key: cord-0880547-2kfmgr7k authors: Gil, Carmen; Ginex, Tiziana; Maestro, Inés; Nozal, Vanesa; Barrado-Gil, Lucía; Cuesta-Geijo, Miguel Ángel; Urquiza, Jesús; Ramírez, David; Alonso, Covadonga; Campillo, Nuria E.; Martinez, Ana title: COVID-19: Drug Targets and Potential Treatments date: 2020-06-08 journal: J Med Chem DOI: 10.1021/acs.jmedchem.0c00606 sha: 258d280490ea476c44692ba10c5f7ea8385689fd doc_id: 880547 cord_uid: 2kfmgr7k [Image: see text] Currently, humans are immersed in a pandemic caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which threatens public health worldwide. To date, no drug or vaccine has been approved to treat the severe disease caused by this coronavirus, COVID-19. In this paper, we will focus on the main virus-based and host-based targets that can guide efforts in medicinal chemistry to discover new drugs for this devastating disease. In principle, all CoV enzymes and proteins involved in viral replication and the control of host cellular machineries are potentially druggable targets in the search for therapeutic options for SARS-CoV-2. This Perspective provides an overview of the main targets from a structural point of view, together with reported therapeutic compounds with activity against SARS-CoV-2 and/or other CoVs. Also, the role of innate immune response to coronavirus infection and the related therapeutic options will be presented. The coronavirus pandemic known as COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This is a highly pathogenic human coronavirus (CoV) first reported in Wuhan, China, where a pneumonia of unknown cause was detected in December 2019. 1 This novel CoV belongs to the Coronaviridae family, along with SARS-CoV and the Middle East respiratory syndrome coronavirus (MERS-CoV). The three of them are zoonotic viruses and have in common their ability to cause severe infection in humans, in contrast to other human CoVs (HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoVHKU1), which are responsible for mild respiratory tract infections. 2 Highly pathogenic CoVs are enveloped, positive polarity, single-stranded RNA betacoronaviruses, and their genomes encode non-structural proteins (nsps), structural proteins, and several accessory proteins. 3, 4 The publication of the genome sequence of SARS-CoV-2 5 has allowed researchers to determine that the new virus is closely related to SARS-CoV (82% sequence identity) and, to a lesser extent, to MERS-CoV. 6 As a starting point, this sequence identity could pave the way to the identification of druggable targets based on previous studies focused on SARS-CoV and MERS-CoV. 7, 8 Knowledge of the life cycle of CoVs is essential to achieve this aim ( Figure 1) . The SARS-CoV-2 infection process starts with the viral entry mediated by the interaction of the spike (S) glycoprotein with the host angiotensin-converting enzyme 2 (ACE2) receptor, 1 and cleavage of the S protein by the host transmembrane serine protease 2 (TMPRSS2) prior to the fusion to the host cell membrane. 9 Entry mechanisms of coronavirus were controversial 15 years ago. In early studies, a non-endosomal pathway was initially thought to be the CoVs mechanism to enter the host cell. In 2004, it was shown that SARS-CoV fused with the cellular surface after attaching the host cell membrane. 10 The nucleocapsids were then blurred after the virions lost their envelopes, and no endocytic-related events were described. However, recent evidence points to the endosomal pathway as the main entry route for CoVs to infect the cells. Remarkably, Ng et al. had published one year earlier a study with a SARS-CoV isolated from a SARS patient in Singapore. 11 They certainly observed fusion events at the plasma membrane, followed by a movement of spherical viral cores into the cytoplasm within large cellular vacuoles during the first 15 min after infection. In 2008, Wang and colleagues established the endocytic pathway as an alternative entry pathway apart from direct fusion with the plasma membrane based on their observations of SARS-CoV. 12 They showed that this virus enters the cell via a pH-and receptor-mediated endocytosis-dependent manner. In fact, the spike (S) protein itself or a pseudovirus bearing S protein induced internalization of SARS-CoV receptor ACE2 from the cell surface to cytoplasmic compartments. Furthermore, lysosomotropic drugs blocked the ACE2 receptor in vesicles, impairing their recycling to the plasma membrane. Pseudoviruses were also affected by inhibition of pH acidification, which indicates that SARS-CoV exploits the endocytic pathway to infect the cells, as they found, in a clathrin-and caveolin-independent manner. Currently, we are immersed in a pandemic caused by the emerging SARS-CoV-2, 13, 14 which is severely threatening the public human health care system worldwide. Some years ago, two other coronaviruses also crossed the species barrier, triggering deadly pneumonia in humans: SARS-CoV 15, 16 and MERS-CoV. 17 Similarities in the entry pathways of these betacoronaviruses need to be elucidated. Coronavirus entry relies on the spike (S) protein, and depending on the viral strain and cell type studied, the S protein is cleaved by several different cellular proteases. 18−24 SARS-CoV-2 presents entry requirements similar to those of SARS-CoV. Both viruses are coincident in the cellular receptor ACE2. Similar to SARS-CoV, SARS-CoV-2 infection is profoundly inhibited by lysosomotropic drugs (99% and 98%, respectively) in cells transduced with pseudovirus. 25 These results point out that the endocytic pathway is the preferred route for SARS-CoV-2 entry into the host cell. Furthermore, SARS-CoV and SARS-CoV-2 pseudovirions' entry is dependent on late endosomal compartments, given their dependence on endosomal acidification. Thus, the inhibition of endosomal maturation would result in the impairment of SARS-CoV-2 infection. In fact, pseudovirions' entry is impaired by treating cells with chemical inhibitors (YM201636 and apilimod) of the PIKfyve enzyme, involved in the phosphoinositides metabolism regulating endosomal maturation. Other viruses depending on PIKfyve enzyme described have been Ebola virus (EBOV) and African swine fever virus, both dependent on the late endosome for entry. 26 −28 SARS-CoV-2 infection is impaired by blocking other proteins characteristic of the late endosomal compartments, like two-pore channel 2 (TPC2) (but not TRMPL1), indicating that TPC2 is important for SARS-CoV-2 pseudovirions' entry. 25 Protease activation of S glycoprotein is crucial for coronavirus entry. Lysosomal cathepsins are necessary for SARS-and MERS-CoV entry via endocytosis. SARS-CoV-2 also requires cathepsin L for a successful infection since its inhibition decreased by 76% the entry of SARS-CoV-2 S pseudovirions, suggesting that cathepsin L should be crucial for SARS-CoV-2 S priming into lysosomes to enter the cells. Conversely, cathepsin B inhibition did not seem to have any effect. 25 Once the virus enters the host cell, it gets disassembled to release the nucleocapsid and the viral genome. Host ribosomes translate the open reading frame (ORF) 1a/b into two polyproteins (pp1a and pp1ab) that encode 16 nsps, while the remaining ORFs encode structural and accessory proteins. Two proteases participate in the cleavage of the polyproteins, the main protease (3CLpro, nsp5) and the papain-like protease (PLpro, nsp3), to produce nsp2−16 involved in the replication−transcription complex (RTC). 29 Some of those are the RNA-dependent RNA polymerase (RdRp, nsp12) and helicase (nsp13). In coronavirus, this process is followed by assembly of the virion components into the endoplasmic reticulum Golgi intermediate compartment complex and release from the infected cells by exocytosis. 30 In principle, all CoV enzymes and proteins involved in viral replication and the control of host cellular machineries are potentially druggable targets in the search for therapeutic options for SARS-CoV-2. In this Perspective, we will give an overview of the main virus-based and host-based targets from a structural point of view, together with reported therapeutic compounds with activity against SARS-CoV-2 and/or other CoVs. Also, the role of innate immune response to coronavirus infection and the therapeutic options based on this response will be presented. 2.1. Structural Proteins. The coronavirus structural proteins that form the viral particle are the spike (S) glycoprotein, envelope (E) protein, membrane (M) protein, and the nucleocapsid (N) protein ( Figure 2 ). These proteins are less conserved than nsps, playing important functions in the viral life cycle. Spike (S) protein has an important role in virus pathogenesis and organ tropism, being responsible for the viral entry through receptor recognition and membrane fusion. 31 The envelope (E) protein is the smallest of the structural proteins but has a crucial role in assembly, budding, envelope formation, and virulence. 32 The main function of the membrane (M) protein is to promote viral assembly due to its membrane-bending properties. 33 The nucleocapsid (N) protein is a multifunctional protein that packages the viral RNA genome into a ribonucleoprotein complex called nucleocapsid to protect the genome. 34 With regard to the search for new therapeutics based on structural proteins of SARS-CoV-2, some computational studies were carried out to identify the structure and function of E protein 35 and the other structural proteins. 36 We will focus our attention on the N protein recently crystallized (PDB codes: 6M3M 37 and 6VYO) and on the S protein due to its fundamental role in viral entry and its available structures that will allow structure-based drug design. 38, 39 2.1.1. Nucleocapsid (N) Protein. As mentioned above, coronavirus N protein is a multifunctional RNA-binding protein considered to be an interesting pharmacological target that merits further attention due to its critical function in viral RNA transcription and replication. 40 This major CoV protein contains two highly conserved domains, an N-terminal RNAbinding domain and a C-terminal dimerization domain, together with a disordered central Ser/Arg-rich linker. Previous studies revealed that the N-terminal domain is responsible for RNA binding, the C-terminal for oligomerization, and the Ser/ Arg-rich linker for primary phosphorylation. 41−43 The crystal structure of SARS-CoV-2 nucleocapsid N-terminal domain has been solved (PDB code: 6M3M), 37 showing an overall similarity with the same domain from other CoVs, although the surface electrostatic potential showed a specific distribution. These important structural findings will significantly stimulate the drug discovery of ligands focused on this appealing target to block coronavirus replication and transcription. The success in the development of compounds that interfere with N proteins of other CoVs, such as the recent discovery of stabilizers of the protein−protein interaction of MERS-CoV N protein, 44 reinforces the potential of the N protein as druggable target for SARS-CoV-2 infection. Remarkably, the N protein is highly immunogenic and is being considered as a potential vaccine target and for the development of COVID-19 diagnostic methods. 45, 46 2.1.2. Spike (S) Glycoprotein. The S glycoprotein is a structural transmembrane protein of about 1200−1400 amino acid residues per monomer located on the outer envelope of the virion. As has been observed for other viruses of the Coronaviridae family, 47 it mediates virus entry by contacting specific host-receptors located on the surface of the cell. Host− guest recognition is virus-specific, and the specificity and selectivity of this process determine both (i) virus tropism and (ii) pathogenesis. 25, 39 In its functional form, S protein assembles as a homotrimer. Each of the three monomeric units are formed by two functional groups, the S1 subunit which is responsible for host recognition and the S2 subunit which guides host−guest membrane fusion. 39 The S1 domain is characterized by an N-terminal domain (NTD) and a Cterminal domain (CTD) (Figure 3 ). The former can recognize carbohydrate moieties and is considered a derivation of ancestral sugar-based recognition domains, while the latter, also known as receptor-binding domain (RBD) or S B , mediates host−guest interaction and promotes virus entry by recognizing protein receptors of the infected organism. In particular, the region directly involved in the recognition process is defined as the receptor-binding motif (RBM). A C-terminal, transmembrane domain in the S2 subunit connects the S glycoprotein to the virus membrane. Several conformational states of S exist in a dynamic equilibrium between them: the uncleaved, S0; the cleaved, prefusogenic S1/S2; and the post-fusogenic forms. S protein is generally biosynthesized in an uncleaved, fusion-incompetent . Structural diagrams of spike glycoproteins of SARS-CoV-2. The spike protein contains an S1 subunit and an S2 subunit, which are divided by the S cleavage sites. Abbreviations: FP, fusion peptide; HR, heptad repeat 1 and heptad repeat 2, RBD, receptor-binding domain, containing the core binding motif in the external subdomain; SP, signal peptide. S0 form, which is then cleaved and activated by proteases. S proteins of less pathogenic CoV are initially exposed uncleaved (S0) on the virus membrane, and their activation is later promoted by host proteases. Conversely, S proteins of the highly pathogenic SARS-CoV-2 are cleaved during protein egress and, thus, are exposed on the virion in its cleaved (S1/ S2) form 48 (Figure 4 ). In the S1/S2 form, a dynamic equilibrium between open ("up", U) and closed ("down", D) conformations characterizes the three RBD domains of the S protein ( Figure 5 ). The receptor-binding site of S is generally occluded when the RBD is in "down" conformation and makes extensive contacts with the NTD of S1. Significant conformational modifications are required for "down-to-up" conversion. A masking and 3D sorting strategy applied on SARS-CoV by Kirchdoerfer et al. 49 suggested that ACE2 does not directly participate in down-toup conversion of S protein and RBD. It is more likely that ACE2 binds an S-RBD in an already "up" conformation. So, according to these data, the S protein of SARS-CoV should sample 56% of the triple "down" and 44% of the single "up" S1 RBD; no double or triple "up" S1 RBD conformations have been observed. Introduction of some stabilizing mutations at positions 968 and 969 of the S protein seems to move the equilibrium toward the open forms: 58% of the single "up", 39%/3% of the double/triple "up", and 0% of the triple "down" S1 RBD. Although intriguing, the exact role and the significance of the dynamic equilibrium of S1 RBD remain unclear. In this regard, it might be interesting to compare with the more pathogenic SARS-CoV-2, but data are not yet available. This strategy has proved useful to produce pre-fusion stabilized S protein for structure-based drug discovery approaches focused on direct inhibition of the interaction between S-RBD and the host protein ACE2 and has been also applied on SARS-CoV-2. 38 Priming of the S protein could occur in several ways, depending on the (guest) type of CoV and the pool of available (host) proteases involved in the event. 39 As previously introduced, the pre-fusion conformation is originated by the proteolytic cleavage of the S1/S2 site, catalyzed by serine-proteases like furin, cathepsin L, or TMPRSS2 in . Schematic diagram of SARS-CoV S2-mediated membrane fusion. In the receptor-binding stage, S protein, which exists as a trimer, binds to the cellular receptor ACE2 via S1-RBD. Representation of the open "up" (PDB code: 6VSB) and closed "down" (PDB code: 6VXX) states for the pre-fusion S1/S2 glycoprotein of SARS-CoV-2. correspondence of the (R/K)-(2X)n-(R/K) motif of S. 50 Given their undoubted role in virus entry by S priming, all these proteases could represent valuable targets for effective antiviral therapies based on protease inhibition or modulation and are presented in different sections of this Perspective. Sequence analyses for this region within CoV of the same clade revealed a significant heterogeneity, posing the bases as the explanation for the higher pathogenicity of SARS-CoV-2. Accordingly, a major exposure of the furin-like (S1/S2) cleavage site and, thus, an efficient priming of S protein would derive from insertion of polybasic amino acids at the S1/S2 cleavage site for SARS-CoV-2. 48 Conversely, the S protein of SARS-CoV is inefficiently cleaved by furin at the S1/S2 cleavage site during biosynthesis due to the low exposure of the cleavage site. Unlike SARS-CoV-2, which is exposed in an already cleaved prefusion form, the uncleaved (S0) form seems to be predominant in the case of SARS-CoV, leading to a lower pathogenicity profile. Conformational changes induced by the first cleavage are not sufficient to promote host−guest membrane fusion, which seems to be finally induced by a second cleavage event at the S2′ site ( Figure 4) . 39, 51 This event appears to be subordinated by recognition of attachment host receptors at the level of the S1 domain. Coronaviruses use different regions of the S1 subdomain to bind the host cell. In SARS-CoVs, recognition is mediated by the RBD of S, which binds an N-terminal α-helical region (peptidase domain) of the ACE2, which is accommodated in a concave region, named a receptor-binding motif (S-RBM, highlighted in violet in Figure 6 ), of the S-RBD. 52 A close look at the contact region for SARS-CoV and SARS-CoV-2 with ACE2 revealed a different interaction pattern among the two viruses. 53 In the case of SARS-CoV-2, a total of 18 residues of the RBD contacting 20 residues of the ACE2 can be detected, whereas a total of 16 residues of the SARS-CoV RBD contact 20 residues of the ACE2. The main polar contacts are reported in Figure 6 . Interestingly, two additional polar interactions, consisting on one hydrogen-bonding interaction between Q439 of S-RBM and E35 of ACE2 and one salt bridge between K417 of S-RBM and D30 of ACE2, can be observed in the central part of the S-RBM−ACE2 surface, as a clear consequence of the different electrostatic profiles envisaged for the two CoVs. These additional contacts are facilitated by the deeper insertion of the peptidase domain of ACE2, 54 as a consequence of rearrangement of the receptorbinding ridge (residues 482−485 of S-RBD). The slight reorientation of the peptide domain (PD) of ACE2 also allows a better stabilization of two other hotspots, Q493(S-RBM)− E35(ACE2) and G496(S-RBM)−K353, previously identified as critical in the stabilization of the complex. 55 No polar contacts can be observed in the central region of the S-RBM−ACE2 interface for SARS-CoV, where K417 is replaced by a valine residue. Taken together, these findings can shed some light on the reasons for the pandemic behavior of the new SARS-CoV-2. More factors need to be examined in the attempts to properly characterize the virus's pathogenicity. In this direction, a novel route for virus entry, involving the immunoglobulin-like host protein CD147, well known to "open the door" to Plasmodium falciparum, the etiological agent of malaria, 56 has been proposed by Wang et al. 57 Preliminary in vitro antiviral tests indicated that meplazumab, an anti-CD147 humanized antibody, could significantly inhibit virus entry. Formation of the CD147−S-RBD complex was then confirmed by coimmunoprecipitation and ELISA tests. Another distinctive trait of obligate parasites such as SARS-CoVs is high-level post-translational modifications. Viral glycans have been proposed to exert a pivotal role contributing to virus functionality and immune selection, and thus to its pathobiology. 58 Viral O-and N-glycosylation, with this last generally occurring on asparagine included in the Asn-X-Ser/ Thr sequences, extensively characterizes envelope glycoproteins of human immunodeficiency virus (HIV-1), 59 EBOV, 60 and hemagglutinin 61 in influenza virus (IFV). These modifications, originated from selective pressure induced by immune evasion, could contribute to regulate glycoprotein folding, assembly, and maturation. The extreme heterogenicity of the number and the distribution of these glycosylated sites makes it difficult to discern their exact role in the virus's life cycle. However, recent advancements on mass spectrometric and chromatographic techniques as well as the availability of recombinant glycosidases have allowed researchers to better study and analyze their impact on folding, structure, sorting, trafficking, and stability of glycoproteins. Glycosylation can help to stabilize protein folding by enhancing its solubility. Moreover, glycoproteins can also participate in intramolecular stabilizing interactions. Another interesting aspect observed for glycoproteins of coronaviruses is that N-glycosylation could help in shielding antigenic sites (as receptor-binding sites) to promote immune evasion. 62 This phenomenon, defined as "glycan shielding", could represent a critical factor during the design of vaccines. A direct comparison between S glycoprotein of SARS-CoV and SARS-CoV-2 revealed a different pattern of glycosylation, 63 which might contribute to the different profiles in trafficking and interaction with adhesion factors. Some of these differences in glycosylation sites correspondence to antibodyaccessible regions of the S protein, thus reinforcing the idea of their effect in hiding the vulnerable region of the protein. 64 Taken together, these aspects highlight the relevance of a probably overlooked aspectthe glycosylationduring vaccine design. For viral-based therapeutic options, S protein and its RBD represent a very interesting and intriguing target for antiviral research. The more efficient recognition of the ACE2 host protein, together with the expanded host cell tropism given by the existence of a potential new entry route mediated by CD147, highlight S protein as an even more important and central target for therapeutic intervention. In this direction, common and trusted antiviral strategies directed to S protein consist of inhibition of host recognition by acting on the S1 RBD and inhibition of the fusion process by acting at the level of the S2 subunit. Both of these effects could be exerted by small inhibitors, peptides, or human monoclonal antibodies, which are able to efficiently recognize these regions of the protein. 65 A good efficiency has been demonstrated by anti-ACE2 and other S1 human antibodies on SARS-CoV. 66, 67 The small inhibitor SSA09E2, a piperazine carboxamide derivative, 68 was also able to interfere with ACE2 recognition (Figure 7) . Systemic peptide mapping on SARS-CoV led to the discovery of the peptide CP-1, which binds with high affinity the heptad-repeat 2 region of S2 and interferes with the conformational changes leading to the 6-helix bundle formation, thus blocking the virus−cell fusion process ( Figure 7) . 69 Recently, a pan-coronavirus fusion inhibitor lipopeptide (EK1C4) has been designed, targeting a structure formed by two specific regions in the S2 subunit. 70 Collectively, all these strategies could be also applied to the discovery of inhibitors specifically designed to act on the SARS-CoV-2 S protein with a more selective and effective antiviral profile. 2.2. Non-structural Proteins (nsps). SARS-CoV-2, like other coronaviruses, has 16 nsps that are highly conserved and present different functions, including the formation of the RTC. The specific roles of most of the nsps have been reported, although the functions of some nsps remain elusive. 6, 29 Focused on reported studies on SARS-CoV-2, it is interesting to highlight the number of nsp structures of this virus available: the main protease (3CLpro, nsp5; PDB code: 6Y2E), 71 the papain-like protease (PLpro, nsp3; PDB code: 6W9C), the RNA-dependent RNA polymerase (RdRp, nsp12) in complex with cofactors nsp7 and nsp8 (PDB code: 6M71), 72 the methyltransferase-stimulatory factor complex of nsp16 and nsp10 (PDB code: 6W61), the complex nspP10-nsp16 (PDB codes: 6W75 and 6W4H), the nsp9-binding protein (PDB code: 6W4B), and the nsp15 endoribonuclease (PDB code: 6VWW). 73 Although any nsp could be exploited as a druggable target, 36 the availability of the crystal structure and described ligand, together with an essential role in viral infection, significantly increase the chances of success. On this premise, we will now focus on the two proteases (3CLpro and PLpro), the RNAdependent RNA polymerase (RdRp), and helicase (nsp13). 2.2.1. Proteases 3CLpro and PLpro. SARS-CoV-2's genomelike most of the Coronaviridae genomeencodes two large polyproteins, pp1a and pp1ab. 74 These polyproteins are cleaved and transformed in mature nsps by the two proteases 3CLpro (3C-like protease or main protease) and PLpro (papain-like protease) encoded by the ORF 1a/b 75 as mentioned above. Both proteins are crucial for virus replication and controlling the host cell response; therefore, they stand as key targets in the development of antiviral drugs. 3CLpro forms a dimer, and each monomer contains two regions, the N-terminal catalytic region and the C-terminal region. 76 The sequences of 3CLpro in SARS-CoV and SARS-CoV-2 share 96% identity, and the minimal differences between the two enzymes are at the surface of the proteins. Therefore, inhibitors against SARS-CoV 3CLpro are expected to inhibit SARS-CoV-2 3CLpro. Recently, the structure of 3CLpro (PDB code: 6Y2E) 71 has been resolved, confirming the high structural similarity between the two enzymes. During recent years, there has been a large development of small molecules, peptides, and peptidomimetics that are able to inhibit SARS-CoV or both SARS-CoV and MERS-CoV 3CLPro, or even both proteases 3CLpro and PLpro. 7, 77, 78 In Figure 8 , some of the most interesting compounds are classified by the proteases they inhibit. As 3CLpro is a cysteine protease, therefore covalent inhibitors have been developed ( Figure 8 ). It is worth mentioning a family of vinylsulfones that inhibit SARS-CoV in the nanomolar range. Indeed, Zhou et al. 79 published an in vivo study in which the combination of the vinylsulfone protease inhibitors and camostat was tested in mice infected with SARS-CoV. Survival of mice treated with the combination therapy significantly increased compared to the control group. Very recently, Yang's group has determined the crystal structure of SARS-CoV-2 virus 3CLpro in complex with an irreversible peptidomimetic inhibitor N3 (PDB code: 6LU7). 80 In addition, they have performed a combination of structurebased virtual and high-throughput screenings of different chemical libraries including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of 3CLpro. As a result, they have identified eight compounds with IC 50 in a range from 0.67 to 21.4 μM ( Figure 9 ). Some of them, such as disulfiram and carmofur, are FDAapproved drugs, while ebselen, shikonin, tideglusib, and PX-12 are currently in clinical trials or preclinical studies. Finally, based on a family of α-ketoamide inhibitors of MERS-CoV, Zhang et al. have designed and crystallized a new family of αketoamide inhibitors of the SARS-CoV-2 3CLpro, among them compound 13b with an IC 50 of 0.65 μM 71,81 ( Figure 9 ). Moreover, there are two SARS-CoV 3CLpro inhibitors approved for the treatment of other viral infections, lopinavir and ritonavir, which are being used in patients in the currently pandemic. 82 PLpro is a key target not only to inhibit viral replication but also to inhibit the dysregulation of signaling cascades in infected cells that may lead to cell death of neighboring, uninfected cells. 7 SARS-CoV-2 PLpro shares 83% sequence identity with SARS-CoV PLpro. That is not as high as it was with 3CLpro; however, the differing residues are located on the surface. It is thus very likely that the SARS-CoV PLpro inhibitors could also be active against PLpro of SARS-CoV-2. Very recently, the structure of PLpro of SARS-CoV-2 has been deciphered (PDB code: 6W9C), but to date, no compound able to inhibit it has been reported. 2.2.2. RNA-Dependent RNA Polymerase (RdRp). RdRp is a crucial enzyme in the coronavirus life cycle as well as in other RNA viruses. This enzyme is conserved in structure and function among viruses with RNA genomes belonging to different families. RdRp mediates the transcription and replication of the RNA genome during infection. The fact that this enzyme has no human counterpart, together with its essentiality for the virus's life cycle, improves its chances as a drug target for antiviral development, 83 as has been done in other viral infections. 84 As mentioned above, the replication−transcription complex in coronavirus is formed after cleavage of the polyproteins pp1a and pp1ab into nsps. Moreover, SARS-CoV RdRp requires nsp7 and nsp8 as cofactors to stimulate its polymerase activity. Also, the association with exoribonuclease/N7guanine cap methyltransferase nsp14 contributes to the formation of a macromolecular assembly for efficient nucleotide polymerization, proofreading, and cap-modifying. Combination of this multifunctional protein assembly with the helicase/RNA triphosphatase nsp13 and the 2′-O-methyltransferase nsp16 could coordinate the replication−transcription machinery of SARS-CoV. 29, 85 All these facts are partially elucidated with the structural characterization of some complexes: nsp10/nsp16 complex, 86 nsp14/nsp10 complex, 87 and RdRp with its cofactors nsp7 and nsp8. 88 The structure of SARS-CoV-2 RdRp in complex with cofactors nsp7 and nsp8 has been recently deposited in the Protein Data Bank (PDB code: 6M71). 72 This structure will significantly accelerate the structure-based drug design toward this essential target. Moreover, a 96% sequence identity between RdRp's from SARS-CoV and SARS-CoV-2 6 contributes to the translation of results with therapeutic agents from one virus to the other. Approved nucleoside analogues acting in other viruses have been successfully used against SARS-CoV and are now being used in the new coronavirus infection. 89 Molecular modeling studies using the structure of RdRp have identified well-known drugs such as ribavirin, remdesivir, sofosbuvir, galidesvir, and tenofovir as inhibitors of RdRp and potential therapies for COVID-19. 90 Of interest is the reported lack of activity of non-nucleoside analogues in SARS-CoV due to the absence of a hydrophobic pocket in the polymerase to allocate this class of compounds present in other viruses, such as HIV-1 or hepatitis C virus (HCV). 91 Remdesivir (GS-5734) (Figure 10 ), an adenosine analogue with broad antiviral spectrum in RNA viruses, has proven to be efficacious against different CoVs in vitro and in a mouse model of SARS-CoV infection. 92 During the COVID-19 pandemic, it has been evaluated against a clinical isolate of the new virus, 93 showing a half-maximal effective concentration (EC 50 ) of 0.77 μM and a selectivity index of 129.87 (Vero E6 cells). Moreover, the crystal structure of this drug with RdRp has been recently elucidated. 94 Furthermore, several clinical trials around the world have been approved to demonstrate its therapeutic potential in patients. 95 During the preparation of this manuscript, results from the first trial in China were published, and remdesivir was not associated with statistically significant clinical benefits for COVID-19 patients. 96 However, the FDA authorized the use of this intravenous antiviral drug for emergency treatment of COVID-19 patients. Remdesivir can be administered only to hospitalized patients with severe illness, defined as patients with low oxygen in blood or needing breathing assistance. 97 This drug initially showed efficacy for the treatment of Ebola disease, being in fact in clinical development for this devastating infectious disease. 98 The mechanism of action of this nucleoside prodrug is through the RdRp, by delaying chain termination in EBOV. In CoVs, remdesivir is able not only to inhibit the RdRp but also to evade the action of the exoribonuclease (nsp14). 99 This fact is of utmost importance, 100 because poor activities of some nucleosides such as ribavirin are attributed to their removal by the exoribonuclease. 101 Together with remdesivir, other broad-spectrum antivirals targeting RdRp were tested in SARS-CoV-2. The half-maximal effective concentration (EC 50 ) and half-cytotoxic concentration (CC 50 2.2.3. Helicase (nsp13). NTPase/helicase (nsp13) is a critical protein in the replication−transcription complex of CoVs that catalyzes the separation of duplex oligonucleotides into single strands in a nucleotide triphosphate (NTP) hydrolysis-dependent manner. Due to the fact that this protein is essential for RNA viral synthesis and one of the most conserved proteins in nidoviruses, it is considered an interesting target for drug development, and several chemical inhibitors have been reported. 103 Bananins are oligo-oxa-adamantanes that, after conjugation with vitamin 6 (pyridoxal), show antiviral activities ( Figure 11 ). Four derivatives, bananin, iodobananin, vanillinbananin, and eubananin, inhibited both the ATPase and the helicase activity of the nsp13 from SARS-CoV. Remarkably, these compounds also inhibited the replication of the virus through a process that occurs after the viral entry. 104 Another family of antivirals acting through the target enzyme are the 5hydroxychromone derivatives, 105 which were synthesized as bioisosteres of the previous reported aryl diketoacids. 106 A screen using a FRET-based helicase assay of the Maybridge Hitfinder chemical library allowed the identification of triazole SSYA10-001 ( Figure 11 ), which specifically blocks the helicase activity of nsp13 and also shows anti-SARS-CoV activity. Kinetic studies to determine the enzyme inhibition mechanism showed that SSYA10-001 acts as a non-competitive inhibitor of nsp13 with respect to nucleic acid and ATP substrates. 107 Further studies with this 1,2,4-triazole showed that it is also able to inhibit two other CoVs (MERS-CoV and mouse hepatitis virus). A putative binding pocket was identified corresponding to a conserved pocket in CoVs nsp13, where Y277 and K508 are the key residues to maintain the interaction. 108 At the time of writing of this manuscript, the structure of nsp13 full-length (PDB code: 6JYT) SARS-CoV is available. 109 So far, no structural information is available on the SARS-CoV-2 helicase, but due to the high sequence similarity (99.8%), the SARS-CoV structure available significantly encourages the search for drugs to treat COVID-19 acting through this target enzyme. Coronavirus entry is mainly achieved by the virus binding to the ACE2 host receptor in the cell surface in a receptormediated endocytosis pathway. Other molecules such as proteases activate the spike (S) protein and facilitate the fusion with the receptor and cell membrane, allowing entry into the host of viral RNA via an endocytic pathway. Host-targeted anti-SARS-CoV-2 agents presented here are based on the molecular study of virus entry, identifying key proteins involved in the process. Targeting host proteins may avoid different limitations frequent in antiviral research, probably offering a genetic barrier to viral resistance. 3.1. Angiotensin I Converting Enzyme 2 (ACE2) Receptor. Coronavirus entry is mainly produced by the virus binding to different host receptors in the cell surface. In SARS-CoV-2, ACE2 has been recently confirmed as the main virus receptor. 9 Therefore, inhibition or modulation of ACE2 represents one of the proposed host-based strategies for treatment of SARS-CoV-2. 110 Activity of ACE2 is related to the renin−angiotensin system (RAS), which is involved in the maintenance of blood pressure homeostasis, fluid, and salt balance in mammals. 111 While renin cleaves angiotensinogen to generate angiotensin (Ang) I, the angiotensin-converting enzyme (ACE) catalyzes the formation of Ang II, a critical signaling molecule, through the proteolytic cleavage of Ang I. Although not exclusive, 112 the activity of ACE has been considered to be of pivotal importance in the regulation of Ang II within the RAS. ACE2, a homologue of ACE, is a multifunctional zinc metalloprotease consisting of 805 amino acids, which can be functionally divided into (i) the amino-terminal catalytic domain and (ii) the carboxy-terminal domain. 110 The enzyme has been observed to negatively regulate RAS through the degradation of Ang II to the heptapeptide Ang 1−7. 112 ACE2 can be found in epithelial cells of lung, liver, and testis. 110 Unlike ACE, ACE2 populates the apical membrane of respiratory epithelial cells, by which infection can occur. ACE2 receptors have been also observed in nasal and mouth epithelial cells. 113 Interestingly, an enhanced expression of ACE2 receptors in the lungs has been correlated with age, thus partially explaining the higher viral load and severity of symptoms observed in older patients infected by SARS-CoV-2. 114, 115 The cryogenic electron microscopy (cryo-EM) structure of the full-length human ACE2 with the transporter B0AT1, with (PDB code: 6M17) or without (PDB code: 6M18) the RBD of the surface S glycoprotein of SARS-CoV-2, has been recently published. 52 The complex appears as a dimer of heterodimers with the collectrin-like domain (CLD) located among B0AT1 and the peptidase extracellular domain (PD) of ACE2 ( Figure 12 ). CLD consists of a small extra-cellular domain, a long linker, and a single transmembrane (TM) helix; the central region (the neck domain) between the PD and the TM helix constitutes the most relevant part of the dimerization surface. Here, coordination between the two dimers is mediated by extensive polar contacts such as R716′-D713, E639′-R710, N636′-Q653, and N638′-R652. A weaker interaction surface can be also observed between the two N-terminal PDs of ACE2, where Q175′ directly interacts with Q139. It was suggested that ACE2 is natively dimeric and thus able to bind two trimeric S proteins. 52 ACE2 was already known for mediating infection of the less pathogenic SARS-CoV, 116 in particular, by recognizing the receptor-binding domain of the S protein (S1-RBD) with an αhelical region located in the peptidase domain. D30, K31, and D35 have been identified as critical residues in the interaction with S1-RBD. 110 Their specific localization with respect to the S-RBD is shown in Figure 12 . More details about the S-RBD−ACE2 interaction pattern are given in the Spike (S) Glycoprotein section. Therapeutic strategies devoted to combat SARS-CoV-2 infection via ACE2 could involve the following: (i) Interfering with the dynamics of the virus−host ACE2− S-RBD interface. This can be achieved by using relatively small molecules able to disrupt or negatively affect the efficiency in the dynamic network of protein−protein interaction that guides viral entry. In this regard, 77 molecular candidates have been identified from the FDA database through an in silico-guided repurposing study, which combines replica exchange molecular dynamics and ensemble docking, although no experimental decrease of virus infection has been reported. 117 Furthermore, machine learning and ensemble docking simulations have also provided new different scaffolds able to interrupt the spike protein−ACE2 interaction. 118 (ii) Inhibiting the ACE receptor. Since myocardial injuries caused by SARS-CoV-2 might be related to ACE2, therapies based on the use of ACE inhibitors or angiotensin blockers might be used for organ-protection purposes. In that sense, telmisartan has been proposed as a real option for COVID-19 therapy, 119 imitates the human enzyme ACE2, which is used by the virus to enter cells. 110 The virus binds to soluble ACE2/ APN01, instead of ACE2 on the cell surface, which means that the virus can no longer infect the cells. At the same time, APN01 reduces harmful inflammatory reactions in the lungs and protects against lung injury due to acute respiratory distress syndrome (ARDS). In this regard, a recombinant human ACE2 (rhACE2; APN01, GSK2586881) has been tested on a small cohort of patients with ARDS, demonstrating encouraging positive results. 122, 123 In a very recent paper, researchers tested the potential of direct treatment with soluble human ACE2 to prevent the entrance of the virus to the host cells in engineered human kidney organoids. 124 In combination with other host-based or virus-based approaches, these strategies offer a vast reservoir of opportunities to combat SARS-CoV-2. Careful examination of their real therapeutic impact and a proper validation are needed to fully address their clinical validity. 3.2. Transmembrane Serine Protease 2 (TMPRSS2). Following receptor interaction, or in addition to it, different host proteases can activate the virus−host cell membrane fusion for subsequent genome delivery. The host cell surface transmembrane serine protease 2 (TMPRSS2) activates S protein present in the highly pathogenic human coronaviruses SARS-CoV and MERS-CoV. 125 Human TMPRSS2 is expressed in the epithelia of the gastrointestinal, urogenital, and respiratory tracts. 126 Cleavage of S protein by TMPRSS2 is preferred for coronavirus infection over other proteases, such as the endosomal cathepsins. 127 Recent research has confirmed that SARS-CoV-2 entry is facilitated by TMPRSS2 and the viral infection is decreased by the use of the protease inhibitor camostat. 9 Moreover, as viral infection is enhanced by TMPRSS2, the Vero E6 cell line overexpressing TMPRSS2 has been described as a useful pharmacological tool for SARS-CoV-2 research. 50 Last, but not least, TMPRSS2 is expressed in different cell types of lung tissue, increasing their vulnerability for SARS-CoV-2 infection. 128 TMPRSS2 has emerged as a useful drug target for antiviral drug discovery, 8 and the lack of influenza and coronavirus infection has been confirmed in TMPRSS2 knock-out mice. 125 COVID-19 may find a potential therapy among different repurposed drugs with inhibitory activity against TMPRSS2. At the moment, only camostat has shown in vitro activity against SARS-CoV-2, but other clinical drugs such as nafamostat and 4-(2-aminoethyl)benzenesulfonyl fluoride, all of them protease inhibitors, 129 may offer some therapeutic options for the pandemic ( Figure 13A ). Repurposing of the mucolytic agent called bromhexine, a TMPRSS2 inhibitor, has been also proposed for COVID-19 therapy. 130 Furthermore, transcriptional inhibition of TMPRSS2 has been proposed as a new therapeutic option. Using computational and experimental methods, estrogen and androgen-related compounds such as genistein, estradiol, and enzatulamide ( Figure 13B ) have been shown to reduce TMPRSS2 expression in different cell lines. 131 As TMPRSS2 expression in the human lungs seems to be modulated by estrogens and androgens, data suggest that the activation of estrogen pathways or inhibition of androgen pathways may be a new target for therapeutic clinical intervention for symptom amelioration in COVID-19 patients. 132 Currently, the crystal structure of TMPRSS2 is not available, and target-based drug discovery and design should be done using different homology models based on other well-known serine protease structures. 3.3. Furin. Recent studies have discovered a "furin-like cleavage site" (FCS) in the S protein of SARS-CoV-2 that may explain the high pathogenicity of the virus. 39, 48 Moreover, this highly cleavable site is not found in closely related CoVs. 133 Furin, a type 1 membrane-bound protease expressed in multiple tissues, belongs to the subtilisin-like proprotein convertase family. This family includes proteases with specific roles in the secretory pathway. The insertion of such cleavage sites in other CoVs, such as the infectious bronchitis virus, increased the pathogenicity, including neural symptoms in infected chickens. 134 As furin is highly expressed in lungs, it is very likely to be involved in SARS-CoV-2 infection, increasing its pathogenicity over other betacoronaviruses, as they lack this cleavage site. 51 Recently, it has been proposed that the FCS may be an important site of coronavirus evolution. In samples isolated from mild COVID-19 patients from Zhejiang Province, China, mutations appeared near FCS (F1−2) that affected the electrostatic distribution of the S protein surface and its structure, and thus its ability to bind to furin. 135 Experimental results in samples from those patients showed that furin had low protein expression levels in the lungs compared with other tissues, such as colon, glands, liver, and kidney. The FCS may contribute to SARS-CoV-2 infection of these organs. Recent reviews describe the close interaction between ACE2 and furin in the viral infection of SARS-CoV-2, 136 showing a potential relationship between furin activities of different populations and the different clinical scenarios of the SARS-CoV-2 infection. 137 Inhibition of furin with peptides and, more recently, with small molecules is a strategy pursued to arrest tumor growth, inflammation, and some viral and bacterial infections. 138 However, due to the pleiotropic role of furin-like enzymes in a large number of cellular processes, side effects are a concern. 139 Determination of the crystal structure of furin will aid the design of specific small molecules. 140, 141 For example, furin's unliganded form (PDB code: 5JXG) suggests activation by a substrate-induced mechanism, 142 while the recent complexes with substrate analogue inhibitors (PDB codes: 6EQV, 6EQW, 6EQX) 143 decipher some new pockets to be exploited by a next generation of furin inhibitors that may be a potential therapy for COVID-19. 3.4. Cathepsin L. Activation of SARS-CoV-2 spike protein by cleavage of proteases is a key step in viral infection. Different lysosomal cathepsins were relevant in human coronavirus entry through endocytosis. 79 In a recent study, it has been shown that only cathepsin L, and not cathepsin B or calpain, is involved in SARS-CoV-2 endocytosis entry. 25 Treatment of HEK 293/hACE2 cells with the cathepsin Lselective inhibitor SID26681509 reduced the entry of SARS-CoV-2 pseudovirus by more than 76%, underscoring the potential role of cathepsin L for priming of SARS-CoV-2 S protein in the lysosome. 9 Previously, the cathepsin L inhibitor named SSAA09E1 was revealed as a novel antiviral agent for SARS-CoV infection ( Figure 14) . 68 Cathepsin L inhibitors may be therapeutic options for COVID-19 also because they can prevent the progression of pulmonary fibrosis. 144 Furthermore, a synergistic effect may be also achieved by targeting simultaneously cathepsin L and TMPRSS2. 145 The main challenge for the design of specific cathepsin inhibitors is to obtain selectivity. In this sense, several computational methods have been developed to solve this problem using information from the known 3D structures. 146 Several crystallographic structures of human cathepsin L are available both in the apo form (PDB code: Journal of Medicinal Chemistry pubs.acs.org/jmc Perspective 4AXL) 147 and in complex with different nitrile molecules (PDB codes: 2YJC, 2YJB, 2YJ9, 2YJ8, 2YJ2) 148 or (PDB codes: 2XU5, 2XU4, 2XU3, 2XU1). 149 3.5. Adaptor-Associated Kinase 1 (AAK1) and Cyclin G-Associated Kinase (GAK). As already explained, the main entry pathway for SARS-CoVs is receptor-mediated endocytosis. AAK1 and GAK are host serine−threonine protein kinases that regulate intracellular viral trafficking during entry, assembly, and release of multiple unrelated RNA viruses such as rabies, Ebola, dengue, or hepatitis C virus. 150,151 AAK1 plays a key role in receptor-mediated endocytosis by specific phosphorylation of adaptor protein 2, which stimulates the binding to cargo proteins. GAK shares some biological functions with AAK1, being a key player in clathrin-mediated trafficking. GAK mediates the binding of clathrin to the plasma membrane and the trans-Golgi network. 152 Although several molecules have been synthesized to inhibit AAK1, none of these compounds have been optimized and developed as antiviral agents. 153 A rationale for repurposing a combination of the pan-kinase inhibitors sunitinib and erlotinib ( Figure 15 ) based on AAK1 and GAK inhibition has been proposed for treatment of dengue and Ebola patients in future outbreaks. However, these have not been included in current clinical trials until now. 154 Recently, baricitinib, a potent AAK1 and GAK inhibitor, has been proposed as an effective therapy for COVID-19, reducing the viral entry, although no experimental work has been done to prove its mechanism of action. 155 Moreover, as this compound targets also the janus kinase (JNK1/2), it may act also to reduce inflammation in these patients, increasing its therapeutic benefit for COVID-19. 156 However, some data coming from the clinical trial program used for baricitinib registration in Europe showed that the most significant side effect seen was a small increase in upper respiratory tract infections, which may be not well tolerated by COVID-19 patients. 157 Several crystal structures of AAK1 have been reported in complex with different inhibitors, such as the pan-kinase inhibitor K252a (PDB code: 4WSQ), 158 BIBF 1120 (nintedanib) (PDB code: 5TE0), and LKB1 (PDB code: 5L4Q), 153 offering useful tools for rational drug discovery and/ or optimization. The GAK 3D structure presents many different conformations when it is bound to nanobodies (PDB codes: 4C57, 4C58, 4C59) 152 or to gefitinib (PDB codes: 5Y7Z, 5Y80). 159 Recently, it has been discovered that AAK1 and GAK share cysteine residues (C193 and C190, respectively) at equivalent positions that may be targeted by covalent inhibition, offering a good opportunity to develop selective covalent inhibitors. 160 Both structures suggest possibilities for the development of selective AAK1 and GAK inhibitors for viral infections. Nowadays, repurposing marketed drugs or optimizing valuable hits such as 3,5-disubstituted pyrrolo[2,3-b]pyridines as potent AAK1 and GAK inhibitors could be a good strategy to prevent SARS-CoV-2 entry. 3.6. Phosphatidylinositol 3-Phosphate 5-Kinase (PIKfyve). In endocytosis, one of the molecules that regulates the dynamic process of endosome maturation is phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2). 161 This phospholipid is synthesized in the late endosome by PIKfyve, an enzyme with lipid and protein kinase activity. 162 PIKfyve plays a key role in several trafficking events associated with the endocytic pathway. 163 Very recently, a significantly reduced entry of SARS-CoV-2 pseudovirus on 293/hACE2 cells was found after the treatment with apilimod ( Figure 16) , a potent inhibitor of PIKfyve, 25 which was previously identified as an inhibitor of production of interleukins IL-12 and IL-23. 164 The same effect was also observed with YM201636 (Figure 16 ), another chemically diverse PIKfyve inhibitor. 165 Like apilimod, YM201636 also blocks viral entry and infection by other viruses like African swine fever virus or EBOV. 26, 27 These data suggest that PIKfyve is a suitable drug target to modulate infection by viruses that enter through endocytosis, including SARS-CoV-2. 166 At the moment, the 3D structure of this lipid kinase has not been determined yet. 3.7. Two-Pore Channel (TPC2). Among different channels in the endolysosomal system, the two-pore channels (TPC1−3) regulate the conductance of sodium and calcium ions across cellular membranes. 167 They are voltage-gated channels, and TPC2 is one of the major downstream effectors of PI(3,5)P2 opening after its binding with such a phosphoinositide. 168 These are involved in the regulation of endolysosomal trafficking and Ebola entry in the host cell. 169 The structure of human TPC2 has been reported using cryo-EM, providing useful structural information about the open, closed, and apo forms for TPC2 (PDB codes: 6NQ0, 6NQ2, and 6NQ1, respectively). 170 By virtual screening, dopamine receptor antagonists and selective estrogen receptor modulators (SERMs) have been recently identified as blockers of TPC2. Specifically, the dopamine antagonists fluphenazine and pimozide, together with the SERMs raloxifene, clomiphene, and tamoxifen, inhibit EBOV infection in experimental models ( Figure 17 ). 171 It has been recently shown that TPC2 plays a relevant role during SARS-CoV-2 infection, and a decrease of SARS-CoV-2 pseudovirus entry was demonstrated after treatment with tetrandrine, a potent calcium blocker used as a pharmacological tool. Altogether, TPC2 emerges as a druggable hosttarget for SARS-CoV-2 infection, and repurposing of dopamine antagonist such fluphenazine or SERMs as raloxifene merits being tested in clinical trials for COVID-19. Innate immune cells display an effective antiviral response to coronavirus infection. This response is based on detection of viral infection by using pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs). PRRs include C-type lectin-like receptors, Tolllike receptor (TLR), NOD-like receptor (NLR), RIG-I-like receptor (RLR), and free-molecule receptors in the cytoplasm, such as cGAS-STING, IFI16, and so on. 172 For RNA viruses such as coronavirus, viral genomic RNA or replication intermediates like double-stranded RNA (dsRNA) are recognized either by the endosomal RNA receptors TLR3 and TLR7 or by the cytosolic RNA sensor RIG-I/MDA5, among others. This recognition induces the activation of several signaling pathways, resulting in inflammation and initiation of cellular immune response. These pathways activate crucial transcription factors including interferon regulatory factor 3 (IRF3), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and AP-1. Consequently, these factors ultimately promote the production of type I interferons (IFN-I), inflammatory cytokines, and chemokines. 173 4.1. Viral Proteins Involved in Innate Immune Modulation. The immune response can be exaggerated, leading to an inordinate inflammatory reaction that causes severe clinical signs of COVID-19. These include pneumonia and lung inflammation, respiratory distress, and a lifethreatening cytokine storm and circulatory shock. 174 Other human CoVs have developed strategies to counteract the immune response, which still need to be deeply studied in SARS-CoV-2. 175 SARS-CoV-2 typically reduces IFN-I response and elevates the expression of IL-6 and IL1RA cytokines. 176 SARS-CoV and MERS-CoV infections also suppress IFN synthesis, 177, 178 which determines their virulence. 179 In this Perspective, we sum up the main CoVs proteins implicated in innate immune response. 4.1.1. Structural Proteins' Modulation of Immune Response. The SARS-CoV and MERS-CoV M protein binds TRAF3 to block its binding and subsequent nuclear translocation of TANK-binding kinase 1 (TBK1), which blocks IRF3-mediated signaling and inhibits type I IFN production. 180, 181 SARS-CoV N protein also interferes with the function of IRF3. 182 The N protein of SARS-CoV targets type I IFN synthesis at early recognition stages of innate immune signaling viral RNA infection. 183 Potentially, it may also act on other viral RNA recognition strategies of the host. The N protein of SARS-CoV also binds to the SPRY domain of TRIM25 and inhibits TRIM25-dependent RIG-I activation, thereby suppressing its ubiquitination and type I IFN production. 184 It was also described that purified SARS-CoV S protein induces an inflammatory response, possibly through TLR2 activation. 185 Finally, the SARS-CoV E protein is a viroporin that aids NLRP3 (NLR family) inflammasome, thereby secreting IL-1β. 32, 186 SARS-CoV-2 viral protein interactions and their role in acting against the immune system response are still unknown. However, the first protein−protein interaction studies have elucidated that N protein could target stress granule protein G3BP1, an essential antiviral protein which is known to induce innate immune response. 187 Indeed, this interactome identifies that N protein binds stress granule-related factors G3BP1/2, the mTOR repressors LARP1, and the kinases CK2. Stress granule induction depends on dsRNA recognition by a protein kinase R (PKR)-mediated pathway, which induces proinflammatory cytokines 188 (Figure 18 ). 4.1.2. Non-structural Proteins' Regulation of the Immune Response. Nsps and accessory proteins of HCoVs also have important roles in the modulation of innate immunity. SARS-CoV nsp1 affects IFN-dependent signaling, 189 and SARS-CoV PLpro domain (nsp3) protein inhibits IRF3 phosphorylation and nuclear translocation, thereby blocking type I IFN production. 190 Apart from its protease activity, SARS-CoV and MERS-CoV PLpro also has de-ubiquitinating activity. 191, 192 PLpro of SARS-CoV inhibits TLR7 signaling by removing K63-linked ubiquitin chains from TRAF3 and TRAF6. 193 A subsequent analysis showed that PLpro of both SARS-CoV and MERS-CoV also recognized another ubiquitin-like modifier, interferon-stimulated gene 15 (ISG15). In those cases, PLpro acts as a de-ISGylating enzyme. This domain downregulates mRNA levels of pro-inflammatory cytokines such as CCL5, IFN-β, and CXCL10. 194 Furthermore, it has been identified that the ADPribose-1-monophosphatase macrodomain encoded within nsp3 in HCoV-229E and SARS-CoV is responsible for suppressing IFN induction. 195 The SARS-unique domain encoded in nsp3 of SARS-CoV can also enhance a cellular E3 ubiquitin ligase called ring-finger and RCHY1, which leads to proteasomal degradation of p53. 196 Endonuclease U (EndoU) domain is encoded in nsp15, and it is an important component of CoVs RTC. EndoU activity prevents dsDNA recognition by PPRs such as MAD5, thereby evading early innate immune response. 197 CoVs also encode ribonucleases that counteract dsRNA antiviral response. This is the case of the exoribonuclease domain of nsp14, which modulates dsRNA levels and innate immune sensing. 198 Despite being dispensable in viral replication, HCoV accessory proteins get involved in processes such as cell proliferation, apoptosis, and interferon signaling. 199 SARS-CoV, ORF3b, and ORF6 are shown to interfere with IFN-β synthesis and IFN signaling by preventing IFN-β-induced activation of interferon-stimulated response element (ISRE), found in the promoter region of ISG. 182 SARS-CoV ORF3a induces TRAF3-mediated ubiquitination of apoptosis-associated speck-like protein containing a caspase recruitment domain, which activates NLRP3 inflammasome and NF-κB pathway. 200 The SARS-CoV ORF6 protein was shown to interact with nuclear pores and blocks p-STAT1 import into the nucleus, reducing innate immune responses and increasing pathogenesis. 201 The accessory proteins of MERS-CoV, ORF4a, ORF4b, and ORF5, could similarly suppress IRF3 nuclear translocation, 181 while P4a suppresses PKR-dependent stress response, an additional antiviral response. 202 Moreover, SARS-CoV ORF9b might also hijack a ubiquitin E3 ligase called AIP4 to trigger the degradation of MAVS, TRAF3, and TRAF6, thereby significantly suppressing IFN responses. 203 Indeed, SARS-CoV accessory protein P6 interacts with the IFN-signaling pathway-mediating protein Nmi and promotes its ubiquitin-dependent proteasomal degradation, thereby potentially modulating the virus-induced innate immune response. 204 SARS-CoV-2 protein−protein interaction maps have shown so far that nsp5 (3CLpro) could interact with the epigenetic regulator histone deacetylase 2 (HDAC2). 188 A previous study demonstrated that HDAC2 mediates inflammation and interferon response. 205 The same study showed that nsp13 interacts with two elements of the IFN signaling pathway, TBK1 and TBK1-binding protein 1 (TBKBP1), modulating the NF-κB inflammatory response. E3 ubiquitin ligase RNF41/ Nrdp1 is targeted by nsp15 protein, which would increase type I interferon production. 206 Two other E3 ubiquitin ligases, TRIM59 and MIB1, regulate antiviral innate immune signaling and are hijacked by ORF3a and nsp9. 207 ORF9c protein was found to modulate IκB kinase and NF-kB signaling pathway, and ORF9b interacts with a mitochondrial import receptor, Tom70, a linker between MAVS and TBK1/IRF3, inducing IRF-3 activation 188 ( Figure 18 ). Nsp3, ORF3b, and ORF6 of SARS-CoV-2with less homology with SARS-CoV pro-teinscould induce IFN-I sensitivity. 208 However, it has been described that truncated ORF3b of SARS-CoV-2 acts as an IFN antagonist more efficiently than does that of SARS-CoV. 209 These studies at the cellular level await further clinical or animal model investigations before the role of the immune response in this disease can be fully understand. 4.2. Therapeutic Targeting of the Innate Immune Response. It has been known that a cytokine storm results from an overreaction of the immune system in SARS and MERS patients. 210 Clinical findings showed exuberant inflammatory responses during SARS-CoV-2 infection, further resulting in uncontrolled pulmonary inflammation, likely leading to fatality. The repurposing of host-based therapeutics to control the immune response may counterattack COVID-19. 211 Some of these treatments include the use of recombinant IFN-α and IFN-β as antiviral cytokines that inhibit viral replication in targeted cells. Some studies have reported that IFN-β alone has more effect against SARS-CoV-2 than IFN-α in vitro. In fact, combinations of IFN-α and IFN-β with other antivirals such as ribavirin ( Figure 19 ) and/or lopinavir/ ritonavir (HIV treatment) ( Figure 9 ) have a synergistic effect in vitro 212,213 and in animal models. 214, 215 Recent results from an open-label, randomized, phase 2 trial (NCT04276688) conducted in China proved that the triple therapy (IFN/βlopinavir/ritonavir) is safe and more effective than lopinavir/ ritonavir alone in alleviating symptoms and shortening the duration of viral shedding in mild to moderate COVID-19 cases. 216 In vitro and in vivo studies show the protective effect of Type III IFN (IFN-γ) treatment against SARS-CoV infection 217,218 that possibly, in combination with IFN-I, could be an effective treatment for SARS-CoV-2. A recent study showed the potential benefits from low-dose corticosteroids treatment in SARS-CoV-2 critically ill patients. 219, 220 Another immunosuppressor, tocilizumab, a humanized monoclonal antibody against the interleukin IL-6 receptor, reduces pro-inflammatory response in COVID-19 patients. 221 Immunosuppressor treatments successfully used against other viruses could be also used for COVID-19. These would include JAK inhibitors such as tofacitinib, baricitinib, and the recently approved upadacitinib (previously used in rheumatoid arthritis), 222 blinatumomab, 223 and HDAC inhibitors, such as vorinostat or belinostat ( Figure 19B ,C). 224 Baricitinib (Figure 15 ), as mentioned before, is also a potent inhibitor of AAK1 and may also lead to a decrease in viral infectivity, making it a good candidate for clinical trials of COVID-19. 155 Another therapy that could control SARS-Cov-2 would be polyinosinic:polycytidylic acid (poly(I:C)), which is a synthetic analogue of dsRNA that induces type I IFN and decreases MERS-CoV load in BALB/c. 225 Nitazoxanide is another potent type I IFN inducer that has been used in humans as an antiparasitic agent ( Figure 20 ). It is a synthetic nitrothiazolyl−salicylamide derivative that exhibits broad-spectrum antiviral activities against both RNA and DNA viruses. 226 It is effective in the treatment of HCV and IFV-A and -B infection, currently in Phase II and Phase III clinical studies, 227, 228 and could be also tested in SARS-CoV-2 infection. This drug could inhibit expression of the viral N protein and pro-inflammatory cytokines (IL-6) in MERS-CoVinfected mice. 229 Other IFV antivirals could be useful in SARS-CoV-2 therapeutic treatments. Glycyrrhizin ( Figure 20) is an antioxidant, anti-inflammatory, immunomodulatory, and antiviral agent 230,231 that inhibits IFV-A infection through inhibiting pro-inflammatory gene expression. 232 Glycyrrhizin can suppress SARS-CoV infection at the early virus entry and the late replication stages in Vero E6 cells. 233 14-Deoxy-11,12dehydroandrographolide suppresses pro-inflammatory cytokines and chemokines as well as IFV-A replication. 234 NSC61610 (Figure 20) induces lanthionine synthetase C-like 2 (LANCL2), a therapeutic target for treating infectious diseases such as IFV-A infection. 235 The Ligustrum purpurascens extract phenylethanoid glycoside (LPG) induces IFN-γ, which can inhibit IFV-A replication in vitro and in vivo. 236 Some of these agents could be considered in clinical trials for treating COVID-19, although adverse effects need to be deeply studied. 237 Other strategies attempting to control host immune response in HCoV include cyclophilin-targeting drugs such as cyclosporin A and alispovir. SARS-CoV-2 uses not only ACE2 receptor for cell entry but also CD147, as described for SARS-CoV. 238, 239 CD147 receptor regulates cytokine secretion and chemotaxis of inflammatory cells through cyclophilin A and cyclophilin B. Another group of drugs with potential applications are other kinase inhibitors used in cancer treatment, e.g., imatinib mesylate, dasatimib, trametinib, saracatinib, etc. 240, 241 or the immunosuppressor mycophenolic acid. 242 Antiviral lopinavir/ritonavir ( Figure 9 ) has been recommended for clinical treatment for COVID-19. Recent results from clinical trials do not confirm any benefit in hospitalized adult patients with severe COVID-19, 243 but the combination of these two antivirals with interferon, as previously mentioned, is more promising. 216 Remdesivir ( Figure 10 ) is an adenosine analogue RdRp inhibitor with antiviral protection against SARS-CoV-2 93 and other viruses. 99 In addition, intravenous administration has been found to be efficacious in an American patient with COVID-19. 244 Based on these results, Gilead Company provided the compound to China to perform the first clinical trials in SARS-CoV-2-infected individuals (Clinical trials NCT04257654/6). As it has been mentioned before, the FDA has approved the emergency use of this drug for COVID-19 patients with severe symptoms. 97 Arbidol (umifenovir) is able to block viral fusion against IFVs. The antiviral activity of umifenovir against SARS-CoV-2 has been confirmed in vitro. 245 The first clinical data from patients with laboratory-confirmed COVID-19 points to a superior efficacy of umifenovir monotherapy over lopinavir/ ritonavit treatment. 246 Also, other drugs used for influenza, such as Avigan (favipiravir) ( Figure 10 ) and Tamiflu (oseltamivir), have been used in COVID-19 patients ( Figure 21 ). Importantly, in this pandemic, clinical trials have been started from the beginning ( Table 1) . The clinical management of the critically ill COVID-19 patient includes not only compounds targeting the viral replication but also therapeutic compounds seeking to modulate the immune dysregulation and the inflammatory storm that cause severe disease and frequent fatalities. Interestingly, some compounds have been shown to control hyper-activated immune response, and simultaneously, they are able to inhibit viral replication to some extent. Antiviral therapeutic options under clinical trials could be classified as follows: (a) Antivirals against viral components: EBOV viral polymerase inhibitor remdesivir (Figure 10 ), 93 developed for EBOV, IFV antiviral favipiravir (Figure 10 ), 90 and others developed for HIV or HCV, such as lopinavir/ritonavir (Figure 9 ), capable of inhibiting 3CLpro. (b) Antivirals developed against host targets: viral endocytosis inhibitors such as chloroquine and hydroxychloroquine ( Figure 22 ), which are drugs for malaria that decrease endosomal acidity, i.e., kinase inhibitors. (c) Anti-inflammatory drugs, corticosteroids, or immunosuppressors. A therapeutic combination (including a and c) has been selected for a randomized large clinical trial launched by WHO, called Solidarity, to collect clinical data from several thousand patients from dozens of countries. 95 Other antivirals used against viral RNA polymerase include ribavirin ( Figure 19 ) 214, 247 and galidesivir (BCX4430) (Figure 22 ). 248 Others are selected on the basis of their protease inhibition activity, such as disulfiram (a PLpro inhibitor) 249 or camostat mesylate (a TMPRSS inhibitor) (Figure 13 ), 116 or their spike protein inhibition, such as griffithsin. 250 Some other antivirals used are clathrin-mediated endocytosis inhibitors. This is the case of chlorpromazine ( Figure 22 ) and fluphenazine (Figure 17) , dopamine inhibitors which are FDA-approved drugs, 171 and miscellaneous compounds ( Figure 22 ) such as resveratrol, 251 gemcitabine, mefloquine, 240 and loperamide. 252 Losartan and, more recently, telmisartan have been used as ACE2 receptor inhibitors. Furthermore, ivermectin has recently shown SARS-CoV-2 inhibition in vitro. 253 As mentioned in the Introduction, a deep knowledge of the life cycle of SARS-CoV-2 is essential to identify druggable targets that will allow the development of effective therapeutics against this coronavirus. Through the journey across the life cycle of the virus presented in this Perspective, we have considered several virus-based and host-based targets as objectives for pharmacological intervention as well as the host immune response. Among the virus-based targets, the importance of the structural spike (S) protein is remarkable due to its key role in SARS-CoV-2 entry through the interaction with the host receptor ACE2. We have also reviewed structural druggable sites and determinants of antibodies' efficacy against S protein. This coronavirus has 16 nsps. Of special relevance for virus replication, and thus relevant as drug targets, are the two proteases (3CLpro and PLpro), the RNA-dependent RNA polymerase (RdRp), and the helicase. Those are highly conserved proteins and represent suitable targets for the development of pan-coronavirus antivirals. Study of the molecular basis of virus entry pointed to key cellular proteins involved in this process. This is the case of the already mentioned host receptor ACE2, host proteases like TMPRSS2, furin, or cathepsin L, or kinases that are implicated in the regulation of intracellular viral trafficking during endocytic entry, such AAK1, GAK, or PIKfyve. TPC2 is also an important host channel involved in the regulation of endolysosomal trafficking. Host immune modulation has proven to be a useful alternative for the clinical management of viral diseases lacking specific treatment. Moreover, targeting human proteins is an excellent alternative to avoid viral scape by mutation. Another promising alternative could be the combination of antiviral drugs acting through different targets in a multi-target strategy that has proven to increase efficacy and overall prevent viral resistance. Given the urgency of the COVID-19 pandemic, the repurposing of approved drugs is the only alternative to find a timely effective treatment. 254 In fact, drugs currently under clinical trials were initially approved for other indications. However, the past and present coronavirus outbreaks require our preparedness not only for the current situation but also for a future potential re-emergence of novel coronaviruses. In this sense, it is of utmost importance to design drugs acting as pancoronavirus antivirals or through a multi-target approach to avoid a lack of effectiveness by viral mutation escape. Addendum (May 29, 2020). Since the submission of this manuscript, the number of tridimensional structures determined for different SARS-CoV-2 proteins available in the PDB has been increasing dramatically. On average, in February 2020, a total of 4 structures were found deposited in the PDB, increasing by 99 in March, by 45 in April, and by 65 in May. As of late May 2020, a total of 214 structures were available in the PDB, although some of them have not yet been published (Table S1 ). Different experimental techniques in addition to X-ray diffraction have been used, including electron microscopy and NMR, and the structure of the SARS-CoV-2 main protease has been the most studied, with more than 136 different crystals deposited. This huge data explosion confirms the relevance of proteomic data in relation with the pandemic. As the number of PDB structures, as well as the compounds with potential antiviral action against SARS-CoV-2 being studied, will increase sharply in the next months, essential Web resources have been made available where the COVID-19 information that may be of utmost importance for medicinal chemists is continuously updated. These Web sites are summarized in Table S2 ■ ABBREVIATIONS USED AAK1, adaptor-associated kinase 1; ACE2, angiotensinconverting enzyme 2; Ang, angiotensin; ARDS, acute respiratory distress syndrome; 3CLpro, 3C-like protease or main protease; COVID-19, coronavirus infection disease (2019 pandemia); CoV, coronavirus; E protein, envelope protein; EBOV, Ebola virus; FCS, furin-like cleavage site; GAK, cyclin G-associated kinase; IFN, interferon; IRF3, interferon regulatory factor 3; ISRE, interferon-stimulated response element; M protein, membrane protein; MERS-CoV, Middle East respiratory syndrome coronavirus; N protein, nucleocapsid protein; nsp, non-structural proteins; NTD, Nterminal domain; ORF, open reading frame; PIKfyve, phosphatidylinositol 3-phosphate 5-kinase; PI(3,5)P2, phosphatidylinositol-3,5-bisphosphate; PLpro, papain-like protease; RAS, renin-angiotensin system; RBD, receptor-binding domain; RBM, receptor-binding motif; RdRp, RNA-dependent RNA polymerase; RTC, replication−transcription complex; S protein, spike protein; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SERMs, selective estrogen receptor modulators; TLR, Toll-like receptor; TMPRSS2, transmembrane serine protease 2; TPC2, two-pore channel 2 ■ REFERENCES SARS-beginning to understand a new virus Advances in MERS-CoV vaccines and therapeutics based on the receptor-binding domain A new coronavirus associated with human respiratory disease in China Genomic characterization of the 2019 novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan Learning from the past: Possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-nCoV Coronaviruses -drug discovery and therapeutic options SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor The life cycle of SARS coronavirus in Vero E6 cells Early events of SARS coronavirus infection in Vero cells SARS coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway A novel coronavirus from patients with pneumonia in China Identification of a novel coronavirus in patients with severe acute respiratory syndrome A novel coronavirus associated with severe acute respiratory syndrome Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein TMPRSS2 activates the human coronavirus 229E for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease The spike protein of the emerging betacoronavirus EMC uses a novel coronavirus receptor for entry, can be activated by TMPRSS2, and is targeted by neutralizing antibodies Middle East respiratory syndrome coronavirus infection mediated by the transmembrane serine protease TMPRSS2 Efficient multiplication of human metapneumovirus in Vero cells expressing the transmembrane serine protease TMPRSS2 Proteolytic processing of Middle East respiratory syndrome coronavirus spikes expands virus tropism Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV The phosphatidylinositol-3-phosphate 5-kinase inhibitor apilimod blocks filoviral entry and infection Ebola virus requires phosphatidylinositol (3,5) bisphosphate production for efficient viral entry Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling The cytoplasmic tail of the severe acute respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds COPI and promotes interaction with membrane protein Coronavirus spike proteins in viral entry and pathogenesis Role of severe acute respiratory syndrome coronavirus viroporins E, 3a, and 8a in peplication and pathogenesis A structural analysis of M protein in coronavirus assembly and morphology The coronavirus nucleocapsid is a multifunctional protein In-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel A guideline for homology modeling of the proteins from newly discovered betacoronavirus, 2019 novel coronavirus (2019-nCoV) Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein Recent insights into the development of therapeutics against coronavirus diseases by targeting N protein Modular organization of SARS coronavirus nucleocapsid protein Crystal structure of the severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona-and arteriviridae Crystallographic analysis of the N-terminal domain of Middle East respiratory syndrome coronavirus nucleocapsid protein Structure-based stabilization of non-native protein-protein interactions of coronavirus nucleocapsid proteins in antiviral drug design Preliminary identification of potential vaccine targets for the COVID-19 coronavirus (SARS-CoV-2) based on SARS-CoV immunological studies Evaluation of nucleocapsid and spike protein-based enzyme-linked immunosorbent assays for detecting antibodies against SARS-CoV-2 Mechanisms of coronavirus cell entry mediated by the viral spike protein The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2 Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor Structural basis of receptor recognition by SARS-CoV-2 Mechanisms of host receptor adaptation by severe acute respiratory syndrome coronavirus Basigin opens the door to malaria SARS-CoV-2 invades host cells via a novel route: CD147-spike protein Exploitation of glycosylation in enveloped virus pathobiology Differential processing of HIV envelope glycans on the virus and soluble recombinant trimer Mechanistic understanding of N-glycosylation in Ebola virus glycoprotein maturation and function Evidence for N-glycan shielding of antigenic sites during evolution of human influenza A virus hemagglutinin Emerging WuHan (COVID-19) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human CD26 Vulnerabilities in coronavirus glycan shields despite extensive glycosylation Potential antivirals and antiviral strategies against SARS coronavirus infections Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association The spike protein of SARS-CoV-a target for vaccine and therapeutic development Novel inhibitors of severe acute respiratory syndrome coronavirus entry that act by three distinct mechanisms Interaction between heptad repeat 1 and 2 regions in spike protein of SARSassociated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors Structure of the RNA-dependent RNA polymerase from COVID-19 virus Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2 Mechanisms and enzymes involved in SARS coronavirus genome expression From SARS to MERS: crystallographic studies on coronaviral proteases enable antiviral drug design Crystal structures of the main peptidase from the SARS coronavirus inhibited by a substrate-like azapeptide epoxide Synergistic inhibitor binding to the papain-like protease of human SARS coronavirus: mechanistic and inhibitor design implications Drug development and medicinal chemistry efforts toward SARS-coronavirus and Covid-19 therapeutics Protease inhibitors targeting coronavirus and filovirus entry Structure of M(pro) from COVID-19 virus and discovery of its inhibitors Hilgenfeld, R. alpha-Ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication: structure-based design, synthesis, and activity assessment Why Are Lopinavir and Ritonavir effective against the newly emerged coronavirus 2019? Atomistic insights into the inhibitory mechanisms Approved antiviral drugs over the past 50 years Imbert, I. One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2′-Omethyltransferase nsp10/nsp16 complex Structural basis and functional analysis of the SARS coronavirus nsp14-nsp10 complex Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors Therapeutic options for the 2019 novel coronavirus (2019-nCoV) Remdesivir, Sofosbuvir, Galidesivir, and Tenofovir against SARS-CoV-2 RNA dependent RNA polymerase (RdRp): A molecular docking study Expression, purification, and characterization of SARS coronavirus RNA polymerase Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir Race to find COVID-19 treatments accelerates COVID-19) Update: FDA Issues Emergency Use Authorization for Potential COVID-19 Treatment the PALM Writing Group. A randomized, controlled trial of Ebola virus disease therapeutics Coronavirus susceptibility to the antiviral Remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease Discovery of an RNA virus 3′->5′ exoribonuclease that is critically involved in coronavirus RNA synthesis Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics Favipiravir: pharmacokinetics and concerns about clinical trials for 2019-nCoV infection What we know but do not understand about nidovirus helicases The adamantane-derived bananins are potent inhibitors of the helicase activities and replication of SARS coronavirus 2,6-Bis-arylmethyloxy-5-hydroxychromones with antiviral activity against both hepatitis C virus (HCV) and SARS-associated coronavirus (SCV) Aryl diketoacids (ADK) selectively inhibit duplex DNA-unwinding activity of SARS coronavirus NTPase/ helicase Severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase Evaluation of SSYA10−001 as a replication inhibitor of severe acute respiratory syndrome, mouse hepatitis, and Middle East respiratory syndrome coronaviruses Delicate structural coordination of the Severe Acute Respiratory Syndrome coronavirus Nsp13 upon ATP hydrolysis Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target Trilogy of ACE2: a peptidase in the renin-angiotensin system, a SARS receptor, and a partner for amino acid transporters Tissue angiotensin II generating system by angiotensin-converting enzyme and chymase Single-cell RNA expression profiling shows that ACE2, the putative receptor of Wuhan 2019-nCoV, has significant expression in the nasal, mouth, lung and colon tissues, and tends to be co-expressed with HLA-DRB1 in the four tissues SARS-CoV-2: virus dynamics and host response ACE2: The key molecule for understanding the pathophysiology of severe and critical conditions of COVID-19: Demon or Angel? Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus Repurposing therapeutics for COVID-19: Supercomputer-based docking to the SARS-CoV-2 viral spike protein and viral spike protein-human ACE2 interface Screening of therapeutic agents for COVID-19 using machine learning and ensemble docking simulations Telmisartan as tentative angiotensin receptor blocker therapeutic for COVID-19 Hypothesis: angiotensin-converting enzyme inhibitors and angiotensin receptor blockers may increase the risk of severe COVID-19 COVID-19: Therapeutics and their toxicities Acute respiratory distress syndrome leads to reduced ratio of ACE/ACE2 activities and is prevented by angiotensin-(1−7) or an angiotensin II receptor antagonist Pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in healthy human subjects Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2 TMPRSS2 contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection Expression of transmembrane serine protease TMPRSS2 in mouse and human tissues Wild-type human coronaviruses prefer cell-surface TMPRSS2 to endosomal cathepsins for cell entry SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells TMPRSS2: A potential target for treatment of influenza virus and coronavirus infections Repurposing the mucolytic cough suppressant and TMPRSS2 protease inhibitor bromhexine for the prevention and management of SARS-CoV-2 infection Transcriptional inhibition of host viral entry proteins as a therapeutic strategy for SARS-CoV-2 Serendipity or opportunity for intervention? Cancer Discovery 2020 A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells The S2 subunit of QX-type infectious bronchitis coronavirus spike protein is an essential determinant of neurotropism Virus strain of a mild COVID-19 A review on the cleavage priming of the spike protein on coronavirus by angiotensin-converting enzyme-2 and furin 2019-nCoV-SARS-CoV-2 (COVID-19) infection: Cruciality of Furin and relevance with cancer Therapeutic uses of furin and its inhibitors: a patent review Optimization of substrateanalogue Furin inhibitors Structural studies revealed active site distortions of human Furin by a small molecule inhibitor X-ray structures of human furin in complex with competitive inhibitors Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism X-ray structures of the proprotein convertase Furin bound with substrate analogue inhibitors reveal substrate specificity determinants beyond the S4 pocket A novel cathepsin L inhibitor prevents the progression of idiopathic pulmonary fibrosis Targeting TMPRSS2 and Cathepsin B/L together may be synergistic against SARS-CoV-2 infection Ligand-induced conformational selection predicts the selectivity of cysteine protease inhibitors Optimization of triazine nitriles as rhodesain inhibitors: structure-activity relationships, bioisosteric imidazopyridine nitriles, and X-ray crystal structure analysis with human cathepsin L Halogen bonding at the active sites of human cathepsin L and MEK1 kinase: efficient interactions in different environments Systematic investigation of halogen bonding in protein-ligand interactions Anticancer kinase inhibitors impair intracellular viral trafficking and exert broadspectrum antiviral effects The serine/threonine kinase AP2-associated kinase 1 plays an important role in Rabies virus entry Structure of cyclin G-associated kinase (GAK) trapped in different conformations using nanobodies Synthesis and structure-activity relationships of 3,5-disubstitutedpyrrolo[2,3-b]pyridines as inhibitors of Adaptor-Associated Kinase 1 with antiviral activity Feasibility and biological rationale of repurposing sunitinib and erlotinib for dengue treatment Baricitinib as potential treatment for 2019-nCoV acute respiratory disease COVID-19: combining antiviral and antiinflammatory treatments Janus kinase inhibitor baricitinib is not an ideal option for management of COVID-19 Family-wide structural analysis of human Numb-associated protein kinases Structural basis for the inhibition of cyclin G-associated kinase by gefitinib Leveraging compound promiscuity to identify targetable cysteines within the kinome Dynamics of phosphoinositide conversion in clathrinmediated endocytic traffic PIKfyve: Partners, significance, debates and paradoxes PIKfyve regulation of endosome-linked pathways Apilimod inhibits the production of IL-12 and IL-23 and reduces dendritic cell infiltration in psoriasis YM201636, an inhibitor of retroviral budding and PIKfyve-catalyzed PtdIns(3,5)P2 synthesis, halts glucose entry by insulin in adipocytes Inhibition of PIKfyve kinase prevents infection by EBOV and SARS-CoV-2 Two-pore channels: Catalyzers of endolysosomal transport and function On the structure and mechanism of two-pore channels Ebola virus. Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment Structural mechanisms of phospholipid activation of the human TPC2 channel Mining of Ebola virus entry inhibitors identifies approved drugs as two-pore channel pore blockers Coronavirus infections and immune responses Human coronaviruses: A review of virus-host interactions Virology, epidemiology, pathogenesis, and control of COVID-19 Immune responses in COVID-19 and potential vaccines: Lessons learned from SARS and MERS epidemic. Asian Pac Imbalanced host response to SARS-CoV-2 drives development of COVID-19 Cytokine responses in severe acute respiratory syndrome coronavirusinfected macrophages in vitro: possible relevance to pathogenesis SARS coronavirus and innate immunity Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures Severe acute respiratory syndrome coronavirus M protein inhibits type I interferon production by impeding the formation of TRAF3.TANK.TBK1/IKKepsilon complex The structural and accessory proteins M, ORF 4a, ORF 4b, and ORF 5 of Middle East respiratory syndrome coronavirus (MERS-CoV) are potent interferon antagonists Severe acute respiratory syndrome coronavirus open reading frame (ORF) 3b, ORF 6, and nucleocapsid proteins function as interferon antagonists SARS-CoV nucleocapsid protein antagonizes IFN-beta response by targeting initial step of IFN-beta induction pathway, and its C-terminal region is critical for the antagonism The severe acute respiratory syndrome coronavirus nucleocapsid inhibits type I Interferon production by interfering with TRIM25-mediated RIG-I ubiquitination SARS coronavirus spike protein-induced innate immune response occurs via activation of the NF-kappaB pathway in human monocyte macrophages in vitro Severe acute respiratory syndrome coronavirus E protein transports calcium ions and activates the NLRP3 inflammasome G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response A SARS-CoV-2 protein interaction map reveals targets for drug repurposing Identification of residues of SARS-CoV nsp1 that differentially affect inhibition of gene expression and antiviral signaling Regulation of IRF-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme Proteolytic processing, deubiquitinase and interferon antagonist activities of Middle East respiratory syndrome coronavirus papain-like protease SARS coronavirus papainlike protease inhibits the TLR7 signaling pathway through removing Lys63-linked polyubiquitination of TRAF3 and TRAF6 MERS-CoV papain-like protease has deISGylating and deubiquitinating activities The ADP-ribose-1′'-monophosphatase domains of severe acute respiratory syndrome coronavirus and human coronavirus 229E mediate resistance to antiviral interferon responses regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1 Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication Mutagenesis of coronavirus nsp14 reveals its potential role in modulation of the innate immune response Accessory proteins of SARS-CoV and other coronaviruses Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC Middle East respiratory coronavirus accessory protein 4a inhibits PKR-mediated antiviral stress responses SARS-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the MAVS/TRAF3/TRAF6 signalosome Severe acute respiratory syndrome coronavirus protein 6 mediates ubiquitin-dependent proteosomal degradation of N-Myc (and STAT) interactor NOS1 inhibits the interferon response of cancer cells by S-nitrosylation of HDAC2 The E3 ubiquitin ligase Nrdp1 'preferentially' promotes TLRmediated production of type I interferon Tle1 tumor suppressor negatively regulates inflammation in vivo and modulates NF-kappaB inflammatory pathway SARS-CoV-2 is sensitive to type I interferon pretreatment SARS-CoV-2 ORF3b is a potent interferon antagonist whose activity is further increased by a naturally occurring elongation variant Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology Pyroptosis, a potential pathogenic mechanism of 2019-nCoV infection In vitro susceptibility of 10 clinical isolates of SARS coronavirus to selected antiviral compounds Ribavirin and interferon-beta synergistically inhibit SARS-associated coronavirus replication in animal and human cell lines Treatment with interferon-alpha2b and ribavirin improves outcome in MERS-CoV-infected rhesus macaques Treatment with Lopinavir/Ritonavir or Interferon-beta1b improves outcome of MERS-CoV infection in a nonhuman primate model of common marmoset Triple combination of interferon beta-1b, lopinavir-ritonavir Lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections Combined action of type I and type III interferon restricts initial replication of severe acute respiratory syndrome coronavirus in the lung but fails to inhibit systemic virus spread Potential benefits of precise corticosteroids therapy for severe 2019-nCoV pneumonia On the use of corticosteroids for 2019-nCoV pneumonia Tocilizumab treatment in severe COVID-19 patients attenuates the inflammatory storm incited by monocyte centric immune interactions revealed by single-cell analysis Cytokine storm syndrome: Looking toward the precision medicine era. Arthritis Rheumatol Cytokine release syndrome HDAC inhibitor reduces cytokine storm and facilitates induction of chimerism that reverses lupus in anti-CD3 conditioning regimen Rapid generation of a mouse model for Middle East respiratory syndrome Nitazoxanide: a first-in-class broad-spectrum antiviral agent Effect of nitazoxanide in adults and adolescents with acute uncomplicated influenza: a double-blind, randomised, placebo-controlled, phase 2b/3 trial Clinical trial: randomized, double-blind, placebo-controlled study of nitazoxanide monotherapy for the treatment of patients with chronic hepatitis C genotype 4 Nitazoxanide, a new drug candidate for the treatment of Middle East respiratory syndrome coronavirus Review of pharmacological effects of Glycyrrhiza sp. and its bioactive compounds Glycyrrhizin inhibits influenza A virus uptake into the cell Glycyrrhizin exerts antioxidative effects in H5N1 influenza A virus-infected cells and inhibits virus replication and pro-inflammatory gene expression Analgesic, antipyretic, anti-inflammatory and toxic effects of andrographolide derivatives in experimental animals 14-Deoxy-11,12-dehydroandrographolide exerts anti-influenza A virus activity and inhibits replication of H5N1 virus by restraining nuclear export of viral ribonucleoprotein complexes Lanthionine synthetase C-like 2 modulates immune responses to Influenza virus infection Anti-influenza virus effects of crude phenylethanoid glycosides isolated from ligustrum purpurascens via inducing endogenous interferon-gamma COVID-19: Immunology and treatment options Function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome coronavirus CD147 as a target for COVID-19 treatment: Suggested effects of Azithromycin and stem cell engagement Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection Antiviral potential of ERK/MAPK and PI3K/AKT/mTOR signaling modulation for Middle East respiratory syndrome coronavirus infection as identified by temporal kinome analysis Interferon-beta and mycophenolic acid are potent inhibitors of Middle East respiratory syndrome coronavirus in cell-based assays First case of 2019 novel coronavirus in the United States Arbidol combined with LPV/r versus LPV/r alone against Corona Virus Disease 2019: A retrospective cohort study Arbidol monotherapy is superior to lopinavir/ritonavir in treating COVID-19 Ribavirin and interferon therapy for critically Ill patients with Middle East respiratory syndrome: A multicenter observational study Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430 Disulfiram can inhibit MERS and SARS coronavirus papain-like proteases via different modes Activity of and effect of subcutaneous treatment with the broad-spectrum antiviral lectin griffithsin in two laboratory rodent models Effective inhibition of MERS-CoV infection by resveratrol Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture The FDA-approved drug Ivermectin inhibits the replication of SARS-CoV-2 in vitro Research and development on therapeutic agents and vaccines for COVID-19 and related human coronavirus diseases