Search Results (21)

The recent coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has exerted considerable impact on global health. To prepare for rapidly mutating viruses and for the forthcoming pandemic, effective therapies targeting the critical stages of the viral life cycle need to be developed. Viruses are dependent on the interaction between the receptor-binding domain (RBD) of the viral Spike (S) protein (S-RBD) and the angiotensin-converting enzyme 2 (ACE2) receptor to efficiently establish infection and the following replicate. Targeting this interaction provides a promising strategy to inhibit the entry process of the virus, which in turn has both preventive and therapeutic effects. In this study, we developed a robust and straightforward assay based on the Yeast-Two Hybrid system (Y2H) for identifying inhibitors targeting the S-RBD-ACE2 interaction of SARS-CoV-2. Through high-throughput screening, two compounds were identified as potential entry inhibitors. Among them, IMB-1C was superior in terms of pseudovirus entry inhibition and toxicity. It could bind to both ACE2 and S-RBD and induce conformational change in the S-RBD+ACE2 complex. This is the first study to verify the feasibility of utilizing the Y2H system to discover potent SARS-CoV-2 inhibitors targeting the receptor recognition stage. This approach may also be applied in the discovery of other virus receptor recognition inhibitors. Full article

Protein–protein interactions (PPIs) are fundamental to many critical biological processes and are crucial in mediating essential cellular functions across diverse organisms, including bacteria, parasites, and viruses. A notable example is the interaction between the SARS-CoV-2 spike (S) protein and the human angiotensin-converting enzyme 2 (hACE2), which initiates a series of events leading to viral replication. Interrupting this interaction offers a promising strategy for blocking or significantly reducing infection, highlighting its potential as a target for anti-SARS-CoV-2 therapies. This review focuses on the hACE2 and SARS-CoV-2 spike protein interaction, exemplifying the latest advancements in peptide-based strategies for developing PPI inhibitors. We discuss various approaches for creating peptide-based inhibitors that target this critical interaction, aiming to provide potential treatments for COVID-19. Full article

Background: The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to viral entry and can cause cardiac injuries. Toll-like receptor 4 (TLR4) and NOD-, LPR-, and pyrin-domain-containing 3 (NLRP3) inflammasome are critical immune system components implicated in cardiac fibrosis. The spike protein activates NLRP3 inflammasome through TLR4 or angiotensin-converting enzyme 2 (ACE2) receptors, damaging various organs. However, the role of spike protein in cardiac fibrosis in humans, as well as its interactions with NLRP3 inflammasomes and TLR4, remain poorly understood. Methods: We utilized scratch assays, Western blotting, and immunofluorescence to evaluate the migration, fibrosis signaling, mitochondrial calcium levels, reactive oxygen species (ROS) production, and cell morphology of cultured human cardiac fibroblasts (CFs) treated with spike (S1) protein for 24 h with or without an anti-ACE2 neutralizing antibody, a TLR4 blocker, or an NLRP3 inhibitor. Results: S1 protein enhanced CFs migration and the expressions of collagen 1, α-smooth muscle actin, transforming growth factor β1 (TGF-β1), phosphorylated SMAD2/3, interleukin 1β (IL-1β), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). S1 protein increased ROS production but did not affect mitochondrial calcium content and cell morphology. Treatment with an anti-ACE2 neutralizing antibody attenuated the effects of S1 protein on collagen 1 and TGF-β1 expressions. Moreover, NLRP3 (MCC950) and NF-kB inhibitors, but not the TLR4 inhibitor TAK-242, prevented the S1 protein-enhanced CFs migration and overexpression of collagen 1, TGF-β1, and IL-1β. Conclusion: S1 protein activates human CFs by priming NLRP3 inflammasomes through NF-κB signaling in an ACE2-dependent manner. Full article

Since the outbreak of COVID-19, researchers have been working tirelessly to discover effective ways to combat coronavirus infection. The use of computational drug repurposing methods and molecular docking has been instrumental in identifying compounds that have the potential to disrupt the binding between the spike glycoprotein of SARS-CoV-2 and human ACE2 (hACE2). Moreover, the pseudovirus approach has emerged as a robust technique for investigating the mechanism of virus attachment to cellular receptors and for screening targeted small molecule drugs. Pseudoviruses are viral particles containing envelope proteins, which mediate the virus’s entry with the same efficiency as that of live viruses but lacking pathogenic genes. Therefore, they represent a safe alternative to screen potential drugs inhibiting viral entry, especially for highly pathogenic enveloped viruses. In this review, we have compiled a list of antiviral plant extracts and natural products that have been extensively studied against enveloped emerging and re-emerging viruses by pseudovirus technology. The review is organized into three parts: (1) construction of pseudoviruses based on different packaging systems and applications; (2) knowledge of emerging and re-emerging viruses; (3) natural products active against pseudovirus-mediated entry. One of the most crucial stages in the life cycle of a virus is its penetration into host cells. Therefore, the discovery of viral entry inhibitors represents a promising therapeutic option in fighting against emerging viruses. Full article

Blocking the interaction between the SARS-CoV-2 spike protein and the human angiotensin-converting enzyme II (hACE2) protein serves as a therapeutic strategy for treating COVID-19. Traditional Chinese medicine (TCM) treatments containing bioactive products could alleviate the symptoms of severe COVID-19. However, the emergence of SARS-CoV-2 variants has complicated the process of developing broad-spectrum drugs. As such, the aim of this study was to explore the efficacy of TCM treatments against SARS-CoV-2 variants through targeting the interaction of the viral spike protein with the hACE2 receptor. Antiviral activity was systematically evaluated using a pseudovirus system. Scutellaria baicalensis (S. baicalensis) was found to be effective against SARS-CoV-2 infection, as it mediated the interaction between the viral spike protein and the hACE2 protein. Moreover, the active molecules of S. baicalensis were identified and analyzed. Baicalein and baicalin, a flavone and a flavone glycoside found in S. baicalensis, respectively, exhibited strong inhibitory activities targeting the viral spike protein and the hACE2 protein, respectively. Under optimized conditions, virus infection was inhibited by 98% via baicalein-treated pseudovirus and baicalin-treated hACE2. In summary, we identified the potential SARS-CoV-2 inhibitors from S. baicalensis that mediate the interaction between the Omicron spike protein and the hACE2 receptor. Future studies on the therapeutic application of baicalein and baicalin against SARS-CoV-2 variants are needed. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus responsible for the coronavirus disease 2019 (COVID-19) pandemic, represents a serious threat to public health. The spike (S) glycoprotein of SARS-CoV-2 mediates viral entry into host cells and is heavily glycosylated. In this study, we systemically analyzed the roles of 22 putative N-linked glycans in SARS-CoV-2 S protein expression, membrane fusion, viral entry, and stability. Using the α-glycosidase inhibitors castanospermine and NB-DNJ, we confirmed that disruption of N-linked glycosylation blocked the maturation of the S protein, leading to the impairment of S protein-mediated membrane fusion. Single-amino-acid substitution of each of the 22 N-linked glycosylation sites with glutamine revealed that 9 out of the 22 N-linked glycosylation sites were critical for S protein folding and maturation. Thus, substitution at these sites resulted in reduced S protein-mediated cell–cell fusion and viral entry. Notably, the N1074Q mutation markedly affected S protein stability and induced significant receptor-independent syncytium (RIS) formation in HEK293T/hACE2-KO cells. Additionally, the removal of the furin cleavage site partially compensated for the instability induced by the N1074Q mutation. Although the corresponding mutation in the SARS-CoV S protein (N1056Q) did not induce RIS in HEK293T cells, the N669Q and N1080Q mutants exhibited increased fusogenic activity and did induce syncytium formation in HEK293T cells. Therefore, N-glycans on the SARS-CoV and SARS-CoV-2 S2 subunits are highly important for maintaining the pre-fusion state of the S protein. This study revealed the critical roles of N-glycans in S protein maturation and stability, information that has implications for the design of vaccines and antiviral strategies. Full article

Although COVID-19 transmission has been reduced by the advent of vaccinations and a variety of rapid monitoring techniques, the SARS-CoV-2 virus itself has shown a remarkable ability to mutate and persist. With this long track record of immune escape, researchers are still exploring prophylactic treatments to curtail future SARS-CoV-2 variants. Specifically, much focus has been placed on the antiviral lectin Griffithsin in preventing spike protein-mediated infection via the hACE2 receptor (direct infection). However, an oft-overlooked aspect of SARS-CoV-2 infection is viral capture by attachment receptors such as DC-SIGN, which is thought to facilitate the initial stages of COVID-19 infection in the lung tissue (called trans-infection). In addition, while immune escape is dictated by mutations in the spike protein, coronaviral virions also incorporate M, N, and E structural proteins within the particle. In this paper, we explored how several structural facets of both the SARS-CoV-2 virion and the antiviral lectin Griffithsin can affect and attenuate the infectivity of SARS-CoV-2 pseudovirus. We found that Griffithsin was a better inhibitor of hACE2-mediated direct infection when the coronaviral M protein is present compared to when it is absent (possibly providing an explanation regarding why Griffithsin shows better inhibition against authentic SARS-CoV-2 as opposed to pseudotyped viruses, which generally do not contain M) and that Griffithsin was not an effective inhibitor of DC-SIGN-mediated trans-infection. Furthermore, we found that DC-SIGN appeared to mediate trans-infection exclusively via binding to the SARS-CoV-2 spike protein, with no significant effect observed when other viral proteins (M, N, and/or E) were present. These results provide etiological data that may help to direct the development of novel antiviral treatments, either by leveraging Griffithsin binding to the M protein as a novel strategy to prevent SARS-CoV-2 infection or by narrowing efforts to inhibit trans-infection to focus on DC-SIGN binding to SARS-CoV-2 spike protein. Full article

SARS-CoV-2 exploits the homotrimer transmembrane Spike glycoproteins (S protein) during host cell invasion. The Omicron XBB subvariant, delta, and prototype SARS-CoV-2 receptor-binding domain show similar binding strength to hACE2 (human Angiotensin-Converting Enzyme 2). Here we utilized multiligand virtual screening to identify small molecule inhibitors for their efficacy against SARS-CoV-2 virus using QPLD, pseudovirus ACE2 Inhibition -Time Resolved Forster/Fluorescence energy transfer (TR-FRET) Assay Screening, and Molecular Dynamics simulations (MDS). Three hundred and fifty thousand compounds were screened against the macrodomain of the nonstructural protein 3 of SARS-CoV-2. Using TR-FRET Assay, we filtered out two of 10 compounds that had no reported activity in in vitro screen against Spike S1: ACE2 binding assay. The percentage inhibition at 30 µM was found to be 79% for “Compound F1877-0839” and 69% for “Compound F0470-0003”. This first of its kind study identified “FILLY” pocket in macrodomains. Our 200 ns MDS revealed stable binding poses of both leads. They can be used for further development of preclinical candidates. Full article

Purslane (Portulaca oleracea L.) is rich in phenolic compounds, protein, and iron. This study aims to produce functional yogurt with enhanced antioxidant, anticancer, antiviral, and antimicrobial properties by including safe purslane extract in yogurt formulation; the yogurt was preserved for 30 days at 4 °C, and then biochemical fluctuations were monitored. The purslane extract (PuE) had high phenolic compounds and flavonoids of 250 and 56 mg/mL, respectively. Therefore, PuE had considerable antioxidant activity, which scavenged 93% of DPPH˙, inhibited the viability of MCF-7, HCT, and HeLa cell lines by 84, 82, and 80%, respectively, and inhibited 82% of the interaction between the binding between Spike and ACE2 compared to a SARS-CoV-2 inhibitor test kit. PuE (20–40 µg/mL) inhibited the growth of tested pathogenic bacteria and Candida strains, these strains isolated from spoild yogurt and identified at gene level by PCR. Caffeic acid glucoside and catechin were the main phenolic compounds in the HPLC profile, while the main flavor compound was carvone and limonene, representing 71% of total volatile compounds (VOCs). PuE was added to rats’ diets at three levels (50, 150, and 250 µg/g) compared to butylated hydroxyanisole (BHA). The body weight of the rats fed the PuE diet (250 µg/g) increased 13% more than the control. Dietary PuE in rats’ diets lowered the levels of low-density lipoprotein (LDL) levels by 72% and increased the levels of high-density lipoprotein (HDL) by 36%. Additionally, liver parameters in rats fed PuE (150 µg/g) decreased aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA) levels by 50, 43, and 25%, respectively, while TP, TA, and GSH were increased by 20, 50, and 40%, respectively, compared to BHA. Additionally, PuE acts as a kidney protector by lowering creatinine and urea. PuE was added to yogurt at three concentrations (50, 150, and 250 µg/g) and preserved for 30 days compared to the control. The yogurt’s pH reduced during storage while acidity, TSS, and fat content increased. Adding PuE increased the yogurt’s water-holding capacity, so syneresis decreased and viscosity increased, which was attributed to enhancing the texture properties (firmness, consistency, and adhesiveness). MDA decreased in PuE yogurt because of the antioxidant properties gained by PuE. Additionally, color parameters L and b were enhanced by PuE additions and sensorial traits, i.e., color, flavor, sugary taste, and texture were enhanced by purslane extract compared to the control yogurt. Concerning the microbial content in the yogurt, the lactic acid bacteria (LAB) count was maintained as a control. Adding PuE at concentrations of 50, 150, and 250 µg/g to the yogurt formulation can enhance the quality of yogurt. Full article

Here, we present evidence that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. Firstly, we found that, while Angiotensin-converting enzyme 2 (ACE2) is expressed in these cells, blocking ACE2 by antibody treatment did not prevent infection by SARS-CoV-2 spike pseudovirions, nor did antibody blockade of extracellular vimentin and other cholesterol-rich lipid raft proteins. Next, we implicated the role of cholesterol homeostasis in infection by showing that incubating cells with different cyclodextrins and oxysterol 25-hydroxycholesterol (25-HC) inhibits pseudovirion infection of ARPE-19. However, the effect of 25-HC is likely not via cholesterol biosynthesis, as incubation with lovastatin did not appreciably affect infection. Additionally, is it not likely to be an agonistic effect of 25-HC on LXR receptors, as the LXR agonist GW3965 had no significant effect on infection of ARPE-19 cells at up to 5 μM GW3965. We probed the role of endocytic pathways but determined that clathrin-dependent and flotillin-dependent rafts were not involved. Furthermore, 20 µM chlorpromazine, an inhibitor of clathrin-mediated endocytosis (CME), also had little effect. In contrast, anti-dynamin I/II antibodies blocked the entry of SARS-CoV-2 spike pseudovirions, as did dynasore, a noncompetitive inhibitor of dynamin GTPase activity. Additionally, anti-caveolin-1 antibodies significantly blocked spike pseudotyped lentiviral infection of ARPE-19. However, nystatin, a classic inhibitor of caveolae-dependent endocytosis, did not affect infection while indomethacin inhibited only at 10 µM at the 48 h time point. Finally, we found that anti-LDLR antibodies block pseudovirion infection to a similar degree as anti-caveolin-1 and anti-dynamin I/II antibodies, while transfection with LDLR-specific siRNA led to a decrease in spike pseudotyped lentiviral infection, compared to scrambled control siRNAs. Thus, we conclude that SARS-CoV-2 spike pseudovirion infection in ARPE-19 cells is a dynamin-dependent process that is primarily mediated by LDLR. Full article

The pandemic outbreak of human coronavirus is a global health concern that affects people of all ages and genders, but there is currently still no effective, approved and potential drug against human coronavirus, as many other coronavirus vaccines have serious side effects while the development of small antiviral inhibitors has gained tremendous attention. For this research, HE was used as a therapeutic target, as the spike protein displays a high binding affinity for both host ACE2 and viral HE glycoprotein. Molecular docking, pharmacophore modelling and virtual screening of 38,000 natural compounds were employed to find out the best natural inhibitor against human coronaviruses with more efficiency and fewer side effects and further evaluated via MD simulation, PCA, DCCR and MMGBSA. The lead compound ‘Calceolarioside B’ was identified on the basis of pharmacophoric features which depict favorable binding (ΔGbind −37.6799 kcal/mol) with the HE(5N11) receptor that describes positive correlation movements in active site residues with better stability, a robust H-bond network, compactness and reliable ADMET properties. The Fraxinus sieboldiana Blume plant containing the Calceolarioside B compound could be used as a potential inhibitor that shows a higher efficacy and potency with fewer side effects. This research work will aid investigators in the testing and identification of chemicals that are effective and useful against human coronavirus. Full article

It is known that the spike protein of human coronaviruses can bind to a secondary receptor, or coreceptor, to facilitate the virus entry. While HCoV-229E uses human aminopeptidase N (hAPN) as a receptor, HCoV-OC43 binds to 9-O-acetyl-sialic acid (9-O-Ac-Sia), which is linked in a terminal way to the oligosaccharides that decorate glycoproteins and gangliosides on the surface of the host cell. Thus, evaluating the possible inhibitory activity of heparan sulfate, a linear polysaccharide found in animal tissues, and enoxaparin sodium on these viral strains can be considered attractive. Therefore, our study also aims to evaluate these molecules’ antiviral activity as possible adsorption inhibitors against non-SARS-CoV. Once the molecules’ activity was verified in in vitro experiments, the binding was studied by molecular docking and molecular dynamic simulations confirming the interactions at the interface of the spike proteins. Full article

The emergence of the recent pandemic causing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created an alarming situation worldwide. It also prompted extensive research on drug repurposing to find a potential treatment for SARS-CoV-2 infection. An active metabolite of the hyperlipidemic drug fenofibrate (also called fenofibric acid or FA) was found to destabilize the receptor-binding domain (RBD) of the viral spike protein and therefore inhibit its binding to human angiotensin-converting enzyme 2 (hACE2) receptor. Despite being considered as a potential drug candidate for SARS-CoV-2, FA’s inhibitory mechanism remains to be elucidated. We used molecular dynamics (MD) simulations to investigate the binding of FA to the RBD of the SARS-CoV-2 spike protein and revealed a potential cryptic FA binding site. Free energy calculations were performed for different FA-bound RBD complexes. The results suggest that the interaction of FA with the cryptic binding site of RBD alters the conformation of the binding loop of RBD and effectively reduces its binding affinity towards ACE2. Our study provides new insights for the design of SARS-CoV-2 inhibitors targeting cryptic sites on the RBD of SARS-CoV-2. Full article

The spike protein is key to SARS-CoV-2 high infectivity because it facilitates the receptor binding domain (RBD) encounter with ACE2. As targeting subunit S1 has not yet delivered an ACE2-binding inhibitor, we have assessed the druggability of the conserved segment of the spike protein stalk within subunit S2 by means of an integrated computational approach that combines the molecular docking of an optimized library of fragments with high-throughput molecular dynamics simulations. The high propensity of the spike protein to mutate in key regions that are responsible for the recognition of the human angiotensin-converting enzyme 2 (hACE2) or for the recognition of antibodies, has made subunit S1 of the spike protein difficult to target. Despite the inherent flexibility of the stalk region, our results suggest two hidden interhelical binding sites, whose accessibility is only partially hampered by glycan residues. Full article

Therapeutically useful small-molecule inhibitors (SMIs) of protein–protein interactions (PPIs) initiating the cell attachment and entry of viruses could provide novel alternative antivirals that act via mechanisms similar to that of neutralizing antibodies but retain the advantages of small-molecule drugs such as oral bioavailability and low likelihood of immunogenicity. From screening our library, which is focused around the chemical space of organic dyes to provide good protein binders, we have identified several promising SMIs of the SARS-CoV-2 spike—ACE2 interaction, which is needed for the attachment and cell entry of this coronavirus behind the COVID-19 pandemic. They included organic dyes, such as Congo red, direct violet 1, and Evans blue, which seem to be promiscuous PPI inhibitors, as well as novel drug-like compounds (e.g., DRI-C23041). Here, we show that in addition to the original SARS-CoV-2 strain, these SMIs also inhibit this PPI for variants of concern including delta (B.1.617.2) and omicron (B.1.1.529) as well as HCoV-NL63 with low- or even sub-micromolar activity. They also concentration-dependently inhibited SARS-CoV-2-S expressing pseudovirus entry into hACE2-expressing cells with low micromolar activity (IC₅₀ < 10 μM) both for the original strain and the delta variant. DRI-C23041 showed good therapeutic (selectivity) index, i.e., separation between activity and cytotoxicity (TI > 100). Specificities and activities require further optimization; nevertheless, these results provide a promising starting point toward novel broad-spectrum small-molecule antivirals that act via blocking the interaction between the spike proteins of coronaviruses and their ACE2 receptor initiating cellular entry. Full article