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1.
J Biol Chem ; 297(6): 101362, 2021 12.
Article in English | MEDLINE | ID: covidwho-1751075

ABSTRACT

The Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study. Using a native mass-spectrometry-based approach to screen a natural product library for Nsp9 binders, we identified an ent-kaurane natural product, oridonin, capable of binding to purified SARS-CoV-2 Nsp9 with micromolar affinities. By determining the crystal structure of the Nsp9-oridonin complex, we showed that oridonin binds through a conserved site near Nsp9's C-terminal GxxxG-helix. In enzymatic assays, oridonin's binding to Nsp9 reduces its potential to act as substrate for Nsp12's Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. We also showed using in vitro cellular assays oridonin, while cytotoxic at higher doses has broad antiviral activity, reducing viral titer following infection with either SARS-CoV-2 or, to a lesser extent, MERS-CoV. Accordingly, these preliminary findings suggest that the oridonin molecular scaffold may have the potential to be developed into an antiviral compound to inhibit the function of Nsp9 during coronaviral replication.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Diterpenes, Kaurane/pharmacology , RNA-Binding Proteins/metabolism , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , Animals , Antiviral Agents/chemistry , Binding Sites/drug effects , Biological Products/chemistry , Biological Products/pharmacology , COVID-19/metabolism , COVID-19/virology , Chlorocebus aethiops , Diterpenes, Kaurane/chemistry , Humans , Molecular Docking Simulation , RNA-Binding Proteins/chemistry , SARS-CoV-2/chemistry , SARS-CoV-2/physiology , Vero Cells , Viral Nonstructural Proteins/chemistry
2.
J Am Soc Mass Spectrom ; 33(1): 181-188, 2022 Jan 05.
Article in English | MEDLINE | ID: covidwho-1596214

ABSTRACT

Affinity selection-mass spectrometry, which includes magnetic microbead affinity selection-screening (MagMASS), is ideal for the discovery of ligands in complex mixtures that bind to pharmacological targets. Therapeutic agents are needed to prevent or treat COVID-19, which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection of human cells by SARS-CoV-2 involves binding of the virus spike protein subunit 1 (S1) to the human cell receptor angiotensin converting enzyme-2 (ACE2). Like antibodies, small molecules have the potential to block the interaction of the viral S1 protein with human ACE2 and prevent SARS-CoV-2 infection. Therefore, a MagMASS assay was developed for the discovery of ligands to the S1 protein. Unlike previous MagMASS approaches, this new assay used robotics for 5-fold enhancement of throughput and sensitivity. The assay was validated using the SBP-1 peptide, which is identical to the ACE2 amino acid sequence recognized by the S1 protein, and then applied to the discovery of natural ligands from botanical extracts. Small molecule ligands to the S1 protein were discovered in extracts of the licorice species, Glycyrrhiza inflata. In particular, the licorice ligand licochalcone A was identified through dereplication and comparison with standards using HPLC with high-resolution tandem mass spectrometry.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Discovery/methods , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Binding Sites/drug effects , COVID-19/metabolism , Chalcones/chemistry , Chalcones/pharmacology , Drug Evaluation, Preclinical/methods , Fabaceae/chemistry , Humans , Ligands , Mass Spectrometry/methods , Molecular Docking Simulation , Protein Binding/drug effects , SARS-CoV-2/metabolism
3.
J Biol Chem ; 297(6): 101362, 2021 12.
Article in English | MEDLINE | ID: covidwho-1545135

ABSTRACT

The Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study. Using a native mass-spectrometry-based approach to screen a natural product library for Nsp9 binders, we identified an ent-kaurane natural product, oridonin, capable of binding to purified SARS-CoV-2 Nsp9 with micromolar affinities. By determining the crystal structure of the Nsp9-oridonin complex, we showed that oridonin binds through a conserved site near Nsp9's C-terminal GxxxG-helix. In enzymatic assays, oridonin's binding to Nsp9 reduces its potential to act as substrate for Nsp12's Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. We also showed using in vitro cellular assays oridonin, while cytotoxic at higher doses has broad antiviral activity, reducing viral titer following infection with either SARS-CoV-2 or, to a lesser extent, MERS-CoV. Accordingly, these preliminary findings suggest that the oridonin molecular scaffold may have the potential to be developed into an antiviral compound to inhibit the function of Nsp9 during coronaviral replication.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Diterpenes, Kaurane/pharmacology , RNA-Binding Proteins/metabolism , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , Animals , Antiviral Agents/chemistry , Binding Sites/drug effects , Biological Products/chemistry , Biological Products/pharmacology , COVID-19/metabolism , COVID-19/virology , Chlorocebus aethiops , Diterpenes, Kaurane/chemistry , Humans , Molecular Docking Simulation , RNA-Binding Proteins/chemistry , SARS-CoV-2/chemistry , SARS-CoV-2/physiology , Vero Cells , Viral Nonstructural Proteins/chemistry
4.
J Med Chem ; 65(4): 2940-2955, 2022 02 24.
Article in English | MEDLINE | ID: covidwho-1475245

ABSTRACT

Antiviral agents that complement vaccination are urgently needed to end the COVID-19 pandemic. The SARS-CoV-2 papain-like protease (PLpro), one of only two essential cysteine proteases that regulate viral replication, also dysregulates host immune sensing by binding and deubiquitination of host protein substrates. PLpro is a promising therapeutic target, albeit challenging owing to featureless P1 and P2 sites recognizing glycine. To overcome this challenge, we leveraged the cooperativity of multiple shallow binding sites on the PLpro surface, yielding novel 2-phenylthiophenes with nanomolar inhibitory potency. New cocrystal structures confirmed that ligand binding induces new interactions with PLpro: by closing of the BL2 loop of PLpro forming a novel "BL2 groove" and by mimicking the binding interaction of ubiquitin with Glu167 of PLpro. Together, this binding cooperativity translates to the most potent PLpro inhibitors reported to date, with slow off-rates, improved binding affinities, and low micromolar antiviral potency in SARS-CoV-2-infected human cells.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Binding Sites/drug effects , COVID-19/metabolism , Coronavirus Papain-Like Proteases/isolation & purification , Coronavirus Papain-Like Proteases/metabolism , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Humans , Microbial Sensitivity Tests , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Models, Molecular , Pandemics , Surface Plasmon Resonance , Tumor Cells, Cultured
5.
Int J Biol Macromol ; 187: 976-987, 2021 Sep 30.
Article in English | MEDLINE | ID: covidwho-1474606

ABSTRACT

Coronavirus 3C-like protease (3CLpro) is a crucial target for treating coronavirus diseases including COVID-19. Our preliminary screening showed that Ampelopsis grossedentata extract (AGE) displayed potent SARS-CoV-2-3CLpro inhibitory activity, but the key constituents with SARS-CoV-2-3CLpro inhibitory effect and their mechanisms were unrevealed. Herein, a practical strategy via integrating bioactivity-guided fractionation and purification, mass spectrometry-based peptide profiling and time-dependent biochemical assay, was applied to identify the crucial constituents in AGE and to uncover their inhibitory mechanisms. The results demonstrated that the flavonoid-rich fractions (10-17.5 min) displayed strong SARS-CoV-2-3CLpro inhibitory activities, while the constituents in these fractions were isolated and their SARS-CoV-2-3CLpro inhibitory activities were investigated. Among all isolated flavonoids, dihydromyricetin, isodihydromyricetin and myricetin strongly inhibited SARS-CoV-2 3CLpro in a time-dependent manner. Further investigations demonstrated that myricetin could covalently bind on SARS-CoV-2 3CLpro at Cys300 and Cys44, while dihydromyricetin and isodihydromyricetin covalently bound at Cys300. Covalent docking coupling with molecular dynamics simulations showed the detailed interactions between the orthoquinone form of myricetin and two covalent binding sites (surrounding Cys300 and Cys44) of SARS-CoV-2 3CLpro. Collectively, the flavonoids in AGE strongly and time-dependently inhibit SARS-CoV-2 3CLpro, while the newly identified SARS-CoV-2 3CLpro inhibitors in AGE offer promising lead compounds for developing novel antiviral agents.


Subject(s)
3C Viral Proteases/chemistry , 3C Viral Proteases/metabolism , Ampelopsis/chemistry , Antiviral Agents/pharmacology , Flavonoids/pharmacology , SARS-CoV-2/enzymology , Antiviral Agents/chemistry , Binding Sites/drug effects , Cysteine/metabolism , Flavonoids/chemistry , Flavonols/chemistry , Flavonols/pharmacology , Mass Spectrometry , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Plant Extracts/chemistry , Plant Extracts/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Binding/drug effects , Protein Conformation/drug effects , SARS-CoV-2/drug effects
6.
J Ovarian Res ; 14(1): 126, 2021 Sep 27.
Article in English | MEDLINE | ID: covidwho-1440942

ABSTRACT

BACKGROUND: Infections by the SARS-CoV-2 virus causing COVID-19 are presently a global emergency. The current vaccination effort may reduce the infection rate, but strain variants are emerging under selection pressure. Thus, there is an urgent need to find drugs that treat COVID-19 and save human lives. Hence, in this study, we identified phytoconstituents of an edible vegetable, Bitter melon (Momordica charantia), that affect the SARS-CoV-2 spike protein. METHODS: Components of Momordica charantia were tested to identify the compounds that bind to the SARS-CoV-2 spike protein. An MTiOpenScreen web-server was used to perform docking studies. The Lipinski rule was utilized to evaluate potential interactions between the drug and other target molecules. PyMol and Schrodinger software were used to identify the hydrophilic and hydrophobic interactions. Surface plasmon resonance (SPR) was employed to assess the interaction between an extract component (erythrodiol) and the spike protein. RESULTS: Our in-silico evaluations showed that phytoconstituents of Momordica charantia have a low binding energy range, -5.82 to -5.97 kcal/mol. A docking study revealed two sets of phytoconstituents that bind at the S1 and S2 domains of SARS-CoV-2. SPR showed that erythrodiol has a strong binding affinity (KD = 1.15 µM) with the S2 spike protein of SARS-CoV-2. Overall, docking, ADME properties, and SPR displayed strong interactions between phytoconstituents and the active site of the SARS-CoV-2 spike protein. CONCLUSION: This study reveals that phytoconstituents from bitter melon are potential agents to treat SARS-CoV-2 viral infections due to their binding to spike proteins S1 and S2.


Subject(s)
COVID-19/drug therapy , Momordica charantia/chemistry , Plant Extracts/pharmacology , Spike Glycoprotein, Coronavirus/genetics , Binding Sites/drug effects , COVID-19/genetics , COVID-19/virology , Humans , Hydrophobic and Hydrophilic Interactions/drug effects , Molecular Docking Simulation , Oleanolic Acid/analogs & derivatives , Oleanolic Acid/chemistry , Oleanolic Acid/pharmacology , Plant Extracts/chemistry , Protein Binding/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Surface Plasmon Resonance
7.
Int J Mol Sci ; 22(18)2021 Sep 10.
Article in English | MEDLINE | ID: covidwho-1409705

ABSTRACT

The inhibition mechanism of the main protease (Mpro) of SARS-CoV-2 by ebselen (EBS) and its analog with a hydroxyl group at position 2 of the benzisoselenazol-3(2H)-one ring (EBS-OH) was studied by using a density functional level of theory. Preliminary molecular dynamics simulations on the apo form of Mpro were performed taking into account both the hydrogen donor and acceptor natures of the Nδ and Nε of His41, a member of the catalytic dyad. The potential energy surfaces for the formation of the Se-S covalent bond mediated by EBS and EBS-OH on Mpro are discussed in detail. The EBS-OH shows a distinctive behavior with respect to EBS in the formation of the noncovalent complex. Due to the presence of canonical H-bonds and noncanonical ones involving less electronegative atoms, such as sulfur and selenium, the influence on the energy barriers and reaction energy of the Minnesota hybrid meta-GGA functionals M06, M06-2X and M08HX, and the more recent range-separated hybrid functional wB97X were also considered. The knowledge of the inhibition mechanism of Mpro by the small protease inhibitors EBS or EBS-OH can enlarge the possibilities for designing more potent and selective inhibitor-based drugs to be used in combination with other antiviral therapies.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Isoindoles/pharmacology , Organoselenium Compounds/pharmacology , Protease Inhibitors/pharmacology , Antiviral Agents/therapeutic use , Binding Sites/drug effects , COVID-19/virology , Catalytic Domain/drug effects , Coronavirus 3C Proteases/metabolism , Drug Design , Humans , Isoindoles/chemistry , Isoindoles/therapeutic use , Molecular Docking Simulation , Molecular Dynamics Simulation , Organoselenium Compounds/chemistry , Organoselenium Compounds/therapeutic use , Protease Inhibitors/chemistry , Protease Inhibitors/therapeutic use , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism
8.
J Med Chem ; 65(4): 2836-2847, 2022 02 24.
Article in English | MEDLINE | ID: covidwho-1333869

ABSTRACT

The SARS-CoV-2 viral spike protein S receptor-binding domain (S-RBD) binds ACE2 on host cells to initiate molecular events, resulting in intracellular release of the viral genome. Therefore, antagonists of this interaction could allow a modality for therapeutic intervention. Peptides can inhibit the S-RBD:ACE2 interaction by interacting with the protein-protein interface. In this study, protein contact atlas data and molecular dynamics simulations were used to locate interaction hotspots on the secondary structure elements α1, α2, α3, ß3, and ß4 of ACE2. We designed a library of discontinuous peptides based upon a combination of the hotspot interactions, which were synthesized and screened in a bioluminescence-based assay. The peptides demonstrated high efficacy in antagonizing the SARS-CoV-2 S-RBD:ACE2 interaction and were validated by microscale thermophoresis which demonstrated strong binding affinity (∼10 nM) of these peptides to S-RBD. We anticipate that such discontinuous peptides may hold the potential for an efficient therapeutic treatment for COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Peptides/pharmacology , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites/drug effects , Cells, Cultured , HEK293 Cells , Humans , Models, Molecular , Peptides/chemical synthesis , Peptides/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
9.
Brief Bioinform ; 22(6)2021 11 05.
Article in English | MEDLINE | ID: covidwho-1284855

ABSTRACT

Targeting the interaction between severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)-receptor-binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) is believed to be an effective strategy for drug design to inhibit the infection of SARS-CoV-2. Herein, several ultrashort peptidase inhibitors against the RBD-ACE2 interaction were obtained by a computer-aided approach based on the RBD-binding residues on the protease domain (PD) of ACE2. The designed peptides were tested on a model coronavirus GX_P2V, which has 92.2 and 86% amino acid identity to the SARS-CoV-2 spike protein and RBD, respectively. Molecular dynamics simulations and binding free energy analysis predicted a potential binding pocket on the RBD of the spike protein, and this was confirmed by the specifically designed peptides SI5α and SI5α-b. They have only seven residues, showing potent antiviral activity and low cytotoxicity. Enzyme-linked immunosorbent assay result also confirmed their inhibitory ability against the RBD-ACE2 interaction. The ultrashort peptides are promising precursor molecules for the drug development of Corona Virus Disease 2019, and the novel binding pocket on the RBD may be helpful for the design of RBD inhibitors or antibodies against SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , COVID-19/drug therapy , Peptides/chemistry , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Antiviral Agents/chemistry , Binding Sites/drug effects , COVID-19/genetics , COVID-19/virology , Drug Design , Humans , Molecular Dynamics Simulation , Peptides/genetics , Peptides/therapeutic use , Protein Binding/drug effects , Protein Domains/drug effects , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
10.
Future Med Chem ; 13(17): 1435-1450, 2021 09.
Article in English | MEDLINE | ID: covidwho-1282696

ABSTRACT

The COVID-19 outbreak has thrown the world into an unprecedented crisis. It has posed a challenge to scientists around the globe who are working tirelessly to combat this pandemic. We herein report a set of molecules that may serve as possible inhibitors of the SARS-CoV-2 main protease. To identify these molecules, we followed a combinatorial structure-based strategy, which includes high-throughput virtual screening, molecular docking and WaterMap calculations. The study was carried out using Protein Data Bank structures 5R82 and 6Y2G. DrugBank, Enamine, Natural product and Specs databases, along with a few known antiviral drugs, were used for the screening. WaterMap analysis aided in the recognition of high-potential molecules that can efficiently displace binding-site waters. This study may help the discovery and development of antiviral drugs against SARS-CoV-2.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus 3C Proteases/chemistry , Protease Inhibitors/chemistry , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Antiviral Agents/pharmacokinetics , Antiviral Agents/therapeutic use , Binding Sites/drug effects , Catalysis , Computer Simulation , Databases, Factual , High-Throughput Screening Assays , Humans , Molecular Docking Simulation , Molecular Structure , Protease Inhibitors/pharmacokinetics , Thermodynamics , Water/chemistry
11.
Biomolecules ; 11(7)2021 06 22.
Article in English | MEDLINE | ID: covidwho-1282440

ABSTRACT

SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) protein is the target for the antiviral drug Remdesivir (RDV). With RDV clinical trials on COVID-19 patients showing a reduced hospitalisation time. During the spread of the virus, the RdRp has developed several mutations, with the most frequent being A97V and P323L. The current study sought to investigate whether A97V and P323L mutations influence the binding of RDV to the RdRp of SARS-CoV-2 compared to wild-type (WT). The interaction of RDV with WT-, A97V-, and P323L-RdRp were measured using molecular dynamic (MD) simulations, and the free binding energies were extracted. Results showed that RDV that bound to WT- and A97V-RdRp had a similar dynamic motion and internal residue fluctuations, whereas RDV interaction with P323L-RdRp exhibited a tighter molecular conformation, with a high internal motion near the active site. This was further corroborated with RDV showing a higher binding affinity to P323L-RdRp (-24.1 kcal/mol) in comparison to WT-RdRp (-17.3 kcal/mol). This study provides insight into the potential significance of administering RDV to patients carrying the SARS-CoV-2 P323L-RdRp mutation, which may have a more favourable chance of alleviating the SARS-CoV-2 illness in comparison to WT-RdRp carriers, thereby suggesting further scientific consensus for the usage of Remdesivir as clinical candidate against COVID-19.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus RNA-Dependent RNA Polymerase/genetics , Point Mutation , SARS-CoV-2/genetics , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Binding Sites/drug effects , COVID-19/virology , Catalytic Domain/drug effects , Humans , Molecular Dynamics Simulation , Point Mutation/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology
12.
Cells ; 10(6)2021 06 07.
Article in English | MEDLINE | ID: covidwho-1259431

ABSTRACT

Coronaviruses such as SARS-CoV-2, which is responsible for COVID-19, depend on virus spike protein binding to host cell receptors to cause infection. The SARS-CoV-2 spike protein binds primarily to ACE2 on target cells and is then processed by membrane proteases, including TMPRSS2, leading to viral internalisation or fusion with the plasma membrane. It has been suggested, however, that receptors other than ACE2 may be involved in virus binding. We have investigated the interactions of recombinant versions of the spike protein with human epithelial cell lines that express low/very low levels of ACE2 and TMPRSS2 in a proxy assay for interaction with host cells. A tagged form of the spike protein containing the S1 and S2 regions bound in a temperature-dependent manner to all cell lines, whereas the S1 region alone and the receptor-binding domain (RBD) interacted only weakly. Spike protein associated with cells independently of ACE2 and TMPRSS2, while RBD required the presence of high levels of ACE2 for interaction. As the spike protein has previously been shown to bind heparin, a soluble glycosaminoglycan, we tested the effects of various heparins on ACE2-independent spike protein interaction with cells. Unfractionated heparin inhibited spike protein interaction with an IC50 value of <0.05 U/mL, whereas two low-molecular-weight heparins were less effective. A mutant form of the spike protein, lacking the arginine-rich putative furin cleavage site, interacted only weakly with cells and had a lower affinity for unfractionated and low-molecular-weight heparin than the wild-type spike protein. This suggests that the furin cleavage site might also be a heparin-binding site and potentially important for interactions with host cells. The glycosaminoglycans heparan sulphate and dermatan sulphate, but not chondroitin sulphate, also inhibited the binding of spike protein, indicating that it might bind to one or both of these glycosaminoglycans on the surface of target cells.


Subject(s)
Angiotensin-Converting Enzyme 2/physiology , Epithelial Cells/metabolism , Heparin/pharmacology , Spike Glycoprotein, Coronavirus/metabolism , A549 Cells , Angiotensin-Converting Enzyme 2/genetics , Animals , Binding Sites/drug effects , Binding Sites/genetics , Caco-2 Cells , Cell Line , Chlorocebus aethiops , Dermatan Sulfate/pharmacology , Down-Regulation/drug effects , Epithelial Cells/drug effects , Epithelial Cells/virology , Glycosaminoglycans/pharmacology , HEK293 Cells , HaCaT Cells , Heparitin Sulfate/pharmacology , Humans , Protein Binding/drug effects , Protein Binding/genetics , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells , Virus Internalization/drug effects
13.
Mol Pharmacol ; 100(2): 155-169, 2021 08.
Article in English | MEDLINE | ID: covidwho-1242189

ABSTRACT

The 14-3-3 proteins constitute a family of adaptor proteins with many binding partners and biological functions, and they are considered promising drug targets in cancer and neuropsychiatry. By screening 1280 small-molecule drugs using differential scanning fluorimetry (DSF), we found 15 compounds that decreased the thermal stability of 14-3-3ζ Among these compounds, ebselen was identified as a covalent, destabilizing ligand of 14-3-3 isoforms ζ, ε, γ, and η Ebselen bonding decreased 14-3-3ζ binding to its partner Ser19-phosphorylated tyrosine hydroxylase. Characterization of site-directed mutants at cysteine residues in 14-3-3ζ (C25, C94, and C189) by DSF and mass spectroscopy revealed covalent modification by ebselen of all cysteines through a selenylsulfide bond. C25 appeared to be the preferential site of ebselen interaction in vitro, whereas modification of C94 was the main determinant for protein destabilization. At therapeutically relevant concentrations, ebselen and ebselen oxide caused decreased 14-3-3 levels in SH-SY5Y cells, accompanied with an increased degradation, most probably by the ubiquitin-dependent proteasome pathway. Moreover, ebselen-treated zebrafish displayed decreased brain 14-3-3 content, a freezing phenotype, and reduced mobility, resembling the effects of lithium, consistent with its proposed action as a safer lithium-mimetic drug. Ebselen has recently emerged as a promising drug candidate in several medical areas, such as cancer, neuropsychiatric disorders, and infectious diseases, including coronavirus disease 2019. Its pleiotropic actions are attributed to antioxidant effects and formation of selenosulfides with critical cysteine residues in proteins. Our work indicates that a destabilization of 14-3-3 may affect the protein interaction networks of this protein family, contributing to the therapeutic potential of ebselen. SIGNIFICANCE STATEMENT: There is currently great interest in the repurposing of established drugs for new indications and therapeutic targets. This study shows that ebselen, which is a promising drug candidate against cancer, bipolar disorder, and the viral infection coronavirus disease 2019, covalently bonds to cysteine residues in 14-3-3 adaptor proteins, triggering destabilization and increased degradation in cells and intact brain tissue when used in therapeutic concentrations, potentially explaining the behavioral, anti-inflammatory, and antineoplastic effects of this drug.


Subject(s)
14-3-3 Proteins/chemistry , 14-3-3 Proteins/metabolism , Cysteine/genetics , Isoindoles/pharmacology , Organoselenium Compounds/pharmacology , 14-3-3 Proteins/genetics , Animals , Binding Sites/drug effects , Brain/metabolism , Cell Line , Circular Dichroism , Down-Regulation , Female , Humans , Male , Models, Molecular , Mutagenesis, Site-Directed , Protein Binding/drug effects , Protein Conformation , Protein Stability/drug effects , Tyrosine 3-Monooxygenase/metabolism , Zebrafish , Zebrafish Proteins/chemistry , Zebrafish Proteins/metabolism
14.
PLoS One ; 16(5): e0251910, 2021.
Article in English | MEDLINE | ID: covidwho-1234593

ABSTRACT

The COVID-19 disease has infected and killed countless people all over the world since its emergence at the end of 2019. No specific therapy for COVID-19 is not currently available, and urgent treatment solutions are needed. Recent studies have found several potential molecular targets, and one of the most critical proteins of the SARS-CoV-2 virus work machine is the Papain-like protease (Plpro). Potential inhibitors are available, and their X-ray crystallographic structures in complex with this enzyme have been determined recently. However, their activities against this enzyme are insufficient and need to be characterized and improved to be of clinical values. Therefore, in this work, by utilizing the Supervised Molecular Dynamics (SuMD) simulation method, we achieved multiple unbinding events of Plpro inhibitors, GRL0617, and its derivates, and captured and understood the details of the unbinding pathway. We found that residues of the BL2 loop, such as Tyr268 and Gln269, play major roles in the unbinding pathways, but the most important contributing factor is the natural movements and behavior of the BL2 loop, which can control the entire process. We believe that the details found in this study can be used to refine and optimize potential inhibitors like GRL0617 and design more efficacious inhibitors as a treatment for the SARS-CoV-2 virus.


Subject(s)
COVID-19/drug therapy , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Protease Inhibitors/pharmacology , SARS-CoV-2/enzymology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Binding Sites/drug effects , COVID-19/enzymology , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Crystallography, X-Ray , Humans , Molecular Dynamics Simulation , Papain/metabolism , Protease Inhibitors/chemistry , Protein Binding/drug effects , SARS-CoV-2/drug effects
15.
PLoS Pathog ; 17(5): e1009519, 2021 05.
Article in English | MEDLINE | ID: covidwho-1232468

ABSTRACT

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.


Subject(s)
Amiloride/pharmacology , COVID-19/drug therapy , Coronavirus Envelope Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Amiloride/pharmacokinetics , Animals , Antiviral Agents/pharmacology , Binding Sites/drug effects , COVID-19/virology , Chlorocebus aethiops , Coronavirus Envelope Proteins/chemistry , Humans , Ion Channels/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Binding/drug effects , Protein Conformation/drug effects , Protein Domains , Vero Cells , Virus Assembly/drug effects
16.
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
17.
Angew Chem Int Ed Engl ; 60(18): 10279-10285, 2021 04 26.
Article in English | MEDLINE | ID: covidwho-1122115

ABSTRACT

The receptor binding domain (RBD) of the spike glycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) binds to the human angiotensin-converting enzyme 2 (ACE2) representing the initial contact point for leveraging the infection cascade. We used an automated selection process and identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and does not block the interaction of CoV2-S with ACE2. Nevertheless, infection studies revealed potent and specific inhibition of pseudoviral infection by the aptamer. The present study opens up new vistas in developing SARS-CoV2 infection inhibitors, independent of blocking the ACE2 interaction of the virus, and harnesses aptamers as potential drug candidates and tools to disentangle hitherto inaccessible infection modalities, which is of particular interest in light of the increasing number of escape mutants that are currently being reported.


Subject(s)
Antiviral Agents/pharmacology , Aptamers, Nucleotide/pharmacology , COVID-19/drug therapy , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Aptamers, Nucleotide/chemistry , Binding Sites/drug effects , COVID-19/metabolism , Drug Discovery , HEK293 Cells , Humans , Protein Binding/drug effects , Protein Interaction Domains and Motifs/drug effects , SARS-CoV-2/chemistry , SARS-CoV-2/physiology , SELEX Aptamer Technique , Spike Glycoprotein, Coronavirus/chemistry
18.
Sci Rep ; 10(1): 16986, 2020 10 12.
Article in English | MEDLINE | ID: covidwho-851312

ABSTRACT

We performed molecular dynamics simulation of the dimeric SARS-CoV-2 (severe acute respiratory syndrome corona virus 2) main protease (Mpro) to examine the binding dynamics of small molecular ligands. Seven HIV inhibitors, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir, were used as the potential lead drugs to investigate access to the drug binding sites in Mpro. The frequently accessed sites on Mpro were classified based on contacts between the ligands and the protein, and the differences in site distributions of the encounter complex were observed among the ligands. All seven ligands showed binding to the active site at least twice in 28 simulations of 200 ns each. We further investigated the variations in the complex structure of the active site with the ligands, using microsecond order simulations. Results revealed a wide variation in the shapes of the binding sites and binding poses of the ligands. Additionally, the C-terminal region of the other chain often interacted with the ligands and the active site. Collectively, these findings indicate the importance of dynamic sampling of protein-ligand complexes and suggest the possibilities of further drug optimisations.


Subject(s)
Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Cysteine Endopeptidases/metabolism , Drug Repositioning/methods , HIV Protease Inhibitors/pharmacology , Pneumonia, Viral/drug therapy , Viral Nonstructural Proteins/metabolism , Betacoronavirus/metabolism , Binding Sites/drug effects , Biophysical Phenomena , COVID-19 , Catalytic Domain/drug effects , Computational Biology , Coronavirus 3C Proteases , Darunavir/metabolism , Darunavir/pharmacology , HIV Protease Inhibitors/metabolism , Humans , Indinavir/metabolism , Indinavir/pharmacology , Lopinavir/metabolism , Lopinavir/pharmacology , Molecular Dynamics Simulation , Nelfinavir/metabolism , Nelfinavir/pharmacology , Pandemics , Ritonavir/metabolism , Ritonavir/pharmacology , SARS-CoV-2 , Saquinavir/metabolism , Saquinavir/pharmacology
19.
Molecules ; 25(6)2020 Mar 12.
Article in English | MEDLINE | ID: covidwho-832671

ABSTRACT

The inhibition of human angiotensin I converting enzyme (ACE) has been regarded as a promising approach for the treatment of hypertension. Despite research attempts over many years, our understanding the mechanisms of activation and inhibition of ACE is still far from complete. Here, we present results of all atom molecular dynamics simulations of ACE with and without ligands. Two types of inhibitors, competitive and mixed non-competitive, were used to model the ligand bound forms. In the absence of a ligand the simulation showed spontaneous large hinge-bending motions of multiple conversions between the closed and open states of ACE, while the ligand bound forms were stable in the closed state. Our simulation results imply that the equilibrium between pre-existing backbone conformations shifts in the presence of a ligand. The hinge-bending motion of ACE is considered as an essential to the enzyme function. A mechanistic model of activation and the inhibition may provide valuable information for novel inhibitors of ACE.


Subject(s)
Hypertension/drug therapy , Peptidyl-Dipeptidase A/chemistry , Protein Binding/drug effects , Protein Conformation , Binding Sites/drug effects , Humans , Hypertension/genetics , Ligands , Molecular Dynamics Simulation , Peptidyl-Dipeptidase A/drug effects , Peptidyl-Dipeptidase A/ultrastructure , Thermodynamics
20.
J Mol Med (Berl) ; 98(12): 1659-1673, 2020 12.
Article in English | MEDLINE | ID: covidwho-784429

ABSTRACT

The outbreak of novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus continually led to infect a large population worldwide. SARS-CoV-2 utilizes its NSP6 and Orf9c proteins to interact with sigma receptors that are implicated in lipid remodeling and ER stress response, to infect cells. The drugs targeting the sigma receptors, sigma-1 and sigma-2, have emerged as effective candidates to reduce viral infectivity, and some of them are in clinical trials against COVID-19. The antipsychotic drug, haloperidol, exerts remarkable antiviral activity, but, at the same time, the sigma-1 benzomorphan agonist, dextromethorphan, showed pro-viral activity. To explore the potential mechanisms of biased binding and activity of the two drugs, haloperidol and dextromethorphan towards NSP6, we herein utilized molecular docking-based molecular dynamics simulation studies. Our extensive analysis of the protein-drug interactions, structural and conformational dynamics, residual frustrations, and molecular switches of NSP6-drug complexes indicates that dextromethorphan binding leads to structural destabilization and increase in conformational dynamics and energetic frustrations. On the other hand, the strong binding of haloperidol leads to minimal structural and dynamical perturbations to NSP6. Thus, the structural insights of stronger binding affinity and favorable molecular interactions of haloperidol towards viral NSP6 suggests that haloperidol can be potentially explored as a candidate drug against COVID-19. KEY MESSAGES: •Inhibitors of sigma receptors are considered as potent drugs against COVID-19. •Antipsychotic drug, haloperidol, binds strongly to NSP6 and induces the minimal changes in structure and dynamics of NSP6. •Dextromethorphan, agonist of sigma receptors, binding leads to overall destabilization of NSP6. •These two drugs bind with NSP6 differently and also induce differences in the structural and conformational changes that explain their different mechanisms of action. •Haloperidol can be explored as a candidate drug against COVID-19.


Subject(s)
COVID-19/drug therapy , Coronavirus Nucleocapsid Proteins/chemistry , Dextromethorphan/chemistry , Haloperidol/chemistry , SARS-CoV-2/drug effects , Binding Sites/drug effects , COVID-19/virology , Computer Simulation , Coronavirus Nucleocapsid Proteins/genetics , Dextromethorphan/therapeutic use , Haloperidol/therapeutic use , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Pandemics , Protein Binding/drug effects , Protein Interaction Domains and Motifs/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity
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