Native Structure-Based Peptides as Potential Protein–Protein Interaction Inhibitors of SARS-CoV-2 Spike Protein and Human ACE2 Receptor

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a positive-strand RNA virus that causes severe respiratory syndrome in humans, which is now referred to as coronavirus disease 2019 (COVID-19). Since December 2019, the new pathogen has rapidly spread globally, with over 65 million cases reported to the beginning of December 2020, including over 1.5 million deaths. Unfortunately, currently, there is no specific and effective treatment for COVID-19. As SARS-CoV-2 relies on its spike proteins (S) to bind to a host cell-surface receptor angiotensin-converting enzyme-2(ACE2), and this interaction is proved to be responsible for entering a virus into host cells, it makes an ideal target for antiviral drug development. In this work, we design three very short peptides based on the ACE2 sequence/structure fragments, which may effectively bind to the receptor-binding domain (RBD) of S protein and may, in turn, disrupt the important virus-host protein–protein interactions, blocking early steps of SARS-CoV-2 infection. Two of our peptides bind to virus protein with affinity in nanomolar range, and as very short peptides have great potential for drug development.     

Degradative Effect of Nattokinase on Spike Protein of SARS-CoV-2

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged as a pandemic and has inflicted enormous damage on the lives of the people and economy of many countries worldwide. However, therapeutic agents against SARS-CoV-2 remain unclear. SARS-CoV-2 has a spike protein (S protein), and cleavage of the S protein is essential for viral entry. Nattokinase is produced by Bacillus subtilis var. natto and is beneficial to human health. In this study, we examined the effect of nattokinase on the S protein of SARS-CoV-2. When cell lysates transfected with S protein were incubated with nattokinase, the S protein was degraded in a dose- and time-dependent manner. Immunofluorescence analysis showed that S protein on the cell surface was degraded when nattokinase was added to the culture medium. Thus, our findings suggest that nattokinase exhibits potential for the inhibition of SARS-CoV-2 infection via S protein degradation.     

Identification of SARS-CoV-2 Receptor Binding Inhibitors by In Vitro Screening of Drug Libraries

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC^®1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.     

Genetic analysis of SARS-CoV-2 isolates collected from Bangladesh: Insights into the origin,mutational spectrum and possible pathomechanism

As the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), rages across the world, killing hundreds of thousands and infecting millions, researchers are racing against time to elucidate the viral genome. Some Bangladeshi institutes are also in this race, sequenced a few isolates of the virus collected from Bangladesh. Here, we present a genomic analysis of these isolates. The analysis revealed that SARS-CoV-2 isolates sequenced from Dhaka and Chittagong were the lineage of Europe and India, respectively. Our analysis identified a total of 42 mutations, including three large deletions, half of which were synonymous. Most of the missense mutations in Bangladeshi isolates found to have weak effects on the pathogenesis. Some mutations may lead the virus to be less pathogenic than the other countries. Molecular docking analysis to evaluate the effect of the mutations on the interaction between the viral spike proteins and the human ACE2 receptor, though no significant difference was observed. This study provides some preliminary insights into the origin of Bangladeshi SARS-CoV-2 isolates, mutation spectrum and its possible pathomechanism, which may give an essential clue for designing therapeutics and management of COVID-19 in Bangladesh.     

In Silico Analysis of the Effects of Omicron Spike Amino Acid Changes on the Interactions with Human Proteins

The SARS-CoV-2 variant Omicron is characterized, among others, by more than 30 amino acid changes occurring on the spike glycoprotein with respect to the original SARS-CoV-2 spike protein. We report a comprehensive analysis of the effects of the Omicron spike amino acid changes in the interaction with human antibodies, obtained by modeling them into selected publicly available resolved 3D structures of spike–antibody complexes and investigating the effects of these mutations at structural level. We predict that the interactions of Omicron spike with human antibodies can be either negatively or positively affected by amino acid changes, with a predicted total loss of interactions only in a few complexes. Moreover, our analysis applied also to the spike-ACE2 interaction predicts that these amino acid changes may increase Omicron transmissibility. Our approach can be used to better understand SARS-CoV-2 transmissibility, detectability, and epidemiology and represents a model to be adopted also in the case of other variants.     

Discovery of Cell‐Permeable Inhibitors That Target the BRCT Domain of BRCA1 Protein by Using a Small‐Molecule Microarray

BRCTs are phosphoserine‐binding domains found in proteins involved in DNA repair, DNA damage response and cell cycle regulation. BRCA1 is a BRCT domain‐containing, tumor‐suppressing protein expressed in the cells of breast and other human tissues. Mutations in BRCA1 have been found in ca. 50 % of hereditary breast cancers. Cell‐permeable, small‐molecule BRCA1 inhibitors are promising anticancer agents, but are not available currently. Herein, with the assist of microarray‐based platforms, we have discovered the first cell‐permeable protein–protein interaction (PPI) inhibitors against BRCA1. By targeting the (BRCT)₂ domain, we showed compound 15 a and its prodrug 15 b inhibited BRCA1 activities in tumor cells, sensitized these cells to ionizing radiation‐induced apoptosis, and showed synergistic inhibitory effect when used in combination with Olaparib (a small‐molecule inhibitor of poly‐ADP‐ribose polymerase) and Etoposide (a small‐molecule inhibitor of topoisomerase II). Unlike previously reported peptide‐based PPI inhibitors of BRCA1, our compounds are small‐molecule‐like and could be directly administered to tumor cells, thus making them useful for future studies of BRCA1/PARP‐related pathways in DNA damage and repair response, and in cancer therapy.     

An Effective MM/GBSA Protocol for Absolute Binding Free Energy Calculations: A Case Study on SARS-CoV-2 Spike Protein and the Human ACE2 Receptor

The binding free energy calculation of protein–ligand complexes is necessary for research into virus–host interactions and the relevant applications in drug discovery. However, many current computational methods of such calculations are either inefficient or inaccurate in practice. Utilizing implicit solvent models in the molecular mechanics generalized Born surface area (MM/GBSA) framework allows for efficient calculations without significant loss of accuracy. Here, GBNSR6, a new flavor of the generalized Born model, is employed in the MM/GBSA framework for measuring the binding affinity between SARS-CoV-2 spike protein and the human ACE2 receptor. A computational protocol is developed based on the widely studied Ras–Raf complex, which has similar binding free energy to SARS-CoV-2/ACE2. Two options for representing the dielectric boundary of the complexes are evaluated: one based on the standard Bondi radii and the other based on a newly developed set of atomic radii (OPT1), optimized specifically for protein–ligand binding. Predictions based on the two radii sets provide upper and lower bounds on the experimental references: −14.7(ΔGbindBondi)<−10.6(ΔGbindExp.)<−4.1(ΔGbindOPT1) kcal/mol. The consensus estimates of the two bounds show quantitative agreement with the experiment values. This work also presents a novel truncation method and computational strategies for efficient entropy calculations with normal mode analysis. Interestingly, it is observed that a significant decrease in the number of snapshots does not affect the accuracy of entropy calculation, while it does lower computation time appreciably. The proposed MM/GBSA protocol can be used to study the binding mechanism of new variants of SARS-CoV-2, as well as other relevant structures.     

Crystallographic and biophysical analysis of the fusion core from SARS-CoV-2 spike protein

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a newly emerging infectious pathogen causing coronavirus disease 2019 (COVID-19). The virus primarily infects cells via its spike glycoprotein, which is cleaved into S1 and S2 subunits to aid in cell attachment and membrane fusion, respectively. Heptad repeat 1 (HR1) and heptad repeat 2 (HR2) of the S2 subunit are essential for membrane fusion, culminating in an expected six-helix bundle termed fusion core. To better understand the structural and biophysical features of the SARS-CoV-2 fusion core, we designed, constructed, and bacterially produced a recombinant single-chain HR1-L6-HR2 protein and conducted a series of biochemical and biophysical experiments. Our findings demonstrate that the HR1-L6-HR2 protein spontaneously assembles into a highly stable trimeric complex, further confirmed by x-ray crystallographic analysis. The crystal structure of the fusion core reveals a trimeric coiled-coil structure of HR1 antiparallelly surrounded by three HR2 to form a six-helical bundle. Additionally, four residues of HR1 that contribute to binding with HR2 through the formation of hydrogen bonds and salt bridges were observed. These results indicate that the SARS-CoV-2 fusion core exhibits similar characteristics to other class I viral glycoproteins, suggesting potential for drug repurposing as an alternative strategy to combat COVID-19.     

Structural Characterization of N-Linked Glycans in the Receptor Binding Domain of the SARS-CoV-2 Spike Protein and their Interactions with Human Lectins

The glycan structures of the receptor binding domain of the SARS-CoV2 spike glycoprotein expressed in human HEK293F cells have been studied by using NMR. The different possible interacting epitopes have been deeply analysed and characterized, providing evidence of the presence of glycan structures not found in previous MS-based analyses. The interaction of the RBD ¹³C-labelled glycans with different human lectins, which are expressed in different organs and tissues that may be affected during the infection process, has also been evaluated by NMR. In particular, ¹⁵N-labelled galectins (galectins-3, -7 and -8 N-terminal), Siglecs (Siglec-8, Siglec-10), and C-type lectins (DC-SIGN, MGL) have been employed. Complementary experiments from the glycoprotein perspective or from the lectin's point of view have permitted to disentangle the specific interacting epitopes in each case. Based on these findings, 3D models of the interacting complexes have been proposed.     

Polyoxygenated Terpenoids and Polyketides from the Roots of Flueggea virosa and Their Inhibitory Effect against SARS-CoV-2-Induced Inflammation

Six new polyoxygenated terpenoids, podovirosanes A–F (1–6), and two known polyketides (7 and 8) were isolated from the roots of F. virosa. Their structures, along with absolute configurations, were deduced using spectroscopic analysis as well as computational calculations, including TDDFT calculation of ECD spectra and GIAO NMR calculations combined with DP4+ probability analysis. Compounds 2, 3, 5, and 8 were found to reduce the phosphorylation levels of NF-κB p65 in SARS-CoV-2 pseudovirus-stimulated PMA-differentiated THP-1 cells.     

Molecular and Structural Analysis of Specific Mutations from Saudi Isolates of SARS-CoV-2 RNA-Dependent RNA Polymerase and their Implications on Protein Structure and Drug–Protein Binding

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has stressed the global health system to a significant level, which has not only resulted in high morbidity and mortality but also poses a threat for future pandemics. This situation warrants efforts to develop novel therapeutics to manage SARS-CoV-2 in specific and other emerging viruses in general. This study focuses on SARS-CoV2 RNA-dependent RNA polymerase (RdRp) mutations collected from Saudi Arabia and their impact on protein structure and function. The Saudi SARS-CoV-2 RdRp sequences were compared with the reference Wuhan, China RdRp using a variety of computational and biophysics-based approaches. The results revealed that three mutations—A97V, P323I and Y606C—may affect protein stability, and hence the relationship of protein structure to function. The apo wild RdRp is more dynamically stable with compact secondary structure elements compared to the mutants. Further, the wild type showed stable conformational dynamics and interaction network to remdesivir. The net binding energy of wild-type RdRp with remdesivir is -50.76 kcal/mol, which is more stable than the mutants. The findings of the current study might deliver useful information regarding therapeutic development against the mutant RdRp, which may further furnish our understanding of SARS-CoV-2 biology.     

Identification of SARS-CoV-2 Main Protease Inhibitors from a Library of Minor Cannabinoids by Biochemical Inhibition Assay and Surface Plasmon Resonance Characterized Binding Affinity

The replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its main protease (M^pro), which is a plausible therapeutic target for coronavirus disease 2019 (COVID-19). Although numerous in silico studies reported the potential inhibitory effects of natural products including cannabis and cannabinoids on SARS-CoV-2 M^pro, their anti-M^pro activities are not well validated by biological experimental data. Herein, a library of minor cannabinoids belonging to several chemotypes including tetrahydrocannabinols, cannabidiols, cannabigerols, cannabichromenes, cannabinodiols, cannabicyclols, cannabinols, and cannabitriols was evaluated for their anti-M^pro activity using a biochemical assay. Additionally, the binding affinities and molecular interactions between the active cannabinoids and the M^pro protein were studied by a biophysical technique (surface plasmon resonance; SPR) and molecular docking, respectively. Cannabinoids tetrahydrocannabutol and cannabigerolic acid were the most active M^pro inhibitors (IC₅₀ = 3.62 and 14.40 μM, respectively) and cannabigerolic acid had a binding affinity KD=2.16×10−4 M). A preliminary structure and activity relationship study revealed that the anti-Mpro effects of cannabinoids were influenced by the decarboxylation of cannabinoids and the length of cannabinoids’ alkyl side chain. Findings from the biochemical, biophysical, and computational assays support the growing evidence of cannabinoids’ inhibitory effects on SARS-CoV-2 M^pro.     

Analyzing the Interaction between Anthocyanins and Native or Heat-Treated Whey Proteins Using Infrared Spectroscopy

The color stability of anthocyanins (ACN) has been shown to be improved by interaction with whey proteins (WP). In this study, we explore the ACN–WP interaction using Fourier transform infrared spectroscopy (IR). ACN from purple corn, grape, and black carrot (50 μM) were evaluated. IR spectra (4000–700 cm⁻¹) were collected for native and preheated (40–80 °C) WP (5 mg/mL) and ACN–WP mixtures at pH 7.4. Soft independent modeling of class analogy was used to analyze the IR data. The WP secondary structure changed after heat treatments and after interaction with ACN. As expected, the WP α-helices decreased, and β-sheet increased after heat treatment. The intensities of the WP amide I and II bands decreased after ACN addition, revealing a decrease in the WP α-helix content. Higher preheating temperatures (70–80 °C) resulted in a more disordered WP structure that favored stronger WP–ACN interactions related to amide III changes. Addition of ACN stabilized WP structure due to heat denaturation, but different ACN structures had different binding affinities with WP. WP structure had less change after interaction with ACN with simpler structures. These results increase our understanding of ACN–WP interactions, providing a potential strategy to extend anthocyanin color stability by WP addition.     

Fruit Bromelain-Derived Peptide Potentially Restrains the Attachment of SARS-CoV-2 Variants to hACE2: A Pharmacoinformatics Approach

Before entering the cell, the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) binds to the human angiotensin-converting enzyme 2 (hACE2) receptor. Hence, this RBD is a critical target for the development of antiviral agents. Recent studies have discovered that SARS-CoV-2 variants with mutations in the RBD have spread globally. The purpose of this in silico study was to determine the potential of a fruit bromelain-derived peptide. DYGAVNEVK. to inhibit the entry of various SARS-CoV-2 variants into human cells by targeting the hACE binding site within the RBD. Molecular docking analysis revealed that DYGAVNEVK interacts with several critical RBD binding residues responsible for the adhesion of the RBD to hACE2. Moreover, 100 ns MD simulations revealed stable interactions between DYGAVNEVK and RBD variants derived from the trajectory of root-mean-square deviation (RMSD), radius of gyration (Rg), and root-mean-square fluctuation (RMSF) analysis, as well as free binding energy calculations. Overall, our computational results indicate that DYGAVNEVK warrants further investigation as a candidate for preventing SARS-CoV-2 due to its interaction with the RBD of SARS-CoV-2 variants.     

Preparation and Characterization of Disulfiram and Beta Cyclodextrin Inclusion Complexes for Potential Application in the Treatment of SARS-CoV-2 via Nebulization

Disulfiram (DS), known as an anti-alcoholism drug, has shown a potent antiviral activity. Still, the potential clinical application of DS is limited by its low water solubility and rapid metabolism. Cyclodextrins (CDs) have been widely used to improve the solubility of drugs in water. In this study, five concentrations of hydroxypropyl β-cyclodextrin (HP) and sulfobutyl ether β-cyclodextrin (SBE) were used to form inclusion complexes of DS for enhanced solubility. Solutions were freeze-dried, and the interaction between DS and CD was characterized using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and Fourier transform infrared spectroscopy (FTIR). In addition, the nebulization properties of the DS–CD solutions were studied. The aqueous solubility of DS increased significantly when loaded to either of both CDs. The phase solubility of both complexes was a linear function of the CD concentration (A_L type). Furthermore, physicochemical characterization studies showed a potent inclusion of the drug in the CD–DS complexes. Aerosolization studies demonstrated that these formulations are suitable for inhalation. Overall, the CD inclusion complexes have great potential for the enhancement of DS solubility. However, further studies are needed to assess the efficacy of DS–CD inclusion complexes against SARS-CoV-2 via nebulization.     

Kinetic Analysis of the Interaction between Nonsteroidal Anti-inflammatory Drugs and Cyclooxygenase-2Using Wavelength Modulation Surface Plasmon Resonance

A surface plasmon resonance (SPR) biosensor based on wavelength modulation technology was developed and validated for the kinetic analysis of the interactions between two nonsteroidal anti‐inflammatory drugs (NSAIDs) and cyclooxygenase‐2 (COX‐2). After the effect of different concentration COX‐2 on the binding capacity of the SPR biosensor surface was studied, the COX‐2 was immobilized covalently onto the biosensor surface using a standard amine coupling method. The affinity constants for indomethacin, ketoprofen binding to COX‐2 are 7.5×10³ L/mol and 9.25×10³ L/mol, respectively. The biosensor surface can be regenerated after being rinsed with 0.01 mol/L NaOH, and the biosensor can be used repeatedly. These indicated that the wavelength modulation SPR biosensor has the potential application in the fields of pharmacokinetics, pharmacodynamics and drug discovery.     

Interaction of small molecules with the SARS-CoV-2 main protease in silico and in vitro validation of potential lead compounds using an enzyme-linked immunosorbent assay

Caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19 pandemic is ongoing, with no proven safe and effective vaccine to date. Further, effective therapeutic agents for COVID-19 are limited, and as a result, the identification of potential small molecule antiviral drugs is of particular importance. A critical antiviral target is the SARS-CoV-2 main protease (M^pro), and our aim was to identify lead compounds with potential inhibitory effects. We performed an initial molecular docking screen of 300 small molecules, which included phenolic compounds and fatty acids from our OliveNet™ library (224), and an additional group of curated pharmacological and dietary compounds. The prototypical α-ketoamide 13b inhibitor was used as a control to guide selection of the top 30 compounds with respect to binding affinity to the M^pro active site. Further studies and analyses including blind docking were performed to identify hypericin, cyanidin-3-O-glucoside and SRT2104 as potential leads. Molecular dynamics simulations demonstrated that hypericin (ΔG = -18.6 and -19.3 kcal/mol), cyanidin-3-O-glucoside (ΔG = -50.8 and -42.1 kcal/mol), and SRT2104 (ΔG = -8.7 and -20.6 kcal/mol), formed stable interactions with the M^pro active site. An enzyme-linked immunosorbent assay indicated that, albeit, not as potent as the covalent positive control (GC376), our leads inhibited the M^pro with activity in the micromolar range, and an order of effectiveness of hypericin and cyanidin-3-O-glucoside > SRT2104 > SRT1720. Overall, our findings, and those highlighted by others indicate that hypericin and cyanidin-3-O-glucoside are suitable candidates for progress to in vitro and in vivo antiviral studies.     

Metallodrug Profiling against SARS-CoV-2 Target Proteins Identifies Highly Potent Inhibitors of the S/ACE2 interaction and the Papain-like Protease PLpro

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has called for an urgent need for dedicated antiviral therapeutics. Metal complexes are commonly underrepresented in compound libraries that are used for screening in drug discovery campaigns, however, there is growing evidence for their role in medicinal chemistry. Based on previous results, we have selected more than 100 structurally diverse metal complexes for profiling as inhibitors of two relevant SARS-CoV-2 replication mechanisms, namely the interaction of the spike (S) protein with the ACE2 receptor and the papain-like protease PL^pro. In addition to many well-established types of mononuclear experimental metallodrugs, the pool of compounds tested was extended to approved metal-based therapeutics such as silver sulfadiazine and thiomersal, as well as polyoxometalates (POMs). Among the mononuclear metal complexes, only a small number of active inhibitors of the S/ACE2 interaction was identified, with titanocene dichloride as the only strong inhibitor. However, among the gold and silver containing complexes many turned out to be very potent inhibitors of PL^pro activity. Highly promising activity against both targets was noted for many POMs. Selected complexes were evaluated in antiviral SARS-CoV-2 assays confirming activity for gold complexes with N-heterocyclic carbene (NHC) or dithiocarbamato ligands, a silver NHC complex, titanocene dichloride as well as a POM compound. These studies might provide starting points for the design of metal-based SARS-CoV-2 antiviral agents.     

Identification of Potential SARS-CoV-2 Main Protease and Spike Protein Inhibitors from the Genus Aloe: An In Silico Study for Drug Development

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) disease is a global rapidly spreading virus showing very high rates of complications and mortality. Till now, there is no effective specific treatment for the disease. Aloe is a rich source of isolated phytoconstituents that have an enormous range of biological activities. Since there are no available experimental techniques to examine these compounds for antiviral activity against SARS-CoV-2, we employed an in silico approach involving molecular docking, dynamics simulation, and binding free energy calculation using SARS-CoV-2 essential proteins as main protease and spike protein to identify lead compounds from Aloe that may help in novel drug discovery. Results retrieved from docking and molecular dynamics simulation suggested a number of promising inhibitors from Aloe. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) calculations indicated that compounds 132, 134, and 159 were the best scoring compounds against main protease, while compounds 115, 120, and 131 were the best scoring ones against spike glycoprotein. Compounds 120 and 131 were able to achieve significant stability and binding free energies during molecular dynamics simulation. In addition, the highest scoring compounds were investigated for their pharmacokinetic properties and drug-likeness. The Aloe compounds are promising active phytoconstituents for drug development for SARS-CoV-2.