Protein Receptor Binding Domain Research Articles

Computationally Guided Liquid Crystal‐Based Competitive Binding Sensing Platform for Optical Detection of Spike Protein

AbstractA liquid crystal (LC)‐based sensing platform for the rapid optical detection of SARS‐CoV‐2′s spike protein's receptor binding domain (RBD) domain is introduced. This platform utilizes a thermotropic LC, hosted on metal‐cation decorated substrates, onto which the spike protein can competitively bind. Density functional theory (DFT) calculations guide the experiments that reveal a homeotropic‐to‐planar transition in the LCs upon exposure to SARS‐CoV‐2 spike‐decorated yeast, providing a basis for sensitive virus detection. The sensor's reversibility/specificity is confirmed through antibody‐induced orientation recovery of the LCs initial orientation. Strikingly, the sensor can detect ≈2000 copies of the spike protein per mL, which is well below the typical concentration of the virus in the saliva of an infected human (104 copies per mL)‐ revealing the practical applicability of the sensor. More broadly, it describes the design principles of the DFT‐guided LC‐based competitive binding platform for the detection of previously unknown pathogens for which antibody‐based detection mechanisms may not be readily available.

Population pharmacokinetics and pharmacodynamics of HFB30132A, a monoclonal antibody against SARS-CoV-2, in healthy Chinese and US subjects.

Development of neutralizing monoclonal antibody (nAb) is a strategy for treatment of infections caused by SARS-CoV-2. This study evaluated the pharmacokinetics (PK) and pharmacodynamics (PD) of HFB30132A, a fully human nAb targeting SARS-CoV-2 spike protein receptor binding domain, in healthy subjects. Randomized, double-blind, placebo-controlled phase I trial was performed in healthy Chinese and US subjects, respectively. The subjects (n=44) received a single ascending dose (400, 1000, 2000 mg) or placebo. Safety and PK data were analysed. PD was evaluated by pseudovirus neutralization test in vitro using serum samples of Chinese subjects. Population PK/PD model was developed using non-linear mixed effects modeling. Effect of covariates was evaluated via covariate screening, Monte Carlo simulation, and randomisation test. The PK profile was consistent with three-compartment model. The clearance (CL) and V1 were 0.38 mL/h and 2.9 L, respectively. Ethnicity and body weight (BW) were factors affecting PK. Compared to the subjects who are not Hispanic or Latino, AUC0-∞ increased by 64% in the healthy subjects of Han nationality. The PD was consistent with effect-compartment model when ND50 titre (reciprocal of 50% neutralization dilution) was used as PD index. Emax reduced along with time, consistent with exponential model. The EC50 was 4590 mg/L. Half-life for reduction of Emax was 133 days. Albumin, lymphocytes, neutrophils or monocytes were covariates on PD. There was ethnic difference in PK, and tolerance in PD of HFB30132A. Population PK/PD model characterized dose-exposure-response relationship of HFB30132A in healthy subjects. These findings are useful for drug development in the future. Clinical trial registration: ClinicalTrial.gov NCT04590430, NCT05275660.

Assessing the Dynamic Interplay of Omicron Spike Protein Receptor-Binding Domain (S-RBD) IgG Antibody Responses in Hospitalized COVID-19 Patients in Egypt: An Original Article

Assessing the Dynamic Interplay of Omicron Spike Protein Receptor-Binding Domain (S-RBD) IgG Antibody Responses in Hospitalized COVID-19 Patients in Egypt: An Original Article

Predictive Insights Into Bioactive Compounds from Streptomyces as Inhibitors of SARS-CoV-2 Mutant Strains by Receptor Binding Domain: Molecular Docking and Dynamics Simulation Approaches

Background: The receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 interacts with the angiotensin-converting enzyme 2 (ACE2) receptor in humans. To date, numerous SARS-CoV-2 variants, particularly those involving mutations in the RBD, have been identified. These variants exhibit differences in transmission, pathogenicity, diagnostics, and vaccine efficacy. Objectives: Although therapeutic agents are currently available to inhibit SARS-CoV-2, most provide supportive and symptomatic relief. Moreover, different variants may exhibit resistance to these treatments. This study aimed to identify a potential compound with favorable antiviral effects against SARS-CoV-2 variants. Methods: The study explored drug discovery through structure-based virtual screening of natural products (NPs) from the StreptomeDB database, targeting the ACE2-binding pocket of the SARS-CoV-2 RBD protein. The analysis included the wild-type protein (PDB ID: 6VW1) as well as the Alpha, Beta, Delta, Lambda, Omicron/BA.1, and Omicron/BA.2 variants. Results: In silico screening identified ‘Stambomycin B’ as a potential compound with the highest binding affinity. Molecular dynamics simulations of the complexes, conducted over 100 ns, confirmed the prediction that ‘Stambomycin B’ could inhibit different SARS-CoV-2 variants effectively. Conclusions: This study concludes that ‘Stambomycin B’, a macrolide compound produced by Streptomycesambofaciens, may be a candidate NP for effectively combating all mutants that occur in the binding of SARS-CoV-2 RBD to ACE2, even those that may arise in the future.

Safety and immunogenicity of a SARS-CoV-2 recombinant protein subunit vaccine adjuvanted with Alum + CpG 1018 in healthy Indonesian adults: A multicenter, randomized, comparative, observer-blind, placebo-controlled phase 2 study

ABSTRACT Globally, dozens of COVID-19 vaccines are licensed under emergency or conditional authorization, but especially in low and middle-income countries, their availability varies. Indonesia decided to become independent and produce its own vaccines locally. This study investigated the safety and immunogenicity of a SARS-CoV-2 recombinant protein subunit vaccine adjuvanted with Alum + CpG 1018. This study involved 360 adults aged 18 years and above. It compared two vaccine dosages, a-12.5 µg and a 25-µg dose of receptor binding domain protein, to a placebo (1:1:1). A total of 40.6% of participants in this study experienced at least one adverse event (AE), with most being mild. There was no statistically significant difference in AEs between the groups. The microneutralization test showed the highest neutralizing antibody titer (IU/mL) in the 25 µg dose vaccine group at day 28 after the second dose (3,300 95%CI 2,215-4,914), although it was not statistically different from the 12.5 µg dose group (3,157 95%CI 2,135-4,669). Similarly, IgG antibody concentrations in the 25 µg dose vaccine group at day 28 were the highest compared to the 12.5 µg dose and placebo. According to protocol, only the formulation with the better antibody profile and comparable reactogenicity was further evaluated at months three and six. Thus, follow-up was only performed for the 25 µg dose vaccine, demonstrating antibody persistence at month six and had a favorable safety profile. These results position this SARS-CoV-2 recombinant protein subunit vaccine adjuvanted with Alum + CpG 1018 as a promising candidate to fight against COVID-19.

Receptor binding and structural basis of raccoon dog ACE2 binding to SARS-CoV-2 prototype and its variants.

Raccoon dog was proposed as a potential host of SARS-CoV-2, but no evidence support such a notion. In our study, we investigated the binding affinities of raccoon dog ACE2 (rdACE2) to the spike (S) protein receptor binding domain (RBD) of SARS-CoV-2 prototype (PT) and its variants. It revealed that the binding affinities of RBD from SARS-CoV-2 variants were generally lower than that of the PT RBD. Through structural and functional analyses, we found amino acids H34 and M82 play pivotal roles in maintaining the binding affinity of ACE2 to different SARS-CoV-2 sub-variants. These results suggest that raccoon dogs exhibit lower susceptibility to SARS-CoV-2 compared to those animal species with a high prevalence of SARS-CoV-2 transmission.

Exploring the antimicrobial and antioxidative potential of Leucas aspera (Willd.) Link: Phytochemical screening, molecular docking, and HR-LC/MS profiling against SARS-CoV-2 protein 3CLPro, Spike and RDB

BackgroundLeucas aspera (Willd.) Link, a member of the Lamiaceae family and commonly known as "Thumbai," is found throughout India and has been utilized in traditional Indian medicine to treat various ailments. PurposeThis study entails methanolic extraction of Leucas aspera (Willd.) Link using a Soxhlet apparatus, followed by phytochemical screening and antioxidant activity assessment. MethodsThe L. aspera methanolic leaf extract displayed a phenolic content of 100 µg/ml, consisting of 3.02 mg of gallic acid dry weight equivalents and a flavonoid content of 100 µg/ml with 3.35 mg quercetin equivalents per dry weight. The extract's free radical scavenging capabilities were at 150 µg/ml in DPPH, showing an 87% effectiveness compared to the standard ascorbic acid 49% inhibition capacity. The ABTS radical scavenging activity in 100 µg/ml extract measured 64.32%, phosphomolybdate assay indicated 86.89%, and hydroxide radical scavenging capacity showed 126.72%. Antibacterial activity was also assessed through agar well diffusion assays against Klebsiella pneumoniae, Salmonella typhi, Staphylococcus aureus, and Leucobacter sp., Where K. pneumoniae exhibits the highest zone of inhibition. The methanolic crude extract of L. aspera was further using TLC, FT-IR and HR-LC/MS to identify functional groups and phytocompound present in the extract. ResultsHRLC-MS was used to conduct metabolite profiling of the methanolic crude extract of L. aspera leaves, discovering 37 positive and 43 negative compounds. To evaluate the ADMET properties and predict the drug-likeness of these 80 phytochemicals, an in silico analysis was performed. The results of this analysis revealed that only 19 of the compounds met the ADMET limitations and had suitable Log P values. Molecular docking of the 19 compounds against the SARS-CoV-2 main protease (3CLPro) protein (PDB ID: 6LU7) and spike protein receptor binding domain (SGp-RBD) protein (PDB ID: 2GHV) revealed Famprofazone and Loxtidine compounds having the highest binding affinity with LibDock scores of 105.53 and 116.70 respectively. ConclusionThus, our findings could serve as potential active molecules of L. aspera against this target protein. This study provides further proof of bioactive compounds produced by the L. aspera leaf extract.

A Novel Circular Delta-XBB15 RBD Dimeric Protein Subunit Vaccine Mediated by Split Intein Elicits an Immune Response and Protection Against Multiple SARS-CoV-2 Variants in Mice.

SARS-CoV-2 continues to mutate, leading to breakthrough infections. The development of new vaccine strategies to combat various strains is crucial. Protein cyclization can enhance thermal stability and may improve immunogenicity. Here, we designed a cyclic tandem dimeric receptor-binding domain protein (cirRBD2) via the split intein Cth-Ter. Cyclization does not affect the antigen epitopes of RBD but results in better thermal stability than that of its linear counterpart (linRBD2). Compared with the miceimmunized with linRBD2, those immunized with two doses of 5 μg of cirRBD2 produced significantly greater levels of broad-spectrum neutralizing antibodies, and generated a considerable cellular immune response. In the VEEV-VRP-hACE2-transduced mouse model, two doses of 5 μg of cirRBD2 provided protection against infection with BA.5, XBB.1.9, and partial protection against EG.5 which has more mutations. This study developed a novel circular RBD dimer subunit vaccine for SARS-CoV-2 that exhibits broad-spectrum neutralizing activity against various variants. A similar strategy can be applied to develop vaccines for other pathogens, especially for thermally stable vaccines.

Adjuvants to the S1-subunit of the SARS-CoV-2 spike protein vaccine improve antibody and T cell responses and surrogate neutralization in mice

Various public health measures have contained outbreaks of SARS-CoV-2, but concerns remain over the possibility of future surges. Improvements in broadening the vaccine response can stifle new and nascent infections. In this study, we tested the effects of different adjuvant combinations on the immunization of mice with the receptor-binding domain (RBD)-containing the S1-subunit of the spike protein (S1 protein) from SARS-CoV-2 to induce a robust humoral and cellular immune response. We showed that subcutaneous immunization of S1 protein co-delivered with IL-15 and TLR-ligands (MALP-2, poly I: C, and CpG) or with IL-12 and GM-CSF in DOTAP, or Alum induced significantly high titers of durable antibodies, predominantly IgG1, IgG2a, and IgG2b, that could bind to RBD, S1-subunit, and the full-length ectodomain of SARS-CoV-2 spike protein in sera compared to the immunization with S1 protein alone in both B6 wild-type (WT) and the K18-hACE2 transgenic mice. In addition, immunization with S1 protein co-delivered with IL-15 and TLR-ligands induced antibody responses against S1 protein in aged mice, and sera from younger mice reduced plaque formation of live SARS-CoV-2, and had effective binding to S1 protein from ten different variants of SARS-CoV-2, including Omicron (B.1.1.529), and greater neutralization activity as early as day 21 post-immunization measured by inhibition of RBD binding to hACE2 than sera from mice immunized with S1 protein alone or co-delivered with Alum. We also identified antibody-binding epitopes using 18-mer peptides with 9-residue overlaps from the S1 protein. CD8+ T-cell responses specific to RBD and S1 protein peptide pools were observed up to day 200 post-immunization by tetramer staining. These data show the efficacy of specific immunologically targeted adjuvants for increasing S1 protein immunogenicity in mice and can contribute to more effective vaccines.

Prothrombotic Antibodies Targeting the Spike Protein's Receptor-Binding Domain in Severe COVID-19

Thromboembolic complication is common in severe coronavirus disease (COVID-19), leading to an investigation into the presence of prothrombotic antibodies akin to those found in heparin-induced thrombocytopenia (HIT). In a study of samples from 130 hospitalized patients collected 3.6 days after COVID-19 diagnosis, 80% had IgG antibodies recognizing complexes of heparin and platelet factor 4 (PF4/H), and 41% had antibodies inducing PF4-dependent P-selectin expression in CpG-treated normal platelets. Unlike HIT, both PF4/H-reactive and platelet-activating antibodies were found in COVID-19 patients regardless of recent heparin exposure. Notably, PF4/H-reactive IgG antibodies correlated with those targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Moreover, introducing exogenous RBD to or removing RBD-reactive IgG from COVID-19 plasma or IgG purified from COVID-19 plasma significantly reduced their ability to activate platelets. RBD-specific antibodies capable of platelet activation were cloned from peripheral blood B cells of COVID-19 patients. These antibodies possessed sequence motifs in the heavy-chain complementarity-determining region 3 (HCDR3) resembling those identified in pathogenic HIT antibodies. Furthermore, IgG+ B cells having these HCDR3 signatures were markedly expanded in severe COVID-19 patients. Importantly, platelet-activating antibodies present in COVID-19 patients were associated with a specific elevation of platelet α-granule proteins in the plasma and showed a positive correlation with markers for inflammation and tissue damage, suggesting functionality of these antibodies in patients. The demonstration of functional and structural similarities between certain RBD-specific antibodies in COVID-19 patients and pathogenic antibodies typical of HIT suggests a novel mechanism whereby RBD-specific antibodies might contribute to thrombosis in COVID-19.

Potent neutralization by a RBD antibody with broad specificity for SARS-CoV-2 JN.1 and other variants

SARS-CoV-2 continues to be a public health burden, driven in-part by its continued antigenic diversification and resulting emergence of new variants. By increasing herd immunity, current vaccines have improved infection outcomes for many. However, prophylactic and treatment interventions that are not compromised by viral evolution of the Spike protein are still needed. Using a differential staining strategy with a rationally designed SARS-CoV-2 Receptor Binding Domain (RBD) – ACE2 fusion protein and a native Omicron RBD protein, we developed a recombinant human monoclonal antibody (hmAb) from a convalescent individual following SARS-CoV-2 Omicron infection. The resulting hmAb, 1301B7 potently neutralized a wide range of SARS-CoV-2 variants including the original Wuhan-1, the more recent Omicron JN.1 strain, and SARS-CoV. 1301B7 contacts the ACE2 binding site of RBD exclusively through its VH1-69 heavy chain. Broad specificity is achieved through 1301B7 binding to many conserved residues of Omicron variants including Y501 and H505. Consistent with its extensive binding epitope, 1301B7 is able to potently diminish viral burden in the upper and lower respiratory tract and protect mice from challenge with Omicron XBB1.5 and Omicron JN.1 viruses. These results suggest 1301B7 has broad potential to prevent or treat clinical SARS-CoV-2 infections and to guide development of RBD-based universal SARS-CoV-2 prophylactic vaccines and therapeutic approaches.

Dynamics of SARS-CoV-2 Spike RBD Protein Mutation and Pathogenicity Consequences in Indonesian Circulating Variants in 2020-2022.

Since the beginning of the coronavirus disease 2019 (COVID-19) outbreak, dynamic mutations in the receptor-binding domain (RBD) in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein have altered the pathogenicity of the variants of the virus circulating in Indonesia. This research analyzes the mutation trend in various RBD samples from Indonesia published in the Global Initiative on Sharing All Influenza Data (GISAID) database using genomic profiling. Patients in Indonesia infected with SARS-CoV-2, whose samples have been published in genomic databases, were selected for this research. The collected data were processed for analysis following several bioinformatics protocols: visualization into phylogenetic trees, 3D rendering, and the assessment of mutational impact. In Indonesia, there are 25 unique SARS-CoV-2 clades and 318 unique SARS-CoV-2 RBD mutations from the earliest COVID-19 sample to samples collected in 2022, with T478K being the most prevalent RBD mutation and 22B being the most abundant clade. The Omicron variant has a lower docking score, higher protein destabilization, and higher KD than the Delta variant and the original virus. The study findings reveal a decreasing trend in virus pathogenicity as a potential trade-off to increase transmissibility via mutations in RBD over the years.

A protein vaccine of RBD integrated with immune evasion mutation shows broad protection against SARS-CoV-2.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge and evade immunity, resulting in breakthrough infections in vaccinated populations. There is an urgent need for the development of vaccines with broad protective effects. In this study, we selected hotspot mutations in the receptor-binding domain (RBD) that contribute to immune escape properties and integrated them into the original RBD protein to obtain a complex RBD protein (cRBD), and we found cRBDs have broad protective effects against SARS-CoV-2 variants. Three cRBDs were designed in our study. Compared with the BA.1 RBD protein, the cRBDs induced the production of higher levels of broader-spectrum neutralizing antibodies, suggesting stronger and broader protective efficacy. In viral challenge experiments, cRBDs were more effective than BA.1 RBD in attenuating lung pathologic injury. Among the three constructs, cRBD3 showed optimal broad-spectrum and protective effects and is a promising candidate for a broad-spectrum SARS-CoV-2 vaccine. In conclusion, immunization with cRBDs triggered immunity against a wide range of variants, including those that emerged after we had completed designing the cRBDs. This study preliminarily explores and validates the feasibility of incorporating hotspot mutations that contribute to immune evasion into the RBD to expand the activity spectrum of antigen-induced antibodies.

Recombinant receptor-binding motif of spike COVID-19 vaccine candidate induces SARS-CoV-2 neutralizing antibody response

Introduction: The SARS-CoV-2 pandemic necessitates effective therapeutic solutions. The receptor-binding motif (RBM) is a subdomain of the spike protein's receptor-binding domain (RBD) and is critical for facilitating the binding of SARS-CoV-2 to the human ACE2 receptor. This study investigates the use of the receptor-binding motif (RBM) domain as an immunogen to produce potent neutralizing antibodies against SARS-CoV-2. Methods: The RBM gene was codon-optimized and cloned into the pET17b vector for expression in E. coli BL21 (DE3) cells, induced with 1 mM IPTG. The recombinant RBM protein was purified using Ni-NTA affinity chromatography. After validating the recombinant RBM by Western blotting with anti-His tag antibodies, BALB/c mice were immunized with 20 µg of the purified RBM protein. Anti-RBM IgG was subsequently purified using protein G resin, and its neutralizing capacity was assessed using the Pishtaz Teb Zaman Neutralization Assay Kit. Results: The recombinant RBM protein, with a molecular weight of 10 kDa, was expressed as inclusion bodies. the typical yield of purification was 27 mg/L of bacterial culture. The neutralization test demonstrated a concentration of 36 µg/mL of neutralizing antibodies in the immunized serum, preventing the spike protein from binding to ACE2. Conclusion: Our study demonstrated that anti-RBM antibodies exhibited neutralization effects on SARS-CoV-2. These findings provide evidence for the development of a vaccine candidate through the induction of antibodies against the RBM, necessitating further studies with adjuvants suitable for human use to evaluate its potential for human vaccination.

Quantitative SARS-CoV-2 Spike Receptor-Binding Domain and Neutralizing Antibody Titers in Previously Infected Persons, United States, January 2021-February 2022.

We studied SARS-CoV-2 binding and neutralizing antibody titers among previously infected persons in the United States over time. We assayed SARS-CoV-2 spike protein receptor-binding domain and neutralizing antibody titers for a convenience sample of residual clinical serum specimens that had evidence of prior SARS-CoV-2 infection gathered during January 2021-February 2022. We correlated titers and examined them by age group (<18, 18-49, 50-64, and >65 years) across 4 different SARS-CoV-2 variant epochs. Among selected specimens, 30,967 had binding antibody titers and 744 had neutralizing titers available. Titers in specimens from children and adults correlated. In addition, mean binding antibody titers increased over time for all age groups, and mean neutralization titers increased over time for persons 16-49 and >65 years of age. Incorporating binding and neutralization antibody titers into infectious disease surveillance could provide a clearer picture of overall immunity and help target vaccination campaigns.

Safety, efficacy and immunogenicity of aerosolized Ad5-nCoV COVID-19 vaccine in a non-inferiority randomized controlled trial

This phase 3, observer-blinded, non-inferiority randomized trial (ClinicalTrials.gov: NCT05517642), conducted from September 2022 to May 2023 at three Malaysian sites, involved 540 adults previously vaccinated with three COVID-19 doses. Participants were randomized 1:1 to receive either one dose of inhaled Recombinant COVID-19 Vaccine (Ad5-nCoV-IH) or intramuscular tozinameran (BNT-IM). The study assessed safety, vaccine efficacy (VE) and immunogenicity against SARS-CoV-2 variants. The primary outcome was the non-inferiority of anti-spike protein receptor-binding domain (S-RBD IgG) antibodies, with a 97.5% confidence interval lower limit for the geometric mean concentration (GMC) ratio >0.67. Ad5-nCoV-IH showed lower immunogenicity than BNT-IM, with a GMC ratio of 0.22 and a seroconversion rate difference of -71.91%. Adverse drug reactions (ADRs) were less frequent with Ad5-nCoV-IH (39.26%) compared to BNT-IM (64.68%). No serious vaccine-related adverse events were reported. Both vaccines had comparable efficacy against COVID-19 variants. This study was funded by Tianjin Biomedical Science and Technology Major Project.

High-throughput molecular simulations of SARS-CoV-2 receptor binding domain mutants quantify correlations between dynamic fluctuations and protein expression.

Prediction of protein fitness from computational modeling is an area of active research in rational protein design. Here, we investigated whether protein fluctuations computed from molecular dynamics simulations can be used to predict the expression levels of SARS-CoV-2 receptor binding domain (RBD) mutants determined in the deep mutational scanning experiment of Starr et al. [Science (New York, N.Y.) 2022, 377, 420] Specifically, we performed more than 0.7 milliseconds of molecular dynamics (MD) simulations of 557 mutant RBDs in triplicate to achieve statistical significance under various simulation conditions. Our results show modest but significant anticorrelation in the range [-0.4, -0.3] between expression and RBD protein flexibility. A simple linear regression machine learning model achieved correlation coefficients in the range [0.7, 0.8], thus outperforming MD-based models, but required about 25 mutations at each residue position for training.

Protein Nanoparticles for Targeted SARS-CoV-2 Trapping and Neutralization.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to challenge global health despite widespread vaccination efforts, underscoring the need for innovative strategies to combat emerging infectious diseases effectively. Herein, LCB1-NPs and LCB3-NPs are engineered as a novel class of protein-only nanoparticles formed through coiled coil-driven self-assembly and tailored to interact specifically with the SARS-CoV-2 spike protein. The multivalency of LCB1-NPs and LCB3-NPs offers a strategy for efficiently targeting and neutralizing SARS-CoV-2 both in solution and when immobilized on surfaces. It is demonstrated that LCB1-NPs and LCB3-NPs bind to the SARS-CoV-2 spike protein's receptor-binding domain (RBD) with high affinity, effectively blocking the entry of SARS-CoV-2 virus-like particles into angiotensin-converting enzyme 2 (ACE2)-coated human cells. The cost-effectiveness, scalability, and straightforward production process of these protein nanoparticles make them suitable for developing novel anti-viral materials. Accordingly, it is shown how these nanostructures can be packed into columns to build up economic and highly potent trapping devices for SARS-CoV-2 adsorption.

Oxoammonium salts exert antiviral effects against coronavirus via denaturation of their spike proteins

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV2) infection has forced social changes worldwide. Development of potent antiviral agents is necessary to prevent future pandemics. Titanium oxide, a photocatalyst, is a long-acting antiviral agent; however, its effects are weakened in the dark. Therefore, new antiviral substances that can be used in the dark are needed. Two types of nitroxyl radicals, 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) and 2-azaadamantane N-oxyl (AZADO), are commonly used as oxidation catalysts utilizing oxygen in the air as the terminal oxidant. Therefore, in this study, we aimed to evaluate the potential of these radicals as antiviral compounds with sustained activity even in the dark. We evaluated the antiviral effects of oxoammonium salts corresponding to TEMPO and AZADO (TEMPO-Oxo and AZADO-Oxo, respectively), which are the active forms of nitroxyl radicals in oxidation reactions. TEMPO-Oxo and AZADO-Oxo inhibited the binding of SARS-CoV2 spike protein receptor-binding domain (S-RBD) to angiotensin-converting enzyme 2. Notably, AZADO-Oxo exhibited a 10-fold stronger inhibitory effect than TEMPO-Oxo. TEMPO-Oxo and AZADO-Oxo also denatured S-RBD; however, effects of AZADO-Oxo were 10-fold stronger than those of TEMPO-Oxo and did not change in the dark. Some S-RBD peptides treated with AZADO-Oxo were cleaved at the N-terminal side of tyrosine residues. TEMPO-Oxo and AZADO-Oxo exhibited concentration-dependent antiviral effects against feline coronavirus. In conclusion, active forms of the nitroxyl radicals, TEMPO-Oxo and AZADO-Oxo, exerted antiviral effects by denaturing S-RBD, regardless of the presence or absence of light, suggesting their potential as novel antiviral agents.

An Analysis of Combined Molecular Weight and Hydrophobicity Similarity between the Amino Acid Sequences of Spike Protein Receptor Binding Domains of Betacoronaviruses and Functionally Similar Sequences from Other Virus Families

Recently, we proposed a new method, based on protein profiles derived from physicochemical dynamic time warping (PCDTW), to functionally/structurally classify coronavirus spike protein receptor binding domains (RBD). Our method, as used herein, uses waveforms derived from two physicochemical properties of amino acids (molecular weight and hydrophobicity (MWHP)) and is designed to reach into the twilight zone of homology, and therefore, has the potential to reveal structural/functional relationships and potentially homologous relationships over greater evolutionary time spans than standard primary sequence alignment-based techniques. One potential application of our method is inferring deep evolutionary relationships such as those between the RBD of the spike protein of betacoronaviruses and functionally similar proteins found in other families of viruses, a task that is extremely difficult, if not impossible, using standard multiple alignment-based techniques. Here, we applied PCDTW to compare members of four divergent families of viruses to betacoronaviruses in terms of MWHP physicochemical similarity of their RBDs. We hypothesized that some members of the families Arteriviridae, Astroviridae, Reoviridae (both from the genera rotavirus and orthoreovirus considered separately), and Toroviridae would show greater physicochemical similarity to betacoronaviruses in protein regions similar to the RBD of the betacoronavirus spike protein than they do to other members of their respective taxonomic groups. This was confirmed to varying degrees in each of our analyses. Three arteriviruses (the glycoprotein-2 sequences) clustered more closely with ACE2-binding betacoronaviruses than to other arteriviruses, and a clade of 33 toroviruses was found embedded within a clade of non-ACE2-binding betacoronaviruses, indicating potentially shared structure/function of RBDs between betacoronaviruses and members of other virus clades.