Total Chemical Synthesis of the SARS-CoV-2 Spike Receptor-Binding Domain.

SARS-CoV-2 and its global spread have created an unprecedented public health crisis. The spike protein of SARS-CoV-2 has gained significant attention due to its crucial role in viral entry into host cells and its potential as both a prophylactic and a target for therapeutic interventions. Herein, we report the first successful total synthesis of the SARS-CoV-2 spike protein receptor binding domain (RBD), highlighting the key challenges and the strategies employed to overcome them. Appropriate utilization of advanced solid phase peptide synthesis and cutting-edge native chemical ligation methods have facilitated the synthesis of this moderately large protein molecule. We discuss the problems encountered during the chemical synthesis and approaches taken to optimize the yield and the purity of the synthetic protein molecule. Furthermore, we demonstrate that the chemically synthesized homogeneous spike RBD efficiently binds to the known mini-protein binder LCB1. The successful chemical synthesis of the spike RBD presented here can be utilized to gain valuable insights into SARS-CoV-2 spike RBD biology, advancing our understanding and aiding the development of intervention strategies to combat future coronavirus outbreaks. The modular synthetic approach described in this study can be effectively implemented in the synthesis of other mutated variants or enantiomer of the spike RBD for mirror-image drug discovery.

In silico binding profile characterization of SARS-CoV-2 spike protein and its mutants bound to human ACE2 receptor.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2), a novel coronavirus, has brought an unprecedented pandemic to the world and affected over 64 million people. The virus infects human using its spike glycoprotein mediated by a crucial area, receptor-binding domain (RBD), to bind to the human ACE2 (hACE2) receptor. Mutations on RBD have been observed in different countries and classified into nine types: A435S, D364Y, G476S, N354D/D364Y, R408I, V341I, V367F, V483A and W436R. Employing molecular dynamics (MD) simulation, we investigated dynamics and structures of the complexes of the prototype and mutant types of SARS-CoV-2 spike RBDs and hACE2. We then probed binding free energies of the prototype and mutant types of RBD with hACE2 protein by using an end-point molecular mechanics Poisson Boltzmann surface area (MM-PBSA) method. According to the result of MM-PBSA binding free energy calculations, we found that V367F and N354D/D364Y mutant types showed enhanced binding affinities with hACE2 compared to the prototype. Our computational protocols were validated by the successful prediction of relative binding free energies between prototype and three mutants: N354D/D364Y, V367F and W436R. Thus, this study provides a reliable computational protocol to fast assess the existing and emerging RBD mutations. More importantly, the binding hotspots identified by using the molecular mechanics generalized Born surface area (MM-GBSA) free energy decomposition approach can guide the rational design of small molecule drugs or vaccines free of drug resistance, to interfere with or eradicate spike-hACE2 binding.

Recombinant ACE2 protein protects against acute lung injury induced by SARS-CoV-2 spike RBD protein.

BACKGROUND: SARS-CoV-2 infection leads to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Both clinical data and animal experiments suggest that the renin-angiotensin system (RAS) is involved in the pathogenesis of SARS-CoV-2-induced ALI. Angiotensin-converting enzyme 2 (ACE2) is the functional receptor for SARS-CoV-2 and a crucial negative regulator of RAS. Recombinant ACE2 protein (rACE2) has been demonstrated to play protective role against SARS-CoV and avian influenza-induced ALI, and more relevant, rACE2 inhibits SARS-CoV-2 proliferation in vitro. However, whether rACE2 protects against SARS-CoV-2-induced ALI in animal models and the underlying mechanisms have yet to be elucidated. METHODS AND RESULTS: Here, we demonstrated that the SARS-CoV-2 spike receptor-binding domain (RBD) protein aggravated lipopolysaccharide (LPS)-induced ALI in mice. SARS-CoV-2 spike RBD protein directly binds and downregulated ACE2, leading to an elevation in angiotensin (Ang) II. AngII further increased the NOX1/2 through AT1R, subsequently causing oxidative stress and uncontrolled inflammation and eventually resulting in ALI/ARDS. Importantly, rACE2 remarkably reversed SARS-CoV-2 spike RBD protein-induced ALI by directly binding SARS-CoV-2 spike RBD protein, cleaving AngI or cleaving AngII. CONCLUSION: This study is the first to prove that rACE2 plays a protective role against SARS-CoV-2 spike RBD protein-aggravated LPS-induced ALI in an animal model and illustrate the mechanism by which the ACE2-AngII-AT1R-NOX1/2 axis might contribute to SARS-CoV-2-induced ALI.

A Novel Human Neutralizing mAb Recognizes Delta, Gamma and Omicron Variants of SARS-CoV-2 and Can Be Used in Combination with Sotrovimab.

The dramatic experience with SARS-CoV-2 has alerted the scientific community to be ready to face new epidemics/pandemics caused by new variants. Among the therapies against the pandemic SARS-CoV-2 virus, monoclonal Antibodies (mAbs) targeting the Spike glycoprotein have represented good drugs to interfere in the Spike/ Angiotensin Converting Enzyme-2 (ACE-2) interaction, preventing virus cell entry and subsequent infection, especially in patients with a defective immune system. We obtained, by an innovative phage display selection strategy, specific binders recognizing different epitopes of Spike. The novel human antibodies specifically bind to Spike-Receptor Binding Domain (RBD) in a nanomolar range and interfere in the interaction of Spike with the ACE-2 receptor. We report here that one of these mAbs, named D3, shows neutralizing activity for virus infection in cell cultures by different SARS-CoV-2 variants and retains the ability to recognize the Omicron-derived recombinant RBD differently from the antibodies Casirivimab or Imdevimab. Since anti-Spike mAbs, used individually, might be unable to block the virus cell entry especially in the case of resistant variants, we investigated the possibility to combine D3 with the antibody in clinical use Sotrovimab, and we found that they recognize distinct epitopes and show additive inhibitory effects on the interaction of Omicron-RBD with ACE-2 receptor. Thus, we propose to exploit these mAbs in combinatorial treatments to enhance their potential for both diagnostic and therapeutic applications in the current and future pandemic waves of coronavirus.

Comparison of Homologous and Heterologous Booster SARS-CoV-2 Vaccination in Autoimmune Rheumatic and Musculoskeletal Patients.

Vaccination against SARS-CoV-2 to prevent COVID-19 is highly recommended for immunocompromised patients with autoimmune rheumatic and musculoskeletal diseases (aiRMDs). Little is known about the effect of booster vaccination or infection followed by previously completed two-dose vaccination in aiRMDs. We determined neutralizing anti-SARS-CoV-2 antibody levels and applied flow cytometric immunophenotyping to quantify the SARS-CoV-2 reactive B- and T-cell mediated immunity in aiRMDs receiving homologous or heterologous boosters or acquired infection following vaccination. Patients receiving a heterologous booster had a higher proportion of IgM+ SARS-CoV-2 S+ CD19+CD27+ peripheral memory B-cells in comparison to those who acquired infection. Biologic therapy decreased the number of S+CD19+; S+CD19+CD27+IgG+; and S+CD19+CD27+IgM+ B-cells. The response rate to a booster event in cellular immunity was the highest in the S-, M-, and N-reactive CD4+CD40L+ T-cell subset. Patients with a disease duration of more than 10 years had higher proportions of CD8+TNF-α+ and CD8+IFN-γ+ T-cells in comparison to patients who were diagnosed less than 10 years ago. We detected neutralizing antibodies, S+ reactive peripheral memory B-cells, and five S-, M-, and N-reactive T-cells subsets in our patient cohort showing the importance of booster events. Biologic therapy and <10 years disease duration may confound anti-SARS-CoV-2 specific immunity in aiRMDs.

The Development of Pharmacophore Models for the Search of New Natural Inhibitors of SARS-CoV-2 Spike RBD-ACE2 Binding Interface.

To date, some succeeding variants of SARS-CoV-2 have become more contagious. This virus is known to enter human cells by binding the receptor-binding domain (RBD) of spike protein with the angiotensin-converting enzyme 2 (ACE2), the latter being a membrane protein that regulates the renin-angiotensin system. Since the host cell receptor plays a critical role in viral entry, inhibition of the RBD-ACE2 complex is a promising strategy for preventing COVID-19 infection. In the present communication, we propose and utilize an approach based on the generation of a complex of pharmacophore models and subsequent Induced Fit Docking (IFD) to identify potential inhibitors of the main binding sites of the Omicron SARS-CoV-2 RBD(S1)-ACE2 complex (PDB ID: 7T9L) among a number of natural products of various types and origins. Several natural compounds have been found to provide a high affinity for the receptor of interest. It is expected that the present results will stimulate further research aimed at the development of specialized drugs against this virus.

SARS-CoV-2 spike protein receptor-binding domain N-glycans facilitate viral internalization in respiratory epithelial cells.

N-glycosylation plays an important role in the pathogenesis of viral infections. However, the role of SARS-CoV-2 RBD N-glycosylation in viral entry remains elusive. In this study, we expressed and purified N331 and N343 N-glycosite mutants of SARS-CoV-2 RBD. We found that de-glycosylation at N331 and N343 drastically reduces the RBD binding to ACE2. More importantly, based on qualitative and quantitative virology research methods, we show that the mutation of RBD N-glycosites interfered with SARS-CoV-2 internalization rather than attachment potentially by decreasing RBD binding to the receptors. Also, the double N-glycosites mutant (N331 + N343) showed significantly increased sensitivity against the designated RBD neutralizing antibodies. Taken together, these results suggest that N-glycosylation of SARS-CoV-2 RBD is not only critical for viral internalization into respiratory epithelial cells but also shields the virus from neutralization. It may provide new insights into the biological process of early-stage SARS-CoV-2 infection with potential therapeutic implications.

Recognition through GRP78 is enhanced in the UK, South African, and Brazilian variants of SARS-CoV-2; An in silico perspective.

New SARS-CoV-2 variants emerged in the United Kingdom and South Africa in December 2020 in concomitant with the Brazillian variant in February 2021 (B.1.1.248 lineage) and currently sparking worldwide during the last few months. The new strain 501.V2 in South Africa bears three mutations in the spike receptor-binding domain (RBD); K417 N, E484K, and N501Y, while the Brazilian B.1.1.248 lineage has 12 mutations. In the current study, we simulate the complex ACE2-SARS-CoV-2 spike RBD system in which the RBD is in the wild-type and mutated isoforms. Additionally, the cell-surface Glucose Regulated Protein 78 (CS-GRP78) associated with the ACE2-SARS-CoV-2 spike RBD complex (ACE2-S RBD) is modeled at the presence of these mutant variants of the viral spike. The results showed that E484K and N501Y are critical in viral spike recognition through either ACE2 or CS-GRP78. The mutated variants (the UK, South African, and Brazilian) of the spike RBD tightly bind to GRP78 more than in the case of the wild-type RBD. These results point to the potent role of GRP78 with ACE2 in the attachment of the new variants, which could be a key for the design of inhibitors to block SARS-CoV-2 attachment and entry to the host cell.

Antibody response to SARS-CoV-2 vaccination is extremely vivacious in subjects with previous SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic calls for rapid actions, now principally oriented to a world-wide vaccination campaign. In this study we verified if, in individuals with a previous SARS-CoV-2 infection, a single dose of messenger RNA (mRNA) vaccine would be immunologically equivalent to a full vaccine schedule in naïve individuals. Health care workers (184) with a previous SARS-CoV-2 infection were sampled soon before the second dose of vaccine and between 7 and 10 days after the second dose, the last sampling time was applied to SARS-CoV-2 naïve individuals, too. Antibodies against SARS-CoV-2 were measured using Elecsys Anti-SARS-CoV-2 S immunoassay. The study was powered for non-inferiority. We used non parametric tests and Pearson correlation test to perform inferential analysis. After a single vaccine injection, the median titer of specific antibodies in individuals with previous coronavirus disease 2019 was 30.527 U/ml (interquartile range [IQR]: 19.992-39.288) and in subjects with previous SARS-CoV-2 asymptomatic infection was 19.367.5 U/ml (IQR: 14.688-31.353) (p = .032). Both results were far above the median titer in naïve individuals after a full vaccination schedule: 1974.5 U/ml (IQR: 895-3455) (p < .0001). Adverse events after vaccine injection were more frequent after the second dose of vaccine (mean: 0.95; 95% confidence interval [CI]: 0.75-1.14 vs. mean: 1.91; 95% CI: 1.63-2.19) (p < .0001) and in exposed compared to naïve (mean: 1.63; 95% CI: 1.28-1.98 vs. mean: 2.35; 95% CI: 1.87-2.82) (p = .015). In SARS-CoV-2 naturally infected individuals a single mRNA vaccine dose seems sufficient to reach immunity. Modifying current dosing schedules would speed-up vaccination campaigns.

Interactions of Spike-RBD of SARS-CoV-2 and Platelet Factor 4: New Insights in the Etiopathogenesis of Thrombosis.

The rare but dangerous adverse events evidenced after massive vaccination against SARS-CoV-2 are represented by thrombosis and thrombocytopenia. The patients diagnosed with severe COVID-19 may develop a pro-thrombotic state with a much higher frequency, thus we decided to investigate the role of Spike protein (the only common product of the two conditions) or the anti-Spike antibodies in the etiopathogenesis of thrombosis. A pathogenic Platelet Factor 4 (PF4)-dependent syndrome, unrelated to the use of heparin therapy, has been reported after the administration of vaccines in the patients manifesting acute thrombocytopenia and thrombosis. Thus, we aimed at shedding light on the structural similarities of Spike of SARS-CoV-2 and PF4 on their eventual biochemical interactions and on the role of their specific antibodies. The similarities between PF4 and Spike-RBD proteins were evaluated by a comparison of the structures and by testing the cross-reactivity of their specific antibodies by ELISA assays. We found that the anti-Spike antibodies do not recognize PF4, on the contrary, the anti-PF4 antibodies show some cross-reactivity for Spike-RBD. More interestingly, we report for the first time that the PF4 and Spike-RBD proteins can bind each other. These data suggest that the interaction of the two proteins could be involved in the generation of anti-PF4 antibodies, their binding to Spike-RBD, which could lead to platelets aggregation due also to their high expression of ACE2.

Establishment of Murine Hybridoma Cells Producing Antibodies against Spike Protein of SARS-CoV-2.

In 2020 the world faced the pandemic of COVID-19 severe acute respiratory syndrome caused by a new type of coronavirus named SARS-CoV-2. To stop the spread of the disease, it is crucial to create molecular tools allowing the investigation, diagnoses and treatment of COVID-19. One of such tools are monoclonal antibodies (mAbs). In this study we describe the development of hybridoma cells that can produce mouse mAbs against receptor binding domain of SARS-CoV-2 spike (S) protein. These mAbs are able to specifically detect native and denatured S proteins in all tested applications, including immunoblotting, enzyme-linked immunosorbent assay, immunofluorescence staining of cells and immunohistochemical staining of paraffin embedded patients' tissue samples. In addition, we showed that the obtained mAbs can efficiently block SARS-CoV-2 infection in in vitro experiments. Finally, we determined the amino acid sequence of light and heavy chains of the mAbs. This information will allow the use of corresponding peptides to establish genetically engineered therapeutic antibodies. To date multiple mAbs against SARS-CoV-2 proteins have been established, however, bigger sets of various antibodies will allow the detection and neutralization of SARS-CoV-2, even if the virus acquires novel mutations.

Screening and optimization of shark nanobodies against SARS-CoV-2 spike RBD.

SARS-CoV-2 continues to threaten human health, antibody therapy is one way to control the infection. Because new SARS-CoV-2 mutations are constantly emerging, there is an urgent need to develop broadly neutralizing antibodies to block the viral entry into host cells. VNAR from sharks is the smallest natural antigen binding domain, with the advantages of small size, flexible paratopes, good stability, and low manufacturing cost. Here, we used recombinant SARS-CoV-2 Spike-RBD to immunize sharks and constructed a VNAR phage display library. VNAR R1C2, selected from the library, efficiently binds to the RBD domain and blocks the infection of ACE2-positive cells by pseudovirus. Next, homologous bivalent VNARs were constructed through the tandem fusion of two R1C2 units, which enhanced both the affinity and neutralizing activity of R1C2. R1C2 was predicted to bind to a relatively conserved region within the RBD. By introducing mutations at four key binding sites within the CDR3 and HV2 regions of R1C2, the affinity and neutralizing activity of R1C2 were significantly improved. Furthermore, R1C2 also exhibits an effective capacity of binding to the Omicron variants (BA.2 and XBB.1). Together, these results suggest that R1C2 could serve as a valuable candidate for preventing and treating SARS-CoV-2 infections.

A live attenuated influenza B virus vaccine expressing RBD elicits protective immunity against SARS-CoV-2 in mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant threat to human health globally. It is crucial to develop a vaccine to reduce the effect of the virus on public health, economy, and society and regulate the transmission of SARS-CoV-2. Influenza B virus (IBV) can be used as a vector that does not rely on the current circulating influenza A strains. In this study, we constructed an IBV-based vector vaccine by inserting a receptor-binding domain (RBD) into a non-structural protein 1 (NS1)-truncated gene (rIBV-NS110-RBD). Subsequently, we assessed its safety, immunogenicity, and protective efficacy against SARS-CoV-2 in mice, and observed that it was safe in a mouse model. Intranasal administration of a recombinant rIBV-NS110-RBD vaccine induced high levels of SARS-CoV-2-specific IgA and IgG antibodies and T cell-mediated immunity in mice. Administering two doses of the intranasal rIBV-NS110-RBD vaccine significantly reduced the viral load and lung damage in mice. This novel IBV-based vaccine offers a novel approach for controlling the SARS-CoV-2 pandemic.

Application of a Receptor-Binding-Domain-Based Simple Immunoassay for Assessing Humoral Immunity against Emerging SARS-CoV-2 Virus Variants.

We have developed a simple, rapid, high-throughput RBD-based ELISA to assess the humoral immunity against emerging SARS-CoV-2 virus variants. The cDNAs of the His-tagged RBD proteins of the virus variants were stably engineered into HEK cells secreting the protein into the supernatant, and RBD purification was performed by Ni-chromatography and buffer exchange by membrane filtration. The simplified assay uses single dilutions of sera from finger-pricked native blood samples, purified RBD in 96-well plates, and a chromogenic dye for development. The results of this RBD-ELISA were confirmed to correlate with those of a commercial immunoassay measuring antibodies against the Wuhan strain, as well as direct virus neutralization assays assessing the cellular effects of the Wuhan and the Omicron (BA.5) variants. Here, we document the applicability of this ELISA to assess the variant-specific humoral immunity in vaccinated and convalescent patients, as well as to follow the time course of selective vaccination response. This simple and rapid assay, easily modified to detect humoral immunity against emerging SARS-CoV-2 virus variants, may help to assess the level of antiviral protection after vaccination or infection.

Accelerating the discovery of the beyond rule of five compounds that have high affinities toward SARS-CoV-2 spike RBD.

The battle against SARS-CoV-2 coronavirus is the focal point for the global pandemic that has affected millions of lives worldwide. The need for effective and selective therapeutics for the treatment of the disease caused by SARS-CoV-2 is critical. Herein, we performed a hierarchical computational approach incorporating molecular docking studies, molecular dynamics simulations, absolute binding energy calculations, and steered molecular dynamics simulations for the discovery of potential compounds with high affinity towards SARS-CoV-2 spike RBD. By leveraging ZINC15 database, a total of 1282 in-clinical and FDA approved drugs were filtered out from nearly 0.5 million protomers of relatively large compounds (MW > 500, and LogP ≤ 5). Our results depict plausible mechanistic aspects related to the blockage of SARS-CoV-2 spike RBD by the top hits discovered. We found that the most promising candidates, namely, ZINC95628821, ZINC95617623, ZINC3979524, and ZINC261494658, strongly bind to the spike RBD and interfere with the human ACE2 receptor. These findings accelerate the rational design of selective inhibitors targeting the spike RBD protein of SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

Neohesperidin and spike RBD interaction in omicron and its sub-variants: In silico, structural and simulation studies.

COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged first around December 2019 in the city of Wuhan, China. Since then, several variants of the virus have emerged with different biological properties. This pandemic has so far led to widespread infection cycles with millions of fatalities and infections globally. In the recent cycle, a new variant omicron and its three sub-variants BA.1, BA.2 and BA.3 have emerged which seems to evade host immune defences and have brisk infection rate. Particularly, BA.2 variant has shown high transmission rate over BA.1 strain in different countries including India. In the present study, we have evaluated a set of eighty drugs/compounds using in silico docking calculations in omicron and its variants. These molecules were reported previously against SARS-CoV-2. Our docking and simulation analyses suggest differences in affinity of these compounds in omicron and BA.2 compared to SARS-CoV-2. These studies show that neohesperidin, a natural flavonoid found in Citrus aurantium makes a stable interaction with spike receptor domain of omicron and BA.2 compared to other variants. Free energy binding analyses further validates that neohesperidin forms a stable complex with spike RBD in omicron and BA.2 with a binding energy of -237.9 ± 18.7 kJ/mol and -164.1 ± 17.5 kJ/mol respectively. Key residual differences in the RBD interface of these variants form the basis for differential interaction affinities with neohesperidin as drug binding site overlaps with RBD-human ACE2 interface. These data might be useful for the design and development of novel scaffolds and pharmacophores to develop specific therapeutic strategies against these novel variants.

Repurposing 1,2,4-oxadiazoles as SARS-CoV-2 PLpro inhibitors and investigation of their possible viral entry blockade potential.

Although vaccines are obviously mitigating the COVID-19 pandemic diffusion, efficient complementary antiviral agents are urgently needed to combat SARS-CoV-2. The viral papain-like protease (PLpro) is a promising therapeutic target being one of only two essential proteases crucial for viral replication. Nevertheless, it dysregulates the host immune sensing response. Here we report repositioning of the privileged 1,2,4-oxadiazole scaffold as promising SARS-CoV-2 PLpro inhibitor with potential viral entry inhibition profile. The design strategy relied on mimicking the general structural features of the lead benzamide PLpro inhibitor GRL0617 with isosteric replacement of its pharmacophoric amide backbone by 1,2,4-oxadiazole core. Inspired by the multitarget antiviral agents, the substitution pattern was rationalized to tune the scaffold's potency against other additional viral targets, especially the spike receptor binding domain (RBD) that is responsible for the viral invasion. The Adopted facial synthetic protocol allowed easy access to various rationally substituted derivatives. Among the evaluated series, the 2-[5-(pyridin-4-yl)-1,2,4-oxadiazol-3-yl]aniline (5) displayed the most balanced dual inhibitory potential against SARS-CoV-2 PLpro (IC50=7.197 µM) and spike protein RBD (IC50 = 8.673 µM), with acceptable ligand efficiency metrics, practical LogP (3.8) and safety profile on Wi-38 (CC50 = 51.78 µM) and LT-A549 (CC50 = 45.77 µM) lung cells. Docking simulations declared the possible structural determinants of activities and enriched the SAR data for further optimization studies.

Impaired Humoral Immunity Identified in Inactivated SARS-CoV-2 Vaccine Recipients without Anti-Spike RBD Antibodies.

Inactivated SARS-CoV-2 vaccines have been deployed in a significant portion of the world population, who have widely varied responses to vaccination. Understanding this differential response would help the development of new vaccines for non-responders. Here, we conducted surveillance of anti-Spike receptor-binding domain (RBD) antibody levels in a large cohort of 534 healthy Chinese subjects vaccinated with two doses of inactivated SARS-CoV-2 vaccines. We show that the positive rate of antibodies among vaccinated subjects rapidly wanes as the interval between antibody testing and vaccination increases (14 to 119 days: 81.03%, 363 of 448 subjects; 120 to 149 days: 46.43%, 13 of 28 subjects; more than 150 days: 20%, 1 of 5 subjects). However, the antibodies were maintained at high levels in 16 convalescent COVID-19 patients at more than 150 days after recovery. We also found that increased age and body mass index are associated with decreased antibody levels. Vaccinated subjects who fail to produce antibodies display impaired B-cell activating humoral immunity, which was confirmed in COVID-19 patients without antibodies detected at 4 to 18 days after diagnosis. IMPORTANCE Our study illustrates the immune responses engaged by encountering antigen, highlighting the critical roles of B-cell activating humoral immunity in the body's antibody production.

Abiotic Synthetic Antibody Inhibitor with Broad-Spectrum Neutralization and Antiviral Efficacy against Escaping SARS-CoV-2 Variants.

The rapid emergence and spread of vaccine/antibody-escaping variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed serious challenges to our efforts in combating corona virus disease 2019 (COVID-19) pandemic. A potent and broad-spectrum neutralizing reagent against these escaping mutants is extremely important for the development of strategies for the prevention and treatment of SARS-CoV-2 infection. We herein report an abiotic synthetic antibody inhibitor as a potential anti-SARS-CoV-2 therapeutic agent. The inhibitor, Aphe-NP14, was selected from a synthetic hydrogel polymer nanoparticle library created by incorporating monomers with functionalities complementary to key residues of the SARS-CoV-2 spike glycoprotein receptor binding domain (RBD) involved in human angiotensin-converting enzyme 2 (ACE2) binding. It has high capacity, fast adsorption kinetics, strong affinity, and broad specificity in biologically relevant conditions to both the wild type and the current variants of concern, including Beta, Delta, and Omicron spike RBD. The Aphe-NP14 uptake of spike RBD results in strong blockage of spike RBD-ACE2 interaction and thus potent neutralization efficacy against these escaping spike protein variant pseudotyped viruses. It also inhibits live SARS-CoV-2 virus recognition, entry, replication, and infection in vitro and in vivo. The Aphe-NP14 intranasal administration is found to be safe due to its low in vitro and in vivo toxicity. These results establish a potential application of abiotic synthetic antibody inhibitors in the prevention and treatment of the infection of emerging or possibly future SARS-CoV-2 variants.

Virtual screening and molecular dynamics simulation study of abyssomicins as potential inhibitors of COVID-19 virus main protease and spike protein.

The lack of any effective cure for the infectious COVID-19 disease has created a sense of urgency and motivated the search for effective antiviral drugs. Abyssomicins are actinomyces-derived spirotetronates polyketides antibiotics known for their promising antibacterial, antitumor, and antiviral activities. In this study, computational approaches were used to investigate the binding mechanism and the inhibitory ability of 38 abyssomicins against the main protease (Mpro) and the spike protein receptor-binding domain (RBD) of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The results identified abyssomicins C, J, W, atrop-O-benzyl abyssomicin C, and atrop-O-benzyl desmethyl abyssomicin C as the most potential inhibitors of Mpro and RBD with binding energy ranges between -8.1 and -9.9 kcal mol-1; and between -6.9 and -8.2 kcal mol-1, respectively. Further analyses of physicochemical properties and drug-likeness suggested that all selected active abyssomicins, with the exception of abyssomicin J, obeyed Lipinski's rule of five. The stability of protein-ligand complexes was confirmed by performing molecular dynamics simulation for 100 ns and evaluating parameters such as such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), total number of contacts, and secondary structure. Prime/MM-GBSA (Molecular Mechanics-General Born Surface Area) and principal component analysis (PCA) analyses also confirmed the stable nature of protein-ligand complexes. Overall, the results showed that the studied abyssomicins have significant interactions with the selected protein targets; therefore, they were deemed viable candidates for further in vitro and in vivo evaluation.Communicated by Ramaswamy H. Sarma.