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1.
FEBS J ; 288(17): 5130-5147, 2021 09.
Article in English | MEDLINE | ID: covidwho-1388264

ABSTRACT

SARS-CoV-2 virus has triggered a global pandemic with devastating consequences. The understanding of fundamental aspects of this virus is of extreme importance. In this work, we studied the viral ribonuclease nsp14, one of the most interferon antagonists from SARS-CoV-2. Nsp14 is a multifunctional protein with two distinct activities, an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), both critical for coronaviruses life cycle, indicating nsp14 as a prominent target for the development of antiviral drugs. In coronaviruses, nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein. We have performed a biochemical characterization of nsp14-nsp10 complex from SARS-CoV-2. We confirm the 3'-5' exoribonuclease and MTase activities of nsp14 and the critical role of nsp10 in upregulating the nsp14 ExoN activity. Furthermore, we demonstrate that SARS-CoV-2 nsp14 N7-MTase activity is functionally independent of the ExoN activity and nsp10. A model from SARS-CoV-2 nsp14-nsp10 complex allowed mapping key nsp10 residues involved in this interaction. Our results show that a stable interaction between nsp10 and nsp14 is required for the nsp14-mediated ExoN activity of SARS-CoV-2. We studied the role of conserved DEDD catalytic residues of SARS-CoV-2 nsp14 ExoN. Our results show that motif I of ExoN domain is essential for the nsp14 function, contrasting to the functionality of these residues in other coronaviruses, which can have important implications regarding the specific pathogenesis of SARS-CoV-2. This work unraveled a basis for discovering inhibitors targeting specific amino acids in order to disrupt the assembly of this complex and interfere with coronaviruses replication.


Subject(s)
COVID-19/genetics , Exoribonucleases/genetics , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Viral Regulatory and Accessory Proteins/genetics , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/virology , Drug Design , Exoribonucleases/antagonists & inhibitors , Humans , Multiprotein Complexes/drug effects , Multiprotein Complexes/genetics , Protein Interaction Maps/genetics , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Virus Replication/genetics
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.
Mol Cell Proteomics ; 20: 100116, 2021.
Article in English | MEDLINE | ID: covidwho-1271716

ABSTRACT

Immunotherapies have emerged to treat diseases by selectively modulating a patient's immune response. Although the roles of T and B cells in adaptive immunity have been well studied, it remains difficult to select targets for immunotherapeutic strategies. Because human leukocyte antigen class II (HLA-II) peptides activate CD4+ T cells and regulate B cell activation, proliferation, and differentiation, these peptide antigens represent a class of potential immunotherapy targets and biomarkers. To better understand the molecular basis of how HLA-II antigen presentation is involved in disease progression and treatment, systematic HLA-II peptidomics combined with multiomic analyses of diverse cell types in healthy and diseased states is required. For this reason, MS-based innovations that facilitate investigations into the interplay between disease pathologies and the presentation of HLA-II peptides to CD4+ T cells will aid in the development of patient-focused immunotherapies.


Subject(s)
Histocompatibility Antigens Class II/immunology , Immunotherapy , Peptides/immunology , Animals , Antigen Presentation , Genomics , Histocompatibility Antigens Class II/genetics , Humans , Mass Spectrometry , Peptides/genetics
4.
Sci Rep ; 11(1): 12448, 2021 06 14.
Article in English | MEDLINE | ID: covidwho-1268001

ABSTRACT

The SARS-CoV-2 spike glycoprotein is a focal point for vaccine immunogen and therapeutic antibody design, and also serves as a critical antigen in the evaluation of immune responses to COVID-19. A common feature amongst enveloped viruses such as SARS-CoV-2 and HIV-1 is the propensity for displaying host-derived glycans on entry spike proteins. Similarly displayed glycosylation motifs can serve as the basis for glyco-epitope mediated cross-reactivity by antibodies, which can have important implications on virus neutralization, antibody-dependent enhancement (ADE) of infection, and the interpretation of antibody titers in serological assays. From a panel of nine anti-HIV-1 gp120 reactive antibodies, we selected two (PGT126 and PGT128) that displayed high levels of cross-reactivity with the SARS-CoV-2 spike. We report that these antibodies are incapable of neutralizing pseudoviruses expressing SARS-CoV-2 spike proteins and are unlikely to mediate ADE via FcγRII receptor engagement. Nevertheless, ELISA and other immunoreactivity experiments demonstrate these antibodies are capable of binding the SARS-CoV-2 spike in a glycan-dependent manner. These results contribute to the growing literature surrounding SARS-CoV-2 S cross-reactivity, as we demonstrate the ability for cross-reactive antibodies to interfere in immunoassays.


Subject(s)
HIV Antibodies/immunology , Polysaccharides/immunology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/immunology , Antigen-Antibody Reactions , COVID-19/pathology , COVID-19/virology , Cross Reactions , Epitopes/immunology , HIV Antibodies/pharmacology , Humans , SARS-CoV-2/isolation & purification , Virus Internalization/drug effects
5.
Commun Biol ; 4(1): 724, 2021 06 11.
Article in English | MEDLINE | ID: covidwho-1265978

ABSTRACT

SARS-CoV-2 infection leads to coronavirus disease 2019 (COVID-19), which is associated with severe and life-threatening pneumonia and respiratory failure. However, the molecular basis of these symptoms remains unclear. SARS-CoV-1 E protein interferes with control of cell polarity and cell-cell junction integrity in human epithelial cells by binding to the PALS1 PDZ domain, a key component of the Crumbs polarity complex. We show that C-terminal PDZ binding motifs of SARS-CoV-1 and SARS-CoV-2 E proteins bind the PALS1 PDZ domain with 29.6 and 22.8 µM affinity, whereas the related sequence from MERS-CoV did not bind. We then determined crystal structures of PALS1 PDZ domain bound to both SARS-CoV-1 and SARS-CoV-2 E protein PDZ binding motifs. Our findings establish the structural basis for SARS-CoV-1/2 mediated subversion of Crumbs polarity signalling and serve as a platform for the development of small molecule inhibitors to suppress SARS-CoV-1/2 mediated disruption of polarity signalling in epithelial cells.


Subject(s)
Coronavirus Envelope Proteins/chemistry , Coronavirus Envelope Proteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Nucleoside-Phosphate Kinase/chemistry , Nucleoside-Phosphate Kinase/metabolism , PDZ Domains , Amino Acid Sequence , Humans , Models, Molecular , Protein Binding
6.
Structure ; 29(7): 655-663.e4, 2021 07 01.
Article in English | MEDLINE | ID: covidwho-1263379

ABSTRACT

Emerging SARS-CoV-2 strains, B.1.1.7 and B.1.351, from the UK and South Africa, respectively, show decreased neutralization by monoclonal antibodies and convalescent or vaccinee sera raised against the original wild-type virus, and are thus of clinical concern. However, the neutralization potency of two antibodies, 1-57 and 2-7, which target the receptor-binding domain (RBD) of the spike, was unaffected by these emerging strains. Here, we report cryo-EM structures of 1-57 and 2-7 in complex with spike, revealing each of these antibodies to utilize a distinct mechanism to bypass or accommodate RBD mutations. Notably, each antibody represented an immune response with recognition distinct from those of frequent antibody classes. Moreover, many epitope residues recognized by 1-57 and 2-7 were outside hotspots of evolutionary pressure for ACE2 binding and neutralizing antibody escape. We suggest the therapeutic use of antibodies, such as 1-57 and 2-7, which target less prevalent epitopes, could ameliorate issues of monoclonal antibody escape.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , Receptors, Virus/chemistry , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , Binding Sites , Cloning, Molecular , Cryoelectron Microscopy , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Epitopes/metabolism , Gene Expression , HEK293 Cells , Humans , Models, Molecular , Mutation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Receptors, Virus/genetics , Receptors, Virus/immunology , Receptors, Virus/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
7.
Nat Commun ; 12(1): 3433, 2021 06 08.
Article in English | MEDLINE | ID: covidwho-1261998

ABSTRACT

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has created global health and economic emergencies. SARS-CoV-2 viruses promote their own spread and virulence by hijacking human proteins, which occurs through viral protein recognition of human targets. To understand the structural basis for SARS-CoV-2 viral-host protein recognition, here we use cryo-electron microscopy (cryo-EM) to determine a complex structure of the human cell junction protein PALS1 and SARS-CoV-2 viral envelope (E) protein. Our reported structure shows that the E protein C-terminal DLLV motif recognizes a pocket formed exclusively by hydrophobic residues from the PDZ and SH3 domains of PALS1. Our structural analysis provides an explanation for the observation that the viral E protein recruits PALS1 from lung epithelial cell junctions. In addition, our structure provides novel targets for peptide- and small-molecule inhibitors that could block the PALS1-E interactions to reduce E-mediated virulence.


Subject(s)
Coronavirus Envelope Proteins/chemistry , Coronavirus Envelope Proteins/metabolism , Intercellular Junctions/metabolism , Membrane Proteins/metabolism , Nucleoside-Phosphate Kinase/metabolism , Amino Acid Sequence , Coronavirus Envelope Proteins/ultrastructure , Cryoelectron Microscopy , Humans , Protein Domains , SARS-CoV-2/physiology , Structural Homology, Protein , Structure-Activity Relationship
8.
Front Mol Biosci ; 8: 653148, 2021.
Article in English | MEDLINE | ID: covidwho-1247882

ABSTRACT

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

9.
Int J Mol Sci ; 22(11)2021 May 21.
Article in English | MEDLINE | ID: covidwho-1244038

ABSTRACT

In late 2019, a new member of the Coronaviridae family, officially designated as "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), emerged and spread rapidly. The Coronavirus Disease-19 (COVID-19) outbreak was accompanied by a high rate of morbidity and mortality worldwide and was declared a pandemic by the World Health Organization in March 2020. Within the Coronaviridae family, SARS-CoV-2 is considered to be the third most highly pathogenic virus that infects humans, following the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV). Four major mechanisms are thought to be involved in COVID-19 pathogenesis, including the activation of the renin-angiotensin system (RAS) signaling pathway, oxidative stress and cell death, cytokine storm, and endothelial dysfunction. Following virus entry and RAS activation, acute respiratory distress syndrome develops with an oxidative/nitrosative burst. The DNA damage induced by oxidative stress activates poly ADP-ribose polymerase-1 (PARP-1), viral macrodomain of non-structural protein 3, poly (ADP-ribose) glycohydrolase (PARG), and transient receptor potential melastatin type 2 (TRPM2) channel in a sequential manner which results in cell apoptosis or necrosis. In this review, blockers of angiotensin II receptor and/or PARP, PARG, and TRPM2, including vitamin D3, trehalose, tannins, flufenamic and mefenamic acid, and losartan, have been investigated for inhibiting RAS activation and quenching oxidative burst. Moreover, the application of organic and inorganic nanoparticles, including liposomes, dendrimers, quantum dots, and iron oxides, as therapeutic agents for SARS-CoV-2 were fully reviewed. In the present review, the clinical manifestations of COVID-19 are explained by focusing on molecular mechanisms. Potential therapeutic targets, including the RAS signaling pathway, PARP, PARG, and TRPM2, are also discussed in depth.


Subject(s)
COVID-19/drug therapy , COVID-19/therapy , Cytokine Release Syndrome/drug therapy , Nanomedicine/methods , Oxidative Stress/drug effects , Poly (ADP-Ribose) Polymerase-1/metabolism , SARS-CoV-2/drug effects , Apoptosis/drug effects , COVID-19/metabolism , COVID-19/physiopathology , Cholecalciferol/pharmacology , GTPase-Activating Proteins/antagonists & inhibitors , GTPase-Activating Proteins/metabolism , Humans , Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors , Renin-Angiotensin System/drug effects , SARS-CoV-2/growth & development , SARS-CoV-2/metabolism , TRPM Cation Channels/antagonists & inhibitors , TRPM Cation Channels/metabolism , Tannins/pharmacology , Trehalose/pharmacology
10.
EMBO J ; 40(16): e107786, 2021 08 16.
Article in English | MEDLINE | ID: covidwho-1239217

ABSTRACT

Pangolins have been suggested as potential reservoir of zoonotic viruses, including SARS-CoV-2 causing the global COVID-19 outbreak. Here, we study the binding of two SARS-CoV-2-like viruses isolated from pangolins, GX/P2V/2017 and GD/1/2019, to human angiotensin-converting enzyme 2 (hACE2), the receptor of SARS-CoV-2. We find that the spike protein receptor-binding domain (RBD) of pangolin CoVs binds to hACE2 as efficiently as the SARS-CoV-2 RBD in vitro. Furthermore, incorporation of pangolin CoV RBDs allows entry of pseudotyped VSV particles into hACE2-expressing cells. A screen for binding of pangolin CoV RBDs to ACE2 orthologs from various species suggests a broader host range than that of SARS-CoV-2. Additionally, cryo-EM structures of GX/P2V/2017 and GD/1/2019 RBDs in complex with hACE2 show their molecular binding in modes similar to SARS-CoV-2 RBD. Introducing the Q498H substitution found in pangolin CoVs into the SARS-CoV-2 RBD expands its binding capacity to ACE2 homologs of mouse, rat, and European hedgehog. These findings suggest that these two pangolin CoVs may infect humans, highlighting the necessity of further surveillance of pangolin CoVs.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Betacoronavirus/physiology , Pangolins/virology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/chemistry , Animals , Binding Sites , HEK293 Cells , Hedgehogs/virology , Host Specificity , Humans , Mice , Models, Molecular , Phylogeny , Protein Binding , Protein Conformation , Rats , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization
11.
Proteins ; 89(9): 1134-1144, 2021 09.
Article in English | MEDLINE | ID: covidwho-1188037

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused substantially more infections, deaths, and economic disruptions than the 2002-2003 SARS-CoV. The key to understanding SARS-CoV-2's higher infectivity lies partly in its host receptor recognition mechanism. Experiments show that the human angiotensin converting enzyme 2 (ACE2) protein, which serves as the primary receptor for both CoVs, binds to the receptor binding domain (RBD) of CoV-2's spike protein stronger than SARS-CoV's spike RBD. The molecular basis for this difference in binding affinity, however, remains unexplained from X-ray structures. To go beyond insights gained from X-ray structures and investigate the role of thermal fluctuations in structure, we employ all-atom molecular dynamics simulations. Microseconds-long simulations reveal that while CoV and CoV-2 spike-ACE2 interfaces have similar conformational binding modes, CoV-2 spike interacts with ACE2 via a larger combinatorics of polar contacts, and on average, makes 45% more polar contacts. Correlation analysis and thermodynamic calculations indicate that these differences in the density and dynamics of polar contacts arise from differences in spatial arrangements of interfacial residues, and dynamical coupling between interfacial and non-interfacial residues. These results recommend that ongoing efforts to design spike-ACE2 peptide blockers will benefit from incorporating dynamical information as well as allosteric coupling effects.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Molecular Dynamics Simulation , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Allosteric Regulation , Humans , Mutation , Protein Binding , Receptors, Virus/chemistry , Receptors, Virus/metabolism , Thermodynamics
12.
J Clin Med ; 10(6)2021 Mar 15.
Article in English | MEDLINE | ID: covidwho-1136512

ABSTRACT

Corona virus disease 2019 (COVID-19) imposes a serious public health pandemic affecting the whole world, as it is spreading exponentially. Besides its high infectivity, SARS-CoV-2 causes multiple serious derangements, where the most prominent is severe acute respiratory syndrome as well as multiple organ dysfunction including heart and kidney injury. While the deleterious impact of SARS-CoV-2 on pulmonary and cardiac systems have attracted remarkable attention, the adverse effects of this virus on the renal system is still underestimated. Kidney susceptibility to SARS-CoV-2 infection is determined by the presence of angiotensin-converting enzyme 2 (ACE2) receptor which is used as port of the viral entry into targeted cells, tissue tropism, pathogenicity and subsequent viral replication. The SARS-CoV-2 cellular entry receptor, ACE2, is widely expressed in proximal epithelial cells, vascular endothelial and smooth muscle cells and podocytes, where it supports kidney integrity and function via the enzymatic production of Angiotensin 1-7 (Ang 1-7), which exerts vasodilatory, anti-inflammatory, antifibrotic and diuretic/natriuretic actions via activation of the Mas receptor axis. Loss of this activity constitutes the potential basis for the renal damage that occurs in COVID-19 patients. Indeed, several studies in a small sample of COVID-19 patients revealed relatively high incidence of acute kidney injury (AKI) among them. Although SARS-CoV-1 -induced AKI was attributed to multiorgan failure and cytokine release syndrome, as the virus was not detectable in the renal tissue of infected patients, SARS-CoV-2 antigens were detected in kidney tubules, suggesting that SARS-CoV-2 infects the human kidney directly, and eventually induces AKI characterized with high morbidity and mortality. The mechanisms underlying this phenomenon are largely unknown. However, the fact that ACE2 plays a crucial role against renal injury, the deprivation of the kidney of this advantageous enzyme, along with local viral replication, probably plays a central role. The current review focuses on the critical role of ACE2 in renal physiology, its involvement in the development of kidney injury during SARS-CoV-2 infection, renal manifestations and therapeutic options. The latter includes exogenous administration of Ang (1-7) as an appealing option, given the high incidence of AKI in this ACE2-depleted disorder, and the benefits of ACE2/Ang1-7 including vasodilation, diuresis, natriuresis, attenuation of inflammation, oxidative stress, cell proliferation, apoptosis and coagulation.

13.
Viruses ; 13(2)2021 02 17.
Article in English | MEDLINE | ID: covidwho-1122299

ABSTRACT

MASP-2, mannose-binding protein-associated serine protease 2, is a key enzyme in the lectin pathway of complement activation. Hyperactivation of this protein by human coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2 has been found to contribute to aberrant complement activation in patients, leading to aggravated lung injury with potentially fatal consequences. This hyperactivation is triggered in the lungs through a conserved, direct interaction between MASP-2 and coronavirus nucleocapsid (N) proteins. Blocking this interaction with monoclonal antibodies and interfering directly with the catalytic activity of MASP-2, have been found to alleviate coronavirus-induced lung injury both in vitro and in vivo. In this study, a virtual library of 8736 licensed drugs and clinical agents has been screened in silico according to two parallel strategies. The first strategy aims at identifying direct inhibitors of MASP-2 catalytic activity, while the second strategy focusses on finding protein-protein interaction inhibitors (PPIs) of MASP-2 and coronaviral N proteins. Such agents could represent promising support treatment options to prevent lung injury and reduce mortality rates of infections caused by both present and future-emerging coronaviruses. Forty-six drug repurposing candidates were purchased and, for the ones selected as potential direct inhibitors of MASP-2, a preliminary in vitro assay was conducted to assess their interference with the lectin pathway of complement activation. Some of the tested agents displayed a dose-response inhibitory activity of the lectin pathway, potentially providing the basis for a viable support strategy to prevent the severe complications of coronavirus infections.


Subject(s)
Coronavirus Nucleocapsid Proteins , Enzyme Inhibitors/chemistry , Mannose-Binding Protein-Associated Serine Proteases/metabolism , Protein Binding/drug effects , Coronavirus Infections/drug therapy , Coronavirus Nucleocapsid Proteins/antagonists & inhibitors , Coronavirus Nucleocapsid Proteins/metabolism , Drug Repositioning , Humans , Structure-Activity Relationship
14.
Biochim Biophys Acta Mol Basis Dis ; 1867(4): 166044, 2021 04 01.
Article in English | MEDLINE | ID: covidwho-1103717

ABSTRACT

Diabetes-associated morbidity and mortality is predominantly due to complications of the disease that may cause debilitating conditions, such as heart and renal failure, hepatic insufficiency, retinopathy or peripheral neuropathy. Fibrosis, the excessive and inappropriate deposition of extracellular matrix in various tissues, is commonly found in patients with advanced type 1 or type 2 diabetes, and may contribute to organ dysfunction. Hyperglycemia, lipotoxic injury and insulin resistance activate a fibrotic response, not only through direct stimulation of matrix synthesis by fibroblasts, but also by promoting a fibrogenic phenotype in immune and vascular cells, and possibly also by triggering epithelial and endothelial cell conversion to a fibroblast-like phenotype. High glucose stimulates several fibrogenic pathways, triggering reactive oxygen species generation, stimulating neurohumoral responses, activating growth factor cascades (such as TGF-ß/Smad3 and PDGFs), inducing pro-inflammatory cytokines and chemokines, generating advanced glycation end-products (AGEs) and stimulating the AGE-RAGE axis, and upregulating fibrogenic matricellular proteins. Although diabetes-activated fibrogenic signaling has common characteristics in various tissues, some organs, such as the heart, kidney and liver develop more pronounced and clinically significant fibrosis. This review manuscript summarizes current knowledge on the cellular and molecular pathways involved in diabetic fibrosis, discussing the fundamental links between metabolic perturbations and fibrogenic activation, the basis for organ-specific differences, and the promises and challenges of anti-fibrotic therapies for diabetic patients.


Subject(s)
Diabetes Mellitus, Type 1/pathology , Diabetes Mellitus, Type 2/pathology , Animals , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/metabolism , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Disease Progression , Epigenesis, Genetic , Fibroblasts/metabolism , Fibroblasts/pathology , Fibrosis , Glucose/genetics , Glucose/metabolism , Humans , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Signal Transduction
15.
Comput Biol Med ; 131: 104293, 2021 04.
Article in English | MEDLINE | ID: covidwho-1101164

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an emerging infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Up to 20%-30% of patients hospitalized with COVID-19 have evidence of cardiac dysfunction. Xuebijing injection is a compound injection containing five traditional Chinese medicine ingredients, which can protect cells from SARS-CoV-2-induced cell death and improve cardiac function. However, the specific protective mechanism of Xuebijing injection on COVID-19-induced cardiac dysfunction remains unclear. METHODS: The therapeutic effect of Xuebijing injection on COVID-19 was validated by the TCM Anti COVID-19 (TCMATCOV) platform. RNA-sequencing (RNA-seq) data from GSE150392 was used to find differentially expressed genes (DEGs) from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) infected with SARS-CoV-2. Data from GSE151879 was used to verify the expression of Angiotensin I Converting Enzyme 2 (ACE2) and central hub genes in both human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) and adult human CMs with SARS-CoV-2 infection. RESULTS: A total of 97 proteins were identified as the therapeutic targets of Xuebijing injection for COVID-19. There were 22 DEGs in SARS-CoV-2 infected hiPSC-CMs overlapped with the 97 therapeutic targets, which might be the therapeutic targets of Xuebijing injection on COVID-19-induced cardiac dysfunction. Based on the bioinformatics analysis, 7 genes (CCL2, CXCL8, FOS, IFNB1, IL-1A, IL-1B, SERPINE1) were identified as central hub genes and enriched in pathways including cytokines, inflammation, cell senescence and oxidative stress. ACE2, the receptor of SARS-CoV-2, and the 7 central hub genes were differentially expressed in at least two kinds of SARS-CoV-2 infected CMs. Besides, FOS and quercetin exhibited the tightest binding by molecular docking analysis. CONCLUSION: Our study indicated the underlying protective effect of Xuebijing injection on COVID-19, especially on COVID19-induced cardiac dysfunction, which provided the theoretical basis for exploring the potential protective mechanism of Xuebijing injection on COVID19-induced cardiac dysfunction.


Subject(s)
COVID-19/metabolism , Drugs, Chinese Herbal/pharmacology , Gene Expression Regulation/drug effects , Myocytes, Cardiac/metabolism , RNA-Seq , SARS-CoV-2/metabolism , COVID-19/drug therapy , Cell Line , Human Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/pathology , Human Embryonic Stem Cells/virology , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Induced Pluripotent Stem Cells/virology , Myocytes, Cardiac/pathology , Myocytes, Cardiac/virology
16.
Int J Mol Sci ; 22(4)2021 Feb 07.
Article in English | MEDLINE | ID: covidwho-1069828

ABSTRACT

The current pandemic of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While this respiratory virus only causes mild symptoms in younger healthy individuals, elderly people and those with cardiovascular diseases such as systemic hypertension are susceptible to developing severe conditions that can be fatal. SARS-CoV-2 infection is also associated with an increased incidence of cardiovascular diseases such as myocardial injury, acute coronary syndrome, and thromboembolism. Understanding the mechanisms of the effects of this virus on the cardiovascular system should thus help develop therapeutic strategies to reduce the mortality and morbidity associated with SARS-CoV-2 infection. Since this virus causes severe and fatal conditions in older individuals with cardiovascular comorbidities, effective therapies targeting specific populations will likely contribute to ending this pandemic. In this review article, the effects of various viruses-including other coronaviruses, influenza, dengue, and human immunodeficiency virus-on the cardiovascular system are described to help provide molecular mechanisms of pathologies associated with SARS-CoV-2 infection and COVID-19. The goal is to provide mechanistic information from the biology of other viral infections in relation to cardiovascular pathologies for the purpose of developing improved vaccines and therapeutic agents effective in preventing and/or treating the acute and long-term consequences of SARS-CoV-2 and COVID-19.


Subject(s)
COVID-19/complications , Cardiovascular Diseases/complications , Virus Diseases/complications , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/metabolism , COVID-19/physiopathology , Cardiovascular Diseases/metabolism , Cardiovascular Diseases/physiopathology , Humans , SARS-CoV-2/physiology , Signal Transduction , Spike Glycoprotein, Coronavirus/metabolism , Virus Diseases/metabolism , Virus Diseases/physiopathology
17.
PLoS One ; 15(11): e0234946, 2020.
Article in English | MEDLINE | ID: covidwho-1067385

ABSTRACT

The human ether-a-go-go-related voltage-gated cardiac ion channel (commonly known as hERG) conducts the rapid outward repolarizing potassium current in cardiomyocytes (IKr). Inadvertent blockade of this channel by drug-like molecules represents a key challenge in pharmaceutical R&D due to frequent overlap between the structure-activity relationships of hERG and many primary targets. Building on our previous work, together with recent cryo-EM structures of hERG, we set about to better understand the energetic and structural basis of promiscuous blocker-hERG binding in the context of Biodynamics theory. We propose a two-step blocker binding process consisting of: The initial capture step: diffusion of a single fully solvated blocker copy into a large cavity lined by the intra-cellular cyclic nucleotide binding homology domain (CNBHD). Occupation of this cavity is a necessary but insufficient condition for ion current disruption.The IKr disruption step: translocation of the captured blocker along the channel axis, such that: The head group, consisting of a quasi-rod-shaped moiety, projects into the open pore, accompanied by partial de-solvation of the binding interface.One tail moiety packs along a kink between the S6 helix and proximal C-linker helix adjacent to the intra-cellular entrance of the pore, likewise accompanied by mutual de-solvation of the binding interface (noting that the association barrier is comprised largely of the total head + tail group de-solvation cost).Blockers containing a highly planar moiety that projects into a putative constriction zone within the closed channel become trapped upon closing, as do blockers terminating prior to this region.A single captured blocker copy may conceivably associate and dissociate to/from the pore many times before exiting the CNBHD cavity. Lastly, we highlight possible flaws in the current hERG safety index (SI), and propose an alternate in vivo-relevant strategy factoring in: Benefit/risk.The predicted arrhythmogenic fractional hERG occupancy (based on action potential (AP) simulations of the undiseased human ventricular cardiomyocyte).Alteration of the safety threshold due to underlying disease.Risk of exposure escalation toward the predicted arrhythmic limit due to patient-to-patient pharmacokinetic (PK) variability, drug-drug interactions, overdose, and use for off-label indications in which the hERG safety parameters may differ from their on-label counterparts.


Subject(s)
ERG1 Potassium Channel/chemistry , ERG1 Potassium Channel/metabolism , Potassium Channel Blockers/pharmacology , Arrhythmias, Cardiac , Binding Sites , Cryoelectron Microscopy , Humans , Models, Biological , Models, Molecular , Potassium Channel Blockers/chemistry , Protein Conformation , Protein Domains
18.
Biochimie ; 182: 177-184, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1039291

ABSTRACT

The main protease (Mpro) of SARS-CoV and SARS-CoV-2 is a key enzyme in viral replication and a promising target for the development of antiviral therapeutics. The understanding of this protein is based on a number of observations derived from earlier x-ray structures, which mostly consider substrates or ligands as the main reason behind modulation of the active site. This lead to the concept of substrate-induced subsite cooperativity as an initial attempt to explain the dual binding specificity of this enzyme in recognizing the cleavage sequences at its N- and C-termini, which are important processing steps in obtaining the mature protease. The presented hypothesis proposes that structural heterogeneity is a property of the enzyme, independent of the presence of a substrate or ligand. Indeed, the analysis of Mpro structures of SARS-CoV and SARS-CoV-2 reveals a conformational diversity for the catalytically competent state in ligand-free structures. Variation in the binding site appears to result from flexibility at residues lining the S1 subpocket and segments incorporating methionine 49 and glutamine 189. The structural evidence introduces "structure-based recognition" as a new paradigm in substrate proteolysis by Mpro. In this concept, the binding space in subpockets of the enzyme varies in a non-cooperative manner, causing distinct conformations, which recognize and process different cleavage sites, as the N- and C-termini. Insights into the recognition basis of the protease provide explanation to the ordered processing of cleavage sites. The hypothesis expands the conformational space of the enzyme and consequently opportunities for drug development and repurposing efforts.


Subject(s)
COVID-19/virology , Protein Conformation , SARS Virus/enzymology , SARS-CoV-2/enzymology , Viral Proteases/chemistry , Viral Proteases/metabolism , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Catalytic Domain , Drug Design , Humans , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Substrate Specificity
19.
Comput Struct Biotechnol J ; 19: 759-766, 2021.
Article in English | MEDLINE | ID: covidwho-1036889

ABSTRACT

The recent emergence of the novel SARS-CoV-2 in China and its rapid spread in the human population has led to a public health crisis worldwide. Like in SARS-CoV, horseshoe bats currently represent the most likely candidate animal source for SARS-CoV-2. Yet, the specific mechanisms of cross-species transmission and adaptation to the human host remain unknown. Here we show that the unsupervised analysis of conservation patterns across the ß-CoV spike protein family, using sequence information alone, can provide valuable insights on the molecular basis of the specificity of ß-CoVs to different host cell receptors. More precisely, our results indicate that host cell receptor usage is encoded in the amino acid sequences of different CoV spike proteins in the form of a set of specificity determining positions (SDPs). Furthermore, by integrating structural data, in silico mutagenesis and coevolution analysis we could elucidate the role of SDPs in mediating ACE2 binding across the Sarbecovirus lineage, either by engaging the receptor through direct intermolecular interactions or by affecting the local environment of the receptor binding motif. Finally, by the analysis of coevolving mutations across a paired MSA we were able to identify key intermolecular contacts occurring at the spike-ACE2 interface. These results show that effective mining of the evolutionary records held in the sequence of the spike protein family can help tracing the molecular mechanisms behind the evolution and host-receptor adaptation of circulating and future novel ß-CoVs.

20.
Sheng Wu Gong Cheng Xue Bao ; 36(4): 605-611, 2020 Apr 25.
Article in Chinese | MEDLINE | ID: covidwho-1024807

ABSTRACT

Cyclophilin A (CypA) is a widely distributed and highly conserved protein in organisms. It has peptidyl-prolyl cis/trans isomerase activity and is a receptor for cyclosporin A (CsA). Coronaviruses are enveloped, single-stranded, positive-sense RNA viruses. Seven types of coronaviruses are currently known to infect humans, among which SARS-CoV, MERS-CoV, and SARS-CoV-2 are fatal for humans. It is well established that CypA is essential for the replication of various coronaviruses such as SARS-CoV, CoV-229E, CoV-NL63, and FCoV. Additionally, CsA and its derivatives (ALV, NIM811, etc.) have obvious inhibitory effects on a variety of coronaviruses. These results suggest that CypA is a potential antiviral target and the existing drug CsA might be used as an anti-coronavirus drug. At the end of 2019, SARS-CoV-2 raged in China, which seriously theatern human health and causes huge economic lases. In view of this, we describe the effects of CypA on the replication of coronaviruses and the antiviral activities of its inhibitors, which will provide the scientific basis and ideas for the development of antiviral drugs for SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus Infections , Coronavirus/drug effects , Coronavirus/growth & development , Cyclophilin A/antagonists & inhibitors , Cyclosporine/pharmacology , Cyclosporine/therapeutic use , Pandemics , Pneumonia, Viral , Antiviral Agents/therapeutic use , Betacoronavirus/drug effects , Betacoronavirus/growth & development , COVID-19 , Coronavirus Infections/drug therapy , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Cyclosporine/chemistry , Humans , Pneumonia, Viral/drug therapy , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , SARS Virus/drug effects , SARS Virus/growth & development , SARS-CoV-2 , Virus Replication/drug effects
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