Possible Link between Higher Transmissibility of Alpha,Kappa and Delta Variants of SARS-CoV-2 and Increased Structural Stability of Its Spike Protein and hACE2 Affinity

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak in December 2019 has caused a global pandemic. The rapid mutation rate in the virus has created alarming situations worldwide and is being attributed to the false negativity in RT-PCR tests. It has also increased the chances of reinfection and immune escape. Recently various lineages namely, B.1.1.7 (Alpha), B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.617.3 have caused rapid infection around the globe. To understand the biophysical perspective, we have performed molecular dynamic simulations of four different spikes (receptor binding domain)-hACE2 complexes, namely wildtype (WT), Alpha variant (N501Y spike mutant), Kappa (L452R, E484Q) and Delta (L452R, T478K), and compared their dynamics, binding energy and molecular interactions. Our results show that mutation has caused significant increase in the binding energy between the spike and hACE2 in Alpha and Kappa variants. In the case of Kappa and Delta variants, the mutations at L452R, T478K and E484Q increased the stability and intra-chain interactions in the spike protein, which may change the interaction ability of neutralizing antibodies to these spike variants. Further, we found that the Alpha variant had increased hydrogen interaction with Lys353 of hACE2 and more binding affinity in comparison to WT. The current study provides the biophysical basis for understanding the molecular mechanism and rationale behind the increase in the transmissivity and infectivity of the mutants compared to wild-type SARS-CoV-2.     

SARS-CoV-2 Virus−Host Interaction: Currently Available Structures and Implications of Variant Emergence on Infectivity and Immune Response

Coronavirus disease 19, or COVID-19, is an infection associated with an unprecedented worldwide pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which has led to more than 215 million infected people and more than 4.5 million deaths worldwide. SARS-CoV-2 cell infection is initiated by a densely glycosylated spike (S) protein, a fusion protein, binding human angiotensin converting enzyme 2 (hACE2), that acts as the functional receptor through the receptor binding domain (RBD). In this article, the interaction of hACE2 with the RBD and how fusion is initiated after recognition are explored, as well as how mutations influence infectivity and immune response. Thus, we focused on all structures available in the Protein Data Bank for the interaction between SARS-CoV-2 S protein and hACE2. Specifically, the Delta variant carries particular mutations associated with increased viral fitness through decreased antibody binding, increased RBD affinity and altered protein dynamics. Combining both existing mutations and mutagenesis studies, new potential SARS-CoV-2 variants, harboring advantageous S protein mutations, may be predicted. These include mutations S13I and W152C, decreasing antibody binding, N460K, increasing RDB affinity, or Q498R, positively affecting both properties.     

Altered Local Interactions and Long-Range Communications in UK Variant (B.1.1.7) Spike Glycoprotein

The COVID-19 pandemic is caused by SARS-CoV-2. Currently, most of the research efforts towards the development of vaccines and antibodies against SARS-CoV-2 were mainly focused on the spike (S) protein, which mediates virus entry into the host cell by binding to ACE2. As the virus SARS-CoV-2 continues to spread globally, variants have emerged, characterized by multiple mutations of the S glycoprotein. Herein, we employed microsecond-long molecular dynamics simulations to study the impact of the mutations of the S glycoprotein in SARS-CoV-2 Variant of Concern 202012/01 (B.1.1.7), termed the “UK variant”, in comparison with the wild type, with the aim to decipher the structural basis of the reported increased infectivity and virulence. The simulations provided insights on the different dynamics of UK and wild-type S glycoprotein, regarding in particular the Receptor Binding Domain (RBD). In addition, we investigated the role of glycans in modulating the conformational transitions of the RBD. The overall results showed that the UK mutant experiences higher flexibility in the RBD with respect to wild type; this behavior might be correlated with the increased transmission reported for this variant. Our work also adds useful structural information on antigenic “hotspots” and epitopes targeted by neutralizing antibodies.     

Targeting SARS-CoV-2 RBD Interface: a Supervised Computational Data-Driven Approach to Identify Potential Modulators

Coronavirus disease 2019 (COVID-19) has spread out as a pandemic threat affecting over 2 million people. The infectious process initiates via binding of SARS-CoV-2 Spike (S) glycoprotein to host angiotensin-converting enzyme 2 (ACE2). The interaction is mediated by the receptor-binding domain (RBD) of S glycoprotein, promoting host receptor recognition and binding to ACE2 peptidase domain (PD), thus representing a promising target for therapeutic intervention. Herein, we present a computational study aimed at identifying small molecules potentially able to target RBD. Although targeting PPI remains a challenge in drug discovery, our investigation highlights that interaction between SARS-CoV-2 RBD and ACE2 PD might be prone to small molecule modulation, due to the hydrophilic nature of the bi-molecular recognition process and the presence of druggable hot spots. The fundamental objective is to identify, and provide to the international scientific community, hit molecules potentially suitable to enter the drug discovery process, preclinical validation and development.     

Characterization of Critical Determinants of ACE2–SARS CoV-2 RBD Interaction

Despite sequence similarity to SARS-CoV-1, SARS-CoV-2 has demonstrated greater widespread virulence and unique challenges to researchers aiming to study its pathogenicity in humans. The interaction of the viral receptor binding domain (RBD) with its main host cell receptor, angiotensin-converting enzyme 2 (ACE2), has emerged as a critical focal point for the development of anti-viral therapeutics and vaccines. In this study, we selectively identify and characterize the impact of mutating certain amino acid residues in the RBD of SARS-CoV-2 and in ACE2, by utilizing our recently developed NanoBiT technology-based biosensor as well as pseudotyped-virus infectivity assays. Specifically, we examine the mutational effects on RBD-ACE2 binding ability, efficacy of competitive inhibitors, as well as neutralizing antibody activity. We also look at the implications the mutations may have on virus transmissibility, host susceptibility, and the virus transmission path to humans. These critical determinants of virus–host interactions may provide more effective targets for ongoing vaccines, drug development, and potentially pave the way for determining the genetic variation underlying disease severity.     

Hidden in Plain Sight: Natural Products of Commensal Microbiota as an Environmental Selection Pressure for the Rise of New Variants of SARS-CoV-2

Since the emergence of SARS-CoV-2, little attention has been paid to the interplay between the interaction of virus and commensal microbiota. Here, we used molecular docking and dynamics simulations to study the interaction of some of the known metabolites and natural products (NPs) produced by commensal microbiota with the receptor binding domain (RBD) of the spike glycoprotein of SARS-CoV-2. The results predict that NPs of commensal microbiota such as bile acids and non-ribosomal peptides (NRPs), of which some are siderophores, bind to the wild-type RBD and interfere with its binding to the ACE2 receptor. N501Y mutation, which is present in many of the emerging variants of the virus, abolishes the predicted binding pocket of bile acids and NRPs. Based on these findings, available experimental data showing that bile acids reduce the binding affinity of wild-type RBD to the ACE2 receptor, and the data suggesting that the respiratory tract microbiota affect viral infection we put forward the following proposal: mutations such as N501Y enable the RBD to bind to the ACE2 receptor more effectively in the presence of NPs produced by the respiratory tract bacteria thereby, increasing the infectivity rate of the virus. We hope our data stimulate future works to better understand the interactions of NPs produced by commensal microbiota with respiratory viruses like SARS-CoV-2.     

Molecular Dynamics Studies on the Structural Characteristics for the Stability Prediction of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affects the COVID-19 pandemic in the world. The spike protein of the various proteins encoded in SARS-CoV-2 binds to human ACE2, fuses, and enters human cells in the respiratory system. Spike protein, however, is highly variable, and many variants were identified continuously. In this study, Korean mutants for spike protein (D614G and D614A-C terminal domain, L455F and F456L-RBD, and Q787H-S2 domain) were investigated in patients. Because RBD in spike protein is related to direct interaction with ACE2, almost all researches were focused on the RBD region or ACE2-free whole domain region. The 3D structure for spike protein complexed with ACE2 was recently released. The stability analysis through RBD distance among each spike protein chain and the binding free energy calculation between spike protein and ACE2 were performed using MD simulation depending on mutant types in 1-, 2-, and 3-open-complex forms. D614G mutant of CT2 domain, showing to be the most prevalent in the global pandemic, showed higher stability in all open-complex forms than the wild type and other mutants. We hope this study will provide an insight into the importance of conformational fluctuation in the whole domain, although RBD is involved in the direct interaction with ACE2.     

Structural Evaluation of the Spike Glycoprotein Variants on SARS-CoV-2 Transmission and Immune Evasion

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents significant social, economic and political challenges worldwide. SARS-CoV-2 has caused over 3.5 million deaths since late 2019. Mutations in the spike (S) glycoprotein are of particular concern because it harbours the domain which recognises the angiotensin-converting enzyme 2 (ACE2) receptor and is the target for neutralising antibodies. Mutations in the S protein may induce alterations in the surface spike structures, changing the conformational B-cell epitopes and leading to a potential reduction in vaccine efficacy. Here, we summarise how the more important variants of SARS-CoV-2, which include cluster 5, lineages B.1.1.7 (Alpha variant), B.1.351 (Beta), P.1 (B.1.1.28/Gamma), B.1.427/B.1.429 (Epsilon), B.1.526 (Iota) and B.1.617.2 (Delta) confer mutations in their respective spike proteins which enhance viral fitness by improving binding affinity to the ACE2 receptor and lead to an increase in infectivity and transmission. We further discuss how these spike protein mutations provide resistance against immune responses, either acquired naturally or induced by vaccination. This information will be valuable in guiding the development of vaccines and other therapeutics for protection against the ongoing coronavirus disease 2019 (COVID-19) pandemic.     

In Silico Investigation of the New UK (B.1.1.7) and South African (501Y.V2) SARS-CoV-2 Variants with a Focus at the ACE2–Spike RBD Interface

SARS-CoV-2 exploits angiotensin-converting enzyme 2 (ACE2) as a receptor to invade cells. It has been reported that the UK and South African strains may have higher transmission capabilities, eventually in part due to amino acid substitutions on the SARS-CoV-2 Spike protein. The pathogenicity seems modified but is still under investigation. Here we used the experimental structure of the Spike RBD domain co-crystallized with part of the ACE2 receptor, several in silico methods and numerous experimental data reported recently to analyze the possible impacts of three amino acid replacements (Spike K417N, E484K, N501Y) with regard to ACE2 binding. We found that the N501Y replacement in this region of the interface (present in both the UK and South African strains) should be favorable for the interaction with ACE2, while the K417N and E484K substitutions (South African strain) would seem neutral or even unfavorable. It is unclear if the N501Y substitution in the South African strain could counterbalance the K417N and E484K Spike replacements with regard to ACE2 binding. Our finding suggests that the UK strain should have higher affinity toward ACE2 and therefore likely increased transmissibility and possibly pathogenicity. If indeed the South African strain has a high transmission level, this could be due to the N501Y replacement and/or to substitutions in regions located outside the direct Spike–ACE2 interface but not so much to the K417N and E484K replacements. Yet, it should be noted that amino acid changes at Spike position 484 can lead to viral escape from neutralizing antibodies. Further, these amino acid substitutions do not seem to induce major structural changes in this region of the Spike protein. This structure–function study allows us to rationalize some observations made for the UK strain but raises questions for the South African strain.     

Computational Hot-Spot Analysis of the SARS-CoV-2 Receptor Binding Domain/ACE2 Complex**

Infection and replication of SARS CoV-2 (the virus that causes COVID-19) requires entry to the interior of host cells. In humans, a protein–protein interaction (PPI) between the SARS CoV-2 receptor-binding domain (RBD) and the extracellular peptidase domain of ACE2 on the surface of cells in the lower respiratory tract is an initial step in the entry pathway. Inhibition of the SARS CoV-2 RBD/ACE2 PPI is currently being evaluated as a target for therapeutic and/or prophylactic intervention. However, relatively little is known about the molecular underpinnings of this complex. Employing multiple computational platforms, we predicted “hot-spot” residues in a positive-control PPI (PMI/MDM2) and the CoV-2 RBD/ACE2 complex. Computational alanine scanning mutagenesis was performed to predict changes in Gibbs’ free energy that are associated with mutating residues at the positive control (PMI/MDM2) or SARS RBD/ACE2 binding interface to alanine. Additionally, we used the Adaptive Poisson-Boltzmann Solver to calculate macromolecular electrostatic surfaces at the interface of the positive-control PPI and SARS CoV-2/ACE2 PPI. Finally, a comparative analysis of hot-spot residues for SARS-CoV and SARS-CoV-2, in complex with ACE2, is provided. Collectively, this study illuminates predicted hot-spot residues, and clusters, at the SARS CoV-2 RBD/ACE2 binding interface, potentially guiding the development of reagents capable of disrupting this complex and halting COVID-19.     

Does the SARS-CoV-2 Spike Protein Receptor Binding Domain Interact Effectively with the DPP4 (CD26) Receptor? A Molecular Docking Study

ACE2 has been established as the main receptor for SARS-CoV-2. Since other human coronaviruses are known to use co-receptors for viral cell entry, it has been suggested that DPP4 (CD26) could be a potential additional binding target or co-receptor, supported by early molecular docking simulation studies. However, recent biophysical studies have shown this interaction to be very weak. We have conducted detailed molecular docking simulations to predict the potential binding interactions between the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 and DPP4 and compare them with the interactions observed in the experimentally determined structure of the complex of MERS-CoV with DPP4. Whilst the overall binding mode of the RBD of SARS-CoV-2 to DPP4 is predicted to be similar to that observed in the MERS-CoV-DPP4 complex, including a number of equivalent interactions, important differences in the amino acid sequences of SARS-CoV-2 and MERS-CoV result in substantially weakened interactions with DPP4. This is shown to arise from differences in the predicted proximity, nature and secondary structure at the binding interface on the RBD of SARS-CoV-2. These findings do not support DPP4 being a significant receptor for SARS-CoV-2.     

Why Does the Novel Coronavirus Spike Protein Interact so Strongly with the Human ACE2? A Thermodynamic Answer

The SARS-CoV-2 pandemic is the biggest health concern today, but until now there is no treatment. One possible drug target is the receptor binding domain (RBD) of the coronavirus’ spike protein, which recognizes the human angiotensin-converting enzyme 2 (hACE2). Our in silico study discusses crucial structural and thermodynamic aspects of the interactions involving RBDs from the SARS-CoV and SARS-CoV-2 with the hACE2. Molecular docking and molecular dynamics simulations explain why the chemical affinity of the new SARS-CoV-2 for hACE2 is much higher than in the case of SARS-CoV, revealing an intricate pattern of hydrogen bonds and hydrophobic interactions and estimating a free energy of binding, which is consistently much more negative in the case of SARS-CoV-2. This work presents a chemical reason for the difficulty in treating the SARS-CoV-2 virus with drugs targeting its spike protein and helps to explain its infectiousness.     

Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid**

Enzymatic beacons, or E-beacons, are 1 : 1 bioconjugates of the nanoluciferase enzyme linked covalently at its C-terminus to hairpin forming ssDNA equipped with a dark quencher. We prepared E-beacons biocatalytically using HhC, the promiscuous Hedgehog C-terminal protein-cholesterol ligase. HhC attached nanoluciferase site-specifically to mono-sterylated hairpin oligonucleotides, called steramers. Three E-beacon dark quenchers were evaluated: Iowa Black, Onyx-A, and dabcyl. Each quencher enabled sensitive, sequence-specific nucleic acid detection through enhanced E-beacon bioluminescence upon target hybridization. We assembled prototype dabcyl-quenched E-beacons specific for SARS-CoV-2. Targeting the E484 codon of the virus Spike protein, E-beacons (80×10⁻¹² M) reported wild-type SARS-CoV-2 nucleic acid at ≥1×10⁻⁹ M by increased bioluminescence of 8-fold. E-beacon prepared for the SARS-CoV-2 E484K variant functioned with similar sensitivity. Both E-beacons could discriminate their target from the E484Q mutation of the SARS-CoV-2 Kappa variant. Along with mismatch specificity, E-beacons are two to three orders of magnitude more sensitive than synthetic molecular beacons.     

SARS-CoV-2 Entry Related Viral and Host Genetic Variations: Implications on COVID-19 Severity,Immune Escape,and Infectivity

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved to display particular patterns of genetic diversity in the genome across geographical regions. These variations in the virus and genetic variation in human populations can determine virus transmissibility and coronavirus disease 2019 (COVID-19) severity. Genetic variations and immune differences in human populations could be the driving forces in viral evolution. Recently emerged SARS-CoV-2 variants show several mutations at the receptor binding domain in the spike (S) glycoprotein and contribute to immune escape and enhanced binding with angiotensin 1-converting enzyme 2 (ACE2). Since ACE2 and transmembrane protease serine 2 (TMPRSS2) play important roles in SARS-CoV-2 entry into the cell, genetic variation in these host entry-related proteins may be a driving force for positive selection in the SARS-CoV-2 S glycoprotein. Dendritic or liver/lymph cell-specific intercellular adhesion molecule (ICAM)-3-grabbing non-integrin is also known to play vital roles in several pathogens. Genetic variations of these host proteins may affect the susceptibility to SARS-CoV-2. This review summarizes the latest research to describe the impacts of genetic variation in the viral S glycoprotein and critical host proteins and aims to provide better insights for understanding transmission and pathogenesis and more broadly for developing vaccine/antiviral drugs and precision medicine strategies, especially for high risk populations with genetic risk variants.     

Challenges of the Omicron (B.1.1.529) Variant and Its Lineages: A Global Perspective

The SARS-CoV-2 virus has shown increased ability to mutate over the past two years, especially in the regions of the spike protein and receptor binding sites. Omicron (B.1.1.529) is the fifth variant of concern (VOC) after the emergence of the Alpha, Beta, Gamma, and Delta VOCs of SARS-CoV-2. This new variant has now circulated in 128 countries and according to the Global Initiative on Sharing All Influenza Data (GISAID), these 128 countries have shared 650,657 Omicron genome sequences as of 26 January, 2022. In this article, we highlight the real challenges of Omicron and its different lineages.     

Multiple Recombination Events and Strong Purifying Selection at the Origin of SARS-CoV-2 Spike Glycoprotein Increased Correlated Dynamic Movements

Our evolutionary and structural analyses revealed that the severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) spike gene is a complex mosaic resulting from several recombination events. Additionally, the fixation of variants has mainly been driven by purifying selection, suggesting the presence of conserved structural features. Our dynamic simulations identified two main long-range covariant dynamic movements of the novel glycoprotein, and showed that, as a result of the evolutionary duality, they are preserved. The first movement involves the receptor binding domain with the N-terminal domain and the C-terminal domain 2 and is maintained across human, bat and pangolin coronaviruses. The second is a complex network of long-range dynamics specific to SARS-CoV-2 involving the novel PRRA and the conserved KR*SF cleavage sites, as well as conserved segments in C-terminal domain 3. These movements, essential for host cell binding, are maintained by hinges conserved across human, bat, and pangolin coronaviruses glycoproteins. The hinges, located around Threonine 333 and Proline 527 within the N-terminal domain and C-terminal domain 2, represent candidate targets for the future development of novel pan-coronavirus inhibitors. In summary, we show that while recombination created a new configuration that increased the covariant dynamic movements of the SARS-CoV-2 glycoprotein, negative selection preserved its inter-domain structure throughout evolution in different hosts and inter-species transmissions.     

More Is Always Better Than One: The N-Terminal Domain of the Spike Protein as Another Emerging Target for Hampering the SARS-CoV-2 Attachment to Host Cells

Although the approved vaccines are proving to be of utmost importance in containing the Coronavirus disease 2019 (COVID-19) threat, they will hardly be resolutive as new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, a single-stranded RNA virus) variants might be insensitive to the immune response they induce. In this scenario, developing an effective therapy is still a dire need. Different targets for therapeutic antibodies and diagnostics have been identified, among which the SARS-CoV-2 spike (S) glycoprotein, particularly its receptor-binding domain, has been defined as crucial. In this context, we aim to focus attention also on the role played by the S N-terminal domain (S1-NTD) in the virus attachment, already recognized as a valuable target for neutralizing antibodies, in particular, building on a cavity mapping indicating the presence of two druggable pockets and on the recent literature hypothesizing the presence of a ganglioside-binding domain. In this perspective, we aim at proposing S1-NTD as a putative target for designing small molecules hopefully able to hamper the SARS-CoV-2 attachment to host cells.     

Geraniin Inhibits the Entry of SARS-CoV-2 by Blocking the Interaction between Spike Protein RBD and Human ACE2 Receptor

The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite the development of vaccines, the emergence of SARS-CoV-2 variants and the absence of effective therapeutics demand the continual investigation of COVID-19. Natural products containing active ingredients may be good therapeutic candidates. Here, we investigated the effectiveness of geraniin, the main ingredient in medical plants Elaeocarpus sylvestris var. ellipticus and Nephelium lappaceum, for treating COVID-19. The SARS-CoV-2 spike protein binds to the human angiotensin-converting enzyme 2 (hACE2) receptor to initiate virus entry into cells; viral entry may be an important target of COVID-19 therapeutics. Geraniin was found to effectively block the binding between the SARS-CoV-2 spike protein and hACE2 receptor in competitive enzyme-linked immunosorbent assay, suggesting that geraniin might inhibit the entry of SARS-CoV-2 into human epithelial cells. Geraniin also demonstrated a high affinity to both proteins despite a relatively lower equilibrium dissociation constant (K_D) for the spike protein (0.63 μM) than hACE2 receptor (1.12 μM), according to biolayer interferometry-based analysis. In silico analysis indicated geraniin’s interaction with the residues functionally important in the binding between the two proteins. Thus, geraniin is a promising therapeutic agent for COVID-19 by blocking SARS-CoV-2’s entry into human cells.