Dendrimer-Peptide Conjugates for Effective Blockade of the Interactions between SARS-CoV-2 Spike Protein and Human ACE2 Receptor.

The coronavirus disease 2019 (COVID-19) pandemic has threatened the stability of global healthcare, which is becoming an endemic issue. Despite the development of various treatment strategies to fight COVID-19, the currently available treatment options have shown varied efficacy. Herein, we have developed an avidity-based SARS-CoV-2 antagonist using dendrimer-peptide conjugates (DPCs) for effective COVID-19 treatment. Two different peptide fragments obtained from angiotensin-converting enzyme 2 (ACE2) were integrated into a single sequence, followed by the conjugation to poly(amidoamine) (PAMAM) dendrimers. We hypothesized that the strong multivalent binding avidity endowed by dendrimers would help peptides effectively block the interaction between SARS-CoV-2 and ACE2, and this antagonist effect would be dependent upon the generation (size) of the dendrimers. To assess this, binding kinetics of the DPCs prepared from generation 4 (G4) and G7 PAMAM dendrimers to spike protein of SARS-CoV-2 were quantitatively measured using surface plasmon resonance. The larger dendrimer-based DPCs exhibited significantly enhanced binding strength by 3 orders of magnitude compared to the free peptides, whereas the smaller one showed a 12.8-fold increase only. An in vitro assay using SARS-CoV-2-mimicking microbeads also showed the improved SARS-CoV-2 blockade efficiency of the G7-peptide conjugates compared to G4. In addition, the interaction between the DPCs and SARS-CoV-2 was analyzed using molecular dynamics (MD) simulation, providing an insight into how the dendrimer-mediated multivalent binding effect can enhance the SARS-CoV-2 blockade. Our findings demonstrate that the DPCs having strong binding to SARS-CoV-2 effectively block the interaction between ACE2 and SARS-CoV-2, providing a potential as a high-affinity drug delivery system to direct anti-COVID payloads to the virus.

Potential use of the S-protein-Angiotensin converting enzyme 2 binding pathway in the treatment of coronavirus disease 2019.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), infects humans through a strong interaction between the viral spike protein (S-protein) and angiotensin converting enzyme 2 (ACE2) receptors on the cell surface. The infection of host lung cells by SARS-CoV-2 leads to clinical symptoms in patients. However, ACE2 expression is not restricted to the lungs; altered receptors have been found in the nasal and oral mucosa, vessel, brain, pancreas, gastrointestinal tract, kidney, and heart. The future of COVID-19 is uncertain, however, new viral variants are likely to emerge. The SARS-CoV-2 Omicron variant has a total of 50 gene mutations compared with the original virus; 15 of which occur in the receptor binding domain (RBD). The RBD of the viral S-protein binds to the human ACE2 receptor for viral entry. Mutations of the ACE2-RBD interface enhance tight binding by increasing hydrogen bond interactions and expanding the accessible surface area. Extracorporeal membrane oxygenation, hyperbaric oxygen, and aggressive dialysis for the treatment of COVID-19 have shown various degrees of clinical success. The use of decoy receptors based on the ACE2 receptor as a broadly potent neutralizer of SARS-CoV-2 variants has potential as a therapeutic mechanism. Drugs such as 3E8 could block binding of the S1-subunit to ACE2 and restrict the infection of ACE2-expressing cells by a variety of coronaviruses. Here, we discuss the development of ACE2-targeted strategies for the treatment and prevention of COVID-19.

High-Throughput Assay for Identifying Diverse Antagonists of the Binding Interaction between the ACE2 Receptor and the Dynamic Spike Proteins of SARS-CoV-2.

SARS-CoV-2, a coronavirus strain that started a worldwide pandemic in early 2020, attaches to human cells by binding its spike (S) glycoprotein to a host receptor protein angiotensin-converting enzyme 2 (ACE2). Blocking the interaction between the S protein and ACE2 has emerged as an important strategy for preventing viral infection. We systematically developed and optimized an AlphaLISA assay to investigate binding events between ACE2 and the ectodomain of the SARS-CoV-2 S protein (S-614G: residues 1-1208 with a D614G mutation). Using S-614G permits discovering potential allosteric inhibitors that stabilize the S protein in a conformation that impedes its access to ACE2. Over 30,000 small molecules were screened in a high-throughput format for activity against S-614G and ACE2 binding using the AlphaLISA assay. A viral entry assay was used to validate hits using lentiviral particles pseudotyped with the full-length S protein of the Wuhan-1 strain. Two compounds identified in the screen, oleic acid and suramin, blocked the attachment of S-614G to ACE2 and S protein-driven cell entry into Calu-3 and ACE2-overexpressing HEK293T cells. Oleic acid inhibits S-614G binding to ACE2 far more potently than to the receptor-binding domain (RBD, residues 319-541 of SARS-CoV-2 S), potentially indicating a noncompetitive mechanism. The results indicate that using the full-length ectodomain of the S protein can be important for identifying allosteric inhibitors of ACE2 binding. The approach reported here represents a rapidly adaptable format for discovering receptor-binding inhibitors to S-proteins of future coronavirus strains.

Enhanced Inactivation of Pseudoparticles Containing SARS-CoV-2 S Protein Using Magnetic Nanoparticles and an Alternating Magnetic Field.

Severe acute respiratory syndrome coronavirus 2's (SARS-CoV-2) rapid global spread has posed a significant threat to human health, and similar outbreaks could occur in the future. Developing effective virus inactivation technologies is critical to preventing and overcoming pandemics. The infection of SARS-CoV-2 depends on the binding of the spike glycoprotein (S) receptor binding domain (RBD) to the host cellular surface receptor angiotensin-converting enzyme 2 (ACE2). If this interaction is disrupted, SARS-CoV-2 infection could be inhibited. Magnetic nanoparticle (MNP) dispersions exposed to an alternating magnetic field (AMF) possess the unique ability for magnetically mediated energy delivery (MagMED); this localized energy delivery and associated mechanical, chemical, and thermal effects are a possible technique for inactivating viruses. This study investigates the MNPs' effect on vesicular stomatitis virus pseudoparticles containing the SARS-CoV-2 S protein when exposed to AMF or a water bath (WB) with varying target steady-state temperatures (45, 50, and 55 °C) for different exposure times (5, 15, and 30 min). In comparison to WB exposures at the same temperatures, AMF exposures resulted in significantly greater inactivation in multiple cases. This is likely due to AMF-induced localized heating and rotation of MNPs. In brief, our findings demonstrate a potential strategy for combating the SARS-CoV-2 pandemic or future ones.

Chemical Compositions of Clove (Syzygium aromaticum (L.) Merr. & L.) Extracts and Their Potentials in Suppressing SARS-CoV-2 Spike Protein-ACE2 Binding, Inhibiting ACE2, and Scavenging Free Radicals.

COVID-19 is initiated by binding the SARS-CoV-2 spike protein to angiotensin-converting enzyme 2 (ACE2) on host cells. Food factors capable of suppressing the binding between the SARS-CoV-2 spike protein and ACE2 or reducing the ACE2 availability through ACE2 inhibitions may potentially reduce the risk of SARS-CoV-2 infection and COVID-19. In this study, the chemical compositions of clove water and ethanol extracts were investigated, along with their potentials in suppressing SARS-CoV-2 spike protein-ACE2 binding, reducing ACE2 availability, and scavenging free radicals. Thirty-four compounds were tentatively identified in the clove water and ethanol extracts, with six reported in clove for the first time. Clove water and ethanol extracts dose-dependently suppressed SARS-CoV-2 spike protein binding to ACE2 and inhibited ACE2 activity. The water extract had stronger inhibitory effects than the ethanol extract on a dry weight basis. The clove water extract also had more potent free radical scavenging activities against DPPH• and ABTS•+ (536.9 and 3525.06 µmol TE/g, respectively) than the ethanol extract (58.44 and 2298.01 µmol TE/g, respectively). In contrast, the ethanol extract had greater total phenolic content (TPC) and relative HO• scavenging capacity (HOSC) values (180.03 mg GAE/g and 2181.08 µmol TE/g, respectively) than the water extract (120.12 mg GAE/g and 1483.02 µmol TE/g, respectively). The present study demonstrated the potential of clove in reducing the risk of SARS-CoV-2 infection and COVID-19 development.

Energetics of Spike Protein Opening of SARS-CoV-1 and SARS-CoV-2 and Its Variants of Concern: Implications in Host Receptor Scanning and Transmission.

The receptor binding domain(s) (RBD) of spike (S) proteins of SARS-CoV-1 and SARS-CoV-2 (severe acute respiratory syndrome coronavirus) undergoes closed to open transition to engage with host ACE2 receptors. In this study, using multi atomistic (equilibrium) and targeted (non-equilibrium) molecular dynamics simulations, we have compared energetics of RBD opening pathways in full-length (modeled from cryo-EM structures) S proteins of SARS-CoV-1 and SARS-CoV-2. Our data indicate that amino acid variations at the RBD interaction interface can culminate into distinct free energy landscapes of RBD opening in these S proteins. We further report that mutations in the S protein of SARS-CoV-2 variants of concern can reduce the protein-protein interaction affinity of RBD(s) with its neighboring domains and could favor its opening to access ACE2 receptors. The findings can also aid in predicting the impact of future mutations on the rate of S protein opening for rapid host receptor scanning.

Host and viral proteins involved in SARS-CoV-2 infection differentially bind heme.

In most severe cases, SARS-CoV-2-induced autoimmune reactions have been associated with hemolytic complications. Hemolysis-derived heme from ruptured red blood cells has been shown to trigger a variety of fatal proinflammatory and procoagulant effects, which might deteriorate the progression of COVID-19. In addition, the virus itself can induce proinflammatory signals via the accessory protein 7a. Direct heme binding to the SARS-CoV-2 protein 7a ectodomain and other COVID-19-related proteins has been suggested earlier. Here, we report the experimental analysis of heme binding to the viral proteins spike glycoprotein, protein 7a as well as the host protein ACE2. Thus, protein 7a chemical synthesis was established, including an in-depth analysis of the three different disulfide-bonded isomers. Surface plasmon resonance spectroscopy and in silico studies confirm a transient, biphasic binding behavior, and heme-binding affinities in the nano- to low micromolar range. These results confirm the presence of the earlier identified heme-binding motifs and emphasize the relevance for consideration of labile heme in preexisting or SARS-CoV-2-induced hemolytic conditions in COVID-19 patients.

Distinct conformational states of SARS-CoV-2 spike protein.

Intervention strategies are urgently needed to control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. The trimeric viral spike (S) protein catalyzes fusion between viral and target cell membranes to initiate infection. Here, we report two cryo-electron microscopy structures derived from a preparation of the full-length S protein, representing its prefusion (2.9-angstrom resolution) and postfusion (3.0-angstrom resolution) conformations, respectively. The spontaneous transition to the postfusion state is independent of target cells. The prefusion trimer has three receptor-binding domains clamped down by a segment adjacent to the fusion peptide. The postfusion structure is strategically decorated by N-linked glycans, suggesting possible protective roles against host immune responses and harsh external conditions. These findings advance our understanding of SARS-CoV-2 entry and may guide the development of vaccines and therapeutics.

Antiviral potential of diminazene aceturate against SARS-CoV-2 proteases using computational and in vitro approaches.

Diminazene aceturate (DIZE), an antiparasitic, is an ACE2 activator, and studies show that activators of this enzyme may be beneficial for COVID-19, disease caused by SARS-CoV-2. Thus, the objective was to evaluate the in silico and in vitro affinity of diminazene aceturate against molecular targets of SARS-CoV-2. 3D structures from DIZE and the proteases from SARS-CoV-2, obtained through the Protein Data Bank and Drug Database (Drubank), and processed in computer programs like AutodockTools, LigPlot, Pymol for molecular docking and visualization and GROMACS was used to perform molecular dynamics. The results demonstrate that DIZE could interact with all tested targets, and the best binding energies were obtained from the interaction of Protein S (closed conformation -7.87 kcal/mol) and Mpro (-6.23 kcal/mol), indicating that it can act both by preventing entry and viral replication. The results of molecular dynamics demonstrate that DIZE was able to promote a change in stability at the cleavage sites between S1 and S2, which could prevent binding to ACE2 and fusion with the membrane. In addition, in vitro tests confirm the in silico results showing that DIZE could inhibit the binding between the spike receptor-binding domain protein and ACE2, which could promote a reduction in the virus infection. However, tests in other experimental models with in vivo approaches are needed.

Electrostatic Features for the Receptor Binding Domain of SARS-COV-2 Wildtype and Its Variants. Compass to the Severity of the Future Variants with the Charge-Rule.

Electrostatic intermolecular interactions are important in many aspects of biology. We have studied the main electrostatic features involved in the interaction of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein with the human receptor Angiotensin-converting enzyme 2 (ACE2). As the principal computational tool, we have used the FORTE approach, capable to model proton fluctuations and computing free energies for a very large number of protein-protein systems under different physical-chemical conditions, here focusing on the RBD-ACE2 interactions. Both the wild-type and all critical variants are included in this study. From our large ensemble of extensive simulations, we obtain, as a function of pH, the binding affinities, charges of the proteins, their charge regulation capacities, and their dipole moments. In addition, we have calculated the pKas for all ionizable residues and mapped the electrostatic coupling between them. We are able to present a simple predictor for the RBD-ACE2 binding based on the data obtained for Alpha, Beta, Gamma, Delta, and Omicron variants, as a linear correlation between the total charge of the RBD and the corresponding binding affinity. This "RBD charge rule" should work as a quick test of the degree of severity of the coming SARS-CoV-2 variants in the future.

Exploring the Binding Affinity and Mechanism between ACE2 and the Trimers of Delta and Omicron Spike Proteins by Molecular Dynamics Simulation and Bioassay.

Five major variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged and posed challenges in controlling the pandemic. Among them, the current dominant variant, viz., Omicron, has raised serious concerns about its infectiousness and antibody neutralization. However, few studies pay attention to the effect of the mutations on the dynamic interaction network of Omicron S protein trimers binding to the host angiotensin-converting enzyme 2 (ACE2). In this study, we conducted molecular dynamics (MD) simulations and enzyme linked immunosorbent assay (ELISA) to explore the binding strength and mechanism of wild type (WT), Delta, and Omicron S protein trimers to ACE2. The results showed that the binding capacities of both the two variants' S protein trimers to ACE2 are enhanced in varying degrees, indicating possibly higher cell infectiousness. Energy decomposition and protein-protein interaction network analysis suggested that both the mutational and conserved sites make effects on the increase in the overall affinity through a variety of interactions. The experimentally determined KD values by biolayer interferometry (BLI) and the predicted binding free energies of the RBDs of Delta and Omicron to mAb HLX70 revealed that the two variants may have the high risk of immune evasion from the mAb. These results are not only helpful in understanding the binding strength and mechanism of S protein trimer-ACE2 but also beneficial for drug, especially for antibody development.

Identification of Potential ACE2-Derived Peptide Mimetics in SARS-CoV-2 Omicron Variant Therapeutics using Computational Approaches.

The COVID-19 pandemic has become a global health challenge because of the emergence of distinct variants. Omicron, a new variant, is recognized as a variant of concern (VOC) by the World Health Organization (WHO) because of its higher mutations and accelerated human infection. The infection rate is strongly dependent on the binding rate of the receptor binding domain (RBD) against human angiotensin converting enzyme-2 (ACE2human) receptor. Inhibition of protein-protein (RBDs(SARS-CoV-2/omicron)-ACE2human) interaction has been already proven to inhibit viral infection. We have systematically designed ACE2human-derived peptides and peptide mimetics that have high binding affinity toward RBDomicron. Our peptide mutational analysis indicated the influence of canonical amino acids on the peptide binding process. Herein, efforts have been made to explore the atomistic details and events of RBDs(SARS-CoV-2/omicron)-ACE2human interactions by using molecular dynamics simulation. Our studies pave a path for developing therapeutic peptidomimetics against omicron.

Computational studies evidenced the potential of steroidal lactone to disrupt surface interaction of SARS-CoV-2 spike protein and hACE2.

The critical event in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis is recognition of host cells by the virus, which is facilitated by protein-protein interaction (PPI) of viral Spike-Receptor Binding Domain (S-RBD) and Human Angiotensin Converting Enzyme 2-Receptor (hACE2-R). Thus, disrupting the interaction between S-RBD and hACE2-R is widely accepted as a primary strategy for managing COVID-19. The purpose of this study is to assess the ability of three steroidal lactones (SL) (4-Dehydrowithaferin A, Withaferin A, and Withalongolide A) derived from plants to disrupt the PPI of S-RBD and hACE2-R under two conditions (CON-I and CON-II) using in-silico methods. Under CON-I, 4-Dehydrowithaferin A destabilizing the interactions between S-RBD and hACE2-R, as indicated by an increase in binding energy (BE) from -1028.5 kJ/mol (control) to -896.12 kJ/mol 4-Dehydrowithaferin A exhibited a strong interaction with S-RBD GLY496 with a hydrogen bond occupancy (HBO) of 37.33%. Under CON-II, Withalongolide A was capable of disrupting all types of PPI, as evidenced by an increased BE from -913 kJ/mol (control) to -133.69 kJ/mol and an increased distance (>3.55 nm) between selected AAR combinations of S-RBD and hACE2-R. Withalongolide A formed a hydrogen bond with TYR453 (97%, HBO) of S-RBD, which is required for interaction with hACE2-R's HIS34. Our studies demonstrated that SL molecules have the potential to disrupt the S-RBD and hACE2-R interaction, thereby preventing SARS-CoV-2 from recognizing host cells. The SL molecules can be considered for additional in-vitro and in-vivo studies with this research evidence.

Expanded ACE2 dependencies of diverse SARS-like coronavirus receptor binding domains.

Viral spillover from animal reservoirs can trigger public health crises and cripple the world economy. Knowing which viruses are primed for zoonotic transmission can focus surveillance efforts and mitigation strategies for future pandemics. Successful engagement of receptor protein orthologs is necessary during cross-species transmission. The clade 1 sarbecoviruses including Severe Acute Respiratory Syndrome-related Coronavirus (SARS-CoV) and SARS-CoV-2 enter cells via engagement of angiotensin converting enzyme-2 (ACE2), while the receptor for clade 2 and clade 3 remains largely uncharacterized. We developed a mixed cell pseudotyped virus infection assay to determine whether various clades 2 and 3 sarbecovirus spike proteins can enter HEK 293T cells expressing human or Rhinolophus horseshoe bat ACE2 proteins. The receptor binding domains from BtKY72 and Khosta-2 used human ACE2 for entry, while BtKY72 and Khosta-1 exhibited widespread use of diverse rhinolophid ACE2s. A lysine at ACE2 position 31 appeared to be a major determinant of the inability of these RBDs to use a certain ACE2 sequence. The ACE2 protein from Rhinolophus alcyone engaged all known clade 3 and clade 1 receptor binding domains. We observed little use of Rhinolophus ACE2 orthologs by the clade 2 viruses, supporting the likely use of a separate, unknown receptor. Our results suggest that clade 3 sarbecoviruses from Africa and Europe use Rhinolophus ACE2 for entry, and their spike proteins appear primed to contribute to zoonosis under the right conditions.

Omicron Binding Mode: Contact Analysis and Dynamics of the Omicron Receptor-Binding Domain in Complex with ACE2.

On 26 November 2021, the WHO classified the Omicron variant of the SARS-CoV-2 virus (B.1.1.529 lineage) as a variant of concern (VOC) (COVID-19 Variant Data, Department of Health, 2022). The Omicron variant contains as many as 26 unique mutations of effects not yet determined (Venkatakrishnan, A., Open Science Framework, 2021). Out of its total of 34 Spike protein mutations, 15 are located on the receptor-binding domain (S-RBD) (Stanford Coronavirus Antiviral & Resistance Database, 2022) that directly contacts the angiotensin-converting enzyme 2 (ACE2) host receptor and is also a primary target for antibodies. Here, we studied the binding mode of the S-RBD domain of the Spike protein carrying the Omicron mutations and the globular domain of human ACE2 using molecular dynamics (MD) simulations. We identified new and key Omicron-specific interactions such as R493 (of mutation Q493R), which forms salt bridges both with E35 and D38 of ACE2, Y501 (N501Y), which forms an edge-to-face aromatic interaction with Y41, and Y505 (Y505H), which makes an H-bond with E37 and K353. The glycan chains of ACE2 also bind differently in the WT and Omicron variants in response to different charge distributions on the surface of Spike proteins. However, while the Omicron mutations considerably improve the overall electrostatic fit of the two interfaces, the total number of specific and favorable interactions between the two does not increase. The dynamics of the complexes are highly affected too, making the Omicron S-RBD:ACE2 complex more rigid; the two main interaction sites, Patches I and II, isolated in the WT complex, become connected in the Omicron complex through the alternating interaction of R493 and R498 with E35 and D38.

Computational repurposing approach for targeting the critical spike mutations in B.1.617.2 (delta), AY.1 (delta plus) and C.37 (lambda) SARS-CoV-2 variants using exhaustive structure-based virtual screening, molecular dynamic simulations and MM-PBSA methods.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the contagious coronavirus disease 2019 (COVID-19) which was first identified in Wuhan, China, in December 2019. Around the world, many researchers focused their research on identifying inhibitors against the druggable SARS-CoV-2 targets. The reported genomic mutations have a direct effect on the receptor-binding domain (RBD), which interacts with host angiotensin-converting enzyme 2 (ACE-2) for viral cell entry. These mutations, some of which are variants of concern (VOC), lead to increased morbidity and mortality rates. The newest variants including B.1.617.2 (Delta), AY.1 (Delta plus), and C.37 (Lambda) were considered in this study. Thus, an exhaustive structure-based virtual screening of a ligand library (in which FDA approved drugs are also present) using the drug-likeness screening, molecular docking, ADMET profiling was performed followed by molecular dynamics (MD) simulation, and Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA) calculation to identify compounds or drugs can be repurposed for inhibiting the wild type, Delta, Delta plus and Lambda variants of RBD of the spike protein. Based on the virtual screening steps, two FDA approved drugs, Atovaquone (atv) and Praziquantel (prz), were selected and repurposed as the best candidates of SARS-CoV-2 RBD inhibitors. Molecular docking results display that both atv and prz contribute in different interaction with binding site residues (Gln493, Asn501 and Gly502 in the hydrogen bond formation, Phe490 and Tyr505 in the π- π stacking and Tyr449, Ser494, and Phe497 in the vdW interactions) in the wild type, Delta, Delta plus and Lambda variants of RBD of the spike protein. MD simulations revealed that among the eight studied complexes, the wild type-atv and Delta-prz complexes have the most structural stability over the simulation time. Furthermore, MM-PBSA calculation showed that in the atv containing complexes, highest binding affinity is related to the wild type-atv complex and in the prz containing complexes, it is related to the Delta-prz complex. The validation of docking results was done by comparing with experimental data (heparin in complex with wild type and Delta variants). Also, comparison of the obtained results with the result of simulation of the k22 with the studied proteins showed that atv and prz are suitable inhibitors for these proteins, especially wild type t and Delta variant, respectively. Thus, we found that atv and prz are the best candidate for inhibition of wild type and Delta variant of the spike protein. Also, atv can be an appropriate inhibitor for the Lambda variant. Obtained in silico results may help the development of new anti-COVID-19 drugs.

The effect of various compounds on the COVID mechanisms, from chemical to molecular aspects.

The novel coronavirus that caused COVID-19 pandemic is SARS-CoV-2. Although various vaccines are currently being used to prevent the disease's severe consequences, there is still a need for medications for those who become infected. The SARS-CoV-2 has a variety of proteins that have been studied extensively since the virus's advent. In this review article, we looked at chemical to molecular aspects of the various structures studied that have pharmaceutical activity and attempted to find a link between drug activity and compound structure. For example, designing of the compounds which bind to the allosteric site and modify hydrogen bonds or the salt bridges can disrupt SARS-CoV2 RBD-ACE2 complex. It seems that quaternary ammonium moiety and quinolin-1-ium structure could act as a negative allosteric modulator to reduce the tendency between spike-ACE2. Pharmaceutical structures with amino heads and hydrophobic tails can block envelope protein to prevent making mature SARS-CoV-2. Also, structures based on naphthalene pharmacophores or isosteres can form a strong bond with the PLpro and form a π-π and the Mpro's active site can be occupied by octapeptide compounds or linear compounds with a similar fitting ability to octapeptide compounds. And for protein RdRp, it is critical to consider pH and pKa so that pKa regulation of compounds to comply with patients is very effective, thus, the presence of tetrazole, phenylpyrazole groups, and analogs of pyrophosphate in the designed drugs increase the likelihood of the RdRp active site inhibition. Finally, it can be deduced that designing hybrid drug molecules along with considering the aforementioned characteristics would be a suitable approach for developing medicines in order to accurate targeting and complete inhibition this virus.

Analysis of the docking property of host variants of hACE2 for SARS-CoV-2 in a large cohort.

The recent novel coronavirus disease (COVID-19) outbreak, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is threatening global health. However, an understanding of the interaction of SARS-CoV-2 with human cells, including the physical docking property influenced by the host's genetic diversity, is still lacking. Here, based on germline variants in the UK Biobank covering 502,543 individuals, we revealed the molecular interactions between human angiotensin-converting enzyme 2 (hACE2), which is the representative receptor for SARS-CoV-2 entry, and COVID-19 infection. We identified six nonsense and missense variants of hACE2 from 2585 subjects in the UK Biobank covering 500000 individuals. Using our molecular dynamics simulations, three hACE2 variants from 2585 individuals we selected showed higher levels of binding free energy for docking in the range of 1.44-3.69 kcal/mol. Although there are diverse contributors to SARS-CoV-2 infections, including the mobility of individuals, we analyzed the diagnosis records of individuals with these three variants of hACE2. Our molecular dynamics simulations combined with population-based genomic data provided an atomistic understanding of the interaction between hACE2 and the spike protein of SARS-CoV-2.

NMR Experiments Provide Insights into Ligand-Binding to the SARS-CoV-2 Spike Protein Receptor-Binding Domain.

We have used chemical shift perturbation (CSP) and saturation transfer difference (STD) NMR experiments to identify and characterize the binding of selected ligands to the receptor-binding domain (RBD) of the spike glycoprotein (S-protein) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We also subjected full-length S-protein to STD NMR experiments, allowing correlations with RBD-based results. CSPs reveal the binding sites for heparin and fondaparinux, and affinities were measured using CSP titrations. We then show that α-2,3-sialyllactose binds to the S-protein but not to the RBD. Finally, combined CSP and STD NMR experiments show that lifitegrast, a compound used for the treatment of dry eye, binds to the linoleic acid (LA) binding pocket with a dissociation constant in the µM range. This is an interesting finding, as lifitegrast lends itself well as a blueprint for medicinal chemistry, eventually furnishing novel entry inhibitors targeting the highly conserved LA binding site.

All-Atom Simulations of Human ACE2-Spike Protein RBD Complexes for SARS-CoV-2 and Some of its Variants: Nature of Interactions and Free Energy Diagrams for Dissociation of the Protein Complexes.

The spike protein of SARS-CoV-2 is known to interact with the human ACE2 protein via its receptor binding domain (RBD). We have investigated the molecular nature of this interprotein interaction and the associated free energy diagrams for the unbinding of the two proteins for SARS-CoV-2 and some of its known variants through all-atom simulations. The present work involves generation and analysis of 2.5 µs of unbiased and 4.2 µs of biased molecular dynamics trajectories in total for five explicitly solvated RBD-ACE2 systems at full atomic level. First, we have made a comparative analysis of the details of residue-wise specific interactions of the spike protein with ACE2 for SARS-CoV-1 and SARS-CoV-2. It is found that the average numbers of both direct interprotein and water-bridged hydrogen bonds between the RBD and ACE2 are higher for SARS-CoV-2 than SARS-CoV-1. These higher hydrogen bonded interactions are further aided by enhanced nonspecific electrostatic attractions between the two protein surfaces for SARS-CoV-2. The free energy calculations reveal that there is an increase in the free energy barrier by ∼1.5 kcal/mol for the unbinding of RBD from ACE2 for SARS-CoV-2 compared to that for SARS-CoV-1. Subsequently, we considered the RBDs of three variants of SARS-CoV-2, namely N501Y, E484Q/L452R, and N440K. The free energy barrier of protein unbinding for the N501Y variant is found to be ∼4 kcal/mol higher than the wild type SARS-CoV-2 which can be attributed to additional specific interactions involving Tyr501 of RBD and Lys353 and Tyr42 of ACE2 and also enhanced nonspecific electrostatic interaction between the protein surfaces. For the other two mutant variants of E484Q/L452R and N440K, the free energy barrier for protein unbinding increases by ∼2 and ∼1 kcal/mol, respectively, compared with the wild type SARS-CoV-2, which can be attributed to an increase in the number of interprotein hydrogen bonds for the former and also to enhanced positive electrostatic potential on the RBD surfaces for both of the variants. The successive breaking of interprotein hydrogen bonds along the free energy pathway of the unbinding process is also found out for all five systems studied here.