Screening of inhibitors against SARS-CoV-2 spike protein and their capability to block the viral entry mechanism: A viroinformatics study

SARS-CoV-2, previously named 2019 novel coronavirus (2019-nCoV), has been associated with the global pandemic of acute respiratory distress syndrome. First reported in December 2019 in the Wuhan province of China, this new RNA virus has several folds higher transmission among humans than its other family member (SARS-CoV and MERS-CoV). The SARS-CoV-2 spike receptor-binding domain (RBD) is the region mediating the binding of the virus to host cells via Angiotensin-converting enzyme 2 (ACE2), a critical step of viral. Here in this study, we have utilized in silico approach for the virtual screening of antiviral library extracted from the Asinex database against the Receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike glycoprotein. Further, the molecules were ranked based on their binding affinity against RBD, and the top 15 molecules were selected. The affinity of these selected molecules to interrupt the ACE2-Spike interaction was also studied. It was found that the chosen molecules were demonstrating excellent binding affinity against spike protein, and these molecules were also very effectively interrupting the ACE2-RBD interaction.Furthermore, molecular dynamics (MD) simulation studies were utilized to investigate the top 3 selected molecules' stability in the ACE2-RBD complexes. To the best of our knowledge, this is the first study where molecules' inhibitory potential against the Receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike glycoprotein and their inhibitory potential against the ACE2-Spike has been studied. We believe that these compounds can be further tested as a potential therapeutic option against COVID-19.     

Therapeutic development by repurposing drugs targeting SARS-CoV-2 spike protein interactions by simulation studies

The human-to-human transmitted respiratory illness in COVID-19 affected by the pathogenic Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2), which appeared in the last of December 2019 in Wuhan, China, and rapidly spread in many countries. Thereon, based on the urgent need for therapeutic molecules, we conducted in silico based docking and simulation molecular interaction studies on repurposing drugs, targeting SARS-CoV-2 spike protein. Further, the best binding energy of doxorubicin interacting with virus spike protein (PDB: 6VYB) was observed to be −6.38 kcal/mol and it was followed by exemestane and gatifloxacin. The molecular simulation dynamics analysis of doxorubicin, Reference Mean Square Deviation (RMSD), Root Mean Square fluctuation (RMSF), Radius of Gyration (Rg), and formation of hydrogen bonds plot interpretation suggested, a significant deviation and fluctuation of Doxorubicin-Spike RBD complex during the whole simulation period. The Rg analysis has stated that the Doxorubicin-Spike RBD complex was stable during 15,000–35,000 ps MDS. The results have suggested that doxorubicin could inhibit the virus spike protein and prevent the access of the SARS-CoV-2 to the host cell. Thus, in-vitro/in-vivo research on these drugs could be advantageous to evaluate significant molecules that control the COVID-19 disease.     

Mechanism and evolution of human ACE2 binding by SARS-CoV-2 spike

Spike glycoprotein of SARS-CoV-2 mediates viral entry into host cells by facilitating virus attachment and membrane fusion. ACE2 is the main receptor of SARS-CoV-2 and its interaction with spike has shaped the virus’ emergence from an animal reservoir and subsequent evolution in the human host. Many structural studies on the spike:ACE2 interaction have provided insights into mechanisms driving viral evolution during the on-going pandemic. This review describes the molecular basis of spike binding to ACE2, outlines mechanisms that have optimised this interaction during viral evolution, and suggests directions for future research.     

High-resolution melting analysis to discriminate between the SARS-CoV-2 Omicron variants BA.1 and BA.2

High-resolution melting (HRM) analysis was conducted to discriminate between SARS-CoV-2 Omicron variant BA.1 (B.1.1.529.1) and subvariant BA.2 (B.1.1.529.2). We performed two-step PCR consisting of the first PCR and the second nested PCR to prepare the amplicon for HRM analysis, which detected G339D, N440K, G446S and D796Y variations in the SARS-CoV-2 spike protein. The melting temperatures (Tms) of the amplicons from the cDNA of the Omicron variant BA.1 and subvariant BA.2 receptor binding domain (RBD) in spike protein were the same: 75.2 °C (G339D variation) and 73.4 °C (D796Y variation). These Tms were distinct from those of SARS-CoV-2 isolate Wuhan-Hu-1, and were specific to the Omicron variant. In HRM analyses that detected the N440K and G446S variations, the Tms of amplicons from the cDNA of the Omicron variant BA.1 and subvariant BA.2 RBDs were 73.0 °C (N440K and G446S variations) and 73.5 °C (G446S variation). This difference indicates that the SARS-CoV-2 Omicron variants BA.1 and BA.2 can be clearly discriminated. Our study demonstrates the usefulness of HRM analysis after two-step PCR for the discrimination of SARS-CoV-2 variants.     

FASN inhibitor TVB-3166 prevents S-acylation of the spike protein of human coronaviruses

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses mediates host cell entry and is S-acylated on multiple phylogenetically conserved cysteine residues. Multiple protein acyltransferase enzymes have been reported to post-translationally modify spike proteins; however, strategies to exploit this modification are lacking. Using resin-assisted capture MS, we demonstrate that the spike protein is S-acylated in SARS-CoV-2-infected human and monkey epithelial cells. We further show that increased abundance of the acyltransferase ZDHHC5 associates with increased S-acylation of the spike protein, whereas ZDHHC5 knockout cells had a 40% reduction in the incorporation of an alkynyl-palmitate using click chemistry detection. We also found that the S-acylation of the spike protein is not limited to palmitate, as clickable versions of myristate and stearate were also labelled the protein. Yet, we observed that ZDHHC5 was only modified when incubated with alkyne-palmitate, suggesting it has specificity for this acyl-CoA, and that other ZDHHC enzymes may use additional fatty acids to modify the spike protein. Since multiple ZDHHC isoforms may modify the spike protein, we also examined the ability of the FASN inhibitor TVB-3166 to prevent S-acylation of the spike proteins of SARS-CoV-2 and human CoV-229E. We show that treating cells with TVB-3166 inhibited S-acylation of expressed spike proteins and attenuated the ability of SARS-CoV-2 and human CoV-229E to spread in vitro. Our findings further substantiate the necessity of CoV spike protein S-acylation and demonstrate that de novo fatty acid synthesis is critical for the proper S-acylation of the spike protein.     

Identification of SARS-CoV-2 RNA-dependent RNA polymerase inhibitors from the major phytochemicals of Nigella sativa: An in silico approach

The coronavirus disease 2019 (COVID-19), which emerged in December 2019, continues to be a serious health concern worldwide. There is an urgent need to develop effective drugs and vaccines to control the spread of this disease. In the current study, the main phytochemical compounds of Nigella sativa were screened for their binding affinity for the active site of the RNA-dependent RNA polymerase (RdRp) enzyme of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The binding affinity was investigated using molecular docking methods, and the interaction of phytochemicals with the RdRp active site was analyzed and visualized using suitable software. Out of the nine phytochemicals of N. sativa screened in this study, a significant docking score was observed for four compounds, namely α-hederin, dithymoquinone, nigellicine, and nigellidine. Based on the findings of our study, we report that α-hederin, which was found to possess the lowest binding energy (–8.6 kcal/mol) and hence the best binding affinity, is the best inhibitor of RdRp of SARS-CoV-2, among all the compounds screened here. Our results prove that the top four potential phytochemical molecules of N. sativa, especially α-hederin, could be considered for ongoing drug development strategies against SARS-CoV-2. However, further in vitro and in vivo testing are required to confirm the findings of this study.     

In silico identification of potential inhibitors against main protease of SARS-CoV-2 6LU7 from Andrographis panniculata via molecular docking,binding energy calculations and molecular dynamics simulation studies

BackgroundThe ongoing global outbreak of new corona virus (SARS-CoV-2) has been recognized as global public health concern since it causes high morbidity and mortality every day. Due to the rapid spreading and re-emerging, we need to find a potent drug against SARS-CoV-2. Synthetic drugs, such as hydroxychloroquine, remdisivir have paid more attention and the effects of these drugs are still under investigation, due to their severe side effects. Therefore, the aim of the present study was performed to identify the potential inhibitor against main protease SARS-CoV-2 6LU7.ObjectiveIn this study, RO5, ADME properties, molecular dynamic simulations and free binding energy prediction were mainly investigated.ResultsThe molecular docking study findings revealed that andrographolide had higher binding affinity among the selected natural diterpenoids compared to co-crystal native ligand inhibitor N3. The persistent inhibition of Ki for diterpenoids was analogous. Furthermore, the simulations of molecular dynamics and free binding energy findings have shown that andrographolide possesses a large amount of dynamic properties such as stability, flexibility and binding energy.ConclusionIn conclusion, findings of the current study suggest that selected diterpenoids were predicted to be the significant phytonutrient-based inhibitor against SARS-CoV-2 6LU7 (M^pro). However, preclinical and clinical trials are needed for the further scientific validation before use.     

Where all the Roads Meet? A Crossover Perspective on Host Factors Regulating SARS-CoV-2 infection

COVID-19 caused by SARS-CoV-2 is the latest pandemic which has thrown the world into an unprecedented social and economic uncertainties along with huge loss to humanity. Identification of the host factors regulating the replication of SARS-CoV-2 in human host may help in the development of novel anti-viral therapies to combat the viral infection and spread. Recently, some research groups used genome-wide CRISPR/Cas screening to identify the host factors critical for the SARS-CoV-2 replication and infection. A comparative analysis of these significant host factors (p < 0.05) identified fifteen proteins common in these studies. Apart from ACE2 (receptor for SARS-CoV-2 attachment), other common host factors were CSNK2B, GDI2, SLC35B2, DDX51, VPS26A, ARPP-19, C1QTNF7, ALG6, LIMA1, COG3, COG8, BCOR, LRRN2 and TLR9. Additionally, viral interactome of these host factors revealed that many of them were associated with several SARS-CoV-2 proteins as well. Interestingly, some of these host factors have already been shown to be critical for the pathogenesis of other viruses suggesting their crucial role in virus-host interactions. Here, we review the functions of these host factors and their role in other diseases with special emphasis on viral diseases.     

Structural Modeling of the SARS-CoV-2 Spike/Human ACE2 Complex Interface can Identify High-Affinity Variants Associated with Increased Transmissibility

The COVID-19 pandemic has triggered concerns about the emergence of more infectious and pathogenic viral strains. As a public health measure, efficient screening methods are needed to determine the functional effects of new sequence variants. Here we show that structural modeling of SARS-CoV-2 Spike protein binding to the human ACE2 receptor, the first step in host-cell entry, predicts many novel variant combinations with enhanced binding affinities. By focusing on natural variants at the Spike-hACE2 interface and assessing over 700 mutant complexes, our analysis reveals that high-affinity Spike mutations (including N440K, S443A, G476S, E484R, G502P) tend to cluster near known human ACE2 recognition sites (K31 and K353). These Spike regions are structurally flexible, allowing certain mutations to optimize interface interaction energies. Although most human ACE2 variants tend to weaken binding affinity, they can interact with Spike mutations to generate high-affinity double mutant complexes, suggesting variation in individual susceptibility to infection. Applying structural analysis to highly transmissible variants, we find that circulating point mutations S477N, E484K and N501Y form high-affinity complexes (~40% more than wild-type). By combining predicted affinities and available antibody escape data, we show that fast-spreading viral variants exploit combinatorial mutations possessing both enhanced affinity and antibody resistance, including S477N/E484K, E484K/N501Y and K417T/E484K/N501Y. Thus, three-dimensional modeling of the Spike/hACE2 complex predicts changes in structure and binding affinity that correlate with transmissibility and therefore can help inform future intervention strategies.     

Open Modification Searching of SARS-CoV-2–Human Protein Interaction Data Reveals Novel Viral Modification Sites

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus 2019 disease, has led to an ongoing global pandemic since 2019. Mass spectrometry can be used to understand the molecular mechanisms of viral infection by SARS-CoV-2, for example, by determining virus–host protein–protein interactions through which SARS-CoV-2 hijacks its human hosts during infection, and to study the role of post-translational modifications. We have reanalyzed public affinity purification–mass spectrometry data using open modification searching to investigate the presence of post-translational modifications in the context of the SARS-CoV-2 virus–host protein–protein interaction network. Based on an over twofold increase in identified spectra, our detected protein interactions show a high overlap with independent mass spectrometry-based SARS-CoV-2 studies and virus–host interactions for alternative viruses, as well as previously unknown protein interactions. In addition, we identified several novel modification sites on SARS-CoV-2 proteins that we investigated in relation to their interactions with host proteins. A detailed analysis of relevant modifications, including phosphorylation, ubiquitination, and S-nitrosylation, provides important hypotheses about the functional role of these modifications during viral infection by SARS-CoV-2.     

Binding Adaptation of GS-441524 Diversifies Macro Domains and Downregulates SARS-CoV-2 de-MARylation Capacity

Viral infection in cells triggers a cascade of molecular defense mechanisms to maintain host-cell homoeostasis. One of these mechanisms is ADP-ribosylation, a fundamental post-translational modification (PTM) characterized by the addition of ADP-ribose (ADPr) on substrates. Poly(ADP-ribose) polymerases (PARPs) are implicated in this process and they perform ADP-ribosylation on host and pathogen proteins. Some viral families contain structural motifs that can reverse this PTM. These motifs known as macro domains (MDs) are evolutionarily conserved protein domains found in all kingdoms of life. They are divided in different classes with the viral belonging to Macro-D-type class because of their properties to recognize and revert the ADP-ribosylation. Viral MDs are potential pharmaceutical targets, capable to counteract host immune response. Sequence and structural homology between viral and human MDs are an impediment for the development of new active compounds against their function. Remdesivir, is a drug administrated in viral infections inhibiting viral replication through RNA-dependent RNA polymerase (RdRp). Herein, GS-441524, the active metabolite of the remdesivir, is tested as a hydrolase inhibitor for several viral MDs and for its binding to human homologs found in PARPs. This study presents biochemical and biophysical studies, which indicate that GS-441524 selectively modifies SARS-CoV-2 MD de-MARylation activity, while it does not interact with hPARP14 MD2 and hPARP15 MD2. The structural investigation of MD?GS-441524 complexes, using solution NMR and X-ray crystallography, discloses the impact of certain amino acids in ADPr binding cavity suggesting that F360 and its adjacent residues tune the selective binding of the inhibitor to SARS-CoV-2 MD.     

基于新型冠状病毒S蛋白结构及入胞机制的抗体与药物研发

新型冠状病毒肺炎,世界卫生组织命名为"2019冠状病毒病"(corona virus disease 2019,COVID-19),是一种由2019新型冠状病毒(2019-nCov)感染导致的肺炎.目前新冠肺炎在全球广泛流行,且疫情尚未得到全部控制.由于新型冠状病毒表面的刺突蛋白(spike protein,S)介导病...     

SARS-CoV-2 Fusion Peptide has a Greater Membrane Perturbating Effect than SARS-CoV with Highly Specific Dependence on Ca2+

Coronaviruses are a major infectious disease threat, and include the zoonotic-origin human pathogens SARS-CoV-2, SARS-CoV, and MERS-CoV (SARS-2, SARS-1, and MERS). Entry of coronaviruses into host cells is mediated by the spike (S) protein. In our previous ESR studies, the local membrane ordering effect of the fusion peptide (FP) of various viral glycoproteins including the S of SARS-1 and MERS has been consistently observed. We previously determined that the sequence immediately downstream from the S2′ cleavage site is the bona fide SARS-1 FP. In this study, we used sequence alignment to identify the SARS-2 FP, and studied its membrane ordering effect. Although there are only three residue differences, SARS-2 FP induces even greater membrane ordering than SARS-1 FP, possibly due to its greater hydrophobicity. This may be a reason that SARS-2 is better able to infect host cells. In addition, the membrane binding enthalpy for SARS-2 is greater. Both the membrane ordering of SARS-2 and SARS-1 FPs are dependent on Ca²⁺, but that of SARS-2 shows a greater response to the presence of Ca²⁺. Both FPs bind two Ca²⁺ ions as does SARS-1 FP, but the two Ca²⁺ binding sites of SARS-2 exhibit greater cooperativity. This Ca²⁺ dependence by the SARS-2 FP is very ion-specific. These results show that Ca²⁺ is an important regulator that interacts with the SARS-2 FP and thus plays a significant role in SARS-2 viral entry. This could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca²⁺ channel.     

Emergence,evolution, and vaccine production approaches of SARS-CoV-2 virus: Benefits of getting vaccinated and common questions

The emergence of coronavirus disease 2019 (COVID-19) pandemic in Wuhan city, China at the end of 2019 made it urgent to identify the origin of the causal pathogen and its molecular evolution, to appropriately design an effective vaccine. This study analyzes the evolutionary background of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or SARS-2) in accordance with its close relative SARS-CoV (SARS-1), which was emerged in 2002. A comparative genomic and proteomic study was conducted on SARS-2, SARS-1, and Middle East respiratory syndrome coronavirus (MERS), which was emerged in 2012. In silico analysis inferred the genetic variability among the tested viruses. The SARS-1 genome harbored 11 genes encoding 12 proteins, while SARS-2 genome contained only 10 genes encoding for 10 proteins. MERS genome contained 11 genes encoding 11 proteins. The analysis also revealed a slight variation in the whole genome size of SARS-2 comparing to its siblings resulting from sequential insertions and deletions (indels) throughout the viral genome particularly ORF1AB, spike, ORF10 and ORF8. The effective indels were observed in the gene encoding the spike protein that is responsible for viral attachment to the angiotensin-converting enzyme 2 (ACE2) cell receptor and initiating infection. These indels are responsible for the newly emerging COVID-19 variants αCoV, βCoV, γCoV and δCoV. Nowadays, few effective COVID-19 vaccines developed based on spike (S) glycoprotein were approved and become available worldwide. Currently available vaccines can relatively prevent the spread of COVID-19 and suppress the disease. The traditional (killed or attenuated virus vaccine and antibody-based vaccine) and innovated vaccine production technologies (RNA- and DNA-based vaccines and viral vectors) are summarized in this review. We finally highlight the most common questions related to COVID-19 disease and the benefits of getting vaccinated.     

Iota-carrageenan extracted from red algae is a potent inhibitor of SARS‐CoV-2 infection in reconstituted human airway epithelia

Iota-carrageenan (IC) nasal spray, a medical device approved for treating respiratory viral infections, has previously been shown to inhibit the ability of a variety of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), to enter and replicate in the cell by interfering with the virus binding to the cell surface. The aim of this study was to further investigate the efficacy and safety of IC in SARS-CoV-2 infection in advanced in vitro models of the human respiratory epithelium, the primary target and entry port for SARS-CoV-2. We extended the in vitro safety assessment of nebulized IC in a 3-dimensional model of reconstituted human bronchial epithelium, and we demonstrated the efficacy of IC in protecting reconstituted nasal epithelium against viral infection and replication of a patient-derived SARS-CoV-2 strain. The results obtained from these two advanced models of human respiratory tract epithelia confirm previous findings from in vitro SARS-CoV-2 infection assays and demonstrate that topically applied IC can effectively prevent SARS-CoV-2 infection and replication. Moreover, the absence of toxicity and functional and structural impairment of the mucociliary epithelium demonstrates that the nebulized IC is well tolerated.     

Variations within the Glycan Shield of SARS-CoV-2 Impact Viral Spike Dynamics

The emergence of SARS-CoV-2 variants alters the efficacy of existing immunity, whether arisen naturally or through vaccination. Understanding the structure of the viral spike assists in determining the impact of mutations on the antigenic surface. One class of mutation impacts glycosylation attachment sites, which have the capacity to influence the antigenic structure beyond the immediate site of attachment. Here, we compare the site-specific glycosylation of recombinant viral spike mimetics of B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), B.1.1.529 (Omicron). The P.1 strain exhibits two additional N-linked glycan sites compared to the other variants analyzed and we investigate the impact of these glycans by molecular dynamics. The acquired N188 site is shown to exhibit very limited glycan maturation, consistent with limited enzyme accessibility. Structural modeling and molecular dynamics reveal that N188 is located within a cavity by the receptor binding domain, which influences the dynamics of these attachment domains. These observations suggest a mechanism whereby mutations affecting viral glycosylation sites have a structural impact across the protein surface.     

Repurposing of approved drugs with potential to interact with SARS-CoV-2 receptor

Respiratory transmission is the primary route of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Angiotensin I converting enzyme 2 (ACE2) is the known receptor of SARS-CoV-2 surface spike glycoprotein for entry into human cells. A recent study reported absent to low expression of ACE2 in a variety of human lung epithelial cell samples. Three bioprojects (PRJEB4337, PRJNA270632 and PRJNA280600) invariably found abundant expression of ACE1 (a homolog of ACE2 and also known as ACE) in human lungs compared to very low expression of ACE2. In fact, ACE1 has a wider and more abundant tissue distribution compared to ACE2. Although it is not obvious from the primary sequence alignment of ACE1 and ACE2, comparison of X-ray crystallographic structures show striking similarities in the regions of the peptidase domains (PD) of these proteins, which is known (for ACE2) to interact with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Critical amino acids in ACE2 that mediate interaction with the viral spike protein are present and organized in the same order in the PD of ACE1. In silico analysis predicts comparable interaction of SARS-CoV-2 spike protein with ACE1 and ACE2. In addition, this study predicts from a list of 1263 already approved drugs that may interact with ACE2 and/or ACE1 and potentially interfere with the entry of SARS-CoV-2 inside the host cells.     

RAAS,ACE2 and COVID-19; a mechanistic review

The use of angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) in coronavirus disease 2019 (COVID-19) patients has been claimed as associated with the risk of COVID-19 infection and its subsequent morbidities and mortalities. These claims were resulting from the possibility of upregulating the expression of angiotensin-converting enzyme 2 (ACE2), facilitation of SARS-CoV-2 entry, and increasing the susceptibility of infection in such treated cardiovascular patients. ACE2 and renin-angiotensin-aldosterone system (RAAS) products have a critical function in controlling the severity of lung injury, fibrosis, and failure following the initiation of the disease. This review is to clarify the mechanisms beyond the possible deleterious effects of angiotensin II (Ang II), and the potential protective role of angiotensin 1–7 (Ang 1–7) against pulmonary fibrosis, with a subsequent discussion of the latest updates on ACEIs/ARBs use and COVID-19 susceptibility in the light of these mechanisms and biochemical explanation.     

Activation of the PDGF β Receptor by a Persistent Artificial Signal Peptide

Most eukaryotic transmembrane and secreted proteins contain N-terminal signal peptides that mediate insertion of the nascent translation products into the membrane of the endoplasmic reticulum. After membrane insertion, signal peptides typically are cleaved from the mature protein and degraded. Here, we tested whether a small hydrophobic protein selected for growth promoting activity in mammalian cells retained transforming activity while also acting as a signal peptide. We replaced the signal peptide of the PDGF β receptor (PDGFβR) with a previously described 29-residue artificial transmembrane protein named 9C3 that can activate the PDGFβR in trans. We showed that a modified version of 9C3 at the N-terminus of the PDGFβR can function as a signal peptide, as assessed by its ability to support high level expression, glycosylation, and cell surface localization of the PDGFβR. The 9C3 signal peptide retains its ability to interact with the transmembrane domain of the PDGFβR and cause receptor activation and cell proliferation. Cleavage of the 9C3 signal peptide from the mature receptor is not required for these activities. However, signal peptide cleavage does occur in some molecules, and the cleaved signal peptide can persist in cells and activate a co-expressed PDGFβR in trans. Our finding that a hydrophobic sequence can display signal peptide and transforming activity suggest that some naturally occurring signal peptides may also display additional biological activities by interacting with the transmembrane domains of target proteins.