Substrate specificity profiling of SARS-CoV-2 main protease enables design of activity-based probes for patient-sample imaging

AbstractIn December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed in Wuhan, China. Due to international travel and human-to-human transmission, the virus spread rapidly inside and outside of China. Currently, there is no effective antiviral treatment for coronavirus disease 2019 (COVID-19); therefore, research efforts are focused on the rapid development of vaccines and antiviral drugs. The SARS-CoV-2 main protease constitutes one of the most attractive antiviral drug targets. To address this emerging problem, we have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 main proteases, using natural and a large panel of unnatural amino acids. On the basis of these findings, we designed and synthesized an inhibitor and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 Mpro. Using this approach we visualized SARS-CoV-2 active Mpro within nasopharyngeal epithelial cells of a patient with active COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents or diagnostic tests.

Download Full-text

Peptide Derivatives of the Zonulin Inhibitor Larazotide (AT1001) as Potential Anti SARS CoV-2: Molecular Modelling, Synthesis and Bioactivity Evaluation

International Journal of Molecular Sciences ◽

10.3390/ijms22179427 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9427

Author(s):

Simone Di Micco ◽

Simona Musella ◽

Marina Sala ◽

Maria C. Scala ◽

Graciela Andrei ◽

...

Keyword(s):

Catalytic Domain ◽

Antiviral Agents ◽

Antiviral Drug ◽

Drug Repurposing ◽

Fast Response ◽

Main Protease ◽

Promising Target ◽

Peptide Derivatives ◽

Novel Coronavirus ◽

Derivatives Of

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the pathogen responsible for the outbreak of a severe, rapidly developing pneumonia (Coronavirus disease 2019, COVID-19). The virus enzyme, called 3CLpro or main protease (Mpro), is essential for viral replication, making it a most promising target for antiviral drug development. Recently, we adopted the drug repurposing as appropriate strategy to give fast response to global COVID-19 epidemic, by demonstrating that the zonulin octapeptide inhibitor AT1001 (Larazotide acetate) binds Mpro catalytic domain. Thus, in the present study we tried to investigate the antiviral activity of AT1001, along with five derivatives, by cell-based assays. Our results provide with the identification of AT1001 peptide molecular framework for lead optimization step to develop new generations of antiviral agents of SARS-CoV-2 with an improved biological activity, expanding the chance for success in clinical trials.

Download Full-text

Recent Advances towards Drug Design Targeting the Protease of 2019 novel coronavirus (2019-nCoV)

Current Medicinal Chemistry ◽

10.2174/0929867327666201027153617 ◽

2020 ◽

Vol 27 ◽

Author(s):

Sehrish Bano ◽

Abdul Hameed ◽

Mariya Al-Rashida ◽

Shafia Iftikhar ◽

Jamshed Iqbal

Keyword(s):

Drug Design ◽

Rna Polymerase ◽

Drug Targets ◽

Antiviral Agents ◽

Structural Features ◽

Antiviral Drugs ◽

Rna Dependent Rna Polymerase ◽

Mortality And Morbidity ◽

Functional Relationships ◽

Novel Coronavirus

Background: The 2019 novel coronavirus (2019-nCoV), also known as coronavirus 2 (SARS-CoV-2) acute respiratory syndrome has recently emerged and continued to spread rapidly with high level of mortality and morbidity rates. Currently, no efficacious therapy is available to relieve coronavirus infections. As new drug design and development takes much time, there is a possibility to find an effective treatment from existing antiviral agents. Objective: In this case, there is a need to find out the relationship between possible drug targets and mechanism of action of antiviral drugs. This review discusses about the efforts to develop drug from known or new molecules. Methods: Viruses usually have two structural integrities, proteins and nucleic acids, both of which can be possible drug targets. Herein, we systemically discuss the structural-functional relationships of the spike, 3-chymotrypsin-like protease (3CLpro), papain like protease (PLpro) and RNA-dependent RNA polymerase (RdRp), as these are prominent structural features of corona virus. Certain antiviral drugs such as Remdesivir are RNA dependent RNA polymerase inhibitor. It has the ability to terminate RNA replication by inhibiting ATP. Results: It is reported that ATP is involved in synthesis of coronavirus non-structural proteins from 3CLpro and PLpro. Similarly, mechanisms of action of many other antiviral agents has been discussed in this review. It will provide new insights into the mechanism of inhibition, and let us develop new therapeutic antiviral approaches against novel SARS-CoV-2 coronavirus. Conclusion: In conclusion, this review summarizes recent progress in developing protease inhibitors for SARS-CoV-2.

Download Full-text

Biochemical and Biophysical characterization of the main protease, 3-chymotrypsin-like protease (3CLpro), from the novel coronavirus disease 19 (COVID-19)

10.21203/rs.3.rs-40945/v1 ◽

2020 ◽

Author(s):

Juliana C. Ferreira ◽

Wael M. Rabeh

Keyword(s):

Antiviral Drug ◽

Analytical Techniques ◽

The Novel ◽

Buffer Solution ◽

Biophysical Characterization ◽

Main Protease ◽

Wide Ph Range ◽

The Stability ◽

Ph Range ◽

Novel Coronavirus

Abstract Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is responsible for the novel coronavirus disease 2019 (COVID-19). An appealing antiviral drug target is the coronavirus 3C-like protease (3CLpro) that is responsible for the processing of the viral polyproteins and liberation of functional proteins essential for the maturation and infectivity of the virus. In this study, multiple thermal analytical techniques have been implemented to acquire the thermodynamic parameters of 3CLpro at different buffer conditions. 3CLpro exhibited relatively high thermodynamic stabilities over a wide pH range; however, the protease was found to be less stable in the presence of salts. Divalent metal cations reduced the thermodynamic stability of 3CLpro more than monovalent cations; however, altering the ionic strength of the buffer solution did not alter the stability of 3CLpro. Furthermore, the most stable thermal kinetic stability of 3CLpro was recorded at pH 7.5, with the highest enthalpy of activation calculated from the slope of Eyring plot. The biochemical and biophysical properties of 3CLpro explored here will improve the solubility and stability of 3CLpro for optimum conditions for the setup of an enzymatic assay for the screening of inhibitors to be used as lead candidates in the drug discovery and antiviral design for therapeutics against COVID-19.

Download Full-text

Inhibition of B Virus (Macacine herpesvirus 1) by Conventional and Experimental Antiviral Compounds

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01435-08 ◽

2009 ◽

Vol 54 (1) ◽

pp. 452-459 ◽

Cited By ~ 6

Author(s):

P. W. Krug ◽

R. F. Schinazi ◽

J. K. Hilliard

Keyword(s):

Antiviral Treatment ◽

Antiviral Agents ◽

Antiviral Drug ◽

Nucleoside Analogs ◽

High Morbidity ◽

Virus Infections ◽

Experimental Drugs ◽

B Virus ◽

Simplex Virus ◽

Virus Isolates

ABSTRACT B virus infection of humans results in high morbidity and mortality in as many as 80% of identified cases. The main objective of this study was to conduct a comparative analysis of conventional and experimental antiviral drug susceptibilities of B virus isolates from multiple macaque species and zoonotically infected humans. We used a plaque reduction assay to establish the effective inhibitory doses of acyclovir, ganciclovir, and vidarabine, as well as those of a group of experimental nucleoside analogs with known anti-herpes simplex virus activity. Four of the experimental drugs tested were 10- to 100-fold more potent inhibitors of B virus replication than conventional antiviral agents. Drug efficacies were similar for multiple B virus isolates tested, with variations within 2-fold of the median effective concentration (EC50) for each drug, and each EC50 was considerably lower than those for B virus thymidine kinase (TK) mutants. We observed no differences in the viral TK amino acid sequence between B virus isolates from rhesus monkeys and those from human zoonoses. Differences in the TK protein sequence between cynomolgus and pigtail macaque B virus isolates did not affect drug sensitivity except in the case of one compound. Taken together, these data suggest that future B virus zoonoses will respond consistently to conventional antiviral treatment. Further, the considerably higher potency of FEAU (2′-fluoro-5-ethyl-Ara-U) than of conventional antiviral drugs argues for its compassionate use in advanced human B virus infections.

Download Full-text

In silico Repositioning for Dual Inhibitor Discovery of SARS-CoV-2 (COVID-19) 3C-like Protease and Papain-like Peptidase

10.20944/preprints202004.0084.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Kowsar Bagherzadeh ◽

Kourosh Daneshvarnejad ◽

Mohammad Abbasinazari ◽

homa azizian

Keyword(s):

Active Sites ◽

Reproduction Number ◽

Antiviral Agents ◽

Binding Energies ◽

Dual Inhibitor ◽

Main Protease ◽

Inhibitor Discovery ◽

Dual Inhibitors ◽

Dual Inhibition ◽

Novel Coronavirus

Aims: In late December 2019, early reports predicted the onset of a potential Coronavirus outbreak in china, given the estimate of a reproduction number for the 2019 Novel Coronavirus (COVID-19). Because of high ability of transmission and widespread prevalence, the mortality of COVID-19 infection is growing fast worldwide. Absent of an anti-COVID-19 has put scientists on the urge to repurpose already approved therapeutics or to find new active compounds against coronavirus. Here in this study, a set of computational approaches were performed in order to repurpose antivirals for dual inhibition of the frontier proteases involved in virus replication, papain-like protease (PLpro; corresponding to nsp3) and a main protease (Mpro), 3C‑like protease (3CLpro; corresponding to nsp5). Materials and Methods: In this regard, a rational virtual screening procedure including exhaustive docking techniques was performed for a database of 160 antiviral agents over 3CLpro and PLpro active sites of SARS-CoV-2. The compounds binding energies and interaction modes over 3CLpro and PLpro active sites were analyzed and ranked with the aid of free Gibbs binding energy. The most potent compounds, based on our filtering process, are then proposed as dual inhibitors of SARSC-CoV-2 proteases. Key findings: Accordingly, seven antiviral agents including two FDA approved (Nelfinavir, Valaganciclovir) and five investigational compounds (Merimepodib, Inarigivir, Remdesivir, Taribavirine and TAS106-106) are proposed as potential dual inhibitors of the enzymes necessary for RNA replication in which Remdesivir as well as Inagrivir have the highest binding affinity for both of the active sites. Significance: The mentioned drug proposed to inhibit both PLpro and 3CLpro enzymes with the aim of finding dual inhibitors of SARSC-CoV-2 proteases.

Download Full-text

Designing Inhibitors for the SARS CoV Main Protease as Anti-SARS Drugs

McGill Journal of Medicine ◽

10.26443/mjm.v8i1.377 ◽

2020 ◽

Vol 8 (1) ◽

Author(s):

Hamza Bari ◽

Arif Ali Awan

Keyword(s):

Substrate Binding ◽

Rapid Development ◽

Respiratory Illness ◽

Significant Inhibition ◽

Computational Techniques ◽

Chemical Structures ◽

Main Protease ◽

Binding Cavity ◽

Antiviral Chemotherapy ◽

Novel Coronavirus

Severe Acute Respiratory Syndrome (SARS) is a serious respiratory illness reported in parts of Asia and Canada. A novel coronavirus (CoV) has been isolated and identified as the cause of the SARS for which there is currently no effective treatment. Given the epidemic, the rapid development of efficacious antiviral drugs is needed. The key replicative enzyme SARS CoV main protease (Mpro) represents an attractive target for antiviral chemotherapy. Detailed structural study of the substrate binding cavity led to the generation of a 3-D pharmacophore. Subsets of chemical structures were extracted from the commercial databases by using the defined pharmacophore. Compound mapping to the pharmacophore were docked into the substrate-binding cavity and scored. The selected chemicals were assayed against the SARS CoV Mpro for their inhibitory activity. Three of the compounds showed significant inhibition of the SARS CoV Mpro at low micromolar concentration. This study provides potential lead compounds for specific SARS CoV protease inhibitors. It also signifies the utility of computational techniques for rapid discovery of inhibitors for novel targets.

Download Full-text

Computational Determination of Potential Inhibitors of SARS-CoV-2 Main Protease

10.26434/chemrxiv.12111297 ◽

2020 ◽

Author(s):

Son Tung Ngo ◽

Ngoc Quynh Anh Pham ◽

Ly Le ◽

Duc-Hung Pham ◽

Van Vu

Keyword(s):

Free Energy ◽

Molecular Docking ◽

Drug Targets ◽

Binding Free Energy ◽

Natural Compounds ◽

Main Protease ◽

Energy Perturbation ◽

Novel Coronavirus ◽

Potential Inhibitors ◽

Hiv 1

The novel coronavirus (SARS-CoV-2) has infected over 850,000 people and caused more than 42000 deaths worldwide as of April 1st, 2020. As the disease is spreading rapidly all over the world, it is urgent to find effective drugs to treat the virus. The main protease (Mpro) of SARS-CoV-2 is one of the potential drug targets. In this work, we used rigorous computational methods, including molecular docking, fast pulling of ligand (FPL), and free energy perturbation (FEP), to investigate potential inhibitors of SARS-CoV-2 Mpro. We first tested our approach with three reported inhibitors of SARS-CoV-2 Mpro; and our computational results are in good agreement with the respective experimental data. Subsequently, we applied our approach on a databases of ~4600 natural compounds found in Vietnamese plants, as well as 8 available HIV-1 protease (PR) inhibitors and an aza-peptide epoxide. Molecular docking resulted in a short list of 35 natural compounds, which was subsequently refined using the FPL scheme. FPL simulations resulted in five potential inhibitors, including 3 natural compounds and two available HIV-1 PR inhibitors. Finally, FEP, the most accurate and precise method, was used to determine the absolute binding free energy of these five compounds. FEP results indicate that two natural compounds, cannabisin A and isoacteoside, and an HIV-1 PR inhibitor, darunavir, exhibit large binding free energy to SARS-CoV-2 Mpro, which is larger than that of 13b, the most reliable SARS-CoV-2 Mpro inhibitor recently reported. The binding free energy largely arises from van der Waals (vdW) interaction. We also found that Glu166 form H-bonds to all the inhibitors. Replacing Glu166 by an alanine residue leads to ~ 2.0 kcal/mol decreases in the affinity of darunavir to SARS-CoV-2 Mpro. Our results could contribute to the development of potentials drugs inhibiting SARS-CoV-2.

Download Full-text

Main protease mutants of SARS-CoV-2 variants remain susceptible to PF-07321332

10.1101/2021.11.28.470226 ◽

2021 ◽

Author(s):

Sven Ullrich ◽

Kasuni B Ekanayake ◽

Gottfried Otting ◽

Christoph Nitsche

Keyword(s):

Public Health ◽

Clinical Relevance ◽

Drug Targets ◽

Antiviral Agents ◽

Spike Protein ◽

Main Protease ◽

Public Health Threat ◽

Vitro Data ◽

In Vitro Data

The COVID-19 pandemic continues to be a public health threat. Multiple mutations in the spike protein of emerging variants of SARS-CoV-2 appear to impact on the effectiveness of available vaccines. Specific antiviral agents are keenly anticipated but their efficacy may also be compromised in emerging variants. One of the most attractive coronaviral drug targets is the main protease (Mpro). A promising Mpro inhibitor of clinical relevance is the peptidomimetic PF-07321332. We expressed Mpro of five SARS-CoV-2 lineages (C.37 Lambda, B.1.1.318, B.1.2, B.1.351 Beta, P.2 Zeta), each of which carries a strongly prevalent missense mutation (G15S, T21I, L89F, K90R, L205V). Enzyme kinetics showed that these Mpro variants are similarly catalytically competent as the wildtype. We show that PF-07321332 has similar potency against the variants as against the wildtype. Our in vitro data suggest that the efficacy of specific Mpro inhibitors such as PF-07321332 is not compromised in current COVID-19 variants.

Download Full-text

Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody

10.1101/2020.01.28.923011 ◽

2020 ◽

Cited By ~ 13

Author(s):

Xiaolong Tian ◽

Cheng Li ◽

Ailing Huang ◽

Shuai Xia ◽

Sicong Lu ◽

...

Keyword(s):

Monoclonal Antibody ◽

Binding Site ◽

Neutralizing Antibodies ◽

Rapid Development ◽

Antiviral Treatment ◽

Human Monoclonal Antibody ◽

Cross Reactivity ◽

Spike Protein ◽

The Cross ◽

Novel Coronavirus

ABSTRACTThe newly identified 2019 novel coronavirus (2019-nCoV) has caused more than 800 laboratory-confirmed human infections, including 25 deaths, posing a serious threat to human health. Currently, however, there is no specific antiviral treatment or vaccine. Considering the relatively high identity of receptor binding domain (RBD) in 2019-nCoV and SARS-CoV, it is urgent to assess the cross-reactivity of anti-SARS-CoV antibodies with 2019-nCoV spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-nCoV. Here, we report for the first time that a SARS-CoV-specific human monoclonal antibody, CR3022, could bind potently with 2019-nCoV RBD (KD of 6.3 nM). The epitope of CR3022 does not overlap with the ACE2 binding site within 2019-nCoV RBD. Therefore, CR3022 has the potential to be developed as candidate therapeutics, alone or in combination with other neutralizing antibodies, for the prevention and treatment of 2019-nCoV infections. Interestingly, some of the most potent SARS-CoV-specific neutralizing antibodies (e.g., m396, CR3014) that target the ACE2 binding site of SARS-CoV failed to bind 2019-nCoV spike protein, indicating that the difference in the RBD of SARS-CoV and 2019-nCoV has a critical impact for the cross-reactivity of neutralizing antibodies, and that it is still necessary to develop novel monoclonal antibodies that could bind specifically to 2019-nCoV RBD.

Download Full-text

Fighting COVID-19

Brazilian Journal of Biology ◽

10.1590/1519-6984.238155 ◽

2020 ◽

Vol 80 (3) ◽

pp. 698-701 ◽

Cited By ~ 2

Author(s):

D. M. O. Campos ◽

C. B. S. Oliveira ◽

J. M. A. Andrade ◽

J. I. N. Oliveira

Keyword(s):

Drug Targets ◽

Vaccine Development ◽

High Rate ◽

The Novel ◽

Rate Of Spread ◽

Specific Effects ◽

Main Protease ◽

Clinical Strategies ◽

Novel Coronavirus ◽

Transmembrane Serine Protease

Abstract The current COVID-19 pandemic caused by the novel coronavirus (SARS-CoV2) poses a threat to global health owing to its high rate of spread and severe forms of respiratory infection. The lack of vaccines and antivirals prevents clinical strategies against the disease, creating an emerging need for the development of safe and effective treatments. Strategies for vaccine development include complete vaccines against viruses, subunits, and nucleic acids, but are still in their early stages. Studies carried out to date on possible SARS-CoV2 drug targets highlight glycoprotein S, Mpro (main protease or protease type 3C), and a member of the transmembrane serine protease II families (TMPRSS2). However, due to the pandemic state, priority is given to marketed drugs. These include chloroquine (CQ), hydroxychloroquine (HCQ), nitazoxanide, remdesivir, Lopinavir/ritonavir (LPV / r), in addition to treatment with convalescent plasma. But, therapeutic specific effects against SARS-CoV2 have not yet been verified. Most of the information obtained about treatment is based on preliminary and limited studies. We conclude that, at this time of emergency, the search for new therapies is more urgent due to the need to save lives. Thus, we point out as interesting targets for future more specific research: glycoprotein S, Mpro, and TMPRSS2.

Download Full-text