values of 100 and 200 nM, respectively. 83\nMLN‐4760 (13; Figure 6) inhibited the catalytic activity of ACE2 with an IC50 of around 440 pM. 85 This is the most potent and selective small‐molecule inhibitor against soluble human ACE2 described to date, thus making it a very promising candidate for SARS‐CoV‐2 interference. It binds to the active site zinc and emulates the transition state peptide. However, no antiviral data for this compound is available at this time.\nThe interference of a virus‐host cell fusion, which is mediated by the viral S protein to its receptor ACE2 on host cells, may be a viable prevention strategy. Umifenovir (14; brand name Arbidol), a broad spectrum antiviral drug used against influenza, prevents viral entry by inhibiting virus‐host cell fusion. 86 It is currently being investigated in a clinical trial for the treatment of SARS‐CoV‐2. 87 , 88\nDo ACEis or ARBs amplify SARS‐CoV‐2 pathogenicity and aggravate the clinical course of COVID‐19? After ACE2 was recognized as the SARS‐CoV‐2 receptor, 14 , 29 speculations emerged about potentially negative consequences of ACEi or ARB therapy in COVID‐19 patients. This theory caused confusion in the public and alarmed patients taking these medicines. One report said that the expression of ACE2 was increased in patients with heart disease compared to healthy individuals. It was also insisted that ACE2 expression could be increased by taking ACEis and ARBs, 89 although there is no supporting report of this happening in the lungs.\nIn another report, it was suggested that patients suffering from high blood pressure receiving “ACE2‐increasing drugs” have a higher risk for severe COVID‐19, since ACEis and ARBs could elevate levels of ACE2. 90\nA joint declaration by the presidents of the HFSA/ACC/AHA on 17 March 2020, 91 followed by a similar statement of the European Medicines Agency, 92 clarified that there was no scientific basis for stopping ACEi or ARB therapy. 93 , 94 , 95 This was in accordance with the editors of the New England Journal of Medicine. 96\nIn case of SARS‐CoV, the experimental data showed that such medications may be beneficial rather than damaging, which led to a new therapeutic approach for lung diseases. 97\n\n2.3 Proteolytic processing inhibitors\nCoVs enter the host cells via both clathrin (endosomal) and nonclathrin pathways (nonendosomal); however, both pathways are dependent upon receptor binding. 98 , 99\nThe clathrin‐mediated pathway involves the binding of CoV S protein to the host receptor followed by the internalization of vesicles that maturate to late endosomes. Acidification of the endosome promotes the H+‐dependent activation of cellular cathepsin L proteinase in late endosomes and lysosomes, which cleaves and activates the S protein, thus initiating viral fusion. Recent research shows that in addition to ACE2 SARS‐CoV‐2 can also use the host cell receptor CD147 to gain access into host cells. 100\nMembrane fusion is also the crucial step for the CoV life cycle in the nonclathrin/endosomal route, in which host proteases such as cathepsin L, TMPRSS2, and TMPRSS11D (airway trypsin‐like protease) cut the S protein at the S1/S2 cleavage site to activate the S protein for membrane fusion. 101 Interference with this process by targeting these proteases could become an attractive strategy for combating CoV infections. A recent study confirms the role of TMPRSS2 for the viral life cycle in SARS‐CoV‐2‐infected VeroE6 cells. 5 Furin (a serine endoprotease) activates MERS‐CoV to initiate the nonclathrin mediated membrane fusion event. 102\nThe neurotransmitter receptor blockers chlorpromazine (15), promethazine (16), and fluphenazine (17; Figure 7), were reported to inhibit MERS‐CoV and SARS‐CoV‐1 most probably by impeding S protein‐induced fusion. 103 Chlorpromazine, a clathrin‐mediated viral entry inhibitor, was already described to inhibit human CoV‐229E, hepatitis C virus, infectious bronchitis virus, as well as mouse hepatitis virus‐2 (MHV2). 104 , 105 , 106 , 107 , 108\nFigure 7 Neurotransmitter inhibitors targeting clathrin/nonclathrin pathways Matsuyama et al. identified the commercially available serine protease inhibitor camostat (18; Figure 8) to be a SARS‐CoV‐1 inhibitor, blocking TMPRSS2 activity at 10 µM. However, at a higher concentration (100 µM), inhibition of viral entry via SARS‐CoV‐1 S protein‐mediated cell fusion never exceeded 65% (inhibition efficiency), indicating that 35% of entry events take place via the endosomal cathepsin pathway. Interestingly, treatment with a combination of EST (a cathepsin inhibitor) and 18 resulted in remarkably blocked infection (\u003e95%) activity of pseudotyped viruses. 109\nFigure 8 Inhibitors targeting TMPRSS2. TMPRSS, transmembrane serine protease A similar approach has been investigated to prevent viral entry of SARS‐CoV‐2. Pöhlmann et al. reported the attainment of full inhibition efficiency with a combination of both 18 and E‐64d (a cathepsin inhibitor). Both studies indicate that SARS‐CoV‐1 and 2 enter cells in a similar manner showing the potential of 18 as a candidate for further development. 15\nRecently, K11777 (19; Figure 8), a cysteine protease inhibitor, was shown in tissue cultures to inhibit SARS‐CoV‐1 and MERS‐CoV replication in the subnanomolar range. 110 , 111 Future tissue culture and animal model studies should be conducted to clarify, whether its antiviral activity is mediated by targeting TMPRSS2.\nTeicoplanin is a glycopeptide antibiotic used to prevent infections with Gram‐positive bacteria like methicillin‐resistant Staphylococcus aureus and Enterococcus faecalis. It was found that teicoplanin inhibits the entry of SARS‐CoV‐1, MERS‐CoV, and Ebola virus by specifically targeting cathepsin L. 112 This knowledge has also been used to block the entry of new SARS‐CoV‐2 pseudoviruses with an IC50 value of 1.66 µM. Therefore, teicoplanin could be considered a potential candidate for the treatment of COVID‐19. 113\n\n2.4 Small‐molecules as cathepsin L inhibitors\nHuman cathepsin L is a cysteine endopeptidase and plays a key role for infection efficiency by activation of the S protein into a fusogenic state to escape the late endosomes. Targeting this protease with small molecules could interfere with virus‐cell entry and therefore be a possible intervention strategy for CoV infection. 114 Bates et al. identified MDL28170 (20; Figure 9) as an antiviral compound that specifically inhibited cathepsin‐L‐mediated substrate cleavage, with an IC50 value of 2.5 nM and EC50 value in the range of 100 nM. However, despite its potent inhibitory activity, no cytotoxicity data for 20 is currently available. 115\nFigure 9 Cathepsin L inhibitors with antiviral activity Diamond et al. reported CID 16725315 (21) and CID 23631927 (22; Figure 9) as viral entry inhibitors of SARS‐CoV in a cathepsin L inhibition assay. Compound 21 could block cathepsin L with an IC50 value of 6.9 nM, while 22 showed slightly weaker potency (IC50, 56 nM). Compound 22 was also found to inhibit Ebola virus infection (EC50, 193 nM) of human embryonic kidney 293T cells. This compound did not show any sign of toxicity to human aortic endothelial cells up to 100 µM. This data offers a new promising point for the treatment of SARS and Ebola virus infections. 116\nScreening of ~14 000 compounds in a cell‐based assay resulted in the identification of SSAA09E1 (23; Figure 9) as inhibitor of cathepsin L proteinase, with an IC50 value of 5.33 µM. In a pseudotype‐based assay in 293T cells, the EC50 value of 23 was around 6.4 µM, and no cytotoxicity was detected below 100 µM. 48\nPhenotypic screening approaches led to the identification of several viral entry inhibitors. This approach has the advantage of finding cellular‐active compounds, providing information on drug solubility and cell uptake. 117 On the other hand, it is limited in terms of capacity compared to in silico target‐based screening. Hsiang et al. identified emodin (24; Figure 9), the active component from Polygonum multiflorum and Rheum officinale, could block the interaction of S protein with ACE2, with an IC50 value of 10 µM and an EC50 value of 200 µM in an S protein‐pseudotyped retrovirus assay using Vero E6 cells. However, the mechanism of action of this compound still needs to be determined. 118 Sarafianos et al. 48 found that SSAA09E3 (25), a benzamide derivative of 24, could prevent virus‐cell membrane fusion in pseudotype‐based and antiviral‐based assays, with an EC50 value of 9.7 µM, but a CC50 value of 20 µM indicates additional unknown cellular targets.\nVE607 (26) was identified among 50 240 structurally diverse small molecules to specifically inhibit SARS‐CoV‐1 entry into cells using a phenotype‐based screening. Its EC50 value was reported at 3.0 µM and it inhibited SARS‐CoV‐1 plaque formation with an EC50 of 1.6 µM. 119 Cathepsin inhibitor E‐64‐D (27) blocked MERS‐CoV and SARS‐CoV‐1 infection as well. 120 , 121"}

{"project":"LitCovid-PD-HP","denotations":[{"id":"T6","span":{"begin":6377,"end":6393},"obj":"Phenotype"},{"id":"T7","span":{"begin":6408,"end":6424},"obj":"Phenotype"},{"id":"T8","span":{"begin":9850,"end":9870},"obj":"Phenotype"},{"id":"T9","span":{"begin":10750,"end":10773},"obj":"Phenotype"},{"id":"T10","span":{"begin":14909,"end":14931},"obj":"Phenotype"},{"id":"T11","span":{"begin":15202,"end":15221},"obj":"Phenotype"},{"id":"T12","span":{"begin":17484,"end":17503},"obj":"Phenotype"},{"id":"T13","span":{"begin":18116,"end":18129},"obj":"Phenotype"},{"id":"T14","span":{"begin":19820,"end":19829},"obj":"Phenotype"},{"id":"T15","span":{"begin":19850,"end":19860},"obj":"Phenotype"},{"id":"T16","span":{"begin":19885,"end":19894},"obj":"Phenotype"}],"attributes":[{"id":"A6","pred":"hp_id","subj":"T6","obj":"http://purl.obolibrary.org/obo/HP_0002721"},{"id":"A7","pred":"hp_id","subj":"T7","obj":"http://purl.obolibrary.org/obo/HP_0002721"},{"id":"A8","pred":"hp_id","subj":"T8","obj":"http://purl.obolibrary.org/obo/HP_0002098"},{"id":"A9","pred":"hp_id","subj":"T9","obj":"http://purl.obolibrary.org/obo/HP_0001626"},{"id":"A10","pred":"hp_id","subj":"T10","obj":"http://purl.obolibrary.org/obo/HP_0004308"},{"id":"A11","pred":"hp_id","subj":"T11","obj":"http://purl.obolibrary.org/obo/HP_0002960"},{"id":"A12","pred":"hp_id","subj":"T12","obj":"http://purl.obolibrary.org/obo/HP_0000822"},{"id":"A13","pred":"hp_id","subj":"T13","obj":"http://purl.obolibrary.org/obo/HP_0002088"},{"id":"A14","pred":"hp_id","subj":"T14","obj":"http://purl.obolibrary.org/obo/HP_0012115"},{"id":"A15","pred":"hp_id","subj":"T15","obj":"http://purl.obolibrary.org/obo/HP_0012387"},{"id":"A16","pred":"hp_id","subj":"T16","obj":"http://purl.obolibrary.org/obo/HP_0012115"}],"text":"2 VIRUS ENTRY INHIBITORS\nBinding of spike protein (S) to its receptor represents the host's first confrontation with the virus and its life cycle, thus providing prophylactic intervention opportunities. The genome of SARS‐CoV‐2 has been recently determined to have an 80% identity to that of SARS‐CoV‐1 and 96% identity to the bat‐CoV RaTG13. 26 The SARS‐CoV‐2 S protein shows nucleotide sequence identities of 75% or less to all other previously described CoVs. However, again, the new SARS‐CoV‐2 S protein shares a 93.1% identity to the S protein of RaTG13. SARS‐CoV‐2 spike (S) recognizes, with its receptor‐binding domain (RBD), the cellular ACE2 receptor with high affinity (K d, 14.7 nM) 27 as judged by surface plasmin resonance spectrometry; and intervention at the RBD‐ACE2 interface can potentially disrupt infection efficiency. It was observed that the RBDs of the SARS‐CoV‐2‐ACE2 and SARS‐CoV‐1‐ACE2 complexes are quite similar (Figure 3). 29\nFigure 3 Comparison of the SARS‐CoV‐2 S and SARS‐CoV‐1 S structures: ribbon diagrams of the (A) SARS‐CoV‐2 S and (D) SARS‐CoV‐1 S [PDB 6NB6] ectodomain cryo‐EM structures. S1 subunits of (B) SARS‐CoV‐2 S and (E) SARS‐CoV‐1 S. S2 subunits of (C) SARS‐CoV‐2 S and (F) SARS‐CoV‐1 S. 28 cryo‐EM, cryogenic electron microscopy; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2 [Color figure can be viewed at wileyonlinelibrary.com] As mentioned previously, Zhang et al. 30 determined the full‐length genome sequence of SARS‐CoV‐2 and revealed that the virus is remarkably similar (89.1% sequence identity) to a group of SARS‐like CoVs. Simultaneously, Shi et al. 26 reported that SARS‐CoV‐2 shares 96% sequence identity at a whole‐genome level with a bat coronavirus—and importantly, like SARS‐CoV‐1, SARS‐CoV‐2 utilizes ACE2 receptor for viral entry. Recently, Yan et al. solved the cryo‐EM structure of full‐length human ACE2 bound to the RBD of SARS‐CoV‐2, providing important structural information for therapeutic intervention strategies. 29\nThe sequence identity of the spike protein between SARS‐CoV‐1 (1273 aa) and SARS‐CoV‐2 (1253 aa) is 76%. The spike protein has two regions, S1 and S2. The S1 region of the SARS‐CoV‐1 has a RBD that forms high‐affinity interactions with ACE2. The prevailing understanding is that SARS‐CoV‐2 employs this RBD to enter its human host cell as well. Aligning the two different RBDs revealed a sequence identity of 73.5%. However, many nonconserved mutations that interact directly with ACE2 are located in the two structural regions. 31 And both crystal and cryo‐EM structures of the SARS‐CoV‐1 spike‐ACE2 complex have shown that merely residues of regions 1 and 2 form hydrogen bonds and hydrophobic interactions with ACE2. The mutations in these two regions of SARS‐CoV‐2 will, therefore, likely reduce the number of those interactions. 32\nStudies also have shown that the RdRP, and the Mpro are highly conserved between SARS‐CoV‐2 and SARS‐CoV‐1. 33 , 34 Therefore, it is widely accepted that SARS‐CoV‐2 behaves similarly to SARS‐CoV‐1 with regard to viral entry and replication. Since the general genomic layout and replication kinetics are so conserved among MERS, SARS‐1, and SARS‐2 CoVs, investigating inhibitors of common structures is a logical step.\nThe inhibitory strength against viral enzyme was expressed as IC50, which is the concentration of the inhibitor needed to inhibit half of the enzyme activity in the tested condition. The K i value is reflective of ligand‐binding affinity to the enzyme. The inhibitory activity for cell‐based bioassays is expressed as EC50, which is a half maximal effective concentration required to induce the biological response. The warhead group means a “reactive group” of the inhibitors that can form both covalent and noncovalent interactions with amino acids in the active site of the enzyme.\n\n2.1 Targeting the RBD\nStructural investigations of the RBD‐ACE2 complex provided information about essential residues for viral entry. Hsiang et al. 35 reported a number of peptides that significantly blocked the interaction of the S protein with ACE2 with IC50 values as low as 1.88 nM. Michael et al. found charged residues between positions 22 and 57 crucial for SARS‐CoV‐1 viral entry. Based on this, they designed peptides P4 (IC50, 50 µM) and P5 (IC50, 6.0 µM) with significant inhibitory activity against SARS‐CoV‐1. The antiviral activity was further improved when they introduced the glycine binding linkage of peptide P4 (residues 22–47) with an ACE2‐derived peptide (residues 351–357) against a SARS‐CoV‐1 pseudovirus with an IC50 of 100 nM and devoid of cytotoxicity up to 200 µM. 36 It is worth highlighting that a similar strategy could work for the new SARS‐CoV‐2. The recently solved cryo‐EM structure of SARS‐CoV‐2 in complex with human ACE2 can provide a structural rationale for the peptide design. 29\nFor viral entry, MERS‐CoV uses its spike protein (S) to interact with the host‐receptor DPP4, 37 , 38 , 39 also known as adenosine deaminase‐complexing protein‐2 or CD26. 37 MERS‐CoV was also the first virus reported to use this particular path. 35 , 37 DPP4 is a type II transmembrane glycoprotein, that forms homodimers on the cell surface, and it is involved in the cleavage of dipeptides. 37 , 40 In humans, DPP4 is predominantly found on the bronchial epithelial and alveolar cells in the lower lungs. 40 , 41\nMERS‐4 and MERS‐27 are monoclonal antibodies targeting the RBD of MERS‐CoV S that were discovered in a nonimmune yeast‐display scFv library screening. The more active MERS‐4 potently blocked the infection of DPP4‐expressing Huh‐7 cells with pseudotyped MERS‐CoV (IC50, 0.056 μg/mL). It also prevented MERS‐CoV‐induced cytopathogenic effects in MERS‐infected Vero E6 cells (IC50, 0.5 μg/mL). 42\nA heptad repeat (HR) is a repeating structural pattern of seven amino acids. A crucial membrane fusion framework of SARS‐CoV is the 6‐helix‐bundle (6‐HB) that is formed by HR1 and HR2 of the viral S protein. Enfuvirtide (T‐20) is an FDA approved HR2 peptide and the first HIV fusion inhibitor. It has opened up new avenues toward identifying and developing peptides as viral entry inhibitors. Such molecules represent a promising strategy against enveloped viruses with class 1 fusion proteins such as Nipah virus, Hendra virus, Ebola virus, and other paramyxoviruses, simian immunodeficiency virus, feline immunodeficiency virus, and respiratory syncytial virus. 43 , 44 , 45 , 46 The HR regions of SARS‐CoV‐1 and SARS‐CoV‐2 S protein share a high degree of conservation, and such fusion inhibitors have potential applications in preventing SARS‐CoV‐2 entry.\nSmall molecule entry inhibitors, on the other hand, are reported to target the RBD. Compared to peptides, proteins, and biologics, small molecules have several advantages due to lower production costs, improved pharmacokinetics, stability, and dosage accuracy. Sarafianos et al. identified the oxazole‐carboxamide derivative SSAA09E2 (1; Figure 4) as an entry inhibitor against SARS‐CoV‐1 by screening a chemical library composed of 3000 compounds. 47 This inhibitor directly blocks ACE2 recognition by interfering with the RBD with an EC50 value of 3.1 µM and a 50% cytotoxic concentration (CC50) value of greater than 100 µM, not affecting ACE2 expression levels. 48\nFigure 4 Inhibitors targeting the receptor‐binding domain Xu et al. 49 identified two small molecules, TGG (2; Figure 4) and luteolin (3; Figure 4), that can bind avidly to the SARS‐CoV‐1 S2 protein and inhibit its entry into Vero E6 cells (EC50: 4.5 µM, 10.6 µM; respectively). Compounds 2 and 3 showed cytotoxicity (CC50) of 1.08 and 0.155 mM, and the selectivity index (SI) values of 2 and 3 were 240.0 and 14.62, respectively. Further studies regarding acute toxicity revealed that the 50% lethal doses of 2 and 3 were ~456 and 232 mg/kg, respectively. These results indicate that these small molecules could be used at relatively high concentrations in mice. 49 Quercetin (4; Figure 4), an analog of 3, also showed antiviral activity against SARS‐CoV‐1, with an EC50 value of 83.4 µM and a CC50 value of 3.32 mM. 50\nNgai et al. reported ADS‐J1 (5; Figure 4) as a potential SARS‐CoV‐1 viral entry inhibitor with an EC50 of 3.89 µM. Molecular docking studies predicted that 5 can bind into a deep pocket of the SARS‐CoV‐1 S HR region and block viral entry into host cells. 51 Imatinib (6; Figure 4), an Abelson kinase inhibitor, could inhibit CoV S protein‐induced fusion with an EC50 value of 10 µM and showed no cytotoxic effects in Vero cells up to 100 µM concentration. 52 , 53\n\n2.2 Inhibitors targeting the cellular receptor\nThe genetic code of SARS‐CoV‐2 shares noticeable similarities with SARS‐CoV‐1, which caused the SARS epidemic in 2002. 26 , 54 More importantly, both viruses have identical mechanisms of infection. SARS‐CoV‐1 uses the host's ACE2 as a portal to infect cells, which has high expression in the vascular endothelium 55 and the lung, particularly in type 2 alveolar epithelial cells. 56 SARS‐CoV‐2 shares 76% of its spike (S) protein with SARS‐CoV‐1. Despite a few amino acid differences in its RBD compared to the SARS‐CoV‐1 S protein, the SARS‐CoV‐2 S protein binds to ACE2 with even greater affinity 27 offering an explanation for its greater virulence and preference for the lung.\nACE, a highly glycosylated type I integral membrane protein, is an essential component of the renin‐angiotensin (Ang) system, which controls blood pressure homeostasis. Both ACE1 and ACE2 cleave Ang peptides. However, they differ markedly: ACE1 cuts and converts the inactive decapeptide Ang I into the octapeptide Ang II by removing the dipeptide His‐Leu. This Ang II induces vaso‐ and bronchoconstriction, increased vascular permeability, inflammation, and fibrosis, thus promoting acute respiratory distress syndrome (ARDS) and lung failure in patients infected with SARS‐CoV‐1 or SARS‐CoV‐2 57 (Figure 5). Therefore, ACE‐inhibitors (ACEis) and angiotensin II receptor blockers (ARBs) could block the disease‐propagating effect of Ang II. 58 , 59 , 60\nFigure 5 The roles of ACE1 and 2 in the renin‐angiotensin system. A, Chemical structures of angiotensin‐related peptides and B, Schematic diagram of roles of ACE1 and 2 in renin‐angiotensin system. ACE, angiotensin‐converting enzyme [Color figure can be viewed at wileyonlinelibrary.com] ACE2, on the other hand, is a zinc‐containing metalloenzyme, and shares merely 42% of its amino acid sequence with ACE1. 61 It cleaves only one amino acid residue (Leu or Phe) from Ang I and Ang II, respectively, generating Ang (1–9) and Ang (1–7) (a vasodilator) (Figure 5). Thus, ACE2 has been considered a potential therapeutic target for cardiovascular diseases.\nVirtual screening combined with a molecular docking approach targeting the ACE2 catalytic site with around 140 000 compounds led to the identification of inhibitor N‐(2‐aminoethyl)‐1 aziridine‐ethanamine (7; Figure 6) with an IC50 value of 57 µM and a K i of 459 µM. However, no information about the cytotoxicity of this compound is available so far. 62\nFigure 6 Inhibitors for SARS‐CoV‐1 and 2 targeting ACE2. ACE, angiotensin‐converting enzyme; SARS‐CoV, severe acute respiratory syndrome coronavirus Chloroquine (8; Figure 6) is a relatively safe, cheap, and effective medication for the treatment of malaria and amebiasis. Savarino et al. 63 reported its antiviral effects. At a molecular level, it increases late endosomal and lysosomal pH, resulting in impaired liberation of virions from endosomes or lysosomes. The virus is therefore unable to release its genetic material into the cell and replicate. 64 , 65 Furthermore, they hypothesize that chloroquine might block the production of proinflammatory cytokines (such as IL‐6), thereby blocking the pathway that subsequently leads to ARDS. 63\nChloroquine is reasonably active in vitro against SARS‐CoV‐1, MERS‐CoV, and SARS‐CoV‐2. It was found to inhibit SARS‐CoV‐2 with an EC50 value of 5.47 µM in vitro. 66 Antiviral activity against SARS‐CoV‐1 was reported with an IC50 of 8.8 μM in Vero cells, but it is unclear how this translates into activity in respiratory epithelial cells and in vivo. 67 , 68 Mechanistic studies of chloroquine for SARS‐CoV‐1 infection revealed that it could also weaken the interaction between the RBD of SARS‐CoV‐1 and ACE2 by interfering with terminal glycosylation of ACE2, thereby reducing its affinity to SARS‐CoV‐1 S. 69\nDuring the SARS‐CoV‐2 pandemic, chloroquine has been recommended by Chinese, South Korean, and Italian health authorities for the experimental treatment of COVID‐19, 70 , 71 despite contraindications for patients with heart disease or diabetes. 72 However, health experts and agencies like the US FDA and European Medicines Agency warned against broad uncontrolled use after reports of misuse of low‐quality versions of chloroquine phosphate intended for fish.\nHydroxychloroquine (9; Figure 6) is being studied as an experimental treatment for COVID‐19. 73 However, the benefits of treatment with this drug are unclear. 74\nHydroxychloroquine was found to inhibit SARS‐CoV‐2 with an EC50 value of 0.74 µM in vitro. 66 Some studies imply synergistic effects of hydroxychloroquine and azithromycin. Azithromycin is active in vitro against Zika and Ebola virus 75 , 76 and can be used to guard against life‐threatening bacterial superinfections when administered to patients suffering from viral infections. 77 A small study that compared hydroxychloroquine monotherapy and combination treatment with azithromycin found a significant advantage of the combination. While evaluating the efficacy of therapeutic intervention with hydroxychloroquine as monotherapy and its impact in combination with azithromycin, the number of patients testing negative in polymerase chain reaction (PCR) tests was substantially different in the two groups with 100% of patients cured (6 days post inclusion) in the combination arm of the study versus 57% in the monotherapy group. At the same time, 12% of patients in the control group receiving only standard care were cured. 78 , 79\nThe WHO declared on 18 March that chloroquine and its derivative hydroxychloroquine will be among the four medicines studied in the solidarity clinical trial 80 for the treatment of COVID‐19. In April 2020, the US National Institutes of Health (NIH) also commenced a study with the drug for treating COVID‐19 patients. 81\nThe recent clinical trial involving 96 032 patients with COVID‐19 concluded that it was unable to confirm a benefit of hydroxychloroquine or chloroquine, when used alone or in combination with a macrolide such as azithromycin (or clarithromycin). 82 The study actually reported decreased survival rates for patients treated with each of these drug regimens. Additionally, patients had an increased risk of developing ventricular arrhythmia under treatment. However, still more evidence is needed to adequately assess the drugs' risks or benefits for the treatment or prevention of COVID‐19 (it is important to note that chloroquine and hydroxychloroquine are still considered safe treatment options in certain autoimmune diseases and malaria). Besides, the WHO announced the premature pause of its clinical trials using hydroxychloroquine as a safety precaution on 24 May 2020.\nOn a different note, it was found that ACE2 undergoes proteolytic shedding; releasing an enzymatic ectodomain during viral entry. 83 A disintegrin and metalloproteinase (ADAM), also known as TNF‐α converting enzyme (TACE), assisted the shedding regulation of ACE2. Inhibition of this enzyme led to reduced shedding of ACE2. GW280264X (10; Figure 6) was found to be a specific inhibitor of ADAM‐induced shedding of ACE2 at 1 nM. 84 Two TACE inhibitors, TAPI‐0 (11) and TAPI‐2 (12; Figure 6), reduced ACE2 shedding, with IC50 values of 100 and 200 nM, respectively. 83\nMLN‐4760 (13; Figure 6) inhibited the catalytic activity of ACE2 with an IC50 of around 440 pM. 85 This is the most potent and selective small‐molecule inhibitor against soluble human ACE2 described to date, thus making it a very promising candidate for SARS‐CoV‐2 interference. It binds to the active site zinc and emulates the transition state peptide. However, no antiviral data for this compound is available at this time.\nThe interference of a virus‐host cell fusion, which is mediated by the viral S protein to its receptor ACE2 on host cells, may be a viable prevention strategy. Umifenovir (14; brand name Arbidol), a broad spectrum antiviral drug used against influenza, prevents viral entry by inhibiting virus‐host cell fusion. 86 It is currently being investigated in a clinical trial for the treatment of SARS‐CoV‐2. 87 , 88\nDo ACEis or ARBs amplify SARS‐CoV‐2 pathogenicity and aggravate the clinical course of COVID‐19? After ACE2 was recognized as the SARS‐CoV‐2 receptor, 14 , 29 speculations emerged about potentially negative consequences of ACEi or ARB therapy in COVID‐19 patients. This theory caused confusion in the public and alarmed patients taking these medicines. One report said that the expression of ACE2 was increased in patients with heart disease compared to healthy individuals. It was also insisted that ACE2 expression could be increased by taking ACEis and ARBs, 89 although there is no supporting report of this happening in the lungs.\nIn another report, it was suggested that patients suffering from high blood pressure receiving “ACE2‐increasing drugs” have a higher risk for severe COVID‐19, since ACEis and ARBs could elevate levels of ACE2. 90\nA joint declaration by the presidents of the HFSA/ACC/AHA on 17 March 2020, 91 followed by a similar statement of the European Medicines Agency, 92 clarified that there was no scientific basis for stopping ACEi or ARB therapy. 93 , 94 , 95 This was in accordance with the editors of the New England Journal of Medicine. 96\nIn case of SARS‐CoV, the experimental data showed that such medications may be beneficial rather than damaging, which led to a new therapeutic approach for lung diseases. 97\n\n2.3 Proteolytic processing inhibitors\nCoVs enter the host cells via both clathrin (endosomal) and nonclathrin pathways (nonendosomal); however, both pathways are dependent upon receptor binding. 98 , 99\nThe clathrin‐mediated pathway involves the binding of CoV S protein to the host receptor followed by the internalization of vesicles that maturate to late endosomes. Acidification of the endosome promotes the H+‐dependent activation of cellular cathepsin L proteinase in late endosomes and lysosomes, which cleaves and activates the S protein, thus initiating viral fusion. Recent research shows that in addition to ACE2 SARS‐CoV‐2 can also use the host cell receptor CD147 to gain access into host cells. 100\nMembrane fusion is also the crucial step for the CoV life cycle in the nonclathrin/endosomal route, in which host proteases such as cathepsin L, TMPRSS2, and TMPRSS11D (airway trypsin‐like protease) cut the S protein at the S1/S2 cleavage site to activate the S protein for membrane fusion. 101 Interference with this process by targeting these proteases could become an attractive strategy for combating CoV infections. A recent study confirms the role of TMPRSS2 for the viral life cycle in SARS‐CoV‐2‐infected VeroE6 cells. 5 Furin (a serine endoprotease) activates MERS‐CoV to initiate the nonclathrin mediated membrane fusion event. 102\nThe neurotransmitter receptor blockers chlorpromazine (15), promethazine (16), and fluphenazine (17; Figure 7), were reported to inhibit MERS‐CoV and SARS‐CoV‐1 most probably by impeding S protein‐induced fusion. 103 Chlorpromazine, a clathrin‐mediated viral entry inhibitor, was already described to inhibit human CoV‐229E, hepatitis C virus, infectious bronchitis virus, as well as mouse hepatitis virus‐2 (MHV2). 104 , 105 , 106 , 107 , 108\nFigure 7 Neurotransmitter inhibitors targeting clathrin/nonclathrin pathways Matsuyama et al. identified the commercially available serine protease inhibitor camostat (18; Figure 8) to be a SARS‐CoV‐1 inhibitor, blocking TMPRSS2 activity at 10 µM. However, at a higher concentration (100 µM), inhibition of viral entry via SARS‐CoV‐1 S protein‐mediated cell fusion never exceeded 65% (inhibition efficiency), indicating that 35% of entry events take place via the endosomal cathepsin pathway. Interestingly, treatment with a combination of EST (a cathepsin inhibitor) and 18 resulted in remarkably blocked infection (\u003e95%) activity of pseudotyped viruses. 109\nFigure 8 Inhibitors targeting TMPRSS2. TMPRSS, transmembrane serine protease A similar approach has been investigated to prevent viral entry of SARS‐CoV‐2. Pöhlmann et al. reported the attainment of full inhibition efficiency with a combination of both 18 and E‐64d (a cathepsin inhibitor). Both studies indicate that SARS‐CoV‐1 and 2 enter cells in a similar manner showing the potential of 18 as a candidate for further development. 15\nRecently, K11777 (19; Figure 8), a cysteine protease inhibitor, was shown in tissue cultures to inhibit SARS‐CoV‐1 and MERS‐CoV replication in the subnanomolar range. 110 , 111 Future tissue culture and animal model studies should be conducted to clarify, whether its antiviral activity is mediated by targeting TMPRSS2.\nTeicoplanin is a glycopeptide antibiotic used to prevent infections with Gram‐positive bacteria like methicillin‐resistant Staphylococcus aureus and Enterococcus faecalis. It was found that teicoplanin inhibits the entry of SARS‐CoV‐1, MERS‐CoV, and Ebola virus by specifically targeting cathepsin L. 112 This knowledge has also been used to block the entry of new SARS‐CoV‐2 pseudoviruses with an IC50 value of 1.66 µM. Therefore, teicoplanin could be considered a potential candidate for the treatment of COVID‐19. 113\n\n2.4 Small‐molecules as cathepsin L inhibitors\nHuman cathepsin L is a cysteine endopeptidase and plays a key role for infection efficiency by activation of the S protein into a fusogenic state to escape the late endosomes. Targeting this protease with small molecules could interfere with virus‐cell entry and therefore be a possible intervention strategy for CoV infection. 114 Bates et al. identified MDL28170 (20; Figure 9) as an antiviral compound that specifically inhibited cathepsin‐L‐mediated substrate cleavage, with an IC50 value of 2.5 nM and EC50 value in the range of 100 nM. However, despite its potent inhibitory activity, no cytotoxicity data for 20 is currently available. 115\nFigure 9 Cathepsin L inhibitors with antiviral activity Diamond et al. reported CID 16725315 (21) and CID 23631927 (22; Figure 9) as viral entry inhibitors of SARS‐CoV in a cathepsin L inhibition assay. Compound 21 could block cathepsin L with an IC50 value of 6.9 nM, while 22 showed slightly weaker potency (IC50, 56 nM). Compound 22 was also found to inhibit Ebola virus infection (EC50, 193 nM) of human embryonic kidney 293T cells. This compound did not show any sign of toxicity to human aortic endothelial cells up to 100 µM. This data offers a new promising point for the treatment of SARS and Ebola virus infections. 116\nScreening of ~14 000 compounds in a cell‐based assay resulted in the identification of SSAA09E1 (23; Figure 9) as inhibitor of cathepsin L proteinase, with an IC50 value of 5.33 µM. In a pseudotype‐based assay in 293T cells, the EC50 value of 23 was around 6.4 µM, and no cytotoxicity was detected below 100 µM. 48\nPhenotypic screening approaches led to the identification of several viral entry inhibitors. This approach has the advantage of finding cellular‐active compounds, providing information on drug solubility and cell uptake. 117 On the other hand, it is limited in terms of capacity compared to in silico target‐based screening. Hsiang et al. identified emodin (24; Figure 9), the active component from Polygonum multiflorum and Rheum officinale, could block the interaction of S protein with ACE2, with an IC50 value of 10 µM and an EC50 value of 200 µM in an S protein‐pseudotyped retrovirus assay using Vero E6 cells. However, the mechanism of action of this compound still needs to be determined. 118 Sarafianos et al. 48 found that SSAA09E3 (25), a benzamide derivative of 24, could prevent virus‐cell membrane fusion in pseudotype‐based and antiviral‐based assays, with an EC50 value of 9.7 µM, but a CC50 value of 20 µM indicates additional unknown cellular targets.\nVE607 (26) was identified among 50 240 structurally diverse small molecules to specifically inhibit SARS‐CoV‐1 entry into cells using a phenotype‐based screening. Its EC50 value was reported at 3.0 µM and it inhibited SARS‐CoV‐1 plaque formation with an EC50 of 1.6 µM. 119 Cathepsin inhibitor E‐64‐D (27) blocked MERS‐CoV and SARS‐CoV‐1 infection as well. 120 , 121"}

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VIRUS ENTRY INHIBITORS\nBinding of spike protein (S) to its receptor represents the host's first confrontation with the virus and its life cycle, thus providing prophylactic intervention opportunities. The genome of SARS‐CoV‐2 has been recently determined to have an 80% identity to that of SARS‐CoV‐1 and 96% identity to the bat‐CoV RaTG13. 26 The SARS‐CoV‐2 S protein shows nucleotide sequence identities of 75% or less to all other previously described CoVs. However, again, the new SARS‐CoV‐2 S protein shares a 93.1% identity to the S protein of RaTG13. SARS‐CoV‐2 spike (S) recognizes, with its receptor‐binding domain (RBD), the cellular ACE2 receptor with high affinity (K d, 14.7 nM) 27 as judged by surface plasmin resonance spectrometry; and intervention at the RBD‐ACE2 interface can potentially disrupt infection efficiency. It was observed that the RBDs of the SARS‐CoV‐2‐ACE2 and SARS‐CoV‐1‐ACE2 complexes are quite similar (Figure 3). 29\nFigure 3 Comparison of the SARS‐CoV‐2 S and SARS‐CoV‐1 S structures: ribbon diagrams of the (A) SARS‐CoV‐2 S and (D) SARS‐CoV‐1 S [PDB 6NB6] ectodomain cryo‐EM structures. S1 subunits of (B) SARS‐CoV‐2 S and (E) SARS‐CoV‐1 S. S2 subunits of (C) SARS‐CoV‐2 S and (F) SARS‐CoV‐1 S. 28 cryo‐EM, cryogenic electron microscopy; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2 [Color figure can be viewed at wileyonlinelibrary.com] As mentioned previously, Zhang et al. 30 determined the full‐length genome sequence of SARS‐CoV‐2 and revealed that the virus is remarkably similar (89.1% sequence identity) to a group of SARS‐like CoVs. Simultaneously, Shi et al. 26 reported that SARS‐CoV‐2 shares 96% sequence identity at a whole‐genome level with a bat coronavirus—and importantly, like SARS‐CoV‐1, SARS‐CoV‐2 utilizes ACE2 receptor for viral entry. Recently, Yan et al. solved the cryo‐EM structure of full‐length human ACE2 bound to the RBD of SARS‐CoV‐2, providing important structural information for therapeutic intervention strategies. 29\nThe sequence identity of the spike protein between SARS‐CoV‐1 (1273 aa) and SARS‐CoV‐2 (1253 aa) is 76%. The spike protein has two regions, S1 and S2. The S1 region of the SARS‐CoV‐1 has a RBD that forms high‐affinity interactions with ACE2. The prevailing understanding is that SARS‐CoV‐2 employs this RBD to enter its human host cell as well. Aligning the two different RBDs revealed a sequence identity of 73.5%. However, many nonconserved mutations that interact directly with ACE2 are located in the two structural regions. 31 And both crystal and cryo‐EM structures of the SARS‐CoV‐1 spike‐ACE2 complex have shown that merely residues of regions 1 and 2 form hydrogen bonds and hydrophobic interactions with ACE2. The mutations in these two regions of SARS‐CoV‐2 will, therefore, likely reduce the number of those interactions. 32\nStudies also have shown that the RdRP, and the Mpro are highly conserved between SARS‐CoV‐2 and SARS‐CoV‐1. 33 , 34 Therefore, it is widely accepted that SARS‐CoV‐2 behaves similarly to SARS‐CoV‐1 with regard to viral entry and replication. Since the general genomic layout and replication kinetics are so conserved among MERS, SARS‐1, and SARS‐2 CoVs, investigating inhibitors of common structures is a logical step.\nThe inhibitory strength against viral enzyme was expressed as IC50, which is the concentration of the inhibitor needed to inhibit half of the enzyme activity in the tested condition. The K i value is reflective of ligand‐binding affinity to the enzyme. The inhibitory activity for cell‐based bioassays is expressed as EC50, which is a half maximal effective concentration required to induce the biological response. The warhead group means a “reactive group” of the inhibitors that can form both covalent and noncovalent interactions with amino acids in the active site of the enzyme.\n\n2.1 Targeting the RBD\nStructural investigations of the RBD‐ACE2 complex provided information about essential residues for viral entry. Hsiang et al. 35 reported a number of peptides that significantly blocked the interaction of the S protein with ACE2 with IC50 values as low as 1.88 nM. Michael et al. found charged residues between positions 22 and 57 crucial for SARS‐CoV‐1 viral entry. Based on this, they designed peptides P4 (IC50, 50 µM) and P5 (IC50, 6.0 µM) with significant inhibitory activity against SARS‐CoV‐1. The antiviral activity was further improved when they introduced the glycine binding linkage of peptide P4 (residues 22–47) with an ACE2‐derived peptide (residues 351–357) against a SARS‐CoV‐1 pseudovirus with an IC50 of 100 nM and devoid of cytotoxicity up to 200 µM. 36 It is worth highlighting that a similar strategy could work for the new SARS‐CoV‐2. The recently solved cryo‐EM structure of SARS‐CoV‐2 in complex with human ACE2 can provide a structural rationale for the peptide design. 29\nFor viral entry, MERS‐CoV uses its spike protein (S) to interact with the host‐receptor DPP4, 37 , 38 , 39 also known as adenosine deaminase‐complexing protein‐2 or CD26. 37 MERS‐CoV was also the first virus reported to use this particular path. 35 , 37 DPP4 is a type II transmembrane glycoprotein, that forms homodimers on the cell surface, and it is involved in the cleavage of dipeptides. 37 , 40 In humans, DPP4 is predominantly found on the bronchial epithelial and alveolar cells in the lower lungs. 40 , 41\nMERS‐4 and MERS‐27 are monoclonal antibodies targeting the RBD of MERS‐CoV S that were discovered in a nonimmune yeast‐display scFv library screening. The more active MERS‐4 potently blocked the infection of DPP4‐expressing Huh‐7 cells with pseudotyped MERS‐CoV (IC50, 0.056 μg/mL). It also prevented MERS‐CoV‐induced cytopathogenic effects in MERS‐infected Vero E6 cells (IC50, 0.5 μg/mL). 42\nA heptad repeat (HR) is a repeating structural pattern of seven amino acids. A crucial membrane fusion framework of SARS‐CoV is the 6‐helix‐bundle (6‐HB) that is formed by HR1 and HR2 of the viral S protein. Enfuvirtide (T‐20) is an FDA approved HR2 peptide and the first HIV fusion inhibitor. It has opened up new avenues toward identifying and developing peptides as viral entry inhibitors. Such molecules represent a promising strategy against enveloped viruses with class 1 fusion proteins such as Nipah virus, Hendra virus, Ebola virus, and other paramyxoviruses, simian immunodeficiency virus, feline immunodeficiency virus, and respiratory syncytial virus. 43 , 44 , 45 , 46 The HR regions of SARS‐CoV‐1 and SARS‐CoV‐2 S protein share a high degree of conservation, and such fusion inhibitors have potential applications in preventing SARS‐CoV‐2 entry.\nSmall molecule entry inhibitors, on the other hand, are reported to target the RBD. Compared to peptides, proteins, and biologics, small molecules have several advantages due to lower production costs, improved pharmacokinetics, stability, and dosage accuracy. Sarafianos et al. identified the oxazole‐carboxamide derivative SSAA09E2 (1; Figure 4) as an entry inhibitor against SARS‐CoV‐1 by screening a chemical library composed of 3000 compounds. 47 This inhibitor directly blocks ACE2 recognition by interfering with the RBD with an EC50 value of 3.1 µM and a 50% cytotoxic concentration (CC50) value of greater than 100 µM, not affecting ACE2 expression levels. 48\nFigure 4 Inhibitors targeting the receptor‐binding domain Xu et al. 49 identified two small molecules, TGG (2; Figure 4) and luteolin (3; Figure 4), that can bind avidly to the SARS‐CoV‐1 S2 protein and inhibit its entry into Vero E6 cells (EC50: 4.5 µM, 10.6 µM; respectively). Compounds 2 and 3 showed cytotoxicity (CC50) of 1.08 and 0.155 mM, and the selectivity index (SI) values of 2 and 3 were 240.0 and 14.62, respectively. Further studies regarding acute toxicity revealed that the 50% lethal doses of 2 and 3 were ~456 and 232 mg/kg, respectively. These results indicate that these small molecules could be used at relatively high concentrations in mice. 49 Quercetin (4; Figure 4), an analog of 3, also showed antiviral activity against SARS‐CoV‐1, with an EC50 value of 83.4 µM and a CC50 value of 3.32 mM. 50\nNgai et al. reported ADS‐J1 (5; Figure 4) as a potential SARS‐CoV‐1 viral entry inhibitor with an EC50 of 3.89 µM. Molecular docking studies predicted that 5 can bind into a deep pocket of the SARS‐CoV‐1 S HR region and block viral entry into host cells. 51 Imatinib (6; Figure 4), an Abelson kinase inhibitor, could inhibit CoV S protein‐induced fusion with an EC50 value of 10 µM and showed no cytotoxic effects in Vero cells up to 100 µM concentration. 52 , 53\n\n2.2 Inhibitors targeting the cellular receptor\nThe genetic code of SARS‐CoV‐2 shares noticeable similarities with SARS‐CoV‐1, which caused the SARS epidemic in 2002. 26 , 54 More importantly, both viruses have identical mechanisms of infection. SARS‐CoV‐1 uses the host's ACE2 as a portal to infect cells, which has high expression in the vascular endothelium 55 and the lung, particularly in type 2 alveolar epithelial cells. 56 SARS‐CoV‐2 shares 76% of its spike (S) protein with SARS‐CoV‐1. Despite a few amino acid differences in its RBD compared to the SARS‐CoV‐1 S protein, the SARS‐CoV‐2 S protein binds to ACE2 with even greater affinity 27 offering an explanation for its greater virulence and preference for the lung.\nACE, a highly glycosylated type I integral membrane protein, is an essential component of the renin‐angiotensin (Ang) system, which controls blood pressure homeostasis. Both ACE1 and ACE2 cleave Ang peptides. However, they differ markedly: ACE1 cuts and converts the inactive decapeptide Ang I into the octapeptide Ang II by removing the dipeptide His‐Leu. This Ang II induces vaso‐ and bronchoconstriction, increased vascular permeability, inflammation, and fibrosis, thus promoting acute respiratory distress syndrome (ARDS) and lung failure in patients infected with SARS‐CoV‐1 or SARS‐CoV‐2 57 (Figure 5). Therefore, ACE‐inhibitors (ACEis) and angiotensin II receptor blockers (ARBs) could block the disease‐propagating effect of Ang II. 58 , 59 , 60\nFigure 5 The roles of ACE1 and 2 in the renin‐angiotensin system. A, Chemical structures of angiotensin‐related peptides and B, Schematic diagram of roles of ACE1 and 2 in renin‐angiotensin system. ACE, angiotensin‐converting enzyme [Color figure can be viewed at wileyonlinelibrary.com] ACE2, on the other hand, is a zinc‐containing metalloenzyme, and shares merely 42% of its amino acid sequence with ACE1. 61 It cleaves only one amino acid residue (Leu or Phe) from Ang I and Ang II, respectively, generating Ang (1–9) and Ang (1–7) (a vasodilator) (Figure 5). Thus, ACE2 has been considered a potential therapeutic target for cardiovascular diseases.\nVirtual screening combined with a molecular docking approach targeting the ACE2 catalytic site with around 140 000 compounds led to the identification of inhibitor N‐(2‐aminoethyl)‐1 aziridine‐ethanamine (7; Figure 6) with an IC50 value of 57 µM and a K i of 459 µM. However, no information about the cytotoxicity of this compound is available so far. 62\nFigure 6 Inhibitors for SARS‐CoV‐1 and 2 targeting ACE2. ACE, angiotensin‐converting enzyme; SARS‐CoV, severe acute respiratory syndrome coronavirus Chloroquine (8; Figure 6) is a relatively safe, cheap, and effective medication for the treatment of malaria and amebiasis. Savarino et al. 63 reported its antiviral effects. At a molecular level, it increases late endosomal and lysosomal pH, resulting in impaired liberation of virions from endosomes or lysosomes. The virus is therefore unable to release its genetic material into the cell and replicate. 64 , 65 Furthermore, they hypothesize that chloroquine might block the production of proinflammatory cytokines (such as IL‐6), thereby blocking the pathway that subsequently leads to ARDS. 63\nChloroquine is reasonably active in vitro against SARS‐CoV‐1, MERS‐CoV, and SARS‐CoV‐2. It was found to inhibit SARS‐CoV‐2 with an EC50 value of 5.47 µM in vitro. 66 Antiviral activity against SARS‐CoV‐1 was reported with an IC50 of 8.8 μM in Vero cells, but it is unclear how this translates into activity in respiratory epithelial cells and in vivo. 67 , 68 Mechanistic studies of chloroquine for SARS‐CoV‐1 infection revealed that it could also weaken the interaction between the RBD of SARS‐CoV‐1 and ACE2 by interfering with terminal glycosylation of ACE2, thereby reducing its affinity to SARS‐CoV‐1 S. 69\nDuring the SARS‐CoV‐2 pandemic, chloroquine has been recommended by Chinese, South Korean, and Italian health authorities for the experimental treatment of COVID‐19, 70 , 71 despite contraindications for patients with heart disease or diabetes. 72 However, health experts and agencies like the US FDA and European Medicines Agency warned against broad uncontrolled use after reports of misuse of low‐quality versions of chloroquine phosphate intended for fish.\nHydroxychloroquine (9; Figure 6) is being studied as an experimental treatment for COVID‐19. 73 However, the benefits of treatment with this drug are unclear. 74\nHydroxychloroquine was found to inhibit SARS‐CoV‐2 with an EC50 value of 0.74 µM in vitro. 66 Some studies imply synergistic effects of hydroxychloroquine and azithromycin. Azithromycin is active in vitro against Zika and Ebola virus 75 , 76 and can be used to guard against life‐threatening bacterial superinfections when administered to patients suffering from viral infections. 77 A small study that compared hydroxychloroquine monotherapy and combination treatment with azithromycin found a significant advantage of the combination. While evaluating the efficacy of therapeutic intervention with hydroxychloroquine as monotherapy and its impact in combination with azithromycin, the number of patients testing negative in polymerase chain reaction (PCR) tests was substantially different in the two groups with 100% of patients cured (6 days post inclusion) in the combination arm of the study versus 57% in the monotherapy group. At the same time, 12% of patients in the control group receiving only standard care were cured. 78 , 79\nThe WHO declared on 18 March that chloroquine and its derivative hydroxychloroquine will be among the four medicines studied in the solidarity clinical trial 80 for the treatment of COVID‐19. In April 2020, the US National Institutes of Health (NIH) also commenced a study with the drug for treating COVID‐19 patients. 81\nThe recent clinical trial involving 96 032 patients with COVID‐19 concluded that it was unable to confirm a benefit of hydroxychloroquine or chloroquine, when used alone or in combination with a macrolide such as azithromycin (or clarithromycin). 82 The study actually reported decreased survival rates for patients treated with each of these drug regimens. Additionally, patients had an increased risk of developing ventricular arrhythmia under treatment. However, still more evidence is needed to adequately assess the drugs' risks or benefits for the treatment or prevention of COVID‐19 (it is important to note that chloroquine and hydroxychloroquine are still considered safe treatment options in certain autoimmune diseases and malaria). Besides, the WHO announced the premature pause of its clinical trials using hydroxychloroquine as a safety precaution on 24 May 2020.\nOn a different note, it was found that ACE2 undergoes proteolytic shedding; releasing an enzymatic ectodomain during viral entry. 83 A disintegrin and metalloproteinase (ADAM), also known as TNF‐α converting enzyme (TACE), assisted the shedding regulation of ACE2. Inhibition of this enzyme led to reduced shedding of ACE2. GW280264X (10; Figure 6) was found to be a specific inhibitor of ADAM‐induced shedding of ACE2 at 1 nM. 84 Two TACE inhibitors, TAPI‐0 (11) and TAPI‐2 (12; Figure 6), reduced ACE2 shedding, with IC50 values of 100 and 200 nM, respectively. 83\nMLN‐4760 (13; Figure 6) inhibited the catalytic activity of ACE2 with an IC50 of around 440 pM. 85 This is the most potent and selective small‐molecule inhibitor against soluble human ACE2 described to date, thus making it a very promising candidate for SARS‐CoV‐2 interference. It binds to the active site zinc and emulates the transition state peptide. However, no antiviral data for this compound is available at this time.\nThe interference of a virus‐host cell fusion, which is mediated by the viral S protein to its receptor ACE2 on host cells, may be a viable prevention strategy. Umifenovir (14; brand name Arbidol), a broad spectrum antiviral drug used against influenza, prevents viral entry by inhibiting virus‐host cell fusion. 86 It is currently being investigated in a clinical trial for the treatment of SARS‐CoV‐2. 87 , 88\nDo ACEis or ARBs amplify SARS‐CoV‐2 pathogenicity and aggravate the clinical course of COVID‐19? After ACE2 was recognized as the SARS‐CoV‐2 receptor, 14 , 29 speculations emerged about potentially negative consequences of ACEi or ARB therapy in COVID‐19 patients. This theory caused confusion in the public and alarmed patients taking these medicines. One report said that the expression of ACE2 was increased in patients with heart disease compared to healthy individuals. It was also insisted that ACE2 expression could be increased by taking ACEis and ARBs, 89 although there is no supporting report of this happening in the lungs.\nIn another report, it was suggested that patients suffering from high blood pressure receiving “ACE2‐increasing drugs” have a higher risk for severe COVID‐19, since ACEis and ARBs could elevate levels of ACE2. 90\nA joint declaration by the presidents of the HFSA/ACC/AHA on 17 March 2020, 91 followed by a similar statement of the European Medicines Agency, 92 clarified that there was no scientific basis for stopping ACEi or ARB therapy. 93 , 94 , 95 This was in accordance with the editors of the New England Journal of Medicine. 96\nIn case of SARS‐CoV, the experimental data showed that such medications may be beneficial rather than damaging, which led to a new therapeutic approach for lung diseases. 97\n\n2.3 Proteolytic processing inhibitors\nCoVs enter the host cells via both clathrin (endosomal) and nonclathrin pathways (nonendosomal); however, both pathways are dependent upon receptor binding. 98 , 99\nThe clathrin‐mediated pathway involves the binding of CoV S protein to the host receptor followed by the internalization of vesicles that maturate to late endosomes. Acidification of the endosome promotes the H+‐dependent activation of cellular cathepsin L proteinase in late endosomes and lysosomes, which cleaves and activates the S protein, thus initiating viral fusion. Recent research shows that in addition to ACE2 SARS‐CoV‐2 can also use the host cell receptor CD147 to gain access into host cells. 100\nMembrane fusion is also the crucial step for the CoV life cycle in the nonclathrin/endosomal route, in which host proteases such as cathepsin L, TMPRSS2, and TMPRSS11D (airway trypsin‐like protease) cut the S protein at the S1/S2 cleavage site to activate the S protein for membrane fusion. 101 Interference with this process by targeting these proteases could become an attractive strategy for combating CoV infections. A recent study confirms the role of TMPRSS2 for the viral life cycle in SARS‐CoV‐2‐infected VeroE6 cells. 5 Furin (a serine endoprotease) activates MERS‐CoV to initiate the nonclathrin mediated membrane fusion event. 102\nThe neurotransmitter receptor blockers chlorpromazine (15), promethazine (16), and fluphenazine (17; Figure 7), were reported to inhibit MERS‐CoV and SARS‐CoV‐1 most probably by impeding S protein‐induced fusion. 103 Chlorpromazine, a clathrin‐mediated viral entry inhibitor, was already described to inhibit human CoV‐229E, hepatitis C virus, infectious bronchitis virus, as well as mouse hepatitis virus‐2 (MHV2). 104 , 105 , 106 , 107 , 108\nFigure 7 Neurotransmitter inhibitors targeting clathrin/nonclathrin pathways Matsuyama et al. identified the commercially available serine protease inhibitor camostat (18; Figure 8) to be a SARS‐CoV‐1 inhibitor, blocking TMPRSS2 activity at 10 µM. However, at a higher concentration (100 µM), inhibition of viral entry via SARS‐CoV‐1 S protein‐mediated cell fusion never exceeded 65% (inhibition efficiency), indicating that 35% of entry events take place via the endosomal cathepsin pathway. Interestingly, treatment with a combination of EST (a cathepsin inhibitor) and 18 resulted in remarkably blocked infection (\u003e95%) activity of pseudotyped viruses. 109\nFigure 8 Inhibitors targeting TMPRSS2. TMPRSS, transmembrane serine protease A similar approach has been investigated to prevent viral entry of SARS‐CoV‐2. Pöhlmann et al. reported the attainment of full inhibition efficiency with a combination of both 18 and E‐64d (a cathepsin inhibitor). Both studies indicate that SARS‐CoV‐1 and 2 enter cells in a similar manner showing the potential of 18 as a candidate for further development. 15\nRecently, K11777 (19; Figure 8), a cysteine protease inhibitor, was shown in tissue cultures to inhibit SARS‐CoV‐1 and MERS‐CoV replication in the subnanomolar range. 110 , 111 Future tissue culture and animal model studies should be conducted to clarify, whether its antiviral activity is mediated by targeting TMPRSS2.\nTeicoplanin is a glycopeptide antibiotic used to prevent infections with Gram‐positive bacteria like methicillin‐resistant Staphylococcus aureus and Enterococcus faecalis. It was found that teicoplanin inhibits the entry of SARS‐CoV‐1, MERS‐CoV, and Ebola virus by specifically targeting cathepsin L. 112 This knowledge has also been used to block the entry of new SARS‐CoV‐2 pseudoviruses with an IC50 value of 1.66 µM. Therefore, teicoplanin could be considered a potential candidate for the treatment of COVID‐19. 113\n\n2.4 Small‐molecules as cathepsin L inhibitors\nHuman cathepsin L is a cysteine endopeptidase and plays a key role for infection efficiency by activation of the S protein into a fusogenic state to escape the late endosomes. Targeting this protease with small molecules could interfere with virus‐cell entry and therefore be a possible intervention strategy for CoV infection. 114 Bates et al. identified MDL28170 (20; Figure 9) as an antiviral compound that specifically inhibited cathepsin‐L‐mediated substrate cleavage, with an IC50 value of 2.5 nM and EC50 value in the range of 100 nM. However, despite its potent inhibitory activity, no cytotoxicity data for 20 is currently available. 115\nFigure 9 Cathepsin L inhibitors with antiviral activity Diamond et al. reported CID 16725315 (21) and CID 23631927 (22; Figure 9) as viral entry inhibitors of SARS‐CoV in a cathepsin L inhibition assay. Compound 21 could block cathepsin L with an IC50 value of 6.9 nM, while 22 showed slightly weaker potency (IC50, 56 nM). Compound 22 was also found to inhibit Ebola virus infection (EC50, 193 nM) of human embryonic kidney 293T cells. This compound did not show any sign of toxicity to human aortic endothelial cells up to 100 µM. This data offers a new promising point for the treatment of SARS and Ebola virus infections. 116\nScreening of ~14 000 compounds in a cell‐based assay resulted in the identification of SSAA09E1 (23; Figure 9) as inhibitor of cathepsin L proteinase, with an IC50 value of 5.33 µM. In a pseudotype‐based assay in 293T cells, the EC50 value of 23 was around 6.4 µM, and no cytotoxicity was detected below 100 µM. 48\nPhenotypic screening approaches led to the identification of several viral entry inhibitors. This approach has the advantage of finding cellular‐active compounds, providing information on drug solubility and cell uptake. 117 On the other hand, it is limited in terms of capacity compared to in silico target‐based screening. Hsiang et al. identified emodin (24; Figure 9), the active component from Polygonum multiflorum and Rheum officinale, could block the interaction of S protein with ACE2, with an IC50 value of 10 µM and an EC50 value of 200 µM in an S protein‐pseudotyped retrovirus assay using Vero E6 cells. However, the mechanism of action of this compound still needs to be determined. 118 Sarafianos et al. 48 found that SSAA09E3 (25), a benzamide derivative of 24, could prevent virus‐cell membrane fusion in pseudotype‐based and antiviral‐based assays, with an EC50 value of 9.7 µM, but a CC50 value of 20 µM indicates additional unknown cellular targets.\nVE607 (26) was identified among 50 240 structurally diverse small molecules to specifically inhibit SARS‐CoV‐1 entry into cells using a phenotype‐based screening. Its EC50 value was reported at 3.0 µM and it inhibited SARS‐CoV‐1 plaque formation with an EC50 of 1.6 µM. 119 Cathepsin inhibitor E‐64‐D (27) blocked MERS‐CoV and SARS‐CoV‐1 infection as well. 120 , 121"}

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VIRUS ENTRY INHIBITORS\nBinding of spike protein (S) to its receptor represents the host's first confrontation with the virus and its life cycle, thus providing prophylactic intervention opportunities. The genome of SARS‐CoV‐2 has been recently determined to have an 80% identity to that of SARS‐CoV‐1 and 96% identity to the bat‐CoV RaTG13. 26 The SARS‐CoV‐2 S protein shows nucleotide sequence identities of 75% or less to all other previously described CoVs. However, again, the new SARS‐CoV‐2 S protein shares a 93.1% identity to the S protein of RaTG13. SARS‐CoV‐2 spike (S) recognizes, with its receptor‐binding domain (RBD), the cellular ACE2 receptor with high affinity (K d, 14.7 nM) 27 as judged by surface plasmin resonance spectrometry; and intervention at the RBD‐ACE2 interface can potentially disrupt infection efficiency. It was observed that the RBDs of the SARS‐CoV‐2‐ACE2 and SARS‐CoV‐1‐ACE2 complexes are quite similar (Figure 3). 29\nFigure 3 Comparison of the SARS‐CoV‐2 S and SARS‐CoV‐1 S structures: ribbon diagrams of the (A) SARS‐CoV‐2 S and (D) SARS‐CoV‐1 S [PDB 6NB6] ectodomain cryo‐EM structures. S1 subunits of (B) SARS‐CoV‐2 S and (E) SARS‐CoV‐1 S. S2 subunits of (C) SARS‐CoV‐2 S and (F) SARS‐CoV‐1 S. 28 cryo‐EM, cryogenic electron microscopy; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2 [Color figure can be viewed at wileyonlinelibrary.com] As mentioned previously, Zhang et al. 30 determined the full‐length genome sequence of SARS‐CoV‐2 and revealed that the virus is remarkably similar (89.1% sequence identity) to a group of SARS‐like CoVs. Simultaneously, Shi et al. 26 reported that SARS‐CoV‐2 shares 96% sequence identity at a whole‐genome level with a bat coronavirus—and importantly, like SARS‐CoV‐1, SARS‐CoV‐2 utilizes ACE2 receptor for viral entry. Recently, Yan et al. solved the cryo‐EM structure of full‐length human ACE2 bound to the RBD of SARS‐CoV‐2, providing important structural information for therapeutic intervention strategies. 29\nThe sequence identity of the spike protein between SARS‐CoV‐1 (1273 aa) and SARS‐CoV‐2 (1253 aa) is 76%. The spike protein has two regions, S1 and S2. The S1 region of the SARS‐CoV‐1 has a RBD that forms high‐affinity interactions with ACE2. The prevailing understanding is that SARS‐CoV‐2 employs this RBD to enter its human host cell as well. Aligning the two different RBDs revealed a sequence identity of 73.5%. However, many nonconserved mutations that interact directly with ACE2 are located in the two structural regions. 31 And both crystal and cryo‐EM structures of the SARS‐CoV‐1 spike‐ACE2 complex have shown that merely residues of regions 1 and 2 form hydrogen bonds and hydrophobic interactions with ACE2. The mutations in these two regions of SARS‐CoV‐2 will, therefore, likely reduce the number of those interactions. 32\nStudies also have shown that the RdRP, and the Mpro are highly conserved between SARS‐CoV‐2 and SARS‐CoV‐1. 33 , 34 Therefore, it is widely accepted that SARS‐CoV‐2 behaves similarly to SARS‐CoV‐1 with regard to viral entry and replication. Since the general genomic layout and replication kinetics are so conserved among MERS, SARS‐1, and SARS‐2 CoVs, investigating inhibitors of common structures is a logical step.\nThe inhibitory strength against viral enzyme was expressed as IC50, which is the concentration of the inhibitor needed to inhibit half of the enzyme activity in the tested condition. The K i value is reflective of ligand‐binding affinity to the enzyme. The inhibitory activity for cell‐based bioassays is expressed as EC50, which is a half maximal effective concentration required to induce the biological response. The warhead group means a “reactive group” of the inhibitors that can form both covalent and noncovalent interactions with amino acids in the active site of the enzyme.\n\n2.1 Targeting the RBD\nStructural investigations of the RBD‐ACE2 complex provided information about essential residues for viral entry. Hsiang et al. 35 reported a number of peptides that significantly blocked the interaction of the S protein with ACE2 with IC50 values as low as 1.88 nM. Michael et al. found charged residues between positions 22 and 57 crucial for SARS‐CoV‐1 viral entry. Based on this, they designed peptides P4 (IC50, 50 µM) and P5 (IC50, 6.0 µM) with significant inhibitory activity against SARS‐CoV‐1. The antiviral activity was further improved when they introduced the glycine binding linkage of peptide P4 (residues 22–47) with an ACE2‐derived peptide (residues 351–357) against a SARS‐CoV‐1 pseudovirus with an IC50 of 100 nM and devoid of cytotoxicity up to 200 µM. 36 It is worth highlighting that a similar strategy could work for the new SARS‐CoV‐2. The recently solved cryo‐EM structure of SARS‐CoV‐2 in complex with human ACE2 can provide a structural rationale for the peptide design. 29\nFor viral entry, MERS‐CoV uses its spike protein (S) to interact with the host‐receptor DPP4, 37 , 38 , 39 also known as adenosine deaminase‐complexing protein‐2 or CD26. 37 MERS‐CoV was also the first virus reported to use this particular path. 35 , 37 DPP4 is a type II transmembrane glycoprotein, that forms homodimers on the cell surface, and it is involved in the cleavage of dipeptides. 37 , 40 In humans, DPP4 is predominantly found on the bronchial epithelial and alveolar cells in the lower lungs. 40 , 41\nMERS‐4 and MERS‐27 are monoclonal antibodies targeting the RBD of MERS‐CoV S that were discovered in a nonimmune yeast‐display scFv library screening. The more active MERS‐4 potently blocked the infection of DPP4‐expressing Huh‐7 cells with pseudotyped MERS‐CoV (IC50, 0.056 μg/mL). It also prevented MERS‐CoV‐induced cytopathogenic effects in MERS‐infected Vero E6 cells (IC50, 0.5 μg/mL). 42\nA heptad repeat (HR) is a repeating structural pattern of seven amino acids. A crucial membrane fusion framework of SARS‐CoV is the 6‐helix‐bundle (6‐HB) that is formed by HR1 and HR2 of the viral S protein. Enfuvirtide (T‐20) is an FDA approved HR2 peptide and the first HIV fusion inhibitor. It has opened up new avenues toward identifying and developing peptides as viral entry inhibitors. Such molecules represent a promising strategy against enveloped viruses with class 1 fusion proteins such as Nipah virus, Hendra virus, Ebola virus, and other paramyxoviruses, simian immunodeficiency virus, feline immunodeficiency virus, and respiratory syncytial virus. 43 , 44 , 45 , 46 The HR regions of SARS‐CoV‐1 and SARS‐CoV‐2 S protein share a high degree of conservation, and such fusion inhibitors have potential applications in preventing SARS‐CoV‐2 entry.\nSmall molecule entry inhibitors, on the other hand, are reported to target the RBD. Compared to peptides, proteins, and biologics, small molecules have several advantages due to lower production costs, improved pharmacokinetics, stability, and dosage accuracy. Sarafianos et al. identified the oxazole‐carboxamide derivative SSAA09E2 (1; Figure 4) as an entry inhibitor against SARS‐CoV‐1 by screening a chemical library composed of 3000 compounds. 47 This inhibitor directly blocks ACE2 recognition by interfering with the RBD with an EC50 value of 3.1 µM and a 50% cytotoxic concentration (CC50) value of greater than 100 µM, not affecting ACE2 expression levels. 48\nFigure 4 Inhibitors targeting the receptor‐binding domain Xu et al. 49 identified two small molecules, TGG (2; Figure 4) and luteolin (3; Figure 4), that can bind avidly to the SARS‐CoV‐1 S2 protein and inhibit its entry into Vero E6 cells (EC50: 4.5 µM, 10.6 µM; respectively). Compounds 2 and 3 showed cytotoxicity (CC50) of 1.08 and 0.155 mM, and the selectivity index (SI) values of 2 and 3 were 240.0 and 14.62, respectively. Further studies regarding acute toxicity revealed that the 50% lethal doses of 2 and 3 were ~456 and 232 mg/kg, respectively. These results indicate that these small molecules could be used at relatively high concentrations in mice. 49 Quercetin (4; Figure 4), an analog of 3, also showed antiviral activity against SARS‐CoV‐1, with an EC50 value of 83.4 µM and a CC50 value of 3.32 mM. 50\nNgai et al. reported ADS‐J1 (5; Figure 4) as a potential SARS‐CoV‐1 viral entry inhibitor with an EC50 of 3.89 µM. Molecular docking studies predicted that 5 can bind into a deep pocket of the SARS‐CoV‐1 S HR region and block viral entry into host cells. 51 Imatinib (6; Figure 4), an Abelson kinase inhibitor, could inhibit CoV S protein‐induced fusion with an EC50 value of 10 µM and showed no cytotoxic effects in Vero cells up to 100 µM concentration. 52 , 53\n\n2.2 Inhibitors targeting the cellular receptor\nThe genetic code of SARS‐CoV‐2 shares noticeable similarities with SARS‐CoV‐1, which caused the SARS epidemic in 2002. 26 , 54 More importantly, both viruses have identical mechanisms of infection. SARS‐CoV‐1 uses the host's ACE2 as a portal to infect cells, which has high expression in the vascular endothelium 55 and the lung, particularly in type 2 alveolar epithelial cells. 56 SARS‐CoV‐2 shares 76% of its spike (S) protein with SARS‐CoV‐1. Despite a few amino acid differences in its RBD compared to the SARS‐CoV‐1 S protein, the SARS‐CoV‐2 S protein binds to ACE2 with even greater affinity 27 offering an explanation for its greater virulence and preference for the lung.\nACE, a highly glycosylated type I integral membrane protein, is an essential component of the renin‐angiotensin (Ang) system, which controls blood pressure homeostasis. Both ACE1 and ACE2 cleave Ang peptides. However, they differ markedly: ACE1 cuts and converts the inactive decapeptide Ang I into the octapeptide Ang II by removing the dipeptide His‐Leu. This Ang II induces vaso‐ and bronchoconstriction, increased vascular permeability, inflammation, and fibrosis, thus promoting acute respiratory distress syndrome (ARDS) and lung failure in patients infected with SARS‐CoV‐1 or SARS‐CoV‐2 57 (Figure 5). Therefore, ACE‐inhibitors (ACEis) and angiotensin II receptor blockers (ARBs) could block the disease‐propagating effect of Ang II. 58 , 59 , 60\nFigure 5 The roles of ACE1 and 2 in the renin‐angiotensin system. A, Chemical structures of angiotensin‐related peptides and B, Schematic diagram of roles of ACE1 and 2 in renin‐angiotensin system. ACE, angiotensin‐converting enzyme [Color figure can be viewed at wileyonlinelibrary.com] ACE2, on the other hand, is a zinc‐containing metalloenzyme, and shares merely 42% of its amino acid sequence with ACE1. 61 It cleaves only one amino acid residue (Leu or Phe) from Ang I and Ang II, respectively, generating Ang (1–9) and Ang (1–7) (a vasodilator) (Figure 5). Thus, ACE2 has been considered a potential therapeutic target for cardiovascular diseases.\nVirtual screening combined with a molecular docking approach targeting the ACE2 catalytic site with around 140 000 compounds led to the identification of inhibitor N‐(2‐aminoethyl)‐1 aziridine‐ethanamine (7; Figure 6) with an IC50 value of 57 µM and a K i of 459 µM. However, no information about the cytotoxicity of this compound is available so far. 62\nFigure 6 Inhibitors for SARS‐CoV‐1 and 2 targeting ACE2. ACE, angiotensin‐converting enzyme; SARS‐CoV, severe acute respiratory syndrome coronavirus Chloroquine (8; Figure 6) is a relatively safe, cheap, and effective medication for the treatment of malaria and amebiasis. Savarino et al. 63 reported its antiviral effects. At a molecular level, it increases late endosomal and lysosomal pH, resulting in impaired liberation of virions from endosomes or lysosomes. The virus is therefore unable to release its genetic material into the cell and replicate. 64 , 65 Furthermore, they hypothesize that chloroquine might block the production of proinflammatory cytokines (such as IL‐6), thereby blocking the pathway that subsequently leads to ARDS. 63\nChloroquine is reasonably active in vitro against SARS‐CoV‐1, MERS‐CoV, and SARS‐CoV‐2. It was found to inhibit SARS‐CoV‐2 with an EC50 value of 5.47 µM in vitro. 66 Antiviral activity against SARS‐CoV‐1 was reported with an IC50 of 8.8 μM in Vero cells, but it is unclear how this translates into activity in respiratory epithelial cells and in vivo. 67 , 68 Mechanistic studies of chloroquine for SARS‐CoV‐1 infection revealed that it could also weaken the interaction between the RBD of SARS‐CoV‐1 and ACE2 by interfering with terminal glycosylation of ACE2, thereby reducing its affinity to SARS‐CoV‐1 S. 69\nDuring the SARS‐CoV‐2 pandemic, chloroquine has been recommended by Chinese, South Korean, and Italian health authorities for the experimental treatment of COVID‐19, 70 , 71 despite contraindications for patients with heart disease or diabetes. 72 However, health experts and agencies like the US FDA and European Medicines Agency warned against broad uncontrolled use after reports of misuse of low‐quality versions of chloroquine phosphate intended for fish.\nHydroxychloroquine (9; Figure 6) is being studied as an experimental treatment for COVID‐19. 73 However, the benefits of treatment with this drug are unclear. 74\nHydroxychloroquine was found to inhibit SARS‐CoV‐2 with an EC50 value of 0.74 µM in vitro. 66 Some studies imply synergistic effects of hydroxychloroquine and azithromycin. Azithromycin is active in vitro against Zika and Ebola virus 75 , 76 and can be used to guard against life‐threatening bacterial superinfections when administered to patients suffering from viral infections. 77 A small study that compared hydroxychloroquine monotherapy and combination treatment with azithromycin found a significant advantage of the combination. While evaluating the efficacy of therapeutic intervention with hydroxychloroquine as monotherapy and its impact in combination with azithromycin, the number of patients testing negative in polymerase chain reaction (PCR) tests was substantially different in the two groups with 100% of patients cured (6 days post inclusion) in the combination arm of the study versus 57% in the monotherapy group. At the same time, 12% of patients in the control group receiving only standard care were cured. 78 , 79\nThe WHO declared on 18 March that chloroquine and its derivative hydroxychloroquine will be among the four medicines studied in the solidarity clinical trial 80 for the treatment of COVID‐19. In April 2020, the US National Institutes of Health (NIH) also commenced a study with the drug for treating COVID‐19 patients. 81\nThe recent clinical trial involving 96 032 patients with COVID‐19 concluded that it was unable to confirm a benefit of hydroxychloroquine or chloroquine, when used alone or in combination with a macrolide such as azithromycin (or clarithromycin). 82 The study actually reported decreased survival rates for patients treated with each of these drug regimens. Additionally, patients had an increased risk of developing ventricular arrhythmia under treatment. However, still more evidence is needed to adequately assess the drugs' risks or benefits for the treatment or prevention of COVID‐19 (it is important to note that chloroquine and hydroxychloroquine are still considered safe treatment options in certain autoimmune diseases and malaria). Besides, the WHO announced the premature pause of its clinical trials using hydroxychloroquine as a safety precaution on 24 May 2020.\nOn a different note, it was found that ACE2 undergoes proteolytic shedding; releasing an enzymatic ectodomain during viral entry. 83 A disintegrin and metalloproteinase (ADAM), also known as TNF‐α converting enzyme (TACE), assisted the shedding regulation of ACE2. Inhibition of this enzyme led to reduced shedding of ACE2. GW280264X (10; Figure 6) was found to be a specific inhibitor of ADAM‐induced shedding of ACE2 at 1 nM. 84 Two TACE inhibitors, TAPI‐0 (11) and TAPI‐2 (12; Figure 6), reduced ACE2 shedding, with IC50 values of 100 and 200 nM, respectively. 83\nMLN‐4760 (13; Figure 6) inhibited the catalytic activity of ACE2 with an IC50 of around 440 pM. 85 This is the most potent and selective small‐molecule inhibitor against soluble human ACE2 described to date, thus making it a very promising candidate for SARS‐CoV‐2 interference. It binds to the active site zinc and emulates the transition state peptide. However, no antiviral data for this compound is available at this time.\nThe interference of a virus‐host cell fusion, which is mediated by the viral S protein to its receptor ACE2 on host cells, may be a viable prevention strategy. Umifenovir (14; brand name Arbidol), a broad spectrum antiviral drug used against influenza, prevents viral entry by inhibiting virus‐host cell fusion. 86 It is currently being investigated in a clinical trial for the treatment of SARS‐CoV‐2. 87 , 88\nDo ACEis or ARBs amplify SARS‐CoV‐2 pathogenicity and aggravate the clinical course of COVID‐19? After ACE2 was recognized as the SARS‐CoV‐2 receptor, 14 , 29 speculations emerged about potentially negative consequences of ACEi or ARB therapy in COVID‐19 patients. This theory caused confusion in the public and alarmed patients taking these medicines. One report said that the expression of ACE2 was increased in patients with heart disease compared to healthy individuals. It was also insisted that ACE2 expression could be increased by taking ACEis and ARBs, 89 although there is no supporting report of this happening in the lungs.\nIn another report, it was suggested that patients suffering from high blood pressure receiving “ACE2‐increasing drugs” have a higher risk for severe COVID‐19, since ACEis and ARBs could elevate levels of ACE2. 90\nA joint declaration by the presidents of the HFSA/ACC/AHA on 17 March 2020, 91 followed by a similar statement of the European Medicines Agency, 92 clarified that there was no scientific basis for stopping ACEi or ARB therapy. 93 , 94 , 95 This was in accordance with the editors of the New England Journal of Medicine. 96\nIn case of SARS‐CoV, the experimental data showed that such medications may be beneficial rather than damaging, which led to a new therapeutic approach for lung diseases. 97\n\n2.3 Proteolytic processing inhibitors\nCoVs enter the host cells via both clathrin (endosomal) and nonclathrin pathways (nonendosomal); however, both pathways are dependent upon receptor binding. 98 , 99\nThe clathrin‐mediated pathway involves the binding of CoV S protein to the host receptor followed by the internalization of vesicles that maturate to late endosomes. Acidification of the endosome promotes the H+‐dependent activation of cellular cathepsin L proteinase in late endosomes and lysosomes, which cleaves and activates the S protein, thus initiating viral fusion. Recent research shows that in addition to ACE2 SARS‐CoV‐2 can also use the host cell receptor CD147 to gain access into host cells. 100\nMembrane fusion is also the crucial step for the CoV life cycle in the nonclathrin/endosomal route, in which host proteases such as cathepsin L, TMPRSS2, and TMPRSS11D (airway trypsin‐like protease) cut the S protein at the S1/S2 cleavage site to activate the S protein for membrane fusion. 101 Interference with this process by targeting these proteases could become an attractive strategy for combating CoV infections. A recent study confirms the role of TMPRSS2 for the viral life cycle in SARS‐CoV‐2‐infected VeroE6 cells. 5 Furin (a serine endoprotease) activates MERS‐CoV to initiate the nonclathrin mediated membrane fusion event. 102\nThe neurotransmitter receptor blockers chlorpromazine (15), promethazine (16), and fluphenazine (17; Figure 7), were reported to inhibit MERS‐CoV and SARS‐CoV‐1 most probably by impeding S protein‐induced fusion. 103 Chlorpromazine, a clathrin‐mediated viral entry inhibitor, was already described to inhibit human CoV‐229E, hepatitis C virus, infectious bronchitis virus, as well as mouse hepatitis virus‐2 (MHV2). 104 , 105 , 106 , 107 , 108\nFigure 7 Neurotransmitter inhibitors targeting clathrin/nonclathrin pathways Matsuyama et al. identified the commercially available serine protease inhibitor camostat (18; Figure 8) to be a SARS‐CoV‐1 inhibitor, blocking TMPRSS2 activity at 10 µM. However, at a higher concentration (100 µM), inhibition of viral entry via SARS‐CoV‐1 S protein‐mediated cell fusion never exceeded 65% (inhibition efficiency), indicating that 35% of entry events take place via the endosomal cathepsin pathway. Interestingly, treatment with a combination of EST (a cathepsin inhibitor) and 18 resulted in remarkably blocked infection (\u003e95%) activity of pseudotyped viruses. 109\nFigure 8 Inhibitors targeting TMPRSS2. TMPRSS, transmembrane serine protease A similar approach has been investigated to prevent viral entry of SARS‐CoV‐2. Pöhlmann et al. reported the attainment of full inhibition efficiency with a combination of both 18 and E‐64d (a cathepsin inhibitor). Both studies indicate that SARS‐CoV‐1 and 2 enter cells in a similar manner showing the potential of 18 as a candidate for further development. 15\nRecently, K11777 (19; Figure 8), a cysteine protease inhibitor, was shown in tissue cultures to inhibit SARS‐CoV‐1 and MERS‐CoV replication in the subnanomolar range. 110 , 111 Future tissue culture and animal model studies should be conducted to clarify, whether its antiviral activity is mediated by targeting TMPRSS2.\nTeicoplanin is a glycopeptide antibiotic used to prevent infections with Gram‐positive bacteria like methicillin‐resistant Staphylococcus aureus and Enterococcus faecalis. It was found that teicoplanin inhibits the entry of SARS‐CoV‐1, MERS‐CoV, and Ebola virus by specifically targeting cathepsin L. 112 This knowledge has also been used to block the entry of new SARS‐CoV‐2 pseudoviruses with an IC50 value of 1.66 µM. Therefore, teicoplanin could be considered a potential candidate for the treatment of COVID‐19. 113\n\n2.4 Small‐molecules as cathepsin L inhibitors\nHuman cathepsin L is a cysteine endopeptidase and plays a key role for infection efficiency by activation of the S protein into a fusogenic state to escape the late endosomes. Targeting this protease with small molecules could interfere with virus‐cell entry and therefore be a possible intervention strategy for CoV infection. 114 Bates et al. identified MDL28170 (20; Figure 9) as an antiviral compound that specifically inhibited cathepsin‐L‐mediated substrate cleavage, with an IC50 value of 2.5 nM and EC50 value in the range of 100 nM. However, despite its potent inhibitory activity, no cytotoxicity data for 20 is currently available. 115\nFigure 9 Cathepsin L inhibitors with antiviral activity Diamond et al. reported CID 16725315 (21) and CID 23631927 (22; Figure 9) as viral entry inhibitors of SARS‐CoV in a cathepsin L inhibition assay. Compound 21 could block cathepsin L with an IC50 value of 6.9 nM, while 22 showed slightly weaker potency (IC50, 56 nM). Compound 22 was also found to inhibit Ebola virus infection (EC50, 193 nM) of human embryonic kidney 293T cells. This compound did not show any sign of toxicity to human aortic endothelial cells up to 100 µM. This data offers a new promising point for the treatment of SARS and Ebola virus infections. 116\nScreening of ~14 000 compounds in a cell‐based assay resulted in the identification of SSAA09E1 (23; Figure 9) as inhibitor of cathepsin L proteinase, with an IC50 value of 5.33 µM. In a pseudotype‐based assay in 293T cells, the EC50 value of 23 was around 6.4 µM, and no cytotoxicity was detected below 100 µM. 48\nPhenotypic screening approaches led to the identification of several viral entry inhibitors. This approach has the advantage of finding cellular‐active compounds, providing information on drug solubility and cell uptake. 117 On the other hand, it is limited in terms of capacity compared to in silico target‐based screening. Hsiang et al. identified emodin (24; Figure 9), the active component from Polygonum multiflorum and Rheum officinale, could block the interaction of S protein with ACE2, with an IC50 value of 10 µM and an EC50 value of 200 µM in an S protein‐pseudotyped retrovirus assay using Vero E6 cells. However, the mechanism of action of this compound still needs to be determined. 118 Sarafianos et al. 48 found that SSAA09E3 (25), a benzamide derivative of 24, could prevent virus‐cell membrane fusion in pseudotype‐based and antiviral‐based assays, with an EC50 value of 9.7 µM, but a CC50 value of 20 µM indicates additional unknown cellular targets.\nVE607 (26) was identified among 50 240 structurally diverse small molecules to specifically inhibit SARS‐CoV‐1 entry into cells using a phenotype‐based screening. Its EC50 value was reported at 3.0 µM and it inhibited SARS‐CoV‐1 plaque formation with an EC50 of 1.6 µM. 119 Cathepsin inhibitor E‐64‐D (27) blocked MERS‐CoV and SARS‐CoV‐1 infection as well. 120 , 121"}