key: cord-0709219-jc8yewv7
authors: He, Jun; Hu, Lijun; Huang, Xiaojun; Wang, Chenran; Zhang, Zhimin; Wang, Ying; Zhang, Dongmei; Ye, Wencai
title: Potential of coronavirus 3C-like protease inhibitors for the development of new anti-SARS-CoV-2 drugs: Insights from structures of protease and inhibitors
date: 2020-06-11
journal: Int J Antimicrob Agents
DOI: 10.1016/j.ijantimicag.2020.106055
sha: aaddc4f72efc3fe51306295f4be4256ad2f963cb
doc_id: 709219
cord_uid: jc8yewv7

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), similar to SARS-CoV and the Middle East respiratory syndrome coronavirus (MERS-CoV), which belong to the same β-coronavirus group, induces sever acute respiratory disease, threatening human health. Since the outbreak of SARS-CoV-2 infection began, the disease has rapidly spread worldwide. Thus, a search for effective drugs, able to inhibit the coronavirus, has become a global pursuit. The 3C-like protease (3CL(pro)), which hydrolyzes the polyprotein to produce functional proteins, is essential for coronavirus replication and considered an important therapeutic target for diseases caused by coronaviruses, including coronavirus disease 2019 (COVID-19). Many 3CL(pro) inhibitors have been proposed, and some new drug candidates have achieved success in preclinical studies. In this review, we briefly describe the recent developments in the structure of 3CL(pro) and its function in coronavirus replication and summarize new insights into 3CL(pro) inhibitors and their mechanisms of action. We also discuss the clinical application prospects and limitations of 3CL(pro) inhibitors for COVID-19 treatment.

Since the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection began in 2019, the disease has rapidly spread worldwide [1] . SARS-CoV-2 infection causes dry cough, fever, shortness of breath, and acute respiratory distress syndrome, which may lead to death [2] . As on April 26, 2020, 2,774,135 SARS-CoV-2-infected cases and 190,871 related deaths have been confirmed worldwide (https://who.sprinklr.com/). SARS-CoV-2 has a high transmission efficiency, with the reproduction number (R0) estimated to be 2.5 [3] . Many experts predict that SARS-CoV-2 may persist for a long time and will cause at least 500,000 deaths worldwide [4] . The World Health Organization has announced the coronavirus disease 2019 (COVID- 19) as an international public health emergency [5] . Therefore, developing safe and effective anti-SARS-CoV-2 drugs is urgently needed. SARS-CoV-2 is a member of the family Coronaviridae, which comprises the largest positive-sense, single-stranded RNA viruses [6] . These viruses are classified into four genera (α, β, γ, and δ). SARS-CoV, the Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 are β-coronaviruses [7] . Analysis of the genome sequences of these three viruses has revealed that SARS-CoV-2 has a higher identity to SARS-CoV (89.1% nucleotide similarity) than to MERS-CoV [8] . The 3C-like protease (3CL pro ) is a cysteine protease that hydrolyzes the polyproteins pp1a and pp1ab to produce functional proteins during coronavirus replication. Because of its highly conserved sequence and essential functional properties, 3CL pro has been validated as a potential target for the development of drugs to treat SARS, MERS, and COVID-19 [9] [10] [11] [12] . At present, a variety of natural and synthetic inhibitors that target different sites and regions of 3CL pro have been developed [13] [14] [15] . As the highly conserved catalytic sites of 3CL pro are shared by the three coronaviruses [12, 16] , tremendous efforts have been made to study this target to speed up the search for anti-SARS-CoV-2 drugs among previously approved drugs, clinical trial candidates, and bioactive agents that were identified in preclinical studies as potential treatments for SARS and MERS [17] . These studies may provide more potential active compounds or drug candidates for the development of new drugs against COVID-19 [18] .

In this review, we briefly describe the recent developments in the crystal structure of 3CL pro and highlight its structural differences among SARS-CoV-2, MERS-CoV, and SARS-CoV.

We further summarize new insights into 3CL pro inhibitors and their mechanisms of action, with a particular focus on newly reported potential SARS-CoV-2 3CL pro inhibitors discovered through virtual screening and in in vitro experiments. In addition, we discuss prospective clinical applications and limitations of 3CL pro inhibitors for COVID-19 treatment. 

In SARS-CoV, 3CL pro cleaves 11 sites in the polyproteins, with the recognition sequence of Leu-Gln↓(Ser, Ala, Gly), including its own Nand C-terminal autoprocessing sites, by recognizing the P1′ and P1-P4 sites [19] . A recent study has indicated that 3CL pro cleaves its C-terminal autoprocessing site through the subsite cooperativity of Phe P2 and Phe P3′ [15] . Three types of SARS-CoV 3CL pro crystal structures have been elucidated, including the wild-type active dimer, monomeric forms with G11A, S139A, or R298A mutation on the dimer interface [18] , and a superactive octamer [20] . In these structures, there are three domains in each protomer, domains I (residues 8-101) and II (residues 102-184), containing N-terminal residues, and domain III (residues 201-303). N-terminal residues form a typical chymotrypsin fold, and C-terminal residues form an extra domain [21] ( Figure 1 ). Active residues, which are located in a gap between domain I and domain II, can be divided into subsites S1-S6. The catalytic dyad His41-Cys145 is at the S1 subsite [20] . The crucial role of the S1 subsite includes the formation of an oxyanion hole when the carboxylate anion of a conserved Gln at the cleavage site interacts with Cys145, Ser144, and Gly143, which can stabilize the transition during proteolysis [22, 23] . The hydrophobic side chains are located at the S2 and S4 subsites. Subsites S5 and S6 are far from the catalytic dyad and close to surface of the structure, thus, contribute little to the substrate binding [10] . In the homodimer structure, seven residues at the very N-terminus (also as known as N-finger) are squeezed between protomers A and B and interact with the two terminal domains of each protomer. These interactions have been proven essential for dimerization. Further, the regions around residues Asn214, Glu288-Glu290, and Arg298-Gln299 at the C-terminus have been confirmed to be important for enzyme dimerization [24] .

The 3CL pro sequences of MERS-CoV and SARS-CoV have 51% similarity [25] . In contrast to the tightly associated dimer of SARS-CoV 3CL pro , the MERS-CoV 3CL pro requires a ligand to form a weakly associated dimer [26] . All of the available MERS-CoV 3CL pro structures have been solved in the presence of a ligand and adopt a conformation similar to that of SARS-CoV 3CL pro , with a backbone root-mean-square deviation (RMSD) of 1.06 Å over 232 Cα atoms in the protomers (Figure 1 ). In the active site, a preferred small amino acid residue at the P2 position induces a narrow S2 pocket of MERS-CoV 3CL pro .

Consistently, none of the 11 cleavage sites contains a phenylalanine residue in MERS-CoV.

Instead, Leu is primarily favored at the P2 position, followed by methionine [26] . These differences between the active sites in the enzyme structures may explain why previously reported inhibitors of SARS-CoV 3CL pro could not potently suppress the activity of MERS-CoV 3CL pro , without structural modifications. On the dimer interface of SARS-CoV 3CL pro , two arginine residues, Arg4 and Arg298, are required to form some indispensable interactions for dimerization. The corresponding residues, Val4 and Met298, are not involved in the dimer formation in MERS-CoV 3CL pro . The substrate binding and dimer formation are affected by some nonconserved residues, which are adjacent to the key residues [27] .

The similarity between the 3CL pro sequences of SARS-CoV-2 and SARS-CoV has been shown to be 96%; out of the 306 residues, only 12 residues are different, namely, T35V, A46S, S65N, L86V, R88K, S94A, H134F, K180N, L202V, A267S, T285A, and I286L [18] .

As expected, the 3CL pro structure of the new coronavirus is a contact dimer, which is similar to the 3CL pro structure of SARS-CoV. When over lapped, the two 3CL pro structures of SARS-CoV-2 (PDB ID: 6M03) and SARS-CoV (PDB ID: 2C3S) (Figure 1 ) present a r.m.s. deviation of 0.53 Å over the 277 Cα atoms. Furthermore, all the residues surrounding both active sites are oriented in almost the same direction, except for Ala46 in the S2 subsite of SARS-CoV-2 3CL pro . Ala46 is located close to the surface of the SARS-CoV-2 3CL pro structure, but this residue is replaced by a serine in SARS-CoV 3CL pro . Another striking difference is observed on the dimer interface. In the SARS-CoV 3CL pro dimer, a polar interaction is formed by the hydroxyl groups of the Thr285 residue in domain III of each protomer, which is owing to a hydrophobic interaction between Thr285 and Ile286, while in the SARS-CoV-2 3CL pro dimer, Thr285 and Ile286 are substituted by alanine and leucine, respectively. These mutations lead to a slightly closer packing of the two domains III of the dimer against one another, and transitions in the catalytic center, which get a higher catalytic activity [28] .

3CL pro is indispensable for coronavirus replication but has not been found in host cells, which makes this enzyme an ideal target for antiviral agents. Based on the crystal structure of 3CL pro , a variety of inhibitors have been developed in the past 5 years. Numerous 3CL pro inhibitors have been reported, including peptide mimetics [29] and small molecules.

However, most studies have mainly focused on small-molecule compounds, through virtual screening, based on the crystal structure of 3CL pro , and through verification of the effects of candidate compounds on the enzyme activity or viral load in vitro.

Based on the conserved active sites of the main protease of coronaviruses, a structure-based design of α-ketoamides was carried out to obtain broad-spectrum antiviral activities against alpha-and betacoronaviruses, as well as enteroviruses [30] . It was found that the optimization of the functional group at the P2 site of α-ketoamides was crucial to find the best compromise for different sizes of the S2 pocket of 3CL pro . The designed compound 1 (P2 = cyclohexylmethyl) showed the most potent inhibition of SARS-CoV 3CL pro (IC 50 = 0.71 μM). The crystal structure (PDB ID: 5N5O) of SARS-CoV 3CL pro in complex with compound 2 (P2 = cyclopropylmethyl) showed that the P2 substituent fitted snugly into the flexible S2 pocket, resulting in hydrophobic interactions with the Met49, Met165, and Asp187 residues and forming hydrogen bonds (H-bonds) between Gln189 and the P2 residue (Figure 2) . Notably, owing to the high similarity between the 3CL pro enzymes of SARS-CoV and SARS-CoV-2, compound 1 is expected to be a potential antiviral candidate against SARS-CoV-2.

A collection of serine derivatives, derived from tetrapeptide inhibitor 3 (IC 50 = 90 nM) and D-serine derivative 4, which was identified as a non-peptidyl small-molecule inhibitor (IC 50 = 30 μM) (Figure 3) , was designed and screened against a SARS-CoV 3CL R188I mutant protease [31] . Compound 4 showed practically no cytotoxicity towards HeLa cells (CC 50 > 200 μM). A detailed docking simulation analysis (4 with 3CL pro ; PDB ID: 3AW1) suggested that the P1′, P1, and P4 substituents in the D-serine skeleton fitted well into the S1′, S1, and S4 pockets, respectively. Further optimization of 4 led to the generation of a series of new scaffolds of phenylisoserine derivatives [32] . Among them, SK80 (5) showed an inhibitory effect on the 3CL mutant protease (IC 50 = 43 μM) (Figure 3) . The binding mode of compound 5 with 3CL pro (PDB ID: 3AW1) indicated that the functional groups at the P1′, P1, P2, and P4 sites of 5 formed hydrophobic interactions with the S1′, S1, S2, and S4 pockets of the protease, respectively. Additionally, two amide groups of 5 formed H-bonds with the His164 and Gln189 residues. 

A library of flavonoids, which consisted of 10 different scaffolds, was tested in vitro for the inhibitory effects on SARS-CoV 3CL pro [9] . Among the compounds, herbacetin (6, PubChem CID: 5280544), rhoifolin (7, PubChem CID: 5282150), and pectolinarin (8, PubChem CID: 168849) were found to exert prominent inhibitory effects, with the IC 50 values of 33.2, 27.5, and 37.8 μM, respectively (Figure 4 ). An induced-fit docking study of compounds 6, 7, and 8 with SARS-CoV 3CL pro (PDB ID: 4WY3) showed that herbacetin formed four H-bonds at the S2 site, and the 8-hydroxyl group was essential for the formation of H-bonds with Glu166 and Gln 189. Interestingly, the binding modes of rhoifolin and pectolinarin were different from that of herbacetin. The bulky carbohydrates attached to the chromen-4-one core skeleton fitted well into the S1 and S2 pockets of the protease. The binding interactions with the S1, S2, and S3′ subsites might contribute to the high affinity of rhoifolin to SARS-CoV 3CL pro . 

Based on reported neuraminidase (NA) inhibitors, a library of pyrazolone derivatives was synthesized and screened against SARS-CoV 3CL pro [10] (Figure 6 ). Compound 18, with a pyrazolone ring surrounded by three hydrophobic groups, showed the most potent inhibition, with an IC 50 of 5.8 ± 1.5 μM. Structure activity relationship analysis suggested that the phenyl pharmacophore in ring C and carboxylate in ring A were essential for the inhibitory activity. A detailed docking simulation analysis of 18 with 3CL pro (PDB ID: 2ALV) showed that the carboxylate group of ring A formed H-bond interactions with the vital residues Gly143, Ser144, and Cys145 at the S1 subsite. The furan B ring was found to interact with the hydrophobic Leu residue in the S1′ pocket, and ring D fitted well in the hydrophobic S2 subsite.

Wang and coworkers [34] reported novel unsymmetrical aromatic disulfide inhibitors against SARS-CoV 3CL pro , with excellent IC 50 values (0.52-5.9 μM). Among them, 1,3,4-oxadiazole disulfide (19) exhibited the most potent inhibition, with an IC 50 value of 0.516 ± 0.06 μM (Figure 7) . Subsequent enzymatic kinetics studies showed that compound 19 acted as a reversible and non-competitive inhibitor. The binding mode of 19 was predicted using simulation models in a docking study with 3CL pro (PDB ID: 2AMD).

Compound 19 formed hydrophobic interactions with Phe140, Leu141, His163, Glu166, and His172, while the 1,3,4-oxadiazole group formed multiple H-bonds with Asn142, Gly143, and Cys145. As part of an ongoing investigation of novel inhibitors [35] , a new class of 55 pyrithiobac derivatives were synthesized and evaluated for their inhibitory activities. Among these synthetic compounds, sulfide 20, with a 1,3,5-triazin ring and a phenyl ring, exhibited a promising inhibitory activity, with an IC 50 value of 4.47 μM (Figure 7) . Molecular docking studies of 20 with 3CL pro (PDB ID: 2AMD) showed that multiple hydrophobic interactions were generated between 20 and the His172, Glu166, His163, Gly143, Leu141, Phe140, and Thr26 residues. Furthermore, four H-bonds were formed between the 1,3,5-triazin ring and Asn142, Ser144, and Cys145. 

It was reported that GC376 (21, PubChem CID: 71481119) displayed potent inhibition toward MERS-CoV in cell-based systems [36] . 

Because the Cys148 residue is highly conserved in the active sites of both proteases, enterovirus 71 3C pro inhibitors were screened for their inhibitory effects against MERS-CoV 3CL pro . The peptide aldehyde 25 exhibited a prominent inhibitory activity, with an IC 50 value of 1.7 ± 0.3 μM, and also suppressed viral replication, with an EC 50 value of 0.6 ± 0.0 μM (Figure 9) . A docking model revealed that a covalent bond was formed between the γ-sulfur of Cys148 and the aldehyde carbon of 25, and the generated oxyanion was stabilized by His41. Moreover, multiple H-bonds were formed to enhance the direct binding, e.g., the P1 lactam moiety of 25 interacted with His166 and Glu169 at the S1 subsite, and the amide group between the cinnamoyl and phenylalanine groups interacted with Gln192 and Glu169 [16] . 

The NA inhibitor 3k (26) is also known as the best inhibitor of MERS-CoV 3CL pro (IC 50 = 5.8 ± 1.6 μM) (Figure 10) . The carboxylate group of 26 was shown to interact with Ser147 and His166 to destabilize the oxyanion hole at the S1 subsite. Moreover, the lactam group in the pyrazolone core interacted with Glu169 through H-bonds, and the phenyl group in ring C formed a π-π stacking interaction with His41. The S2 pocket of MERS-CoV 3CL pro is smaller than that of SARS-CoV 3CL pro , making the phenyl group in ring C of 26 deeply incorporated into the S2 pocket of MERS-CoV 3CL pro [10] . 

The emergence of SARS-CoV-2 has put drug repurposing on the fast track. An anti-HIV drug, lopinavir/ritonavir (PubChem CID: 11979606), has been approved for phase III clinical trials for COVID-19 [37] [38] [39] [40] . In virtual docking experiments, lopinavir (27, PubChem CID: 92727) and ritonavir (28, PubChem CID: 392622) were predicted to bind to the key residues Thr24, Thr26, and Asn119 in the 3CL pro pocket to form two H-bonds each [41] . Additionally, ASC09F, as a fixed-dose combination of ASC09 (29) and ritonavir for HIV infection, is undergoing phase III clinical trials in combination with oseltamivir (30, PubChem CID: 65028) (NCT04261270) (Figure 11) . Given the similarity of the genomic sequences encoding the 3CL pro catalytic sites of SARS-CoV-2, MERS-CoV, and SARS-CoV, previously reported 3CL pro inhibitors for SARS and MERS have been widely investigated for their action on COVID-19 [42] . Examples include pyrithiobac derivatives, unsymmetrical aromatic disulfides, and SK80 (5) for SARS and GC813 (22) for MERS, as wells as GC376 (21), a peptidomimetic inhibitor (1) [15, 16] , and an NA inhibitor analog (26) [10] for SARS and MERS, which are being investigated in preclinical studies as potential candidates for COVID-19 treatment [43] . 

A competitive inhibitor that binds to the active site of a protease may block the enzyme activity by competing with a specific substrate. Peptidic inhibitors usually mimic natural substrates and can be further optimized by attaching chemical agents such as epoxy ketones, aldehydes, Michael acceptors, and halomethyl ketones [44] . The Michael acceptor inhibitor N3 (31), based on specific computer-aided drug design for inhibiting 3CL pro , is a potent irreversible inhibitor that fits inside the substrate-binding pocket of the enzyme [45] .

Compound 31 showed multiple H-bonds with the main chain of the residues in the substrate-binding pocket and significantly prevented (a concentration of 10 μM) the SARS-CoV-2-induced cytopathic effects in infected cells [46] (Figure 12) . Two preferred substrates of SARS-CoV-2 3CL pro , Ac-Abu-Tle-Leu-Gln-ACC and Ac-Thz-Tle-Leu-Gln-ACC, were identified through HyCoSuL screening, and the best recognized natural amino acid substrate was Ac-Val-Lys-Leu-Gln-ACC, with the kinetic parameter of 228.4 ± 9.9 μM [47] . The poor oral bioavailability and metabolic stability of peptides or peptidomimetics are major obstacles for their drug development. Therefore, further studies of small molecules with favorable pharmacokinetic properties may offer a promising alternative.

The natural product 5,7,3′,4′-tetrahydroxy-2′-(3,3-dimethylallyl) isoflavone (32), extracted from Psorothamnus arborescens (Figure 13) . Homology modeling and molecular dynamic simulations showed that compound 32 formed H-bonds with the Cys145 and His41 catalytic dyad of SARS-CoV-2 3CL pro and interacted with Gln189, Glu166, His164, Gly143, Asn142, Met49, Ser46, Thr45, Cys44, Thr26, Thr25, and Thr24 receptor-binding sites, with a docking score of −16.35 and a binding affinity of −29.57 kcal/mol [25] .

Docking procedures based on a crystal structure of SARS-CoV-2 3CL pro (PDB ID: 6LU7) demonstrated that two hydropyrimidinedione compounds, CP-1 (33) and CP-2 (34), had binding energies of −70.6 ± 3.9 and −69.9 ± 4.0 kcal/mol, respectively. These compounds formed H-bonds with the Gly143 and Glu166 residues and a π-π stacking interaction with His41 [48] . Pseudotheonamide C (35) and D (36) , isolated from the marine sponge Theonella swinhoei, formed a covalent bond with the Cys145 residue of SARS-CoV-2 3CL pro . These two compounds occupied a similar position within the catalytic site and formed H-bonds with the Asn142, Ser144, and Glu166 residues, while their benzyl groups fitted into the hydrophobic pockets in the enzyme structure. These compounds acted similarly to Michael acceptor covalent inhibitors, with the binding energies of −14.4 and −14.9 kcal/mol, respectively [49] . The phlorotannins 8,8′-bieckol (37) and 6,6′-bieckol (38) from Ecklonia cava formed an extensive network of H-bonds with the His41 and Cys145 residues of SARS-CoV-2 3CL pro , with the binding energies of −12.9 and −12.1 kcal/mol, respectively [49] . In the case of an improved α-ketoamide inhibitor (39) , its amide oxygen interacted with the main-chain amides of Cys145, Ser144, and Gly143 to form an oxyanion hole and to bind to the P3-P2 amide bond integrated into a pyridone ring. Compound 39

showed inhibition of purified recombinant 3CL pro enzymes from SARS-CoV-2, MERS-CoV, and SARS-CoV, with IC 50 values from 0.58 to 0.90 μM [28] (Figure 14 ). (5) helicase [50] . High-resolution crystal structures of 3CL pro exhibited highly conserved cleavage sites, and numerous 3CL pro inhibitors have been reported, which may hasten the process of anti-COVID-19 drug discovery. Molecular docking methods and binding mode analyses are feasible and practical options for virtual screening of inhibitors targeting the key sites of SARS-CoV-2 3CL pro . Many 3CL pro inhibitors that were previously reported for SARS and MERS may be candidate inhibitors for SARS-CoV-2 3CL pro .

Repurposing of antiviral drugs, as well as screening and modification of existing 3CL pro inhibitors, may provide a fast-track approach for COVID-19 treatment. Kaletra® (lopinavir/ritonavir), an anti-HIV drug, has received much attention amid the current COVID-19 outbreak [42, 51] . In addition, ASC09F, lopinavir, and ritonavir, which are currently in clinical trials, and GC376, GC813, and SK80, which are in preclinical studies, may deserve even more attention. Studies on 3CL pro inhibitors are usually focused on substrate-binding sites (S1′−S1−S2−S3−S4), cleavage sites (P1−P4 and P1′−P4′), the catalytic dyad (His41 and Cys145), and other key residues, such as Thr190, Gln189, Glu166, Met165, Ser144, Gly143, Asn142, Leu141, and Phe140. Because the catalytic sites are highly conserved in the SARS-CoV, SARS-CoV-2, and MERS-CoV 3CL pro structures, the existing inhibitors of the 3CL pro enzymes of the two other coronaviruses may also be effective against SARS-CoV-2 3CL pro . Even though virtual screening makes it possible to discover inhibitor molecules within a relatively short time, the antiviral activities of these agents still need to be experimentally tested in relevant cell and animal models. Additionally, cocrystallization experiments would provide insights into the mechanisms of inhibitor binding to 3CL pro of SARS-CoV-2.

Taken together, 3CL pro inhibitors have a great potential for the development of new drugs against SARS-CoV-2. Although some known 3CL pro inhibitors will accelerate the discovery and development of anti-SARS-CoV-2 drug candidates, there is a long way to go. 

High contagiousness and rapid spread of severe acute respiratory syndrome coronavirus 2, Emerg

A rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-nCoV) infected pneumonia

The reproductive number of COVID-19 is higher compared to SARS coronavirus

Geographical tracking and mapping of coronavirus disease COVID-19/severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic and associated events around the world: how 21st century GIS technologies are supporting the global fight against outbreaks and epidemics，

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): The epidemic and the challenges

Discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest RNA virus genomes

Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of MERS-CoV infection

A new coronavirus associated with human respiratory disease in China

Inhibition of SARS-CoV 3CL protease by flavonoids

Identification, synthesis and evaluation of SARS-CoV and MERS-CoV 3C-like protease inhibitors

Identification of novel proteolytically inactive mutations in coronavirus 3C-like protease using a combined approach

A guideline for homology modeling of the proteins from newly discovered betacoronavirus

Structural basis for inhibiting porcine epidemic diarrhea virus replication with the 3C-Like protease inhibitor GC376

In silico and in vitro analysis of small molecules and natural compounds targeting the 3CL protease of feline infectious peritonitis virus

SARS-CoV 3CL protease cleaves its C-terminal autoprocessing site by novel subsite cooperativity

Identification and evaluation of potent Middle East respiratory syndrome coronavirus (MERS-CoV) 3CL Pro inhibitors

Rapid identification of potential inhibitors of SARS-CoV-2 main protease by deep docking of 1.3 billion compounds

Prediction of the SARS-CoV-2 (2019-nCoV) 3C-like protease (3CL pro ) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates

Profiling of substrate specificity of SARS-CoV 3CL

Three-dimensional domain swapping as a mechanism to lock the active conformation in a super-active octamer of SARS-CoV main protease

3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism

Mechanism of the maturation process of SARS-CoV 3CL protease

Two adjacent mutations on the dimer interface of SARS coronavirus 3C-like protease cause different conformational changes in crystal structure

Structurally-and dynamically-driven allostery of the chymotrypsin-like proteases of SARS, Dengue and Zika viruses

Structural basis of SARS-CoV-2 3CL pro and anti-COVID-19 drug discovery from medicinal plants

Ligand-induced dimerization of Middle East respiratory syndrome (MERS) Coronavirus nsp5 protease (3CLpro): implications for nsp5 regulation and the development of antivirals

Critical assessment of the important residues involved in the dimerization and catalysis of MERS coronavirus main protease

Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors

Structure-based design, synthesis, and evaluation of peptide-mimetic SARS 3CL protease inhibitors

α-Ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication: structure-based design, synthesis, and activity assessment

Design and synthesis of a series of serine derivatives as small molecule inhibitors of the SARS coronavirus 3CL protease

Synthesis and evaluation of phenylisoserine derivatives for the SARS-CoV 3CL protease inhibitor

Chalcones isolated from Angelica keiskei inhibit cysteine proteases of SARS-CoV

Discovery of unsymmetrical aromatic disulfides as novel inhibitors of SARS-CoV main protease: Chemical synthesis, biological evaluation, molecular docking and 3D-QSAR study

Chemical synthesis, crystal structure, versatile evaluation of their biological activities and molecular simulations of novel pyrithiobac derivatives

Structure-guided design of potent and permeable inhibitors of MERS coronavirus 3CL protease that utilize a piperidine moiety as a novel design element

Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV

Treatment of Middle East respiratory syndrome with a combination of lopinavir-ritonavir and interferon-beta1b (MIRACLE trial): study protocol for a randomized controlled trial

Combination therapy with lopinavir/ritonavir, ribavirin and interferon-alpha for Middle East respiratory syndrome

Coronavirus puts drug repurposing on the fast track

Potential inhibitors for 2019-nCoV coronavirus M protease from clinically approved medicines

Research and development on therapeutic agents and vaccines for COVID-19 and related human coronavirus diseases

Therapeutic options for the 2019 novel coronavirus (2019-nCoV)

An overview of severe acute respiratory syndrome-coronavirus (SARS-CoV) 3CL protease inhibitors: Peptidomimetics and small molecule chemotherapy

Structure of main protease from human coronavirus NL63: Insights for wide spectrum anti-coronavirus drug design

Structure of M pro from 1 COVID-19 virus and discovery of its inhibitors

Substrate specificity profiling of SARS-CoV-2 M pro protease provides basis for anti-COVID-19 drug design

Inhibitors for novel coronavirus protease identified by virtual screening of 687 million compounds

Inhibitors of SARS-CoV-2 main protease from a library of marine natural products: A virtual screening and molecular modeling study

Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods

3CL pro Inhibitors as a potential therapeutic option for COVID-19: available evidence and ongoing clinical trials