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1
Ziebuhr, John, Sonja Bayer, Jeff A. Cowley, and Alexander E. Gorbalenya. "The 3C-Like Proteinase of an Invertebrate Nidovirus Links Coronavirus and Potyvirus Homologs." Journal of Virology 77, no. 2 (January 15, 2003): 1415–26. http://dx.doi.org/10.1128/jvi.77.2.1415-1426.2003.
Abstract:
ABSTRACT Gill-associated virus (GAV), a positive-stranded RNA virus of prawns, is the prototype of newly recognized taxa (genus Okavirus, family Roniviridae) within the order Nidovirales. In this study, a putative GAV cysteine proteinase (3C-like proteinase [3CLpro]), which is predicted to be the key enzyme involved in processing of the GAV replicase polyprotein precursors, pp1a and pp1ab, was characterized. Comparative sequence analysis indicated that, like its coronavirus homologs, 3CLpro has a three-domain organization and is flanked by hydrophobic domains. The putative 3CLpro domain including flanking regions (pp1a residues 2793 to 3143) was fused to the Escherichia coli maltose-binding protein (MBP) and, when expressed in E. coli, was found to possess N-terminal autoprocessing activity that was not dependent on the presence of the 3CLpro C-terminal domain. N-terminal sequence analysis of the processed protein revealed that cleavage occurred at the location 2827LVTHE↓VRTGN2836. The trans-processing activity of the purified recombinant 3CLpro (pp1a residues 2832 to 3126) was used to identify another cleavage site, 6441KVNHE↓LYHVA6450, in the C-terminal pp1ab region. Taken together, the data tentatively identify VxHE↓(L,V) as the substrate consensus sequence for the GAV 3CLpro. The study revealed that the GAV and potyvirus 3CLpros possess similar substrate specificities which correlate with structural similarities in their respective substrate-binding sites, identified in sequence comparisons. Analysis of the proteolytic activities of MBP-3CLpro fusion proteins carrying replacements of putative active-site residues provided evidence that, in contrast to most other 3C/3CLpros but in common with coronavirus 3CLpros, the GAV 3CLpro employs a Cys2968-His2879 catalytic dyad. The properties of the GAV 3CLpro define a novel RNA virus proteinase variant that bridges the gap between the distantly related chymotrypsin-like cysteine proteinases of coronaviruses and potyviruses.
2
Tsu, Brian V., Rimjhim Agarwal, Nandan S. Gokhale, Jessie Kulsuptrakul, Andrew P. Ryan, Elizabeth J. Fay, Lennice K. Castro, et al. "Host-specific sensing of coronaviruses and picornaviruses by the CARD8 inflammasome." PLOS Biology 21, no. 6 (June 8, 2023): e3002144. http://dx.doi.org/10.1371/journal.pbio.3002144.
Abstract:
Hosts have evolved diverse strategies to respond to microbial infections, including the detection of pathogen-encoded proteases by inflammasome-forming sensors such as NLRP1 and CARD8. Here, we find that the 3CL protease (3CLpro) encoded by diverse coronaviruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), cleaves a rapidly evolving region of human CARD8 and activates a robust inflammasome response. CARD8 is required for cell death and the release of pro-inflammatory cytokines during SARS-CoV-2 infection. We further find that natural variation alters CARD8 sensing of 3CLpro, including 3CLpro-mediated antagonism rather than activation of megabat CARD8. Likewise, we find that a single nucleotide polymorphism (SNP) in humans reduces CARD8’s ability to sense coronavirus 3CLpros and, instead, enables sensing of 3C proteases (3Cpro) from select picornaviruses. Our findings demonstrate that CARD8 is a broad sensor of viral protease activities and suggests that CARD8 diversity contributes to inter- and intraspecies variation in inflammasome-mediated viral sensing and immunopathology.
3
Rawson, Jonathan M. O., Alice Duchon, Olga A. Nikolaitchik, Vinay K. Pathak, and Wei-Shau Hu. "Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors." Viruses 13, no. 2 (January 24, 2021): 173. http://dx.doi.org/10.3390/v13020173.
Abstract:
The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.
4
Zhang, Jingjing, Yingpei Jiang, Chunxiu Wu, Dan Zhou, Jufang Gong, Tiejun Zhao, and Zhigang Jin. "Development of FRET and Stress Granule Dual-Based System to Screen for Viral 3C Protease Inhibitors." Molecules 28, no. 7 (March 28, 2023): 3020. http://dx.doi.org/10.3390/molecules28073020.
Abstract:
3C proteases (3Cpros) of picornaviruses and 3C-like proteases (3CLpros) of coronaviruses and caliciviruses represent a group of structurally and functionally related viral proteases that play pleiotropic roles in supporting the viral life cycle and subverting host antiviral responses. The design and screening for 3C/3CLpro inhibitors may contribute to the development broad-spectrum antiviral therapeutics against viral diseases related to these three families. However, current screening strategies cannot simultaneously assess a compound’s cytotoxicity and its impact on enzymatic activity and protease-mediated physiological processes. The viral induction of stress granules (SGs) in host cells acts as an important antiviral stress response by blocking viral translation and stimulating the host immune response. Most of these viruses have evolved 3C/3CLpro-mediated cleavage of SG core protein G3BP1 to counteract SG formation and disrupt the host defense. Yet, there are no SG-based strategies screening for 3C/3CLpro inhibitors. Here, we developed a fluorescence resonance energy transfer (FRET) and SG dual-based system to screen for 3C/3CLpro inhibitors in living cells. We took advantage of FRET to evaluate the protease activity of poliovirus (PV) 3Cpro and live-monitor cellular SG dynamics to cross-verify its effect on the host antiviral response. Our drug screen uncovered a novel role of Telaprevir and Trifluridine as inhibitors of PV 3Cpro. Moreover, Telaprevir and Trifluridine also modulated 3Cpro-mediated physiological processes, including the cleavage of host proteins, inhibition of the innate immune response, and consequent facilitation of viral replication. Taken together, the FRET and SG dual-based system exhibits a promising potential in the screening for inhibitors of viral proteases that cleave G3BP1.
5
Sanachai, Kamonpan, Tuanjai Somboon, Patcharin Wilasluck, Peerapon Deetanya, Peter Wolschann, Thierry Langer, Vannajan Sanghiran Lee, Kittikhun Wangkanont, Thanyada Rungrotmongkol, and Supot Hannongbua. "Identification of repurposing therapeutics toward SARS-CoV-2 main protease by virtual screening." PLOS ONE 17, no. 6 (June 30, 2022): e0269563. http://dx.doi.org/10.1371/journal.pone.0269563.
Abstract:
SARS-CoV-2 causes the current global pandemic coronavirus disease 2019. Widely-available effective drugs could be a critical factor in halting the pandemic. The main protease (3CLpro) plays a vital role in viral replication; therefore, it is of great interest to find inhibitors for this enzyme. We applied the combination of virtual screening based on molecular docking derived from the crystal structure of the peptidomimetic inhibitors (N3, 13b, and 11a), and experimental verification revealed FDA-approved drugs that could inhibit the 3CLpro of SARS-CoV-2. Three drugs were selected using the binding energy criteria and subsequently performed the 3CLpro inhibition by enzyme-based assay. In addition, six common drugs were also chosen to study the 3CLpro inhibition. Among these compounds, lapatinib showed high efficiency of 3CLpro inhibition (IC50 value of 35 ± 1 μM and Ki of 23 ± 1 μM). The binding behavior of lapatinib against 3CLpro was elucidated by molecular dynamics simulations. This drug could well bind with 3CLpro residues in the five subsites S1’, S1, S2, S3, and S4. Moreover, lapatinib’s key chemical pharmacophore features toward SAR-CoV-2 3CLpro shared important HBD and HBA with potent peptidomimetic inhibitors. The rational design of lapatinib was subsequently carried out using the obtained results. Our discovery provides an effective repurposed drug and its newly designed analogs to inhibit SARS-CoV-2 3CLpro.
6
Glab-ampai, Kittirat, Kanasap Kaewchim, Thanatsaran Saenlom, Watayagorn Thepsawat, Kodchakorn Mahasongkram, Nitat Sookrung, Wanpen Chaicumpa, and Monrat Chulanetra. "Human Superantibodies to 3CLpro Inhibit Replication of SARS-CoV-2 across Variants." International Journal of Molecular Sciences 23, no. 12 (June 13, 2022): 6587. http://dx.doi.org/10.3390/ijms23126587.
Abstract:
Broadly effective and safe anti-coronavirus agent is existentially needed. Major protease (3CLpro) is a highly conserved enzyme of betacoronaviruses. The enzyme plays pivotal role in the virus replication cycle. Thus, it is a good target of a broadly effective anti-Betacoronavirus agent. In this study, human single-chain antibodies (HuscFvs) of the SARS-CoV-2 3CLpro were generated using phage display technology. The 3CLpro-bound phages were used to infect Escherichia coli host for the production the 3CLpro-bound HuscFvs. Computerized simulation was used to guide the selection of the phage infected-E. coli clones that produced HuscFvs with the 3CLpro inhibitory potential. HuscFvs of three phage infected-E. coli clones were predicted to form contact interface with residues for 3CLpro catalytic activity, substrate binding, and homodimerization. These HuscFvs were linked to a cell-penetrating peptide to make them cell-penetrable, i.e., became superantibodies. The superantibodies blocked the 3CLpro activity in vitro, were not toxic to human cells, traversed across membrane of 3CLpro-expressing cells to co-localize with the intracellular 3CLpro and most of all, they inhibited replication of authentic SARS-CoV-2 Wuhan wild type and α, β, δ, and Omicron variants that were tested. The superantibodies should be investigated further towards clinical application as a safe and broadly effective anti-Betacoronavirus agent.
7
Ye, Gang, Xiaowei Wang, Xiaohan Tong, Yuejun Shi, Zhen F. Fu, and Guiqing Peng. "Structural Basis for Inhibiting Porcine Epidemic Diarrhea Virus Replication with the 3C-Like Protease Inhibitor GC376." Viruses 12, no. 2 (February 21, 2020): 240. http://dx.doi.org/10.3390/v12020240.
Abstract:
Porcine epidemic diarrhea virus (PEDV), being highly virulent and contagious in piglets, has caused significant damage to the pork industries of many countries worldwide. There are no commercial drugs targeting coronaviruses (CoVs), and few studies on anti-PEDV inhibitors. The coronavirus 3C-like protease (3CLpro) has a conserved structure and catalytic mechanism and plays a key role during viral polyprotein processing, thus serving as an appealing antiviral drug target. Here, we report the anti-PEDV effect of the broad-spectrum inhibitor GC376 (targeting 3Cpro or 3CLpro of viruses in the picornavirus-like supercluster). GC376 was highly effective against the PEDV 3CLpro and exerted similar inhibitory effects on two PEDV strains. Furthermore, the structure of the PEDV 3CLpro in complex with GC376 was determined at 1.65 Å. We elucidated structural details and analyzed the differences between GC376 binding with the PEDV 3CLpro and GC376 binding with the transmissible gastroenteritis virus (TGEV) 3CLpro. Finally, we explored the substrate specificity of PEDV 3CLpro at the P2 site and analyzed the effects of Leu group modification in GC376 on inhibiting PEDV infection. This study helps us to understand better the PEDV 3CLpro substrate specificity, providing information on the optimization of GC376 for development as an antiviral therapeutic against coronaviruses.
8
Chen, Chia-Nan, Coney P. C. Lin, Kuo-Kuei Huang, Wei-Cheng Chen, Hsin-Pang Hsieh, Po-Huang Liang, and John T. A. Hsu. "Inhibition of SARS-CoV 3C-like Protease Activity by Theaflavin-3,3'-digallate (TF3)." Evidence-Based Complementary and Alternative Medicine 2, no. 2 (2005): 209–15. http://dx.doi.org/10.1093/ecam/neh081.
Abstract:
SARS-CoV is the causative agent of severe acute respiratory syndrome (SARS). The virally encoded 3C-like protease (3CLPro) has been presumed critical for the viral replication of SARS-CoV in infected host cells. In this study, we screened a natural product library consisting of 720 compounds for inhibitory activity against 3CLPro. Two compounds in the library were found to be inhibitive: tannic acid (IC50 = 3 µM) and 3-isotheaflavin-3-gallate (TF2B) (IC50 = 7 µM). These two compounds belong to a group of natural polyphenols found in tea. We further investigated the 3CLPro-inhibitory activity of extracts from several different types of teas, including green tea, oolong tea, Puer tea and black tea. Our results indicated that extracts from Puer and black tea were more potent than that from green or oolong teas in their inhibitory activities against 3CLPro. Several other known compositions in teas were also evaluated for their activities in inhibiting 3CLPro. We found that caffeine, (—)-epigallocatechin gallte (EGCg), epicatechin (EC), theophylline (TP), catechin (C), epicatechin gallate (ECg) and epigallocatechin (EGC) did not inhibit 3CLPro activity. Only theaflavin-3,3′-digallate (TF3) was found to be a 3CLPro inhibitor. This study has resulted in the identification of new compounds that are effective 3CLPro inhibitors.
9
Rana, Shiwani, Prateek Kumar, Anchal Sharma, Sanjay Sharma, Rajanish Giri, and Kalyan S. Ghosh. "Identification of Naturally Occurring Antiviral Molecules for SARS-CoV-2 Mitigation." Open COVID Journal 1, no. 1 (June 10, 2021): 38–46. http://dx.doi.org/10.2174/2666958702101010038.
Abstract:
Aim: This study aimed to virtually screen the naturally occurring antiviral molecules for SARS-CoV-2 mitigation based on multiple molecular targets using docking and molecular dynamics simulations. Background: The coronavirus catastrophe (COVID-19) caused by a novel strain of coronavirus (SARS-CoV-2) has turned the world upside down at an unprecedented level and has been declared a pandemic by the World Health Organization. It has resulted in a huge number of infections as well as fatalities due to severe lower respiratory tract sickness. Objective: The objective of this study was the identification of inhibitors against the crucial molecular targets linked with viral infection caused by SARS-CoV-2. Materials and Methods: In silico screening of twenty naturally occurring antiviral molecules was performed using the Autodock docking tool. Further, molecular dynamics (MD) simulations were performed on the most stable docked complex between cysteine-like protease or 3CL protease (3CLpro) and the best-identified inhibitor (bilobetin). Results: 3CLpros is one of the very important molecular targets as it is involved in the replication process of the virus. In the present study, we have initially investigated the inhibitory potential of naturally occurring antiviral molecules against the activity of main viral protease (3CLpro) to put a halt to viral replication. The investigation had been carried out through docking of the molecules with 3CLpro. Based on the results, the three most potential molecules (bilobetin, ginkgetin and sciadopitysin) have been screened. Further, these molecules were subjected to checking their activity on other molecular targets like papain-like protease (PLpro), spike protein S1, RNA dependent RNA polymerase (RdRp), and Angiotensin-Converting Enzyme 2 (ACE2) receptor. In addition to 3CLpro inhibition, ginkgetin was also predicted as an inhibitor of PLpro. However, none of these three compounds was found to be effective on the rest of the molecular targets. Molecular Dynamics (MD) simulations of the most stable docked complex between 3CLpro and its best inhibitor (bilobetin) confirmed notable conformational stability of the docked complex under a dynamic state. Conclusion: Bilobetin alone or a combination of bilobetin and ginkgetin may be used to impede viral replication. These observations are solely based on the results from blind docking with protein molecules and need to be further corroborated with experimental results.
10
Wu, Jing, Bo Feng, Li-Xin Gao, Chun Zhang, Jia Li, Da-Jun Xiang, Yi Zang, and Wen-Long Wang. "Synthesis and Biochemical Evaluation of 8H-Indeno[1,2-d]thiazole Derivatives as Novel SARS-CoV-2 3CL Protease Inhibitors." Molecules 27, no. 10 (May 23, 2022): 3359. http://dx.doi.org/10.3390/molecules27103359.
Abstract:
The COVID-19 pandemic caused by SARS-CoV-2 is a global burden on human health and economy. The 3-Chymotrypsin-like cysteine protease (3CLpro) becomes an attractive target for SARS-CoV-2 due to its important role in viral replication. We synthesized a series of 8H-indeno[1,2-d]thiazole derivatives and evaluated their biochemical activities against SARS-CoV-2 3CLpro. Among them, the representative compound 7a displayed inhibitory activity with an IC50 of 1.28 ± 0.17 μM against SARS-CoV-2 3CLpro. Molecular docking of 7a against 3CLpro was performed and the binding mode was rationalized. These preliminary results provide a unique prototype for the development of novel inhibitors against SARS-CoV-2 3CLpro.
11
Kim, Yunjeong, Vinay Shivanna, Sanjeev Narayanan, Allan M. Prior, Sahani Weerasekara, Duy H. Hua, Anushka C. Galasiti Kankanamalage, William C. Groutas, and Kyeong-Ok Chang. "Broad-Spectrum Inhibitors against 3C-Like Proteases of Feline Coronaviruses and Feline Caliciviruses." Journal of Virology 89, no. 9 (February 18, 2015): 4942–50. http://dx.doi.org/10.1128/jvi.03688-14.
Abstract:
ABSTRACTFeline infectious peritonitis and virulent, systemic calicivirus infection are caused by certain types of feline coronaviruses (FCoVs) and feline caliciviruses (FCVs), respectively, and are important infectious diseases with high fatality rates in members of the Felidae family. While FCoV and FCV belong to two distinct virus families, theCoronaviridaeand theCaliciviridae, respectively, they share a dependence on viral 3C-like protease (3CLpro) for their replication. Since 3CLpro is functionally and structurally conserved among these viruses and essential for viral replication, 3CLpro is considered a potential target for the design of antiviral drugs with broad-spectrum activities against these distinct and highly important viral infections. However, small-molecule inhibitors against the 3CLpro enzymes of FCoV and FCV have not been previously identified. In this study, derivatives of peptidyl compounds targeting 3CLpro were synthesized and evaluated for their activities against FCoV and FCV. The structures of compounds that showed potent dual antiviral activities with a wide margin of safety were identified and are discussed. Furthermore, thein vivoefficacy of 3CLpro inhibitors was evaluated using a mouse model of coronavirus infection. Intraperitoneal administration of two 3CLpro inhibitors in mice infected with murine hepatitis virus A59, a hepatotropic coronavirus, resulted in significant reductions in virus titers and pathological lesions in the liver compared to the findings for the controls. These results suggest that the series of 3CLpro inhibitors described here may have the potential to be further developed as therapeutic agents against these important viruses in domestic and wild cats. This study provides important insights into the structure and function relationships of 3CLpro for the design of antiviral drugs with broader antiviral activities.IMPORTANCEFeline infectious peritonitis virus (FIPV) is the leading cause of death in young cats, and virulent, systemic feline calicivirus (vs-FCV) causes a highly fatal disease in cats for which no preventive or therapeutic measure is available. The genomes of these distinct viruses, which belong to different virus families, encode a structurally and functionally conserved 3C-like protease (3CLpro) which is a potential target for broad-spectrum antiviral drug development. However, no studies have previously reported a structural platform for the design of antiviral drugs with activities against these viruses or on the efficacy of 3CLpro inhibitors against coronavirus infection in experimental animals. In this study, we explored the structure-activity relationships of the derivatives of 3CLpro inhibitors and identified inhibitors with potent dual activities against these viruses. In addition, the efficacy of the 3CLpro inhibitors was demonstrated in mice infected with a murine coronavirus. Overall, our study provides the first insight into a structural platform for anti-FIPV and anti-FCV drug development.
12
Naumovich, Vladislav, Maria Grishina, and Vladimir Potemkin. "Establishment of models for reliability evaluation of 3CLpro ligand-receptor complexes with different binding sites." Future Medicinal Chemistry 14, no. 7 (April 2022): 501–10. http://dx.doi.org/10.4155/fmc-2021-0271.
Abstract:
Aim: Recent research shows that 3CLpro enzyme of SARS-CoV-2 is a significant target against COVID-19. Drug modeling allows the design of inhibitors of 3CLpro, but the accuracy of those methods remains unclear. Therefore, it is important to determine the trustworthiness of the designed ligand–receptor complexes. Method & materials: The authors built models for the reliability evaluation of 3CLpro complexes with ligands using an in-house developed AlteQ approach and complementarity principles. The models were based on 145 experimentally found 3CLpro complexes with ligands for five different binding sites. Result & conclusion: The obtained models correspond to linear regression with high values of correlation coefficients and can be successfully used to determine the reliability of the docked 3CLpro complexes with ligands.
13
Zhang, Yue, Huijie Chen, Mengmeng Zou, Rick Oerlemans, Changhao Shao, Yudong Ren, Ruili Zhang, Xiaodan Huang, Guangxing Li, and Yingying Cong. "Hypericin Inhibit Alpha-Coronavirus Replication by Targeting 3CL Protease." Viruses 13, no. 9 (September 14, 2021): 1825. http://dx.doi.org/10.3390/v13091825.
Abstract:
The porcine epidemic diarrhea virus (PEDV) is an Alphacoronavirus (α-CoV) that causes high mortality in infected piglets, resulting in serious economic losses in the farming industry. Hypericin is a dianthrone compound that has been shown as an antiviral activity on several viruses. Here, we first evaluated the antiviral effect of hypericin in PEDV and found the viral replication and egression were significantly reduced with hypericin post-treatment. As hypericin has been shown in SARS-CoV-2 that it is bound to viral 3CLpro, we thus established a molecular docking between hypericin and PEDV 3CLpro using different software and found hypericin bound to 3CLpro through two pockets. These binding pockets were further verified by another docking between hypericin and PEDV 3CLpro pocket mutants, and the fluorescence resonance energy transfer (FRET) assay confirmed that hypericin inhibits the PEDV 3CLpro activity. Moreover, the alignments of α-CoV 3CLpro sequences or crystal structure revealed that the pockets mediating hypericin and PEDV 3CLpro binding were highly conserved, especially in transmissible gastroenteritis virus (TGEV). We then validated the anti-TGEV effect of hypericin through viral replication and egression. Overall, our results push forward that hypericin was for the first time shown to have an inhibitory effect on PEDV and TGEV by targeting 3CLpro, and it deserves further attention as not only a pan-anti-α-CoV compound but potentially also as a compound of other coronaviral infections.
14
Ahmad, Bilal, Maria Batool, Qurat ul Ain, Moon Suk Kim, and Sangdun Choi. "Exploring the Binding Mechanism of PF-07321332 SARS-CoV-2 Protease Inhibitor through Molecular Dynamics and Binding Free Energy Simulations." International Journal of Molecular Sciences 22, no. 17 (August 24, 2021): 9124. http://dx.doi.org/10.3390/ijms22179124.
Abstract:
The novel coronavirus disease, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), rapidly spreading around the world, poses a major threat to the global public health. Herein, we demonstrated the binding mechanism of PF-07321332, α-ketoamide, lopinavir, and ritonavir to the coronavirus 3-chymotrypsin-like-protease (3CLpro) by means of docking and molecular dynamic (MD) simulations. The analysis of MD trajectories of 3CLpro with PF-07321332, α-ketoamide, lopinavir, and ritonavir revealed that 3CLpro–PF-07321332 and 3CLpro–α-ketoamide complexes remained stable compared with 3CLpro–ritonavir and 3CLpro–lopinavir. Investigating the dynamic behavior of ligand–protein interaction, ligands PF-07321332 and α-ketoamide showed stronger bonding via making interactions with catalytic dyad residues His41–Cys145 of 3CLpro. Lopinavir and ritonavir were unable to disrupt the catalytic dyad, as illustrated by increased bond length during the MD simulation. To decipher the ligand binding mode and affinity, ligand interactions with SARS-CoV-2 proteases and binding energy were calculated. The binding energy of the bespoke antiviral PF-07321332 clinical candidate was two times higher than that of α-ketoamide and three times than that of lopinavir and ritonavir. Our study elucidated in detail the binding mechanism of the potent PF-07321332 to 3CLpro along with the low potency of lopinavir and ritonavir due to weak binding affinity demonstrated by the binding energy data. This study will be helpful for the development and optimization of more specific compounds to combat coronavirus disease.
15
Lu, Xiao Tao, Amy C. Sims, and Mark R. Denison. "Mouse Hepatitis Virus 3C-Like Protease Cleaves a 22-Kilodalton Protein from the Open Reading Frame 1a Polyprotein in Virus-Infected Cells and In Vitro." Journal of Virology 72, no. 3 (March 1, 1998): 2265–71. http://dx.doi.org/10.1128/jvi.72.3.2265-2271.1998.
Abstract:
ABSTRACT The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.
16
Ibrahim, Mahmoud A. A., Alaa H. M. Abdelrahman, Dina E. M. Mohamed, Khlood A. A. Abdeljawaad, Mohamed Ahmed Naeem, Gamal A. Gabr, Ahmed M. Shawky, et al. "Chetomin, a SARS-CoV-2 3C-like Protease (3CLpro) Inhibitor: In Silico Screening, Enzyme Docking, Molecular Dynamics and Pharmacokinetics Analysis." Viruses 15, no. 1 (January 15, 2023): 250. http://dx.doi.org/10.3390/v15010250.
Abstract:
The emergence of the Coronavirus Disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to over 6 million deaths. The 3C-like protease (3CLpro) enzyme of the SARS-CoV-2 virus is an attractive druggable target for exploring therapeutic drug candidates to combat COVID-19 due to its key function in viral replication. Marine natural products (MNPs) have attracted considerable attention as alternative sources of antiviral drug candidates. In looking for potential 3CLpro inhibitors, the MNP database (>14,000 molecules) was virtually screened against 3CLpro with the assistance of molecular docking computations. The performance of AutoDock and OEDocking software in anticipating the ligand-3CLpro binding mode was first validated according to the available experimental data. Based on the docking scores, the most potent MNPs were further subjected to molecular dynamics (MD) simulations, and the binding affinities of those molecules were computed using the MM-GBSA approach. According to MM-GBSA//200 ns MD simulations, chetomin (UMHMNP1403367) exhibited a higher binding affinity against 3CLpro than XF7, with ΔGbinding values of −55.5 and −43.7 kcal/mol, respectively. The steadiness and tightness of chetomin with 3CLpro were evaluated, revealing the high stabilization of chetomin (UMHMNP1403367) inside the binding pocket of 3CLpro throughout 200 ns MD simulations. The physicochemical and pharmacokinetic features of chetomin were also predicted, and the oral bioavailability of chetomin was demonstrated. Furthermore, the potentiality of chetomin analogues –namely, chetomin A-D– as 3CLpro inhibitors was investigated. These results warrant further in vivo and in vitro assays of chetomin (UMHMNP1403367) as a promising anti-COVID-19 drug candidate.
17
Fakih, Taufik Muhammad, and Dwi Syah Fitra Ramadhan. "Prediction of SARS-CoV-2 3C-like protease (3CLpro) crystal structure to provide COVID-19 inhibitor design through computational studies." Biogenesis: Jurnal Ilmiah Biologi 9, no. 2 (December 30, 2021): 213–19. http://dx.doi.org/10.24252/bio.v9i2.24520.
Abstract:
Infectious diseases have lately become pandemic, posing a threat to global public health with the introduction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), previously provisionally named 2019 novel coronavirus or 2019-nCoV). Technological advancements have increased the possibility of discovering natural inhibitor candidates capable of preventing and controlling COVID-19 infections. The SARS-CoV-2 3C-like protease (3CLpro) is critical for SARS-CoV-2 replication and is a prospective therapeutic target. This study aims to identify, evaluate, and explore the 3CLpro macromolecular structures from SARS-CoV and SARS-CoV-2, as well as their impact on angiotensin-converting enzyme 2 (ACE-2). The discovery of the two 3CLpro macromolecules revealed structural similarities in several regions. These findings were subsequently confirmed by performing protein-protein docking simulations to observe the interaction of 3CLpro with the active site ACE-2. With an ACE score of 701.41 kJ/mol, SARS-COV-2 3CLpro forms the strongest binding with ACE-2. As a result, the findings of this research can be used to guide the development of potential SARS-CoV-2 3CLpro inhibitors for the treatment of COVID-19 infectious diseases.
18
Ma, Ling, Yongli Xie, Mei Zhu, Dongrong Yi, Jianyuan Zhao, Saisai Guo, Yongxin Zhang, et al. "Identification of Darunavir Derivatives for Inhibition of SARS-CoV-2 3CLpro." International Journal of Molecular Sciences 23, no. 24 (December 16, 2022): 16011. http://dx.doi.org/10.3390/ijms232416011.
Abstract:
The effective antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed around the world. The 3C-like protease (3CLpro) of SARS-CoV-2 plays a pivotal role in virus replication; it also has become an important therapeutic target for the infection of SARS-CoV-2. In this work, we have identified Darunavir derivatives that inhibit the 3CLpro through a high-throughput screening method based on a fluorescence resonance energy transfer (FRET) assay in vitro. We found that the compounds 29# and 50# containing polyphenol and caffeine derivatives as the P2 ligand, respectively, exhibited favorable anti-3CLpro potency with EC50 values of 6.3 μM and 3.5 μM and were shown to bind to SARS-CoV-2 3CLpro in vitro. Moreover, we analyzed the binding mode of the DRV in the 3CLpro through molecular docking. Importantly, 29# and 50# exhibited a similar activity against the protease in Omicron variants. The inhibitory effect of compounds 29# and 50# on the SARS-CoV-2 3CLpro warrants that they are worth being the template to design functionally improved inhibitors for the treatment of COVID-19.
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Valipour, Mehdi, Silvia Di Giacomo, Antonella Di Sotto, and Hamid Irannejad. "Discovery of Chalcone-Based Hybrid Structures as High Affinity and Site-Specific Inhibitors against SARS-CoV-2: A Comprehensive Structural Analysis Based on Various Host-Based and Viral Targets." International Journal of Molecular Sciences 24, no. 10 (May 15, 2023): 8789. http://dx.doi.org/10.3390/ijms24108789.
Abstract:
Previous studies indicated that natural-based chalcones have significant inhibitory effects on the coronavirus enzymes 3CLpro and PLpro as well as modulation of some host-based antiviral targets (HBATs). In this study, a comprehensive computational and structural study was performed to investigate the affinity of our compound library consisting of 757 chalcone-based structures (CHA-1 to CHA-757) for inhibiting the 3CLpro and PLpro enzymes and against twelve selected host-based targets. Our results indicated that CHA-12 (VUF 4819) is the most potent and multi-target inhibitor in our chemical library over all viral and host-based targets. Correspondingly, CHA-384 and its congeners containing ureide moieties were found to be potent and selective 3CLpro inhibitors, and benzotriazole moiety in CHA-37 was found to be a main fragment for inhibiting the 3CLpro and PLpro. Surprisingly, our results indicate that the ureide and sulfonamide moieties are integral fragments for the optimum 3CLpro inhibition while occupying the S1 and S3 subsites, which is fully consistent with recent reports on the site-specific 3CLpro inhibitors. Finding the multi-target inhibitor CHA-12, previously reported as an LTD4 antagonist for the treatment of inflammatory pulmonary diseases, prompted us to suggest it as a concomitant agent for relieving respiratory symptoms and suppressing COVID-19 infection.
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Guijarro-Real, Carla, Mariola Plazas, Adrián Rodríguez-Burruezo, Jaime Prohens, and Ana Fita. "Potential In Vitro Inhibition of Selected Plant Extracts against SARS-CoV-2 Chymotripsin-Like Protease (3CLPro) Activity." Foods 10, no. 7 (June 29, 2021): 1503. http://dx.doi.org/10.3390/foods10071503.
Abstract:
Antiviral treatments inhibiting Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication may represent a strategy complementary to vaccination to fight the ongoing Coronavirus disease 19 (COVID-19) pandemic. Molecules or extracts inhibiting the SARS-CoV-2 chymotripsin-like protease (3CLPro) could contribute to reducing or suppressing SARS-CoV-2 replication. Using a targeted approach, we identified 17 plant products that are included in current and traditional cuisines as promising inhibitors of SARS-CoV-2 3CLPro activity. Methanolic extracts were evaluated in vitro for inhibition of SARS-CoV-2 3CLPro activity using a quenched fluorescence resonance energy transfer (FRET) assay. Extracts from turmeric (Curcuma longa) rhizomes, mustard (Brassica nigra) seeds, and wall rocket (Diplotaxis erucoides subsp. erucoides) at 500 µg mL−1 displayed significant inhibition of the 3CLPro activity, resulting in residual protease activities of 0.0%, 9.4%, and 14.9%, respectively. Using different extract concentrations, an IC50 value of 15.74 µg mL−1 was calculated for turmeric extract. Commercial curcumin inhibited the 3CLPro activity, but did not fully account for the inhibitory effect of turmeric rhizomes extracts, suggesting that other components of the turmeric extract must also play a main role in inhibiting the 3CLPro activity. Sinigrin, a major glucosinolate present in mustard seeds and wall rocket, did not have relevant 3CLPro inhibitory activity; however, its hydrolysis product allyl isothiocyanate had an IC50 value of 41.43 µg mL−1. The current study identifies plant extracts and molecules that can be of interest in the search for treatments against COVID-19, acting as a basis for future chemical, in vivo, and clinical trials.
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Jukič, Marko, Blaž Škrlj, Gašper Tomšič, Sebastian Pleško, Črtomir Podlipnik, and Urban Bren. "Prioritisation of Compounds for 3CLpro Inhibitor Development on SARS-CoV-2 Variants." Molecules 26, no. 10 (May 18, 2021): 3003. http://dx.doi.org/10.3390/molecules26103003.
Abstract:
COVID-19 represents a new potentially life-threatening illness caused by severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2 pathogen. In 2021, new variants of the virus with multiple key mutations have emerged, such as B.1.1.7, B.1.351, P.1 and B.1.617, and are threatening to render available vaccines or potential drugs ineffective. In this regard, we highlight 3CLpro, the main viral protease, as a valuable therapeutic target that possesses no mutations in the described pandemically relevant variants. 3CLpro could therefore provide trans-variant effectiveness that is supported by structural studies and possesses readily available biological evaluation experiments. With this in mind, we performed a high throughput virtual screening experiment using CmDock and the “In-Stock” chemical library to prepare prioritisation lists of compounds for further studies. We coupled the virtual screening experiment to a machine learning-supported classification and activity regression study to bring maximal enrichment and available structural data on known 3CLpro inhibitors to the prepared focused libraries. All virtual screening hits are classified according to 3CLpro inhibitor, viral cysteine protease or remaining chemical space based on the calculated set of 208 chemical descriptors. Last but not least, we analysed if the current set of 3CLpro inhibitors could be used in activity prediction and observed that the field of 3CLpro inhibitors is drastically under-represented compared to the chemical space of viral cysteine protease inhibitors. We postulate that this methodology of 3CLpro inhibitor library preparation and compound prioritisation far surpass the selection of compounds from available commercial “corona focused libraries”.
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Huynh, Thi Ngoc Thanh, Thi Thanh Thu Tran, Thi My Hanh Pham, and Kha Quang Quach. "Study on the interaction mechanism of penciclovir drug on 3CLpro of SAR-COV-2 by simulation methods." Dong Thap University Journal of Science 12, no. 5 (June 23, 2023): 42–47. http://dx.doi.org/10.52714/dthu.12.5.2023.1070.
Abstract:
Since the outbreak of SAR-CoV-2 infections in Wuhan (China), researched communication is on race to investigate the specific antiviral drug for Covid-19 treatment. 3CLpro main protease is chosen as a protein target because of its high value in preventing the SAR-CoV-2 viral replications. In this study, we hereby aim to clarify the efficiency of Penciclovir in inhibiting the mechanic of 3CLpro target of SAR-CoV-2. Using docking simulation and molecular dynamic simulation (SMD), the interaction of Penciclovir with 3CLpro target was investigated. The results show that Penciclovir strongly interacts with 3CLpro target, and the non-binding interaction plays a more important role than hydrogen bonding in the steady state of the receptor-ligand conformation.
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Chen, Lili, Shuai Chen, Chunshan Gui, Jianhua Shen, Xu Shen, and Hualiang Jiang. "Discovering Severe Acute Respiratory Syndrome Coronavirus 3CL Protease Inhibitors: Virtual Screening, Surface Plasmon Resonance, and Fluorescence Resonance Energy Transfer Assays." Journal of Biomolecular Screening 11, no. 8 (December 2006): 915–21. http://dx.doi.org/10.1177/1087057106293295.
Abstract:
An integrated system has been developed for discovering potent inhibitors of severe acute respiratory syndrome coronavirus 3C-like protease (SARS-CoV 3CLpro) 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 3CLpro. The enzymatic inhibition activities of these 52 SARS-CoV 3CLpro binders were further applied to FRET-based assay, and IC50 values were determined. Based on this integrated assay platform, 8 new SARS-CoV 3CLpro inhibitors were discovered. The fact that the obtained IC50 values for the inhibitors are in good accordance with the discovered dissociation equilibrium constants (KDs) 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 3CLpro inhibitors, and the determined inhibitors could be used as possible lead compounds for further research.
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Hamill, Pamela, Derek Hudson, Richard Y. Kao, Polly Chow, Meera Raj, Hongyan Xu, Martin J. Richer, and François Jean. "Development of a red-shifted fluorescence-based assay for SARS-coronavirus 3CL protease: identification of a novel class of anti-SARS agents from the tropical marine sponge Axinella corrugata." Biological Chemistry 387, no. 8 (August 1, 2006): 1063–74. http://dx.doi.org/10.1515/bc.2006.131.
Abstract:
Abstract 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 (K m and k cat values of 14 μM 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 μM) and anti-SARS agent (EC50=112 μM; median toxic concentration >800 μM) from the tropical marine sponge Axinella corrugata.
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Jo, Seri, Hwa Young Kim, Dong Hae Shin, and Mi-Sun Kim. "Dimerization Tendency of 3CLpros of Human Coronaviruses Based on the X-ray Crystal Structure of the Catalytic Domain of SARS-CoV-2 3CLpro." International Journal of Molecular Sciences 23, no. 9 (May 9, 2022): 5268. http://dx.doi.org/10.3390/ijms23095268.
Abstract:
3CLpro of SARS-CoV-2 is a promising target for developing anti-COVID19 agents. In order to evaluate the catalytic activity of 3CLpros according to the presence or absence of the dimerization domain, two forms had been purified and tested. Enzyme kinetic studies with a FRET method revealed that the catalytic domain alone presents enzymatic activity, despite it being approximately 8.6 times less than that in the full domain. The catalytic domain was crystallized and its X-ray crystal structure has been determined to 2.3 Å resolution. There are four protomers in the asymmetric unit. Intriguingly, they were packed as a dimer though the dimerization domain was absent. The RMSD of superimposed two catalytic domains was 0.190 for 182 Cα atoms. A part of the long hinge loop (LH-loop) from Gln189 to Asp197 was not built in the model due to its flexibility. The crystal structure indicates that the decreased proteolytic activity of the catalytic domain was due to the incomplete construction of the substrate binding part built by the LH-loop. A structural survey with other 3CLpros showed that SARS-CoV families do not have interactions between DM-loop due to the conformational difference at the last turn of helix α7 compared with others. Therefore, we can conclude that the monomeric form contains nascent enzyme activity and that its efficiency increases by dimerization. This new insight may contribute to understanding the behavior of SARS-CoV-2 3CLpro and thus be useful in developing anti-COVID-19 agents.
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Garland, Gavin D., Robert F. Harvey, Thomas E. Mulroney, Mie Monti, Stewart Fuller, Richard Haigh, Pehuén Pereyra Gerber, Michael R. Barer, Nicholas J. Matheson, and Anne E. Willis. "Development of a colorimetric assay for the detection of SARS-CoV-2 3CLpro activity." Biochemical Journal 479, no. 8 (April 21, 2022): 901–20. http://dx.doi.org/10.1042/bcj20220105.
Abstract:
Diagnostic testing continues to be an integral component of the strategy to contain the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) global pandemic, the causative agent of Coronavirus Disease 2019 (COVID-19). The SARS-CoV-2 genome encodes the 3C-like protease (3CLpro) which is essential for coronavirus replication. This study adapts an in vitro colorimetric gold nanoparticle (AuNP) based protease assay to specifically detect the activity of SARS-CoV-2 3CLpro as a purified recombinant protein and as a cellular protein exogenously expressed in HEK293T human cells. We also demonstrate that the specific sensitivity of the assay for SARS-CoV-2 3CLpro can be improved by use of an optimised peptide substrate and through hybrid dimerisation with inactive 3CLpro mutant monomers. These findings highlight the potential for further development of the AuNP protease assay to detect SARS-CoV-2 3CLpro activity as a novel, accessible and cost-effective diagnostic test for SARS-CoV-2 infection at the point-of-care. Importantly, this versatile assay could also be easily adapted to detect specific protease activity associated with other viruses or diseases conditions.
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Li, Zhonghua, Hua Cao, Yufang Cheng, Xiaoqian Zhang, Wei Zeng, Yumei Sun, Shuhua Chen, Qigai He, and Heyou Han. "Inhibition of Porcine Epidemic Diarrhea Virus Replication and Viral 3C-Like Protease by Quercetin." International Journal of Molecular Sciences 21, no. 21 (October 30, 2020): 8095. http://dx.doi.org/10.3390/ijms21218095.
Abstract:
For the last decade, porcine epidemic diarrhea virus (PEDV) variant strains have caused severe damage to the global pig industry. Until now, no effective antivirals have been developed for the therapeutic treatment of PEDV infection. In the present study, we found that quercetin significantly suppressed PEDV infection at noncytotoxic concentrations. A molecular docking study indicated that quercetin might bind the active site and binding pocket of PEDV 3C-like protease (3CLpro). Surface plasmon resonance (SPR) analysis revealed that quercetin exhibited a binding affinity to PEDV 3CLpro. Based on the results of the fluorescence resonance energy transfer (FRET) assay, quercetin was proven to exert an inhibitory effect on PEDV 3CLpro. Since coronavirus 3CLpro is an important drug target and participates in the viral replication process, quercetin should be developed as a novel drug in the control of PEDV infection.
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Fitriana, Adita Silvia, and Sri Royani. "Molecular Docking Study of Chalcone Derivatives as Potential Inhibitors of SARS-CoV-2 Main Protease." Indo. J. Chem. Res. 9, no. 3 (January 29, 2022): 150–62. http://dx.doi.org/10.30598//ijcr.2022.9-fit.
Abstract:
SARS-CoV-2 main protease is a potential target for the development of AntiCOVID-19. Several chalcones have inhibitory activity against 3CLpro SARS-CoV and 3CLpro MERS-CoV. This study aims to predict the potential of chalcones in inhibiting 3CLpro SARS-CoV-2, which plays a role in the viral replication process. In silico research carried the prediction through molecular docking toward proteins with PDB ID 6LU7 and 6Y2F. Compound K27 has a docking score more negative than lopinavir. This result indicates that compound K27 is predicted to inhibit the SARS-CoV-2 replication.
29
Hegyi, Annette, Agnes Friebe, Alexander E. Gorbalenya, and John Ziebuhr. "Mutational analysis of the active centre of coronavirus 3C-like proteases." Journal of General Virology 83, no. 3 (March 1, 2002): 581–93. http://dx.doi.org/10.1099/0022-1317-83-3-581.
Abstract:
Formation of the coronavirus replication–transcription complex involves the synthesis of large polyprotein precursors that are extensively processed by virus-encoded cysteine proteases. In this study, the coding sequence of the feline infectious peritonitis virus (FIPV) main protease, 3CLpro, was determined. Comparative sequence analyses revealed that FIPV 3CLpro and other coronavirus main proteases are related most closely to the 3C-like proteases of potyviruses. The predicted active centre of the coronavirus enzymes has accepted unique replacements that were probed by extensive mutational analysis. The wild-type FIPV 3CLpro domain and 25 mutants were expressed in Escherichia coli and tested for proteolytic activity in a peptide-based assay. The data strongly suggest that, first, the FIPV 3CLpro catalytic system employs His41 and Cys144 as the principal catalytic residues. Second, the amino acids Tyr160 and His162, which are part of the conserved sequence signature Tyr160–Met161–His162 and are believed to be involved in substrate recognition, were found to be indispensable for proteolytic activity. Third, replacements of Gly83 and Asn64, which were candidates to occupy the position spatially equivalent to that of the catalytic Asp residue of chymotrypsin-like proteases, resulted in proteolytically active proteins. Surprisingly, some of the Asn64 mutants even exhibited strongly increased activities. Similar results were obtained for human coronavirus (HCoV) 3CLpro mutants in which the equivalent Asn residue (HCoV 3CLpro Asn64) was substituted. These data lead us to conclude that both the catalytic systems and substrate-binding pockets of coronavirus main proteases differ from those of other RNA virus 3C and 3C-like proteases.
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Saquib, Quaiser, Ahmed H. Bakheit, Sarfaraz Ahmed, Sabiha M. Ansari, Abdullah M. Al-Salem, and Abdulaziz A. Al-Khedhairy. "Identification of Phytochemicals from Arabian Peninsula Medicinal Plants as Strong Binders to SARS-CoV-2 Proteases (3CLPro and PLPro) by Molecular Docking and Dynamic Simulation Studies." Molecules 29, no. 5 (February 25, 2024): 998. http://dx.doi.org/10.3390/molecules29050998.
Abstract:
We provide promising computational (in silico) data on phytochemicals (compounds 1–10) from Arabian Peninsula medicinal plants as strong binders, targeting 3-chymotrypsin-like protease (3CLPro) and papain-like proteases (PLPro) of SARS-CoV-2. Compounds 1–10 followed the Lipinski rules of five (RO5) and ADMET analysis, exhibiting drug-like characters. Non-covalent (reversible) docking of compounds 1–10 demonstrated their binding with the catalytic dyad (CYS145 and HIS41) of 3CLPro and catalytic triad (CYS111, HIS272, and ASP286) of PLPro. Moreover, the implementation of the covalent (irreversible) docking protocol revealed that only compounds 7, 8, and 9 possess covalent warheads, which allowed the formation of the covalent bond with the catalytic dyad (CYS145) in 3CLPro and the catalytic triad (CYS111) in PLPro. Root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and radius of gyration (Rg) analysis from molecular dynamic (MD) simulations revealed that complexation between ligands (compounds 7, 8, and 9) and 3CLPro and PLPro was stable, and there was less deviation of ligands. Overall, the in silico data on the inherent properties of the above phytochemicals unravel the fact that they can act as reversible inhibitors for 3CLPro and PLPro. Moreover, compounds 7, 8, and 9 also showed their novel properties to inhibit dual targets by irreversible inhibition, indicating their effectiveness for possibly developing future drugs against SARS-CoV-2. Nonetheless, to confirm the theoretical findings here, the effectiveness of the above compounds as inhibitors of 3CLPro and PLPro warrants future investigations using suitable in vitro and in vivo tests.
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Komissarov, Alexey, Maria Karaseva, Marina Roschina, Sergey Kostrov, and Ilya Demidyuk. "The SARS-CoV-2 main protease doesn’t induce cell death in human cells in vitro." PLOS ONE 17, no. 5 (May 24, 2022): e0266015. http://dx.doi.org/10.1371/journal.pone.0266015.
Abstract:
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19) which has extremely rapidly spread worldwide. In order to develop the effective antiviral therapies, it is required to understand the molecular mechanisms of the SARS-CoV-2 pathogenesis. The main protease, or 3C-like protease (3CLpro), plays the essential role in the coronavirus replication that makes the enzyme a promising therapeutic target. Viral enzymes are known to be multifunctional. Particularly, 3CLpro of SARS-CoV was shown to induce apoptosis in addition to its main function. In the present study we analyzed the cytotoxicity of active SARS-CoV-2 3CLpro and its inactivated form upon their individual expression in four human cell lines. For this purpose, we constructed a protein biosensor which allows to detect the proteolytic activity of SARS-CoV-2 3CLpro and confirmed the expression of the active protease in all cell lines used. We studied viability and morphology of the cells and found that both active and inactivated enzyme variants induce no cell death in contrast to the homologous 3CL protease of SARS-CoV. These results indicate that SARS-CoV-2 3CLpro is unlikely contribute to the cytopathic effect observed during viral infection directly.
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Zhang, Shilei, Jingfeng Wang, and Genhong Cheng. "Protease cleavage of RNF20 facilitates coronavirus replication via stabilization of SREBP1." Proceedings of the National Academy of Sciences 118, no. 37 (August 27, 2021): e2107108118. http://dx.doi.org/10.1073/pnas.2107108118.
Abstract:
COVID-19, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has presented a serious risk to global public health. The viral main protease Mpro (also called 3Clpro) encoded by NSP5 is an enzyme essential for viral replication. However, very few host proteins have been experimentally validated as targets of 3Clpro. Here, through bioinformatics analysis of 300 interferon stimulatory genes (ISGs) based on the prediction method NetCorona, we identify RNF20 (Ring Finger Protein 20) as a novel target of 3Clpro. We have also provided evidence that 3Clpro, but not the mutant 3ClproC145A without catalytic activity, cleaves RNF20 at a conserved Gln521 across species, which subsequently prevents SREBP1 from RNF20-mediated degradation and promotes SARS-CoV-2 replication. We show that RNA interference (RNAi)-mediated depletion of either RNF20 or RNF40 significantly enhances viral replication, indicating the antiviral role of RNF20/RNF40 complex against SARS-CoV-2. The involvement of SREBP1 in SARS-CoV-2 infection is evidenced by a decrease of viral replication in the cells with SREBP1 knockdown and inhibitor AM580. Taken together, our findings reveal RNF20 as a novel host target for SARS-CoV-2 main protease and indicate that 3Clpro inhibitors may treat COVID-19 through not only blocking viral polyprotein cleavage but also enhancing host antiviral response.
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Duarte Filho, Luiz Antonio Miranda de Souza, Cintia Emi Yanaguibashi Leal, Pierre-Edouard Bodet, Edilson Beserra de Alencar Filho, Jackson Roberto Guedes da Silva Almeida, Manon Porta Zapata, Oussama Achour, et al. "The Identification of Peptide Inhibitors of the Coronavirus 3CL Protease from a Fucus ceranoides L. Hydroalcoholic Extract Using a Ligand-Fishing Strategy." Marine Drugs 22, no. 6 (May 27, 2024): 244. http://dx.doi.org/10.3390/md22060244.
Abstract:
Brown seaweeds of the Fucus genus represent a rich source of natural antiviral products. In this study, a Fucus ceranoides hydroalcoholic extract (FCHE) was found to inhibit 74.2 ± 1.3% of the proteolytic activity of the free SARS-CoV-2 3CL protease (3CLpro), an enzyme that plays a pivotal role in polyprotein processing during coronavirus replication and has been identified as a relevant drug discovery target for SARS- and MERS-CoVs infections. To purify and identify 3CLpro ligands with potential inhibitory activity using a one-step approach, we immobilized the enzyme onto magnetic microbeads (3CLpro-MPs), checked that the enzymatic activity was maintained after grafting, and used this bait for a ligand-fishing strategy followed by a high-resolution mass spectrometry analysis of the fished-out molecules. Proof of concept for the ligand-fishing capacity of the 3CLpro-MPs was demonstrated by doping the FCHE extract with the substrate peptide TSAVLQ-pNA, resulting in the preferential capture of this high-affinity peptide within the macroalgal complex matrix. Ligand fishing in the FCHE alone led to the purification and identification via high-resolution mass spectrometry (HRMS) of seven hepta-, octa-, and decapeptides in an eluate mix that significantly inhibited the free 3CLpro more than the starting FCHE (82.7 ± 2.2% inhibition). Molecular docking simulations of the interaction between each of the seven peptides and the 3CLpro demonstrated a high affinity for the enzyme’s proteolytic active site surpassing that of the most affine peptide ligand identified so far (a co-crystallographic peptide). Testing of the corresponding synthetic peptides demonstrated that four out of seven significantly inhibited the free 3CLpro (from 46.9 ± 6.4 to 76.8 ± 3.6% inhibition at 10 µM). This study is the first report identifying peptides from Fucus ceranoides with high inhibitory activity against the SARS-CoV-2 3CLprotease which bind with high affinity to the protease’s active site. It also confirms the effectiveness of the ligand-fishing strategy for the single-step purification of enzyme inhibitors from complex seaweed matrices.
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Rajeswari, Kalepu, W. Jun Chen, A. Aashika, T. Xian Ying, C. Choon Hoong, S. Kuha, and Diya Rajasekhar Chinta. "Binding Interaction Analysis of Phytoconstituents of Commiphora mukul with 3CLPro and PlPro Enzymes of SARS-CoV-2 Virus." ECS Transactions 107, no. 1 (April 24, 2022): 7509–30. http://dx.doi.org/10.1149/10701.7509ecst.
Abstract:
The SARS-CoV-2 belongs to a family of coronaviruses, responsible for the COVID-19 pandemic. The Chymotrypsin-like protease (3CLpro) and Papain-like protease (PLPro), two important enzymes of the SARS-CoV-2, play a key role in translating the viral RNA genome into functional proteins. This study aimed to evaluate and analyze the binding interactions of phytochemicals of oleoresin of Commiphora mukul plant with 3CLPro and PLPro enzymes of SARS-CoV-2 virus. Docking studies were performed on 3CLPro and PLPro with 30 phytoconstituents using AutoDock Vina. A total of 12 compounds were shortlisted based on their minimum binding energies. Their minimum binding energy ranged from -9.2 to -7.1 kcal and the analysis and interpretations are tabulated. Further, the ADME, drug-likeness, and bioactivity scores were documented. The current study enlightened the binding abilities, and the interactions of constituents present in the oleoresin of Commiphora mukul plant with the active sites of 3CLPro and PLPro enzymes.
35
Wang, Yaxin, Binghong Xu, Sen Ma, Hao Wang, Luqing Shang, Cheng Zhu, and Sheng Ye. "Discovery of SARS-CoV-2 3CLPro Peptidomimetic Inhibitors through the Catalytic Dyad Histidine-Specific Protein–Ligand Interactions." International Journal of Molecular Sciences 23, no. 4 (February 21, 2022): 2392. http://dx.doi.org/10.3390/ijms23042392.
Abstract:
As the etiological agent for the coronavirus disease 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenges the ongoing efforts of vaccine development and drug design. Due to the accumulating cases of breakthrough infections, there are urgent needs for broad-spectrum antiviral medicines. Here, we designed and examined five new tetrapeptidomimetic anti-SARS-CoV-2 inhibitors targeting the 3C-Like protease (3CLPro), which is highly conserved among coronaviruses and essential for viral replications. We significantly improved the efficacy of a ketoamide lead compound based on high-resolution co-crystal structures, all-atom simulations, and binding energy calculations. The inhibitors successfully engaged the catalytic dyad histidine residue (H41) of 3CLPro as designed, and they exhibited nanomolar inhibitory capacity as well as mitigated the viral loads of SARS-CoV-2 in cellular assays. As a widely applicable design principle, our results revealed that the potencies of 3CLPro-specific drug candidates were determined by the interplay between 3CLPro H41 residue and the peptidomimetic inhibitors.
36
Olubiyi, Olujide O., Maryam Olagunju, Monika Keutmann, Jennifer Loschwitz, and Birgit Strodel. "High Throughput Virtual Screening to Discover Inhibitors of the Main Protease of the Coronavirus SARS-CoV-2." Molecules 25, no. 14 (July 13, 2020): 3193. http://dx.doi.org/10.3390/molecules25143193.
Abstract:
We use state-of-the-art computer-aided drug design (CADD) techniques to identify prospective inhibitors of the main protease enzyme, 3CLpro of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19. From our screening of over one million compounds including approved drugs, investigational drugs, natural products, and organic compounds, and a rescreening protocol incorporating enzyme dynamics via ensemble docking, we have been able to identify a range of prospective 3CLpro inhibitors. Importantly, some of the identified compounds had previously been reported to exhibit inhibitory activities against the 3CLpro enzyme of the closely related SARS-CoV virus. The top-ranking compounds are characterized by the presence of multiple bi- and monocyclic rings, many of them being heterocycles and aromatic, which are flexibly linked allowing the ligands to adapt to the geometry of the 3CLpro substrate site and involve a high amount of functional groups enabling hydrogen bond formation with surrounding amino acid residues, including the catalytic dyad residues H41 and C145. Among the top binding compounds we identified several tyrosine kinase inhibitors, which include a bioflavonoid, the group of natural products that binds best to 3CLpro. Another class of compounds that decently binds to the SARS-CoV-2 main protease are steroid hormones, which thus may be endogenous inhibitors and might provide an explanation for the age-dependent severity of COVID-19. Many of the compounds identified by our work show a considerably stronger binding than found for reference compounds with in vitro demonstrated 3CLpro inhibition and anticoronavirus activity. The compounds determined in this work thus represent a good starting point for the design of inhibitors of SARS-CoV-2 replication.
37
Yang, Cheng-Wei, Yung-Ning Yang, Po-Huang Liang, Chi-Min Chen, Wei-Liang Chen, Hwan-You Chang, Yu-Sheng Chao, and Shiow-Ju Lee. "Novel Small-Molecule Inhibitors of Transmissible Gastroenteritis Virus." Antimicrobial Agents and Chemotherapy 51, no. 11 (August 20, 2007): 3924–31. http://dx.doi.org/10.1128/aac.00408-07.
Abstract:
ABSTRACT We used swine testicle (ST) cells infected with transmissible gastroenteritis virus (TGEV) and an indirect immunofluorescent assay with antibodies against TGEV spike and nucleocapsid proteins to screen small-molecule compounds that inhibit TGEV replication. Analogues of initial hits were collected and subjected to a 3CL protease (3CLpro) inhibition assay with recombinant 3CLpro and a fluorogenic peptide substrate. A series of benzothiazolium compounds were found to have inhibitory activity against TGEV 3CLpro and to exert anti-TGEV activities in terms of viral protein and RNA replication in TGEV-infected ST cells, with consequent protection of TGEV-infected ST cells from cytopathic effect by blocking the activation of caspase-3.
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Sobhy, Remah, Asad Nawaz, Mohammad Fikry, Rokayya Sami, Eman Algarni, Nada Benajiba, Sameer H. Qari, Alaa T. Qumsani, and Ibrahim Khalifa. "In-Silico Evaluation of 10 Structurally Different Glucosinolates on the Key Enzyme of SARS-CoV-2." Science of Advanced Materials 14, no. 1 (January 1, 2022): 162–74. http://dx.doi.org/10.1166/sam.2022.4190.
Abstract:
The novel coronavirus (2019-nCoV) triggered a worldwide rise in the prevalence of the coronavirus outbreak (COVID-19) and surfaced as a universal wellbeing matter. Analogous with SARS-CoV and MERS-CoV, the main 3-chymotrypsin-alike cysteine protease (3CLPro) virus enzyme that manages the replications of 2019-nCoV and regulates its existence span, possibly will be considered like a medication break through focus. In this study, the binding potential of 10 glucosinolates (Glu) having a variety of structures was studied with the catalytic dyad remains of 2019-nCoV-3CLPro by molecular cutting developing. The outcomes have shown that Glu containing sinigrin (SN) have been shown to be realistically bound to the 2019-nCoV-3CLPro receptor and catalytic dyad binding sites (Cys145 and His41). Our simulation results have shown that sinigrin have a potential activity against 2019-nCoV and could be further used for drug production and optimization in the battle against COVID-19. In details, SN-SARS-CoV-2-3CLPro-facilityacted without exhibit whichever observable variations, with reference to the constancy of Glu-enzyme complexes by means of average RMSD of 1.5±0.02 Å. Meanwhile, the ordinary behavior of a SN-SARS-CoV-2-3CLPro complex continued as compact and steady during (50 ns) MD simulations. Current investigation has revealed that Glu with a specific structure could be successful against COVID-19 as natural components.
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Du, Weian, Liang Zhao, Rong Wu, Boning Huang, Si Liu, Yufeng Liu, Huaiqiu Huang, and Ge Shi. "Predicting drug–Protein interaction with deep learning framework for molecular graphs and sequences: Potential candidates against SAR-CoV-2." PLOS ONE 19, no. 5 (May 10, 2024): e0299696. http://dx.doi.org/10.1371/journal.pone.0299696.
Abstract:
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the COVID-19 disease, which represents a new life-threatening disaster. Regarding viral infection, many therapeutics have been investigated to alleviate the epidemiology such as vaccines and receptor decoys. However, the continuous mutating coronavirus, especially the variants of Delta and Omicron, are tended to invalidate the therapeutic biological product. Thus, it is necessary to develop molecular entities as broad-spectrum antiviral drugs. Coronavirus replication is controlled by the viral 3-chymotrypsin-like cysteine protease (3CLpro) enzyme, which is required for the virus’s life cycle. In the cases of severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV), 3CLpro has been shown to be a promising therapeutic development target. Here we proposed an attention-based deep learning framework for molecular graphs and sequences, training from the BindingDB 3CLpro dataset (114,555 compounds). After construction of such model, we conducted large-scale screening the in vivo/vitro dataset (276,003 compounds) from Zinc Database and visualize the candidate compounds with attention score. geometric-based affinity prediction was employed for validation. Finally, we established a 3CLpro-specific deep learning framework, namely GraphDPI-3CL (AUROC: 0.958) achieved superior performance beyond the existing state of the art model and discovered 10 molecules with a high binding affinity of 3CLpro and superior binding mode.
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Haniyya, M. Ulfah, A. Riswoko, L. Mulyawati, T. Ernawati, and I. Helianti. "Production of recombinant SARS-CoV-2 3CL-protease: The key for the development of protease inhibitors screening kit in search of potential herb cure for COVID-19." IOP Conference Series: Earth and Environmental Science 976, no. 1 (February 1, 2022): 012051. http://dx.doi.org/10.1088/1755-1315/976/1/012051.
Abstract:
Abstract Coronavirus disease-19 (COVID-19) pandemic caused millions of deaths and socio-economics damage worldwide. Corresponding to this, many studies on antiviral drugs exploration are rising to investigate the potential of drug compounds that can repress the replication of the SARS-CoV-2 virus by targeting its main protease named 3-chymotrypsin like protease (3CLpro). Without 3CLpro splicing, polyproteins of SARS-CoV-2 will not function to form new virions. We conducted an in silico thermodynamic study, and found several already known compounds from natural sources such as lovastatin, quinidine, and quinine were potential in inhibiting 3CLpro. However, most of the findings still need further wet-lab experiments to confirm their activity against it. To facilitate rapid screening of protease inhibitors, we aim to develop a screening kit for researchers with focus studies on herbal sources for COVID-19 treatments. Hence, the development of 3CLpro as the target of inhibition screening is essential. Our current work focused on the expression of the SARS-CoV-2 3CL Pro gene harbored by pET-32b(+) in E. coli BL21(DE3) and its purification. The recombinant E.coli were cultivated in LB media at 37 °C with 5 hours IPTG-induced phase followed by crude protein lysate extraction by sonication, and then Ni-NTA purification. A band of recombinant 3CLpro protein (33.8 kDa) detected by SDS PAGE and Western blotting after Ni-NTA purification was specific and clear, showing the right size of our protein interest. A final concentrated protein mixture obtained by 10-kDa membrane filtration displayed >90% purity with a total protein of 2.497 mg mL−1. Further functional assays and inhibitory tests with several natural compounds showed that 3CLpro functioned well as a drug target.
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Ziebuhr, John, and Stuart G. Siddell. "Processing of the Human Coronavirus 229E Replicase Polyproteins by the Virus-Encoded 3C-Like Proteinase: Identification of Proteolytic Products and Cleavage Sites Common to pp1a and pp1ab." Journal of Virology 73, no. 1 (January 1, 1999): 177–85. http://dx.doi.org/10.1128/jvi.73.1.177-185.1999.
Abstract:
ABSTRACT Replicase gene expression by the human coronavirus 229E involves the synthesis of two large polyproteins, pp1a and pp1ab. Experimental evidence suggests that these precursor molecules are subject to extensive proteolytic processing. In this study, we show that a chymotrypsin-like enzyme, the virus-encoded 3C-like proteinase (3CLpro), cleaves within a common region of pp1a and pp1ab (amino acids 3490 to 4068) at four sites. trans-cleavage assays revealed that polypeptides of 5, 23, 12, and 16 kDa are processed from pp1a/pp1ab by proteolysis of the peptide bonds Q3546/S3547, Q3629/S3630, Q3824/N3825, and Q3933/A3934. Relative rate constants for the 3CLpro-mediated cleavages Q2965/A2966, Q3267/S3268, Q3824/N3825, and Q3933/A3934 were derived by competition experiments using synthetic peptides and recombinant 3CLpro. The results indicate that coronavirus cleavage sites differ significantly with regard to their susceptibilities to proteolysis by 3CLpro. Finally, immunoprecipitation with specific rabbit antisera was used to detect the pp1a/pp1ab processing end products in virus-infected cells, and immunofluorescence data that suggest an association of these polypeptides with intracellular membranes were obtained.
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Cheng, Jin, Yixuan Hao, Qin Shi, Guanyu Hou, Yanan Wang, Yong Wang, Wen Xiao, et al. "Discovery of Novel Chinese Medicine Compounds Targeting 3CL Protease by Virtual Screening and Molecular Dynamics Simulation." Molecules 28, no. 3 (January 17, 2023): 937. http://dx.doi.org/10.3390/molecules28030937.
Abstract:
The transmission and infectivity of COVID-19 have caused a pandemic that has lasted for several years. This is due to the constantly changing variants and subvariants that have evolved rapidly from SARS-CoV-2. To discover drugs with therapeutic potential for COVID-19, we focused on the 3CL protease (3CLpro) of SARS-CoV-2, which has been proven to be an important target for COVID-19 infection. Computational prediction techniques are quick and accurate enough to facilitate the discovery of drugs against the 3CLpro of SARS-CoV-2. In this paper, we used both ligand-based virtual screening and structure-based virtual screening to screen the traditional Chinese medicine small molecules that have the potential to target the 3CLpro of SARS-CoV-2. MD simulations were used to confirm these results for future in vitro testing. MCCS was then used to calculate the normalized free energy of each ligand and the residue energy contribution. As a result, we found ZINC15676170, ZINC09033700, and ZINC12530139 to be the most promising antiviral therapies against the 3CLpro of SARS-CoV-2.
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Razali, Rafida, Vijay Kumar Subbiah, and Cahyo Budiman. "Technical Data of Heterologous Expression and Purification of SARS-CoV-2 Proteases Using Escherichia coli System." Data 6, no. 9 (September 16, 2021): 99. http://dx.doi.org/10.3390/data6090099.
Abstract:
The SARS-CoV-2 coronavirus expresses two essential proteases: firstly, the 3Chymotrypsin-like protease (3CLpro) or main protease (Mpro), and secondly, the papain-like protease (PLpro), both of which are considered as viable drug targets for the inhibition of viral replication. In order to perform drug discovery assays for SARS-CoV-2, it is imperative that efficient methods are established for the production and purification of 3CLpro and PLpro of SARS-CoV-2, designated as 3CLpro-CoV2 and PLpro-CoV2, respectively. This article expands the data collected in the attempts to express SARS-CoV-2 proteases under different conditions and purify them under single-step chromatography. Data showed that the use of E. coli BL21(DE3) strain was sufficient to express 3CLpro-CoV2 in a fully soluble form. Nevertheless, the single affinity chromatography step was only applicable for 3CLpro-CoV2 expressed at 18 °C, with a yield and purification fold of 92% and 49, respectively. Meanwhile, PLpro-CoV2 was successfully expressed in a fully soluble form in either BL21(DE3) or BL21-CodonPlus(DE3) strains. In contrast, the single affinity chromatography step was only applicable for PLpro-CoV2 expressed using E. coli BL21-CodonPlus(DE3) at 18 or 37 °C, with a yield and purification fold of 86% (18 °C) or 83.36% (37 °C) and 112 (18 °C) or 71 (37 °C), respectively. The findings provide a guide for optimizing the production of SARS-CoV-2 proteases of E. coli host cells.
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Rizzuti, Bruno, Laura Ceballos-Laita, David Ortega-Alarcon, Ana Jimenez-Alesanco, Sonia Vega, Fedora Grande, Filomena Conforti, Olga Abian, and Adrian Velazquez-Campoy. "Sub-Micromolar Inhibition of SARS-CoV-2 3CLpro by Natural Compounds." Pharmaceuticals 14, no. 9 (September 1, 2021): 892. http://dx.doi.org/10.3390/ph14090892.
Abstract:
Inhibiting the main protease 3CLpro is the most common strategy in the search for antiviral drugs to fight the infection from SARS-CoV-2. We report that the natural compound eugenol is able to hamper in vitro the enzymatic activity of 3CLpro, the SARS-CoV-2 main protease, with an inhibition constant in the sub-micromolar range (Ki = 0.81 μM). Two phenylpropene analogs were also tested: the same effect was observed for estragole with a lower potency (Ki = 4.1 μM), whereas anethole was less active. The binding efficiency index of these compounds is remarkably favorable due also to their small molecular mass (MW < 165 Da). We envision that nanomolar inhibition of 3CLpro is widely accessible within the chemical space of simple natural compounds.
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Morita, Takeshi, Kei Miyakawa, Sundararaj Stanleyraj Jeremiah, Yutaro Yamaoka, Mitsuru Sada, Tomoko Kuniyoshi, Jinwei Yang, Hirokazu Kimura, and Akihide Ryo. "All-Trans Retinoic Acid Exhibits Antiviral Effect against SARS-CoV-2 by Inhibiting 3CLpro Activity." Viruses 13, no. 8 (August 23, 2021): 1669. http://dx.doi.org/10.3390/v13081669.
Abstract:
The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread despite the global efforts taken to control it. The 3C-like protease (3CLpro), the major protease of SARS-CoV-2, is one of the most interesting targets for antiviral drug development because it is highly conserved among SARS-CoVs and plays an important role in viral replication. Herein, we developed high throughput screening for SARS-CoV-2 3CLpro inhibitor based on AlphaScreen. We screened 91 natural product compounds and found that all-trans retinoic acid (ATRA), an FDA-approved drug, inhibited 3CLpro activity. The 3CLpro inhibitory effect of ATRA was confirmed in vitro by both immunoblotting and AlphaScreen with a 50% inhibition concentration (IC50) of 24.7 ± 1.65 µM. ATRA inhibited the replication of SARS-CoV-2 in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 2.69 ± 0.09 µM in the former and 0.82 ± 0.01 µM in the latter. Further, we showed the anti-SARS-CoV-2 effect of ATRA on the currently circulating variants of concern (VOC); alpha, beta, gamma, and delta. These results suggest that ATRA may be considered as a potential therapeutic agent against SARS-CoV-2.
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AHMED, N. ZAHEER, DICKY JOHN DAVIS, NOMAN ANWAR, ASIM ALI KHAN, RAM PRATAP MEENA, ZEBA AFNAAN, and MEERA DEVI. "In-Silico Evaluation of Tiryaq-E-Wabai, an Unani Formulation for its Potency against SARS-CoV-2 Spike Glycoprotein and Main Protease." Journal of Drug Delivery and Therapeutics 11, no. 4-S (August 15, 2021): 86–100. http://dx.doi.org/10.22270/jddt.v11i4-s.4993.
Abstract:
COVID-19 was originated in Wuhan, China, in December 2019 and has been declared a pandemic disease by WHO. The number of infected cases continues unabated and so far, no specific drug approved for targeted therapy. Hence, there is a need for drug discovery from traditional medicine. Tiryaq-e-Wabai is a well-documented formulation in Unani medicine for its wide use as prophylaxis during epidemics of cholera, plague and other earlier epidemic diseases. The objective of the current study is to generate in-silico evidence and evaluate the potency of Tiryaq-e-Wabai against SARS-CoV-2 spike (S) glycoprotein and main protease (3CLpro). The structures of all phytocompounds used in this study were retrieved from PubChem database and some were built using Marvin Sketch. The protein structure of the SARS-CoV-2 S glycoprotein and 3CLpro was retrieved from the PDB ID: 6LZG and 7BQY respectively. AutoDock Vina was used to predict top ranking poses with best scores. The results of the molecular docking showed that phytocompounds of Tiryaq-e-Wabai exhibited good docking power with spike glycoprotein and 3CLpro. Among tested compounds Crocin from Zafran and Aloin A from Sibr showed strong binding to spike glycoprotein and 3CLpro respectively. Molecular dynamics simulation confirmed the stability of the S glycoprotein-Crocin and 3CLpro-Aloin A complexes. The Unani formulation Tiryaq-e-Wabai has great potential to inhibit the SARS-CoV-2, which have to be substantiated with further in-vitro and in-vivo studies.
Keywords: In-silico study, SARS-CoV-2, Tiryaq-e-Wabai, Unani formulation, Crocin, Aloin A
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Rizma, Baiq Ressa Puspita, Yek Zen Mubarok, Dian Fathita Dwi Lestari, and Agus Dwi Ananto. "Molecular Study of Antiviral Compound of Indonesian Herbal Medicine as 3CLpro and PLpro Inhibitor in SARS-COV-2." Acta Chimica Asiana 4, no. 2 (October 29, 2021): 127–34. http://dx.doi.org/10.29303/aca.v4i2.74.
Abstract:
Rapid transmission of COVID-19 disease and the fatal effects of the disease lead researchers to use various way to find potential anti-COVID-19 compounds, including using modern approaches. Molecular docking is one of the methods that can be used to analyse antiviral compounds and its molecular target from Indonesian herbs that are believed to have properties as anti-COVID-19. This study aims to analyse antiviral compounds from 5 herbs that have the potential as inhibitors of PLpro and 3CLpro, which both are a non-structural protein in SARS-CoV-2 by molecular docking approach using PLANTS. Remdesivir triphosphate, the active metabolite of remdesivir, was used as the comparison compound in studies. The results showed docking scores obtained from interactions between natural ligands, remdesivir trifospat, curcumin, demetoksikurkumin, bisdemetoksikurkumin, luteolin, apigenin, kuersetin, kaempferol, formononrtin-7-O-glucoronide, androgafolide, and neoandrogafolide with PLpro are as follows -111,441, -103,827, -103,609, -102,363, -100,27,-79,6655, -78.6901, -80.9337, -79.4686, -82.1124, -79.1789, and -97.2452.Combination between quercetin, neoandrographolide, bisdemethoxycurcumin, demetoxycurcumin, and curcumin showed a synergy effect by reduce its docking score. Meanwhile its interaction with the protein 3CLpro showed docking score for those compounds as follows 64.0074, -86.1811, -81.428, -87.1625, -78.2899, -73.4345,-70,3368, -71.5539, -68.4321, -72.0154, -75.9777 and -93.7746.Combination between andrographolide, neoandrographolide, bisdemethoxycurcumin, demetoxycurcumin and curcumin, also shows synegy effect in 3CLpro allow them to reduce the docking score.This study concludes that curcumin was known as the most potent compound that act as a PLpro inhibitor based on a docking score of -103,609, while in 3CLpro all the compound have a potential to inhibit 3CLpro with demethosxycurcumin and neoandrogafolide as the most potent compound with a docking score -87,126 and -93.7746.
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Herlah, Barbara, Andrej Hoivik, Luka Jamšek, Katja Valjavec, Norio Yamamoto, Tyuji Hoshino, Krištof Kranjc, and Andrej Perdih. "Design, Synthesis and Evaluation of Fused Bicyclo[2.2.2]octene as a Potential Core Scaffold for the Non-Covalent Inhibitors of SARS-CoV-2 3CLpro Main Protease." Pharmaceuticals 15, no. 5 (April 27, 2022): 539. http://dx.doi.org/10.3390/ph15050539.
Abstract:
The emergence of SARS-CoV-2, responsible for the global COVID-19 pandemic, requires the rapid development of novel antiviral drugs that would contribute to an effective treatment alongside vaccines. Drug repurposing and development of new molecules targeting numerous viral targets have already led to promising drug candidates. To this end, versatile molecular scaffolds with high functionalization capabilities play a key role. Starting with the clinically used conformationally flexible HIV-1 protease inhibitors that inhibit replication of SARS-CoV-2 and bind major protease 3CLpro, we designed and synthesized a series of rigid bicyclo[2.2.2]octenes fused to N-substituted succinimides to test whether this core scaffold could support the development of non-covalent 3CLpro inhibitors. Inhibition assays confirmed that some compounds can inhibit the SARS-CoV-2 main protease; the most promising compound 11a inhibited 3CLpro in micromolar range (IC50 = 102.2 μM). Molecular simulations of the target-ligand complex in conjunction with dynophore analyses and endpoint free energy calculations provide additional insight and first recommendations for future optimization. The fused bicyclo[2.2.2]octenes can be used as a new potential starting point in the development of non-covalent SARS-CoV-2 3CLpro protease inhibitors and the study also substantiates the potential of this versatile scaffold for the development of biologically active molecules.
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Fagnani, Lorenza, Lisaurora Nazzicone, Pierangelo Bellio, Nicola Franceschini, Donatella Tondi, Andrea Verri, Sabrina Petricca, et al. "Protocetraric and Salazinic Acids as Potential Inhibitors of SARS-CoV-2 3CL Protease: Biochemical, Cytotoxic, and Computational Characterization of Depsidones as Slow-Binding Inactivators." Pharmaceuticals 15, no. 6 (June 4, 2022): 714. http://dx.doi.org/10.3390/ph15060714.
Abstract:
The study investigated the inhibitory activity of protocetraric and salazinic acids against SARS-CoV-2 3CLpro. The kinetic parameters were determined by microtiter plate-reading fluorimeter using a fluorogenic substrate. The cytotoxic activity was tested on murine Sertoli TM4 cells. In silico analysis was performed to ascertain the nature of the binding with the 3CLpro. The compounds are slow-binding inactivators of 3CLpro with a Ki of 3.95 μM and 3.77 μM for protocetraric and salazinic acid, respectively, and inhibitory efficiency kinact/Ki at about 3 × 10−5 s−1µM−1. The mechanism of inhibition shows that both compounds act as competitive inhibitors with the formation of a stable covalent adduct. The viability assay on epithelial cells revealed that none of them shows cytotoxicity up to 80 μM, which is well below the Ki values. By molecular modelling, we predicted that the catalytic Cys145 makes a nucleophilic attack on the carbonyl carbon of the cyclic ester common to both inhibitors, forming a stably acyl-enzyme complex. The computational and kinetic analyses confirm the formation of a stable acyl-enzyme complex with 3CLpro. The results obtained enrich the knowledge of the already numerous biological activities exhibited by lichen secondary metabolites, paving the way for developing promising scaffolds for the design of cysteine enzyme inhibitors.
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Mohammad, Firdous Sayeed, Mohsina F. Patwekar, and Faheem I. Patwekar. "Are Plant-derived Flavonoids the Emerging Anti-coronavirus Agents?" INNOSC Theranostics and Pharmacological Sciences 4, no. 2 (April 29, 2022): 11–16. http://dx.doi.org/10.36922/itps.v4i2.42.
Abstract:
The current outbreak of coronavirus disease 2019 (COVID-19), which is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has negatively impacted the global health and economy. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shares many similarities with SARS-CoV and Middle East respiratory syndrome-related coronavirus. It has been identified that the 3C-like protease (3CLpro) enzyme in coronaviruses can be a major therapeutic target for combating these serious infections. Therefore, flavonoids are believed to hold high potential in eliminating the viruses and infections. Flavonoids are polyphenolic secondary metabolites found in plants and have been demonstrated for their notable benefits for health. The antiviral activity of flavonoids has been reported in recent studies. Flavonoids, such as apigenin, quercetin, luteolin, amentoflavone, epigallocatechin gallate, gallocatechin gallate, and kaempferol, are known to be able to fight against coronaviruses by reducing the 3CLpro activity, according to the docking studies. Besides, we also found that several flavonoids have the potential to suppress the inflammatory cytokines, which are generally expressed in the lungs of coronavirus-infected individuals. However, the studies utilizing 3CLpro using various scaffolds of flavonoids need to be performed for better understanding on the antiviral potential of flavonoid derivatives against 3CLpro.