Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro): IMPLICATIONS FOR nsp5 REGULATION AND THE DEVELOPMENT OF ANTIVIRALS*

All coronaviruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) from the β-CoV subgroup, require the proteolytic activity of the nsp5 protease (also known as 3C-like protease, 3CL^pro) during virus replication, making it a high value target for the development of anti-coronavirus therapeutics. Kinetic studies indicate that in contrast to 3CL^pro from other β-CoV 2c members, including HKU4 and HKU5, MERS-CoV 3CL^pro is less efficient at processing a peptide substrate due to MERS-CoV 3CL^pro being a weakly associated dimer. Conversely, HKU4, HKU5, and SARS-CoV 3CL^pro enzymes are tightly associated dimers. Analytical ultracentrifugation studies support that MERS-CoV 3CL^pro is a weakly associated dimer (K_d ∼52 μm) with a slow off-rate. Peptidomimetic inhibitors of MERS-CoV 3CL^pro were synthesized and utilized in analytical ultracentrifugation experiments and demonstrate that MERS-CoV 3CL^pro undergoes significant ligand-induced dimerization. Kinetic studies also revealed that designed reversible inhibitors act as activators at a low compound concentration as a result of induced dimerization. Primary sequence comparisons and x-ray structural analyses of two MERS-CoV 3CLpro and inhibitor complexes, determined to 1.6 Å, reveal remarkable structural similarity of the dimer interface with 3CL^pro from HKU4-CoV and HKU5-CoV. Despite this structural similarity, substantial differences in the dimerization ability suggest that long range interactions by the nonconserved amino acids distant from the dimer interface may control MERS-CoV 3CL^pro dimerization. Activation of MERS-CoV 3CL^pro through ligand-induced dimerization appears to be unique within the genogroup 2c and may potentially increase the complexity in the development of MERS-CoV 3CL^pro inhibitors as antiviral agents.     

Evaluation of a non-prime site substituent and warheads combined with a decahydroisoquinolin scaffold as a SARS 3CL protease inhibitor

A non-prime site substituent and warheads combined with a decahydroisoquinolin scaffold was evaluated as a novel inhibitor for severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CL^pro). The decahydroisoquinolin scaffold has been demonstrated to be an effective hydrophobic center to interact with S2 site of SARS 3CL^pro, but the lack of interactions at S3 to S4 site is thought to be a major reason for the moderate inhibitory activity. In this study, the effects of an additional non-prime site substituent on the scaffold as well as effects of several warheads are evaluated. For the introduction of a desired non-prime site substituent, amino functionality was introduced on the decahydroisoquinolin scaffold, and the scaffold was constructed by Pd(II) catalyzed diastereoselective ring formation. The synthesized decahydroisoquinolin inhibitors showed about 2.4 times potent inhibitory activities for SARS 3CL^pro when combined with a non-prime site substituent. The present results indicated not only the expected additional interactions with the SARS 3CL^pro but also the possibility of new inhibitors containing a fused-ring system as a hydrophobic scaffold and a new warhead such as thioacetal.     

Molecular dynamic simulations analysis of ritonavir and lopinavir as SARS-CoV 3CL(pro) inhibitors

Since the emergence of the severe acute respiratory syndrome (SARS) to date, neither an effective antiviral drug nor a vaccine against SARS is available. However, it was found that a mixture of two HIV-1 proteinase inhibitors, lopinavir and ritonavir, exhibited some signs of effectiveness against the SARS virus. To understand the fine details of the molecular interactions between these proteinase inhibitors and the SARS virus via complexation, molecular dynamics simulations were carried out for the SARS-CoV 3CL^pro free enzyme (free SARS) and its complexes with lopinavir (SARS-LPV) and ritonavir (SARS-RTV). The results show that flap closing was clearly observed when the inhibitors bind to the active site of SARS-CoV 3CL^pro. The binding affinities of LPV and RTV to SARS-CoV 3CL^pro do not show any significant difference. In addition, six hydrogen bonds were detected in the SARS-LPV system, while seven hydrogen bonds were found in SARS-RTV complex.     

Synthesis,modification and docking studies of 5-sulfonyl isatin derivatives as SARS-CoV 3C-like protease inhibitors

The Severe Acute Respiratory Syndrome (SARS) is a serious life-threatening and strikingly mortal respiratory illness caused by SARS-CoV. SARS-CoV which contains a chymotrypsin-like main protease analogous to that of the main picornavirus protease, 3CL^pro. 3CL^pro plays a pivotal role in the viral replication cycle and is a potential target for SARS inhibitor development. A series of isatin derivatives as possible SARS-CoV 3CL^pro inhibitors was designed, synthesized, and evaluated by in vitro protease assay using fluorogenic substrate peptide, in which several showed potent inhibition against the 3CL^pro. Structure–activity relationship was analyzed, and possible binding interaction modes were proposed by molecular docking studies. Among all compounds, 8k₁ showed most potent inhibitory activity against 3CL^pro (IC₅₀ = 1.04 μM). These results indicated that these inhibitors could be potentially developed into anti-SARS drugs.     

Crystal Structure of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Papain-like Protease Bound to Ubiquitin Facilitates Targeted Disruption of Deubiquitinating Activity to Demonstrate Its Role in Innate Immune Suppression

Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging human pathogen that was first isolated in 2012. MERS-CoV replication depends in part on a virus-encoded papain-like protease (PL^pro) that cleaves the viral replicase polyproteins at three sites releasing non-structural protein 1 (nsp1), nsp2, and nsp3. In addition to this replicative function, MERS-CoV PL^pro was recently shown to be a deubiquitinating enzyme (DUB) and to possess deISGylating activity, as previously reported for other coronaviral PL^pro domains, including that of severe acute respiratory syndrome coronavirus. These activities have been suggested to suppress host antiviral responses during infection. To understand the molecular basis for ubiquitin (Ub) recognition and deconjugation by MERS-CoV PL^pro, we determined its crystal structure in complex with Ub. Guided by this structure, mutations were introduced into PL^pro to specifically disrupt Ub binding without affecting viral polyprotein cleavage, as determined using an in trans nsp3↓4 cleavage assay. Having developed a strategy to selectively disable PL^pro DUB activity, we were able to specifically examine the effects of this activity on the innate immune response. Whereas the wild-type PL^pro domain was found to suppress IFN-β promoter activation, PL^pro variants specifically lacking DUB activity were no longer able to do so. These findings directly implicate the DUB function of PL^pro, and not its proteolytic activity per se, in the inhibition of IFN-β promoter activity. The ability to decouple the DUB activity of PL^pro from its role in viral polyprotein processing now provides an approach to further dissect the role(s) of PL^pro as a viral DUB during MERS-CoV infection.     

Virtual screening identification of novel severe acute respiratory syndrome 3C-like protease inhibitors and in vitro confirmation

The 3C-like protease (3CL^pro) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Structure based virtual screening of 308 307 chemical compounds was performed using the computation tool Autodock 3.0.5 on a WISDOM Production Environment. The top 1468 ranked compounds with free binding energy ranging from −14.0 to −17.09 kcal mol⁻¹ were selected to check the hydrogen bond interaction with amino acid residues in the active site of 3CL^pro. Fifty-three compounds from 35 main groups were tested in an in vitro assay for inhibition of 3CL^pro expressed by Escherichia coli. Seven of the 53 compounds were selected; their IC₅₀ ranged from 38.57 ± 2.41 to 101.38 ± 3.27 μM. Two strong 3CL^pro inhibitors were further identified as competitive inhibitors of 3CL^pro with K_i values of 9.11 ± 1.6 and 9.93 ± 0.44 μM. Hydrophobic and hydrogen bond interactions of compound with amino acid residues in the active site of 3CL^pro were also identified.     

Allosteric Regulation of 3CL Protease of SARS-CoV-2 and SARS-CoV Observed in the Crystal Structure Ensemble

The 3C-like protease (3CL^pro) of SARS-CoV-2 is a potential therapeutic target for COVID-19. Importantly, it has an abundance of structural information solved as a complex with various drug candidate compounds. Collecting these crystal structures (83 Protein Data Bank (PDB) entries) together with those of the highly homologous 3CL^pro of SARS-CoV (101 PDB entries), we constructed the crystal structure ensemble of 3CL^pro to analyze the dynamic regulation of its catalytic function. The structural dynamics of the 3CL^pro dimer observed in the ensemble were characterized by the motions of four separate loops (the C-loop, E-loop, H-loop, and Linker) and the C-terminal domain III on the rigid core of the chymotrypsin fold. Among the four moving loops, the C-loop (also known as the oxyanion binding loop) causes the order (active)–disorder (collapsed) transition, which is regulated cooperatively by five hydrogen bonds made with the surrounding residues. The C-loop, E-loop, and Linker constitute the major ligand binding sites, which consist of a limited variety of binding residues including the substrate binding subsites. Ligand binding causes a ligand size dependent conformational change to the E-loop and Linker, which further stabilize the C-loop via the hydrogen bond between the C-loop and E-loop. The T285A mutation from SARS-CoV 3CL^pro to SARS-CoV-2 3CL^pro significantly closes the interface of the domain III dimer and allosterically stabilizes the active conformation of the C-loop via hydrogen bonds with Ser1 and Gly2; thus, SARS-CoV-2 3CL^pro seems to have increased activity relative to that of SARS-CoV 3CL^pro.     

Aryl methylene ketones and fluorinated methylene ketones as reversible inhibitors for severe acute respiratory syndrome (SARS) 3C-like proteinase

The severe acute respiratory syndrome (SARS) virus depends on a chymotrypsin-like cysteine proteinase (3CL^pro) to process the translated polyproteins to functional viral proteins. This enzyme is a target for the design of potential anti-SARS drugs. A series of ketones and corresponding mono- and di-fluoro ketones having two or three aromatic rings were synthesized as possible reversible inhibitors of SARS 3CL^pro. The design was based on previously established potent inhibition of the enzyme by oxa analogues (esters), which also act as substrates. Structure-activity relationships and modeling studies indicate that three aromatic rings, including a 5-bromopyridin-3-yl moiety, are key features for good inhibition of SARS 3CL^pro. Compound 11d, 2-(5-bromopyridin-3-yl)-1-(5-(4-chlorophenyl)furan-2-yl)ethanone and its α-monofluorinated analogue 12d, gave the best reversible inhibition with IC₅₀ values of 13 μM and 28 μM, respectively. In contrast to inhibitors having two aromatic rings, α-fluorination of compounds with three rings unexpectedly decreased the inhibitory activity.     

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

Severe acute respiratory syndrome (SARS) led to a life-threatening form of atypical pneumonia in late 2002. Following that, Middle East Respiratory Syndrome (MERS-CoV) has recently emerged, killing about 36% of patients infected globally, mainly in Saudi Arabia and South Korea. Based on a scaffold we reported for inhibiting neuraminidase (NA), we synthesized the analogues and identified compounds with low micromolar inhibitory activity against 3CL^pro of SARS-CoV and MERS-CoV. Docking studies show that a carboxylate present at either R¹ or R⁴ destabilizes the oxyanion hole in the 3CL^pro. Interestingly, 3f, 3g and 3m could inhibit both NA and 3CL^pro and serve as a starting point to develop broad-spectrum antiviral agents.     

Dieckol,a SARS-CoV 3CLpro inhibitor,isolated from the edible brown algae Ecklonia cava

SARS-CoV 3CL^pro plays an important role in viral replication. In this study, we performed a biological evaluation on nine phlorotannins isolated from the edible brown algae Ecklonia cava. The nine isolated phlorotannins (1–9), except phloroglucinol (1), possessed SARS-CoV 3CL^pro inhibitory activities in a dose-dependently and competitive manner. Of these phlorotannins (1–9), two eckol groups with a diphenyl ether linked dieckol (8) showed the most potent SARS-CoV 3CL^pro trans/cis-cleavage inhibitory effects (IC₅₀s = 2.7 and 68.1 μM, respectively). This is the first report of a (8) phlorotannin chemotype significantly blocking the cleavage of SARS-CoV 3CL^pro in a cell-based assay with no toxicity. Furthermore, dieckol (8) exhibited a high association rate in the SPR sensorgram and formed extremely strong hydrogen bonds to the catalytic dyad (Cys145 and His41) of the SARS-CoV 3CL^pro.     

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

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus. Viral maturation requires a main protease (3CL(pro)) to cleave the virus-encoded polyproteins. We report here that the 3CL(pro) containing additional N- and/or C-terminal segments of the polyprotein sequences undergoes autoprocessing and yields the mature protease in vitro. The dimeric three-dimensional structure of the C145A mutant protease shows that the active site of one protomer binds with the C-terminal six amino acids of the protomer from another asymmetric unit, mimicking the product-bound form and suggesting a possible mechanism for maturation. The P1 pocket of the active site binds the Gln side chain specifically, and the P2 and P4 sites are clustered together to accommodate large hydrophobic side chains. The tagged C145A mutant protein served as a substrate for the wild-type protease, and the N terminus was first digested (55-fold faster) at the Gln(-1)-Ser1 site followed by the C-terminal cleavage at the Gln306-Gly307 site. Analytical ultracentrifuge of the quaternary structures of the tagged and mature proteases reveals the remarkably tighter dimer formation for the mature enzyme (K(d) = 0.35 nm) than for the mutant (C145A) containing 10 extra N-terminal (K(d) = 17.2 nM) or C-terminal amino acids (K(d) = 5.6 nM). The data indicate that immature 3CL(pro) can form dimer enabling it to undergo autoprocessing to yield the mature enzyme, which further serves as a seed for facilitated maturation. Taken together, this study provides insights into the maturation process of the SARS 3CL(pro) from the polyprotein and design of new structure-based inhibitors.     

Specific Cleavage of the Nuclear Pore Complex Protein Nup62 by a Viral Protease

Previous work has shown that several nucleoporins, including Nup62 are degraded in cells infected with human rhinovirus (HRV) and poliovirus (PV) and that this contributes to the disruption of certain nuclear transport pathways. In this study, the mechanisms underlying proteolysis of Nup62 have been investigated. Analysis of Nup62 in lysates from HRV-infected cells revealed that Nup62 was cleaved at multiple sites during viral infection. The addition of purified HRV2 2A protease (2A^pro) to uninfected HeLa whole cell lysates resulted in the cleavage of Nup62, suggesting that 2A^pro is a major contributor to Nup62 processing. The ability of purified 2A^pro to cleave bacterially expressed and purified Nup62 demonstrated that 2A^pro directly cleaves Nup62 in vitro. Site-directed mutagenesis of putative cleavage sites in Nup62 identified six different positions that are cleaved by 2A^pro in vitro. This analysis revealed that 2A^pro cleavage sites were located between amino acids 103 and 298 in Nup62 and suggested that the N-terminal FG-rich region of Nup62 was released from the nuclear pore complex in infected cells. Analysis of HRV- and PV-infected cells using domain-specific antibodies confirmed that this was indeed the case. These results are consistent with a model whereby PV and HRV disrupt nucleo-cytoplasmic trafficking by selectively removing FG repeat domains from a subset of nuclear pore complex proteins.     

Foot and mouth disease leader protease (Lbpro): Investigation of prime side specificity allows the synthesis of a potent inhibitor

Foot and mouth disease virus expresses its genetic information as a single polyprotein that is translated from the single-stranded RNA genome. Proteinases contained within the polyprotein then generate the mature viral proteins. The leader protease (Lb^pro) performs the initial cleavage by freeing itself from the growing polypeptide chain; subsequently, Lb^pro cleaves the two homologues of the host cell protein eukaryotic initiation factor 4G (eIF4G). We showed that Lb^pro possesses specific binding sites at the non prime side from S₁ down to S₇ [Santos et al. (2009) Biochemistry, 48, 7948–7958]. Here, we demonstrate that Lb^pro has high prime side specificity at least down to the S′₅ site. Lb^pro is thus not only one of the smallest papain-like cysteine peptidases but also one of the most specific. It can still however cleave between both K↓G and G↓R pairs. We further determined the two-step irreversible inhibition (E + I ↔ EI→ E − I) kinetic parameters of two known irreversible epoxide-based inhibitors of cysteine proteinases, E64 and CA074 on Lb^pro that show for the reversible step (E + I ↔ EI) K_i = 3.4 μM and 11.6 μM, and for the irreversible step (EI→E−I) k₄ = 0.16 and 0.06 min⁻¹, respectively. Knowledge of the Lb^pro specificity led us to extend E64 by addition of the dipeptide R–P. This compound, termed E64-R-P-NH₂, irreversibly inhibited Lb^pro with a K_i = 30 nM and k₄ = 0.01 min⁻¹ and can serve as the basis for design of specific inhibitors of FMDV replication.     

Design and synthesis of new tripeptide-type SARS-CoV 3CL protease inhibitors containing an electrophilic arylketone moiety

We describe here the design, synthesis and biological evaluation of a series of molecules toward the development of novel peptidomimetic inhibitors of SARS-CoV 3CL^pro. A docking study involving binding between the initial lead compound 1 and the SARS-CoV 3CL^pro motivated the replacement of a thiazole with a benzothiazole unit as a warhead moiety at the P1′ site. This modification led to the identification of more potent derivatives, including 2i, 2k, 2m, 2o, and 2p, with IC₅₀ or K_i values in the submicromolar to nanomolar range. In particular, compounds 2i and 2p exhibited the most potent inhibitory activities, with K_i values of 4.1 and 3.1 nM, respectively. The peptidomimetic compounds identified through this process are attractive leads for the development of potential therapeutic agents against SARS. The structural requirements of the peptidomimetics with potent inhibitory activities against SARS-CoV 3CL^pro may be summarized as follows: (i) the presence of a benzothiazole warhead at the S1′-position; (ii) hydrogen bonding capabilities at the cyclic lactam of the S1-site; (iii) appropriate stereochemistry and hydrophobic moiety size at the S2-site and (iv) a unique folding conformation assumed by the phenoxyacetyl moiety at the S4-site.     

C‐terminal domain of SARS‐CoV main protease can form a 3D domain‐swapped dimer

SARS coronavirus main protease (M^pro) plays an essential role in the extensive proteolytic processing of the viral polyproteins (pp1a and pp1ab), and it is an important target for anti-SARS drug development. We have reported that both the M^pro C-terminal domain alone (M^pro-C) and the N-finger deletion mutant of M^pro (M^pro-Δ7) exist as a stable dimer and a stable monomer (Zhong et al., J Virol 2008; 82:4227-4234). Here, we report structures of both M^pro-C monomer and dimer. The structure of the M^pro-C monomer is almost identical to that of the C-terminal domain in the crystal structure of M^pro. Interestingly, the M^pro-C dimer structure is characterized by 3D domain-swapping, in which the first helices of the two protomers are interchanged and each is enwrapped by four other helices from the other protomer. Each folding subunit of the M^pro-C domain-swapped dimer still has the same general fold as that of the M^pro-C monomer. This special dimerization elucidates the structural basis for the observation that there is no exchange between monomeric and dimeric forms of M^pro-C and M^pro-Δ7.     

新型冠状病毒3C样蛋白酶结构和功能特征分析

采用生物信息学方法分析新型冠状病毒(Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)3C样蛋白酶(3-chymotrypsin-like protease, 3CL^pro)的理化性质、结构与功能,为抗SARS-CoV-2药物研发提供参考。通过ProtParam、ProtScale、Bioedit服务器对3CL^pro进行一级结构如氨基酸理化性质、疏水性的预测分析;COILS Server、SignalP、TMPred、TargetP Server、NetPhos Server、NetNGlyc Server服务器对3CL^pro结构进行如卷曲螺旋区、信号肽、跨膜结构域、亚细胞定位、磷酸化位点、糖基化位点的预测分析;SOPMA、SWISS MODEL服务器对3CL^pro进行二级结构、三级结构的预测分析;IEBD对3CL^pro进行B细胞表位的预测分析。3CL^pro由306个氨基酸组成,其中亮氨酸占比最高,分子质量为33 796.64,理论等电点值为5.95,半衰期为1.9 h,脂肪系数为82.12;亲水性较高,不具有卷曲螺旋区与信号肽特点,含一个跨膜区;具有4个磷酸化位点,2个糖基化修饰点;二级结构中无规则卷曲占据主导地位,三级结构能与已知的6y2g.1（SMTL ID）模型同源建模;存在4个潜在的B细胞表位,位于92~101位的氨基酸区域应答频率最高。利用生物信息学技术分析3CL^pro的结构和功能特征,可为新型冠状肺炎药物的研发提供参考。     

Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications

The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1–168) joined to an RNA helicase (residues 180–618) by an 11-amino acid linker (169–179). The structure at 3.15 Å of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B₁₈NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173–183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by ∼161° with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn²⁺ refined to a resolution of 2.2 Å. The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu¹⁷³ and Pro¹⁷⁴ or replacing Pro¹⁷⁴ with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro¹⁷⁶ to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication.     

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

Two viral proteases of severe acute respiratory syndrome coronavirus (SARS-CoV), a chymotrypsin-like protease (3CL^pro) and a papain-like protease (PL^pro) are attractive targets for the development of anti-SARS drugs. In this study, nine alkylated chalcones (1–9) and four coumarins (10–13) were isolated from Angelica keiskei, and the inhibitory activities of these constituents against SARS-CoV proteases (3CL^pro and PL^pro) were determined (cell-free/based). Of the isolated alkylated chalcones, chalcone 6, containing the perhydroxyl group, exhibited the most potent 3CL^pro and PL^pro inhibitory activity with IC₅₀ values of 11.4 and 1.2?µM. Our detailed protein-inhibitor mechanistic analysis of these species indicated that the chalcones exhibited competitive inhibition characteristics to the SARS-CoV 3CL^pro, whereas noncompetitive inhibition was observed with the SARS-CoV PL^pro.     

Profiling of substrate specificities of 3C-like proteases from group 1, 2a, 2b, and 3 coronaviruses

Background

Coronaviruses (CoVs) can be classified into alphacoronavirus (group 1), betacoronavirus (group 2), and gammacoronavirus (group 3) based on diversity of the protein sequences. Their 3C-like protease (3CL^pro), which catalyzes the proteolytic processing of the polyproteins for viral replication, is a potential target for anti-coronaviral infection.

Methodology/Principal Findings

Here, we profiled the substrate specificities of 3CL^pro from human CoV NL63 (group 1), human CoV OC43 (group 2a), severe acute respiratory syndrome coronavirus (SARS-CoV) (group 2b) and infectious bronchitis virus (IBV) (group 3), by measuring their activity against a substrate library of 19×8 of variants with single substitutions at P5 to P3'' positions. The results were correlated with structural properties like side chain volume, hydrophobicity, and secondary structure propensities of substituting residues. All 3CL^pro prefer Gln at P1 position, Leu at P2 position, basic residues at P3 position, small hydrophobic residues at P4 position, and small residues at P1'' and P2'' positions. Despite 3CL^pro from different groups of CoVs share many similarities in substrate specificities, differences in substrate specificities were observed at P4 positions, with IBV 3CL^pro prefers P4-Pro and SARS-CoV 3CL^pro prefers P4-Val. By combining the most favorable residues at P3 to P5 positions, we identified super-active substrate sequences ‘VARLQ↓SGF’ that can be cleaved efficiently by all 3CL^pro with relative activity of 1.7 to 3.2, and ‘VPRLQ↓SGF’ that can be cleaved specifically by IBV 3CL^pro with relative activity of 4.3.

Conclusions/Significance

The comprehensive substrate specificities of 3CL^pro from each of the group 1, 2a, 2b, and 3 CoVs have been profiled in this study, which may provide insights into a rational design of broad-spectrum peptidomimetic inhibitors targeting the proteases.     

Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro

COVID-19 has become a global pandemic and there is an urgent call for developing drugs against the virus (SARS-CoV-2). The 3C-like protease (3CL^pro) of SARS-CoV-2 is a preferred target for broad spectrum anti-coronavirus drug discovery. We studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredients. We found that the ethanol extract of S. baicalensis and its major component, baicalein, inhibit SARS-CoV-2 3CL^pro activity in vitro with IC₅₀’s of 8.52 µg/ml and 0.39 µM, respectively. Both of them inhibit the replication of SARS-CoV-2 in Vero cells with EC₅₀’s of 0.74 µg/ml and 2.9 µM, respectively. While baicalein is mainly active at the viral post-entry stage, the ethanol extract also inhibits viral entry. We further identified four baicalein analogues from other herbs that inhibit SARS-CoV-2 3CL^pro activity at µM concentration. All the active compounds and the S. baicalensis extract also inhibit the SARS-CoV 3CL^pro, demonstrating their potential as broad-spectrum anti-coronavirus drugs.