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1.
J Biomol Struct Dyn ; 40(1): 86-100, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1597994

ABSTRACT

Novel Coronavirus or SARS-CoV-2 outbreak has developed a pandemic condition all over the world. The virus is highly infectious and spreads by human to human local transmission mode. Till date, there is no vaccination or drugs been approved for the treatment by the World Health Organisation. Henceforth, the discovery of the potential drugs is an urgent and utmost requirement for the medical fraternity. Since, the side effects of plant-derived compounds will be lower compared to synthetic/chemical drugs. The Main protease (3CLpro or NSP5) and endoribonuclease (NSP15) proteins are necessity for viral replication and its survival in the host cell. In the present study, in-silico approach of drug development was used to search for potential antiviral plant-derived compounds as inhibitors against SARS-CoV-2 replication proteins. Eight plant-derived compounds of which the antiviral activity was known and available, and two reported drugs against SARS-CoV-2 selected for the molecular docking analysis. The docking results suggested that bisdemethoxycurcumin, demethoxycurcumin, scutellarin, quercetin and myricetin showed least binding energy, i.e., greater than -6.5 Kcal/mol against 3CLpro and endoribonuclease of SARS-CoV-2. Further studies of ADME-Tox and bioavailability of drugs were also performed that exhibited efficient parameters of drug likeness. Molecular dynamics simulation calculations were performed for the most negative binding affinity of the compound to evaluate the dynamic behavior,and stability of protein-ligand complex. Our findings suggest that these compounds could be potential inhibitors of SARS-CoV-2 main protease and endoribonuclease. However, further in-vitro and pre-clinical experiments would validate the potential inhibitors of SARS-CoV-2 proteins.


Subject(s)
Antiviral Agents , Phytochemicals/pharmacology , Protease Inhibitors , SARS-CoV-2 , Antiviral Agents/pharmacology , COVID-19 , Coronavirus 3C Proteases/antagonists & inhibitors , Endoribonucleases/antagonists & inhibitors , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors
2.
J Virol ; 94(13)2020 06 16.
Article in English | MEDLINE | ID: covidwho-1583223

ABSTRACT

Fusion with, and subsequent entry into, the host cell is one of the critical steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca2+) play an important role in fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homology, and here, we investigated whether Ca2+ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca2+ environments. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV wild-type (WT) PP infection by approximately 2-fold and that E891 is a crucial residue for Ca2+ interaction. Subsequent electron spin resonance (ESR) experiments revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approximate 1:1 MERS-CoV FP to Ca2+ ratio, as opposed to an 1:2 SARS-CoV FP to Ca2+ ratio, suggesting significant differences in FP Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging infectious disease with zoonotic potential and has reservoirs in dromedary camels and bats. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans, with 2,468 confirmed cases causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. In order to develop novel drugs and vaccines, it is important to understand the molecular mechanisms that enable the virus to infect host cells. Our data have found that calcium is an important regulator of viral fusion by interacting with negatively charged residues in the MERS-CoV FP region. This information can guide therapeutic solutions to block this calcium interaction and also repurpose already approved drugs for this use for a fast response to MERS-CoV outbreaks.


Subject(s)
Calcium/metabolism , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Host-Pathogen Interactions , Ions/metabolism , Membrane Fusion , Middle East Respiratory Syndrome Coronavirus/physiology , Virus Internalization , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Line , Chlorocebus aethiops , Humans , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Models, Molecular , Mutation , Protein Binding , Proteolysis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship , Vero Cells , Virulence , Virus Assembly
3.
J Virol ; 95(17): e0080721, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1486516

ABSTRACT

The membrane fusion between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host cells is essential for the initial step of infection; therefore, the host cell membrane components, including sphingolipids, influence the viral infection. We assessed several inhibitors of the enzymes pertaining to sphingolipid metabolism, against SARS-CoV-2 spike protein (S)-mediated cell-cell fusion and viral infection. N-(4-Hydroxyphenyl) retinamide (4-HPR), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1), suppressed cell-cell fusion and viral infection. The analysis of sphingolipid levels revealed that the inhibition efficiencies of cell-cell fusion and viral infection in 4-HPR-treated cells were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids. We investigated the relationship of DES1 with the inhibition efficiencies of cell-cell fusion. The changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous cell-permeative ceramide; however, the reduced cell-cell fusion could not be reversed. The efficiency of cell-cell fusion in DES1 knockout (KO) cells was at a level comparable to that in wild-type (WT) cells; however, the ratio of saturated sphinganine-based lipids to the total sphingolipids was higher in DES1 KO cells than in WT cells. 4-HPR reduced cell membrane fluidity without any significant effects on the expression or localization of angiotensin-converting enzyme 2, the SARS-CoV-2 receptor. Therefore, 4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion through a DES1-independent mechanism, and this decrease in membrane fluidity induced by 4-HPR could be the major cause for the inhibition of SARS-CoV-2 infection. IMPORTANCE Sphingolipids could play an important role in SARS-CoV-2 S-mediated membrane fusion with host cells. We studied the cell-cell fusion using SARS-CoV-2 S-expressing cells and sphingolipid-manipulated target cells, with an inhibitor of the sphingolipid metabolism. 4-HPR (also known as fenretinide) is an inhibitor of DES1, and it exhibits antitumor activity and suppresses cell-cell fusion and viral infection. 4-HPR suppresses membrane fusion through a decrease in membrane fluidity, which could possibly be the cause for the inhibition of SARS-CoV-2 infection. There is accumulating clinical data on the safety of 4-HPR. Therefore, it could be a potential candidate drug against COVID-19.


Subject(s)
Cell Membrane/metabolism , Fenretinide/pharmacology , Membrane Fluidity/drug effects , Oxidoreductases/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Cell Fusion , Cell Membrane/genetics , Gene Knockout Techniques , HEK293 Cells , Humans , Membrane Fluidity/genetics , Oxidoreductases/deficiency , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
4.
Future Microbiol ; 16: 107-118, 2021 01.
Article in English | MEDLINE | ID: covidwho-1389067

ABSTRACT

Viruses have caused the death of millions of people worldwide. Specifically, human viruses are grouped into 21 families, including the family of coronaviruses (CoVs). In December 2019, in Wuhan, China, a new human CoV was identified, SARS-CoV-2. The first step of the infection mechanism of the SARS-CoV-2 in the human host is adhesion, which occurs through the S glycoprotein that is found in diverse human organs. Another way through which SARS-CoV-2 could possibly attach to the host's cells is by means of the histo-blood group antigens. In this work, we have reviewed the mechanisms by which some viruses bind to the histo-blood group antigens, which could be related to the susceptibility of the individual and are dependent on the histo-blood group.


Subject(s)
Blood Group Antigens/metabolism , COVID-19/pathology , Spike Glycoprotein, Coronavirus/metabolism , Virus Attachment , Animals , Chiroptera/virology , Coronavirus Envelope Proteins/metabolism , Disease Susceptibility/blood , Genome, Viral/genetics , Glycoproteins/metabolism , Humans , SARS-CoV-2/genetics
5.
Mol Biol Evol ; 38(2): 702-715, 2021 01 23.
Article in English | MEDLINE | ID: covidwho-1387955

ABSTRACT

Despite SARS-CoV and SARS-CoV-2 being equipped with highly similar protein arsenals, the corresponding zoonoses have spread among humans at extremely different rates. The specific characteristics of these viruses that led to such distinct outcomes remain unclear. Here, we apply proteome-wide comparative structural analysis aiming to identify the unique molecular elements in the SARS-CoV-2 proteome that may explain the differing consequences. By combining protein modeling and molecular dynamics simulations, we suggest nonconservative substitutions in functional regions of the spike glycoprotein (S), nsp1, and nsp3 that are contributing to differences in virulence. Particularly, we explain why the substitutions at the receptor-binding domain of S affect the structure-dynamics behavior in complexes with putative host receptors. Conservation of functional protein regions within the two taxa is also noteworthy. We suggest that the highly conserved main protease, nsp5, of SARS-CoV and SARS-CoV-2 is part of their mechanism of circumventing the host interferon antiviral response. Overall, most substitutions occur on the protein surfaces and may be modulating their antigenic properties and interactions with other macromolecules. Our results imply that the striking difference in the pervasiveness of SARS-CoV-2 and SARS-CoV among humans seems to significantly derive from molecular features that modulate the efficiency of viral particles in entering the host cells and blocking the host immune response.


Subject(s)
Molecular Dynamics Simulation , Proteomics , SARS Virus/chemistry , SARS Virus/pathogenicity , SARS-CoV-2/chemistry , SARS-CoV-2/pathogenicity , Viral Proteins/chemistry , Animals , Humans , Protein Domains , SARS Virus/metabolism , SARS-CoV-2/metabolism , Species Specificity , Viral Proteins/metabolism
6.
ACS Chem Biol ; 16(4): 642-650, 2021 04 16.
Article in English | MEDLINE | ID: covidwho-1387141

ABSTRACT

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 µM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 µM. There was no toxicity to any of the host cell lines at 10-100 µM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.


Subject(s)
Antiviral Agents/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Phenylalanine/pharmacology , Piperazines/pharmacology , SARS-CoV-2/drug effects , Tosyl Compounds/pharmacology , Animals , Cathepsin L/antagonists & inhibitors , Cathepsin L/metabolism , Cell Line, Tumor , Chlorocebus aethiops , Humans , Microbial Sensitivity Tests , Protein Domains , Proteolysis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Internalization/drug effects
7.
J King Saud Univ Sci ; 33(2): 101344, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1386058

ABSTRACT

Camptothetin (CPT) is a quinoline alkaloid originally isolated from the Chinese tree, Camptotheca acuminata Decne. CPT was found to have anticancerous and antiviral properties. Derivatives of natural CPT, including topothecan and irinotecan are used clinically to treat a variety of cancers. Apart from Camptotheca acuminata Decne, CPT production was also found in the perennial plant Ophiorrhiza mungos. In this study we attempted the immobilization of the tissue culture grown callus of Ophiorrhiza mungos for the continuous production of a higher concentration of CPT. As evident from previous studies about the antiviral effects of CPT, we wanted to bioinformatically analyze the binding potential of CPT towards two important proteins of SARS-CoV-2, protease (Mpro) and RNA dependent RNA polymerase (RdRp). Further docking analysis of the CPT against the exterior spike glycoprotein of SARS-CoV-2 was also done to determine their potential interaction. The immobilized callus of Ophiorrhiza mungos produced CPT at a concentration of 420 µg/l by the end of 12 days of growth. The HPLC analysis was done to determine the purity of the CPT synthesized by the immobilization technique. The bioinformatic analysis revealed a higher binding efficiency of CPT and its derivatives, toptecan and irinotecan against Mpro and RdRp. The docking analysis of CPT against the spike glycoprotein of SARS-CoV-2 showed hydrogen bonding with the amino acids at K466 with a bond distance of 2.56A° and K355 with a bond distance of 2.40A°. This finding was of particular importance that other compounds including hydroxychloroquine sulphate, lopinavir and ivermectin could bind with the spike protein only by weak Vander wall bonds and no hydrogen bond formation was noticed. Our studies hence evaluate the efficiency of CPT against SARS-CoV-2, by potentially blocking the interaction of the spike glycoprotein with the angiotensin-converting enzyme 2 (ACE2) receptor found on host cells.

8.
Biomol NMR Assign ; 15(1): 165-171, 2021 04.
Article in English | MEDLINE | ID: covidwho-1384622

ABSTRACT

SARS-CoV-2 RNA, nsP3c (non-structural Protein3c) spans the sequence of the so-called SARS Unique Domains (SUDs), first observed in SARS-CoV. Although the function of this viral protein is not fully elucidated, it is believed that it is crucial for the formation of the replication/transcription viral complex (RTC) and of the interaction of various viral "components" with the host cell; thus, it is essential for the entire viral life cycle. The first two SUDs, the so-called SUD-N (the N-terminal domain) and SUD-M (domain following SUD-N) domains, exhibit topological and conformational features that resemble the nsP3b macro (or "X") domain. Indeed, they are all folded in a three-layer α/ß/α sandwich structure, as revealed through crystallographic structural investigation of SARS-CoV SUDs, and they have been attributed to different substrate selectivity as they selectively bind to oligonucleotides. On the other hand, the C-terminal SUD (SUD-C) exhibit much lower sequence similarities compared to the SUD-N & SUD-M, as reported in previous crystallographic and NMR studies of SARS-CoV. In the absence of the 3D structures of SARS-CoV-2, we report herein the almost complete NMR backbone and side-chain resonance assignment (1H,13C,15N) of SARS-CoV-2 SUD-M and SUD-C proteins, and the NMR chemical shift-based prediction of their secondary structure elements. These NMR data will set the base for further understanding at the atomic-level conformational dynamics of these proteins and will allow the effective screening of a large number of small molecules as binders with potential biological impact on their function.


Subject(s)
Coronavirus Papain-Like Proteases/chemistry , Magnetic Resonance Spectroscopy , SARS-CoV-2/chemistry , Carbon Isotopes , Hydrogen , Nitrogen Isotopes , Protein Binding , Protein Domains , Protein Structure, Secondary
9.
Biomol NMR Assign ; 15(1): 85-89, 2021 04.
Article in English | MEDLINE | ID: covidwho-1384621

ABSTRACT

Among the proteins encoded by the SARS-CoV-2 RNA, nsP3 (non-structural Protein3) is the largest multi-domain protein. Its role is multifaceted and important for the viral life cycle. Nonetheless, regarding the specific role of each domain there are many aspects of their function that have to be investigated. SARS Unique Domains (SUDs), constitute the nsP3c region of the nsP3, and were observed for the first time in SARS-CoV. Two of them, namely SUD-N (the first SUD) and the SUD-M (sequential to SUD-N), exhibit structural homology with nsP3b ("X" or macro domain); indeed all of them are folded in a three-layer α/ß/α sandwich. On the contrary, they do not exhibit functional similarities, like ADP-ribose binding properties and ADP-ribose hydrolase activity. There are reports that suggest that these two SUDs may exhibit a binding selectivity towards G-oligonucleotides, a feature which may contribute to the characterization of their role in the formation of the replication/transcription viral complex (RTC) and of the interaction of various viral "components" with the host cell. While the structures of these domains of SARS-CoV-2 have not been determined yet, SUDs interaction with oligonucleotides and/or RNA molecules may provide a platform for drug discovery. Here, we report the almost complete NMR backbone and side-chain resonance assignment (1H,13C,15N) of SARS-CoV-2 SUD-N protein, and the NMR chemical shift-based prediction of the secondary structure elements. These data may be exploited for its 3D structure determination and the screening of chemical compounds libraries, which may alter SUD-N function.


Subject(s)
Coronavirus Papain-Like Proteases/chemistry , Magnetic Resonance Spectroscopy , SARS-CoV-2/chemistry , Carbon Isotopes , Drug Design , Hydrogen , Nitrogen Isotopes , Oligonucleotides/chemistry , Protein Domains , Protein Structure, Secondary , Virus Replication
10.
Front Mol Biosci ; 7: 568954, 2020.
Article in English | MEDLINE | ID: covidwho-1389212

ABSTRACT

Because ACE2 is a host cell receptor of the SARS-CoV-2, an investigation of ACE2 expression in normal and virus-infected human tissues is crucial for understanding the mechanism of SARS-CoV-2 infection. We identified pathways associated with ACE2 expression and gene co-expression networks of ACE2 in pan-tissue based on the gene expression profiles in normal human tissues. We found that the pathways significantly associated with ACE2 upregulation were mainly involved in immune, stromal signature, metabolism, cell growth and proliferation, and cancer and other diseases. The number of genes having a significant positive expression correlation with ACE2 in females far exceeded that in males. The estrogen receptors (ESR1 and ESR2) and androgen receptor (AR) genes had a significant positive expression correlation with ACE2. Meanwhile, the enrichment levels of immune cells were positively associated with the expression levels of ESR1 and ESR2, while they were inversely associated with the expression levels of AR in pan-tissue and multiple individual tissues. It suggests that females are likely to have a more robust immune defense system against SARS-CoV-2 than males. ACE2 was upregulated in SARS-CoV-2-infected tissues relative to normal tissues and in SARS-CoV-2-infected males relative to females, while its expression levels had no significant difference between healthy females and males. Numerous immune-related pathways were highly enriched in SARS-CoV-2-infected males relative to females. These data indicate that males are more susceptible and more likely to have an excessive immune response to SARS-CoV-2 infection than females. This study furnishes potentially cues explaining why females have better clinical outcomes of SARS-CoV-2 infections than males and warrant further investigation for understanding the mechanism of SARS-CoV-2 infection.

11.
Front Mol Biosci ; 7: 222, 2020.
Article in English | MEDLINE | ID: covidwho-1389211

ABSTRACT

Zinc plays a crucial role in the process of virion maturation inside the host cell. The accessory Cys-rich proteins expressed in SARS-CoV-2 by genes ORF7a and ORF8 are likely involved in zinc binding and in interactions with cellular antigens activated by extensive disulfide bonds. In this report we provide a proof of concept for the feasibility of a structural study of orf7a and orf8 proteins. A conceivable hypothesis is that lack of cellular zinc, or substitution thereof, might lead to a significant slowing down of viral maturation.

12.
J Virol ; 95(17): e0080721, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1381151

ABSTRACT

The membrane fusion between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host cells is essential for the initial step of infection; therefore, the host cell membrane components, including sphingolipids, influence the viral infection. We assessed several inhibitors of the enzymes pertaining to sphingolipid metabolism, against SARS-CoV-2 spike protein (S)-mediated cell-cell fusion and viral infection. N-(4-Hydroxyphenyl) retinamide (4-HPR), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1), suppressed cell-cell fusion and viral infection. The analysis of sphingolipid levels revealed that the inhibition efficiencies of cell-cell fusion and viral infection in 4-HPR-treated cells were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids. We investigated the relationship of DES1 with the inhibition efficiencies of cell-cell fusion. The changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous cell-permeative ceramide; however, the reduced cell-cell fusion could not be reversed. The efficiency of cell-cell fusion in DES1 knockout (KO) cells was at a level comparable to that in wild-type (WT) cells; however, the ratio of saturated sphinganine-based lipids to the total sphingolipids was higher in DES1 KO cells than in WT cells. 4-HPR reduced cell membrane fluidity without any significant effects on the expression or localization of angiotensin-converting enzyme 2, the SARS-CoV-2 receptor. Therefore, 4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion through a DES1-independent mechanism, and this decrease in membrane fluidity induced by 4-HPR could be the major cause for the inhibition of SARS-CoV-2 infection. IMPORTANCE Sphingolipids could play an important role in SARS-CoV-2 S-mediated membrane fusion with host cells. We studied the cell-cell fusion using SARS-CoV-2 S-expressing cells and sphingolipid-manipulated target cells, with an inhibitor of the sphingolipid metabolism. 4-HPR (also known as fenretinide) is an inhibitor of DES1, and it exhibits antitumor activity and suppresses cell-cell fusion and viral infection. 4-HPR suppresses membrane fusion through a decrease in membrane fluidity, which could possibly be the cause for the inhibition of SARS-CoV-2 infection. There is accumulating clinical data on the safety of 4-HPR. Therefore, it could be a potential candidate drug against COVID-19.


Subject(s)
Cell Membrane/metabolism , Fenretinide/pharmacology , Membrane Fluidity/drug effects , Oxidoreductases/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Cell Fusion , Cell Membrane/genetics , Gene Knockout Techniques , HEK293 Cells , Humans , Membrane Fluidity/genetics , Oxidoreductases/deficiency , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
13.
Brief Bioinform ; 22(2): 1239-1253, 2021 03 22.
Article in English | MEDLINE | ID: covidwho-1352106

ABSTRACT

The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the binding to the permissive cells. The receptor-binding domain (RBD) of SARS-CoV-2 S protein directly interacts with the human angiotensin-converting enzyme 2 (ACE2) on the host cell membrane. In this study, we used computational saturation mutagenesis approaches, including structure-based energy calculations and sequence-based pathogenicity predictions, to quantify the systemic effects of missense mutations on SARS-CoV-2 S protein structure and function. A total of 18 354 mutations in S protein were analyzed, and we discovered that most of these mutations could destabilize the entire S protein and its RBD. Specifically, residues G431 and S514 in SARS-CoV-2 RBD are important for S protein stability. We analyzed 384 experimentally verified S missense variations and revealed that the dominant pandemic form, D614G, can stabilize the entire S protein. Moreover, many mutations in N-linked glycosylation sites can increase the stability of the S protein. In addition, we investigated 3705 mutations in SARS-CoV-2 RBD and 11 324 mutations in human ACE2 and found that SARS-CoV-2 neighbor residues G496 and F497 and ACE2 residues D355 and Y41 are critical for the RBD-ACE2 interaction. The findings comprehensively provide potential target sites in the development of drugs and vaccines against COVID-19.


Subject(s)
Mutation, Missense , Spike Glycoprotein, Coronavirus/genetics , COVID-19/metabolism , COVID-19/virology , Humans , Protein Binding , SARS-CoV-2/metabolism , Thermodynamics
14.
J Virol ; 95(17): e0080721, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1350004

ABSTRACT

The membrane fusion between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host cells is essential for the initial step of infection; therefore, the host cell membrane components, including sphingolipids, influence the viral infection. We assessed several inhibitors of the enzymes pertaining to sphingolipid metabolism, against SARS-CoV-2 spike protein (S)-mediated cell-cell fusion and viral infection. N-(4-Hydroxyphenyl) retinamide (4-HPR), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1), suppressed cell-cell fusion and viral infection. The analysis of sphingolipid levels revealed that the inhibition efficiencies of cell-cell fusion and viral infection in 4-HPR-treated cells were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids. We investigated the relationship of DES1 with the inhibition efficiencies of cell-cell fusion. The changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous cell-permeative ceramide; however, the reduced cell-cell fusion could not be reversed. The efficiency of cell-cell fusion in DES1 knockout (KO) cells was at a level comparable to that in wild-type (WT) cells; however, the ratio of saturated sphinganine-based lipids to the total sphingolipids was higher in DES1 KO cells than in WT cells. 4-HPR reduced cell membrane fluidity without any significant effects on the expression or localization of angiotensin-converting enzyme 2, the SARS-CoV-2 receptor. Therefore, 4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion through a DES1-independent mechanism, and this decrease in membrane fluidity induced by 4-HPR could be the major cause for the inhibition of SARS-CoV-2 infection. IMPORTANCE Sphingolipids could play an important role in SARS-CoV-2 S-mediated membrane fusion with host cells. We studied the cell-cell fusion using SARS-CoV-2 S-expressing cells and sphingolipid-manipulated target cells, with an inhibitor of the sphingolipid metabolism. 4-HPR (also known as fenretinide) is an inhibitor of DES1, and it exhibits antitumor activity and suppresses cell-cell fusion and viral infection. 4-HPR suppresses membrane fusion through a decrease in membrane fluidity, which could possibly be the cause for the inhibition of SARS-CoV-2 infection. There is accumulating clinical data on the safety of 4-HPR. Therefore, it could be a potential candidate drug against COVID-19.


Subject(s)
Cell Membrane/metabolism , Fenretinide/pharmacology , Membrane Fluidity/drug effects , Oxidoreductases/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Cell Fusion , Cell Membrane/genetics , Gene Knockout Techniques , HEK293 Cells , Humans , Membrane Fluidity/genetics , Oxidoreductases/deficiency , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
15.
Viral Immunol ; 34(5): 352-357, 2021 06.
Article in English | MEDLINE | ID: covidwho-1343610

ABSTRACT

Intense immunological dysregulation including immune cell lesions has been characteristically observed in severe cases of coronavirus disease-2019 (COVID-19), for which molecular mechanisms are not properly understood. A study of physiological expressions of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) host cell entry-related factors in immune system components may help explain molecular mechanisms involved in COVID-19 immunopathology. We analyzed transcriptomic and proteomic expression metadata for SARS-CoV-2 host cell entry receptor ACE2 and entry associated proteases (TMPRSS2, CTSL, and FURIN) in silico across immune system components including the blood lineage cells. ACE2 was not detected in any of the studied immune cell components; however, varying transcriptomic and proteomic expressions were observed for TMPRSS2, CTSL, and FURIN. Nondetectable expressions of SARS-CoV-2 host cell entry receptor ACE2 in immune system components or blood lineage cells indicate it does not mediate immune cell lesions in COVID-19. Alternative mechanisms need to be explored for COVID-19 immunopathogenesis.


Subject(s)
COVID-19/immunology , COVID-19/pathology , SARS-CoV-2/immunology , Virus Internalization , Angiotensin-Converting Enzyme 2/genetics , Cathepsin L/genetics , Furin/genetics , Healthy Volunteers , Humans , Immune System , Metadata , Proteomics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Serine Endopeptidases/genetics , Transcriptome
16.
J Biomol Struct Dyn ; 39(11): 4175-4184, 2021 07.
Article in English | MEDLINE | ID: covidwho-1343548

ABSTRACT

SARS coronavirus (COVID-19) is a real health challenge of the 21st century for scientists, health workers, politicians, and all humans that has severe cause epidemic worldwide. The virus exerts its pathogenic activity through by mechanism and gains the entry via spike proteins (S) and Angiotensin-Converting Enzyme 2 (ACE2) receptor proteins on host cells. The present work is an effort for a computational target to block the residual binding protein (RBP) on spike proteins (S), Angiotensin-Converting Enzyme 2 (ACE2) receptor proteins by probiotics namely Plantaricin BN, Plantaricin JLA-9, Plantaricin W, Plantaricin D along with RNA-dependent RNA polymerase (RdRp). Docking studies were designed in order to obtain the binding energies for Plantaricin metabolites. The binding energies for Plantaricin W were -14.64, -11.1 and -12.68 for polymerase, RBD and ACE2 respectively comparatively very high with other compounds. Plantaricin W, D, and JLA-9 were able to block the residues (THR556, ALA558) surrounding the deep grove catalytic site (VAL557) of RdRp making them more therapeutically active for COVID-19. Molecular dynamics studies further strengthen stability of the complexes of plantaricin w and SARS-CoV-2 RdRp enzyme, RBD of spike protein, and human ACE2 receptor. The present study present multi-way options either by blocking RBD on S proteins or interaction of S protein with ACE2 receptor proteins or inhibiting RdRp to counter any effect of COVID-19 by Plantaricin molecules paving a way that can be useful in the treatment of COVID-19 until some better option will be available.Communicated by Ramaswamy H. Sarma.


Subject(s)
COVID-19 , Probiotics , Antiviral Agents/pharmacology , Humans , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism
17.
Retrovirology ; 18(1): 13, 2021 06 05.
Article in English | MEDLINE | ID: covidwho-1257950

ABSTRACT

Humanized mice model human disease and as such are used commonly for research studies of infectious, degenerative and cancer disorders. Recent models also reflect hematopoiesis, natural immunity, neurobiology, and molecular pathways that influence disease pathobiology. A spectrum of immunodeficient mouse strains permit long-lived human progenitor cell engraftments. The presence of both innate and adaptive immunity enables high levels of human hematolymphoid reconstitution with cell susceptibility to a broad range of microbial infections. These mice also facilitate investigations of human pathobiology, natural disease processes and therapeutic efficacy in a broad spectrum of human disorders. However, a bridge between humans and mice requires a complete understanding of pathogen dose, co-morbidities, disease progression, environment, and genetics which can be mirrored in these mice. These must be considered for understanding of microbial susceptibility, prevention, and disease progression. With known common limitations for access to human tissues, evaluation of metabolic and physiological changes and limitations in large animal numbers, studies in mice prove important in planning human clinical trials. To these ends, this review serves to outline how humanized mice can be used in viral and pharmacologic research emphasizing both current and future studies of viral and neurodegenerative diseases. In all, humanized mouse provides cost-effective, high throughput studies of infection or degeneration in natural pathogen host cells, and the ability to test transmission and eradication of disease.


Subject(s)
Disease Models, Animal , Immunity, Innate , Mice, SCID , Neurodegenerative Diseases/immunology , Animals , HIV-1/immunology , Mice
18.
Tetrahedron ; 77: 131761, 2021 Jan 01.
Article in English | MEDLINE | ID: covidwho-1279699

ABSTRACT

Originated in China, coronavirus disease 2019 (COVID-19)- the highly contagious and fatal respiratory disease caused by SARS-CoV-2 has already infected more than 29 million people worldwide with a mortality rate of 3.15% (according to World Health Organization's (WHO's) report, September 2020) and the number is exponentially increasing with no remedy whatsoever discovered till date. But it is not the first time this infectious viral disease has appeared, in 2002 SARS-CoV infected more than 8000 individuals of which 9.6% patients died and in 2012 approximately 35% of MERS-CoV infected patients have died. Literature reports indicate that a chymotripsin-like cystein protease (3CLpro) is responsible for the replication of the virus inside the host cell. Therefore, design and synthesis of 3CLpro inhibitor molecules play a great impact in drug development against this COVID-19 pandemic. In this review, we are discussing the anti-SARS effect of some small molecule 3CLpro inhibitors with their various binding modes of interactions to the target protein.

19.
Nat Nanotechnol ; 16(8): 942-951, 2021 08.
Article in English | MEDLINE | ID: covidwho-1275929

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has grown into a global pandemic, and only a few antiviral treatments have been approved to date. Angiotensin-converting enzyme 2 (ACE2) plays a fundamental role in SARS-CoV-2 pathogenesis because it allows viral entry into host cells. Here we show that ACE2 nanodecoys derived from human lung spheroid cells (LSCs) can bind and neutralize SARS-CoV-2 and protect the host lung cells from infection. In mice, these LSC-nanodecoys were delivered via inhalation therapy and resided in the lungs for over 72 h post-delivery. Furthermore, inhalation of the LSC-nanodecoys accelerated clearance of SARS-CoV-2 mimics from the lungs, with no observed toxicity. In cynomolgus macaques challenged with live SARS-CoV-2, four doses of these nanodecoys delivered by inhalation promoted viral clearance and reduced lung injury. Our results suggest that LSC-nanodecoys can serve as a potential therapeutic agent for treating COVID-19.


Subject(s)
COVID-19/drug therapy , Lung Injury/prevention & control , Nanostructures/administration & dosage , SARS-CoV-2/drug effects , Administration, Inhalation , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/virology , Cell-Derived Microparticles/metabolism , Cell-Derived Microparticles/transplantation , Disease Models, Animal , Humans , Lung Injury/virology , Macaca fascicularis , Mice , Protein Binding , SARS-CoV-2/metabolism , Spheroids, Cellular/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Viral Load/drug effects
20.
J Proteins Proteom ; 12(3): 161-175, 2021.
Article in English | MEDLINE | ID: covidwho-1267535

ABSTRACT

COVID-19, the current global pandemic has caused immense damage to human lives and the global economy. It is instigated by the SARS-CoV-2 virus and there is an immediate need for the identification of effective drugs against this deadly virus. SARS-CoV-2 genome codes for four structural proteins, sixteen non-structural proteins (NSPs) and several accessory proteins for its survival inside the host cells. In the present study, through in silico approaches, we aim to identify compounds that are effective against the four NSPs namely, NSP1, NSP4, NSP6 and NSP13 of SARS-CoV-2. The selection criteria of these four NSP proteins are they are least explored and potential targets. First, we have modeled the 3D structures of these proteins using homology modeling methods. Further, through molecular docking studies, we have screened the FDA-approved compounds against these modeled proteins and reported their docking scores. To gain dynamic insights, molecular dynamics studies have also been carried out for the best scored ligand against the NSPs. This study can further pave way for exposing more number of compounds against these proteins and enhance COVID-19 treatment. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42485-021-00067-w.

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