Search Results (231)

The rapid evolution of SARS-CoV-2 has underscored the need for a detailed understanding of antibody binding mechanisms to combat immune evasion by emerging variants. In this study, we investigated the interactions between Class I neutralizing antibodies—BD55-1205, BD-604, OMI-42, P5S-1H1, and P5S-2B10—and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein using multiscale modeling, which combined molecular simulations with the ensemble-based mutational scanning of the binding interfaces and binding free energy computations. A central theme emerging from this work is that the unique binding strength and resilience to immune escape of the BD55-1205 antibody are determined by leveraging a broad epitope footprint and distributed hotspot architecture, additionally supported by backbone-mediated specific interactions, which are less sensitive to amino acid substitutions and together enable exceptional tolerance to mutational escape. In contrast, BD-604 and OMI-42 exhibit localized binding modes with strong dependence on side-chain interactions, rendering them particularly vulnerable to escape mutations at K417N, L455M, F456L and A475V. Similarly, P5S-1H1 and P5S-2B10 display intermediate behavior—effective in some contexts but increasingly susceptible to antigenic drift due to narrower epitope coverage and concentrated hotspots. Our computational predictions show strong agreement with experimental deep mutational scanning data, validating the accuracy of the models and reinforcing the value of binding hotspot mapping in predicting antibody vulnerability. This work highlights that neutralization breadth and durability are not solely dictated by epitope location, but also by how binding energy is distributed across the interface. The results provide atomistic insight into mechanisms driving resilience to immune escape for broadly neutralizing antibodies targeting the ACE2 binding interface—which stems from cumulative effects of structural diversity in binding contacts, redundancy in interaction patterns and reduced vulnerability to mutation-prone positions. Full article

Nucleolin (NCL), an RNA-binding protein which regulates critical cellular processes, is frequently dysregulated in human cancers, including breast cancer, making it an attractive therapeutic target. However, molecular details of the RNA-NCL interaction have not been investigated yet. A tRNA fragment named tRF3E, displaying tumor suppressor roles in breast cancer, was found to bind NCL with high affinity displacing NCL-controlled transcripts. Here, we investigated the determinants and cooperativity of tRF3E-NCL interaction by Electrophoretic Mobility Shift Assays and in silico docking analysis, using wild-type or mutated tRF3E. We found that NCL, through its RNA-binding domains (RBD1–2 and RBD3–4), binds simultaneously two tRF3E molecules, giving rise to an energetically favored complex. Instead, a mutant form of tRF3E (M19–24), in which the NCL recognition element in position 19–24 has been disrupted, contacts NCL exclusively at RBD3–4, causing the loss of cooperativity among RBDs. Importantly, when expressed in MCF7 breast cancer cells, tRF3E significantly reduced cell proliferation and colony formation, confirming its role as tumor suppressor, but tRF3E functional properties were lost when the 19–24 motif was mutated, suggesting that cooperativity among multiple domains is required for the NCL-mediated tRF3E antitumor function. This study sheds light on the dynamic of RNA-NCL interaction and lays the foundations for using tRF3E as a promising NCL-targeted biodrug candidate. Full article

Glycyrrhizic acid (GA) and its derivatives have been reported to have potent pharmacological effects against viral infections, including SARS-CoV and SARS-CoV-2. However, their antiviral mechanisms against coronaviruses are not fully understood. In this study, we found that diammonium glycyrrhizinate (DG) can effectively reduce infections of several human coronaviruses, including HCoV-OC43, HCoV-229E, and SARS-CoV-2, as well as newly emerged variants, with EC₅₀ values ranging from 115 to 391 μg/mL being recorded. Time-of-addition and pseudotype virus infection studies indicated that DG treatment dramatically inhibits the process of virus entry into cells. Furthermore, we demonstrated that DG broadly binds to the RBD of human coronaviruses, thereby blocking spike-mediated cellular entry, by using TR-FRET-based receptor-binding domain (RBD)-ACE2 interaction assay, capillary electrophoresis (CE), and surface plasmon resonance (SPR) assay. In support of this notion, studies of molecular docking and amino acid mutation showed that DG may directly bind to a conserved hydrophobic pocket of the RBD of coronaviruses. Importantly, intranasal administration of DG had a significant protective effect against viral infection in a HCoV-OC43 mouse model. Finally, we found that combinations of DG and other coronavirus inhibitors exhibited antiviral synergy. In summary, our studies strongly reveal that DG exerts broad-spectrum antiviral activity against human coronaviruses by interrupting spike-mediated cellular entry, demonstrating the pharmacological feasibility of using DG as a candidate for alternative treatment and prevention of coronavirus infection. Full article

Background/Objectives: Identification and characterization of broadly neutralizing monoclonal antibodies from individuals exposed to SARS-CoV-2, either by infection or vaccination, can inform the development of next-generation vaccines and antibody therapeutics with pan-SARS-CoV-2 protection. Methods: Through single B cell sorting and RT-PCR, monoclonal antibodies (mAbs) were isolated from a donor who experienced a BA.5 or BF.7 breakthrough infection after three doses of inactivated vaccines. Their binding and neutralizing capacities were measured with ELISA and a pseudovirus-based neutralization assay, respectively. Their epitopes were mapped by competition ELISA and site-directed mutation. Results: Among a total of 67 spike-specific mAbs cloned from the donor, four mAbs (KXD643, KXD652, KXD681, and KXD686) can neutralize all tested SARS-CoV-2 variants from wild-type to KP.3. Moreover, KXD643, KXD652, and KXD681 belong to a clonotype encoded by IGHV5-51 and IGKV1-13 and recognize the cryptic and conserved RBD-8 epitope on the receptor-binding domain (RBD). In contrast, KXD686 is encoded by IGHV1-69 and IGKV3-20 and targets a conserved epitope (NTD Site iv) outside the antigenic supersite (NTD Site i) of the N-terminal domain (NTD). Notably, antibody cocktails containing these two groups of mAbs can neutralize SARS-CoV-2 more potently due to synergistic effects. In addition, bispecific antibodies derived from KXD643 and KXD686 demonstrate further improved neutralizing potency compared to antibody cocktails. Conclusions: These four mAbs can be developed as candidates of pan-SARS-CoV-2 antibody therapeutics through further antibody engineering. On the other hand, vaccines designed to simultaneously elicit neutralizing antibodies towards RBD-8 and NTD Site iv have the potential to provide pan-SARS-CoV-2 protection. Full article

In this study, we conducted a comprehensive analysis of the interactions between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and four neutralizing antibodies—S309, S304, CYFN1006, and VIR-7229. Using integrative computational modeling that combined all-atom molecular dynamics (MD) simulations, mutational scanning, and MM-GBSA binding free energy calculations, we elucidated the structural, energetic, and dynamic determinants of antibody binding. Our findings reveal distinct dynamic binding mechanisms and evolutionary adaptation driving the broad neutralization effect of these antibodies. We show that S309 targets conserved residues near the ACE2 interface, leveraging synergistic van der Waals and electrostatic interactions, while S304 focuses on fewer but sensitive residues, making it more susceptible to escape mutations. The analysis of CYFN-1006.1 and CYFN-1006.2 antibody binding highlights broad epitope coverage with critical anchors at T345, K440, and T346, enhancing its efficacy against variants carrying the K356T mutation, which caused escape from S309 binding. Our analysis of broadly potent VIR-7229 antibody binding to XBB.1.5 and EG.5 Omicron variants emphasized a large and structurally complex epitope, demonstrating certain adaptability and compensatory effects to F456L and L455S mutations. Mutational profiling identified key residues crucial for antibody binding, including T345, P337, and R346 for S309 as well as T385 and K386 for S304, underscoring their roles as evolutionary “weak spots” that balance viral fitness and immune evasion. The results of the energetic analysis demonstrate a good agreement between the predicted binding hotspots, reveal distinct energetic mechanisms of binding, and highlight the importance of targeting conserved residues and diverse epitopes to counteract viral resistance. Full article

The protein sequence space is vast. This fact, together with the prevalence of epistasis, hampers the engineering of novel enzymes through library screening and is a major obstacle to any attempt to predict natural protein evolution. Recently, specialized methodologies have been used to determine fitness data on ~260,000 sequences for the gene of the enzyme dihydrofolate reductase and antibody affinity data for all combinations of the mutations present in the receptor-binding domain (RBD) of the Omicron strain of SARS-CoV-2 (~30,000 variants). We show that upon iterative training on a total of just a few hundred variants, various state-of-the-art AI tools (multi-layer perceptron, random forest, and XGBoost algorithms) find very high fitness variants of the enzyme and predict the antibody evasion patterns of the RBD. This work provides a basis for efficient, widely applicable, low-throughput experimental approaches to assess viral protein evolution and to engineer enzymes for biotechnological applications. Full article

In the past 5 years, the COVID-19 pandemic has experienced frequently changing variants contextualizing immune evasion. The emergence of Omicron with >30–50 mutations on the spike gene has shown a sharp divergence from its relative VOCs, such as WT, Alpha, Beta, Gamma, and Delta. The requisition of prime boosting was essential within 3–6 months to improve the Nab response that had been not lasted for longer. Omicron subvariant BA.1.1 was less transmissible, but with an extra nine mutations in next variant BA.2 made it more transmissible. This remarkable heterogeneity was reported in ORF1ab or TRS sites, ORF7a, and 10 regions in the genomic sequences of Omicron BA.2 and its evolving subvariants BA.4.6, BF.7, BQ.2, BF. 7, BA.2.75.2, and BA.5 (BQ.1 and BQ.1.1). The mutational stability of subvariants XBB, XBB 1, XBB 1.5, and XBB 1.6 conferred a similar affinity towards ACE-2. This phenomenon has been reported in breakthrough infections and after booster vaccinations producing hybrid immunity. The reduced pathogenic nature of Omicron has implicated its adaptation either through immunocompromised individuals or other animal hosts. The binding capacity of RBD and ACE-2, including the proteolytic priming via TMPRSS2, reveals its (in-vitro) transmissibility behavior. RBD mutations signify transmissibility, S1/S2 enhances virulence, while S2 infers the effective immunogenic response. Initial mutations D614G, E484A, N501Y, Q493K, K417N, S477N, Y505H, and G496S were found to increase the Ab escape. Some mutations such as, R346K, L452R, and F486Vwere seen delivering immune pressure. HR2 region (S2) displayed mutations R436S, K444T, F486S, and D1199N with altered spike positions. Later on, the booster dose or breakthrough infections contributed to elevating the immune profile. Several other mutations in BA.1.1-N460K, R346T, K444T, and BA.2.75.2-F486S have also conferred the neutralization resistance. The least studied T-cell response in SARS-CoV-2 affects HLA- TCR interactions, thus, it plays a role in limiting the virus clearance. Antigenic cartographic analysis has also shown Omicron’s drift from its predecessor variants. The rapidly evolving SARS-CoV-2 variants and subvariants have driven the population-based immunity escape in fully immunized individuals within short period. This could be an indication that Omicron is heading towards endemicity and may evolve in future with subvariants could lead to outbreaks, which requires regular surveillance. Full article

SARS-CoV-2 remains a severe threat to worldwide public health, particularly as the virus continues to evolve and diversify into variants of concern (VOCs). Among these VOCs, Omicron variants exhibit unique phenotypic traits, such as immune evasion, transmissibility, and severity, due to numerous spike protein mutations and the rapid subvariant evolution. These Omicron subvariants have more than 15 mutations in the receptor-binding domain (RBD), a region of the SARS-CoV-2 spike protein that is important for recognition and binding with the angiotensin-converting enzyme 2 (ACE2) human receptor. To address the impact of these high numbers of Omicron mutations on the binding process, we have developed a novel method to precisely quantify amino acid interactions via the amino acid–amino acid bond pair (AABP). We applied this concept to investigate the interface interactions of the RBD–ACE2 complex in four Omicron Variants (BA.1, BA.2, BA.5, and XBB.1.16) with its Wild Type counterpart. Based on the AABP analysis, we have identified all the sites that are affected by mutation and have provided evidence that unmutated sites are also impacted by mutation. We have calculated that the binding between RBD and ACE2 is strongest in OV BA.1, followed by OV BA.2, WT, OV BA.5, and OV XBB.1.16. We also present the partial charge values for all 311 residues across these five models. Our analysis provides a detailed understanding of changes caused by mutation in each Omicron interface complex. Full article

Mesothelioma is a rare and aggressive cancer linked to asbestos exposure and characterized by rapid metastasis and poor prognosis. Inhibition of adenosine deaminase acting on dsRNA 2 (ADAR2) RNA binding but not ADAR2 editing has shown antitumor effects in mesothelioma. Natural compounds from the Traditional Chinese Medicine (TCM) database were docked to the RNA-binding interface of ADAR2’s second dsRNA binding domain (dsRBD2), and their drug-likeness and predicted safety were assessed. Eight ligands (ZINC000085597263, ZINC000085633079, ZINC000014649947, ZINC000034512861, ZINC000070454124, ZINC000085594944, ZINC000085633008, and ZINC000095909822) showed high binding affinity to dsRBD2 from molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) calculations. Protein–ligand interactions were analyzed to identify key residues contributing to these binding affinities. Molecular dynamics (MD) simulations of dsRBD–ligand–RNA complexes revealed that four compounds (ZINC000085597263, ZINC000085633079, ZINC000014649947, and ZINC000034512861) had negative binding affinities to dsRBD2 in the presence of the RNA substrate GluR-2. Key residues, including Val164, Met165, Lys209, and Lys212, were crucial for ligand binding, even with RNA present, suggesting these compounds could inhibit dsRBD2’s RNA-binding function. The predicted biological activities of these compounds indicate potential anticancer properties, particularly for the treatment of mesothelioma. These compounds are structurally similar to known anti-mesothelioma agents or anticancer drugs, highlighting their therapeutic potential. Current mesothelioma treatments are limited. Optimization of these compounds, alone or in combination with current therapeutics, has potential for mesothelioma treatment. Additionally, five high-quality full-length ADAR2 models were developed. These models provide insights into ADAR2 function, mutation impacts, and potential areas for protein engineering to enhance stability, RNA-binding specificity, or protein interactions, particularly concerning dimerization or complex formation with other proteins and RNAs. Full article

The evolution of SARS-CoV-2, particularly the emergence of Omicron variants, has raised questions regarding changes in its binding affinity to the human angiotensin-converting enzyme 2 receptor (hACE2). Understanding the impact of mutations on the interaction between the receptor-binding domain (RBD) of the spike protein and hACE2 is critical for evaluating viral transmissibility, immune evasion, and the efficacy of therapeutic strategies. Here, we used molecular dynamics (MD) simulations and binding energy calculations to investigate the structural and energetic differences between the hACE2- RBD complexes of wild-type (WT), Delta, and Omicron subvariants. Our results indicate that the Delta and the first Omicron variants showed the highest and the second-highest binding energy among the variants studied. Furthermore, while Omicron variants exhibit increased structural stability and altered electrostatic potential at the hACE2–RBD interface when compared to the ancestral WT, their binding strength to hACE2 does not consistently increase with viral evolution. Moreover, newer Omicron subvariants like JN.1 exhibit a bimodal conformational strategy, alternating between a high-affinity state for hACE2 and a low-affinity state, which could potentially facilitate immune evasion. These findings suggest that, in addition to enhanced hACE2 binding affinity, other factors, such as immune evasion and structural adaptability, shape SARS-CoV-2 evolution. Full article

The SARS-CoV-2 Omicron variants show different behavior compared to the previous variants, especially with respect to the Delta variant, which promotes a lower morbidity despite being much more contagious. In this perspective, we performed molecular dynamics (MD) simulations of the different spike RBD/hACE2 complexes corresponding to the WT, Delta and four Omicron variants. Carrying out a comprehensive analysis of residue interactions within and between the two partners allowed us to draw the profile of each variant by using complementary methods (PairInt, hydrophobic potential, contact PCA). PairInt calculations highlighted the residues most involved in electrostatic interactions, which make a strong contribution to the binding with highly stable interactions between spike RBD and hACE2. Apolar contacts made a substantial and complementary contribution in Omicron with the detection of two hydrophobic patches. Contact networks and cross-correlation matrices were able to detect subtle changes at point mutations as the S375F mutation occurring in all Omicron variants, which is likely to confer an advantage in binding stability. This study brings new highlights on the dynamic binding of spike RBD to hACE2, which may explain the final persistence of Omicron over Delta. Full article

We performed a comprehensive structural analysis of the conformational space of several spike (S) protein variants using molecular dynamics (MD) simulations. Specifically, we examined four well-known variants (Delta, BA.1, XBB.1.5, and JN.1) alongside the wild-type (WT) form of SARS-CoV-2. The conformational states of each variant were characterized by analyzing their distributions within a selected space of collective variables (CVs), such as inter-domain distances between the receptor-binding domain (RBD) and the N-terminal domain (NTD). Our primary focus was to identify conformational states relevant to potential structural transitions and to determine the set of native contacts (NCs) that stabilize these conformations. The results reveal that genetically more distant variants, such as XBB.1.5, BA.1, and JN.1, tend to adopt more compact conformational states compared to the WT. Additionally, these variants exhibit novel NC profiles, characterized by an increased number of specific contacts distributed among ionic, polar, and nonpolar residues. We further analyzed the impact of specific mutations, including T478K, N500Y, and Y504H. These mutations not only enhance interactions with the human host receptor but also alter inter-chain stability by introducing additional NCs compared to the WT. Consequently, these mutations may influence the accessibility of certain protein regions to neutralizing antibodies. Overall, these findings contribute to a deeper understanding of the structural and functional variations among S protein variants. Full article

Background: Since the emergence of SARS-CoV-2 in the human population, the virus genome has undergone numerous mutations, enabling it to enhance transmissibility and evade acquired immunity. As a result of these mutations, most monoclonal neutralizing antibodies have lost their efficacy, as they are unable to neutralize new variants. Antibodies that neutralize a broad range of SARS-CoV-2 variants are of significant value in combating both current and potential future variants, making the identification and development of such antibodies an ongoing critical goal. This study discusses the strategy of using heterologous antigens in biopanning rounds. Methods: After four rounds of biopanning, nanobody variants were selected from a phage display library. Immunochemical methods were used to evaluate their specificity to the S protein of various SARS-CoV-2 variants, as well as to determine their competitive ability against ACE2. Viral neutralization activity was analyzed. A three-dimensional model of nanobody interaction with RBD was constructed. Results: Four nanobodies were obtained that specifically bind to the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein and exhibit neutralizing activity against various SARS-CoV-2 strains. Conclusions: The study demonstrates that performing several rounds of biopanning with heterologous antigens allows the selection of nanobodies with a broad reactivity spectrum. However, the fourth round of biopanning does not lead to the identification of nanobodies with improved characteristics. Full article

Within-host evolution plays a critical role in shaping the diversity of SARS-CoV-2. However, understanding the primary factors contributing to the prevalence of intra-host single nucleotide variants (iSNVs) in the viral population remains elusive. Here, we conducted a comprehensive analysis of over 556,000 SARS-CoV-2 sequencing data and prevalence data of different SARS-CoV-2 S protein amino acid mutations to elucidate key factors influencing the prevalence of iSNVs in the SARS-CoV-2 S gene. Within-host diversity analysis revealed the presence of mutational hotspots within the S gene, mainly located in NTD, RBD, TM, and CT domains. Additionally, we generated a single amino acid resolution selection status map of the S protein. We observed a significant variance in within-host fitness among iSNVs in the S protein. The majority of iSNVs exhibited low to no within-host fitness and displayed low alternate allele frequency (AAF), suggesting that they will be eliminated due to the narrow transmission bottleneck of SARS-CoV-2. Notably, iSNVs with moderate AAFs (0.06–0.12) were found to be more prevalent than those with high AAFs. Furthermore, iSNVs with the potential to alter antigenicity were more prevalent. These findings underscore the significance of within-host fitness and antigenicity shift as two key factors influencing the prevalence of iSNVs in the SARS-CoV-2 S gene. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, continually undergoes mutation, leading to variants with altered pathogenicity and transmissibility. The Omicron variant (B.1.1.529), first identified in South Africa in 2021, has become the dominant strain worldwide. It harbors approximately 50 mutations compared to the original strain, with 15 located in the receptor-binding domain (RBD) of the spike protein that facilitates viral entry via binding to the human angiotensin-converting enzyme 2 (ACE2) receptor. How do these mutated residues modulate the intermolecular interactions and binding affinity between the RBD and ACE2? This is a question of great theoretical importance and practical implication. In this study, we employed quantum chemical calculations at the B2PLYP-D3/def2-TZVP level of theory to investigate the molecular determinants governing Omicron’s ACE2 interaction. Comparative analysis of the Omicron and wild-type RBD–ACE2 interfaces revealed that mutations including S477N, Q493R, Q498R, and N501Y enhance binding through the formation of bifurcated hydrogen bonds, π–π stacking, and cation–π interactions. These favorable interactions counterbalance such destabilizing mutations as K417N, G446S, G496S, and Y505H, which disrupt salt bridges and hydrogen bonds. Additionally, allosteric effects improve the contributions of non-mutated residues (notably A475, Y453, and F486) via structural realignment and novel hydrogen bonding with ACE2 residues such as S19, leading to an overall increase in the electrostatic and π-system interaction energy. In conclusion, our findings provide a mechanistic basis for Omicron’s increased infectivity and offer valuable insights for the development of targeted antiviral therapies. Moreover, from a methodological perspective, we directly calculated mutation-induced binding energy changes at the residue level using advanced quantum chemical methods rather than relying on the indirect decomposition schemes typical of molecular dynamics-based free energy analyses. The strong correlation between calculated energy differences and experimental deep mutational scanning (DMS) data underscores the robustness of the theoretical framework in predicting the effects of RBD mutations on ACE2 binding affinity. This demonstrates the potential of quantum chemical methods as predictive tools for studying mutation-induced changes in protein–protein interactions and guiding rational therapeutic design. Full article