3  PROTEASES AS A DRUG TARGETS
Papain‐like protease (PLpro), and, predominantly, Mpro are required for the proteolytic cleavage of polyproteins produced by the virus. Together they produce 16 nsp that are involved in viral replication and transcription. 122  PLpro is responsible for cleavage at the first three positions of its polyprotein to produce three nsp, while Mpro cleaves at no less than 11 conserved sites, releasing nsp4 to nsp16. Mpro‐mediated cleavage generates functional proteins like RdRP, RNA binding proteins, exoribonuclease, helicase, and methyltransferase. 123  The indispensable role of Mpro for the viral life cycle and infection process makes Mpro an ideal target for anti‐coronaviral therapy.

3.1  Mpro inhibitors

3.1.1  Structure and function of CoV Mpro
Mpro is a homodimeric cysteine protease. The SARS‐CoV‐1 Mpro consists of three domains: I (residues 8–101), and II (residues 102–184), which are β‐barrel domains that shape the chymotrypsin‐like structure, while domain III (residues 201–306) is made up by α‐helices. 124  The CoV Mpro active site uses a catalytic dyad (Cys145‐His41), in which cysteine acts as the nucleophile in the proteolysis while histidine behaves as general acid‐base. The peptide substrate or inhibitor binds in a cleft between domains I and II. 125
As far as the development of new therapeutics against SARS‐ and MERS‐CoV infection is concerned, efforts have mainly focused on protease inhibitors. These enzymes are highly attractive drug targets because they are so essential to the virus. Peptides, peptidomimetics, and even small molecules can inhibit them, which leads to markedly reduced viral transmission and pathogenicity. Although most of the reported molecules display only weak anti‐CoV activity, several of studies elucidated structure–activity relationships that can be used to further improve their activity. 100 ,  126 ,  127 ,  128

3.1.2  Substrate‐derived Mpro inhibitors
To date, no approved drugs or vaccines are available for treating a coronavirus infection. In a race to identify chemotherapeutic options, various approaches, such as chemical synthesis, testing of natural products, and virtual screening of compound libraries, have been used. The systematic design of inhibitors of CoV Mpro was essentially based on the enzyme's substrate. In general, a substrate can be transformed into a good inhibitor by modifying part of its sequence such that it binds to the catalytic cysteine in either a reversible or an irreversible manner. Peptide inhibitors are designed by attaching a reactive group (also known as warhead group) to peptides that mimic the natural substrate. The partial peptide substrate sequence for SARS‐CoV‐1 Mpro is mentioned in Figure 10, indicating the specific subsite of each amino acid residue.
Figure 10  A, SARS‐CoV‐1 Mpro partial substrate sequence. B, (Overlay) structures of SARS‐CoV Mpro inhibitors. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus [Color figure can be viewed at wileyonlinelibrary.com]

3.1.3  Inhibitors with Michael acceptor as a warhead group
The disclosure of the first crystal structure of the SARS‐CoV‐1 Mpro in complex with a peptidic inhibitor Cbz‐Val‐Asn‐Ser‐Thr‐Leu‐Gln‐chloromethyl ketone (also known as hexapeptide chloromethyl ketone; 28) 125  provided clues for the substrate‐based design. Although it is a substrate analog for the porcine transmissible gastroenteritis CoV (TGEV) Mpro, it offers a structural explanation for the P1‐Gln entering into the specific subsite S1 pocket and decreased P2‐leucine specificity in the hydrophobic S2 site of SARS‐CoV‐1 Mpro. Additionally, rupintrivir (29; AG7088), a peptidomimetic inhibitor of human rhinovirus 3C protease is oriented similar to inhibitor 28 in the binding pocket of TGEV Mpro. 129  These two molecules became prototype compounds for the development of SARS‐CoV‐1 Mpro inhibitors.
Compound 29 was only weakly active against SARS‐CoV‐1 Mpro (IC50, 800 µM) also in cellular antiviral assays. 130  However, systematic structural modifications led to a series of analogs that show moderate to good activity. 131  For example, compound 30 (Figure 11), in which the P1‐lactam was replaced by a phenyl ring, showed moderate activity. Compound 31, in which the larger P2 p‐fluorophenyl was replaced with a phenyl group, was even more effective. By taking 29 as a lead, Ghosh et al. designed new molecules mainly focusing on the replacement of the large P2 p‐fluorobenzyl group. Two of the resulting structures with P2‐benzyl (32) and prenyl (33) moieties showed decent inhibitory potencies at both enzymatic (K inact, 0.014 and 0.045 min−1, respectively) and cell‐based (IC50, 45 and 70 µM) assays. 132  Besides, no cytotoxicity was observed for these compounds up to 100 µM concentration. However, 32 and 33 were inactive at MERS‐CoV Mpro. 133  The same research group further modified the molecule with the introduction of P4 Boc‐serine, to establish additional hydrogen bond interactions as described in compound 34 (IC50, 75 µM). Unfortunately, the activity of the resulting compound was not improved. Further modification of the isobutyl group in compound 34 to isoprenyl group in compound 35 displayed potent activity with K i = 3.6 µM (Figure 11). 14
Figure 11  SARS‐CoV Mpro inhibitors containing Michael acceptor as a warhead group. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus On the other hand, Yang et al. 134  reported a series of peptide inhibitors with a greater inhibitory potency. In general, they systematically changed the backbone of inhibitor 29. As a result, they were able to identify more specific residues for each subsite (compounds 36–38; Figure 12): At first, the P1‐lactam ring was identified as a more specific moiety for the S1‐site, forming multiple hydrogen‐bond interactions with the enzyme as can be seen in the crystal structure (36); P2‐leucine showed a fourfold increased inhibitory activity when compared to the P2‐phenylalanine or ‐4‐fluorophenylalanine (37). A lipophilic tert‐butyl residue was recognized to be a better P3‐moiety than the P3‐valine (38). Finally, the replacement of P4‐methylisoxazole with a benzyloxy group was the best option for activity enhancement (compare 29 vs 36). They all showed moderate to high antiviral activity against HCoV‐229E in cell‐based assays.
Figure 12  Broad‐spectral antiviral compounds containing a Michael acceptor Shie et al. 131  reported another series of peptide inhibitors with comparatively reduced molecular weight to increase drug‐like properties. These pseudo‐C2‐symmetric inhibitors consist of a Phe‐Phe‐dipeptidic α,β‐unsaturated ester. One of these inhibitors (39) had an outstanding inhibitory activity with an EC50 value of 0.52 µM (see Figure 12). Besides, it displayed remarkable antiviral activity with an EC50 value of 0.18 µM. Structurally, the presence of 4‐dimethylamine on the phenyl ring was found to be crucial for activity enhancement.
Another peptidic drug with a Michael acceptor was N3 (40), which was reported to inhibit SARS‐CoV‐1 3CLpro (K i, 9.0 µM) by Yang et al. It was observed to be a broad‐spectrum antiviral compound, also inhibiting other CoVs, such as MERS‐CoV Mpro (IC50, 0.28µM), 135  HCoV‐229E, HCoV‐NL63, and HCoV‐HKU1 Mpro. 135 ,  136 ,  137 ,  138  It has also exhibited high antiviral activity in an animal model of infectious bronchitis virus. 137  The CC50 of 40 is greater than 133 μM.
SARS‐CoV‐2 shares only 82% of its genome with its relative SARS‐CoV‐1. However, essential viral enzymes of both species show sequence similarities of greater than 90%. 137 ,  139 ,  140 ,  141 ,  142  SARS‐CoV‐2 3CLpro is highly similar to SARS‐CoV‐1 3CLpro, sharing 96% of its sequence. Therefore, one could expect that SARS‐CoV‐1 Mpro inhibitors are active against SARS‐CoV‐2 Mpro. Compound 40 was found to be active against SARS‐CoV‐2 Mpro and its value of kobs/[I] for the COVID‐19 virus Mpro was determined to be 11 300 ± 880 M−1·s−1. 143  Peptide N3 was co‐crystalized with SARS‐CoV‐1 Mpro at 2.1 Å resolution (see Figure 13). Its binding mode to SARS‐CoV‐2 Mpro is highly similar to that of other CoV main proteases. Some key features include the Cys‐His catalytic dyad and the substrate‐binding pocket situated in a gap between domain I and II.
Figure 13  The crystal structure of COVID‐19 virus Mpro in complex with N3. (A) Representation of the dimeric Mpro‐inhibitor complex. (B) Surface representation of the homodimer of Mpro. Protomer A (blue), protomer B (salmon), compound N3 is presented as green sticks. (C) Schematic view of compound N3 (40) in the substrate‐binding pocket. 143  Mpro, main protease [Color figure can be viewed at wileyonlinelibrary.com] In general, inhibitors possessing a Michael acceptor group as a warhead moiety could form an irreversible (covalent) bond with the catalytic cysteine residue in the following manner (Figure 14): First, the cysteine residue undergoes 1,4‐addition at the inhibitor's Michael acceptor group (warhead). Rapid protonation of the α‐carbanion from His‐H+ leads to the covalent bond formation between the warhead of the inhibitor and the cysteine residue.
Figure 14  Mechanism of inhibitors with Michael acceptor group [Color figure can be viewed at wileyonlinelibrary.com]

3.1.4  Inhibitors with aldehyde as a warhead group
Although the above‐described inhibitors with 1,4‐Michael acceptors (e.g., α,β‐vinyl ethyl ester, –CH═CH–C(O)–OEt) showed enzymatic or cell‐based in‐vitro activities, they can be cleaved to their carboxylic acids by plasma esterases; for instance, AG7088 (29) was inactive in the plasma of rodents and rabbits. 144 ,  145  Therefore, scientists explored different reactive groups that are stable in vivo.
Based on the highly potent 1,4‐Michael‐acceptor‐based inhibitor 38, which they had previously developed (see Figure 15), Yang et al. 134  designed a peptide with a new efficient cysteine‐reactive group, using an aldehyde moiety. In addition, the P2‐leucine and the Michael groups of 38 were modified by a cyclohexyl unit and aldehyde group respectively to improve cellular activity. Indeed, the resulting peptide‐aldehyde 41 (Figure 15) showed remarkable activity against SARS‐CoV‐1 and HCoV‐229E Mpro. 134  It displayed promising antiviral activities decreasing viral load by 4.7 log (at 5 µM) for SARS‐CoV‐1 and 5.2 log (at 1.2 µM) for HCoV‐229E. This compound was stable in rat, mouse, and human plasma (even after 120 min, more than 70% of it remained in respective cells).
Figure 15  SARS‐CoV‐1 and MERS‐CoV Mpro inhibitors with peptide aldehyde functionality. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus Kumar et al. 146  reported another series of peptide‐aldehyde inhibitors with reduced molecular weight. Selected examples (42, 43) are depicted in Figure 15. They were potent, cell‐membrane permeable, dual Mpro inhibitors of SARS‐CoV‐1 and MERS‐CoV, without cytotoxicity (CC50 > 100 µM). Compound 43, in particular, revealed highly potent activity against SARS‐CoV‐1 Mpro (IC50, 0.2 µM) and MERS‐CoV Mpro (IC50, 1.7 µM). It displayed antiviral activity (EC50, 0.06 µM) lowering the viral load and the secretion of virus particles in MERS‐CoV‐infected cells. Also, it displayed broad‐spectrum antiviral activity against other human α‐ and β‐CoVs.
Akaji et al. discovered a series of SARS‐CoV‐1 Mpro inhibitors derived from its natural peptide substrate. Initially, they designed a pentapeptide (Ac‐Ser‐Val‐Leu‐N(CH3)2Gln‐CHO, 44) with Mpro inhibitory activity of 37 µM. 147  SAR studies of 44 led to inhibitor containing P1‐imidazole with improved potency (45; IC50, 5.7 µM). Further systematic structural modifications, primarily concentrating on P1‐, P2‐, and P4‐moieties, driven by X‐ray structure‐based analyses of the Mpro‐inhibitor complex, led to the identification of inhibitor 46 with remarkable inhibitory activity (IC50, 98 nM). The crystal structure of Mpro with 46 revealed significant binding interactions in the active site. The P1‐imidazole nitrogen atom created a hydrogen bond with the histidine residue's imidazole nitrogen, and the P2‐cyclohexyl moiety fitted well into the S2‐subsite. This compound was characterized as a competitive inhibitor without covalent bond formation.
The same research group disclosed a novel series of peptide inhibitors containing a decahydroisoquinoline moiety in place of P2‐cyclohexyl of 46 to reduce the peptidic nature of the inhibitors. A few examples (47–51) are shown in Figure 16. Among them, 49 was moderately more active against SARS‐CoV Mpro when compared to 46. 148  The X‐ray structure of Mpro in complex with 49 revealed that the P2‐decahydroisoquinoline moiety was fittingly placed in the S2‐subsite, while the P1‐imidazole moiety occupied the S1‐subsite. With these key residues located appropriately in their respective pockets, the terminal functional group fits tightly into the active site.
Figure 16  Peptide inhibitors containing cyclohexyl and decahydroisoquinoline groups [Color figure can be viewed at wileyonlinelibrary.com] This group further extended their study to find inhibitors that interact with S2 to S4 subsites. Taking 49 as a lead, they designed a new compound, by combining a nonprime substituent at the decahydroisoquinoline moiety, as shown in example 52. 149  The resulting 52 showed more than twofold increased Mpro inhibitory activity compared to 49. This indicates that the additional interactions at S2–S4 sites enhance inhibitory activity.
Rather recently, the same research group explored the ability of octahydroisochromene to interact with the hydrophobic S2 pocket as an innovative P2‐moiety. 150  To identify the best specific configuration, all possible diastereomers were evaluated. It was found that the molecule with (1S,3S)‐octahydroisochromene 53–56 could secure the optimal position of the P1‐imidazole as well as the aldehyde functional group at the active site. Additionally, the N‐butyl side chain attached to the 1‐position of the fused ring system was recognized to be important for establishing hydrophobic interactions.
In 2018, Groutas et al. 151  disclosed a novel class of dual MERS‐CoV and SARS‐CoV‐1 Mpro inhibitors that contain a P3‐piperidine moiety (58–59; Figure 17). These inhibitors were derived from the dipeptidic‐aldehyde bisulfite adduct 57 (GC376), which was clinically studied as a protease inhibitor for its efficacy against CoVs such as the feline infectious peritonitis virus (FIPV). Compounds 58 and 59 showed potent antiviral activity toward MERS‐CoV in cell‐based bioassays (EC50, 0.5 µM for 58 and 0.8 µM for 59). SAR studies revealed that the piperidine moiety engaged in favorable hydrophobic interactions at the S3 and S4 pockets of the protease.
Figure 17  Inhibitors with aldehyde, aldehyde bisulfite adduct, and epoxide warhead group The X‐ray crystal structures of MERS‐CoV 3CLpro in complex with inhibitor 59 showed that the piperidine ring is likely projecting toward the S4 subsite. Additionally, 59 was engaged in backbone H‐bonds with Gln192, Gln167, and Glu169.
Azapeptide epoxides (APEs) are another class of SARS‐CoV‐1 Mpro inhibitors, although they were originally developed for clan CD cysteine peptidases. 152 ,  153  The epoxide S,S‐diastereomer 60 (K inact/K i, 1900 (±400) M−1·s−1; Figure 17) exhibited the best inhibitory activity against SARS‐CoV Mpro. 154  The X‐ray structure of Mpro in complex with 60 confirmed the formation of a covalent bond between the cysteine‐S atom and the epoxide C‐3. It is worth noting that the S,S‐configured epoxide is required for the activity.
Very recently, Dai et al. designed and synthesized two novel peptidomimetic SARS‐CoV‐2 Mpro inhibitors 61 and 62 (Figure 18) which exhibited extremely high inhibitory activity on purified Mpro with IC50 values of 50 and 40 nM, respectively. Furthermore, the group observed high antiviral activity of both compounds in cell‐based assays (61: EC50, 0.42 µM; 62: EC50, 0.33 µM). X‐ray structures were determined for both derivatives in complex with SARS‐CoV‐2 Mpro at 1.5 Å, providing detailed information about the binding pockets. Similar to related molecules that employ the aldehyde moiety as a warhead, a covalent bond with the active‐site Cys145 was demonstrated for both structures. Cytotoxicity assays revealed CC50 values greater than 100 µM. 155
Figure 18  Peptidomimetic SARS‐CoV‐2 Mpro inhibitors with P3‐indole moiety. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus

3.1.5  Ketoamide inhibitors
Liu et al. reported dipeptidic α‐ketoamides as broad‐spectrum antiviral agents against the main proteases of human α and β‐CoVs as well as the 3C protease of enterovirus. The α‐ketoamide warhead group was promising, as it provides two hydrogen bond acceptors—one from the keto and one from the amide oxygen—whereas other warhead groups, such as Michael acceptor esters and aldehydes, provide only one hydrogen bond acceptor. Compound 63 was identified as SARS‐CoV Mpro inhibitor with an IC50 value of 1.95 µM (Figure 19). 156
Figure 19  Ketoamide inhibitors targeting SARS‐CoV‐2 Mpro. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus Taking 63 as a lead, aided by its X‐ray structure in complex with SARS‐CoV‐1, HCoV‐NL63, and coxsackievirus Mpros, systematic structural modifications were investigated, focusing on the P2‐moiety. As a result, the replacement of P2‐phenyl with P2‐cyclohexyl (64) was found to be the best substitution, while P2‐cyclopentyl (65) showed similar potency against the enzyme SARS‐CoV‐1 Mpro. In Huh7 cells, 64 also showed strong antiviral activity with an EC50 of 400 pM, but in Vero cells the antiviral activity of 64 was drastically reduced to 5 µM. This compound also exhibited antiviral activity against a range of enteroviruses in various cell lines.
Due to the high similarity between SARS‐CoV‐1 Mpro and SARS‐CoV‐2 Mpro authors speculated that 64 was likely to inhibit the new virus as well. Zhang et al. recently reported this molecule as a SARS‐CoV‐2 Mpro inhibitor with an IC50 value of 0.18 µM. They first resolved the unliganded crystal structure of SARS‐CoV‐2 Mpro (Figure 20), 157  which is largely identical to that of SARS‐CoV‐1 Mpro with a 96% sequence identity. Compound 64 was docked to SARS‐CoV‐2 Mpro, and a series of structural modifications were performed to improve its pharmacokinetic properties. Specifically, masking the P2‐P3 amide bond with the pyridone ring could improve plasma half‐life; and exchanging the lipophilic cinnamoyl residue for the less lipophilic Boc group, could increase plasma solubility and reduce its binding to plasma proteins.
Figure 20  Crystal structure of SARS‐CoV‐2 Mpro. 157  Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus [Color figure can be viewed at wileyonlinelibrary.com] Indeed, the resulting 66 had a ~3‐fold improved plasma half‐life in mice when compared to the lead 65 (from 18 min to 1 h). The in vitro kinetic plasma solubility has been increased by a factor of ~19 (from 6 µM for the lead to 112 µM for best derivative), and the thermodynamic solubility by a factor of ~13 (from 41 to 530 µM). Compound 66 also showed reduced binding to mouse plasma protein. However, compared to the lead (IC50, 0.18 µM), the structural modifications caused a reduction of activity against SARS‐CoV‐2 Mpro (IC50, 2.39 µM) and enteroviral 3 C proteases. Nevertheless, the introduction of a cyclopropyl group as in 67 instead of P2‐cyclohexyl enhanced the antiviral activity against β‐coronaviruses.
Compound 67 (Figure 19) inhibited purified SARS‐CoV‐2 Mpro with an IC50 of 0.67 µM. It also inhibited SARS‐CoV‐1 Mpro (IC50, 0.90 µM) and MERS‐CoV Mpro (IC50, 0.58 µM) with similar potency. It was effective against SARS‐CoV‐1 replication with an EC50 value of 1.75 µM. In SARS‐CoV‐2 infected human Calu3 cells, it inhibited the viral replication with an EC50 of 4–5 µM, when in fact the Boc‐unprotected 68 was inactive, suggesting a bulky hydrophobic group is necessary for cellular membrane penetration. On the other hand, increasing hydrophobicity of molecules should be pondered carefully, as it can increase plasma protein binding as it was described for 64. The pharmacokinetic properties of 67 revealed striking lung tropism and was suitable for inhalation in mice without any perceived adverse effects.
Compound 67 was cocrystallized with the enzyme in two different forms at 1.95 and 2.20 Å (Figure 21). The key feature observed from this crystal structure was that the inhibitor binds to the shallow substrate‐binding site at the surface of each protomer, between domains I and II. The thioketal that resulted from the nucleophilic Cys145 attacking the inhibitor, is stabilized by a H‐bond from His41, whereas the amide oxygen of 67 accepts a H‐bond from the main‐chain amides of Gly143, Cys145, and in part, Ser144 that make up the cysteine protease's canonical oxyanion hole. 157  The P1 lactam moiety is deeply embedded in the S1 pocket where the lactam nitrogen donates a three‐center H‐bond to the main chain oxygen of the Phe140 and the carboxylate of Glu166. The carbonyl oxygen forms a H‐bond to His163. The P2‐cyclopropyl moiety fits into the S2 subsite. The P3‐P2 pyridone moiety occupies the space normally filled by the substrate's main chain. The Boc group is not situated in the canonical S4 site, rather it is located near Pro168, which explains why the removal of the Boc group as in 68 weakened the inhibitory activity.
Figure 21  Crystal structure of 67 with SARS‐CoV‐2 Mpro. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus [Color figure can be viewed at wileyonlinelibrary.com]

3.1.6  Inhibitors with electrophilic ketone
It was envisioned that a fluorinated ketone moiety could be utilized as a warhead for targeting proteases, because it forms a thermodynamically stable hemiketal or hemithioketal after nucleophilic attack by Ser‐OH or Cys‐SH residues, which are present in the active sites of serine or cysteine proteases, respectively (see Figure 22).
Figure 22  Peptide inhibitors containing electrophilic ketone warheads [Color figure can be viewed at wileyonlinelibrary.com] Initially, Hayashi et al. reported a series of natural‐substrate‐derived peptide inhibitors containing a trifluoromethyl ketone warhead targeting SARS‐CoV‐1 Mpro. Compound 69 (Figure 22) was the best of the series with a K i value of 116 µM against SARS‐CoV‐1 Mpro. 158  It was sequentially modified mainly focusing on the warhead moiety since the formation of a cyclic structure prevented the nucleophilic attack by cysteine at the active site. This study led to the discovery of 70 containing a P1‐lactam and P1'‐thiazole moiety with a >50‐fold increase in inhibitory activity compared to 69. 159  Docking studies of 70 to Mpro highlighted key H‐bond interactions with backbone amino acid residues Cys143, Ser144, and Cys145. The nitrogen atom of the thiazole warhead moiety also engaged in H‐bond interactions, and the P1‐lactam nicely fitted into the S1‐pocket.
Continued computer‐assisted structural design led to a tripeptide containing benzothiazole as a warhead group and an m‐N,N‐dimethylaminophenyl group as P4‐moiety (71). 160  This compound was extremely potent in inhibiting Mpro of SARS‐CoV‐1 with a K i value of 3.1 nM. Docking studies of 71 confirmed that the benzothiazole group was tightly bound to the active site. Consequently, the same research group disclosed a series of dipeptides with reduced molecular weight in an attempt to improve drug‐like properties. The P3‐valine in the tripeptide 71 was exchanged for a variety of functional groups. 161  The study determined N‐arylglycyl to be the optimal P3‐moiety. Compound 72 displayed the best inhibitory activity. Docking studies of 72 to the protease highlighted the amino hydrogen of the P3‐N‐phenyl glycyl forming a H‐bond with backbone Glu166 of Mpro, in addition to the best P2‐leucine and P1'‐benzthiazole moieties (see Figure 23A). Further structural optimization at the P3‐N‐arylglycyl moiety found the indole‐2 carbonyl group to be one of the best P3‐moeities, thus reaching inhibitors with low nanomolar potency, for example 73 (K i, 0.006 µM) against SARS‐CoV‐1 Mpro. 162  Docking studies of compound 73 to the protease revealed that the indole amino hydrogen and the carbonyl group attached to the 2‐position formed H‐bond interactions with the backbone Glu166 (see Figure 23B). These interactions are of great importance, seeing as shifting the position of the carbonyl group from position 2 to 3, or replacing the indole with benzofuran drastically reduced inhibitory potency.
Figure 23  (A) Docking poses of 72 and (B) 73 with SARS‐CoV‐1 Mpro. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus [Color figure can be viewed at wileyonlinelibrary.com] Zhao et al. reported a series of trifluoromethyl ketones. Among them, 74, which has the same sequence as the peptide substrate from sites P1 to P4, exhibited moderate inhibitory activity with an IC50value of 10 µM. Inhibitor 74 also displayed time‐dependent inhibition (K i, 0.3 µM). 163
Zhang et al. described a series of dipeptides containing difluoromethyl ketone as SARS‐CoV‐1 Mpro inhibitors. Compound 75 displayed the best inhibitory activity in infected Vero and Caco‐2 cell cultures with an IC50 value of 2.5 µM. It also exhibited little toxicity. 164
A library of small peptide‐anilides was developed as anti‐SARS‐CoV‐1 Mpro agents (77–80; Figure 23). These inhibitors were basically designed from niclosamide (76) which was inactive at Mpro of SARS‐CoV‐1. Proper structural modifications led to the discovery of 77 (IC50, 0.06 µM). It behaved as a competitive, noncovalent inhibitor (K i, 0.03 µM). SAR investigations pointed out that the N,N‐dimethyl group on the phenyl ring, and electron‐withdrawing groups at the warhead phenyl are important. Structural modification of 77 resulted in compounds 78 – 80 displaying reduced potency. 165
A novel series of ketoglutamide tripeptides bearing a phthalhydrazido warhead group were identified as reversible SARS‐CoV‐1 Mpro inhibitors (81–84; Figure 24). 166  Among them, compound 83 showed the best inhibition (IC50, 0.6 µM). SAR studies revealed the presence of β and β'‐amino functionality adjacent to the keto and the intramolecular hydrogen bond to the carbonyl group made the keto center more electrophilic and inclined to build a hemithioacetal with Cys‐SH at the active site. Additionally, the hydrophobic P3‐benzyloxy moiety, the P1‐lactam, and the nitro group significantly contributed to the activity increment.
Figure 24  Small peptide anilides and ketoglutamide tripeptides as SARS‐CoV‐1 inhibitors. SARS‐CoV, severe acute respiratory syndrome coronavirus Wang et al. 167  described the development of selective and reversible SARS‐CoV‐1 Mpro inhibitors derived from HIV proteases inhibitors (Figure 25). The compound 85 as a SARS‐CoV‐1 Mpro lead inhibitor was continuously modified to obtain 86 and 87. These derivatives were highly selective toward SARS‐CoV‐1 Mpro versus HIV protease. Docking studies of 87 to Mpro demonstrated that both indole amino hydrogens establish H‐bond networks with side chain His142 and His41.
Figure 25  SARS‐CoV‐1 Mpro inhibitors derived from HIV proteases inhibitors. HIV, human immunodeficiency virus; Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus

3.1.7  Small molecule inhibitors of Mpro
Benzotriazole esters (88–91; Figure 26) were discovered as novel nonpeptidic irreversible inhibitors of SARS‐CoV‐1 Mpro. 168  Among them, 91 exhibited the best enzymatic inhibitory activity, but no antiviral activity in cell‐based assays. The covalent binding mode of 91 was confirmed by electrospray ionization mass spectrometry (ESI‐MS) analyses.
Figure 26  Active esters as SARS‐CoV‐1 Mpro inhibitors. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus With a slight structural modification from benzotriazole ester, Zhang et al. reported a series of active halopyridyl esters containing thiophene, furan, and indole moieties (92–95; Figure 26). Among them, 93 displayed the highest enzymatic inhibitory activity at SARS‐CoV‐1 Mpro. 169  However, no antiviral activity for this compound was communicated. The irreversible binding mode of 93 was confirmed by ESI‐MS analysis. 170 ,  171
Ghosh et al. 172  studied the SARs of halopyridinyl indole carboxylates and identified a series of analogs (96–101; Figure 27) as SARS‐CoV‐1 Mpro inhibitors in the nanomolar potency range. The best derivative (100) had high enzymatic inhibitory potency (IC50, 0.030 µM) and antiviral activity (EC50, 6.9 µM). Compound 97 was also observed to inhibit the MERS‐CoV Mpro both in enzymatic and cell‐based (EC50, 12.5 µM) bioassays. 173  This molecule covalently modified Mpro, which was confirmed by MALDI‐TOF studies.
Figure 27  SAR of halopyridinyl indole carboxylates as SARS‐CoV‐1 Mpro inhibitors. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus 5‐Halopyridinyl esters are troublesome drug candidates because of their potential for rapid hydrolysis by various esterases and other enzymes in mammalian cells. They can potentially also react nonspecifically with other thiols and nucleophiles, a recipe for cytotoxicity. To bypass this problem by developing stable noncovalent inhibitors, Zhang et al. 174  reported a group of methylene ketones and analogous mono‐ and di‐fluorinated methylene ketones based on pyridinyl esters (102 and 103; Figure 28) as SARS‐CoV‐1 Mpro inhibitors. Enzymatic investigations and ESI‐MS experiments illustrate that those inhibitors bind to their target in a noncovalent, reversible manner.
Figure 28  Etacrynic acid and isatin derivatives as SARS‐CoV‐1 Mpro inhibitors. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus An HPLC‐based screening of electrophilic compounds identified the etacrynic acid‐derived amide 106 and ester 107 as SARS‐CoV‐1 Mpro inhibitors with moderate potency. 175  Etacrynic carboxamide (105; K i, 35.3 µM) bound more strongly to SARS‐CoV‐1 Mpro than to papain protease, while etacrynic acid ester 104 was more active at papain protease (K i, 3.2 µM) than at SARS‐CoV‐1 Mpro (K i, 45.8 µM; Figure 28). SAR studies suggested that chloro substituents were necessary for protease inhibition. Docking studies of 105 to Mpro revealed that it forms hydrogen bonds with Gln189, Glu166, Thr190, and Gln192 with its terminal amino group. The Michael system carbonyl group interacts with Gly143, and the reactive double bond remained next to the Cys145 sulfur.
Previously, isatin (2,3‐dioxoindole) derivatives were observed to inhibit rhinovirus 3C protease. 176  Due to the structural similarity between the rhinovirus 3C protease and SARS‐CoV‐1 Mpro, these derivatives were tested against SARS‐CoV‐1 Mpro. Among them, 106 (IC50, 0.95 µM) and 107 (IC50, 0.98 µM) exhibited the best SARS‐CoV‐1 Mpro inhibitory activity in the low micromolar range. 176  SAR studies suggested that the inhibition efficiency was mainly reliant on hydrophobic and electronic properties of the isatin core substitution pattern. Docking studies revealed that the molecules fit well in the active site of the protease. Both carbonyl groups of the isatin core engaged in H‐bonds with NH of Gly143, Ser144, Cys145, and His41. Compounds 106 and 107  176  were more selective for SARS‐CoV‐1 Mpro than other proteases like papain (106, 103 µM; 107, 87.24 µM), chymotrypsin (106, ~1 mM; 107, 10.4 µM), and trypsin (106, 362 µM; 107, 243 µM; Figure 28).
Zhou et al. extended the SAR studies for further activity improvement. Compound 108 bearing carboxamide showed the best SARS‐CoV‐1 Mpro inhibitory activity. However, this derivative did not bind covalently to the Cys145 residue of the active site. 177  Further structural investigations at the carboxamide of 108 with a variety of substituted sulfonamides did not improve the activity. Compound 109 was the best one of that series (Figure 28). 178
The modification of 110, identified by high‐throughput screening (HTS; Figure 29), led to pyrazolone and pyrazole derivatives 111 and 112 as SARS‐CoV‐1 Mpro inhibitors. 179 ,  180  Taking these as leads, Ramajeyam et al. 181  reported compounds 112–114 to be the best‐performing inhibitors of the series(IC50 5.5, 6.8, 8.4 µM, respectively). They also observed moderate inhibitory activity against CVB3 3Cpro. Structure‐functionality analyses illustrated that the benzylidene ring next to pyrazolone C4 in addition to electron‐withdrawing groups, favors inhibitory activity. Molecular modeling studies of 112 predicted that for its inhibitory function, the N1‐phenyl residue in the Mpro S1 site as well as the carboxyl benzylidene moiety in the S3 pocket are important.
Figure 29  Pyrazoles and pyrimidines as SARS‐CoV‐1 Mpro inhibitors. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus Kumar et al. described furan‐inserted pyrazolone derivatives as dual SARS‐CoV‐1 Mpro and MERS‐CoV Mpro inhibitors (115–118; Figure 29). 182  Compounds 115, 117, and 118 exhibited the best dual inhibitory activities. Compounds 115 and 116 also displayed inhibitory activity against H5N1 neuraminidase (IC50 2.8, 2.9 µM, respectively). 183  Ramajeyam et al. also disclosed a range of pyrimidine derivatives as SARS‐CoV‐1 Mpro inhibitors (119–121). Compound 121 showed high inhibitory potency with an IC50 value 6.1 µM. 181
HTS of NIH molecular libraries (~293 000 substances) yielded the dipeptide 122 containing 3‐pyridyl as hit compound against SARS‐CoV‐1 Mpro with an IC50 value of 2.2 µM (Figure 30). Preliminary SAR studies identified 123 and 124 as the most promising inhibitors of the series. 184 ,  185
Figure 30  Simple dipeptide derivatives as SARS‐CoV‐1 Mpro inhibitors. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus The X‐ray crystal structure of 123 attached to SARS‐CoV‐1 Mpro highlighted the compound's identical orientation in the pocket to that of established covalent peptidomimetic inhibitors (Figure 31). The compound with an R‐configuration occupied the S3‐S1' subsites of SARS‐CoV‐1 Mpro. Indeed, only (R)‐123 was able to inhibit the Mpro enzyme with an IC50 value of 1.5 µM, while the (S)‐enantiomer was inactive. (R)‐123 inhibited SARS‐CoV‐1 Mpro in a competitive manner (K i, 1.6 µM) with a noncovalent mode of inhibition. (R)‐123 also showed antiviral activity (12.9 µM) in mock infected and SARS‐CoV‐1 infected Vero E6 cells.
Figure 31  The X‐ray crystal structure of 123 bound to the binding pocket of SARS‐CoV‐1 Mpro (PDB ID: 3V3M). Pockets S1'–S3 are highlighted. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus [Color figure can be viewed at wileyonlinelibrary.com] To enhance the inhibitory activity, SAR study efforts around P1' of 123 provided compounds containing imidazole (125) and 5‐chlorofuran (126) with equipotent activity to lead 123 (Figure 30). Next, the exploration of P1 3‐pyridyl unit of 123 revealed pyridazine (127) and pyrazine (128) which were only tolerated, albeit without any improvement.
The same group of researchers discovered potent, noncovalent SARS‐CoV‐1 Mpro blockers based on a benzotriazole scaffold in an MLPCN screening, 186  resulting in hit compound 129 (Figure 32) with a SARS‐CoV‐1 Mpro IC50 value of 6.2 µM.
Figure 32  SARS‐CoV‐1 Mpro inhibitors containing the benzotriazole scaffold. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus SAR studies focusing on the benzotriazole moiety of 129 were performed to improve activity. The replacement of this group with 4‐phenyl‐1,2,3‐triazole (as in 130) was somewhat tolerated (IC50 of 11 µM; Figure 32). Further modifications to the acetamide (P2‐P1' region) resulted in molecules bearing a thiophene ring on one side and a branched i‐propyl amide (131) or cyclobutylamide (132) on the other—reaching IC50 values below 5 µM.
To cut overall molecular weight of the inhibitors, P3‐truncation was performed, which led to potent derivatives (133–137; Figure 32). Compound 137 displayed extremely high inhibition (IC50, 51 nM).
SARS‐CoV‐1 Mpro inhibitors were also discovered from medicinal plants. In 2011, Ryu et al. 187  disclosed a range of inhibitors obtained from Torreya nucifera leaves. Of all the isolated chemicals, the biflavone, amentoflavone (138; Figure 33), was identified as a potent noncompetitive inhibitor with an IC50 of 8.3 µM. Docking studies of 138 identified the interactions of Val186 and Gln192 as major sites at the target.
Figure 33  Flavone and terpenoid derivatives with inhibitory activity against SARS‐CoV‐1 3CLpro. 3CLpro, 3C‐like protease; SARS‐CoV, severe acute respiratory syndrome coronavirus They also isolated a series of terpenoids from T. nucifera as anti‐SARS‐CoV Mpro agents (Figure 33). 187  Among them, ferruginol (139; IC50 49.6 µM) was the most active compound. Additionally, they isolated quinone‐methide triterpenoids celastrol (140), pritimererin (141), and tingenone (142) from methanol extracts of Tripterygium regelii which exhibited fair inhibition activity (IC50 2.6, 9.9, 5.5 µM, respectively). SAR studies indicated that for effective inhibition, the quinone‐methide group in ring A and the more lipophilic ring E were critical. All compounds were characterized as competitive inhibitors using kinetic analyses.
Wen et al. 188  reported abietane‐type diterpenoids and lignoids with a powerful anti‐SARS‐CoV‐1 Mpro effect. Especially betulinic acid (143) and savinin (144) effectively inhibited SARS‐CoV‐1 Mpro (K i 8.2 µM, 9.1 µM, respectively) (Figure 33). These inhibitors acted in a competitive manner.
Lu et al. discovered two hit SARS‐CoV‐1 3CLpro inhibitors, sulfone 145 and dihydroimidazole 146, by structure‐based virtual screening of a compound library of 58 855 chemicals (Figure 34). 189  The central structural elements of the hits, determined in docking experiments, were then used for additional analog searches.
Figure 34  Structure of SARS‐CoV‐1 Mpro inhibitors 145–149. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus Computational similarity screening discovered 21 analogs from these hits. Among them, the two best compounds 147 and 148 display IC50 values of 0.3 and 3 µM, respectively. A variety of SARS‐CoV‐1 Mpro inhibitors have been identified through virtual screening (VS) as an alternative to HTS. VS of 50 240 structurally diverse small molecules allowed to identify 104 molecules with anti‐SARS‐CoV‐1 activity. Compound 149 (Figure 34) demonstrated potent enzyme inhibition (IC50, 2.5 μM) and an EC50 of 7 μM in Vero cell‐based SARS‐CoV‐1 plaque reduction assays
Virtual screening identified the serotonin antagonist cinanserin (150, Figure 35) as a potential inhibitor of Mpro. It had previously shown activity against SARS‐CoV‐1 Mpro with an IC50 value of 5 µM. 190  Subsequent tests revealed its anti‐SARS‐CoV‐2 activity (EC50, 20.6 µM) and an IC50 value of 125 µM (SARS‐CoV‐2 Mpro).
Figure 35  Covalent bond inhibitors of Mpro. Mpro, main protease Their HTS yielded seven primary hits including the approved drugs disulfiram (151) and carmofur (152), as well as ebselen (153), shikonin (154), tideglusib (155), and PX‐12 (156) (Figure 35).
Using MS/MS analysis, they deduced that ebselen (153) and 156 are irreversible inhibitors of Mpro by covalently attaching to Cys145 of the catalytic dyad. Molecular docking was used to illustrate how 151, 154, and 155 bind to Mpro. Antiviral activity assays, using real‐time reverse transcription‐PCR, indicated that ebselen and inhibitor “N3” (40; Figure 12) had the strongest antiviral effects. Ebselen displayed an EC50 value of 4.67 µM, and “N3” showed an EC50 value of 16.77 µM in a plaque‐reduction assay. Ebselen's IC50 value for SARS‐CoV‐2 Mpro was reported at 0.67 µM. The activity data of remaining compounds is summarized in Figure 35.
Ebselen has been studied for an array of diseases and has a very low toxicity. 191 ,  192 ,  193  Its safety has been demonstrated in clinical trials. 191 ,  192 ,  194  It can therefore be considered a promising molecule for the treatment or prevention of CoV infections.

3.2  Coronavirus PLpro inhibitors
Along with the Mpro, papain‐like protease (PLpro) also cleaves polyproteins which is an important process for viral replication. PLpro cleaves at the first three positions creating three nonstructural functional proteins (nsp1‐nsp3). In particular, nsp3 is central for the generation of the viral replication complex. The multifunctionality of PLpro in deubiquitinating, de‐ISGylation (ISG: interferon‐stimulated gene), 195 ,  196  and in the evasion of the innate immune response make PLpro an attractive antiviral drug target.
PLpro is a cysteine protease and its active site contains a catalytic triad composing of Cys112‐His273‐Asp287. Cys112 behaves as a nucleophile, and His273 is a general acid‐base. Asp287 helps His273 to align perfectly, thus promoting His to deprotonate Cys‐SH.
Ghosh et al. 197  contributed significantly to the development of SARS‐CoV‐1 PLpro inhibitors based on the naphthalene scaffold. Two lead compounds 157 and 158 (Figure 36) were identified by an HTS of a chemical library containing greater than 50 000 compounds. They both inhibit PLpro of SARS‐CoV‐1 at a moderate potency (IC50 20.1 and 59 µM, respectively). The (R)‐enantiomer of compound 157 was found to be a greater than twofold more potent inhibitor of PLpro when compared with its racemic mixture (157). Subsequent SAR studies highlighted the 2‐naphthyl substitution as an important structural requirement rather than at the position 1 of the naphthyl ring in addition to the presence of o‐methyl and m‐amino groups, in the other phenyl ring. Compound 159 displayed the best inhibitory activity of PLpro (IC50, 0.6 µM) and acts in a noncovalent reversible manner with a K i value of 0.49 µM. 198  Compound 159 also showed moderate antiviral activity in Vero cells with an EC50 value of 14.5 µM.
Figure 36  SARS‐CoV‐1 PLpro inhibitors based on naphthalene scaffold. PLpro, papain‐like protease; SARS‐CoV, severe acute respiratory syndrome coronavirus Compound 159 was further scrutinized by investigating the importance of the amide NH, and the effect of the substituent on the benzamide ring (160–164). Among them, compounds 163 and 164 exhibited the most potent enzymatic (163: IC50, 0.46 µM; 164: IC50, 1.23 µM) and cell‐based antiviral (163: EC50, 6.0 µM; 164: EC50, 5.2 µM) activities.
Next, the same group studied the SARs for compound 158 further. This led to the discovery of compound 165 with high PLpro inhibitory activity of SARS‐CoV‐1 (IC50, 0.32 µM) and antiviral activity (EC50, 9.1 µM) in Vero cells. 199  The mode of action of 165 was found to be a noncovalent, competitive inhibition of PLpro. Unlike the previous series, the stereochemistry at the α‐methyl group did not make a significant difference in inhibition of PLpro. For example, both (S)‐ and (R)‐methyl inhibitors, 165 (IC50, 0.32 µM; EC50, 9.1 µM and 166 (IC50, 0.56 µM; EC50, 9.1 µM), respectively, shared equipotent inhibitory activity in enzymatic and cell‐based assays.
Further SARs of 159 and 165 were investigated to improve the activity. However, no significant improvement in the activity was observed for the prepared compounds either in the enzymatic or cell‐based bioassay. Compounds 167–169 (Figure 36) displayed the best inhibitory activities. Especially, the m‐fluoro‐substituted benzamide derivative 168 (IC50, 0.15 µM; EC50, 5.4 µM) showed the best inhibition activity against PLpro. It also inhibited SARS‐CoV‐1 in the cell‐based bioassay. Both compounds 168 and 169 were metabolically more stable when compared to 167.
HTS of a chemical library of 25000 molecules identified 170 (Figure 37) as a dual SARS‐CoV‐1 PLpro (IC50, 10.9 µM) and MERS‐CoV PLpro (IC50, 6.2 µM) inhibitor. 200  This compound acts via competitive inhibition against MERS‐CoV PLpro, yet via allosteric inhibition against SARS‐CoV‐1 PLpro. This compound also exhibited a preference for SARS‐CoV‐1 PLpro and MERS‐CoV PLpro versus two human homologs of the PLpro, ubiquitin C‐terminal hydrolase, (hUCH‐L1) and (hUCH‐L3).
Figure 37  Broad spectral PLpro inhibitors from different sources. PLpro, papain‐like protease; SARS‐CoV, severe acute respiratory syndrome coronavirus Chou et al. 201  identified thiopurine (171) and 6‐thioguanine (172) as SARS‐CoV‐1 PLpro inhibitors by the screening of a library containing 160 compounds. The thiocarbonyl group was important for PLpro inhibition. However, the toxicity of these anticancer agents limits their therapeutic utility as anti‐SARS agents.
In 2012, Park et al. 202  reported a tanshinone derivative 173 as a SARS‐CoV‐1 PLpro inhibitor with an IC50 value of 0.8 µM. The same research group also described diarylheptanoids blocking SARS‐CoV‐1 PLpro. In particular compound 174 performed as the best inhibitor of SARS‐CoV PLpro with an IC50 value of 4.1 µM. An α,β‐unsaturated carbonyl functionality was crucial for effective inhibition. The geranylated flavonoid 175 was another plant‐derived natural product, which displayed SARS‐CoV‐1 PLpro inhibition with an IC50 value of 5.0 µM. 203
In 2017, Park et al. 204  assessed the inhibitory activity of polyphenols isolated from B. Papyrifera against SARS‐CoV PLpro and MERS‐CoV PLpro. Two of them (176 and 177 Figure 37) displayed moderate inhibition at both SARS‐CoV‐1 PLpro and MERS‐CoV PLpro with a noncompetitive mechanism of action.
Disulfiram (151; Figure 35) was also reported as a SARS‐CoV‐1 PLpro inhibitor (IC50, 24.1 µM), 205  probably by reacting with the active site cysteine, thereby covalently modifying the enzyme target, as was reported for other targets.