Binding Of Receptor-binding Domain Research Articles

Dipeptidyl peptidase 4 interacts with porcine coronavirus PHEV spikes and mediates host range expansion.

Porcine hemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus, is prevalent in natural reservoir pigs and infects mice. This raises concerns about host jumping or spillover, but little is known about the cause of occurrence. Here, we revealed that dipeptidyl peptidase 4 (DPP4) is a candidate binding target of PHEV spikes and works as a broad barrier to overcome. Investigations of the host breadth of PHEV confirmed that cells derived from pigs and mice are permissive to virus propagation. Both porcine DPP4 and murine DPP4 have high affinity for the viral spike receptor-binding domain (RBD), independent of their catalytic activity. Loss of DPP4 expression results in limited PHEV infection. Structurally, PHEV spike protein binds to the outer surface of blades IV and V of the DPP4 β-propeller domain, and the DPP4 residues N229 and N321 (relative to human DPP4 numbering) participate in RBD binding via its linked carbohydrate entities. Removal of these N-glycosylations profoundly enhanced the RBD-DPP4 interaction and viral invasion, suggesting they act as shielding in PHEV infection. Furthermore, we found that glycosylation, rather than structural differences or surface charges, is more responsible for DPP4 recognition and species barrier formation. Overall, our findings shed light on virus-receptor interactions and highlight that PHEV tolerance to DPP4 orthologs is a putative determinant of its cross-species transmission or host range expansion.IMPORTANCEPHEV is a neurotropic betacoronavirus that is circulating worldwide and has raised veterinary and economic concerns. In addition to being a reservoir species of pigs, PHEV can also infect wild-type mice, suggesting a "host jump" event. Understanding cross-species transmission is crucial for disease prevention and control but remains to be addressed. Herein, we show that the multifunctional receptor DPP4 plays a pivotal role in the host tropism of PHEV and identifies the conserved glycosylation sites in DPP4 responsible for this restriction. These findings highlight that the ability of PHEV to utilize DPP4 orthologs potentially affects its natural host expansion.

Network pharmacology, molecular docking, and dynamics analyses to predict the antiviral activity of ginger constituents against coronavirus infection.

COVID-19 is a global pandemic that caused a dramatic loss of human life worldwide, leading to accelerated research for antiviral drug discovery. Herbal medicine is one of the most commonly used alternative medicine for the prevention and treatment of many conditions including respiratory system diseases. In this study, a computational pipeline was employed, including network pharmacology, molecular docking simulations, and molecular dynamics simulations, to analyze the common phytochemicals of ginger rhizomes and identify candidate constituents as viral inhibitors. Furthermore, experimental assays were performed to analyze the volatile and non-volatile compounds of ginger and to assess the antiviral activity of ginger oil and hydroalcoholic extract. Network pharmacology analysis showed that ginger compounds target human genes that are involved in related cellular processes to the viral infection. Docking analysis highlighted five pungent compounds and zingiberenol as potential inhibitors for the main protease (Mpro), spike receptor-binding domain (RBD), and human angiotensin-converting enzyme 2 (ACE2). Then, (6)-gingerdiacetate was selected for molecular dynamics (MD) simulations as it exhibited the best binding interactions and free energies over the three target proteins. Trajectories analysis of the three complexes showed that RBD and ACE2 complexes with the ligand preserved similar patterns of root mean square deviation (RMSD) and radius of gyration (Rg) values to their respective native structures. Finally, experimental validation of the ginger hydroalcoholic extract confirmed the existence of (6)-gingerdiacetate and revealed the strong antiviral activity of the hydroalcoholic extract with IC of 2.727 . Our study provides insights into the potential antiviral activity of (6)-gingerdiacetate that may enhance the host immune response and block RBD binding to ACE2, thereby, inhibiting SARS-CoV-2 infection.

Sites and Zones of Maximum Reactivity of the most Stable Structure of the Receptor-binding Domain of Wild-type SARS-CoV-2 Spike Protein: A Quantum Density Functional Theory Study

Today, it is well known that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has four types of proteins within its structure, between them the spike protein (S). The infection mechanism is carried out by the entry of the virus into the human host cell through the S protein, which strongly interacts with the human cell receptor angiotensin-converting enzyme 2 (ACE2). In this work, we propose an atomic model of the Receptor Binding Domain (RBD) of the S spike protein of the wild-type SARS-CoV-2 virus. The molecular structure of the model was composed of 50 amino acids that were chemically bonded, starting with Leucine and ending with one amino acid Tyrosine. The novelty of our work lies in the importance of knowing the sites and zones of maximum reactivity of the RBD from the fundamental levels of quantum mechanics considering the atomic structure of matter. For this, the local and global reactivity indices of the RBD were calculated, such as frontier orbitals, Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO), Fukui indices, chemical potential, chemical hardness, electrophilicity index; with this, it will be possible to know what type of molecules are more likely to interact with the RBD structure, and in this way, new knowledge will be generated at the quantum, atomic and molecular level to inhibit the virulent effects of wild-type SARS-CoV-2. Finally, in order to identify the functional groups within the most stable structure and thereby verify the future reactions that can be carried out between the RBD structure and biomolecules, the Infrared (IR) absorption spectrum was calculated. For this work, we used Material Studio v6.0 which uses the density functional theory (DFT) implemented in its DMol3 computational code. The IR spectrum was obtained using the Spartan ‘94 computer code. One novelty would be that we found nine amino acids more that could make the RBD and ACE2 binding further the already known. Thus, the Mulliken charge distribution indicates that the highest concentrations of positive and negative charge are found in the zones 477S, 478T, 484E, and 501N amino acids letting ionic or Van der Waals possible interactions with other structures.

Kaempferia parviflora Wall. ex Baker against SARS-CoV-2 spike protein: In silico and in vitro studies

Context: SARS-CoV-2 spike (S) protein, governed by its receptor binding domain (RBD), is a key player in mediating viral attachment and fusion into host cells, which leads to infection and transmission of COVID-19. Searching for effective inhibitors against S RBD protein is essential to stop the virus infection. Aims: To evaluate Kaempferia parviflora’s bioactive compounds as inhibitors against SARS-CoV-2 S RBD protein and its complex with human angiotensin-converting enzyme 2 (ACE2) receptor through in silico, and to determine the ability of K. parviflora’s extract to inhibit the binding of S RBD and ACE2. Methods: Molecular docking was performed to evaluate the inhibition potentials of K. parviflora’s bioactive compounds against S RBD protein and its complex with ACE2. The inhibitory activity of K. parviflora’s extract against the binding of S RBD and ACE2 was determined using an in vitro inhibition assay. Results: K. parviflora’s compounds had inhibition potentials against S RBD protein in both closed and open states. In the open RBD, these compounds were bound to the key amino acids that were involved in the binding of RBD with ACE2, suggesting their possible roles in preventing the RBD-ACE2 association. K. parviflora’s compounds also had strong affinities towards the S RBD-ACE2 complex by interacting with the paired RBD-ACE2 amino acids, which were crucial for the S RBD-ACE2 complex’s stability. In addition, K. parviflora extract also demonstrated inhibitory activity against the binding of S RBD and ACE2. Conclusions: This study proposes K. parviflora as a promising inhibitor against SARS-CoV-2 S protein for the treatment of COVID-19.

Raman spectroscopy study of 7,8-dihydrofolate inhibition on the Wuhan strain SARS-CoV-2 binding to human ACE2 receptor

Emerging evidence suggests that elevated levels of folic acid in the bloodstream may confer protection against Wuhan-SARS-CoV-2 infection and mitigate its associated symptoms. Notably, two comprehensive studies of COVID-19 patients in Israel and UK uncovered a remarkable trend, wherein individuals with heightened folic acid levels exhibited only mild symptoms and necessitated no ventilatory support. In parallel, research has underscored the potential connection between decreased folic acid levels and the severity of Covid-19 among hospitalized patients. Yet, the underlying mechanisms governing this intriguing inhibition remain elusive. In a quest to elucidate these mechanisms, we conducted a molecular dynamics simulation approach followed by a Raman spectroscopy study to delve into the intricate interplay between the folic acid metabolite, 7,8-dihydrofolate (DHF), and the angiotensin-converting enzyme ACE2 receptor, coupled with its interaction with the receptor-binding domain (RBD) of the Wuhan strain of SARS-CoV-2. Through a meticulous exploration, we scrutinized the transformation of the ACE2 + RBD complex, allowing these reactants to form bonds. This was juxtaposed with a similar investigation where ACE2 was initially permitted to react with DHF, followed by the exposure of the ACE2 + DHF complex to RBD. We find that DHF, when bonded to ACE2, functions as a physical barrier, effectively inhibiting the binding of the Wuhan strain RBD. This physicochemical process offers a cogent explanation for the observed inhibition of host cell infection in subjects receiving supplementary folic acid doses, as epidemiologically substantiated in multiple studies. This study not only sheds light on a potential avenue for mitigating SARS-CoV-2 infection but also underscores the crucial role of folic acid metabolites in host-virus interactions. This research paves the way for novel therapeutic strategies in the battle against COVID-19 and reinforces the significance of investigating the molecular mechanisms underlying the protective effects of folic acid in the context of viral infections.

Binding behavior of receptor binding domain of the SARS-CoV-2 virus and ivermectin

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), sparked an international debate on effective ways to prevent and treat the virus. Specifically, there were many varying opinions on the use of ivermectin (IVM) throughout the world, with minimal research to support either side. IVM is an FDA-approved antiparasitic drug that was discovered in the 1970s and was found to show antiviral activity. The objective of this study is to examine the binding behavior and rates of association and dissociation between SARS-CoV-2 receptor binding domain (RBD), IVM, and their combination using aminopropylsilane (APS) biosensors as surrogates for the hydrophobic interaction between the viral protein and human angiotensin-converting enzyme 2 (ACE2) receptors to determine the potential of IVM as a repurposed drug for SARS-CoV-2 prevention and treatment. The IVM, RBD, and combination binding kinetics were analyzed using biolayer interferometry (BLI) and validated with multiple in silico techniques including protein–ligand docking, molecular dynamics simulation, molecular mechanics-generalized Born surface area (MM-GBSA), and principal component analysis (PCA). Our results suggest that with increasing IVM concentrations the association rate with the hydrophobic biosensor increases with a simultaneous decrease in dissociation. Significant kinetic changes to RBD, when combined with IVM, were found only at a concentration a thousand times the approved dosage with minimal changes found over a 35-min time period. Our study suggests that IVM is not an effective preventative or treatment method at the currently approved dosage.

Rapid Profiling of the Glycosylation Effects on the Binding of SARS-CoV-2 Spike Protein to Angiotensin-Converting Enzyme 2 Using MALDI-MS with High Mass Detection.

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 binds directly to angiotensin-converting enzyme 2 (ACE2), mediating the host cell entry of SARS-CoV-2. Both spike protein and ACE2 are highly glycosylated, which can regulate the binding. Here, we utilized high-mass MALDI-MS with chemical cross-linking for profiling the glycosylation effects on the binding between RBD and ACE2. Overall, it was found that ACE2 glycosylation affects the binding more strongly than does RBD glycosylation. The binding affinity was improved after desialylation or partial deglycosylation (N690) of ACE2, while it decreased after degalactosylation. ACE2 can form dimers in solution, which bind more tightly to the RBD than the ACE2 monomers. The ACE2 dimerization and the binding of RBD to dimeric ACE2 can also be improved by the desialylation or deglycosylation of ACE2. Partial deglycosylation of ACE2 increased the dimerization of ACE2 and the binding affinity of RBD and ACE2 by more than a factor of 2, suggesting its high potential for neutralizing SARS-CoV-2. The method described in the work provided a simple way to analyze the protein-protein interaction without sample purification. It can be widely used for rapid profiling of glycosylation effects on protein-protein interaction for glycosylation-related diseases and the study of multiple interactions between protein and protein aggregates in a single system.

Design of protein-binding peptides with controlled binding affinity: the case of SARS-CoV-2 receptor binding domain and angiotensin-converting enzyme 2 derived peptides.

The development of methods able to modulate the binding affinity between proteins and peptides is of paramount biotechnological interest in view of a vast range of applications that imply designed polypeptides capable to impair or favour Protein-Protein Interactions. Here, we applied a peptide design algorithm based on shape complementarity optimization and electrostatic compatibility and provided the first experimental in vitro proof of the efficacy of the design algorithm. Focusing on the interaction between the SARS-CoV-2 Spike Receptor-Binding Domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) receptor, we extracted a 23-residues long peptide that structurally mimics the major interacting portion of the ACE2 receptor and designed in silico five mutants of such a peptide with a modulated affinity. Remarkably, experimental KD measurements, conducted using biolayer interferometry, matched the in silico predictions. Moreover, we investigated the molecular determinants that govern the variation in binding affinity through molecular dynamics simulation, by identifying the mechanisms driving the different values of binding affinity at a single residue level. Finally, the peptide sequence with the highest affinity, in comparison with the wild type peptide, was expressed as a fusion protein with human H ferritin (HFt) 24-mer. Solution measurements performed on the latter constructs confirmed that peptides still exhibited the expected trend, thereby enhancing their efficacy in RBD binding. Altogether, these results indicate the high potentiality of this general method in developing potent high-affinity vectors for hindering/enhancing protein-protein associations.

Microwave-assisted synthesis of highly sulfated mannuronate glycans as potential inhibitors against SARS-CoV-2.

Algae-based marine carbohydrate drugs are typically decorated with negative ion groups such as carboxylate and sulfate groups. However, the precise synthesis of highly sulfated alginates is challenging, thus impeding their structure-activity relationship studies. Herein we achieve a microwave-assisted synthesis of a range of highly sulfated mannuronate glycans with up to 17 sulfation sites by overcoming the incomplete sulfation due to the electrostatic repulsion of crowded polyanionic groups. Although the partially sulfated tetrasaccharide had the highest affinity for the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant, the fully sulfated octasaccharide showed the most potent interference with the binding of the RBD to angiotensin-converting enzyme 2 (ACE2) and Vero E6 cells, indicating that the sulfated oligosaccharides might inhibit the RBD binding to ACE2 in a length-dependent manner.

Heteromultimeric sarbecovirus receptor binding domain immunogens primarily generate variant-specific neutralizing antibodies.

Vaccination will likely be a key component of strategies to curtail or prevent future sarbecovirus pandemics and to reduce the prevalence of infection and disease by future SARS-CoV-2 variants. A "pan-sarbecovirus" vaccine, that provides maximum possible mitigation of human disease, should elicit neutralizing antibodies with maximum possible breadth. By positioning multiple different receptor binding domain (RBD) antigens in close proximity on a single immunogen, it is postulated that cross-reactive B cell receptors might be selectively engaged. Heteromultimeric vaccines could therefore elicit individual antibodies that neutralize a broad range of viral species. Here, we use model systems to investigate the ability of multimeric sarbecovirus RBD immunogens to expand cross-reactive B cells and elicit broadly reactive antibodies. Homomultimeric RBD immunogens generated higher serum neutralizing antibody titers than the equivalent monomeric immunogens, while heteromultimeric RBD immunogens generated neutralizing antibodies recognizing each RBD component. Moreover, RBD heterodimers elicited a greater fraction of cross-reactive germinal center B cells and cross-reactive RBD binding antibodies than did homodimers. However, when serum antibodies from RBD heterodimer-immunized mice were depleted using one RBD component, neutralization activity against the homologous viral pseudotype was removed, but neutralization activity against pseudotypes corresponding to the other RBD component was unaffected. Overall, simply combining divergent RBDs in a single immunogen generates largely separate sets of individual RBD-specific neutralizing serum antibodies that are mostly incapable of neutralizing viruses that diverge from the immunogen components.

Exploring the disruption of SARS-CoV-2 RBD binding to hACE2.

The COVID-19 pandemic was declared due to the spread of the novel coronavirus, SARS-CoV-2. Viral infection is caused by the interaction between the SARS-CoV-2 receptor binding domain (RBD) and the human ACE2 receptor (hACE2). Previous computational studies have identified repurposed small molecules that target the RBD, but very few have screened drugs in the RBD-hACE2 interface. When studies focus solely on the binding affinity between the drug and the RBD, they ignore the effect of hACE2, resulting in an incomplete analysis. We screened ACE inhibitors and previously identified SARS-CoV-2 inhibitors for binding to the RBD-hACE2 interface, and then conducted 500ns of unrestrained molecular dynamics (MD) simulations of fosinopril, fosinoprilat, lisinopril, emodin, diquafosol, and physcion bound to the interface to assess the binding characteristics of these ligands. Based on MM-GBSA analysis, all six ligands bind favorably in the interface and inhibit the RBD-hACE2 interaction. However, when we repeat our simulation by first binding the drug to the RBD before interacting with hACE2, we find that fosinopril, fosinoprilat, and lisinopril result in a strongly interacting trimeric complex (RBD-drug-hACE2). Hydrogen bonding and pairwise decomposition analyses further suggest that fosinopril is the best RBD inhibitor. However, when lisinopril is bound, it stabilizes the trimeric complex and, therefore, is not an ideal potential drug candidate. Overall, these results reveal important atomistic interactions critical to the binding of the RBD to hACE2 and highlight the significance of including all protein partners in the evaluation of a potential drug candidate.

Molecular dynamic simulation reveals spider antimicrobial peptide Latarcin-1 and human eosinophil cationic protein as peptide inhibitors of SARS-CoV-2 variants

COVID-19 has rapidly proliferated around 180 countries, and new cases are reported frequently. No peptide medication has been developed that can reliably block SARS-CoV-2 infection. The investigation focuses on the crucial host receptors angiotensin-converting enzyme 2 (ACE2) , which can bind receptor-binding domain (RBD) on the SARS-CoV-2 spike protein (S). To investigate the inhibitory effects of human Eosinophil Cationic Protein (hECP) and Latarcin-1 (L1)on SARS-CoV-2 infection, we have selected them as research subjects. Further, we ran extensive molecular dynamics simulations to bring the docked peptide-ACE2 complex into its equilibrium state. The outcomes were then evaluated with g_MMPBSA and interaction analysis. We have also considered the Delta and Omicron variants to examine these peptides’ inhibitory effects. The experimental findings revealed an enhanced capability of L1 and hECP as SARS-CoV-2 inhibitors, occupying hot spots and numerous key residues in ACE2. These include ASP30, ASP38, GLU35 and GLU75, which significantly inhibit the binding of RBD and ACE2 and are effective against two common variants in a similar manner. In addition, this study can serve as a springboard for future research on SARS-CoV-2 inhibitors. Communicated by Ramaswamy H. Sarma

Interface-Based Design of High-Affinity Affibody Ligands for the Purification of RBD from Spike Proteins.

The outbreak of coronavirus disease 2019 (COVID-19) has sparked an urgent demand for advanced diagnosis and vaccination worldwide. The discovery of high-affinity ligands is of great significance for vaccine and diagnostic reagent manufacturing. Targeting the receptor binding domain (RBD) from the spike protein of severe acute respiratory syndrome-coronavirus 2, an interface at the outer surface of helices on the Z domain from protein A was introduced to construct a virtual library for the screening of ZRBD affibody ligands. Molecular docking was performed using HADDOCK software, and three potential ZRBD affibodies, ZRBD-02, ZRBD-04, and ZRBD-07, were obtained. Molecular dynamics (MD) simulation verified that the binding of ZRBD affibodies to RBD was driven by electrostatic interactions. Per-residue free energy decomposition analysis further substantiated that four residues with negative-charge characteristics on helix α1 of the Z domain participated in this process. Binding affinity analysis by microscale thermophoresis showed that ZRBD affibodies had high affinity for RBD binding, and the lowest dissociation constant was 36.3 nmol/L for ZRBD-07 among the three potential ZRBD affibodies. Herein, ZRBD-02 and ZRBD-07 affibodies were selected for chromatographic verifications after being coupled to thiol-activated Sepharose 6 Fast Flow (SepFF) gel. Chromatographic experiments showed that RBD could bind on both ZRBD SepFF gels and was eluted by 0.1 mol/L NaOH. Moreover, the ZRBD-07 SepFF gel had a higher affinity for RBD. This research provided a new idea for the design of affibody ligands and validated the potential of affibody ligands in the application of RBD purification from complex feedstock.

SARS-CoV-2 hijacks neutralizing dimeric IgA for nasal infection and injury in Syrian hamsters1

ABSTRACT Prevention of robust severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in nasal turbinate (NT) requires in vivo evaluation of IgA neutralizing antibodies. Here, we report the efficacy of receptor binding domain (RBD)-specific monomeric B8-mIgA1 and B8-mIgA2, and dimeric B8-dIgA1, B8-dIgA2 and TH335-dIgA1 against intranasal SARS-CoV-2 challenge in Syrian hamsters. These antibodies exhibited comparable neutralization potency against authentic virus by competing with human angiotensin converting enzyme-2 (ACE2) receptor for RBD binding. While reducing viral loads in lungs significantly, prophylactic intranasal B8-dIgA unexpectedly led to high amount of infectious viruses and extended damage in NT compared to controls. Mechanistically, B8-dIgA failed to inhibit SARS-CoV-2 cell-to-cell transmission, but was hijacked by the virus through dendritic cell-mediated trans-infection of NT epithelia leading to robust nasal infection. Cryo-EM further revealed B8 as a class II antibody binding trimeric RBDs in 3-up or 2-up/1-down conformation. Neutralizing dIgA, therefore, may engage an unexpected mode of SARS-CoV-2 nasal infection and injury.

The structure of the RBD-E77 Fab complex reveals neutralization and immune escape of SARS-CoV-2.

The spike protein (S) of SARS-CoV-2 is the major target of neutralizing antibodies and vaccines. Antibodies that target the receptor-binding domain (RBD) of S have high potency in preventing viral infection. The ongoing evolution of SARS-CoV-2, especially mutations occurring in the RBD of new variants, has severely challenged the development of neutralizing antibodies and vaccines. Here, a murine monoclonal antibody (mAb) designated E77 is reported which engages the prototype RBD with high affinity and potently neutralizes SARS-CoV-2 pseudoviruses. However, the capability of E77 to bind RBDs vanishes upon encountering variants of concern (VOCs) which carry the N501Y mutation, such as Alpha, Beta, Gamma and Omicron, in contrast to its performance with the Delta variant. To explain the discrepancy, cryo-electron microscopy was used to analyze the structure of an RBD-E77 Fab complex, which reveals that the binding site of E77 on RBD belongs to the RBD-1 epitope, which largely overlaps with the binding site of human angiotensin-converting enzyme 2 (hACE2). Both the heavy chain and the light chain of E77 interact extensively with RBD and contribute to the strong binding of RBD. E77 employs CDRL1 to engage Asn501 of RBD and the Asn-to-Tyr mutation could generate steric hindrance, abolishing the binding. In sum, the data provide the landscape for an in-depth understanding of immune escape of VOCs and rational antibody engineering against emerging variants of SARS-CoV-2.

Lung Surfactant Protein B Peptide Mimics Interact with the Human ACE2 Receptor

Lung surfactant is a complex mixture of phospholipids and surfactant proteins that is produced in alveolar type 2 cells. It prevents lung collapse by reducing surface tension and is involved in innate immunity. Exogenous animal-derived and, more recently, synthetic lung surfactant has shown clinical efficacy in surfactant-deficient premature infants and in critically ill patients with acute respiratory distress syndrome (ARDS), such as those with severe COVID-19 disease. COVID-19 pneumonia is initiated by the binding of the viral receptor-binding domain (RBD) of SARS-CoV-2 to the cellular receptor angiotensin-converting enzyme 2 (ACE2). Inflammation and tissue damage then lead to loss and dysfunction of surface activity that can be relieved by treatment with an exogenous lung surfactant. Surfactant protein B (SP-B) is pivotal for surfactant activity and has anti-inflammatory effects. Here, we study the binding of two synthetic SP-B peptide mimics, Super Mini-B (SMB) and B-YL, to a recombinant human ACE2 receptor protein construct using molecular docking and surface plasmon resonance (SPR) to evaluate their potential as antiviral drugs. The SPR measurements confirmed that both the SMB and B-YL peptides bind to the rhACE2 receptor with affinities like that of the viral RBD–ACE2 complex. These findings suggest that synthetic lung surfactant peptide mimics can act as competitive inhibitors of the binding of viral RBD to the ACE2 receptor.

Targeting DPP4-RBD interactions by sitagliptin and linagliptin delivers a potential host-directed therapy against pan-SARS-CoV-2 infections

Highly mutated SARS-CoV-2 is known aetiological factor for COVID-19. Here, we have demonstrated that the receptor binding domain (RBD) of the spike protein can interact with human dipeptidyl peptidase 4 (DPP4) to facilitate virus entry, in addition to the usual route of ACE2-RBD binding. Significant number of residues of RBD makes hydrogen bonds and hydrophobic interactions with α/β-hydrolase domain of DPP4. With this observation, we created a strategy to combat COVID-19 by circumventing the catalytic activity of DPP4 using its inhibitors. Sitagliptin, linagliptin or in combination disavowed RBD to establish a heterodimer complex with both DPP4 and ACE2 which is requisite strategy for virus entry into the cells. Both gliptins not only impede DPP4 activity, but also prevent ACE2-RBD interaction, crucial for virus growth. Sitagliptin, and linagliptin alone or in combination have avidity to impede the growth of pan-SARS-CoV-2 variants including original SARS-CoV-2, alpha, beta, delta, and kappa in a dose dependent manner. However, these drugs were unable to alter enzymatic activity of PLpro and Mpro. We conclude that viruses hijack DPP4 for cell invasion via RBD binding. Impeding RBD interaction with both DPP4 and ACE2 selectively by sitagliptin and linagliptin is an potential strategy for efficiently preventing viral replication.

CD62L expression marks SARS-CoV-2 memory B cell subset with preference for neutralizing epitopes

Severe acute respiratory syndrome coronavirus 2–neutralizing antibodies primarily target the spike receptor binding domain (RBD). However, B cell antigen receptors (BCRs) on RBD-binding memory B (B mem ) cells have variation in the neutralizing activities. Here, by combining single B mem cell profiling with antibody functional assessment, we dissected the phenotype of B mem cell harboring the potently neutralizing antibodies in coronavirus disease 2019 (COVID-19)–convalescent individuals. The neutralizing subset was marked by an elevated CD62L expression and characterized by distinct epitope preference and usage of convergent V H (variable region of immunoglobulin heavy chain) genes, accounting for the neutralizing activities. Concordantly, the correlation was observed between neutralizing antibody titers in blood and CD62L + subset, despite the equivalent RBD binding of CD62L + and CD62L − subset. Furthermore, the kinetics of CD62L + subset differed between the patients who recovered from different COVID-19 severities. Our B mem cell profiling reveals the unique phenotype of B mem cell subset that harbors potently neutralizing BCRs, advancing our understanding of humoral protection.

Persistent topological Laplacian analysis of SARS-CoV-2 variants.

Topological data analysis (TDA) is an emerging field in mathematics and data science. Its central technique, persistent homology, has had tremendous success in many science and engineering disciplines. However, persistent homology has limitations, including its inability to handle heterogeneous information, such as multiple types of geometric objects; being qualitative rather than quantitative, e.g., counting a 5-member ring the same as a 6-member ring, and a failure to describe non-topological changes, such as homotopic changes in protein-protein binding. Persistent topological Laplacians (PTLs), such as persistent Laplacian and persistent sheaf Laplacian, were proposed to overcome the limitations of persistent homology. In this work, we examine the modeling and analysis power of PTLs in the study of the protein structures of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor binding domain (RBD). First, we employ PTLs to study how the RBD mutation-induced structural changes of RBD-angiotensin-converting enzyme 2 (ACE2) binding complexes are captured in the changes of spectra of the PTLs among SARS-CoV-2 variants. Additionally, we use PTLs to analyze the binding of RBD and ACE2-induced structural changes of various SARS-CoV-2 variants. Finally, we explore the impacts of computationally generated RBD structures on a topological deep learning paradigm and predictions of deep mutational scanning datasets for the SARS-CoV-2 Omicron BA.2 variant. Our results indicate that PTLs have advantages over persistent homology in analyzing protein structural changes and provide a powerful new TDA tool for data science.

The molecular basis of the neutralization breadth of the RBD-specific antibody CoV11.

SARS-CoV-2, the virus behind the COVID-19 pandemic, has changed over time to the extent that the current virus is substantially different from what originally led to the pandemic in 2019-2020. Viral variants have modified the severity and transmissibility of the disease and continue do so. How much of this change is due to viral fitness versus a response to immune pressure is hard to define. One class of antibodies that continues to afford some level of protection from emerging variants are those that closely overlap the binding site for angiotensin-converting enzyme 2 (ACE2) on the receptor binding domain (RBD). Some members of this class that were identified early in the course of the pandemic arose from the VH 3-53 germline gene (IGHV3-53*01) and had short heavy chain complementarity-determining region 3s (CDR H3s). Here, we describe the molecular basis of the SARS-CoV-2 RBD recognition by the anti-RBD monoclonal antibody CoV11 isolated early in the COVID-19 pandemic and show how its unique mode of binding the RBD determines its neutralization breadth. CoV11 utilizes a heavy chain VH 3-53 and a light chain VK 3-20 germline sequence to bind to the RBD. Two of CoV11's four heavy chain changes from the VH 3-53 germline sequence, to Ile and to Arg, and some unique features in its CDR H3 increase its affinity to the RBD, while the four light chain changes from the VK 3-20 germline sequence sit outside of the RBD binding site. Antibodies of this type can retain significant affinity and neutralization potency against variants of concern (VOCs) that have diverged significantly from original virus lineage such as the prevalent omicron variant. We also discuss the mechanism by which VH 3-53 encoded antibodies recognize spike antigen and show how minimal changes to their sequence, their choice of light chain, and their mode of binding influence their affinity and impact their neutralization breadth.