Identification of non-covalent 3C-like protease inhibitors against severe acute respiratory syndrome coronavirus-2 via virtual screening of a Korean compound library.
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ABSTRACT:
SUBMITTER: Lee JY
PROVIDER: S-EPMC8091729 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome. Site-specific proteolysis plays a critical role in regulating a number of cellular and viral processes. Since the main protease of SCoV, also termed 3C-like protease, is an attractive target for drug therapy, we have developed a safe, simple, and rapid genetic screen assay to monitor the activity of the SCoV 3C-like protease. This genetic system is based on the bacteriophage lambda regulatory circuit, in which the viral repressor cI is specifically cleaved to initiate the lysogenic-to-lytic switch. A specific target for the SCoV 3C-like protease, P1/P2 (SAVLQ/SGFRK), was inserted into the lambda phage cI repressor. The target specificity of the SCoV P1/P2 repressor was evaluated by coexpression of this repressor with a chemically synthesized SCoV 3C-like protease gene construct. Upon infection of Escherichia coli cells containing the two plasmids encoding the cI. SCoV P1/P2-cro and the beta-galactosidase-SCoV 3C-like protease constructs, lambda phage replicated up to 2,000-fold more efficiently than in cells that did not express the SCoV 3C-like protease. This simple and highly specific assay can be used to monitor the activity of the SCoV 3C-like protease, and it has the potential to be used for screening specific inhibitors.
| S-EPMC533918 | biostudies-literature
Project description: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 308307 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 IC(50) ranged from 38.57±2.41 to 101.38±3.27 ?M. 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 ?M. Hydrophobic and hydrogen bond interactions of compound with amino acid residues in the active site of 3CL(pro) were also identified.
| S-EPMC7126700 | biostudies-literature
Project description:Coronavirus 3C-like protease (3CLPro) is a highly conserved cysteine protease employing a catalytic dyad for its functions. 3CLPro is essential to the viral life cycle and, therefore, is an attractive target for developing antiviral agents. However, the detailed catalytic mechanism of coronavirus 3CLPro remains largely unknown. We took an integrated approach of employing X-ray crystallography, mutational studies, enzyme kinetics study, and inhibitors to gain insights into the mechanism. Such experimental work is supplemented by computational studies, including the prereaction state analysis, the ab initio calculation of the critical catalytic step, and the molecular dynamic simulation of the wild-type and mutant enzymes. Taken together, such studies allowed us to identify a residue pair (Glu-His) and a conserved His as critical for binding; a conserved GSCGS motif as important for the start of catalysis, a partial negative charge cluster (PNCC) formed by Arg-Tyr-Asp as essential for catalysis, and a conserved water molecule mediating the remote interaction between PNCC and catalytic dyad. The data collected and our insights into the detailed mechanism have allowed us to achieve a good understanding of the difference in catalytic efficiency between 3CLPro from SARS and MERS, conduct mutational studies to improve the catalytic activity by 8-fold, optimize existing inhibitors to improve the potency by 4-fold, and identify a potential allosteric site for inhibitor design. All such results reinforce each other to support the overall catalytic mechanism proposed herein.
| S-EPMC7202269 | biostudies-literature
Project description:Recently, inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) have been proposed as potential therapeutic agents for COVID-19. Studying effects of amino acid mutations in the conformation of drug targets is necessary for anticipating drug resistance. In this study, with the structure of the SARS-CoV-2 Mpro complexed with a non-covalent inhibitor, we performed molecular dynamics (MD) simulations to determine the conformation of the complex when single amino acid residue in the active site is mutated. As a model of amino acid mutation, we constructed mutant proteins with one residue in the active site mutated to alanine. This method is called virtual alanine scan. The results of the MD simulations showed that the conformation and configuration of the ligand was changed for mutants H163A and E166A, although the structure of the whole protein and of the catalytic dyad did not change significantly, suggesting that mutations in His163 and Glu166 may be linked to drug resistance.
| S-EPMC8466562 | biostudies-literature
Project description: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.
| S-EPMC2934678 | biostudies-literature
Project description: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.
| S-EPMC2293028 | biostudies-literature
Project description:Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic virus that causes severe respiratory illness accompanied by multi-organ dysfunction, resulting in a case fatality rate of approximately 40%. As found in other coronaviruses, the majority of the positive-stranded RNA MERS-CoV genome is translated into two polyproteins, one created by a ribosomal frameshift, that are cleaved at three sites by a papain-like protease and at 11 sites by a 3C-like protease (3 CL(pro)). Since 3 CL(pro) is essential for viral replication, it is a leading candidate for therapeutic intervention. To accelerate the development of 3 CL(pro) inhibitors, three crystal structures of a catalytically inactive variant (C148A) of the MERS-CoV 3 CL(pro) enzyme were determined. The aim was to co-crystallize the inactive enzyme with a peptide substrate. Fortuitously, however, in two of the structures the C-terminus of one protomer is bound in the active site of a neighboring molecule, providing a snapshot of an enzyme-product complex. In the third structure, two of the three protomers in the asymmetric unit form a homodimer similar to that of SARS-CoV 3 CL(pro); however, the third protomer adopts a radically different conformation that is likely to correspond to a crystallographic monomer, indicative of substantial structural plasticity in the enzyme. The results presented here provide a foundation for the structure-based design of small-molecule inhibitors of the MERS-CoV 3 CL(pro) enzyme.
| S-EPMC4427198 | biostudies-literature
Project description:The papain-like protease of severe acute respiratory syndrome coronavirus (PLpro) (EC 3.4.22.46) is essential for the viral life cycle and therefore represents an important antiviral target. We have identified 6MP and 6TG as reversible and slow-binding inhibitors of SARS-CoV PLpro, which is the first report about small molecule reversible inhibitors of PLpro. The inhibition mechanism was investigated by kinetic measurements and computer docking. Both compounds are competitive, selective, and reversible inhibitors of the PLpro with K(is) values approximately 10 to 20 microM. A structure-function relationship study has identified the thiocarbonyl moiety of 6MP or 6TG as the active pharmacophore essential for these inhibitions, which has not been reported before. The inhibition is selective because these compounds do not exert significant inhibitory effects against other cysteine proteases, including SARS-CoV 3CLpro and several cathepsins. Thus, our results present the first potential chemical leads against SARS-CoV PLpro, which might be used as lead compounds for further optimization to enhance their potency against SARS-CoV. Both 6MP and 6TG are still used extensively in clinics, especially for children with acute lymphoblastic or myeloblastic leukemia. In light of the possible inhibition against subset of cysteine proteases, our study has emphasized the importance to study in depth these drug actions in vivo.
| S-EPMC7092826 | biostudies-literature
Project description:Replication of the genomic RNA of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by replicase polyproteins that are processed by two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro). Previously, we showed that SARS-CoV PLpro processes the replicase polyprotein at three conserved cleavage sites. Here, we report the identification and characterization of a 316-amino-acid catalytic core domain of PLpro that can efficiently cleave replicase substrates in trans-cleavage assays and peptide substrates in fluorescent resonance energy transfer-based protease assays. We performed bioinformatics analysis on 16 papain-like protease domains from nine different coronaviruses and identified a putative catalytic triad (Cys1651-His1812-Asp1826) and zinc-binding site. Mutagenesis studies revealed that Asp1826 and the four cysteine residues involved in zinc binding are essential for SARS-CoV PLpro activity. Molecular modeling of SARS-CoV PLpro suggested that this catalytic core may also have deubiquitinating activity. We tested this hypothesis by measuring the deubiquitinating activity of PLpro by two independent assays. SARS CoV-PLpro hydrolyzed both diubiquitin and ubiquitin-7-amino-4-methylcoumarin (AMC) substrates, and hydrolysis of ubiquitin-AMC is approximately 180-fold more efficient than hydrolysis of a peptide substrate that mimics the PLpro replicase recognition sequence. To investigate the critical determinants recognized by PLpro, we performed site-directed mutagenesis on the P6 to P2' residues at each of the three PLpro cleavage sites. We found that PLpro recognizes the consensus cleavage sequence LXGG, which is also the consensus sequence recognized by cellular deubiquitinating enzymes. This similarity in the substrate recognition sites should be considered during the development of SARS-CoV PLpro inhibitors.
| S-EPMC1316023 | biostudies-literature
Project description:Abstract The main protease of SARS‐associated coronavirus (SARS‐CoV), also called 3C‐like protease (3CLpro), is vital for the viral replication. It cleaves the replicase polyproteins at 11 sites and is a promising drug target. Several groups of inhibitors have been identified through high‐throughput screening and rational drug design. In addition to the pharmaceutical applications, a mutant 3CLpro (T25G) with an expanded S1′ space has been demonstrated to tolerate larger residues at P1′, facilitating the cleavage behind the recognition sequence. This review summarizes current developments in anti‐SARS agents targeting 3CLpro and the application of the mutant protease as a tag‐cleavage endopeptidase. Severe acute respiratory syndrome (SARS)‐associated coronavirus (CoV) has disappeared, but might re‐emerge in the future, together with other new and existing CoV. The 3CL protease of SARS‐CoV, a key enzyme for viral replication, is a promising drug target for inhibitor molecules in order to combat CoVs. This review summarizes current developments and research on inhibitors against proteases and the engineering of the mutant protease as an endopeptidase. WILEY-VCH
| S-EPMC7159133 | biostudies-literature