A Comprehensive Survey of Prospective Structure-Based Virtual Screening for Early Drug Discovery in the Past Fifteen Years.

Structure-based virtual screening (SBVS), also known as molecular docking, has been increasingly applied to discover small-molecule ligands based on the protein structures in the early stage of drug discovery. In this review, we comprehensively surveyed the prospective applications of molecular docking judged by solid experimental validations in the literature over the past fifteen years. Herein, we systematically analyzed the novelty of the targets and the docking hits, practical protocols of docking screening, and the following experimental validations. Among the 419 case studies we reviewed, most virtual screenings were carried out on widely studied targets, and only 22% were on less-explored new targets. Regarding docking software, GLIDE is the most popular one used in molecular docking, while the DOCK 3 series showed a strong capacity for large-scale virtual screening. Besides, the majority of identified hits are promising in structural novelty and one-quarter of the hits showed better potency than 1 µM, indicating that the primary advantage of SBVS is to discover new chemotypes rather than highly potent compounds. Furthermore, in most studies, only in vitro bioassays were carried out to validate the docking hits, which might limit the further characterization and development of the identified active compounds. Finally, several successful stories of SBVS with extensive experimental validations have been highlighted, which provide unique insights into future SBVS drug discovery campaigns.

Multivalent IgM scaffold enhances the therapeutic potential of variant-agnostic ACE2 decoys against SARS-CoV-2.

As immunological selection for escape mutants continues to give rise to future SARS-CoV-2 variants, novel universal therapeutic strategies against ACE2-dependent viruses are needed. Here we present an IgM-based decavalent ACE2 decoy that has variant-agnostic efficacy. In immuno-, pseudovirus, and live virus assays, IgM ACE2 decoy had potency comparable or superior to leading SARS-CoV-2 IgG-based mAb therapeutics evaluated in the clinic, which were variant-sensitive in their potency. We found that increased ACE2 valency translated into increased apparent affinity for spike protein and superior potency in biological assays when decavalent IgM ACE2 was compared to tetravalent, bivalent, and monovalent ACE2 decoys. Furthermore, a single intranasal dose of IgM ACE2 decoy at 1 mg/kg conferred therapeutic benefit against SARS-CoV-2 Delta variant infection in a hamster model. Taken together, this engineered IgM ACE2 decoy represents a SARS-CoV-2 variant-agnostic therapeutic that leverages avidity to drive enhanced target binding, viral neutralization, and in vivo respiratory protection against SARS-CoV-2.

Balancing Functional Tradeoffs between Protein Stability and ACE2 Binding in the SARS-CoV-2 Omicron BA.2, BA.2.75 and XBB Lineages: Dynamics-Based Network Models Reveal Epistatic Effects Modulating Compensatory Dynamic and Energetic Changes.

Evolutionary and functional studies suggested that the emergence of the Omicron variants can be determined by multiple fitness trade-offs including the immune escape, binding affinity for ACE2, conformational plasticity, protein stability and allosteric modulation. In this study, we systematically characterize conformational dynamics, structural stability and binding affinities of the SARS-CoV-2 Spike Omicron complexes with the host receptor ACE2 for BA.2, BA.2.75, XBB.1 and XBB.1.5 variants. We combined multiscale molecular simulations and dynamic analysis of allosteric interactions together with the ensemble-based mutational scanning of the protein residues and network modeling of epistatic interactions. This multifaceted computational study characterized molecular mechanisms and identified energetic hotspots that can mediate the predicted increased stability and the enhanced binding affinity of the BA.2.75 and XBB.1.5 complexes. The results suggested a mechanism driven by the stability hotspots and a spatially localized group of the Omicron binding affinity centers, while allowing for functionally beneficial neutral Omicron mutations in other binding interface positions. A network-based community model for the analysis of epistatic contributions in the Omicron complexes is proposed revealing the key role of the binding hotspots R498 and Y501 in mediating community-based epistatic couplings with other Omicron sites and allowing for compensatory dynamics and binding energetic changes. The results also showed that mutations in the convergent evolutionary hotspot F486 can modulate not only local interactions but also rewire the global network of local communities in this region allowing the F486P mutation to restore both the stability and binding affinity of the XBB.1.5 variant which may explain the growth advantages over the XBB.1 variant. The results of this study are consistent with a broad range of functional studies rationalizing functional roles of the Omicron mutation sites that form a coordinated network of hotspots enabling a balance of multiple fitness tradeoffs and shaping up a complex functional landscape of virus transmissibility.

Recombinant SARS-CoV-2 Spike Protein Stimulates Secretion of Chymase, Tryptase, and IL-1ß from Human Mast Cells, Augmented by IL-33.

SARS-CoV-2 infects cells via its spike (S) protein binding to its surface receptor angiotensin-converting enzyme 2 (ACE2) and results in the production of multiple proinflammatory cytokines, especially in the lungs, leading to what is known as COVID-19. However, the cell source and the mechanism of secretion of such cytokines have not been adequately characterized. In this study, we used human cultured mast cells that are plentiful in the lungs and showed that recombinant SARS-CoV-2 full-length S protein (1-10 ng/mL), but not its receptor-binding domain (RBD), stimulates the secretion of the proinflammatory cytokine interleukin-1ß (IL-1ß) as well as the proteolytic enzymes chymase and tryptase. The secretion of IL-1ß, chymase, and tryptase is augmented by the co-administration of interleukin-33 (IL-33) (30 ng/mL). This effect is mediated via toll-like receptor 4 (TLR4) for IL-1ß and via ACE2 for chymase and tryptase. These results provide evidence that the SARS-CoV-2 S protein contributes to inflammation by stimulating mast cells through different receptors and could lead to new targeted treatment approaches.

Supramolecular Nanofibers Block SARS-CoV-2 Entry into Human Host Cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection relies on its spike protein binding to angiotensin-converting enzyme 2 (ACE2) on host cells to initiate cellular entry. Blocking the interactions between the spike protein and ACE2 offers promising therapeutic opportunities to prevent infection. We report here on peptide amphiphile supramolecular nanofibers that display a sequence from ACE2 in order to promote interactions with the SARS-CoV-2 spike receptor binding domain. We demonstrate that displaying this sequence on the surface of supramolecular assemblies preserves its α-helical conformation and blocks the entry of a pseudovirus and its two variants into human host cells. We also found that the chemical stability of the bioactive structures was enhanced in the supramolecular environment relative to the unassembled peptide molecules. These findings reveal unique advantages of supramolecular peptide therapies to prevent viral infections and more broadly for other targets as well.

Zn2+ and Cu2+ Interaction with the Recognition Interface of ACE2 for SARS-CoV-2 Spike Protein.

The spike protein (S) of SARS-CoV-2 is able to bind to the human angiotensin-converting enzyme 2 (ACE2) receptor with a much higher affinity compared to other coronaviruses. The binding interface between the ACE2 receptor and the spike protein plays a critical role in the entry mechanism of the SARS-CoV-2 virus. There are specific amino acids involved in the interaction between the S protein and the ACE2 receptor. This specificity is critical for the virus to establish a systemic infection and cause COVID-19 disease. In the ACE2 receptor, the largest number of amino acids playing a crucial role in the mechanism of interaction and recognition with the S protein is located in the C-terminal part, which represents the main binding region between ACE2 and S. This fragment is abundant in coordination residues such as aspartates, glutamates, and histidine that could be targeted by metal ions. Zn2+ ions bind to the ACE2 receptor in its catalytic site and modulate its activity, but it could also contribute to the structural stability of the entire protein. The ability of the human ACE2 receptor to coordinate metal ions, such as Zn2+, in the same region where it binds to the S protein could have a crucial impact on the mechanism of recognition and interaction of ACE2-S, with consequences on their binding affinity that deserve to be investigated. To test this possibility, this study aims to characterize the coordination ability of Zn2+, and also Cu2+ for comparison, with selected peptide models of the ACE2 binding interface using spectroscopic and potentiometric techniques.

Allosteric communication between ACE2 active site and binding interface with SARS-CoV-2.

SARS-CoV-2, the virus causing COVID-19, initiates cell invasion by deploying a receptor binding domain (RBD) to recognize the host transmembrane peptidase angiotensin-converting enzyme 2 (ACE2). Numerous experimental and theoretical studies have adopted high-throughput and structure-guided approaches to (i) understand how the RBD recognizes ACE2, (ii) rationalize, and (iii) predict the effect of viral mutations on the binding affinity. Here, we investigate the allosteric signal triggered by the dissociation of the ACE2-RBD complex. To this end, we construct an Elastic Network Model (ENM), and we use the Structural Perturbation Method (SPM). Our key result is that complex dissociation opens the ACE2 substrate-binding cleft located away from the interface and that fluctuations of the ACE2 binding cleft are facilitated by RBD binding. These and other observations provide a structural and dynamical basis for the influence of SARS-CoV-2 on ACE2 enzymatic activity. In addition, we identify a conserved glycine (G502 in SARS-CoV-2) as a key participant in complex disassembly.

Correlating the differences in the receptor binding domain of SARS-CoV-2 spike variants on their interactions with human ACE2 receptor.

Spike glycoprotein of SARS-CoV-2 variants plays a critical role in infection and transmission through its interaction with human angiotensin converting enzyme 2 (hACE2) receptors. Prior findings using molecular docking and biomolecular studies reported varied findings on the difference in the interactions among the spike variants with the hACE2 receptors. Hence, it is a prerequisite to understand these interactions in a more precise manner. To this end, firstly, we performed ELISA with trimeric spike glycoproteins of SARS-CoV-2 variants including Wuhan Hu-1(Wild), Delta, C.1.2 and Omicron. Further, to study the interactions in a more specific manner by mimicking the natural infection, we developed hACE2 receptors expressing HEK-293T cell line, evaluated their binding efficiencies and competitive binding of spike variants with D614G spike pseudotyped virus. In line with the existing findings, we observed that Omicron had higher binding efficiency compared to Delta in both ELISA and Cellular models. Intriguingly, we found that cellular models could differentiate the subtle differences between the closely related C.1.2 and Delta in their binding to hACE2 receptors. Our study using the cellular model provides a precise method to evaluate the binding interactions between spike sub-lineages to hACE2 receptors.

Molecular mechanism study of the structural regulation of the N-terminal domain binding antibody on the receptor binding domain of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the global spread of the coronavirus disease (COVID-19), which has caused great loss of life and property worldwide. We investigated the regulatory mechanism with the antibody targeting the N-terminal domain (NTD) of the S protein by molecular dynamic simulation. It was found that the structure of the S1-4A8 complex experienced the largest change when the receptor binding domain (RBD) of S1 was in the Up state. By calculating the angle between domains of S1 in the Down and Up states, we found that the RBD angle changed more in the Up state. We further performed binding free energy calculations for S1-4A8 complexes in both Up and Down states, and the results showed that 4A8 has a stronger affinity with NTD in the Up state. These results indicate that 4A8 plays a stronger regulatory role in the RBD Up state. The N3 and N5 loops on the NTD are the main antigen-antibody binding sites, and residues on the antibody complementarity determining region 3 (CDR3) in the Up state can penetrate deeper into the hydrophobic pocket at the bottom of the N5 loop to form a tighter binding. Through the tICA method, we found that except the residues at the binding interface, distant residues including A609, V610, G652, and A653 at the linker region of subdomain 2, and residues S359 and N360 near the bottom of RBD are able to influence the regulatory effect in the long-range. This work provides new insights into the neutralization mechanism of targeting NTD antibodies in SARS-CoV-2.

Novel Polymyxin-Inspired Peptidomimetics Targeting the SARS-CoV-2 Spike:hACE2 Interface.

Though the bulk of the COVID-19 pandemic is behind, the search for effective and safe anti-SARS-CoV-2 drugs continues to be relevant. A highly pursued approach for antiviral drug development involves targeting the viral spike (S) protein of SARS-CoV-2 to prevent its attachment to the cellular receptor ACE2. Here, we exploited the core structure of polymyxin B, a naturally occurring antibiotic, to design and synthesize unprecedented peptidomimetics (PMs), intended to target contemporarily two defined, non-overlapping regions of the S receptor-binding domain (RBD). Monomers 1, 2, and 8, and heterodimers 7 and 10 bound to the S-RBD with micromolar affinity in cell-free surface plasmon resonance assays (KD ranging from 2.31 µM to 2.78 µM for dimers and 8.56 µM to 10.12 µM for monomers). Although the PMs were not able to fully protect cell cultures from infection with authentic live SARS-CoV-2, dimer 10 exerted a minimal but detectable inhibition of SARS-CoV-2 entry in U87.ACE2+ and A549.ACE2.TMPRSS2+ cells. These results validated a previous modeling study and provided the first proof-of-feasibility of using medium-sized heterodimeric PMs for targeting the S-RBD. Thus, heterodimers 7 and 10 may serve as a lead for the development of optimized compounds, which are structurally related to polymyxin, with improved S-RBD affinity and anti-SARS-CoV-2 potential.

Design and Identification of Inhibitors for the Spike-ACE2 Target of SARS-CoV-2.

When an epidemic started in the Chinese city of Wuhan in December 2019, coronavirus was identified as the cause. Infection by the virus occurs through the interaction of viral S protein with the hosts' angiotensin-converting enzyme 2. By leveraging resources such as the DrugBank database and bioinformatics techniques, ligands with potential activity against the SARS-CoV-2 spike protein were designed and identified in this investigation. The FTMap server and the Molegro software were used to determine the active site of the Spike-ACE2 protein's crystal structure. Virtual screening was performed using a pharmacophore model obtained from antiparasitic drugs, obtaining 2000 molecules from molport®. The ADME/Tox profiles were used to identify the most promising compounds with desirable drug characteristics. The binding affinity investigation was then conducted with selected candidates. A molecular docking study showed five structures with better binding affinity than hydroxychloroquine. Ligand_003 showed a binding affinity of -8.645 kcal·mol-1, which was considered an optimal value for the study. The values presented by ligand_033, ligand_013, ligand_044, and ligand_080 meet the profile of novel drugs. To choose compounds with favorable potential for synthesis, synthetic accessibility studies and similarity analyses were carried out. Molecular dynamics and theoretical IC50 values (ranging from 0.459 to 2.371 µM) demonstrate that these candidates are promising for further tests. Chemical descriptors showed that the candidates had strong molecule stability. Theoretical analyses here show that these molecules have potential as SARS-CoV-2 antivirals and therefore warrant further investigation.

Investigation on the binding behavior of human α1-acid glycoprotein with Janus Kinase inhibitor baricitinib: Multi-spectroscopic and molecular simulation methodologies.

Baricitinib is a Janus Kinase (JAK) inhibitor that is primarily used to treat moderately to severely active rheumatoid arthritis in adults and has recently been reported for the treatment of patients with severe COVID-19. This paper describes the investigation of the binding behavior of baricitinib to human α1-acid glycoprotein (HAG) employing a variety of spectroscopic techniques, molecular docking and dynamics simulations. Baricitinib can quench the fluorescence from amino acids in HAG through a mix of dynamic and static quenching, according to steady-state fluorescence and UV spectra observations, but it is mainly static quenching at low concentration. The binding constant (Kb) of baricitinib to HAG at 298 K was at the level of 104 M-1, indicating a moderate affinity of baricitinib to HAG. Hydrogen bonding and hydrophobic interactions conducted the main effect, according to thermodynamic characteristics, competition studies between ANS and sucrose, and molecular dynamics simulations. For the change in HAG conformation, the results of multiple spectra showed that baricitinib was able to alter the secondary structure of HAG as well as increase the polarity of the microenvironment around the Trp amino acid. Furthermore, the binding behavior of baricitinib to HAG was investigated by molecular docking and molecular dynamics simulations, which validated experimental results. Also explored is the influence of K+, Co2+, Ni2+, Ca2+, Fe3+, Zn2+, Mg2+ and Cu2+plasma on binding affinity.

Identification and characterization of 7-azaindole derivatives as inhibitors of the SARS-CoV-2 spike-hACE2 protein interaction.

The COVID-19 pandemic, caused by SARS-CoV-2, has become a global public health crisis. The entry of SARS-CoV-2 into host cells is facilitated by the binding of its spike protein (S1-RBD) to the host receptor hACE2. Small molecule compounds targeting S1-RBD-hACE2 interaction could provide an alternative therapeutic strategy sensitive to viral mutations. In this study, we identified G7a as a hit compound that targets the S1-RBD-hACE2 interaction, using high-throughput screening in the SARS2-S pseudovirus model. To enhance the antiviral activity of G7a, we designed and synthesized a series of novel 7-azaindole derivatives that bind to the S1-RBD-hACE2 interface. Surprisingly, ASM-7 showed excellent antiviral activity and low cytotoxicity, as confirmed by pseudovirus and native virus assays. Molecular docking and molecular dynamics simulations revealed that ASM-7 could stably bind to the binding interface of S1-RBD-hACE2, forming strong non-covalent interactions with key residues. Furthermore, the binding of ASM-7 caused alterations in the structural dynamics of both S1-RBD and hACE2, resulting in a decrease in their binding affinity and ultimately impeding the viral invasion of host cells. Our findings demonstrate that ASM-7 is a promising lead compound for developing novel therapeutics against SARS-CoV-2.

How helpful were molecular dynamics simulations in shaping our understanding of SARS-CoV-2 spike protein dynamics?

The SARS-CoV-2 spike protein (S) represents an important viral component that is required for successful viral infection in humans owing to its essential role in recognition of and entry to host cells. The spike is also an appealing target for drug designers who develop vaccines and antivirals. This article is important as it summarizes how molecular simulations successfully shaped our understanding of spike conformational behavior and its role in viral infection. MD simulations found that the higher affinity of SARS-CoV-2-S to ACE2 is linked to its unique residues that add extra electrostatic and van der Waal interactions in comparison to the SARS-CoV S. This illustrates the spread potential of the pandemic SARS-CoV-2 relative to the epidemic SARS-CoV. Different mutations at the S-ACE2 interface, which is believed to increase the transmission of the new variants, affected the behavior and binding interactions in different simulations. The contributions of glycans to the opening of S were revealed via simulations. The immune evasion of S was linked to the spatial distribution of glycans. This help the virus to escape the immune system recognition. This article is important as it summarizes how molecular simulations successfully shaped our understanding of spike conformational behavior and its role in viral infection. This will pave the way to us preparing for the next pandemic as the computational tools are tailored to help fight new challenges.

Different electrostatic forces drive the binding kinetics of SARS-CoV, SARS-CoV-2 and MERS-CoV Envelope proteins with the PDZ2 domain of ZO1.

The Envelope protein (E) is a structural protein encoded by the genome of SARS-CoV, SARS-CoV-2 and MERS-CoV Coronaviruses. It is poorly present in the virus but highly expressed in the host cell, with prominent role in virus assembly and virulence. The E protein possesses a PDZ-binding motif (PBM) at its C terminus that allows it to interact with host PDZ domain containing proteins. ZO1 is a key protein in assembling the cytoplasmic plaque of epithelial and endothelial Tight Junctions (TJs) as well as in determining cell differentiation, proliferation and polarity. The PDZ2 domain of ZO1 is known to interact with the Coronaviruses Envelope proteins, however the molecular details of such interaction have not been established. In this paper we directly measured, through Fluorescence Resonance Energy Transfer and Stopped-Flow methodology, the binding kinetics of the PDZ2 domain of ZO1 with peptides mimicking the C-terminal portion of the Envelope protein from SARS-CoV, SARS-CoV-2 and MERS-CoV in different ionic strength conditions. Interestingly, the peptide mimicking the E protein from MERS-CoV display much higher microscopic association rate constant with PDZ2 compared to SARS-CoV and SARS-CoV-2 suggesting a stronger contribution of electrostatic forces in the early events of binding. A comparison of thermodynamic and kinetic data obtained at increasing ionic strengths put in evidence different contribution of electrostatics in the recognition and complex formation events for the three peptides. Our data are discussed under the light of available structural data of PDZ2 domain of ZO1 and of previous works about these protein systems.

Molecular Insights into the Binding Behavior of Imidazolium Ionic Liquids to the Receptor Binding Domain of the SARS-CoV-2 Spike Protein.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is considered as a key target for the design and development of COVID-19 drugs and inhibitors. Due to their unique structure and properties, ionic liquids (ILs) have many special interactions with proteins, showing great potential in biomedicine. Nevertheless, few research studies have been carried out on ILs and the spike RBD protein. Here, we explore the interaction of ILs and the RBD protein through large-scale molecular dynamics simulations (4 µs in total). It was found that IL cations with long alkyl chain lengths (nchain) could spontaneously bind to the cavity region of the RBD protein. The longer the alkyl chain is, the stabler the cations bind to the protein. The binding free energy (ΔG) had the same trend, peaking at nchain = 12 with -101.19 kJ/mol. The cationic chain lengths and their fit to the pocket are decisive factors that influence the binding strength of cations and proteins. The cationic imidazole ring has a high contact frequency with phenylalanine and tryptophan, and the hydrophobic residues phenylalanine, valine, leucine, and isoleucine are the most interacting residues with side chains of cations. Meanwhile, through analysis of the interaction energy, the hydrophobic and π-π interactions are the main contributors to the high affinity between cations and the RBD protein. In addition, the long-chain ILs would also act on the protein through clustering. These studies not only provide insights into the molecular interaction between ILs and the RBD of SARS-CoV-2 but also contribute to the rational design of IL-based drugs, drug carriers, and selective inhibitors as a therapeutic for SARS-CoV-2.

Discovery of dual S-RBD/NRP1-targeting peptides: structure-based virtual screening, synthesis, biological evaluation, and molecular dynamics simulation studies.

Both receptor-binding domain in spike protein (S-RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human neuropilin-1 (NRP1) are important in the virus entry, and their concomitant inhibition may become a potential strategy against the SARS-CoV-2 infection. Herein, five novel dual S-RBD/NRP1-targeting peptides with nanomolar binding affinities were identified by structure-based virtual screening. Particularly, RN-4 was found to be the most promising peptide targeting S-RBD (Kd = 7.4 ± 0.5 nM) and NRP1-BD (the b1 domain of NRP1) (Kd = 16.1 ± 1.1 nM) proteins. Further evidence in the pseudovirus infection assay showed that RN-4 can significantly inhibit the SARS-CoV-2 pseudovirus entry into 293 T cells (EC50 = 0.39 ± 0.09 µM) without detectable side effects. These results suggest that RN-4, a novel dual S-RBD/NRP1-targeting agent, holds potential as an effective therapeutic to combat the SARS-CoV-2 infection.

Inhibition of SARS-CoV-2-mediated thromboinflammation by CLEC2.Fc.

Thromboinflammation is the major cause of morbidity and mortality in COVID-19 patients, and post-mortem examination demonstrates the presence of platelet-rich thrombi and microangiopathy in visceral organs. Moreover, persistent microclots were detected in both acute COVID-19 and long COVID plasma samples. However, the molecular mechanism of SARS-CoV-2-induced thromboinflammation is still unclear. We found that the spleen tyrosine kinase (Syk)-coupled C-type lectin member 2 (CLEC2), which was highly expressed in platelets and alveolar macrophages, interacted with the receptor-binding domain (RBD) of SARS-CoV-2 spike protein (SARS-CoV-2 RBD) directly. Unlike the thread-like NETs, SARS-CoV-2-induced aggregated NET formation in the presence of wild-type (WT), but not CLEC2-deficient platelets. Furthermore, SARS-CoV-2 spike pseudotyped lentivirus was able to induce NET formation via CLEC2, indicating SARS-CoV-2 RBD engaged CLEC2 to activate platelets to enhance NET formation. Administration of CLEC2.Fc inhibited SARS-CoV-2-induced NET formation and thromboinflammation in AAV-ACE2-infected mice. Thus, CLEC2 is a novel pattern recognition receptor for SARS-CoV-2, and CLEC2.Fc and may become a promising therapeutic agent to inhibit SARS-CoV-2-induced thromboinflammation and reduced the risk of post-acute sequelae of COVID-19 (PASC) in the future.

In silico design of miniprotein to inhibit SARS-CoV-2 variant Omicron spike protein.

Omicron is a novel variant of SARS-CoV-2 that is currently spreading globally as the dominant strain. The virus first enters the host cell through the receptor binding domain (RBD) of the spike protein by interacting with the angiotensin-converting enzyme 2 (ACE2). Thus, the RBD protein is an ideal target for the design of drugs against the Omicron variant. Here, we designed several miniprotein inhibitors in silico to combat the SARS-CoV-2 Omicron variant using single- and double-point mutation approaches, based on the structure of the initial inhibitor AHB2. Also, two parallel molecular dynamics (MD) simulations were performed for each system to reproduce the calculated results, and the binding free energy was evaluated with the MM/PBSA method. The evaluated values showed that all inhibitors, including AHB2, M7E, M7E + M43W, and M7E + M43Y, were energetically more beneficial to the binding with the RBD than ACE2. In particular, the mutant inhibitor M7E + M43Y possessed the highest binding affinity to RBD and was selected as the most promising "best" inhibitor among all inhibitors. In addition, the combination of multiple analysis methods, such as free energy landscape analysis (FEL), principal component analysis (PCA), dynamic cross-correlation matrix analysis (DCCM), and hydrogen bond, salt bridge, and hydrophobic interaction analysis, also demonstrated that the mutations significantly affect the dynamical behavior and binding pattern of the inhibitor binding to the RBD protein. The current work suggested that miniprotein inhibitors can form stable complex structures with the RBD protein and exert a blocking or inhibitory effect on the SARS-CoV-2 variant Omicron. In conclusion, this study has identified several novel mutant inhibitors with enhanced affinity to the RBD protein, and provided potential guidance and insights for the rational design of therapeutic approaches for the new SARS-CoV-2 variant Omicron.

Oxalic acid blocked the binding of spike protein from SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variants to human angiotensin-converting enzymes 2.

An epidemic of Corona Virus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading worldwide. Moreover, the emergence of SARS-CoV-2 variants of concern, such as Delta and Omicron, has seriously challenged the application of current therapeutics including vaccination and drugs. Relying on interaction of spike protein with receptor angiotensin-converting enzymes 2 (ACE2), SARS-CoV-2 successfully invades to the host cells, which indicates a strategy that identification of small-molecular compounds to block the entry is of great significance for COVID-19 prevention. Our study evaluated the potential efficacy of natural compound oxalic acid (OA) as an inhibitory agent against SARS-CoV-2 invasion, particular on the interaction of the receptor binding domain (RBD) of Delta and Omicron variants to ACE2. By employing a competitive binding assay in vitro, OA significantly blocked the binding of RBDs from Delta B.1.617.2 and Omicron B.1.1.529 to ACE2, but has no effect on the wide-type SARS-CoV-2 strain. Furthermore, OA inhibited the entries of Delta and Omicron pseudovirus into ACE2 high expressing-HEK293T cells. By surface plasmon resonance (SPR) assay, the direct bindings of OA to RBD and ACE2 were analyzed and OA had both affinities with RBDs of B.1.617.2 and B.1.1.529 and with ACE2. Molecular docking predicted the binding sites on the RBD-ACE2 complex and it showed similar binding abilities to both complex of variant Delta or Omicron RBD and ACE2. In conclusion, we provided a promising novel small-molecule compound OA as an antiviral candidate by blocking the cellular entries of SARS-CoV-2 variants.