Two Novel Angiotensin-Converting Enzyme (ACE) Inhibitory and ACE2 Upregulating Peptides from the Hydrolysate of Pumpkin (Cucurbita moschata) Seed Meal.

Pumpkin (Cucurbita moschata) seed meal (PSM), the major byproduct of pumpkin seed oil industry, was used to prepare angiotensin-converting enzyme (ACE) inhibitory and angiotensin-converting enzyme 2 (ACE2) upregulating peptides. These peptides were isolated and purified from the PSM hydrolysate prepared using Neutrase 5.0 BG by ultrafiltration, Sephadex G-15 column chromatography, and reversed-phase high-performance liquid chromatography. Two peptides with significant ACE inhibition activity were identified as SNHANQLDFHP and PVQVLASAYR with IC50 values of 172.07 and 90.69 µM, respectively. The C-terminal tripeptides of the two peptides contained Pro, Phe, and Tyr, respectively, and PVQVLASAYR also had Val in its N-terminal tripeptide, which was a favorable structure for ACE inhibition. Molecular docking results declared that the two peptides could interact with ACE through hydrogen bonds and hydrophobic interactions. Furthermore, the two peptides performed protective function on EA.hy926 cells by decreasing the secretion of endothelin-1, increasing the release of nitric oxide, and regulating the ACE2 activity. In vitro simulated gastrointestinal digestion showed the two peptides exhibited good stability against gastrointestinal enzyme digestion. In conclusion, PSM is a promising material for preparing antihypertensive peptides.

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.

Analysis the molecular similarity of least common amino acid sites in ACE2 receptor to predict the potential susceptible species for SARS-CoV-2.

SARS-CoV-2 infections in animals have been reported globally. However, the understanding of the complete spectrum of animals susceptible to SARS-CoV-2 remains limited. The virus's dynamic nature and its potential to infect a wide range of animals are crucial considerations for a One Health approach that integrates both human and animal health. This study introduces a bioinformatic approach to predict potential susceptibility to SARS-CoV-2 in both domestic and wild animals. By examining genomic sequencing, we establish phylogenetic relationships between the virus and its potential hosts. We focus on the interaction between the SARS-CoV-2 genome sequence and specific regions of the host species' ACE2 receptor. We analyzed and compared ACE2 receptor sequences from 29 species known to be infected, selecting 10 least common amino acid sites (LCAS) from key binding domains based on similarity patterns. Our analysis included 49 species across primates, carnivores, rodents, and artiodactyls, revealing complete consistency in the LCAS and identifying them as potentially susceptible. We employed the LCAS similarity pattern to predict the likelihood of SARS-CoV-2 infection in unexamined species. This method serves as a valuable screening tool for assessing infection risks in domestic and wild animals, aiding in the prevention of disease outbreaks.

Some mechanistic underpinnings of molecular adaptations of SARS-COV-2 spike protein by integrating candidate adaptive polymorphisms with protein dynamics.

We integrate evolutionary predictions based on the neutral theory of molecular evolution with protein dynamics to generate mechanistic insight into the molecular adaptations of the SARS-COV-2 spike (S) protein. With this approach, we first identified candidate adaptive polymorphisms (CAPs) of the SARS-CoV-2 S protein and assessed the impact of these CAPs through dynamics analysis. Not only have we found that CAPs frequently overlap with well-known functional sites, but also, using several different dynamics-based metrics, we reveal the critical allosteric interplay between SARS-CoV-2 CAPs and the S protein binding sites with the human ACE2 (hACE2) protein. CAPs interact far differently with the hACE2 binding site residues in the open conformation of the S protein compared to the closed form. In particular, the CAP sites control the dynamics of binding residues in the open state, suggesting an allosteric control of hACE2 binding. We also explored the characteristic mutations of different SARS-CoV-2 strains to find dynamic hallmarks and potential effects of future mutations. Our analyses reveal that Delta strain-specific variants have non-additive (i.e., epistatic) interactions with CAP sites, whereas the less pathogenic Omicron strains have mostly additive mutations. Finally, our dynamics-based analysis suggests that the novel mutations observed in the Omicron strain epistatically interact with the CAP sites to help escape antibody binding.

Molecular basis of hippopotamus ACE2 binding to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a wide range of hosts, including hippopotami, which are semi-aquatic mammals and phylogenetically closely related to Cetacea. In this study, we characterized the binding properties of hippopotamus angiotensin-converting enzyme 2 (hiACE2) to the spike (S) protein receptor binding domains (RBDs) of the SARS-CoV-2 prototype (PT) and variants of concern (VOCs). Furthermore, the cryo-electron microscopy (cryo-EM) structure of the SARS-CoV-2 PT S protein complexed with hiACE2 was resolved. Structural and mutational analyses revealed that L30 and F83, which are specific to hiACE2, played a crucial role in the hiACE2/SARS-CoV-2 RBD interaction. In addition, comparative and structural analysis of ACE2 orthologs suggested that the cetaceans may have the potential to be infected by SARS-CoV-2. These results provide crucial molecular insights into the susceptibility of hippopotami to SARS-CoV-2 and suggest the potential risk of SARS-CoV-2 VOCs spillover and the necessity for surveillance. IMPORTANCE: The hippopotami are the first semi-aquatic artiodactyl mammals wherein SARS-CoV-2 infection has been reported. Exploration of the invasion mechanism of SARS-CoV-2 will provide important information for the surveillance of SARS-CoV-2 in hippopotami, as well as other semi-aquatic mammals and cetaceans. Here, we found that hippopotamus ACE2 (hiACE2) could efficiently bind to the RBDs of the SARS-CoV-2 prototype (PT) and variants of concern (VOCs) and facilitate the transduction of SARS-CoV-2 PT and VOCs pseudoviruses into hiACE2-expressing cells. The cryo-EM structure of the SARS-CoV-2 PT S protein complexed with hiACE2 elucidated a few critical residues in the RBD/hiACE2 interface, especially L30 and F83 of hiACE2 which are unique to hiACE2 and contributed to the decreased binding affinity to PT RBD compared to human ACE2. Our work provides insight into cross-species transmission and highlights the necessity for monitoring host jumps and spillover events on SARS-CoV-2 in semi-aquatic/aquatic mammals.

Ensemble-Based Mutational Profiling and Network Analysis of the SARS-CoV-2 Spike Omicron XBB Lineages for Interactions with the ACE2 Receptor and Antibodies: Cooperation of Binding Hotspots in Mediating Epistatic Couplings Underlies Binding Mechanism and Immune Escape.

In this study, we performed a computational study of binding mechanisms for the SARS-CoV-2 spike Omicron XBB lineages with the host cell receptor ACE2 and a panel of diverse class one antibodies. The central objective of this investigation was to examine the molecular factors underlying epistatic couplings among convergent evolution hotspots that enable optimal balancing of ACE2 binding and antibody evasion for Omicron variants BA.1, BA2, BA.3, BA.4/BA.5, BQ.1.1, XBB.1, XBB.1.5, and XBB.1.5 + L455F/F456L. By combining evolutionary analysis, molecular dynamics simulations, and ensemble-based mutational scanning of spike protein residues in complexes with ACE2, we identified structural stability and binding affinity hotspots that are consistent with the results of biochemical studies. In agreement with the results of deep mutational scanning experiments, our quantitative analysis correctly reproduced strong and variant-specific epistatic effects in the XBB.1.5 and BA.2 variants. It was shown that Y453W and F456L mutations can enhance ACE2 binding when coupled with Q493 in XBB.1.5, while these mutations become destabilized when coupled with the R493 position in the BA.2 variant. The results provided a molecular rationale of the epistatic mechanism in Omicron variants, showing a central role of the Q493/R493 hotspot in modulating epistatic couplings between convergent mutational sites L455F and F456L in XBB lineages. The results of mutational scanning and binding analysis of the Omicron XBB spike variants with ACE2 receptors and a panel of class one antibodies provide a quantitative rationale for the experimental evidence that epistatic interactions of the physically proximal binding hotspots Y501, R498, Q493, L455F, and F456L can determine strong ACE2 binding, while convergent mutational sites F456L and F486P are instrumental in mediating broad antibody resistance. The study supports a mechanism in which the impact on ACE2 binding affinity is mediated through a small group of universal binding hotspots, while the effect of immune evasion could be more variant-dependent and modulated by convergent mutational sites in the conformationally adaptable spike regions.

The Recognition Pathway of the SARS-CoV-2 Spike Receptor-Binding Domain to Human Angiotensin-Converting Enzyme 2.

COVID-19 caused by SARS-CoV-2 has spread around the world. The receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 is a critical component that directly interacts with host ACE2. Here, we simulate the ACE2 recognition processes of RBD of the WT, Delta, and OmicronBA.2 variants using our recently developed supervised Gaussian accelerated molecular dynamics (Su-GaMD) approach. We show that RBD recognizes ACE2 through three contact regions (regions I, II, and III), which aligns well with the anchor-locker mechanism. The higher binding free energy in State d of the RBDOmicronBA.2-ACE2 system correlates well with the increased infectivity of OmicronBA.2 in comparison with other variants. For RBDDelta, the T478K mutation affects the first step of recognition, while the L452R mutation, through its nearby Y449, affects the RBDDelta-ACE2 binding in the last step of recognition. For RBDOmicronBA.2, the E484A mutation affects the first step of recognition, the Q493R, N501Y, and Y505H mutations affect the binding free energy in the last step of recognition, mutations in the contact regions affect the recognition directly, and other mutations indirectly affect recognition through dynamic correlations with the contact regions. These results provide theoretical insights for RBD-ACE2 recognition and may facilitate drug design against SARS-CoV-2.

Antibiotics daptomycin interacts with S protein of SARS-CoV-2 to promote cell invasion of Omicron (B1.1.529) pseudovirus.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has posed enormous challenges to global public health. The use of antibiotics has greatly increased during the SARS-CoV-2 epidemic owing to the presence of bacterial co-infection and secondary bacterial infections. The antibiotics daptomycin (DAP) is widely used in the treatment of infectious diseases caused by gram-positive bacteria owing to its highly efficient antibacterial activity. It is pivotal to study the antibiotics usage options for patients of coronavirus infectious disease (COVID-19) with pneumonia those need admission to receive antibiotics treatment for bacterial co-infection in managing COVID-19 disease. Herein, we have revealed the interactions of DAP with the S protein of SARS-CoV-2 and the variant Omicron (B1.1.529) using the molecular docking approach and Omicron (B1.1.529) pseudovirus (PsV) mimic invasion. Molecular docking analysis shows that DAP has a certain degree of binding ability to the S protein of SARS-CoV-2 and several derived virus variants, and co-incubation of 1-100 µM DAP with cells promotes the entry of the PsV into human angiotensin-converting enzyme 2 (hACE2)-expressing HEK-293T cells (HEK-293T-hACE2), and this effect is related to the concentration of extracellular calcium ions (Ca2+). The PsV invasion rate in the HEK-293T-hACE2 cells concurrently with DAP incubation was 1.7 times of PsV infection alone. In general, our findings demonstrate that DAP promotes the infection of PsV into cells, which provides certain reference of antibiotics selection and usage optimization for clinicians to treat bacterial coinfection or secondary infection during SARS-CoV-2 infection.

Multifunctional Self-Signaling nanoMIP and Its Application for a Washing-Free Assay of Human Angiotensin-Converting Enzyme 2.

Molecular imprinting techniques have attracted a lot of attention as a potential biomimetic technology, but there are still challenges in protein imprinting. Herein, multifunctional nanosized molecularly imprinted polymers (nanoMIPs) for human angiotensin-converting enzyme 2 (ACE2) were prepared by epitope imprinting of magnetic nanoparticles-anchored peptide (magNP-P) templates, which were further applied to construct a competitive displacement fluorescence assay toward ACE2. A cysteine-flanked dodecapeptide sequence was elaborately selected as an epitope for ACE2, which was immobilized onto the surface of magnetic nanoparticles and served as a magNP-P template for imprinting. During polymerization, fluorescent monomers were introduced to endow fluorescence responsiveness to the prepared self-signaling nanoMIPs. A competitive displacement fluorescence assay based on the nanoMIPs was established and operated in a washing-free manner, yielding a wide range for ACE2 (0.1-6.0 pg/mL) and a low detection limit (0.081 pg/mL). This approach offers a promising avenue in the preparation of nanoMIPs for macromolecule recognition and expands potential application of an MIP in the detection of proteins as well as peptides.

Microfluidic Diffusional Sizing (MDS) Measurements of Secretory Neutralizing Antibody Affinity Against SARS-CoV-2.

SARS-CoV-2 has rampantly spread around the globe and continues to cause unprecedented loss through ongoing waves of (re)infection. Increasing our understanding of the protection against infection with SARS-CoV-2 is critical to ending the pandemic. Serological assays have been widely used to assess immune responses, but secretory antibodies, the essential first line of defense, have been studied to only a limited extent. Of particular interest and importance are neutralizing antibodies, which block the binding of the spike protein of SARS-CoV-2 to the human receptor angiotensin-converting enzyme-2 (ACE2) and thus are essential for immune defense. Here, we employed Microfluidic Diffusional Sizing (MDS), an immobilization-free technology, to characterize neutralizing antibody affinity to SARS-CoV-2 spike receptor-binding domain (RBD) and spike trimer in saliva. Affinity measurement was obtained through a contrived sample and buffer using recombinant SARS-CoV-2 RBD and monoclonal antibody. Limited saliva samples demonstrated that MDS applies to saliva neutralizing antibody measurement. The ability to disrupt a complex of ACE2-Fc and spike trimer is shown. Using a quantitative assay on the patient sample, we determined the affinity and binding site concentration of the neutralizing antibodies.

Molecular Insights into the Variability in Infection and Immune Evasion Capabilities of SARS-CoV-2 Variants: A Sequence and Structural Investigation of the RBD Domain.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continuously emerge, an increasing number of mutations are accumulating in the Spike protein receptor-binding domain (RBD) region. Through sequence analysis of various Variants of Concern (VOC), we identified that they predominantly fall within the ο lineage although recent variants introduce any novel mutations in the RBD. Molecular dynamics simulations were employed to compute the binding free energy of these variants with human Angiotensin-converting enzyme 2 (ACE2). Structurally, the binding interface of the ο RBD displays a strong positive charge, complementing the negatively charged binding interface of ACE2, resulting in a significant enhancement in the electrostatic potential energy for the ο variants. Although the increased potential energy is partially offset by the rise in polar solvation free energy, enhanced electrostatic potential contributes to the long-range recognition between the ο variant's RBD and ACE2. We also conducted simulations of glycosylated ACE2-RBD proteins. The newly emerged ο (JN.1) variant has added a glycosylation site at N-354@RBD, which significantly weakened its binding affinity with ACE2. Further, our interaction studies with three monoclonal antibodies across multiple SARS-CoV-2 strains revealed a diminished neutralization efficacy against the ο variants, primarily attributed to the electrostatic repulsion between the antibodies and RBD interface. Considering the characteristics of the ο variant and the trajectory of emerging strains, we propose that newly developed antibodies against SARS-CoV-2 RBD should have surfaces rich in negative potential and, postbinding, exhibit strong van der Waals interactions. These findings provide invaluable guidance for the formulation of future therapeutic strategies.

Structural basis and analysis of hamster ACE2 binding to different SARS-CoV-2 spike RBDs.

Pet golden hamsters were first identified being infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) delta variant of concern (VOC) and transmitted the virus back to humans in Hong Kong in January 2022. Here, we studied the binding of two hamster (golden hamster and Chinese hamster) angiotensin-converting enzyme 2 (ACE2) proteins to the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 prototype and eight variants, including alpha, beta, gamma, delta, and four omicron sub-variants (BA.1, BA.2, BA.3, and BA.4/BA.5). We found that the two hamster ACE2s present slightly lower affinity for the RBDs of all nine SARS-CoV-2 viruses tested than human ACE2 (hACE2). Furthermore, the similar infectivity to host cells expressing hamster ACE2s and hACE2 was confirmed with the nine pseudotyped SARS-CoV-2 viruses. Additionally, we determined two cryo-electron microscopy (EM) complex structures of golden hamster ACE2 (ghACE2)/delta RBD and ghACE2/omicron BA.3 RBD. The residues Q34 and N82, which exist in many rodent ACE2s, are responsible for the lower binding affinity of ghACE2 compared to hACE2. These findings suggest that all SARS-CoV-2 VOCs may infect hamsters, highlighting the necessity of further surveillance of SARS-CoV-2 in these animals.IMPORTANCESARS-CoV-2 can infect many domestic animals, including hamsters. There is an urgent need to understand the binding mechanism of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants to hamster receptors. Herein, we showed that two hamster angiotensin-converting enzyme 2s (ACE2s) (golden hamster ACE2 and Chinese hamster ACE2) can bind to the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 prototype and eight variants and that pseudotyped SARS-CoV-2 viruses can infect hamster ACE2-expressing cells. The binding pattern of golden hamster ACE2 to SARS-CoV-2 RBDs is similar to that of Chinese hamster ACE2. The two hamster ACE2s present slightly lower affinity for the RBDs of all nine SARS-CoV-2 viruses tested than human ACE2. We solved the cryo-electron microscopy (EM) structures of golden hamster ACE2 in complex with delta RBD and omicron BA.3 RBD and found that residues Q34 and N82 are responsible for the lower binding affinity of ghACE2 compared to hACE2. Our work provides valuable information for understanding the cross-species transmission mechanism of SARS-CoV-2.

Asialoglycoprotein receptor 1 promotes SARS-CoV-2 infection of human normal hepatocytes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes multi-organ damage, which includes hepatic dysfunction, as observed in over 50% of COVID-19 patients. Angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (ACE2) is the primary receptor for SARS-CoV-2 entry into host cells, and studies have shown the presence of intracellular virus particles in human hepatocytes that express ACE2, but at extremely low levels. Consequently, we asked if hepatocytes might express receptors other than ACE2 capable of promoting the entry of SARS-CoV-2 into cells. To address this question, we performed a genome-wide CRISPR-Cas9 activation library screening and found that Asialoglycoprotein receptor 1 (ASGR1) promoted SARS-CoV-2 pseudovirus infection of HeLa cells. In Huh-7 cells, simultaneous knockout of ACE2 and ASGR1 prevented SARS-CoV-2 pseudovirus infection. In the immortalized THLE-2 hepatocyte cell line and primary hepatic parenchymal cells, both of which barely expressed ACE2, SARS-CoV-2 pseudovirus could successfully establish an infection. However, after treatment with ASGR1 antibody or siRNA targeting ASGR1, the infection rate significantly dropped, suggesting that SARS-CoV-2 pseudovirus infects hepatic parenchymal cells mainly through an ASGR1-dependent mechanism. We confirmed that ASGR1 could interact with Spike protein, which depends on receptor binding domain (RBD) and N-terminal domain (NTD). Finally, we also used Immunohistochemistry and electron microscopy to verify that SARS-CoV-2 could infect primary hepatic parenchymal cells. After inhibiting ASGR1 in primary hepatic parenchymal cells by siRNA, the infection efficiency of the live virus decreased significantly. Collectively, these findings indicate that ASGR1 is a candidate receptor for SARS-CoV-2 that promotes infection of hepatic parenchymal cells.

Mechanistic study of the transmission pattern of the SARS-CoV-2 omicron variant.

The omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) characterized by 30 mutations in its spike protein, has rapidly spread worldwide since November 2021, significantly exacerbating the ongoing COVID-19 pandemic. In order to investigate the relationship between these mutations and the variant's high transmissibility, we conducted a systematic analysis of the mutational effect on spike-angiotensin-converting enzyme-2 (ACE2) interactions and explored the structural/energy correlation of key mutations, utilizing a reliable coarse-grained model. Our study extended beyond the receptor-binding domain (RBD) of spike trimer through comprehensive modeling of the full-length spike trimer rather than just the RBD. Our free-energy calculation revealed that the enhanced binding affinity between the spike protein and the ACE2 receptor is correlated with the increased structural stability of the isolated spike protein, thus explaining the omicron variant's heightened transmissibility. The conclusion was supported by our experimental analyses involving the expression and purification of the full-length spike trimer. Furthermore, the energy decomposition analysis established those electrostatic interactions make major contributions to this effect. We categorized the mutations into four groups and established an analytical framework that can be employed in studying future mutations. Additionally, our calculations rationalized the reduced affinity of the omicron variant towards most available therapeutic neutralizing antibodies, when compared with the wild type. By providing concrete experimental data and offering a solid explanation, this study contributes to a better understanding of the relationship between theories and observations and lays the foundation for future investigations.

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.

Antiviral activity of curcumin and its analogs selected by an artificial intelligence-supported activity prediction system in SARS-CoV-2-infected VeroE6 cells.

Curcumin has been reported to exert its anti-SARS-CoV-2 activity by inhibiting the binding of spike receptor-binding domain (RBD) to angiotensin-converting enzyme-2 (ACE2). To identify more potent compounds, we evaluated the antiviral activities of curcumin and its analogs in SARS-CoV-2-infected cells. An artificial intelligence-supported activity prediction system was used to select the compounds, and 116 of the 334 curcumin analogs were proposed to have spike RBD-ACE2 binding inhibitory activity. These compounds were narrowed down to eight compounds for confirmatory studies. Six out of the eight compounds showed antiviral activity with EC50 values of less than 30 µM and binding inhibitory activity with IC20 values of less than 30 µM. Structure-activity relationship analyses revealed that the double bonds in the carbon chain connecting the two phenolic groups were essential for both activities. X-ray co-crystallography studies are needed to clarify the true binding pose and design more potent derivatives.

Discovery of High Affinity Cyclic Peptide Ligands for Human ACE2 with SARS-CoV-2 Entry Inhibitory Activity.

The development of effective antiviral compounds is essential for mitigating the effects of the COVID-19 pandemic. Entry of SARS-CoV-2 virions into host cells is mediated by the interaction between the viral spike (S) protein and membrane-bound angiotensin-converting enzyme 2 (ACE2) on the surface of epithelial cells. Inhibition of this viral protein-host protein interaction is an attractive avenue for the development of antiviral molecules with numerous spike-binding molecules generated to date. Herein, we describe an alternative approach to inhibit the spike-ACE2 interaction by targeting the spike-binding interface of human ACE2 via mRNA display. Two consecutive display selections were performed to direct cyclic peptide ligand binding toward the spike binding interface of ACE2. Through this process, potent cyclic peptide binders of human ACE2 (with affinities in the picomolar to nanomolar range) were identified, two of which neutralized SARS-CoV-2 entry. This work demonstrates the potential of targeting ACE2 for the generation of anti-SARS-CoV-2 therapeutics as well as broad spectrum antivirals for the treatment of SARS-like betacoronavirus infection.

AI-Driven Design System for Fabrication of Inhalable Nanocatchers for Virus Capture and Neutralization.

The global pandemic presents a critical threat to humanity, with no effective rapid-response solutions for early-stage virus dissemination. This study aims to create an AI-driven entry-blocker design system (AIEB) to fabricate inhalable virus-like nanocatchers (VLNCs) fused with entry-blocking peptides (EBPs) to counter pandemic viruses and explore therapeutic applications. This work focuses on developing angiotensin-converting enzyme 2 (ACE2)-mimic domain-fused VLNCs (ACE2@VLNCs) using AIEB and analyzing their interaction with the SARS-CoV-2 receptor binding domain (RBD), demonstrating their potential to hinder SARS-CoV-2 infection. Aerosol-based tests show ACE2@VLNCs persist over 70 min in the air and neutralize pseudoviruses within 30 min, indicating their utility in reducing airborne virus transmission. In vivo results reveal ACE2@VLNCs mitigate over 67% of SARS-CoV-2 infections. Biosafety studies confirm their safety, causing no damage to eyes, skin, lungs, or trachea, and not eliciting significant immune responses. These findings offer crucial insights into pandemic virus prevention and treatment, highlighting the potential of the ACE2@VLNCs system as a promising strategy against future pandemics.

Dramatic Differences between the Structural Susceptibility of the S1 Pre- and S2 Postfusion States of the SARS-CoV-2 Spike Protein to External Electric Fields Revealed by Molecular Dynamics Simulations.

In its prefusion state, the SARS-CoV-2 spike protein (similarly to other class I viral fusion proteins) is metastable, which is considered to be an important feature for optimizing or regulating its functions. After the binding process of its S1 subunit (S1) with ACE2, the spike protein (S) undergoes a dramatic conformational change where S1 splits from the S2 subunit, which then penetrates the membrane of the host cell, promoting the fusion of the viral and cell membranes. This results in the infection of the host cell. In a previous work, we showed-using large-scale molecular dynamics simulations-that the application of external electric fields (EFs) induces drastic changes and damage in the receptor-binding domain (RBD) of the wild-type spike protein, as well of the Alpha, Beta, and Gamma variants, leaving a structure which cannot be recognized anymore by ACE2. In this work, we first extend the study to the Delta and Omicron variants and confirm the high sensitivity and extreme vulnerability of the RBD of the prefusion state of S to moderate EF (as weak as 104 V/m), but, more importantly, we also show that, in contrast, the S2 subunit of the postfusion state of the spike protein does not suffer structural damage even if electric field intensities four orders of magnitude higher are applied. These results provide a solid scientific basis to confirm the connection between the prefusion-state metastability of the SARS-CoV-2 spike protein and its susceptibility to be damaged by EF. After the virus docks to the ACE2 receptor, the stable and robust postfusion conformation develops, which exhibits a similar resistance to EF (damage threshold higher than 108 V/m) like most globular proteins.

Mapping immunological and host receptor binding determinants of SARS-CoV spike protein utilizing the Qubevirus platform.

The motifs involved in tropism and immunological interactions of SARS-CoV spike (S) protein were investigated utilizing the Qubevirus platform. We showed that separately, 14 overlapping peptide fragments representing the S protein (F1-14 of 100 residues each) could be inserted into the C terminus of A1 on recombinant Qubevirus without affecting its viability. Additionally, recombinant phage expression resulted in the surface exposure of different engineered fragments in an accessible manner. The F6 from S425-525 was found to contain the binding determinant of the recombinant human angiotensin-converting enzyme 2, with the shortest active binding motif situated between residues S437-492. Upstream, another fragment, F7, containing an overlapping portion of F6 would not bind to recombinant human angiotensin-converting enzyme 2, confirming that a contiguous stretch of residues could adopt the appropriate structural orientation of F6 as an insertion within the Qubevirus. The F6 (S441-460) and other inserts, including F7/F8 (S601-620) and F10 (S781-800), were demonstrated to contain important immunological determinants through recognition and binding of S protein specific (anti-S) antibodies. An engineered chimeric insert bearing the fusion of all three anti-S reactive epitopes improved substantially the recognition and binding to their cognate antibodies. These results provide insights into humoral immune relevant epitopes and tropism characteristics of the S protein with implications for the development of subunit vaccines or other biologics against SARS-CoV.