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.

AlphaFold2 Predictions of Conformational Ensembles and Atomistic Simulations of the SARS-CoV-2 Spike XBB Lineages Reveal Epistatic Couplings between Convergent Mutational Hotspots that Control ACE2 Affinity.

In this study, we combined AlphaFold-based atomistic structural modeling, microsecond molecular simulations, mutational profiling, and network analysis to characterize binding mechanisms of the SARS-CoV-2 spike protein with the host receptor ACE2 for a series of Omicron XBB variants including XBB.1.5, XBB.1.5+L455F, XBB.1.5+F456L, and XBB.1.5+L455F+F456L. AlphaFold-based structural and dynamic modeling of SARS-CoV-2 Spike XBB lineages can accurately predict the experimental structures and characterize conformational ensembles of the spike protein complexes with the ACE2. Microsecond molecular dynamics simulations identified important differences in the conformational landscapes and equilibrium ensembles of the XBB variants, suggesting that combining AlphaFold predictions of multiple conformations with molecular dynamics simulations can provide a complementary approach for the characterization of functional protein states and binding mechanisms. Using the ensemble-based mutational profiling of protein residues and physics-based rigorous calculations of binding affinities, we identified binding energy hotspots and characterized the molecular basis underlying epistatic couplings between convergent mutational hotspots. Consistent with the experiments, the results revealed the mediating role of the Q493 hotspot in the synchronization of epistatic couplings between L455F and F456L mutations, providing a quantitative insight into the energetic determinants underlying binding differences between XBB lineages. We also proposed a network-based perturbation approach for mutational profiling of allosteric communications and uncovered the important relationships between allosteric centers mediating long-range communication and binding hotspots of epistatic couplings. The results of this study support a mechanism in which the binding mechanisms of the XBB variants may be determined by epistatic effects between convergent evolutionary hotspots that control ACE2 binding.

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.

Biophysical principles predict fitness of SARS-CoV-2 variants.

SARS-CoV-2 employs its spike protein's receptor binding domain (RBD) to enter host cells. The RBD is constantly subjected to immune responses, while requiring efficient binding to host cell receptors for successful infection. However, our understanding of how RBD's biophysical properties contribute to SARS-CoV-2's epidemiological fitness remains largely incomplete. Through a comprehensive approach, comprising large-scale sequence analysis of SARS-CoV-2 variants and the identification of a fitness function based on binding thermodynamics, we unravel the relationship between the biophysical properties of RBD variants and their contribution to viral fitness. We developed a biophysical model that uses statistical mechanics to map the molecular phenotype space, characterized by dissociation constants of RBD to ACE2, LY-CoV016, LY-CoV555, REGN10987, and S309, onto an epistatic fitness landscape. We validate our findings through experimentally measured and machine learning (ML) estimated binding affinities, coupled with infectivity data derived from population-level sequencing. Our analysis reveals that this model effectively predicts the fitness of novel RBD variants and can account for the epistatic interactions among mutations, including explaining the later reversal of Q493R. Our study sheds light on the impact of specific mutations on viral fitness and delivers a tool for predicting the future epidemiological trajectory of previously unseen or emerging low-frequency variants. These insights offer not only greater understanding of viral evolution but also potentially aid in guiding public health decisions in the battle against COVID-19 and future pandemics.

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 50 of 2.727 µ g / ml . 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.

Structural basis for raccoon dog receptor recognition by SARS-CoV-2.

Since the COVID-19 outbreak, raccoon dogs have been suggested as a potential intermediary in transmitting SARS-CoV-2 to humans. To understand their role in the COVID-19 pandemic and the species barrier for SARS-CoV-2 transmission to humans, we analyzed how their ACE2 protein interacts with SARS-CoV-2 spike protein. Biochemical data showed that raccoon dog ACE2 is an effective receptor for SARS-CoV-2 spike protein, though not as effective as human ACE2. Structural comparisons highlighted differences in the virus-binding residues of raccoon dog ACE2 compared to human ACE2 (L24Q, Y34H, E38D, T82M, R353K), explaining their varied effectiveness as receptors for SARS-CoV-2. These variations contribute to the species barrier that exists between raccoon dogs and humans regarding SARS-CoV-2 transmission. Identifying these barriers can help assess the susceptibility of other mammals to SARS-CoV-2. Our research underscores the potential of raccoon dogs as SARS-CoV-2 carriers and identifies molecular barriers that affect the virus's ability to jump between species.

A Piecewise Design Approach to Engineering a Miniature ACE2 Mimic to Bind SARS-CoV-2.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues its global spread, the exploration of novel therapeutic and diagnostic strategies is still needed. The virus enters host cells by binding the angiotensin-converting enzyme 2 (ACE2) receptor through the spike protein. Here, we develop an engineered, small, stable, and catalytically inactive version of ACE2, termed miniature ACE2 (mACE2), designed to bind the spike protein with high affinity. Employing a magnetic nanoparticle-based assay, we harnessed the strong binding affinity of mACE2 to develop a sensitive and specific platform for the detection or neutralization of SARS-CoV-2. Our findings highlight the potential of engineered mACE2 as a valuable tool in the fight against SARS-CoV-2. The success of developing such a small reagent based on a piecewise molecular design serves as a proof-of-concept approach for the rapid deployment of such agents to diagnose and fight other viral diseases.

A systematic mutation analysis of 13 major SARS-CoV-2 variants.

SARS-CoV-2 evolves constantly with various novel mutations. Due to their enhanced infectivity, transmissibility and immune evasion, a comprehensive understanding of the association between these mutations and the respective functional changes is crucial. However, previous mutation studies of major SARS-CoV-2 variants remain limited. Here, we performed systematic analyses of full-length amino acids mutation, phylogenetic features, protein physicochemical properties, molecular dynamics and immune escape as well as pseudotype virus infection assays among thirteen major SARS-CoV-2 variants. We found that Omicron exhibited the most abundant and complex mutation sites, higher indices of hydrophobicity and flexibility than other variants. The results of molecular dynamics simulation suggest that Omicron has the highest number of hydrogen bonds and strongest binding free energy between the S protein and ACE2 receptor. Furthermore, we revealed 10 immune escape sites in 13 major variants, some of them were reported previously, but four of which (i.e. 339/373/477/496) are first reported to be specific to Omicron, whereas 462 is specific to Epslion. The infectivity of these variants was confirmed by the pseudotype virus infection assays. Our findings may help us understand the functional consequences of the mutations within various variants and the underlying mechanisms of the immune escapes conferred by the S proteins.

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.

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.

A structure-function analysis shows SARS-CoV-2 BA.2.86 balances antibody escape and ACE2 affinity.

BA.2.86, a recently described sublineage of SARS-CoV-2 Omicron, contains many mutations in the spike gene. It appears to have originated from BA.2 and is distinct from the XBB variants responsible for many infections in 2023. The global spread and plethora of mutations in BA.2.86 has caused concern that it may possess greater immune-evasive potential, leading to a new wave of infection. Here, we examine the ability of BA.2.86 to evade the antibody response to infection using a panel of vaccinated or naturally infected sera and find that it shows marginally less immune evasion than XBB.1.5. We locate BA.2.86 in the antigenic landscape of recent variants and look at its ability to escape panels of potent monoclonal antibodies generated against contemporary SARS-CoV-2 infections. We demonstrate, and provide a structural explanation for, increased affinity of BA.2.86 to ACE2, which may increase transmissibility.

Pseudotyped virus infection of multiplexed ACE2 libraries reveals SARS-CoV-2 variant shifts in receptor usage.

Pairwise compatibility between virus and host proteins can dictate the outcome of infection. During transmission, both inter- and intraspecies variabilities in receptor protein sequences can impact cell susceptibility. Many viruses possess mutable viral entry proteins and the patterns of host compatibility can shift as the viral protein sequence changes. This combinatorial sequence space between virus and host is poorly understood, as traditional experimental approaches lack the throughput to simultaneously test all possible combinations of protein sequences. Here, we created a pseudotyped virus infection assay where a multiplexed target-cell library of host receptor variants can be assayed simultaneously using a DNA barcode sequencing readout. We applied this assay to test a panel of 30 ACE2 orthologs or human sequence mutants for infectability by the original SARS-CoV-2 spike protein or the Alpha, Beta, Gamma, Delta, and Omicron BA1 variant spikes. We compared these results to an analysis of the structural shifts that occurred for each variant spike's interface with human ACE2. Mutated residues were directly involved in the largest shifts, although there were also widespread indirect effects altering interface structure. The N501Y substitution in spike conferred a large structural shift for interaction with ACE2, which was partially recreated by indirect distal substitutions in Delta, which does not harbor N501Y. The structural shifts from N501Y greatly influenced the set of animal orthologs the variant spike was capable of interacting with. Out of the thirteen non-human orthologs, ten exhibited unique patterns of variant-specific compatibility, demonstrating that spike sequence changes during human transmission can toggle ACE2 compatibility and potential susceptibility of other animal species, and cumulatively increase overall compatibilities as new variants emerge. These experiments provide a blueprint for similar large-scale assessments of protein compatibility during entry by diverse viruses. This dataset demonstrates the complex compatibility relationships that occur between variable interacting host and virus proteins.

Molecular modeling of some commercially available antiviral drugs and their derivatives against SARS-CoV-2 infection.

Numerous prior studies have identified therapeutic targets that could effectively combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, including the angiotensin-converting enzyme 2 (ACE2) receptor, RNA-dependent RNA polymerase (RdRp), and Main protease (Mpro). In parallel, antiviral compounds like abacavir, acyclovir, adefovir, amantadine, amprenavir, darunavir, didanosine, oseltamivir, penciclovir, and tenofovir are under investigation for their potential in drug repurposing to address this infection. The aim of the study was to determine the effect of modifying the functional groups of the aforementioned antivirals in silico. Using the genetic optimization for ligand docking algorithm on software Maestro (version 11.1), the modified antivirals were docked onto ACE2 receptor, RdRp, and Mpro. Using QuickProp (Maestro v11.1), PASS (prediction of activity spectra for the substances), and altogether with SwissADME, the ADMET (absorption, distribution, metabolism, excretion, and toxicity) of the modified antivirals, as well as their bioavailability and the predicted activity spectra, were determined. Discovery studio software was used to undertake post-docking analysis. Among the 10 antivirals, N(CH3)2 derivative of darunavir, N(CH3)2 derivative of amprenavir and NCH3 derivative of darunavir exhibited best binding affinities with ACE2 receptor (docking scores: -10.333, -9.527 and -9.695 kJ/mol, respectively). Moreover, NCH3 derivative of abacavir (-6.506 kJ/mol), NO2 derivative of didanosine (-6.877 kJ/mol), NCH3 derivative of darunavir (-7.618 kJ/mol) exerted promising affinity to Mpro. In conclusion, the results of the in silico screenings can serve as a useful information for future experimental works.

A Rationally Designed Synthetic Antiviral Peptide Binder Targeting the Receptor-Binding Domain of SARS-CoV-2.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a novel coronavirus, is the causative agent responsible for the spread of the COVID19 pandemic across the globe. The global impact of the COVID19 pandemic, the successful approval of vaccines for controlling the pandemic, and the further resurgence of COVID19 necessitate the exploration and validation of alternative therapeutic avenues targeting SARS-CoV-2. The initial entry and further invasion by SARS-CoV-2 require strong protein-protein interactions (PPIs) between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptors expressed on the cell surfaces of various tissues. In principle, disruption of the PPIs between the RBD of SARS-CoV-2 and the ACE2 receptor by designer peptides with optimized pharmacology appears to be an ideal choice for potentially preventing viral entry with minimal immunogenicity. In this context, the current study describes a short, synthetic designer peptide (codenamed SR16, ≤18 aa, molecular weight ≤2.5 kDa), which has a few noncoded amino acids, demonstrates a helical conformation in solution, and also engages the RBD of SARS-CoV-2 through a high-affinity interaction, as judged from a battery of biophysical studies. Further, the designer peptide demonstrates resistance to trypsin degradation, appears to be nontoxic to mammalian cells, and also does not induce hemolysis in freshly isolated human erythrocytes. In summary, SR16 appears to be an ideal peptide binder targeting the RBD of SARS-CoV-2, which has the potential for further optimization and development as an antiviral agent targeting SARS-CoV-2.

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.

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.

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.

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.

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.

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.