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The coronavirus disease 2019 (COVID-19) pandemic caused by 2019 novel coronavirus (2019-nCoV) has been a crisis of global health, whereas the effective vaccines against 2019-nCoV are still under development. Alternatively, utilization of old drugs or available medicine that can suppress the viral activity or replication may provide an urgent solution to suppress the rapid spread of 2019-nCoV. Andrographolide is a highly abundant natural product of the medicinal plant, Andrographis paniculata, which has been clinically used for inflammatory diseases and anti-viral therapy. We herein demonstrate that both andrographolide and its fluorescent derivative, the nitrobenzoxadiazole-conjugated andrographolide (Andro- NBD), suppressed the main protease (M pro ) activities of 2019-nCoV and severe acute respiratory syndrome coronavirus (SARS-CoV). Moreover, Andro-NBD was shown to covalently link its fluorescence to these proteases. Further mass spectrometry (MS) analysis suggests that andrographolide formed a covalent bond with the active site Cys 145 of either 2019-nCoV M pro or SARS-CoV M pro . Consistently, molecular modeling analysis supported the docking of andrographolide within the catalytic pockets of both viral M pro s. Considering that andrographolide is used in clinical practice with acceptable safety and its diverse pharmacological activities that could be beneficial for attenuating COVID-19 symptoms, extensive investigation of andrographolide on the suppression of 2019-nCoV as well as its application in COVID-19 therapy is suggested., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aims: In December 2019, the Coronavirus disease-2019 (COVID-19) virus has emerged in Wuhan, China. In this research, the first resolved COVID-19 crystal structure (main protease) was targeted in a virtual screening study by of FDA approved drugs dataset. In addition, a knowledge gap in relations of COVID-19 with the previously known fatal Coronaviruses (CoVs) epidemics, SARS and MERS CoVs, was covered by investigation of sequence statistics and phylogenetics., Materials and Methods: Molecular modeling, virtual screening, docking, sequence comparison statistics and phylogenetics of the COVID-19 main protease were investigated., Key Findings: COVID-19 Mpro formed a phylogenetic group with SARS CoV that was distant from MERS CoV. The identity% was 96.061 and 51.61 for COVID-19/SARS and COVID-19/MERS CoV sequence comparisons, respectively. The top 20 drugs in the virtual screening studies comprised a broad-spectrum antiviral (ribavirin), anti-hepatitis B virus (telbivudine), two vitamins (vitamin B12 and nicotinamide) and other miscellaneous systemically acting drugs. Of special interest, ribavirin had been used in treating cases of SARS CoV., Significance: The present study provided a comprehensive targeting of the first resolved COVID+19 structure of Mpro and found a suitable save drugs for repurposing against the viral Mpro. Ribavirin, telbivudine, vitamin B12 and nicotinamide can be combined and used for COVID treatment. This initiative relocates already marketed and approved safe drugs for potential use in COVID-treatment., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The novel coronavirus whose outbreak took place in December 2019 continues to spread at a rapid rate worldwide. In the absence of an effective vaccine, inhibitor repurposing or de novo drug design may offer a longer-term strategy to combat this and future infections due to similar viruses. Here, we report on detailed classical and mixed-solvent molecular dynamics simulations of the main protease (Mpro) enriched by evolutionary and stability analysis of the protein. The results were compared with those for a highly similar severe acute respiratory syndrome (SARS) Mpro protein. In spite of a high level of sequence similarity, the active sites in both proteins showed major differences in both shape and size, indicating that repurposing SARS drugs for COVID-19 may be futile. Furthermore, analysis of the binding site's conformational changes during the simulation time indicated its flexibility and plasticity, which dashes hopes for rapid and reliable drug design. Conversely, structural stability of the protein with respect to flexible loop mutations indicated that the virus' mutability will pose a further challenge to the rational design of small-molecule inhibitors. However, few residues contribute significantly to the protein stability and thus can be considered as key anchoring residues for Mpro inhibitor design.

SARS-coronavirus (CoV) is a zoonotic agent derived from rhinolophid bats, in which a plethora of SARS-related, conspecific viral lineages exist. Whereas the variability of virulence among reservoir-borne viruses is unknown, it is generally assumed that the emergence of epidemic viruses from animal reservoirs requires human adaptation. To understand the influence of a viral factor in relation to interspecies spillover, we studied the papain-like protease (PLP) of SARS-CoV. This key enzyme drives the early stages of infection as it cleaves the viral polyprotein, deubiquitinates viral and cellular proteins, and antagonizes the interferon (IFN) response. We identified a bat SARS-CoV PLP, which shared 86% amino acid identity with SARS-CoV PLP, and used reverse genetics to insert it into the SARS-CoV genome. The resulting virus replicated like SARS-CoV in Vero cells but was suppressed in IFN competent MA-104 (3.7-fold), Calu-3 (2.6-fold) and human airway epithelial cells (10.3-fold). Using ectopically-expressed PLP variants as well as full SARS-CoV infectious clones chimerized for PLP, we found that a protease-independent, anti-IFN function exists in SARS-CoV, but not in a SARS-related, bat-borne virus. This PLP-mediated anti-IFN difference was seen in primate, human as well as bat cells, thus independent of the host context. The results of this study revealed that coronavirus PLP confers a variable virulence trait among members of the species SARS-CoV, and that a SARS-CoV lineage with virulent PLPs may have pre-existed in the reservoir before onset of the epidemic., Competing Interests: The authors have declared that no competing interests exist.

Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) encode multifunctional papain-like proteases (PLPs) that have the ability to process the viral polyprotein to facilitate RNA replication and antagonize the host innate immune response. The latter function involves reversing the post-translational modification of cellular proteins conjugated with either ubiquitin (Ub) or Ub-like interferon-stimulated gene product 15 (ISG15). Ub is known to be highly conserved among eukaryotes, but surprisingly, ISG15 is highly divergent among animals. The ramifications of this sequence divergence to the recognition of ISG15 by coronavirus PLPs at a structural and biochemical level are poorly understood. Therefore, the activity of PLPs from SARS-CoV, MERS-CoV, and mouse hepatitis virus was evaluated against seven ISG15s originating from an assortment of animal species susceptible, and not, to certain coronavirus infections. Excitingly, our kinetic, thermodynamic, and structural analysis revealed an array of different preferences among PLPs. Included in these studies is the first insight into a coronavirus PLP's interface with ISG15 via SARS-CoV PLpro in complex with the principle binding domain of human ISG15 (hISG15) and mouse ISG15s (mISG15s). The first X-ray structure of the full-length mISG15 protein is also reported and highlights a unique, twisted hinge region of ISG15 that is not conserved in hISG15, suggesting a potential role in differential recognition. Taken together, this new information provides a structural and biochemical understanding of the distinct specificities among coronavirus PLPs observed and addresses a critical gap of how PLPs can interact with ISG15s from a wide variety of species., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Design of inhibitors against severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CL(pro) ) is a potentially important approach to fight against SARS. We have developed several synthetic inhibitors by structure-based drug design. In this report, we reveal two crystal structures of SARS 3CL(pro) complexed with two new inhibitors based on our previous work. These structures combined with six crystal structures complexed with a series of related ligands reported by us are collectively analyzed. To these eight complexes, the structural basis for inhibitor binding was analyzed by the COMBINE method, which is a chemometrical analysis optimized for the protein-ligand complex. The analysis revealed that the first two latent variables gave a cumulative contribution ratio of r(2)  = 0.971. Interestingly, scores using the second latent variables for each complex were strongly correlated with root mean square deviations (RMSDs) of side-chain heavy atoms of Met(49) from those of the intact crystal structure of SARS-3CL(pro) (r = 0.77) enlarging the S2 pocket. The substantial contribution of this side chain (∼10%) for the explanation of pIC50 s was dependent on stereochemistry and the chemical structure of the ligand adapted to the S2 pocket of the protease. Thus, starting from a substrate mimic inhibitor, a design for a central scaffold for a low molecular weight inhibitor was evaluated to develop a further potent inhibitor. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 391-403, 2016., (© 2015 Wiley Periodicals, Inc.)

Deubiquitinating enzymes (DUBs) recognize and cleave linkage-specific polyubiquitin (polyUb) chains, but mechanisms underlying specificity remain elusive in many cases. The severe acute respiratory syndrome (SARS) coronavirus papain-like protease (PLpro) is a DUB that cleaves ISG15, a two-domain Ub-like protein, and Lys48-linked polyUb chains, releasing diUb(Lys48) products. To elucidate this specificity, we report the 2.85 Å crystal structure of SARS PLpro bound to a diUb(Lys48) activity-based probe. SARS PLpro binds diUb(Lys48) in an extended conformation via two contact sites, S1 and S2, which are proximal and distal to the active site, respectively. We show that specificity for polyUb(Lys48) chains is predicated on contacts in the S2 site and enhanced by an S1-S1' preference for a Lys48 linkage across the active site. In contrast, ISG15 specificity is dominated by contacts in the S1 site. Determinants revealed for polyUb(Lys48) specificity should prove useful in understanding PLpro deubiquitinating activities in coronavirus infections., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Over 10 years have passed since the deadly human coronavirus that causes severe acute respiratory syndrome (SARS-CoV) emerged from the Guangdong Province of China. Despite the fact that the SARS-CoV pandemic infected over 8500 individuals, claimed over 800 lives and cost billions of dollars in economic loss worldwide, there still are no clinically approved antiviral drugs, vaccines or monoclonal antibody therapies to treat SARS-CoV infections. The recent emergence of the deadly human coronavirus that causes Middle East respiratory syndrome (MERS-CoV) is a sobering reminder that new and deadly coronaviruses can emerge at any time with the potential to become pandemics. Therefore, the continued development of therapeutic and prophylactic countermeasures to potentially deadly coronaviruses is warranted. The coronaviral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), are attractive antiviral drug targets because they are essential for coronaviral replication. Although the primary function of PLpro and 3CLpro are to process the viral polyprotein in a coordinated manner, PLpro has the additional function of stripping ubiquitin and ISG15 from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells. This review provides an up-to-date discussion on the SARS-CoV papain-like protease including a brief overview of the SARS-CoV genome and replication followed by a more in-depth discussion on the structure and catalytic mechanism of SARS-CoV PLpro, the multiple cellular functions of SARS-CoV PLpro, the inhibition of SARS-CoV PLpro by small molecule inhibitors, and the prospect of inhibiting papain-like protease from other coronaviruses. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses.", (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: The outcome of a viral infection is regulated by complex interactions of viral and host factors. SARS coronavirus (SARS-CoV) engages and regulates several innate immune response pathways during infection. We have previously shown that the SARS-CoV Papain-like Protease (PLpro) inhibits type I interferon (IFN) by inhibiting IRF3 phosphorylation thereby blocking downstream Interferon induction. This finding prompted us to identify other potential mechanisms of inhibition of PLpro on IFN induction., Methods: We have used plasmids expressing PLpro and IRF3 including an IRF3 mutant that is constitutively active, called IRF3(5D). In these experiments we utilize transfections, chromatin immunoprecipitation, Electro-mobility Shift Assays (EMSA) and protein localization to identify where IRF3 and IRF3(5D) are inhibited by PLpro., Results: Here we show that PLpro also inhibits IRF3 activation at a step after phosphorylation and that this inhibition is dependent on the de-ubiquitination (DUB) activity of PLpro. We found that PLpro is able to block the type I IFN induction of a constitutively active IRF3, but does not inhibit IRF3 dimerization, nuclear localization or DNA binding. However, inhibition of PLpro's DUB activity by mutagenesis blocked the IRF3 inhibition activity of PLpro, suggesting a role for IRF3 ubiquitination in induction of a type I IFN innate immune response., Conclusion: These results demonstrate an additional mechanism that PLpro is able to inhibit IRF3 signaling. These data suggest novel innate immune antagonism activities of PLpro that may contribute to SARS-CoV pathogenesis.

Previously we revealed that the extra domain of SARS 3CLpro mediated the catalysis via different mechanisms. While the R298A mutation completely abolished the dimerization, thus resulting in the inactive catalytic machinery, N214A inactivated the enzyme by altering its dynamics without significantly perturbing its structure. Here we studied another mutant with S284-T285-I286 replaced by Ala (STI/A) with a 3.6-fold activity increase and slightly enhanced dimerization. We determined its crystal structure, which still adopts the dimeric structure almost identical to that of the wild-type (WT), except for slightly tighter packing between two extra-domains. We then conducted 100-ns molecular dynamics (MD) simulations for both STI/A and WT, the longest reported so far for 3CLpro. In the simulations, two STI/A extra domains become further tightly packed, leading to a significant volume reduction of the nano-channel formed by residues from both catalytic and extra domains. The enhanced packing appears to slightly increase the dynamic stability of the N-finger and the first helix residues, which subsequently triggers the redistribution of dynamics over residues directly contacting them. This ultimately enhances the dynamical stability of the residues constituting the catalytic dyad and substrate-binding pockets. Further correlation analysis reveals that a global network of the correlated motions exists in the protease, whose components include all residues identified so far to be critical for the dimerization and catalysis. Most strikingly, the N214A mutation globally decouples this network while the STI/A mutation alters the correlation pattern. Together with previous results, the present study establishes that besides the classic structural allostery, the dynamic allostery also operates in the SARS 3CLpro, which is surprisingly able to relay the perturbations on the extra domain onto the catalytic machinery to manifest opposite catalytic effects. Our results thus imply a promising avenue to design specific inhibitors for 3CL proteases by disrupting their dynamic correlation network.

Backgrounds: A new highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries since September 2012. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally. Studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent. In the present study, MERS-CoV papain-like protease (PLpro) is expressed, and its structural and functional consequences are elucidated., Results: Circular dichroism and Tyr/Trp fluorescence analyses indicated that the secondary and tertiary structure of MERS-CoV PLpro is well organized and folded. Analytical ultracentrifugation analyses demonstrated that MERS-CoV PLpro is a monomer in solution. The steady-state kinetic and deubiquitination activity assays indicated that MERS-CoV PLpro exhibits potent deubiquitination activity but lower proteolytic activity, compared with SARS-CoV PLpro. A natural mutation, Leu105, is the major reason for this difference., Conclusions: Overall, MERS-CoV PLpro bound by an endogenous metal ion shows a folded structure and potent proteolytic and deubiquitination activity. These findings provide important insights into the structural and functional properties of coronaviral PLpro family, which is applicable to develop strategies inhibiting PLpro against highly pathogenic coronaviruses.

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a papain-like protease (PLpro) with both deubiquitinating (DUB) and deISGylating activities that are proposed to counteract the post-translational modification of signaling molecules that activate the innate immune response. Here we examine the structural basis for PLpro's ubiquitin chain and interferon stimulated gene 15 (ISG15) specificity. We present the X-ray crystal structure of PLpro in complex with ubiquitin-aldehyde and model the interaction of PLpro with other ubiquitin-chain and ISG15 substrates. We show that PLpro greatly prefers K48- to K63-linked ubiquitin chains, and ISG15-based substrates to those that are mono-ubiquitinated. We propose that PLpro's higher affinity for K48-linked ubiquitin chains and ISG15 stems from a bivalent mechanism of binding, where two ubiquitin-like domains prefer to bind in the palm domain of PLpro with the most distal ubiquitin domain interacting with a "ridge" region of the thumb domain. Mutagenesis of residues within this ridge region revealed that these mutants retain viral protease activity and the ability to catalyze hydrolysis of mono-ubiquitin. However, a select number of these mutants have a significantly reduced ability to hydrolyze the substrate ISG15-AMC, or be inhibited by K48-linked diubuiquitin. For these latter residues, we found that PLpro antagonism of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway is abrogated. This identification of key and unique sites in PLpro required for recognition and processing of diubiquitin and ISG15 versus mono-ubiquitin and protease activity provides new insight into ubiquitin-chain and ISG15 recognition and highlights a role for PLpro DUB and deISGylase activity in antagonism of the innate immune response.

Like many other RNA viruses, severe acute respiratory syndrome coronavirus (SARS-CoV) produces polyproteins containing several non-structural proteins, which are then processed by the viral proteases. These proteases often exist within the polyproteins, and are excised by their own proteolytic activity ('autoprocessing'). It is important to investigate the autoprocessing mechanism of these proteases from the point of view of anti-SARS-CoV drug design. In this paper, we describe a new method for investigating the autoprocessing mechanism of the main protease (M(pro)), which is also called the 3C-like protease (3CL(pro)). Using our method, we measured the activities, under the same conditions, of the mature form and pro-forms with the N-terminal pro-sequence, the C-terminal pro-sequence or both pro-sequences, toward the pro-form with both N- and C-terminal pro-sequences. The data indicate that the pro-forms of the enzyme have proteolytic activity, and are stimulated by the same proteolytic activity. The stimulation occurs in two steps, with approximately eightfold stimulation by N-terminal cleavage, approximately fourfold stimulation by C-terminal cleavage, and 23-fold stimulation by the cleavage of both termini, compared to the pro-form with both the N- and C-terminal pro-sequences. Such cleavage mainly occurs in a trans manner; i.e. the pro-form dimer cleaves the monomeric form. The stimulation by N-terminal pro-sequence removal is due to the cis (intra-dimer and inter-protomer) effect of formation of the new N-terminus, whereas that by C-terminal cleavage is due to removal of its trans (inter-dimer) inhibitory effect. A numerical simulation of the maturation pathway is presented., (© 2013 The Authors Journal compilation © 2013 FEBS.)

The Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (M(pro)) cleaves two virion polyproteins (pp1a and pp1ab); this essential process represents an attractive target for the development of anti-SARS drugs. The functional unit of M(pro) is a homodimer and each subunit contains a His41/Cys145 catalytic dyad. Large amounts of biochemical and structural information are available on M(pro); nevertheless, the mechanism by which monomeric M(pro) is converted into a dimer during maturation still remains poorly understood. Previous studies have suggested that a C-terminal residue, Arg298, interacts with Ser123 of the other monomer in the dimer, and mutation of Arg298 results in a monomeric structure with a collapsed substrate-binding pocket. Interestingly, the R298A mutant of M(pro) shows a reversible substrate-induced dimerization that is essential for catalysis. Here, the conformational change that occurs during substrate-induced dimerization is delineated by X-ray crystallography. A dimer with a mutual orientation of the monomers that differs from that of the wild-type protease is present in the asymmetric unit. The presence of a complete substrate-binding pocket and oxyanion hole in both protomers suggests that they are both catalytically active, while the two domain IIIs show minor reorganization. This structural information offers valuable insights into the molecular mechanism associated with substrate-induced dimerization and has important implications with respect to the maturation of the enzyme.

Viruses have evolved elaborate mechanisms to evade or inactivate the complex system of sensors and signaling molecules that make up the host innate immune response. Here we show that human coronavirus (HCoV) NL63 and severe acute respiratory syndrome (SARS) CoV papain-like proteases (PLP) antagonize innate immune signaling mediated by STING (stimulator of interferon genes, also known as MITA/ERIS/MYPS). STING resides in the endoplasmic reticulum and upon activation, forms dimers which assemble with MAVS, TBK-1 and IKKε, leading to IRF-3 activation and subsequent induction of interferon (IFN). We found that expression of the membrane anchored PLP domain from human HCoV-NL63 (PLP2-TM) or SARS-CoV (PLpro-TM) inhibits STING-mediated activation of IRF-3 nuclear translocation and induction of IRF-3 dependent promoters. Both catalytically active and inactive forms of CoV PLPs co-immunoprecipitated with STING, and viral replicase proteins co-localize with STING in HCoV-NL63-infected cells. Ectopic expression of catalytically active PLP2-TM blocks STING dimer formation and negatively regulates assembly of STING-MAVS-TBK1/IKKε complexes required for activation of IRF-3. STING dimerization was also substantially reduced in cells infected with SARS-CoV. Furthermore, the level of ubiquitinated forms of STING, RIG-I, TBK1 and IRF-3 are reduced in cells expressing wild type or catalytic mutants of PLP2-TM, likely contributing to disruption of signaling required for IFN induction. These results describe a new mechanism used by CoVs in which CoV PLPs negatively regulate antiviral defenses by disrupting the STING-mediated IFN induction.

SARS coronavirus main protease (SARS-CoV M(pro)) is essential for the replication of the virus and regarded as a major antiviral drug target. The enzyme is a cysteine protease, with a catalytic dyad (Cys-145/His-41) in the active site. Aldehyde inhibitors can bind reversibly to the active-site sulfhydryl of SARS-CoV M(pro). Previous studies using peptidic substrates and inhibitors showed that the substrate specificity of SARS-CoV M(pro) requires glutamine in the P1 position and a large hydrophobic residue in the P2 position. We determined four crystal structures of SARS-CoV M(pro) in complex with pentapeptide aldehydes (Ac-ESTLQ-H, Ac-NSFSQ-H, Ac-DSFDQ-H, and Ac-NSTSQ-H). Kinetic data showed that all of these aldehydes exhibit inhibitory activity towards SARS-CoV M(pro), with K(i) values in the μM range. Surprisingly, the X-ray structures revealed that the hydrophobic S2 pocket of the enzyme can accommodate serine and even aspartic-acid side-chains in the P2 positions of the inhibitors. Consequently, we reassessed the substrate specificity of the enzyme by testing the cleavage of 20 different tetradecapeptide substrates with varying amino-acid residues in the P2 position. The cleavage efficiency for the substrate with serine in the P2 position was 160-times lower than that for the original substrate (P2=Leu); furthermore, the substrate with aspartic acid in the P2 position was not cleaved at all. We also determined a crystal structure of SARS-CoV M(pro) in complex with aldehyde Cm-FF-H, which has its P1-phenylalanine residue bound to the relatively hydrophilic S1 pocket of the enzyme and yet exhibits a high inhibitory activity against SARS-CoV M(pro), with K(i)=2.24±0.58 μM. These results show that the stringent substrate specificity of the SARS-CoV M(pro) with respect to the P1 and P2 positions can be overruled by the highly electrophilic character of the aldehyde warhead, thereby constituting a deviation from the dogma that peptidic inhibitors need to correspond to the observed cleavage specificity of the target protease., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The worldwide outbreak of the severe acute respiratory syndrome (SARS) in 2003 was due to the transmission of SARS coronavirus (SARS-CoV). The main protease (M(pro)) of SARS-CoV is essential for the viral life cycle, and is considered to be an attractive target of anti-SARS drug development. As a key enzyme for proteolytic processing of viral polyproteins to produce functional non-structure proteins, M(pro) is first auto-cleaved out of polyproteins. The monomeric form of M(pro) is enzymatically inactive, and it is activated through homo-dimerization which is strongly affected by extra residues to both ends of the mature enzyme. This review provides a summary of the related literatures on the study of the quaternary structure, activation, and self-maturation of M(pro) over the past years.

The 3C-like peptidase of the severe acute respiratory syndrome virus (SARS-CoV) is strictly required for viral replication, thus being a potential target for the development of antiviral agents. In contrast to monomeric picornavirus 3C peptidases, SARS-CoV 3CLpro exists in equilibrium between the monomer and dimer forms in solution, and only the dimer is proteolytically active in dilute buffer solutions. In this study, the increase of SARS-CoV 3CLpro peptidase activity in presence of kosmotropic salts and crowding agents is described. The activation followed the Hofmeister series of anions, with two orders of magnitude enhancement in the presence of Na₂SO₄, whereas the crowding agents polyethylene glycol and bovine serum albumin increased the hydrolytic rate up to 3 times. Kinetic determinations of the monomer dimer dissociation constant (K(d)) indicated that activation was a result of a more active dimer, without significant changes in K(d) values. The activation was found to be independent of substrate length and was derived from both k(cat) increase and K(m) decrease. The viral peptidase activation described here could be related to the crowded intracellular environment and indicates a further fine-tuning mechanism for biological control, particularly in the microenvironment of the vesicles that are induced in host cells during positive strand RNA virus infection.

Background: The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome-coronavirus is required for autoprocessing of the polyprotein, and is a potential target for treating coronaviral infection., Methodology/principal Findings: To obtain a thorough understanding of substrate specificity of the protease, a substrate library of 198 variants was created by performing saturation mutagenesis on the autocleavage sequence at P5 to P3' positions. The substrate sequences were inserted between cyan and yellow fluorescent proteins so that the cleavage rates were monitored by in vitro fluorescence resonance energy transfer. The relative cleavage rate for different substrate sequences was correlated with various structural properties. P5 and P3 positions prefer residues with high β-sheet propensity; P4 prefers small hydrophobic residues; P2 prefers hydrophobic residues without β-branch. Gln is the best residue at P1 position, but observable cleavage can be detected with His and Met substitutions. P1' position prefers small residues, while P2' and P3' positions have no strong preference on residue substitutions. Noteworthy, solvent exposed sites such as P5, P3 and P3' positions favour positively charged residues over negatively charged one, suggesting that electrostatic interactions may play a role in catalysis. A super-active substrate, which combined the preferred residues at P5 to P1 positions, was found to have 2.8 fold higher activity than the wild-type sequence., Conclusions/significance: Our results demonstrated a strong structure-activity relationship between the 3CL(pro) and its substrate. The substrate specificity profiled in this study may provide insights into a rational design of peptidomimetic inhibitors.

The 3C-like proteinase (3CL(pro)) of the severe acute respiratory syndrome (SARS) coronavirus plays a vital role in virus maturation and is proposed to be a key target for drug design against SARS. Various in vitro studies revealed that only the dimer of the matured 3CL(pro) is active. However, as the internally encoded 3CL(pro) gets matured from the replicase polyprotein by autolytic cleavage at both the N-terminal and the C-terminal flanking sites, it is unclear whether the polyprotein also needs to dimerize first for its autocleavage reaction. We constructed a large protein containing the cyan fluorescent protein (C), the N-terminal flanking substrate peptide of SARS 3CL(pro) (XX), SARS 3CL(pro) (3CLP), and the yellow fluorescent protein (Y) to study the autoprocessing of 3CL(pro) using fluorescence resonance energy transfer. In contrast to the matured 3CL(pro), the polyprotein, as well as the one-step digested product, 3CLP-Y-His, were shown to be monomeric in gel filtration and analytic ultracentrifuge analysis. However, dimers can still be induced and detected when incubating these large proteins with a substrate analog compound in both chemical cross-linking experiments and analytic ultracentrifuge analysis. We also measured enzyme activity under different enzyme concentrations and found a clear tendency of substrate-induced dimer formation. Based on these discoveries, we conclude that substrate-induced dimerization is essential for the activity of SARS-3CL(pro) in the polyprotein, and a modified model for the 3CL(pro) maturation process was proposed. As many viral proteases undergo a similar maturation process, this model might be generally applicable.

The main protease of the coronavirus causing severe acute respiratory syndrome performs proteolytic processing of the viral polyproteins. The active form of the enzyme is a homodimer with each subunit consisting of three structural domains. Domains I and II, hosting the complete catalytic machinery, constitute the N-terminal chymotrypsin-like folding scaffold and connect to the extra C-terminal domain III by a long loop. Previously, the domain III-truncated enzyme was demonstrated to fold independently into an intact chymotrypsin-like fold, but it showed no enzyme activity. To further delineate the structure-function relationships of the domain III and the long loop, we generated some truncated and mutated M(pro) forms bearing various combinations of the loop with other structural parts of the enzyme. Their conformational and association properties were investigated in detail. Far-ultraviolet circular dichroism (CD) measurements revealed that these fragments could fold independently. The secondary, tertiary and quaternary structures of these mixtures were monitored by CD, fluorescence spectroscopy and analytical ultracentrifugation. However, no enzyme activity was observed for any mutant or mixtures. These observations indicate that the covalent linkage between the chymotrypsin like and the extra domain is essential for enzymatic activity of the main coronavirus protease and for the integrity of its quaternary structure.

The main protease (M(pro)) plays a vital role in proteolytic processing of the polyproteins in the replicative cycle of SARS coronavirus (SARS-CoV). Dimerization of this enzyme has been shown to be indispensable for trans-cleavage activity. However, the auto-processing mechanism of M(pro), i.e. its own release from the polyproteins through autocleavage, remains unclear. This study elucidates the relationship between the N-terminal autocleavage activity and the dimerization of "immature" M(pro). Three residues (Arg4, Glu290, and Arg298), which contribute to the active dimer conformation of mature M(pro), are selected for mutational analyses. Surprisingly, all three mutants still perform N-terminal autocleavage, while the dimerization of mature protease and trans-cleavage activity following auto-processing are completely inhibited by the E290R and R298E mutations and partially so by the R4E mutation. Furthermore, the mature E290R mutant can resume N-terminal autocleavage activity when mixed with the "immature" C145A/E290R double mutant whereas its trans-cleavage activity remains absent. Therefore, the N-terminal auto-processing of M(pro) appears to require only two "immature" monomers approaching one another to form an "intermediate" dimer structure and does not strictly depend on the active dimer conformation existing in mature protease. In conclusion, an auto-release model of M(pro) from the polyproteins is proposed, which will help understand the auto-processing mechanism and the difference between the autocleavage and trans-cleavage proteolytic activities of SARS-CoV M(pro).

The outcome of a viral infection is regulated in part by the complex coordination of viral and host interactions that compete for the control and optimization of virus replication. Severe acute respiratory syndrome coronavirus (SARS-CoV) intimately engages and regulates the host innate immune responses during infection. Using a novel interferon (IFN) antagonism screen, we show that the SARS-CoV proteome contains several replicase, structural, and accessory proteins that antagonize the IFN pathway. In this study, we focus on the SARS-CoV papain-like protease (PLP), which engages and antagonizes the IFN induction and NF-kappaB signaling pathways. PLP blocks these pathways by affecting activation of the important signaling proteins in each pathway, IRF3 and NF-kappaB. We also show that the ubiquitin-like domain of PLP is necessary for pathway antagonism but not sufficient by itself to block these pathways regardless of the enzymatic activity of the protease. The potential mechanism of PLP antagonism and its role in pathogenesis are discussed.

The 3C-like protease of SARS coronavirus (SARS-CoV 3CL(pro)) is vital for SARS-CoV replication and is a promising drug target. It has been extensively proved that only the dimeric enzyme is active. Here we discovered that two adjacent mutations (Ser139_Ala and Phe140_Ala) on the dimer interface resulted in completely different crystal structures of the enzyme, demonstrating the distinct roles of these two residues in maintaining the active conformation of SARS-CoV 3CL(pro). S139A is a monomer that is structurally similar to the two reported monomers G11A and R298A. However, this mutant still retains a small fraction of dimer in solution, which might account for its remaining activity. F140A is a dimer with the most collapsed active pocket discovered so far, well-reflecting the stabilizing role of this residue. Moreover, a plausible dimerization mechanism was also deduced from structural analysis. Our work is expected to provide insight on the dimerization-function relationship of SARS-CoV 3CL(pro).

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)-Delta7) 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)-Delta7.

Human coxsackievirus (CV) belongs to the picornavirus family, which consists of over 200 medically relevant viruses. In picornavirus, a chymotrypsin-like protease (3C(pro)) is required for viral replication by processing the polyproteins, and thus it is regarded as an antiviral drug target. A 3C-like protease (3CL(pro)) also exists in human coronaviruses (CoV) such as 229E and the one causing severe acute respiratory syndrome (SARS). To combat SARS, we previously had developed peptidomimetic and zinc-coordinating inhibitors of 3CL(pro). As shown in the present study, some of these compounds were also found to be active against 3C(pro) of CV strain B3 (CVB3). Several crystal structures of 3C(pro) from CVB3 and 3CL(pro) from CoV-229E and SARS-CoV in complex with the inhibitors were solved. The zinc-coordinating inhibitor is tetrahedrally coordinated to the His(40)-Cys(147) catalytic dyad of CVB3 3C(pro). The presence of specific binding pockets for the residues of peptidomimetic inhibitors explains the binding specificity. Our results provide a structural basis for inhibitor optimization and development of potential drugs for antiviral therapies.

Background: SARS coronavirus main proteinase (SARS CoVMpro) is an important enzyme for the replication of Severe Acute Respiratory Syndrome virus. The active site region of SARS CoVMpro is divided into 8 subsites. Understanding the binding mode of SARS CoVMpro with a specific substrate is useful and contributes to structural-based drug design. The purpose of this research is to investigate the binding mode between the SARS CoVMpro and two octapeptides, especially in the region of the S3 subsite, through a molecular docking and molecular dynamics (MD) simulation approach., Results: The one turn alpha-helix chain (residues 47-54) of the SARS CoVMpro was directly involved in the induced-fit model of the enzyme-substrate complex. The S3 subsite of the enzyme had a negatively charged region due to the presence of Glu47. During MD simulations, Glu47 of the enzyme was shown to play a key role in electrostatic bonding with the P3Lys of the octapeptide., Conclusion: MD simulations were carried out on the SARS CoVMpro-octapeptide complex. The hypothesis proposed that Glu47 of SARS CoVMpro is an important residue in the S3 subsite and is involved in binding with P3Lys of the octapeptide.

The 3C-like (3CL) protease of the severe acute respiratory syndrome (SARS) coronavirus is a key enzyme for the virus maturation. We found for the first time that the mature SARS 3CL protease is subject to degradation at 188Arg/189Gln. Replacing Arg with Ile at position 188 rendered the protease resistant to proteolysis. The R188I mutant digested a conserved undecapeptide substrate with a K(m) of 33.8 microM and k(cat) of 4753 s(-1). Compared with the value reported for the mature protease containing a C-terminal His-tag, the relative activity of the mutant was nearly 10(6). Novel peptide-aldehyde derivatives containing a side-chain-protected C-terminal Gln efficiently inhibited the catalytic activity of the R188I mutant. The results indicated for the first time that the tetrapeptide sequence is enough for inhibitory activities of peptide-aldehyde derivatives.

Severe acute respiratory syndrome (SARS) is an illness caused by a novel corona virus wherein the main proteinase called 3CL(Pro) has been established as a target for drug design. The mechanism of action involves nucleophilic attack by Cys145 present in the active site on the carbonyl carbon of the scissile amide bond of the substrate and the intermediate product is stabilized by hydrogen bonds with the residues of the oxyanion hole. Based on the X-ray structure of 3CL(Pro) co-crystallized with a trans-alpha,beta-unsaturated ethyl ester (Michael acceptor), a set of QM/QM and QM/MM calculations were performed, yielding three models with increasingly higher the number of atoms. A previous validation step was performed using classical theoretical calculation and PROCHECK software. The Michael reaction studies show an exothermic process with -4.5 kcal/mol. During the reaction pathway, an intermediate is formed by hydrogen and water molecule migration from a histidine residue and solvent, respectively. In addition, similar with experimental results, the complex between N3 and 3CL(Pro) is 578 kcal/mol more stable than N1-3CL(Pro) using Own N-layer Integrated molecular Orbital molecular Mechanics (ONIOM) approach. We suggest 3CL(Pro) inhibitors need small polar groups to decrease the energy barrier for alkylation reaction. These results can be useful for the development of new compounds against SARS.

Unlike 3C protease, the severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CLpro) is only enzymatically active as a homodimer and its catalysis is under extensive regulation by the unique extra domain. Despite intense studies, two puzzles still remain: (i) how the dimer-monomer switch is controlled and (ii) why dimerization is absolutely required for catalysis. Here we report the monomeric crystal structure of the SARS-CoV 3CLpro mutant R298A at a resolution of 1.75 A. Detailed analysis reveals that Arg298 serves as a key component for maintaining dimerization, and consequently, its mutation will trigger a cooperative switch from a dimer to a monomer. The monomeric enzyme is irreversibly inactivated because its catalytic machinery is frozen in the collapsed state, characteristic of the formation of a short 3(10)-helix from an active-site loop. Remarkably, dimerization appears to be coupled to catalysis in 3CLpro through the use of overlapped residues for two networks, one for dimerization and another for the catalysis.

A series of trifluoromethyl ketones as SARS-CoV 3CL protease inhibitors was developed. The inhibitors were synthesized in four steps from commercially available compounds. Three different amino acids were explored in the P1-position and in the P2-P4 positions varying amino acids and long alkyl chain were incorporated. All inhibitors were evaluated in an in vitro assay using purified enzyme and fluorogenic substrate peptide. One of the inhibitors showed a time-dependent inhibition, with a K(i) value of 0.3 microM after 4h incubation.

Although the initial outbreaks of the deadly coronavirus that causes severe acute respiratory syndrome (SARS-CoV) were controlled by public health measures, the development of vaccines and antiviral agents for SARS-CoV is essential for improving control and treatment of future outbreaks. One potential target for SARS-CoV antiviral drug development is the 3C-like protease (3CLpro). This enzyme is an attractive target since it is essential for viral replication, and since there are now a number of high resolution X-ray structures of SARS-CoV 3CLpro available making structure-based drug-design possible. As a result, SARS-CoV 3CLpro has become the focus of numerous drug discovery efforts worldwide, but as a consequence, a variety of different 3CLpro expression constructs and kinetic assays have been independently developed making evaluation and comparison between potential inhibitors problematic. Here, we review the literature focusing on different SARS-CoV 3CLpro expression constructs and assays used to measure enzymatic activity. Moreover, we provide experimental evidence showing that the activity of 3CLpro enzymatic is significantly reduced when non-native sequences or affinity-tags are added to the N- or C-termini of the enzyme, or when the enzyme used in assays is at concentrations below the equilibrium dissociation constant of the 3CLpro dimer. We demonstrate for the first time the utility of a highly sensitive and novel Alexa488-QSY7 FRET-based peptide substrate designed for routine analysis and high-throughput screening, and show that kinetic constants determined from FRET-based assays that are uncorrected for inner-filter effects can lead to artifacts. Finally, we evaluated the effects of common assay components including DTT, NaCl, EDTA and DMSO on enzymatic activity, and we recommend standardized assay conditions and constructs for routine SARS-CoV 3CLpro assays to facilitate direct comparisons between SARS-CoV 3CLpro inhibitors under development worldwide.

Severe acute respiratory syndrome is a highly infectious upper respiratory tract disease caused by SARS-CoV, a previously unidentified human coronavirus. SARS-3CL(pro) is a viral cysteine protease critical to the pathogen's life cycle and hence a therapeutic target of importance. The recently elucidated crystal structures of this enzyme provide an opportunity for the discovery of inhibitors through rational drug design. In the current study, Gold docking program was utilized to conduct extensive docking studies against the target crystal structure to develop a robust and predictive docking protocol. The validated docking protocol was used to conduct a structure-based virtual screening of the Asinex Platinum collection. Biological evaluation of a screened selection of compounds was carried out to identify novel inhibitors of the viral protease.

Severe acute respiratory syndrome (SARS) was a worldwide epidemic caused by a coronavirus that has a cysteine protease (3CLpro) essential to its life cycle. Steady-state and pre-steady-state kinetic methods were used with highly active 3CLpro to characterize the reaction mechanism. We show that 3CLpro has mechanistic features common and disparate to the archetypical proteases papain and chymotrypsin. The kinetic mechanism for 3CLpro-mediated ester hydrolysis, including the individual rate constants, is consistent with a simple double displacement mechanism. The pre-steady-state burst rate was independent of ester substrate concentration indicating a high commitment to catalysis. When homologous peptidic amide and ester substrates were compared, a series of interesting observations emerged. Despite a 2000-fold difference in nonenzymatic reactivity, highly related amide and ester substrates were found to have similar kinetic parameters in both the steady-state and pre-steady-state. Steady-state solvent isotope effect (SIE) studies showed an inverse SIE for the amide but not ester substrates. Evaluation of the SIE in the pre-steady-state revealed normal SIEs for both amide and ester burst rates. Proton inventory (PI) studies on amide peptide hydrolysis were consistent with two proton-transfer reactions in the transition state while the ester data was consistent with a single proton-transfer reaction. Finally, the pH-inactivation profile of 3CLpro with iodoacetamide is indicative of an ion-pair mechanism. Taken together, the data are consistent with a 3CLpro mechanism that utilizes an "electrostatic" trigger to initiate the acylation reaction, a cysteine-histidine catalytic dyad ion pair, an enzyme-facilitated release of P1, and a general base-catalyzed deacylation reaction.

SARS-CoV 3C-like protease (3CL(pro)) is an attractive target for anti-severe acute respiratory syndrome (SARS) drug discovery, and its dimerization has been extensively proved to be indispensable for enzymatic activity. However, the reason why the dissociated monomer is inactive still remains unclear due to the absence of the monomer structure. In this study, we showed that mutation of the dimer-interface residue Gly-11 to alanine entirely abolished the activity of SARS-CoV 3CL(pro). Subsequently, we determined the crystal structure of this mutant and discovered a complete crystallographic dimer dissociation of SARS-CoV 3CL(pro). The mutation might shorten the alpha-helix A' of domain I and cause a mis-oriented N-terminal finger that could not correctly squeeze into the pocket of another monomer during dimerization, thus destabilizing the dimer structure. Several structural features essential for catalysis and substrate recognition are severely impaired in the G11A monomer. Moreover, domain III rotates dramatically against the chymotrypsin fold compared with the dimer, from which we proposed a putative dimerization model for SARS-CoV 3CL(pro). As the first reported monomer structure for SARS-CoV 3CL(pro), the crystal structure of G11A mutant might provide insight into the dimerization mechanism of the protease and supply direct structural evidence for the incompetence of the dissociated monomer.

The 3C-like main peptidase 3CL(pro) is a viral polyprotein processing enzyme essential for the viability of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). While it is generalized that 3CL(pro) and the structurally related 3C(pro) viral peptidases cleave their substrates via a mechanism similar to that underlying the peptide hydrolysis by chymotrypsin-like serine proteinases (CLSPs), some of the hypothesized key intermediates have not been structurally characterized. Here, we present three crystal structures of SARS 3CL(pro) in complex with each of two members of a new class of peptide-based phthalhydrazide inhibitors. Both inhibitors form an unusual thiiranium ring with the nucleophilic sulfur atom of Cys145, trapping the enzyme's catalytic residues in configurations similar to the intermediate states proposed to exist during the hydrolysis of native substrates. Most significantly, our crystallographic data are consistent with a scenario in which a water molecule, possibly via indirect coordination from the carbonyl oxygen of Thr26, has initiated nucleophilic attack on the enzyme-bound inhibitor. Our data suggest that this structure resembles that of the proposed tetrahedral intermediate during the deacylation step of normal peptidyl cleavage.

Chemical denaturant sensitivity of the dimeric main protease from severe acute respiratory syndrome (SARS) coronavirus to guanidinium chloride was examined in terms of fluorescence spectroscopy, circular dichroism, analytical ultracentrifuge, and enzyme activity change. The dimeric enzyme dissociated at guanidinium chloride concentration of <0.4 M, at which the enzymatic activity loss showed close correlation with the subunit dissociation. Further increase in guanidinium chloride induced a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme, on the other hand, followed a monophasic unfolding curve. Different mutants of the full-length protease (W31 and W207/W218), with tryptophanyl residue(s) mutated to phenylalanine at the C-terminal or N-terminal domain, respectively, were constructed. Unfolding curves of these mutants were monophasic but corresponded to the first and second phases of the protease, respectively. The unfolding intermediate of the protease thus represented a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties. The various enzyme forms were characterized in terms of hydrophobicity and size-and-shape distributions. We provide direct evidence for the functional role of C-terminal domain in stabilization of the catalytic N-terminal domain of SARS coronavirus main protease.

The phenomenon that SARS coronavirus main protease (SARS M(pro)) dimer is the main functional form has been confirmed by experiment. However, because of the absence of structural information of the monomer, the reasons for this remain unknown. To investigate it, two molecular dynamics (MD) simulations in water for dimer and monomer models have been carried out, using the crystal structure of protomer A of the dimer as the starting structure for the monomer. During the MD simulation of dimer, three interest phenomena of protomer A have been observed: (i) the distance between NE2 of His41 and SG of Cys145 averages 3.72 A, which agrees well with the experimental observations made by X-ray crystallography; (ii) His163 and Glu166 form the "tooth" conformational properties, resulting in the specificity for glutamine at substrate P1 site; and (iii) the substrate-binding pocket formed by loop 140-146 and loop 184-197 is large enough to accommodate the substrate analog. However, during the MD simulation of the monomer complex, the three structural characteristics are all absent, which results directly in the inactivation of the monomer. Throughout the MD simulation of the dimer, the N-terminus of protomer B forms stable hydrogen bonds with Phe140 and Glu166, through which His163, Glu166, and loop 140-146 are kept active form. Furthermore, a water-bridge has been found between the N-terminus of protomer B and Gly170, which stabilizes His172 and avoids it moving toward Tyr161 to disrupt the H-bond between Tyr161 and His163, stabilizing the conformation of His163. The interactions between the N-terminus and another monomer maintain the activity of dimer., (Copyright 2006 Wiley-Liss, Inc.)

Severe acute respiratory syndrome (SARS) is an infectious disease caused by the human coronavirus, SARS-CoV. The main viral protease, SARS 3CLpro, is a validated target for the development of antiviral therapies. Since the enzyme is a homodimer and the individual monomers are inactive, two approaches are being used to develop inhibitors: enzyme activity inhibitors that target the active site and dimerization inhibitors. Dimerization inhibitors are usually targeted to the dimerization interface and need to compete with the attractive forces between subunits to be effective. In this paper, we show that the dimerization of SARS 3CLpro is also under allosteric control and that additional and energetically more favorable target sites away from the dimerization interface may also lead to subunit dissociation. We previously identified a cluster of conserved serine residues (Ser139, Ser144, and Ser147) located adjacent to the active site of 3CLpro that could effectively be targeted to inactivate the protease [Bacha, U et al. (2004) Biochemistry 43, 4906-4912]. Mutation of any of these serine residues to alanine had a debilitating effect on the catalytic activity of 3CLpro. In particular, the mutation of Ser147, which does not make any contact with the opposing subunit and is located approximately 9 A away from the dimer interface, totally inhibited dimerization and resulted in a complete loss of enzymatic activity. The finding that residues away from the dimer interface are able to control dimerization defines alternative targets for the design of dimerization inhibitors.

The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for discovery of drugs against SARS, because of its critical role in the viral life cycle. In this study, a natural compound called quercetin-3-beta-galactoside was identified as an inhibitor of the protease by molecular docking, SPR/FRET-based bioassays, and mutagenesis studies. Both molecular modeling and Q189A mutation revealed that Gln189 plays a key role in the binding. Furthermore, experimental evidence showed that the secondary structure and enzymatic activity of SARS-CoV 3CL(pro) were not affected by the Q189A mutation. With the help of molecular modeling, eight new derivatives of the natural product were designed and synthesized. Bioassay results reveal salient features of the structure-activity relationship of the new compounds: (1) removal of the 7-hydroxy group of the quercetin moiety decreases the bioactivity of the derivatives; (2) acetoxylation of the sugar moiety abolishes inhibitor action; (3) introduction of a large sugar substituent on 7-hydroxy of quercetin can be tolerated; (4) replacement of the galactose moiety with other sugars does not affect inhibitor potency. This study not only reveals a new class of compounds as potential drug leads against the SARS virus, but also provides a solid understanding of the mechanism of inhibition against the target enzyme.

The SARS coronavirus main proteinase (SARS CoV main proteinase) is required for the replication of the severe acute respiratory syndrome coronavirus (SARS CoV), the virus that causes SARS. One function of the enzyme is to process viral polyproteins. The active form of the SARS CoV main proteinase is a homodimer. In the literature, estimates of the monomer-dimer equilibrium dissociation constant, KD, have varied more than 65,0000-fold, from <1 nM to more than 200 microM. Because of these discrepancies and because compounds that interfere with activation of the enzyme by dimerization may be potential antiviral agents, we investigated the monomer-dimer equilibrium by three different techniques: small-angle X-ray scattering, chemical cross-linking, and enzyme kinetics. Analysis of small-angle X-ray scattering data from a series of measurements at different SARS CoV main proteinase concentrations yielded KD values of 5.8 +/- 0.8 microM (obtained from the entire scattering curve), 6.5 +/- 2.2 microM (obtained from the radii of gyration), and 6.8 +/- 1.5 microM (obtained from the forward scattering). The KD from chemical cross-linking was 12.7 +/- 1.1 microM, and from enzyme kinetics, it was 5.2 +/- 0.4 microM. While each of these three techniques can present different, potential limitations, they all yielded similar KD values.

An integrated system has been developed for discovering potent inhibitors of severe acute respiratory syndrome coronavirus 3C-like protease (SARS-CoV 3CL(pro)) by virtual screening correlating with surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) technologies-based assays. The authors screened 81,287 small molecular compounds against SPECS database by virtual screening; 256 compounds were subsequently selected for biological evaluation. Through SPR technology-based assay, 52 from these 256 compounds were discovered to show binding to SARS-CoV 3CL(pro). The enzymatic inhibition activities of these 52 SARS-CoV 3CL(pro) binders were further applied to FRET-based assay, and IC(50) values were determined. Based on this integrated assay platform, 8 new SARS-CoV 3CL(pro) inhibitors were discovered. The fact that the obtained IC(50) values for the inhibitors are in good accordance with the discovered dissociation equilibrium constants (K(D)s) assayed by SPR implied the reliability of this platform. Our current work is hoped to supply a powerful approach in the discovery of potent SARS-CoV 3CL(pro) inhibitors, and the determined inhibitors could be used as possible lead compounds for further research.

SARS-coronavirus (SARS-CoV) encodes a main protease, 3CLpro, which plays an essential role in the viral life cycle and is currently the prime target for discovering new anti-coronavirus agents. In this article, we report our success in developing a novel red-shifted (RS) fluorescence-based assay for 3CLpro and its application for identifying small-molecule anti-SARS agents from marine organisms. We have synthesised and characterised the first generation of a red-shifted internally quenched fluorogenic substrate (RS-IQFS) for 3CLpro based on resonance energy transfer between the donor and acceptor pair CAL Fluor Red 610 and Black Hole Quencher-1 (Km and kcat values of 14 microM and 0.65 min-1). The RS-IQFS primary sequence was selected based on the results of our screening analysis of 3CLpro performed using a series of blue-shifted (BS)-IQFSs corresponding to the 3CLpro-mediated cleavage junctions of the SARS-CoV polyproteins. In contrast to BS-IQFSs, the RS-IQFS was not susceptible to fluorescence interference from coloured samples and allowed for successful screening of marine natural products and identification of a coumarin derivative, esculetin-4-carboxylic acid ethyl ester, a novel 3CLpro inhibitor (IC50=46 microM) and anti-SARS agent (EC50=112 microM; median toxic concentration>800 microM) from the tropical marine sponge Axinella corrugata.

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) 3C-like protease (3CL(pro) or M(pro)) is an attractive target for the development of anti-SARS drugs because of its crucial role in the viral life cycle. In this study, a compound database was screened by the structure-based virtual screening approach to identify initial hits as inhibitors of SARS-CoV 3CL(pro). Out of the 59,363 compounds docked, 93 were selected for the inhibition assay, and 21 showed inhibition against SARS-CoV 3CL(pro) (IC(50)

The severe acute respiratory syndrome coronavirus 3C-like protease has been proposed to be a key target for structurally based drug design against SARS. The enzyme exists as a mixture of dimer and monomer, and only the dimer was considered to be active. In this report, we have investigated, using molecular dynamics simulation and mutational studies, the problems as to why only the dimer is active and whether both of the two protomers in the dimer are active. The molecular dynamics simulations show that the monomers are always inactive, that the two protomers in the dimer are asymmetric, and that only one protomer is active at a time. The enzyme activity of the hybrid severe acute respiratory syndrome coronavirus 3C-like protease of the wild-type protein and the inactive mutant proves that the dimerization is important for enzyme activity and only one active protomer in the dimer is enough for the catalysis. Our simulations also show that the right conformation for catalysis in one protomer can be induced upon dimer formation. These results suggest that the enzyme may follow the association, activation, catalysis, and dissociation mechanism for activity control.

The enzymatic activity of the SARS coronavirus main proteinase dimer was characterized by a sensitive, quantitative assay. The new, fluorogenic substrate, (Ala-Arg-Leu-Gln-NH)(2)-Rhodamine, contained a severe acute respiratory syndrome coronavirus (SARS CoV) main proteinase consensus cleavage sequence and Rhodamine 110, one of the most detectable compounds known, as the reporter group. The gene for the enzyme was cloned in the absence of purification tags, expressed in Escherichia coli and the enzyme purified. Enzyme activity from the SARS CoV main proteinase dimer could readily be detected at low pM concentrations. The enzyme exhibited a high K(m), and is unusually sensitive to ionic strength and reducing agents.

Replication of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) requires proteolytic processing of the replicase polyprotein by two viral cysteine proteases, a chymotrypsin-like protease (3CLpro) and a papain-like protease (PLpro). These proteases are important targets for development of antiviral drugs that would inhibit viral replication and reduce mortality associated with outbreaks of SARS-CoV. In this work, we describe the 1.85-A crystal structure of the catalytic core of SARS-CoV PLpro and show that the overall architecture adopts a fold closely resembling that of known deubiquitinating enzymes. Key features, however, distinguish PLpro from characterized deubiquitinating enzymes, including an intact zinc-binding motif, an unobstructed catalytically competent active site, and the presence of an intriguing, ubiquitin-like N-terminal domain. To gain insight into the active-site recognition of the C-terminal tail of ubiquitin and the related LXGG motif, we propose a model of PLpro in complex with ubiquitin-aldehyde that reveals well defined sites within the catalytic cleft that help to account for strict substrate-recognition motifs.

The pathogenesis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is an important issue for the treatment and prevention of severe acute respiratory syndrome. Recently, SARS-CoV has been demonstrated to induce cell apoptosis in Vero-E6 cells. The possible role of SARS-CoV 3C-like protease (3CLpro) in virus-induced apoptosis is characterized in this study. Growth arrest and apoptosis via caspase-3 and caspase-9 activities were demonstrated in SARS-CoV 3CLpro -expressing human promonocyte cells. The fluorescence intensity of dihydrorhodamine 123 staining indicated that cellular reactive oxygen species were markedly increased in SARS-CoV 3CLpro -expressing cells. Moreover, in vivo signalling pathway assay indicated that 3CLpro increased the activation of the nuclear factor-kappa B-dependent reporter, but inhibited activator protein-1-dependent transcription. This finding is likely to be responsible for virus-induced apoptotic signalling.

The 3C-like protease of the severe acute respiratory syndrome (SARS) coronavirus has a C-terminal extra domain in addition to the chymotrypsin-fold adopted by picornavirus 3C proteases hosting the complete catalytic machinery. Previously we identified the extra domain to be involved in enzyme dimerization which has been considered essential for the catalytic activity. In an initial attempt to map out the extra-domain residues critical for dimerization, we have systematically generated 15 point mutations, five deletions and one triple mutation and subsequently characterized them by enzymatic assay, dynamic light scattering, CD and NMR spectroscopy. The results led to identification of four regions critical for enzyme dimerization. Interestingly, Asn214Ala mutant with a significant tendency to form a monomer still retained approximately 30% activity, indicating that the relationship between the activity and dimerization might be very complex. Very surprisingly, two regions (one over Ser284-Thr285-Ile286 and another around Phe291) were discovered on which Ala-mutations significantly increased the enzymatic activities. Based on this, a super-active triple-mutant STI/A with a 3.7-fold activity enhancement was thus engineered by mutating residues Ser284, Thr285 and Ile286 to Ala. The dynamic light scattering, CD and NMR characterizations indicate that the wild-type (WT) and STI/A mutant share similar structural and dimerization properties, thus implying that in addition to dimerization, the extra domain might have other mechanisms to regulate the catalytic machinery. We rationalized these results based on the enzyme structure and consequently observed an interesting picture: the majority of the dimerization-critical residues plus Ser284-Thr285-Ile286 and Phe291 are clustered together to form a nano-scale channel passing through the central region of the enzyme. We therefore speculate that this channel might play a role in relaying regulatory effects from the extra domain to the catalytic machinery.