Spike RBD Research Articles

I2-Catalyzed Carbonylation of α-Methylene Ketones to Synthesize 1,2-Diaryl Diketones and Antiviral Quinoxalines in One Pot.

An efficient approach for the synthesis of 1,2-diaryl diketones was developed from readily available α-methylene ketones by catalysis of I2. In the same oxidation system, a novel one-pot procedure was established for the construction of antiviral and anticancer quinoxalines. The reactions proceeded well with a wide variety of substrates and good functional group tolerance, affording desired compounds in moderate to excellent yields. Quinoxalines 4ca and 4ad inhibited viral entry of SARS-CoV-2 spike pseudoviruses into HEK-293T-ACE2h host cells as dual blockers of both human ACE2 receptor and viral spike RBD with IC50 values of 19.70 and 21.28 μM, respectively. In addition, cytotoxic evaluation revealed that 4aa, 4ba, 4ia, and 4ab suppressed four cancer cells with IC50 values ranging from 6.25 to 28.55 μM.

Microarray-Based Detection of Antibodies against SARS-CoV-2 Proteins, Common Respiratory Viruses and Type I Interferons.

A microarray-based assay to detect IgG and IgM antibodies against betacoronaviruses (SARS-CoV-2, SARS, MERS, OC43, and HKU1), other respiratory viruses and type I interferons (IFN-Is) was developed. This multiplex assay was applied to track antibody cross-reactivity due to previous contact with similar viruses and to identify antibodies against IFN-Is as the markers for severe COVID-19. In total, 278 serum samples from convalescent plasma donors, COVID-19 patients in the intensive care unit (ICU) and patients who recovered from mild/moderate COVID-19, vaccine recipients, prepandemic and pandemic patients with autoimmune endocrine disorders, and a heterogeneous prepandemic cohort including healthy individuals and chronically ill patients were analyzed. The anti-SARS-CoV-2 microarray results agreed well with the ELISA results. Regarding ICU patients, autoantibodies against IFN-Is were detected in 10.5% of samples, and 10.5% of samples were found to simultaneously contain IgM antibodies against more than two different viruses. Cross-reactivity between IgG against the SARS-CoV-2 nucleocapsid and IgG against the OC43 and HKU1 spike proteins was observed, resulting in positive signals for the SARS-CoV-2 nucleocapsid in prepandemic samples from patients with autoimmune endocrine disorders. The presence of IgG against the SARS-CoV-2 nucleocapsid in the absence of IgG against the SARS-CoV-2 spike RBD should be interpreted with caution.

Lymphocytopenia and Anti-CD38 Directed Treatment Impact the Serological SARS-CoV-2 Response after Prime Boost Vaccination in Patients with Multiple Myeloma.

Even though several SARS-CoV-2 vaccines have shown high effectiveness in the prevention of COVID-19 in healthy subjects, vaccination response in patients with plasma-cell-related disorders (PCD) remains widely unknown. Here, we report on an analysis describing the serological response after prime-boost SARS-CoV-2 vaccination in PCD patients, as compared to a healthy control group, and on possible influencing factors of serological responses. Blood samples were analyzed for the presence of quantitative anti-SARS-CoV-2 spike RBD Ig. A total of 82 patients were included; 67 received mRNA-, eight vector-based and four heterologous vaccinations. SARS-CoV-2 antibody titers (SP-AbT) were assessed in a mean of 23 days (SD ± 11 days) after the first and in a mean 21 days (SD ± 9) after prime-boost vaccination. A positive SP-AbT was detected in 31.9% of PCD patients after the first vaccination, and in 88.9% (44/49) after prime-boost vaccination, which was significantly less likely than that in the control group (100%, 78/78) (p = 0.008). Furthermore, we have been able to validate our previously suggested threshold of 30 CD19+ B lymphocytes/µL as being predictive for SP-AbT development. Despite anti-CD38 directed therapy, quadruplet treatment, higher age and missing deep remission, which correlated negatively with SP-AbT appearance, SP-AbT formation is possible in a majority of myeloma patients after prime-boost vaccination.

An outbreak of SARS-CoV-2 with high mortality in mink (Neovison vison) on multiple Utah farms.

The breadth of animal hosts that are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and may serve as reservoirs for continued viral transmission are not known entirely. In August 2020, an outbreak of SARS-CoV-2 occurred on five mink farms in Utah and was associated with high mink mortality (35–55% of adult mink) and rapid viral transmission between animals. The premise and clinical disease information, pathology, molecular characterization, and tissue distribution of virus within infected mink during the early phase of the outbreak are provided. Infection spread rapidly between independently housed animals and farms, and caused severe respiratory disease and death. Disease indicators were most notably sudden death, anorexia, and increased respiratory effort. Gross pathology examination revealed severe pulmonary congestion and edema. Microscopically there was pulmonary edema with moderate vasculitis, perivasculitis, and fibrinous interstitial pneumonia. Reverse transcriptase polymerase chain reaction (RT-PCR) of tissues collected at necropsy demonstrated the presence of SARS-CoV-2 viral RNA in multiple organs including nasal turbinates, lung, tracheobronchial lymph node, epithelial surfaces, and others. Localization of viral RNA by in situ hybridization revealed a more localized infection, particularly of the upper respiratory tract. Whole genome sequencing from multiple mink was consistent with published SARS-CoV-2 genomes with few polymorphisms. The Utah mink SARS-CoV-2 strains fell into Clade GH, which is unique among mink and other animal strains sequenced to date. While sharing the N501T mutation which is common in mink, the Utah strains did not share other spike RBD mutations Y453F and F486L found in nearly all mink from the United States. Mink in the outbreak reported herein had high levels of SARS-CoV-2 in the upper respiratory tract associated with symptomatic respiratory disease and death.

Epidemiological and Immunological Features of Obesity and SARS-CoV-2.

Obesity is a key correlate of severe SARS-CoV-2 outcomes while the role of obesity on risk of SARS-CoV-2 infection, symptom phenotype, and immune response remain poorly defined. We examined data from a prospective SARS-CoV-2 cohort study to address these questions. Serostatus, body mass index, demographics, comorbidities, and prior COVID-19 compatible symptoms were assessed at baseline and serostatus and symptoms monthly thereafter. SARS-CoV-2 immunoassays included an IgG ELISA targeting the spike RBD, multiarray Luminex targeting 20 viral antigens, pseudovirus neutralization, and T cell ELISPOT assays. Our results from a large prospective SARS-CoV-2 cohort study indicate symptom phenotype is strongly influenced by obesity among younger but not older age groups; we did not identify evidence to suggest obese individuals are at higher risk of SARS-CoV-2 infection; and remarkably homogenous immune activity across BMI categories suggests immune protection across these groups may be similar.

Phylogenetic and Ancestral Sequence Reconstruction of SARS-CoV-2 Reveals Latent Capacity to Bind Human ACE2 Receptor.

SARS-CoV-2 is a unique event, having emerged suddenly as a highly infectious viral pathogen for human populations. Previous phylogenetic analyses show its closest known evolutionary relative to be a virus detected in bats (RaTG13), with a common assumption that SARS-CoV-2 evolved from a zoonotic ancestor via recent genetic changes (likely in the Spike protein receptor-binding domain or RBD) that enabled it to infect humans. We used detailed phylogenetic analysis, ancestral sequence reconstruction, and in situ molecular dynamics simulations to examine the Spike-RBD’s functional evolution, finding that the common ancestral virus with RaTG13, dating to no later than 2013, possessed high binding affinity to the human ACE2 receptor. This suggests that SARS-CoV-2 likely possessed a latent capacity to bind to human cellular targets (though this may not have been sufficient for successful infection) and emphasizes the importance of expanding efforts to catalog and monitor viruses circulating in both human and non-human populations.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00239-021-10034-0.

CD19+ B Lymphocytes Are Predictive for Anti-Sars-Cov-2 Vaccination Response in Multiple Myeloma Patients

CD19+ B Lymphocytes Are Predictive for Anti-Sars-Cov-2 Vaccination Response in Multiple Myeloma Patients

Intradermal-delivered DNA vaccine induces durable immunity mediating a reduction in viral load in a rhesus macaque SARS-CoV-2 challenge model

SummaryCoronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health and social and economic infrastructures. Here, we assess the immunogenicity and anamnestic protective efficacy in rhesus macaques of an intradermal (i.d.)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800, currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and induced spike antigen and RBD binding antibodies with ADCP and ADCD activity. Sera from the animals neutralized both the D614 and G614 SARS-CoV-2 pseudotype viruses. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T cell and neutralizing antibody responses. These responses were associated with lower viral loads in the lung. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system, which are likely important for providing durable protection against COVID-19 disease.

A bioluminescent and homogeneous SARS-CoV-2 spike RBD and hACE2 interaction assay for antiviral screening and monitoring patient neutralizing antibody levels

Here we describe a homogeneous bioluminescent immunoassay based on the interaction between Fc-tagged SARS-CoV-2 Spike RBD and human ACE2, and its detection by secondary antibodies labeled with NanoLuc luciferase fragments LgBit and SmBit. The assay utility for the discovery of novel inhibitors was demonstrated with a panel of anti-RBD antibodies, ACE2-derived miniproteins and soluble ACE2. Studying the effect of RBD mutations on ACE2 binding showed that the N501Y mutation increased RBD apparent affinity toward ACE2 tenfold that resulted in escaping inhibition by some anti-RBD antibodies. In contrast, while E484K mutation did not highly change the binding affinity, it still escaped antibody inhibition likely due to changes in the epitope recognized by the antibody. Also, neutralizing antibodies (NAbs) from COVID-19 positive samples from two distinct regions (USA and Brazil) were successfully detected and the results further suggest the persistence of NAbs for at least 6 months post symptom onset. Finally, sera from vaccinated individuals were tested for NAbs and showed varying neutralizing activity after first and second doses, suggesting the assay can be used to assess immunity of vaccinated populations. Our results demonstrate the broad utility and ease of use of this methodology both for drug discovery and clinical research applications.

Use of Outpatient-Derived COVID-19 Convalescent Plasma in COVID-19 Patients Before Seroconversion.

BackgroundTransfusion of COVID-19 convalescent plasma (CCP) containing high titers of anti-SARS-CoV-2 antibodies serves as therapy for COVID-19 patients. Transfusions early during disease course was found to be beneficial. Lessons from the SARS-CoV-2 pandemic could inform early responses to future pandemics and may continue to be relevant in lower resource settings. We sought to identify factors correlating to high antibody titers in convalescent plasma donors and understand the magnitude and pharmacokinetic time course of both transfused antibody titers and the endogenous antibody titers in transfused recipients.MethodsPlasma samples were collected up to 174 days after convalescence from 93 CCP donors with mild disease, and from 16 COVID-19 patients before and after transfusion. Using ELISA, anti-SARS-CoV-2 Spike RBD, S1, and N-protein antibodies, as well as capacity of antibodies to block ACE2 from binding to RBD was measured in an in vitro assay. As an estimate for viral load, viral RNA and N-protein plasma levels were assessed in COVID-19 patients.ResultsAnti-SARS-CoV-2 antibody levels and RBD-ACE2 blocking capacity were highest within the first 60 days after symptom resolution and markedly decreased after 120 days. Highest antibody titers were found in CCP donors that experienced fever. Effect of transfused CCP was detectable in COVID-19 patients who received high-titer CCP and had not seroconverted at the time of transfusion. Decrease in viral RNA was seen in two of these patients.ConclusionOur results suggest that high titer CCP should be collected within 60 days after recovery from donors with past fever. The much lower titers conferred by transfused antibodies compared to endogenous production in the patient underscore the importance of providing CCP prior to endogenous seroconversion.

Characterization of humoral response to COVID mRNA vaccines in multiple sclerosis patients on disease modifying therapies

Little is known about COVID-19 mRNA vaccine humoral immune responses in patients with central nervous system autoimmune demyelinating diseases, multiple sclerosis (MS) and neuromyelitis optica (NMO), who are on B-cell depleting therapies (BCDT) and other disease modifying therapies (DMTs). We conducted a single center prospective study to identify the clinical and immunological features associated with vaccine-induced antibody response in 53 participants before and after COVID-19 mRNA vaccination. This is the first report on the anti-spike RBD and anti-nucleocapsid antibody response, along with pre- and post-vaccine absolute lymphocyte counts (ALC) and flow cytometry analysis of CD19 and CD20 lymphocytes in patients with MS and NMO. We tested the hypothesis that patients on BCDT may have impaired COVID-19 vaccine humoral responses. Among patients on BCDT, 36.4% demonstrated a positive antibody response to spike RBD, in comparison to 100% in all other groups such as healthy controls, untreated MS, and patients on non-B cell depleting DMTs (p < 0.0001). Immunological data revealed lower baseline (pre-vaccination) levels of IgM in patients on BCDT (p = 0.003). Low CD19 and CD20 counts and a shorter interval from the last B cell depleting therapy infusion to the first vaccine dose were associated with a negative spike RBD antibody response (non-seroconverter) in patients on BCDT. Age, body mass index (BMI) and total treatment duration did not differ between seroconverters and non-seroconverters.

Recent advances in SARS-CoV-2 Spike protein and RBD mutations comparison between new variants Alpha (B.1.1.7, United Kingdom), Beta (B.1.351, South Africa), Gamma (P.1, Brazil) and Delta (B.1.617.2, India)

New variants of SARS-CoV-2 Alpha (B.1.1.7); Beta (B.1.351) Gamma (P.1) and Delta (B.1.617.2) quickly spread in the UK, South Africa, Brazil and India, respectively. To address whether mutations in SARS-CoV-2 RBD spike protein could affect virus infectivity, peptides containing RBD amino acids mutations have been constructed and interacted with human ACE2 by computational methods. Our results suggest that mutations in RBD amino acids K417, E484, L452, T478 and N501 are expressively increasing the affinity of this protein with human angiotensin-converting enzyme 2 (ACE2), consequently, variants Alpha (B.1.1.7), Beta (B1.351), Gamma (P.1) and Delta (B.1.617.2) could be more infective in human cells compared with SARS-CoV-2 isolated in Wuhan-2019 and the Gamma and Delta variants could be the most infective among them.

Synthesis and Biological Evaluation of Honokiol Derivatives Bearing 3-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)oxazol-2(3H)-ones as Potential Viral Entry Inhibitors against SARS-CoV-2.

The 2019 coronavirus disease (COVID-19) caused by SARS-CoV-2 virus infection has posed a serious danger to global health and the economy. However, SARS-CoV-2 medications that are specific and effective are still being developed. Honokiol is a bioactive component from Magnoliae officinalis Cortex with damp-drying effect. To develop new potent antiviral molecules, a series of novel honokiol analogues were synthesized by introducing various 3-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)oxazol-2(3H)-ones to its molecule. In a SARS-CoV-2 pseudovirus model, all honokiol derivatives were examined for their antiviral entry activities. As a result, 6a and 6p demonstrated antiviral entry effect with IC50 values of 29.23 and 9.82 µM, respectively. However, the parental honokiol had a very weak antiviral activity with an IC50 value more than 50 µM. A biolayer interfero-metry (BLI) binding assay and molecular docking study revealed that 6p binds to human ACE2 protein with higher binding affinity and lower binding energy than the parental honokiol. A competitive ELISA assay confirmed the inhibitory effect of 6p on SARS-CoV-2 spike RBD’s binding with ACE2. Importantly, 6a and 6p (TC50 > 100 μM) also had higher biological safety for host cells than honokiol (TC50 of 48.23 μM). This research may contribute to the discovery of potential viral entrance inhibitors for the SARS-CoV-2 virus, although 6p’s antiviral efficacy needs to be validated on SARS-CoV-2 viral strains in a biosafety level 3 facility.

Targeting CoV-2 spike RBD and ACE-2 interaction with flavonoids of Anatolian propolis by in silico and in vitro studies in terms of possible COVID-19 therapeutics.

Propolis is a multi-functional bee product rich in polyphenols. In this study, the inhibitory effect of Anatolian propolis against SARS-coronavirus-2 (SARS-CoV-2) was investigated in vitro and in silico. Raw and commercial propolis samples were used, and both samples were found to be rich in caffeic acid, p-coumaric acid, ferulic acid, t-cinnamic acid, hesperetin, chrysin, pinocembrin, and caffeic acid phenethyl ester (CAPE) at HPLC-UV analysis. Ethanolic propolis extracts (EPE) were used in the ELISA screening test against the spike S1 protein (SARS-CoV-2): ACE-2 interaction for in vitro study. The binding energy values of these polyphenols to the SARS-CoV-2 spike and ACE-2 protein were calculated separately with a molecular docking study using the AutoDock 4.2.6 program. In addition, the pharmacokinetics and drug-likeness properties of these eight polyphenols were calculated according to the SwissADME tool. The binding energy value of pinocembrin was highest in both receptors, followed by chrysin, CAPE, and hesperetin. Based on the in silico modeling and ADME (absorption, distribution, metabolism, and excretion) behaviors of the eight polyphenols, the compounds exhibited the potential ability to act effectively as novel drugs. The findings of both studies showed that propolis has a high inhibitory potential against the Covid-19 virus. However, further studies are now needed.

SARS-COV-2 recombinant Receptor-Binding-Domain (RBD) induces neutralizing antibodies against variant strains of SARS-CoV-2 and SARS-CoV-1

SARS-CoV-2 is the etiological agent of COVID19. There are currently several licensed vaccines approved for human use and most of them target the spike protein in the virion envelope to induce protective immunity. Recently, variants that spread more quickly have emerged. There is evidence that some of these variants are less sensitive to neutralization in vitro, but it is not clear whether they can evade vaccine induced protection. In this study, we tested SARS-CoV-2 spike RBD as a vaccine antigen and explored the effect of formulation with Alum/MPLA or AddaS03 adjuvants. Our results show that RBD induces high titers of neutralizing antibodies and activates strong cellular immune responses. There is also significant cross-neutralization of variants B.1.1.7 and B.1.351 and to a lesser extent, SARS-CoV-1. These results indicate that recombinant RBD can be a viable candidate as a stand-alone vaccine or as a booster shot to diversify our strategy for COVID19 protection.

Synthesis of novel calcium channel blockers with ACE2 inhibition and dual antihypertensive/anti-inflammatory effects: A possible therapeutic tool for COVID-19

Hypertension has been recognized as one of the most frequent comorbidities and risk factors for the seriousness and adverse consequences in COVID-19 patients. 3,4-dihydropyrimidin-2(1H) ones have attracted researchers to be synthesized via Beginilli reaction and evaluate their antihypertensive activities as bioisosteres of nifedipine a well-known calcium channel blocker. In this study, we report synthesis of some bioisosteres of pyrimidines as novel CCBs with potential ACE2 inhibitory effect as antihypertensive agents with protective effect against COVID-19 infection by suppression of ACE2 binding to SARS-CoV-2 Spike RBD. All compounds were evaluated for their antihypertensive and calcium channel blocking activities using nifedipine as a reference standard. Furthermore, they were screened for their ACE2 inhibition potential in addition to their anti‐inflammatory effects on LPS-stimulated THP‐1 cells. Most of the tested compounds exhibited significant antihypertensive activity, where compounds 7a, 8a and 9a exhibited the highest activity compared to nifedipine. Moreover, compounds 4a,b, 5a,b, 7a,b, 8a,c and 9a showed promising ACE2:SARS-CoV-2 Spike RBD inhibitory effect. Finally, compounds 5a, 7b and 9a exerted a promising anti-inflammatory effect by inhibition of CRP and IL-6 production. Ultimately, compound 9a may be a promising antihypertensive candidate with anti-inflammatory and potential efficacy against COVID-19 via ACE2 receptor inhibition.

Myxobacterial depsipeptide chondramides interrupt SARS-CoV-2 entry by targeting its broad, cell tropic spike protein

The severity of the COVID-19 pandemic has necessitated the search for drugs against SARS-CoV-2. In this study, we explored via in silico approaches myxobacterial secondary metabolites against various receptor-binding regions of SARS-CoV-2 spike which are responsible in recognition and attachment to host cell receptors mechanisms, namely ACE2, GRP78, and NRP1. In general, cyclic depsipeptide chondramides conferred high affinities toward the spike RBD, showing strong binding to the known viral hot spots Arg403, Gln493 and Gln498 and better selectivity compared to most host cell receptors studied. Among them, chondramide C3 (1) exhibited a binding energy which remained relatively constant when docked against most of the spike variants. Chondramide C (2) on the other hand exhibited strong affinity against spike variants identified in the United Kingdom (N501Y), South Africa (N501Y, E484K, K417N) and Brazil (N501Y, E484K, K417T). Chondramide C6 (9) showed highest BE towards GRP78 RBD. Molecular dynamics simulations were also performed for chondramides 1 and 2 against SARS-CoV-2 spike RBD of the Wuhan wild-type and the South African variant, respectively, where resulting complexes demonstrated dynamic stability within a 120-ns simulation time. Protein-protein binding experiments using HADDOCK illustrated weaker binding affinity for complexed chondramide ligands in the RBD against the studied host cell receptors. The chondramide derivatives in general possessed favorable pharmacokinetic properties, highlighting their potential as prototypic anti-COVID-19 drugs limiting viral attachment and possibly minimizing viral infection. Communicated by Ramaswamy H. Sarma

Antibodies against Receptor Binding Domain of SARS-CoV-2 spike protein induced by BNT162b2 vaccine: results from a pragmatic, real-life study

BackgroundAs part of the fight against SARS CoV2 infection, vaccination program for health workers at Giannina Gaslini pediatric hospital (IGG) in Genoa, Italy, started on December 2020. We evaluated the anti-Spike protein response in healthcare workers after a complete vaccination scheme of 2 doses spaced by 3 weeks. MethodsImmunoglobulin class G (IgG) against SARS-CoV-2 spike RBD were detected by means of a chemiluminescence immunoassay for quantitative IgG antibodies using Maglumi SARS-CoV-2-S-RBD IgG kit during the 3rd week after vaccination completion. ResultsIgG anti SARS-CoV-2 spike protein were detected in 99.88% of 1765 healthcare workers 3 weeks after 2nd dose of BNT162b2. Higher median IgG values were observed in younger subjects (807 UA/mL in under 30 vs 429 UA/mL in over 60; p < 0.001) and those with previous COVID-19 (1284 vs 574 UA/mL; p < 0.001). ConclusionBNT162b2 is effective in inducing anti SARS-CoV-2 antibodies even in real-life setting. The higher antibody title observed in workers with a previous documented SARS CoV2 infection confirms the possibility to carry out only one dose of BNT162b2 in a context of vaccines shortage.

V367F Mutation in SARS-CoV-2 Spike RBD Emerging during the Early Transmission Phase Enhances Viral Infectivity through Increased Human ACE2 Receptor Binding Affinity.

ABSTRACTThe current pandemic of COVID-19 is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 spike protein receptor-binding domain (RBD) is the critical determinant of viral tropism and infectivity. To investigate whether naturally occurring RBD mutations during the early transmission phase have altered the receptor binding affinity and infectivity, we first analyzed in silico the binding dynamics between SARS-CoV-2 RBD mutants and the human angiotensin-converting enzyme 2 (ACE2) receptor. Among 32,123 genomes of SARS-CoV-2 isolates (December 2019 through March 2020), 302 nonsynonymous RBD mutants were identified and clustered into 96 mutant types. The six dominant mutations were analyzed applying molecular dynamics simulations (MDS). The mutant type V367F continuously circulating worldwide displayed higher binding affinity to human ACE2 due to the enhanced structural stabilization of the RBD beta-sheet scaffold. The MDS also indicated that it would be difficult for bat SARS-like CoV to infect humans. However, the pangolin CoV is potentially infectious to humans. The increased infectivity of V367 mutants was further validated by performing receptor-ligand binding enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance, and pseudotyped virus assays. Phylogenetic analysis of the genomes of V367F mutants showed that during the early transmission phase, most V367F mutants clustered more closely with the SARS-CoV-2 prototype strain than the dual-mutation variants (V367F+D614G), which may derivate from recombination. The analysis of critical RBD mutations provides further insights into the evolutionary trajectory of early SARS-CoV-2 variants of zoonotic origin under negative selection pressure and supports the continuing surveillance of spike mutations to aid in the development of new COVID-19 drugs and vaccines.IMPORTANCE A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused the pandemic of COVID-19. The origin of SARS-CoV-2 was associated with zoonotic infections. The spike protein receptor-binding domain (RBD) is identified as the critical determinant of viral tropism and infectivity. Thus, whether mutations in the RBD of the circulating SARS-CoV-2 isolates have altered the receptor binding affinity and made them more infectious has been the research hot spot. Given that SARS-CoV-2 is a novel coronavirus, the significance of our research is in identifying and validating the RBD mutant types emerging during the early transmission phase and increasing human angiotensin-converting enzyme 2 (ACE2) receptor binding affinity and infectivity. Our study provides insights into the evolutionary trajectory of early SARS-CoV-2 variants of zoonotic origin. The continuing surveillance of RBD mutations with increased human ACE2 affinity in human or other animals is critical to the development of new COVID-19 drugs and vaccines against these variants during the sustained COVID-19 pandemic.

Plausible blockers of Spike RBD in SARS-CoV2-molecular design and underlying interaction dynamics from high-level structural descriptors.

COVID-19 is characterized by an unprecedented abrupt increase in the viral transmission rate (SARS-CoV-2) relative to its pandemic evolutionary ancestor, SARS-CoV (2003). The complex molecular cascade of events related to the viral pathogenicity is triggered by the Spike protein upon interacting with the ACE2 receptor on human lung cells through its receptor binding domain (RBDSpike). One potential therapeutic strategy to combat COVID-19 could thus be limiting the infection by blocking this key interaction. In this current study, we adopt a protein design approach to predict and propose non-virulent structural mimics of the RBDSpike which can potentially serve as its competitive inhibitors in binding to ACE2. The RBDSpike is an independently foldable protein domain, resilient to conformational changes upon mutations and therefore an attractive target for strategic re-design. Interestingly, in spite of displaying an optimal shape fit between their interacting surfaces (attributed to a consequently high mutual affinity), the RBDSpike–ACE2 interaction appears to have a quasi-stable character due to a poor electrostatic match at their interface. Structural analyses of homologous protein complexes reveal that the ACE2 binding site of RBDSpike has an unusually high degree of solvent-exposed hydrophobic residues, attributed to key evolutionary changes, making it inherently “reaction-prone.” The designed mimics aimed to block the viral entry by occupying the available binding sites on ACE2, are tested to have signatures of stable high-affinity binding with ACE2 (cross-validated by appropriate free energy estimates), overriding the native quasi-stable feature. The results show the apt of directly adapting natural examples in rational protein design, wherein, homology-based threading coupled with strategic “hydrophobic ↔ polar” mutations serve as a potential breakthrough.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1007/s00894-021-04779-0.