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1.
J Med Chem ; 64(8): 4991-5000, 2021 04 22.
Article in English | MEDLINE | ID: covidwho-1574766

ABSTRACT

The main protease (3CL Mpro) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is an essential enzyme for viral replication with no human counterpart, making it an attractive drug target. To date, no small-molecule clinical drugs are available that specifically inhibit SARS-CoV-2 Mpro. To aid rational drug design, we determined a neutron structure of Mpro in complex with the α-ketoamide inhibitor telaprevir at near-physiological (22 °C) temperature. We directly observed protonation states in the inhibitor complex and compared them with those in the ligand-free Mpro, revealing modulation of the active-site protonation states upon telaprevir binding. We suggest that binding of other α-ketoamide covalent inhibitors can lead to the same protonation state changes in the Mpro active site. Thus, by studying the protonation state changes induced by inhibitors, we provide crucial insights to help guide rational drug design, allowing precise tailoring of inhibitors to manipulate the electrostatic environment of SARS-CoV-2 Mpro.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Oligopeptides/chemistry , Binding Sites , Coronavirus 3C Proteases/metabolism , Crystallography/methods , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/metabolism , Models, Molecular , Neutrons , Oligopeptides/metabolism , Protein Conformation , Protons
2.
Int J Mol Sci ; 21(20)2020 Oct 16.
Article in English | MEDLINE | ID: covidwho-1298153

ABSTRACT

The non-structural protein 2 (nsP2) of alphavirus Venezuelan equine encephalitis virus (VEEV) is a cysteine protease that is responsible for processing of the viral non-structural polyprotein and is an important drug target owing to the clinical relevance of VEEV. In this study we designed two recombinant VEEV nsP2 constructs to study the effects of an N-terminal extension on the protease activity and to investigate the specificity of the elongated enzyme in vitro. The N-terminal extension was found to have no substantial effect on the protease activity. The amino acid preferences of the VEEV nsP2 protease were investigated on substrates representing wild-type and P5, P4, P2, P1, P1', and P2' variants of Semliki forest virus nsP1/nsP2 cleavage site, using a His6-MBP-mEYFP recombinant substrate-based protease assay which has been adapted for a 96-well plate-based format. The structural basis of enzyme specificity was also investigated in silico by analyzing a modeled structure of VEEV nsP2 complexed with oligopeptide substrate. To our knowledge, in vitro screening of P1' amino acid preferences of VEEV nsP2 protease remains undetermined to date, thus, our results may provide valuable information for studies and inhibitor design of different alphaviruses or other Group IV viruses.


Subject(s)
Encephalitis Virus, Venezuelan Equine/enzymology , Viral Proteases/chemistry , Catalytic Domain , Molecular Dynamics Simulation , Oligopeptides/chemistry , Oligopeptides/metabolism , Substrate Specificity , Viral Proteases/genetics , Viral Proteases/metabolism
3.
J Chem Inf Model ; 60(12): 5803-5814, 2020 12 28.
Article in English | MEDLINE | ID: covidwho-1065781

ABSTRACT

The main protease (Mpro) of the SARS-CoV-2 virus is one focus of drug development efforts for COVID-19. Here, we show that interactive molecular dynamics in virtual reality (iMD-VR) is a useful and effective tool for creating Mpro complexes. We make these tools and models freely available. iMD-VR provides an immersive environment in which users can interact with MD simulations and so build protein complexes in a physically rigorous and flexible way. Recently, we have demonstrated that iMD-VR is an effective method for interactive, flexible docking of small molecule drugs into their protein targets (Deeks et al. PLoS One 2020, 15, e0228461). Here, we apply this approach to both an Mpro inhibitor and an oligopeptide substrate, using experimentally determined crystal structures. For the oligopeptide, we test against a crystallographic structure of the original SARS Mpro. Docking with iMD-VR gives models in agreement with experimentally observed (crystal) structures. The docked structures are also tested in MD simulations and found to be stable. Different protocols for iMD-VR docking are explored, e.g., with and without restraints on protein backbone, and we provide recommendations for its use. We find that it is important for the user to focus on forming binding interactions, such as hydrogen bonds, and not to rely on using simple metrics (such as RMSD), in order to create realistic, stable complexes. We also test the use of apo (uncomplexed) crystal structures for docking and find that they can give good results. This is because of the flexibility and dynamic response allowed by the physically rigorous, atomically detailed simulation approach of iMD-VR. We make our models (and interactive simulations) freely available. The software framework that we use, Narupa, is open source, and uses commodity VR hardware, so these tools are readily accessible to the wider research community working on Mpro (and other COVID-19 targets). These should be widely useful in drug development, in education applications, e.g., on viral enzyme structure and function, and in scientific communication more generally.


Subject(s)
Antiviral Agents/chemistry , Benzeneacetamides/chemistry , COVID-19/metabolism , Coronavirus 3C Proteases/metabolism , Imidazoles/chemistry , SARS-CoV-2/enzymology , Viral Protease Inhibitors/chemistry , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Benzeneacetamides/pharmacokinetics , Benzeneacetamides/pharmacology , Coronavirus 3C Proteases/genetics , Crystallization , Cyclohexylamines , Drug Design , Humans , Hydrogen Bonding , Imidazoles/pharmacokinetics , Imidazoles/pharmacology , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Oligopeptides/chemistry , Oligopeptides/metabolism , Protein Conformation , Pyridines , Structure-Activity Relationship , Viral Protease Inhibitors/pharmacokinetics , Viral Protease Inhibitors/pharmacology
4.
Endocr Res ; 45(3): 210-215, 2020 Aug.
Article in English | MEDLINE | ID: covidwho-1050038

ABSTRACT

BACKGROUND: Uptake of coronaviruses by target cells involves binding of the virus by cell ectoenzymes. For the etiologic agent of COVID-19 (SARS-CoV-2), a receptor has been identified as angiotensin-converting enzyme-2 (ACE2). Recently it has been suggested that plasma membrane integrins may be involved in the internalization and replication of clinically important coronaviruses. For example, integrin αvß3 is involved in the cell uptake of a model porcine enteric α-coronavirus that causes human epidemics. ACE2 modulates the intracellular signaling generated by integrins. OBJECTIVE: We propose that the cellular internalization of αvß3 applies to uptake of coronaviruses bound to the integrin, and we evaluate the possibility that clinical host T4 may contribute to target cell uptake of coronavirus and to the consequence of cell uptake of the virus. DISCUSSION AND CONCLUSIONS: The viral binding domain of the integrin is near the Arg-Gly-Asp (RGD) peptide-binding site and RGD molecules can affect virus binding. In this same locale on integrin αvß3 is the receptor for thyroid hormone analogues, particularly, L-thyroxine (T4). By binding to the integrin, T4 has been shown to modulate the affinity of the integrin for other proteins, to control internalization of αvß3 and to regulate the expression of a panel of cytokine genes, some of which are components of the 'cytokine storm' of viral infections. If T4 does influence coronavirus uptake by target cells, other thyroid hormone analogues, such as deaminated T4 and deaminated 3,5,3'-triiodo-L-thyronine (T3), are candidate agents to block the virus-relevant actions of T4 at integrin αvß3 and possibly restrict virus uptake.


Subject(s)
Coronavirus Infections/virology , Integrin alphaVbeta3/metabolism , Porcine epidemic diarrhea virus/metabolism , Receptors, Virus/drug effects , Thyroid Hormones/pharmacology , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/metabolism , Binding Sites , COVID-19 , Cytokines/physiology , Epithelial Cells/virology , Humans , Oligopeptides/metabolism , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , Receptors, Virus/chemistry , Receptors, Virus/metabolism , SARS-CoV-2 , Swine , Thyroid Hormones/physiology , Thyroxine/physiology , Virus Internalization
5.
Acta Crystallogr C Struct Chem ; 76(Pt 12): 1043-1050, 2020 12 01.
Article in English | MEDLINE | ID: covidwho-1040909

ABSTRACT

The first example of molecular docking of the SARS-CoV-2 main protease for COVID-19 [Mpro, Protein Data Bank (PDB) code 7BQY] by a chalcone-based ligand, namely, (E)-1-(2,4-dichlorophenyl)-3-[4-(morpholin-4-yl)phenyl]prop-2-en-1-one, C19H17Cl2NO2, I, is presented. Two-dimensional (2D) LIGPLOT representations calculated for the inhibitor N3, viz. N-{[(5-methylisoxazol-3-yl)carbonyl]alanyl}-L-valyl-N1-((1R,2Z)-4-(benzyloxy)-4-oxo-1-{[(3R)-2-oxopyrrolidin-3-yl]methyl}but-2-enyl)-L-leucinamide, and 7BQY are included for comparison with our chalcone-based complexes. The binding affinity of our chalcone ligand with 7BQY is -7.0 kcal mol-1, a high value which was attributed to the presence of a hydrogen bond, together with many hydrophobic interactions between the drug and the active amino acid residues of the receptor. Docking studies were also performed, employing rigid and flexible binding modes for the ligand. The superposition of N3 and the chalcone docked into the binding pocket of 7BQY is also presented. The synthesis, single-crystal structure, Hirshfeld surface analysis (HSA) and spectral characterization of heterocyclic chalcone-based compound I, are also presented. The molecules are stacked, with normal π-π interactions, in the crystal.


Subject(s)
Antiviral Agents/metabolism , COVID-19/enzymology , Chalcones/metabolism , Coronavirus 3C Proteases/metabolism , SARS-CoV-2/enzymology , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Catalytic Domain , Chalcones/chemical synthesis , Chalcones/chemistry , Coronavirus 3C Proteases/chemistry , Crystallography, X-Ray , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Molecular Docking Simulation , Oligopeptides/metabolism , Protein Binding , Stereoisomerism
6.
Immunobiology ; 226(1): 152021, 2021 01.
Article in English | MEDLINE | ID: covidwho-908903

ABSTRACT

SARS-CoV-2 is a highly contagious virus that has caused serious health crisis world-wide resulting into a pandemic situation. As per the literature, the SARS-CoV-2 is known to exploit humanACE2 receptors (similar toprevious SARS-CoV-1) for gaining entry into the host cell for invasion, infection, multiplication and pathogenesis. However, considering the higher infectivity of SARS-CoV-2 along with the complex etiology and pathophysiological outcomes seen in COVID-19 patients, it seems that there may be an alternate receptor for SARS-CoV-2. I performed comparative protein sequence analysis, database based gene expression profiling, bioinformatics based molecular docking using authentic tools and techniques for unveiling the molecular basis of high infectivity of SARS-CoV-2 as compared to previous known coronaviruses. My study revealed that SARS-CoV-2 (previously known as 2019-nCoV) harbors a RGD motif in its receptor binding domain (RBD) and the motif is absent in all other previously known SARS-CoVs. The RGD motif is well known for its role in cell-attachment and cell-adhesion. My hypothesis is that the SARS-CoV-2 may be (via RGD) exploiting integrins, that have high expression in lungs and all other vital organs, for invading host cells. However, an experimental verification is required. The expression of ACE2, which is a known receptor for SARS-CoV-2, was found to be negligible in lungs. I assume that higher infectivity of SARS-CoV-2 could be due to this RGD-integrin mediated acquired cell-adhesive property. Gene expression profiling revealed that expression of integrins is significantly high in lung cells, in particular αvß6, α5ß1, αvß8 and an ECM protein, ICAM1. The molecular docking experiment showed the RBD of spike protein binds with integrins precisely at RGD motif in a similar manner as a synthetic RGD peptide binds to integrins as found by other researchers. SARS-CoV-2 spike protein has a number of phosphorylation sites that can induce cAMP, PKC, Tyr signaling pathways. These pathways either activate calcium ion channels or get activated by calcium. In fact, integrins have calcium & metal binding sites that were predicted around and in vicinity of RGD-integrin docking site in our analysis which suggests that RGD-integrins interaction possibly occurs in calcium-dependent manner. The higher expression of integrins in lungs along with their previously known high binding affinity (~KD = 4.0 nM) for virus RGD motif could serve as a possible explanation for high infectivity of SARS-CoV-2. On the contrary, human ACE2 has lower expression in lungs and its high binding affinity (~KD = 15 nM) for spike RBD alone could not manifest significant virus-host attachment. This suggests that besides human ACE2, an additional or alternate receptor for SARS-CoV-2 is likely to exist. A highly relevant evidence never reported earlier which corroborate in favor of RGD-integrins mediated virus-host attachment is an unleashed cytokine storm which causes due to activation of TNF-α and IL-6 activation; and integrins role in their activation is also well established. Altogether, the current study has highlighted possible role of calcium and other divalent ions in RGD-integrins interaction for virus invasion into host cells and suggested that lowering divalent ion in lungs could avert virus-host cells attachment.


Subject(s)
COVID-19/virology , Calcium/metabolism , Chelation Therapy , Edetic Acid/therapeutic use , Integrins/metabolism , Receptors, Immunologic/metabolism , Receptors, Peptide/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites/genetics , COVID-19/drug therapy , Calcium Channels/metabolism , Gene Expression Profiling , Humans , Integrins/chemistry , Intercellular Adhesion Molecule-1/metabolism , Interleukin-6/metabolism , Lung/metabolism , Molecular Docking Simulation , Oligopeptides/chemistry , Oligopeptides/metabolism , Protein Binding , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , Sequence Alignment , Signal Transduction/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Tumor Necrosis Factor-alpha/metabolism , Virus Attachment
7.
Sci Rep ; 10(1): 17716, 2020 10 19.
Article in English | MEDLINE | ID: covidwho-880701

ABSTRACT

In the rapidly evolving coronavirus disease (COVID-19) pandemic, repurposing existing drugs and evaluating commercially available inhibitors against druggable targets of the virus could be an effective strategy to accelerate the drug discovery process. The 3C-Like proteinase (3CLpro) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as an important drug target due to its role in viral replication. The lack of a potent 3CLpro inhibitor and the availability of the X-ray crystal structure of 3CLpro (PDB-ID 6LU7) motivated us to perform computational studies to identify commercially available potential inhibitors. A combination of modeling studies was performed to identify potential 3CLpro inhibitors from the protease inhibitor database MEROPS ( https://www.ebi.ac.uk/merops/index.shtml ). Binding energy evaluation identified key residues for inhibitor design. We found 15 potential 3CLpro inhibitors with higher binding affinity than that of an α-ketoamide inhibitor determined via X-ray structure. Among them, saquinavir and three other investigational drugs aclarubicin, TMC-310911, and faldaprevir could be suggested as potential 3CLpro inhibitors. We recommend further experimental investigation of these compounds.


Subject(s)
Betacoronavirus/enzymology , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Aclarubicin/chemistry , Aclarubicin/metabolism , Aminoisobutyric Acids , Betacoronavirus/isolation & purification , Binding Sites , COVID-19 , Coronavirus 3C Proteases , Coronavirus Infections/pathology , Coronavirus Infections/virology , Cysteine Endopeptidases/metabolism , Databases, Factual , Humans , Hydrogen Bonding , Leucine/analogs & derivatives , Oligopeptides/chemistry , Oligopeptides/metabolism , Pandemics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Proline/analogs & derivatives , Protease Inhibitors/metabolism , Quinolines , SARS-CoV-2 , Thermodynamics , Thiazoles/chemistry , Thiazoles/metabolism , Viral Nonstructural Proteins/metabolism
8.
Sci Adv ; 6(42)2020 10.
Article in English | MEDLINE | ID: covidwho-873433

ABSTRACT

Viral papain-like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library and performed comprehensive activity profiling of SARS-CoV-2 PLpro. On the scaffold of the best hits from positional scanning, we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro. We determined crystal structures of two of these inhibitors in complex with SARS-CoV-2 PLpro that reveals their inhibitory mechanisms and provides a molecular basis for the observed substrate specificity profiles. Last, we demonstrate that SARS-CoV-2 PLpro harbors deISGylating activity similar to SARSCoV-1 PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Together, this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repurposing.


Subject(s)
Betacoronavirus/enzymology , Drug Design , Protease Inhibitors/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Amino Acid Sequence , Betacoronavirus/isolation & purification , Binding Sites , COVID-19 , Catalytic Domain , Coronavirus 3C Proteases , Coronavirus Infections/pathology , Coronavirus Infections/virology , Crystallography, X-Ray , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Humans , Kinetics , Molecular Dynamics Simulation , Oligopeptides/chemistry , Oligopeptides/metabolism , Pandemics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Protease Inhibitors/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SARS-CoV-2 , Substrate Specificity , Ubiquitins/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
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