Assessing Dietary Polyphenols' Inhibitory Potential Against SARS-CoV-2 RNA-Dependent RNA Polymerase: Structural Insights and Implications for Indian Variants

The first Chinese oral anti-COVID-19 drug Azvudine launched

Coronavirus disease 2019 (COVID-19) is caused by a novel strain of coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has caused a global pandemic rapidly s...

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.

Structural Basis of SARS-CoV-2 Polymerase Inhibition by Favipiravir

Does COVID-19 cause pre-eclampsia?

SNX27-mediated endocytic recycling of GLUT1 is suppressed by SARS-CoV-2 spike, possibly explaining neuromuscular disorders in patients with COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide has increased the importance of computational tools to design a drug or vaccine in reduced time with minimum risk. Earlier studies have emphasized the important role of RNA-dependent RNA polymerase (RdRp) in SARS-CoV-2 replication as a potential drug target. In our study, comprehensive computational approaches were applied to identify potential compounds targeting RdRp of SARS-CoV-2. To study the binding affinity and stability of the phytocompounds from Phyllanthus emblica and Aegel marmelos within the defined binding site of SARS-CoV-2 RdRp, they were subjected to molecular docking, 100 ns molecular dynamics (MD) simulation followed by post-simulation analysis. Furthermore, to assess the importance of features involved in the strong binding affinity, molecular field-based similarity analysis was performed. Based on comparative molecular docking and simulation studies of the selected phytocompounds with SARS-CoV-2 RdRp revealed that EBDGp possesses a stronger binding affinity (−23.32 kcal/mol) and stability than other phytocompounds and reference compound, Remdesivir (−19.36 kcal/mol). Molecular field-based similarity profiling has supported our study in the validation of the importance of the presence of hydroxyl groups in EBDGp, involved in increasing its binding affinity toward SARS-CoV-2 RdRp. Molecular docking and dynamic simulation results confirmed that EBDGp has better inhibitory potential than Remdesivir and can be an effective novel drug for SARS-CoV-2 RdRp. Furthermore, binding free energy calculations confirmed the higher stability of the SARS-CoV-2 RdRp-EBDGp complex. These results suggest that the EBDGp compound may emerge as a promising drug against SARS-CoV-2 and hence requires further experimental validation.

SARS-CoV-2 infection and ACE2 inhibition.

SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike N-terminal domain (NTD) remains poorly characterized despite enrichment of mutations in this region across variants of concern (VOCs). Here, we examine the contribution of the NTD to infection and cell-cell fusion by constructing chimeric spikes bearing B.1.617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus. We find that the Delta NTD on a Kappa or wild-type (WT) background increases S1/S2 cleavage efficiency and virus entry, specifically in lung cells and airway organoids, through use of TMPRSS2. Delta exhibits increased cell-cell fusogenicity that could be conferred to WT and Kappa spikes by Delta NTD transfer. However, chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD do not show more efficient TMPRSS2 use or fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions in a spike context-dependent manner, and allosteric interactions may be lost when combining regions from more distantly related VOCs.

Tackling COVID19 by Exploiting Pre-existing Cross-Reacting Spike-Specific Immunity

The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3′ of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involved in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA.

The rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mainly due to its high mutation rate and rapid viral replication, has led to new variants resistant to the available vaccines and monoclonal antibodies. In contrast, oral clinical drugs targeting viral protease and RNA polymerase remain effective against Omicron variants [1]. Main protease (Mpro) plays a crucial role in the maturation and replication of viral strains, making it an attractive target for developing antiviral drugs. Nirmatrelvir (NTV) is the first-in-class Mpro peptidomimetic covalent inhibitor known as "Paxlovid" approved in 2021 by the Food and Drug Administration [2]. Nevertheless, NTV-resistant Mpro mutants particularly the E166V mutation, have been characterized in the Global Initiative on Sharing Avian Influenza Data (GISAID) database [3] and reported in COVID-19 patients [4, 5]. Additionally, viral passage experiments have identified other mutations such as L50F and T21I, which can restore the viral fitness reduced by E166V [6]. The second-generation Mpro drug, ensitrelvir (ETV), is a non-covalent inhibitor approved in 2022 with the brand name "Xocova" [7]. Besides, leritrelvir (LTV) is another covalent inhibitor that was approved in China last year [8]. Preclinical studies showed that ETV and LTV exhibited comparable antiviral activity as NTV and improved pharmacokinetics. However, the effectiveness of these clinical drugs against NTV-resistant Mpro mutants has yet to be fully assessed. Here, we analyzed the inhibition efficiency of four inhibitors, NTV, ETV, LTV, and a veterinary drug, GC376 (Figure 1A), against the Mpro of SARS-CoV-2 variants and other pathogenic coronaviruses. Assessment of drug inhibition against Mpro of SARS-CoV-2 mutants and other coronaviruses. (A) Chemical structure of four Mpro inhibitors. Their pocket-binding moieties including P1′ and P1–4 are labeled in gradient colors. (B) Binding mode of nirmatrelvir (PDB code: 7VH8), ensitrelvir (PDB code: 7VU6), and leritrelvir (PDB code: 8IGN) in the SARS-CoV-2 Mpro pocket. The surrounding residues analyzed in this study are labeled and two active sites, H41 and C145, are colored in red. (C) Longitudinal analysis of the accumulation of global mutations at six residues of Mpro from January 2020 to September 2023, as per the GISAID database. Note the overlap of two curves, E166 and M165. (D) Distribution of different mutation types at six residues, with frequent mutation types labeled in the center. (E) Fold-change in IC50 relative to wild-type Mpro for the indicated mutants, determined through a FRET-based enzymatic assay with three replicates. (F) Longitudinal analysis of three Mpro mutations using the same method as in (C). (G) Fold-change (log base 2) in Kcat/Km of double mutations that consist of additional mutations, T21I, L50F, and P132H, relative to single mutations, as determined by a FRET-based enzymatic assay with three replicates. (H) Phylogenetic relationship and protein sequence alignment of seven β- and α-coronaviral Mpro. The conserved amino acids are colored with gradual degrees of red and two active sites are designated with asterisks above. (I) IC50 of four Mpro inhibitors against various coronaviral proteases. The data represent mean ± SEM from three experiments. FRET, fluorescence resonance energy transfer; GISAID, Global Initiative on Sharing Avian Influenza Data. The Mpro drugs interact tightly with the amino acids of the active pocket (Figure 1B), and nonsynonymous mutations of pocket residues have the potential to induce severe resistance than mutations in other locations [3]. Particularly, six pocket residues, G143, S144, M165, E166, H172, and Q192S have been reported to confer SARS-CoV-2 resistance to NTV [3]. Based on the GISAID database, we investigated the occurrence and frequency of mutations at these six residues. Results showed that all of these sites have been consistently mutating since 2020 (Figure 1C) and adjacent residues, such as M165/E166 and G143/S144, exhibited similar mutational rates. The substitutions at these residues demonstrated a preference for specific types of amino acid, for example, the substitution of glutamine (Q) or histidine (H) at position E166 (Figure 1D). These mutations are thought to confer resistance to SARS-CoV-2 and simultaneously compromise viral fitness [3]. To assess the impact of substitutions at the six residues on drug inhibition, we purified 30 tag-free recombinant Mpro mutants that exhibited high frequencies at these mutation sites (Figure S1A). Fluorescence resonance energy transfer (FRET) assays were then conducted to determine their IC50, Ki, and Kcat/Km (Figure S2 and Table S1). The results showed that the IC50 of NTV increased remarkably in H172T, S144L, M165Y, and E166H mutants (Figure 1E). Their IC50 values exceeded 10 μM (>500-fold of wild-type Mpro), indicating a severe resistance to NTV. By contrast, these mutants remained susceptible to ETV (except S144L), LTV, and GC376. Further analysis revealed that mutations at E166 and S144 significantly confer resistance to NTV and ETV, respectively (Figure S3). The resistance of E166D/V/H substitutions to NTV is consistent with previous studies and could be attributed to the hydrogen bond between E166 and the lactam nitrogen of NTV at the P1 position [2]. Unlike NTV, the central triazine moiety of ETV forms a distinctive hydrogen bond with the backbone nitrogen of E166, thereby preserving the binding affinity of ETV regardless of substitutions at E166 (Figure S4A). Nevertheless, hydrophobic S144/V/L substitutions significantly impeded the efficacy of ETV, resulting from the diminished interaction between its triazine moiety and the hydroxyl group of S144 at the S1 pocket [7]. By contrast, LTV and GC376 remained effective to S144 and E166 mutants. Although they share a common lactam ring at the P1 position as NTV, they differ especially in their warheads. LTV and GC376 possess ketoamide and aldehyde warheads, respectively, as opposed to NTV, which utilizes a nitrile warhead. Previously resolved co-structure of Mpro with LTV [8] showed that the α-ketoamide warhead of LTV can interact with the S1′ pocket, forming a hydrogen bond with H41 and hydrophobic contact with L27. Our molecular docking indicated that this warhead at the P1′ position potentially contributed to greater binding affinity than the pharmacophore at the P1 position (Figure S4). Additionally, GC376 remained effective to various types of Mpro mutants in our study. The potency of GC376 probably benefits from its bisulfite warhead which is more reactive than that of NTV and LTV. To validate the resistance of these mutants in cellular models, we further assessed the inhibition efficiency of Mpro inhibitors using the flip green fluorescent protein (FlipGFP) assay [9]. The results showed that the GFP/mCherry fluorescence ratio associated with the activity of E166V/H mutants cannot be attenuated by NTV, which means that these mutants are resistant to NTV, instead of ETV and LTV (Figure S5). Nevertheless, it is difficult for the FlipGFP model to analyze Mpro mutants with lower enzymatic activity, such as S144L, because they were not able to produce detectable fluorescence signals. Future studies should consider using alternative cellular models such as those utilizing luciferase or recombinant viruses. Overall, four Mpro inhibitors, NTV, ETV, LTV, and GC376, showed different resistance profiles. Importantly, LTV and GC376 remained effective for some NTV- and ETV-resistant mutants. In addition to structural molecular interactions between Mpro and inhibitors, pharmacokinetics also plays a crucial role in determining antiviral activity. NTV, a representative peptidomimetic covalent inhibitor, requires co-administration with ritonavir, a CYP3A4 inhibitor, to enhance its in vivo half-life [2]. In contrast, ETV, a second-generation Mpro inhibitor, the only non-covalent drug in our study, exhibits more favorable pharmacokinetic profiles and no longer necessitates ritonavir co-administration [7]. Furthermore, α-ketoamide inhibitors, such as LTV, demonstrate improved pharmacokinetics [8], with a longer half-life (4.8 h) than NTV (0.5 h) and ETV (2.4 h) [7] in male rat models following intravenous administration. Previous research studies indicate that LTV exhibits "slow-on, slow-off" kinetic behavior, forming a stable enzyme-inhibitor complex. This characteristic prolongs the drug-target residence time of LTV, potentially enhancing its inhibitory activity against Mpro mutants [8]. Given the distinct pharmacokinetic profiles of these Mpro inhibitors, further studies are warranted to evaluate their antiviral efficacy against SARS-CoV-2 with resistance mutations in animal models. The replication of SARS-CoV-2 depends on the enzymatic activity of Mpro. Our FRET experiments show that the wild-type Mpro has a higher Kcat/Km value than other resistant mutants (Figure S6), which means that resistant mutations could attenuate the catalytic activity. Particularly, we observed that H172T and G143V mutants exhibited severe resistance to all drugs (Figure 1E), but their enzymatic activities significantly declined (Kcat/Km < 10). This result implies that the resistance level of Mpro mutants is inversely correlated with the enzymatic activity to some degree. Previous research studies found that impaired activity of resistant Mpro can be compensated by secondary mutations, such as T21I and L50F, increasing the viral replication efficiency [3]. Furthermore, P132H is the most prevalent substitution mutation in Mpro, and demonstrates comparable enzymatic activity as the wild-type Mpro [10]. But it remains unclear whether it would synergize with other resistant mutants to affect enzymatic activity. To evaluate the effect of three additional mutations, we prepared 45 purified Mpro with double mutations, composed of T21, L50F, and P132H, respectively (Figure 1F and Figure S1B). By comparing the enzymatic activities of resistant mutants with or without three additional mutations, we found that T21I and L50F, instead of P132H, were able to enhance the enzymatic activity of all mutants studied (Figure 1G). Moreover, the introduction of T21, L50F, and P132H had little impact on drug resistance (Figure S7). These results highlight the compensatory function of secondary mutations for the activity of resistant Mpro. Apart from SARS-CoV-2, the main protease is also an attractive target for other pathogenic α- and β-coronaviruses (Figure 1H). To assess the anti-coronaviral activity of NTV, ETV, LTV, and GC376, we purified six coronaviral Mpro and conducted the FRET assay to compare their drug inhibition. The results showed that four inhibitors exhibited comparable efficacy against the β-coronaviral Mpro, but LTV showed better broad-spectrum activity, especially for α-coronaviruses, 229E and NL63 (Figure 1I and Figure S8). The effectiveness of LTV probably benefits from its α-ketoamide warhead that forms two hydrogen bonds with conserved active residues, His and Cys, which are necessary for the activity of coronaviral Mpro (Figure S9B). However, ETV showed less efficacy for α-coronavirus (Figure 1I), which was consistent with reported cytopathic effect assays, showing that the EC50 value of ETV against 229E is higher than other β-coronaviruses [7]. The reduced binding affinity of ETV could be attributed to the difference between α- and β-coronaviral Mpro pockets. Though the substrate-binding residues of Mpro are highly conserved among different coronaviruses, the α-coronaviral proteases possess a more negatively charged and open pocket than β-coronaviruses, including SARS-CoV-2 and Middle East respiratory syndrome coronavirus (MERS-CoV) (Figure S9A). In addition, α-coronaviruses carry different residues from SARS-CoV-2 at S144 and M49, which play a vital role in ETV–pocket interaction (Figure S9C). Interestingly, ETV was also more sensitive to SARS-CoV-2 mutants at residue S144 and M49 than other drugs, as revealed by our study and previous research studies [11]. These results implied that the resistance profiles of Mpro inhibitors could reflect their inhibitor–pocket interactions. Taken together, our study characterized the resistance profiles of four Mpro inhibitors. LTV, a newly approved drug, and GC376 remained effective for the Mpro mutants that were resistant to NTV and ETV. Although resistant mutations compromised the enzymatic activity, additional mutations, T21I and L50F, were able to broadly compensate the Mpro activity associated with viral replication efficiency. Moreover, LTV showed better broad-spectrum activity for other pathogenic coronaviruses, probably due to its α-ketoamide warhead interacting with conserved residues. Overall, this study offered valuable insights for the development of next-generation Mpro inhibitors for SARS-CoV-2 variants as well as other coronavirus diseases and highlighted the importance of monitoring resistant variants harboring compensatory mutations. Wenlong Zhao: Conceptualization; methodology; writing – original draft. Cecylia S. Lupala: Methodology; writing – review & editing. Shifeng Hou: Investigation; validation. Shuxin Yang: Methodology; validation. Ziqi Yan: Validation. Shujie Liao: Validation. Xuefei Li: Project administration; supervision; writing – review & editing. Nan Li: Project administration; supervision; writing – review & editing. The research was supported by the National Key Research and Development Program of China (2023YFA0913900) and the National Natural Science Foundation of China (32271501, 32170672, 32100146, and 31971354). The authors declare no competing interests. All data in this study are available within the article or from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The rapidly spreading Omicron variant is highly resistant to vaccines, convalescent sera, and neutralizing antibodies (nAbs), highlighting the urgent need for potent therapeutic nAbs. Here, a panel of human nAbs from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) convalescent patients show diverse neutralization against Omicron, of which XMA01 and XMA04 maintain nanomolar affinities and excellent neutralization (half maximal inhibitory concentration [IC50]: ∼20 ng/mL). nAb XMA09 shows weak but unattenuated neutralization against all variants of concern (VOCs) as well as SARS-CoV. Structural analysis reveals that the above three antibodies could synergistically bind to the receptor-binding domains (RBDs) of both wild-type and Omicron spikes and defines the critical determinants for nAb-mediated broad neutralizations. Three nAbs confer synergistic neutralization against Omicron, resulting from the inter-antibody interaction between XMA04 and XMA01(or XMA09). Furthermore, the XMA01/XMA04 cocktail provides synergistic protection against Beta and Omicron variant infections in hamsters. In summary, our results provide insights for the rational design of antibody cocktail therapeutics or universal vaccines against Omicron.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people worldwide. Currently, many clinical trials in search of effective COVID-19 drugs are underway. Viral RNA-dependent RNA polymerase (RdRp) remains the target of choice for prophylactic or curative treatment of COVID-19. Nucleoside analogs are the most promising RdRp inhibitors and have shown effectiveness in vitro, as well as in clinical settings. One limitation of such RdRp inhibitors is the removal of incorporated nucleoside analogs by SARS-CoV-2 exonuclease (ExoN). Thus, ExoN proofreading activity accomplishes resistance to many of the RdRp inhibitors. We hypothesize that in the absence of highly efficient antivirals to treat COVID-19, combinatorial drug therapy with RdRp and ExoN inhibitors will be a promising strategy to combat the disease. To repurpose drugs for COVID-19 treatment, 10,397 conformers of 2,240 approved drugs were screened against the ExoN domain of nsp14 using AutoDock VINA. The molecular docking approach and detailed study of interactions helped us to identify dexamethasone metasulfobenzoate, conivaptan, hesperidin, and glycyrrhizic acid as potential inhibitors of ExoN activity. The results were further confirmed using molecular dynamics (MD) simulations and molecular mechanics combined with generalized Born model and solvent accessibility method (MM-GBSA) calculations. Furthermore, the binding free energy of conivaptan and hesperidin, estimated using MM-GBSA, was −85.86 ± 0.68 and 119.07 ± 0.69 kcal/mol, respectively. Based on docking, MD simulations and known antiviral activities, and conivaptan and hesperidin were identified as potential SARS-CoV-2 ExoN inhibitors. We recommend further investigation of this combinational therapy using RdRp inhibitors with a repurposed ExoN inhibitor as a potential COVID-19 treatment.

D614G mutation eventuates in all VOI and VOC in SARS-CoV-2: Is it part of the positive selection pioneered by Darwin?