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1.
Nat Commun ; 13(1): 868, 2022 02 14.
Article in English | MEDLINE | ID: covidwho-1684025

ABSTRACT

SARS-CoV-2 infection is a major global public health concern with incompletely understood pathogenesis. The SARS-CoV-2 spike (S) glycoprotein comprises a highly conserved free fatty acid binding pocket (FABP) with unknown function and evolutionary selection advantage1,2. Deciphering FABP impact on COVID-19 progression is challenged by the heterogenous nature and large molecular variability of live virus. Here we create synthetic minimal virions (MiniVs) of wild-type and mutant SARS-CoV-2 with precise molecular composition and programmable complexity by bottom-up assembly. MiniV-based systematic assessment of S free fatty acid (FFA) binding reveals that FABP functions as an allosteric regulatory site enabling adaptation of SARS-CoV-2 immunogenicity to inflammation states via binding of pro-inflammatory FFAs. This is achieved by regulation of the S open-to-close equilibrium and the exposure of both, the receptor binding domain (RBD) and the SARS-CoV-2 RGD motif that is responsible for integrin co-receptor engagement. We find that the FDA-approved drugs vitamin K and dexamethasone modulate S-based cell binding in an FABP-like manner. In inflammatory FFA environments, neutralizing immunoglobulins from human convalescent COVID-19 donors lose neutralization activity. Empowered by our MiniV technology, we suggest a conserved mechanism by which SARS-CoV-2 dynamically couples its immunogenicity to the host immune response.


Subject(s)
COVID-19/immunology , Fatty Acids/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Virion/immunology , A549 Cells , Allosteric Site/genetics , Amino Acid Sequence , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites/genetics , COVID-19/metabolism , COVID-19/virology , Cells, Cultured , Cryoelectron Microscopy/methods , Electron Microscope Tomography/methods , Fatty Acid-Binding Proteins/immunology , Fatty Acid-Binding Proteins/metabolism , Fatty Acids/metabolism , Humans , MCF-7 Cells , Microscopy, Confocal/methods , Protein Binding , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Sequence Homology, Amino Acid , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virion/metabolism , Virion/ultrastructure
2.
J Chem Inf Model ; 62(3): 618-626, 2022 02 14.
Article in English | MEDLINE | ID: covidwho-1671473

ABSTRACT

In this study, we target the main protease (Mpro) of the SARS-CoV-2 virus as it is a crucial enzyme for viral replication. Herein, we report three plausible allosteric sites on Mpro that can expand structure-based drug discovery efforts for new Mpro inhibitors. To find these sites, we used mixed-solvent molecular dynamics (MixMD) simulations, an efficient computational protocol that finds binding hotspots through mapping the surface of unbound proteins with 5% cosolvents in water. We have used normal mode analysis to support our claim of allosteric control for these sites. Further, we have performed virtual screening against the sites with 361 hits from Mpro screenings available through the National Center for Advancing Translational Sciences (NCATS). We have identified the NCATS inhibitors that bind to the remote sites better than the active site of Mpro, and we propose these molecules may be allosteric regulators of the system. After identifying our sites, new X-ray crystal structures were released that show fragment molecules in the sites we found, supporting the notion that these sites are accurate and druggable.


Subject(s)
COVID-19 , SARS-CoV-2 , Allosteric Site , Antiviral Agents , Coronavirus 3C Proteases , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/pharmacology
3.
Molecules ; 27(1)2021 Dec 30.
Article in English | MEDLINE | ID: covidwho-1580564

ABSTRACT

The COVID-19 pandemic has caused millions of fatalities since 2019. Despite the availability of vaccines for this disease, new strains are causing rapid ailment and are a continuous threat to vaccine efficacy. Here, molecular docking and simulations identify strong inhibitors of the allosteric site of the SARS-CoV-2 virus RNA dependent RNA polymerase (RdRp). More than one hundred different flavonoids were docked with the SARS-CoV-2 RdRp allosteric site through computational screening. The three top hits were Naringoside, Myricetin and Aureusidin 4,6-diglucoside. Simulation analyses confirmed that they are in constant contact during the simulation time course and have strong association with the enzyme's allosteric site. Absorption, distribution, metabolism, excretion and toxicity (ADMET) data provided medicinal information of these top three hits. They had good human intestinal absorption (HIA) concentrations and were non-toxic. Due to high mutation rates in the active sites of the viral enzyme, these new allosteric site inhibitors offer opportunities to drug SARS-CoV-2 RdRp. These results provide new information for the design of novel allosteric inhibitors against SARS-CoV-2 RdRp.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Computational Biology/methods , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Drug Evaluation, Preclinical , Flavonoids/pharmacology , SARS-CoV-2/enzymology , Allosteric Site , COVID-19/virology , Catalytic Domain , Drug Design , Humans , Intestinal Absorption , Molecular Docking Simulation
4.
J Biochem Mol Toxicol ; 36(2): e22948, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1508784

ABSTRACT

The outbreak of coronavirus disease 2019 (COVID-19) has induced a large number of deaths worldwide. Angiotensin-converting enzyme 2 (ACE2) is the entry receptor for the 2019 novel coronavirus (2019-nCoV) to infect the host cells. Therefore, ACE2 may be an important target for the prevention and treatment of COVID-19. The aim of this study was to investigate the inhibition effect of valaciclovir hydrochloride (VACV), zidovudine (ZDV), saquinavir (SQV), and efavirenz (EFV) on 2019-nCoV infection. The results of molecule docking and surface plasmon resonance showed that VACV, ZDV, SQV, and EFV could bind to ACE2 protein, with the KD value of (4.33 ± 0.09) e-8 , (6.29 ± 1.12) e-6 , (2.37 ± 0.59) e-5 , and (4.85 ± 1.57) e-5 M, respectively. But only ZDV and EFV prevent the 2019-nCoV spike pseudotyped virus to enter ACE2-HEK293T cells with an EC50 value of 4.30 ± 1.46 and 3.92 ± 1.36 µM, respectively. ZDV and EFV also have a synergistic effect on preventing entry of virus into cells. In conclusion, ZDV and EFV suppress 2019-nCoV infection of ACE2-HEK293T cells by interacting with ACE2.


Subject(s)
Antiviral Agents/pharmacology , Peptidyl-Dipeptidase A/drug effects , SARS-CoV-2/drug effects , Allosteric Site , Antiviral Agents/metabolism , COVID-19/drug therapy , COVID-19/prevention & control , COVID-19/virology , HEK293 Cells , Humans , Molecular Docking Simulation , Peptidyl-Dipeptidase A/metabolism , Protein Binding , Surface Plasmon Resonance
5.
Angew Chem Int Ed Engl ; 60(44): 23492-23494, 2021 10 25.
Article in English | MEDLINE | ID: covidwho-1427056

ABSTRACT

This article highlights recent pioneering work by Günther et al. towards the discovery of potential repurposed antiviral compounds (peptidomimetic and non-peptidic) against the SARS-CoV-2 main protease (Mpro ). The antiviral activity of the most potent drugs is discussed along with their binding mode to Mpro as observed through X-ray crystallographic screening.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Drug Repositioning , Protease Inhibitors/pharmacology , SARS-CoV-2/enzymology , Allosteric Site , Animals , Antiviral Agents/chemistry , Chlorocebus aethiops , Crystallography, X-Ray , Molecular Structure , Protease Inhibitors/chemistry , Vero Cells
6.
J Phys Chem Lett ; 12(26): 6218-6226, 2021 Jul 08.
Article in English | MEDLINE | ID: covidwho-1387122

ABSTRACT

Following our previous work ( Chem. Sci. 2021, 12, 4889-4907), we study the structural dynamics of the SARS-CoV-2 Main Protease dimerization interface (apo dimer) by means of microsecond adaptive sampling molecular dynamics simulations (50 µs) using the AMOEBA polarizable force field (PFF). This interface is structured by a complex H-bond network that is stable only at physiological pH. Structural correlations analysis between its residues and the catalytic site confirms the presence of a buried allosteric site. However, noticeable differences in allosteric connectivity are observed between PFFs and non-PFFs. Interfacial polarizable water molecules are shown to appear at the heart of this discrepancy because they are connected to the global interface H-bond network and able to adapt their dipole moment (and dynamics) to their diverse local physicochemical microenvironments. The water-interface many-body interactions appear to drive the interface volume fluctuations and to therefore mediate the allosteric interactions with the catalytic cavity.


Subject(s)
Molecular Dynamics Simulation , SARS-CoV-2/metabolism , Viral Matrix Proteins/chemistry , Water/chemistry , Allosteric Site , COVID-19/pathology , COVID-19/virology , Catalytic Domain , Dimerization , Humans , Hydrogen Bonding , Hydrogen-Ion Concentration , SARS-CoV-2/isolation & purification , Viral Matrix Proteins/metabolism
7.
J Am Chem Soc ; 143(30): 11349-11360, 2021 08 04.
Article in English | MEDLINE | ID: covidwho-1316702

ABSTRACT

The SARS-CoV-2 coronavirus is an enveloped, positive-sense single-stranded RNA virus that is responsible for the COVID-19 pandemic. The spike is a class I viral fusion glycoprotein that extends from the viral surface and is responsible for viral entry into the host cell and is the primary target of neutralizing antibodies. The receptor binding domain (RBD) of the spike samples multiple conformations in a compromise between evading immune recognition and searching for the host-cell surface receptor. Using atomistic simulations of the glycosylated wild-type spike in the closed and 1-up RBD conformations, we map the free energy landscape for RBD opening and identify interactions in an allosteric pocket that influence RBD dynamics. The results provide an explanation for experimental observation of increased antibody binding for a clinical variant with a substitution in this pocket. Our results also suggest the possibility of allosteric targeting of the RBD equilibrium to favor open states via binding of small molecules to the hinge pocket. In addition to potential value as experimental probes to quantify RBD conformational heterogeneity, small molecules that modulate the RBD equilibrium could help explore the relationship between RBD opening and S1 shedding.


Subject(s)
SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Allosteric Site , Molecular Dynamics Simulation , Protein Domains , Thermodynamics
8.
Antiviral Res ; 190: 105075, 2021 06.
Article in English | MEDLINE | ID: covidwho-1290345

ABSTRACT

The emerging SARS-CoV-2 infection is the cause of the global COVID-19 pandemic. To date, there are limited therapeutic options available to fight this disease. Here we examined the inhibitory abilities of two broad-spectrum antiviral natural products chebulagic acid (CHLA) and punicalagin (PUG) against SARS-CoV-2 viral replication. Both CHLA and PUG reduced virus-induced plaque formation in Vero-E6 monolayer at noncytotoxic concentrations, by targeting the enzymatic activity of viral 3-chymotrypsin-like cysteine protease (3CLpro) as allosteric regulators. Our study demonstrates the potential use of CHLA and PUG as novel COVID-19 therapies.


Subject(s)
Antiviral Agents/pharmacology , Benzopyrans/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Glucosides/pharmacology , Hydrolyzable Tannins/pharmacology , SARS-CoV-2/drug effects , Allosteric Site , Animals , Antiviral Agents/chemistry , Benzopyrans/chemistry , COVID-19/drug therapy , COVID-19/virology , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Drug Discovery , Glucosides/chemistry , Molecular Docking Simulation , Protease Inhibitors/pharmacology , SARS-CoV-2/metabolism , Vero Cells , Virus Replication/drug effects
9.
Proteins ; 89(11): 1425-1441, 2021 11.
Article in English | MEDLINE | ID: covidwho-1281247

ABSTRACT

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still has serious negative effects on health, social life, and economics. Recently, vaccines from various companies have been urgently approved to control SARS-CoV-2 infections. However, any specific antiviral drug has not been confirmed so far for regular treatment. An important target is the main protease (Mpro ), which plays a major role in replication of the virus. In this study, Gaussian and residue network models are employed to reveal two distinct potential allosteric sites on Mpro that can be evaluated as drug targets besides the active site. Then, Food and Drug Administration (FDA)-approved drugs are docked to three distinct sites with flexible docking using AutoDock Vina to identify potential drug candidates. Fourteen best molecule hits for the active site of Mpro are determined. Six of these also exhibit high docking scores for the potential allosteric regions. Full-atom molecular dynamics simulations with MM-GBSA method indicate that compounds docked to active and potential allosteric sites form stable interactions with high binding free energy (∆Gbind ) values. ∆Gbind values reach -52.06 kcal/mol for the active site, -51.08 kcal/mol for the potential allosteric site 1, and - 42.93 kcal/mol for the potential allosteric site 2. Energy decomposition calculations per residue elucidate key binding residues stabilizing the ligands that can further serve to design pharmacophores. This systematic and efficient computational analysis successfully determines ivermectine, diosmin, and selinexor currently subjected to clinical trials, and further proposes bromocriptine, elbasvir as Mpro inhibitor candidates to be evaluated against SARS-CoV-2 infections.


Subject(s)
Antiviral Agents/metabolism , Benzofurans/chemistry , Coronavirus 3C Proteases/metabolism , Drug Repositioning/methods , Imidazoles/chemistry , Allosteric Site , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Benzofurans/metabolism , Benzofurans/pharmacology , Binding Sites , Bromocriptine/chemistry , Bromocriptine/metabolism , Bromocriptine/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Diosmin/chemistry , Diosmin/metabolism , Hydrazines/chemistry , Hydrazines/metabolism , Hydrazines/pharmacology , Imidazoles/metabolism , Imidazoles/pharmacology , Ivermectin/chemistry , Ivermectin/metabolism , Ivermectin/pharmacology , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Triazoles/chemistry , Triazoles/metabolism , Triazoles/pharmacology , United States , United States Food and Drug Administration
10.
Molecules ; 26(9)2021 Apr 23.
Article in English | MEDLINE | ID: covidwho-1238919

ABSTRACT

The CB1 cannabinoid receptor (CB1R) contains one of the longest N termini among class A G protein-coupled receptors. Mutagenesis studies suggest that the allosteric binding site of cannabidiol (CBD) involves residues from the N terminal domain. In order to study the allosteric binding of CBD to CB1R we modeled the whole N-terminus of this receptor using the replica exchange molecular dynamics with solute tempering (REST2) approach. Then, the obtained structures of CB1R with the N terminus were used for ligand docking. A natural cannabinoid receptor agonist, Δ9-THC, was docked to the orthosteric site and a negative allosteric modulator, CBD, to the allosteric site positioned between extracellular ends of helices TM1 and TM2. The molecular dynamics simulations were then performed for CB1R with ligands: (i) CBD together with THC, and (ii) THC-only. Analyses of the differences in the residue-residue interaction patterns between those two cases allowed us to elucidate the allosteric network responsible for the modulation of the CB1R by CBD. In addition, we identified the changes in the orthosteric binding mode of Δ9-THC, as well as the changes in its binding energy, caused by the CBD allosteric binding. We have also found that the presence of a complete N-terminal domain is essential for a stable binding of CBD in the allosteric site of CB1R as well as for the allosteric-orthosteric coupling mechanism.


Subject(s)
Cannabidiol/metabolism , Receptor, Cannabinoid, CB1/metabolism , Allosteric Regulation/physiology , Allosteric Site , Animals , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Dynamics Simulation , Protein Binding , Protein Structure, Secondary , Receptor, Cannabinoid, CB1/chemistry
11.
J Phys Chem B ; 125(15): 3763-3780, 2021 04 22.
Article in English | MEDLINE | ID: covidwho-1180209

ABSTRACT

While the pervasiveness of allostery in proteins is commonly accepted, we further show the generic nature of allosteric mechanisms by analyzing here transmembrane ion-channel viroporin 3a and RNA-dependent RNA polymerase (RdRp) from SARS-CoV-2 along with metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and fumarate hydratase (FH) implicated in cancers. Using the previously developed structure-based statistical mechanical model of allostery (SBSMMA), we share our experience in analyzing the allosteric signaling, predicting latent allosteric sites, inducing and tuning targeted allosteric response, and exploring the allosteric effects of mutations. This, yet incomplete list of phenomenology, forms a complex and unique allosteric territory of protein function, which should be thoroughly explored. We propose a generic computational framework, which not only allows one to obtain a comprehensive allosteric control over proteins but also provides an opportunity to approach the fragment-based design of allosteric effectors and drug candidates. The advantages of allosteric drugs over traditional orthosteric compounds, complemented by the emerging role of the allosteric effects of mutations in the expansion of the cancer mutational landscape and in the increased mutability of viral proteins, leave no choice besides further extensive studies of allosteric mechanisms and their biomedical implications.


Subject(s)
COVID-19 , Allosteric Regulation , Allosteric Site , Humans , Models, Molecular , SARS-CoV-2
12.
Science ; 372(6542): 642-646, 2021 05 07.
Article in English | MEDLINE | ID: covidwho-1166347

ABSTRACT

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.


Subject(s)
Allosteric Site , Antiviral Agents/chemistry , Catalytic Domain , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Drug Development , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , Animals , Antiviral Agents/pharmacology , Chlorocebus aethiops , Crystallography, X-Ray , Drug Evaluation, Preclinical , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Vero Cells , Virus Replication/drug effects
13.
FEBS Lett ; 595(4): 442-451, 2021 02.
Article in English | MEDLINE | ID: covidwho-1098832

ABSTRACT

The pathogenesis of the SARS-CoV-2 virus initiates through recognition of the angiotensin-converting enzyme 2 (ACE2) receptor of the host cells by the receptor-binding domain (RBD) located at the spikes of the virus. Here, using molecular dynamics simulations, we have demonstrated the allosteric crosstalk within the RBD in the apo- and the ACE2 receptor-bound states, revealing the contribution of the dynamics-based correlated motions and the electrostatic energy perturbations to this crosstalk. While allostery, based on correlated motions, dominates inherent distal communication in the apo-RBD, the electrostatic energy perturbations determine favorable pairwise crosstalk within the RBD residues upon binding to ACE2. Interestingly, the allosteric path is composed of residues which are evolutionarily conserved within closely related coronaviruses, pointing toward the biological relevance of the communication and its potential as a target for drug development.


Subject(s)
COVID-19/virology , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Allosteric Site , Angiotensin-Converting Enzyme 2/chemistry , Host Microbial Interactions , Humans , Molecular Dynamics Simulation , Protein Domains , Receptors, Virus/chemistry , Static Electricity
14.
PLoS One ; 16(2): e0246181, 2021.
Article in English | MEDLINE | ID: covidwho-1088753

ABSTRACT

The 2019 emergence of, SARS-CoV-2 has tragically taken an immense toll on human life and far reaching impacts on society. There is a need to identify effective antivirals with diverse mechanisms of action in order to accelerate preclinical development. This study focused on five of the most established drug target proteins for direct acting small molecule antivirals: Nsp5 Main Protease, Nsp12 RNA-dependent RNA polymerase, Nsp13 Helicase, Nsp16 2'-O methyltransferase and the S2 subunit of the Spike protein. A workflow of solvent mapping and free energy calculations was used to identify and characterize favorable small-molecule binding sites for an aromatic pharmacophore (benzene). After identifying the most favorable sites, calculated ligand efficiencies were compared utilizing computational fragment screening. The most favorable sites overall were located on Nsp12 and Nsp16, whereas the most favorable sites for Nsp13 and S2 Spike had comparatively lower ligand efficiencies relative to Nsp12 and Nsp16. Utilizing fragment screening on numerous possible sites on Nsp13 helicase, we identified a favorable allosteric site on the N-terminal zinc binding domain (ZBD) that may be amenable to virtual or biophysical fragment screening efforts. Recent structural studies of the Nsp12:Nsp13 replication-transcription complex experimentally corroborates ligand binding at this site, which is revealed to be a functional Nsp8:Nsp13 protein-protein interaction site in the complex. Detailed structural analysis of Nsp13 ZBD conformations show the role of induced-fit flexibility in this ligand binding site and identify which conformational states are associated with efficient ligand binding. We hope that this map of over 200 possible small-molecule binding sites for these drug targets may be of use for ongoing discovery, design, and drug repurposing efforts. This information may be used to prioritize screening efforts or aid in the process of deciphering how a screening hit may bind to a specific target protein.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Methyltransferases/metabolism , RNA Helicases/metabolism , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/metabolism , Allosteric Site , Binding Sites , COVID-19/drug therapy , COVID-19/metabolism , Computational Biology/methods , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Humans , Methyltransferases/antagonists & inhibitors , Methyltransferases/chemistry , Models, Molecular , Molecular Targeted Therapy , Protein Binding , RNA Helicases/antagonists & inhibitors , RNA Helicases/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Virus Replication/drug effects
15.
Elife ; 102021 02 08.
Article in English | MEDLINE | ID: covidwho-1069944

ABSTRACT

The spike (S) protein is the main handle for SARS-CoV-2 to enter host cells via surface angiotensin-converting enzyme 2 (ACE2) receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, using amide hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations, we have mapped the S:ACE2 interaction interface and uncovered long-range allosteric propagation of ACE2 binding to sites necessary for host-mediated proteolysis of S protein, critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking protease docking site ~27 Å away while dampening dynamics of the stalk hinge (central helix and heptad repeat [HR]) regions ~130 Å away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the prefusion state. Our findings provide a dynamics map of the S:ACE2 interface in solution and also offer mechanistic insights into how ACE2 binding is allosterically coupled to distal proteolytic processing sites and viral-host membrane fusion. Thus, protease docking sites flanking the S1/S2 cleavage site represent alternate allosteric hotspot targets for potential therapeutic development.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Allosteric Site , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/chemistry , Binding Sites , COVID-19/metabolism , Humans , Mass Spectrometry/methods , Molecular Dynamics Simulation , Protein Binding , Protein Processing, Post-Translational , Proteolysis , Receptors, Virus/chemistry , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization
16.
Curr Opin Struct Biol ; 65: 209-216, 2020 12.
Article in English | MEDLINE | ID: covidwho-1065572

ABSTRACT

Understanding allosteric regulation of proteins is fundamental to our study of protein structure and function. Moreover, allosteric binding pockets have become a major target of drug discovery efforts in recent years. However, even though the function of almost every protein can be influenced by allostery, it remains a challenge to discover, rationalise and validate putative allosteric binding pockets. This review examines how the discovery and analysis of putative allosteric binding sites have been influenced by the availability of centralised facilities for crystallographic fragment screening, along with newly developed computational methods for modelling low occupancy features. We discuss the experimental parameters required for success, and how new methods could influence the field in the future. Finally, we reflect on the general problem of how to translate these findings into actual ligand development programs.


Subject(s)
Proteins , Allosteric Regulation , Allosteric Site , Humans , Ligands , Protein Binding , Protein Conformation , Proteins/chemistry , Proteins/metabolism
17.
J Proteome Res ; 19(11): 4576-4586, 2020 11 06.
Article in English | MEDLINE | ID: covidwho-960267

ABSTRACT

SARS-CoV-2 has caused the largest pandemic of the twenty-first century (COVID-19), threatening the life and economy of all countries in the world. The identification of novel therapies and vaccines that can mitigate or control this global health threat is among the most important challenges facing biomedical sciences. To construct a long-term strategy to fight both SARS-CoV-2 and other possible future threats from coronaviruses, it is critical to understand the molecular mechanisms underlying the virus action. The viral entry and associated infectivity stems from the formation of the SARS-CoV-2 spike protein complex with angiotensin-converting enzyme 2 (ACE2). The detection of putative allosteric sites on the viral spike protein molecule can be used to elucidate the molecular pathways that can be targeted with allosteric drugs to weaken the spike-ACE2 interaction and, thus, reduce viral infectivity. In this study, we present the results of the application of different computational methods aimed at detecting allosteric sites on the SARS-CoV-2 spike protein. The adopted tools consisted of the protein contact networks (PCNs), SEPAS (Affinity by Flexibility), and perturbation response scanning (PRS) based on elastic network modes. All of these methods were applied to the ACE2 complex with both the SARS-CoV2 and SARS-CoV spike proteins. All of the adopted analyses converged toward a specific region (allosteric modulation region [AMR]), present in both complexes and predicted to act as an allosteric site modulating the binding of the spike protein with ACE2. Preliminary results on hepcidin (a molecule with strong structural and sequence with AMR) indicated an inhibitory effect on the binding affinity of the spike protein toward the ACE2 protein.


Subject(s)
Allosteric Site/genetics , Coronavirus Infections/virology , Pneumonia, Viral/virology , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2 , Betacoronavirus/genetics , Binding Sites , COVID-19 , Drug Discovery , Humans , Models, Molecular , Neural Networks, Computer , Pandemics , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
18.
Molecules ; 25(17)2020 Aug 23.
Article in English | MEDLINE | ID: covidwho-727434

ABSTRACT

The SARS-CoV-2 outbreak caused an unprecedented global public health threat, having a high transmission rate with currently no drugs or vaccines approved. An alternative powerful additional approach to counteract COVID-19 is in silico drug repurposing. The SARS-CoV-2 main protease is essential for viral replication and an attractive drug target. In this study, we used the virtual screening protocol with both long-range and short-range interactions to select candidate SARS-CoV-2 main protease inhibitors. First, the Informational spectrum method applied for small molecules was used for searching the Drugbank database and further followed by molecular docking. After in silico screening of drug space, we identified 57 drugs as potential SARS-CoV-2 main protease inhibitors that we propose for further experimental testing.


Subject(s)
Antiviral Agents/chemistry , Betacoronavirus/drug effects , Cysteine Endopeptidases/chemistry , Mezlocillin/chemistry , Protease Inhibitors/chemistry , Raltegravir Potassium/chemistry , Viral Nonstructural Proteins/chemistry , Allosteric Site , Antiviral Agents/pharmacology , Betacoronavirus/enzymology , Betacoronavirus/pathogenicity , COVID-19 , Catalytic Domain , Coronavirus 3C Proteases , Coronavirus Infections/drug therapy , Coronavirus Infections/enzymology , Coronavirus Infections/virology , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Drug Repositioning , Gene Expression , High-Throughput Screening Assays , Humans , Mezlocillin/pharmacology , Molecular Docking Simulation , Pandemics , Pneumonia, Viral/drug therapy , Pneumonia, Viral/enzymology , Pneumonia, Viral/virology , Protease Inhibitors/pharmacology , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Raltegravir Potassium/pharmacology , SARS-CoV-2 , Thermodynamics , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
19.
Cell Host Microbe ; 28(3): 445-454.e6, 2020 09 09.
Article in English | MEDLINE | ID: covidwho-615004

ABSTRACT

There are as yet no licensed therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2, initiating conformational changes that drive membrane fusion. We find that the monoclonal antibody CR3022 binds the RBD tightly, neutralizing SARS-CoV-2, and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilizing CR3022 epitope is inaccessible in the prefusion spike, suggesting that CR3022 binding facilitates conversion to the fusion-incompetent post-fusion state. Cryogenic electron microscopy (cryo-EM) analysis confirms that incubation of spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope could be useful therapeutically, possibly in synergy with an antibody that blocks receptor attachment.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/chemistry , Betacoronavirus/immunology , Coronavirus Infections/therapy , Pneumonia, Viral/therapy , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Allosteric Site , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/therapeutic use , Antigen-Antibody Complex/chemistry , Betacoronavirus/genetics , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/drug therapy , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Cryoelectron Microscopy , Crystallography, X-Ray , Host Microbial Interactions/immunology , Humans , Models, Molecular , Neutralization Tests , Pandemics , Peptidyl-Dipeptidase A/chemistry , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Receptors, Virus/chemistry , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Viral Vaccines/immunology , Viral Vaccines/therapeutic use , Virus Internalization
20.
Nucleic Acids Res ; 48(13): 7520-7531, 2020 07 27.
Article in English | MEDLINE | ID: covidwho-601821

ABSTRACT

2'-5'-Oligoadenylate synthetases (OAS) are innate immune sensors of cytosolic double-stranded RNA (dsRNA) and play a critical role in limiting viral infection. dsRNA binding induces allosteric structural changes in OAS1 that reorganize its catalytic center to promote synthesis of 2'-5'-oligoadenylate and thus activation of endoribonuclease L. Specific RNA sequences and structural motifs can also enhance activation of OAS1 through currently undefined mechanisms. To better understand these drivers of OAS activation, we tested the impact of defined sequence changes within a short dsRNA that strongly activates OAS1. Both in vitro and in human A549 cells, appending a 3'-end single-stranded pyrimidine (3'-ssPy) can strongly enhance OAS1 activation or have no effect depending on its location, suggesting that other dsRNA features are necessary for correct presentation of the motif to OAS1. Consistent with this idea, we also find that the dsRNA binding position is dictated by an established consensus sequence (WWN9WG). Unexpectedly, however, not all sequences fitting this consensus activate OAS1 equivalently, with strong dependence on the identity of both partially conserved (W) and non-conserved (N9) residues. A picture thus emerges in which both specific RNA features and the context in which they are presented dictate the ability of short dsRNAs to activate OAS1.


Subject(s)
2',5'-Oligoadenylate Synthetase/metabolism , Consensus Sequence , RNA/chemistry , 2',5'-Oligoadenylate Synthetase/chemistry , A549 Cells , Allosteric Regulation , Allosteric Site , Catalytic Domain , Humans , Molecular Docking Simulation , Protein Binding , RNA/metabolism
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