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1.
J Virol ; 96(8): e0201321, 2022 04 27.
Article in English | MEDLINE | ID: covidwho-1779314

ABSTRACT

The high mutation rate of COVID-19 and the prevalence of multiple variants strongly support the need for pharmacological options to complement vaccine strategies. One region that appears highly conserved among different genera of coronaviruses is the substrate-binding site of the main protease (Mpro or 3CLpro), making it an attractive target for the development of broad-spectrum drugs for multiple coronaviruses. PF-07321332, developed by Pfizer, is the first orally administered inhibitor targeting the main protease of SARS-CoV-2, which also has shown potency against other coronaviruses. Here, we report three crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome (MERS)-CoV bound to the inhibitor PF-07321332. The structures reveal a ligand-binding site that is conserved among SARS-CoV-2, SARS-CoV, and MERS-CoV, providing insights into the mechanism of inhibition of viral replication. The long and narrow cavity in the cleft between domains I and II of the main protease harbors multiple inhibitor-binding sites, where PF-07321332 occupies subsites S1, S2, and S4 and appears more restricted than other inhibitors. A detailed analysis of these structures illuminated key structural determinants essential for inhibition and elucidated the binding mode of action of the main proteases from different coronaviruses. Given the importance of the main protease for the treatment of SARS-CoV-2 infection, insights derived from this study should accelerate the design of safer and more effective antivirals. IMPORTANCE The current pandemic of multiple variants has created an urgent need for effective inhibitors of SARS-CoV-2 to complement vaccine strategies. PF-07321332, developed by Pfizer, is the first orally administered coronavirus-specific main protease inhibitor approved by the FDA. We solved the crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and MERS-CoV that bound to the PF-07321332, suggesting PF-07321332 is a broad-spectrum inhibitor for coronaviruses. Structures of the main protease inhibitor complexes present an opportunity to discover safer and more effective inhibitors for COVID-19.


Subject(s)
Lactams , Leucine , Nitriles , Peptide Hydrolases , Proline , Antiviral Agents/chemistry , Antiviral Agents/metabolism , COVID-19/drug therapy , Humans , Lactams/chemistry , Lactams/metabolism , Leucine/chemistry , Leucine/metabolism , Middle East Respiratory Syndrome Coronavirus/chemistry , Middle East Respiratory Syndrome Coronavirus/enzymology , Nitriles/chemistry , Nitriles/metabolism , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , Proline/chemistry , Proline/metabolism , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , SARS Virus/chemistry , SARS Virus/enzymology , SARS-CoV-2/chemistry , SARS-CoV-2/enzymology
2.
Phys Chem Chem Phys ; 24(3): 1743-1759, 2022 Jan 19.
Article in English | MEDLINE | ID: covidwho-1606147

ABSTRACT

The outbreak caused by SARS-CoV-2 has received extensive worldwide attention. As the main protease (Mpro) in SARS-CoV-2 has no human homologues, it is feasible to reduce the possibility of targeting the host protein by accidental drugs. Thus, Mpro has been an attractive target of efficient drug design for anti-SARS-CoV-2 treatment. In this work, multiple replica molecular dynamics (MRMD) simulations, principal component analysis (PCA), free energy landscapes (FELs), and the molecular mechanics-generalized Born surface area (MM-GBSA) method were integrated together to decipher the binding mechanism of four inhibitors masitinib, O6K, FJC and GQU to Mpro. The results indicate that the binding of four inhibitors clearly affects the structural flexibility and internal dynamics of Mpro along with dihedral angle changes of key residues. The analysis of FELs unveils that the stability in the relative orientation and geometric position of inhibitors to Mpro is favorable for inhibitor binding. Residue-based free energy decomposition reveals that the inhibitor-Mpro interaction networks involving hydrogen bonding interactions and hydrophobic interactions provide significant information for the design of potent inhibitors against Mpro. The hot spot residues including H41, M49, F140, N142, G143, C145, H163, H164, M165, E166 and Q189 identified by computational alanine scanning are considered as reliable targets of clinically available inhibitors inhibiting the activities of Mpro.


Subject(s)
Antiviral Agents/chemistry , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Proline/chemistry , SARS-CoV-2/drug effects , Viral Protease Inhibitors/chemistry , Antiviral Agents/pharmacology , Drug Design , Humans , Molecular Dynamics Simulation , Principal Component Analysis , Proline/pharmacology , Protein Binding , Protein Conformation , Structure-Activity Relationship , Thermodynamics , Viral Protease Inhibitors/pharmacology
4.
Chem Commun (Camb) ; 57(72): 9096-9099, 2021 Sep 09.
Article in English | MEDLINE | ID: covidwho-1373453

ABSTRACT

We present a detailed computational analysis of the binding mode and reactivity of the novel oral inhibitor PF-07321332 developed against the SARS-CoV-2 3CL protease. Alchemical free energy calculations suggest that positions P3 and P4 could be susceptible to improvement in order to get a larger binding strength. QM/MM simulations unveil the reaction mechanism for covalent inhibition, showing that the nitrile warhead facilitates the recruitment of a water molecule for the proton transfer step.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Molecular Dynamics Simulation , Nitriles/chemistry , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , Binding Sites , COVID-19/pathology , COVID-19/virology , Catalytic Domain , Coronavirus 3C Proteases/metabolism , Humans , Lactams/chemistry , Lactams/metabolism , Leucine/chemistry , Leucine/metabolism , Nitriles/metabolism , Proline/chemistry , Proline/metabolism , Protease Inhibitors/metabolism , Quantum Theory , SARS-CoV-2/isolation & purification , Thermodynamics
5.
Bioorg Med Chem Lett ; 50: 128333, 2021 10 15.
Article in English | MEDLINE | ID: covidwho-1363893

ABSTRACT

Specific anti-coronaviral drugs complementing available vaccines are urgently needed to fight the COVID-19 pandemic. Given its high conservation across the betacoronavirus genus and dissimilarity to human proteases, the SARS-CoV-2 main protease (Mpro) is an attractive drug target. SARS-CoV-2 Mpro inhibitors have been developed at unprecedented speed, most of them being substrate-derived peptidomimetics with cysteine-modifying warheads. In this study, Mpro has proven resistant towards the identification of high-affinity short substrate-derived peptides and peptidomimetics without warheads. 20 cyclic and linear substrate analogues bearing natural and unnatural residues, which were predicted by computational modelling to bind with high affinity and designed to establish structure-activity relationships, displayed no inhibitory activity at concentrations as high as 100 µM. Only a long linear peptide covering residues P6 to P5' displayed moderate inhibition (Ki = 57 µM). Our detailed findings will inform current and future drug discovery campaigns targeting Mpro.


Subject(s)
COVID-19/pathology , Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , COVID-19/virology , Coronavirus 3C Proteases/metabolism , Cysteine/chemistry , Cysteine/metabolism , Humans , Lactams/chemistry , Lactams/metabolism , Leucine/chemistry , Leucine/metabolism , Nitriles/chemistry , Nitriles/metabolism , Peptides/chemistry , Peptides/metabolism , Peptidomimetics/chemistry , Peptidomimetics/metabolism , Proline/chemistry , Proline/metabolism , Protease Inhibitors/metabolism , SARS-CoV-2/isolation & purification , Structure-Activity Relationship , Substrate Specificity
6.
J Enzyme Inhib Med Chem ; 36(1): 1646-1650, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1320278

ABSTRACT

The chemical structure of PF-07321332, the first orally available Covid-19 clinical candidate, has recently been revealed by Pfizer. No information has been provided about the interaction pattern between PF-07321332 and its biomolecular counterpart, the SARS-CoV-2 main protease (Mpro). In the present work, we exploited Supervised Molecular Dynamics (SuMD) simulations to elucidate the key features that characterise the interaction between this drug candidate and the protease, emphasising similarities and differences with other structurally related inhibitors such as Boceprevir and PF-07304814. The structural insights provided by SuMD will hopefully be able to inspire the rational discovery of other potent and selective protease inhibitors.


Subject(s)
Antiviral Agents/chemistry , Lactams/chemistry , Leucine/chemistry , Molecular Dynamics Simulation , Nitriles/chemistry , Proline/chemistry , Protease Inhibitors/chemistry , Antiviral Agents/pharmacology , Humans , Lactams/pharmacology , Leucine/pharmacology , Ligands , Nitriles/pharmacology , Peptide Hydrolases/metabolism , Proline/pharmacology , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Software
7.
J Med Chem ; 65(4): 2848-2865, 2022 02 24.
Article in English | MEDLINE | ID: covidwho-1199254

ABSTRACT

The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II, and XII, with each containing a reactive warhead that covalently modifies the catalytic Cys145. Coupling structure-based drug design with the one-pot Ugi four-component reaction, we discovered one of the most potent noncovalent inhibitors, 23R (Jun8-76-3A) that is structurally distinct from the canonical Mpro inhibitor GC376. Significantly, 23R is highly selective compared with covalent inhibitors such as GC376, especially toward host proteases. The cocrystal structure of SARS-CoV-2 Mpro with 23R revealed a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study discovered 23R, one of the most potent and selective noncovalent SARS-CoV-2 Mpro inhibitors reported to date, and a novel binding pocket in Mpro that can be explored for inhibitor design.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Drug Design , SARS-CoV-2/drug effects , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , COVID-19/drug therapy , COVID-19/metabolism , Chlorocebus aethiops , Coronavirus 3C Proteases/isolation & purification , Coronavirus 3C Proteases/metabolism , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Dose-Response Relationship, Drug , Humans , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Proline/analogs & derivatives , Proline/chemical synthesis , Proline/chemistry , Proline/pharmacology , Pyrrolidines/chemical synthesis , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , SARS-CoV-2/enzymology , Sulfonic Acids/chemical synthesis , Sulfonic Acids/chemistry , Sulfonic Acids/pharmacology , Vero Cells
8.
Biochem Biophys Res Commun ; 555: 147-153, 2021 05 28.
Article in English | MEDLINE | ID: covidwho-1157143

ABSTRACT

Several existing drugs are currently being tested worldwide to treat COVID-19 patients. Recent data indicate that SARS-CoV-2 is rapidly evolving into more transmissible variants. It is therefore highly possible that SARS-CoV-2 can accumulate adaptive mutations modulating drug susceptibility and hampering viral antigenicity. Thus, it is vital to predict potential non-synonymous mutation sites and predict the evolution of protein structural modifications leading to drug tolerance. As two FDA-approved anti-hepatitis C virus (HCV) drugs, boceprevir, and telaprevir, have been shown to effectively inhibit SARS-CoV-2 by targeting the main protease (Mpro), here we used a high-throughput interface-based protein design strategy to identify mutational hotspots and potential signatures of adaptation in these drug binding sites of Mpro. Several mutants exhibited reduced binding affinity to these drugs, out of which hotspot residues having a strong tendency to undergo positive selection were identified. The data further indicated that these anti-HCV drugs have larger footprints in the mutational landscape of Mpro and hence encompass the highest potential for positive selection and adaptation. These findings are crucial in understanding the potential structural modifications in the drug binding sites of Mpro and thus its signatures of adaptation. Furthermore, the data could provide systemic strategies for robust antiviral design and discovery against COVID-19 in the future.


Subject(s)
Adaptation, Physiological/genetics , Antiviral Agents/chemistry , Coronavirus 3C Proteases/chemistry , Drug Design , Drug Resistance, Viral/genetics , Mutation , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Amino Acid Sequence , Antiviral Agents/pharmacology , Binding Sites/drug effects , Binding Sites/genetics , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Genetic Fitness/genetics , Hepacivirus/drug effects , Hepacivirus/enzymology , Ligands , Models, Molecular , Oligopeptides/chemistry , Oligopeptides/pharmacology , Proline/analogs & derivatives , Proline/chemistry , Proline/pharmacology , Reproducibility of Results , SARS-CoV-2/drug effects , Selection, Genetic/genetics , Structure-Activity Relationship
9.
Molecules ; 26(6)2021 Mar 17.
Article in English | MEDLINE | ID: covidwho-1138745

ABSTRACT

The COVID-19 outbreak continues to spread worldwide at a rapid rate. Currently, the absence of any effective antiviral treatment is the major concern for the global population. The reports of the occurrence of various point mutations within the important therapeutic target protein of SARS-CoV-2 has elevated the problem. The SARS-CoV-2 main protease (Mpro) is a major therapeutic target for new antiviral designs. In this study, the efficacy of PF-00835231 was investigated (a Mpro inhibitor under clinical trials) against the Mpro and their reported mutants. Various in silico approaches were used to investigate and compare the efficacy of PF-00835231 and five drugs previously documented to inhibit the Mpro. Our study shows that PF-00835231 is not only effective against the wild type but demonstrates a high affinity against the studied mutants as well.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Indoles/chemistry , Indoles/pharmacology , Leucine/chemistry , Leucine/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Pyrrolidinones/chemistry , Pyrrolidinones/pharmacology , Binding Sites , COVID-19/drug therapy , Computer Simulation , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Databases, Protein , Diarylquinolines/chemistry , Diarylquinolines/pharmacology , Dihydropyridines/chemistry , Dihydropyridines/pharmacology , Humans , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Nitrobenzenes/chemistry , Nitrobenzenes/pharmacology , Nitrophenols/chemistry , Nitrophenols/pharmacology , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/pharmacology , Piperazines/chemistry , Piperazines/pharmacology , Proline/analogs & derivatives , Proline/chemistry , Proline/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics
10.
Cell Host Microbe ; 29(3): 327-333, 2021 03 10.
Article in English | MEDLINE | ID: covidwho-1126778

ABSTRACT

Most viral vaccines are based on inducing neutralizing antibodies (NAbs) against the virus envelope or spike glycoproteins. Many viral surface proteins exist as trimers that transition from a pre-fusion state when key NAb epitopes are exposed to a post-fusion form in which the potential for virus-cell fusion no longer exists. For optimal vaccine performance, these viral proteins are often engineered to enhance stability and presentation of these NAb epitopes. The method involves the structure-guided introduction of proline residues at key positions that maintain the trimer in the pre-fusion configuration. We review how this technique emerged during HIV-1 Env vaccine development and its subsequent wider application to other viral vaccines including SARS-CoV-2.


Subject(s)
Proline/chemistry , Proline/immunology , Viral Vaccines/chemistry , Viral Vaccines/immunology , Antibodies, Neutralizing/immunology , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Humans , Models, Molecular , Proline/genetics , Protein Engineering , Viral Vaccines/genetics
11.
mBio ; 12(2)2021 03 02.
Article in English | MEDLINE | ID: covidwho-1115091

ABSTRACT

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the prime target for vaccine development. The spike protein mediates both binding to host cells and membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus spike proteins are large trimers that are relatively unstable, a feature that might be enhanced by the presence of a polybasic cleavage site in SARS-CoV-2 spike. Exchange of K986 and V987 for prolines has been shown to stabilize the trimers of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus spike proteins. Here, we test multiple versions of a soluble spike protein for their immunogenicity and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human angiotensin-converting enzyme 2 via adenovirus transduction. Variants tested include spike proteins with a deleted polybasic cleavage site, proline mutations, or a combination thereof, besides the wild-type protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the K986P and V987P (PP) mutations completely protected from challenge in this mouse model.IMPORTANCE A vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes that vaccine candidates need to have to induce protective immunity is of high importance. The data presented here validate the choice of antigens that contain the PP mutations and suggest that deletion of the polybasic cleavage site may lead to a further-optimized design.


Subject(s)
Proline/chemistry , SARS-CoV-2/immunology , Animals , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/immunology , COVID-19 Vaccines/therapeutic use , Mice , Mutation , Spike Glycoprotein, Coronavirus/chemistry
12.
Biomolecules ; 10(9)2020 09 21.
Article in English | MEDLINE | ID: covidwho-976281

ABSTRACT

We report the results of our in silico study of approved drugs as potential treatments for COVID-19. The study is based on the analysis of normal modes of proteins. The drugs studied include chloroquine, ivermectin, remdesivir, sofosbuvir, boceprevir, and α-difluoromethylornithine (DMFO). We applied the tools we developed and standard tools used in the structural biology community. Our results indicate that small molecules selectively bind to stable, kinetically active residues and residues adjoining them on the surface of proteins and inside protein pockets, and that some prefer hydrophobic sites over other active sites. Our approach is not restricted to viruses and can facilitate rational drug design, as well as improve our understanding of molecular interactions, in general.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus Infections/drug therapy , Pandemics , Pneumonia, Viral/drug therapy , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/chemistry , Alanine/pharmacology , Angiotensin-Converting Enzyme 2 , Antibodies, Viral/immunology , Antigen-Antibody Reactions , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Betacoronavirus , Binding Sites , COVID-19 , Chloroquine/chemistry , Chloroquine/pharmacology , Coronavirus Infections/prevention & control , Drug Repositioning , Eflornithine/chemistry , Eflornithine/pharmacology , Humans , Hydrophobic and Hydrophilic Interactions , Ivermectin/chemistry , Ivermectin/pharmacology , L-Lactate Dehydrogenase/chemistry , L-Lactate Dehydrogenase/drug effects , Models, Molecular , Molecular Docking Simulation , Pandemics/prevention & control , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/drug effects , Pneumonia, Viral/prevention & control , Proline/analogs & derivatives , Proline/chemistry , Proline/pharmacology , Protein Binding , Protein Conformation , Protein Interaction Mapping , Receptors, Glycine/chemistry , Receptors, Glycine/drug effects , SARS-CoV-2 , Saposins/chemistry , Saposins/drug effects , Sofosbuvir/chemistry , Sofosbuvir/pharmacology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/drug effects , Structure-Activity Relationship
13.
Comb Chem High Throughput Screen ; 24(5): 716-728, 2021.
Article in English | MEDLINE | ID: covidwho-721423

ABSTRACT

AIMS: To predict potential drugs for COVID-19 by using molecular docking for virtual screening of drugs approved for other clinical applications. BACKGROUND: SARS-CoV-2 is the betacoronavirus responsible for the COVID-19 pandemic. It was listed as a potential global health threat by the WHO due to high mortality, high basic reproduction number, and lack of clinically approved drugs and vaccines. The genome of the virus responsible for COVID-19 has been sequenced. In addition, the three-dimensional structure of the main protease has been determined experimentally. OBJECTIVE: To identify potential drugs that can be repurposed for treatment of COVID-19 by using molecular docking based virtual screening of all approved drugs. METHODS: A list of drugs approved for clinical use was obtained from the SuperDRUG2 database. The structure of the target in the apo form, as well as structures of several target-ligand complexes, were obtained from RCSB PDB. The structure of SARS-CoV-2 Mpro determined from X-ray diffraction data was used as the target. Data regarding drugs in clinical trials for COVID-19 was obtained from clinicaltrials.org. Input for molecular docking based virtual screening was prepared by using Obabel and customized python, bash, and awk scripts. Molecular docking calculations were carried out with Vina and SMINA, and the docked conformations were analyzed and visualized with PLIP, Pymol, and Rasmol. RESULTS: Among the drugs that are being tested in clinical trials for COVID-19, Danoprevir and Darunavir were predicted to have the highest binding affinity for the Main protease (Mpro) target of SARS-CoV-2. Saquinavir and Beclabuvir were identified as the best novel candidates for COVID-19 therapy by using Virtual Screening of drugs approved for other clinical indications. CONCLUSION: Protease inhibitors approved for treatment of other viral diseases have the potential to be repurposed for treatment of COVID-19.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/virology , Drug Evaluation, Preclinical , Molecular Docking Simulation , SARS-CoV-2/drug effects , Antiviral Agents/chemistry , Benzazepines/chemistry , Benzazepines/pharmacology , Cyclopropanes/chemistry , Cyclopropanes/pharmacology , Darunavir/chemistry , Darunavir/pharmacology , Drug Repositioning , High-Throughput Screening Assays , Humans , Indoles/chemistry , Indoles/pharmacology , Isoindoles/chemistry , Isoindoles/pharmacology , Lactams, Macrocyclic/chemistry , Lactams, Macrocyclic/pharmacology , Proline/analogs & derivatives , Proline/chemistry , Proline/pharmacology , Saquinavir/chemistry , Saquinavir/pharmacology , Sulfonamides/chemistry , Sulfonamides/pharmacology
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