An interaction-based drug discovery screen explains known SARS-CoV-2 inhibitors and predicts new compound scaffolds.

The recent outbreak of the COVID-19 pandemic caused by severe acute respiratory syndrome-Coronavirus-2 (SARS-CoV-2) has shown the necessity for fast and broad drug discovery methods to enable us to react quickly to novel and highly infectious diseases. A well-known SARS-CoV-2 target is the viral main 3-chymotrypsin-like cysteine protease (Mpro), known to control coronavirus replication, which is essential for the viral life cycle. Here, we applied an interaction-based drug repositioning algorithm on all protein-compound complexes available in the protein database (PDB) to identify Mpro inhibitors and potential novel compound scaffolds against SARS-CoV-2. The screen revealed a heterogeneous set of 692 potential Mpro inhibitors containing known ones such as Dasatinib, Amodiaquine, and Flavin mononucleotide, as well as so far untested chemical scaffolds. In a follow-up evaluation, we used publicly available data published almost two years after the screen to validate our results. In total, we are able to validate 17% of the top 100 predictions with publicly available data and can furthermore show that predicted compounds do cover scaffolds that are yet not associated with Mpro. Finally, we detected a potentially important binding pattern consisting of 3 hydrogen bonds with hydrogen donors of an oxyanion hole within the active side of Mpro. Overall, these results give hope that we will be better prepared for future pandemics and that drug development will become more efficient in the upcoming years.

Docking-Based Evidence for the Potential of ImmunoDefender: A Novel Formulated Essential Oil Blend Incorporating Synergistic Antiviral Bioactive Compounds as Promising Mpro Inhibitors against SARS-CoV-2.

Essential oils (Eos) have demonstrated antiviral activity, but their toxicity can hinder their use as therapeutic agents. Recently, some essential oil components have been used within safe levels of acceptable daily intake limits without causing toxicity. The "ImmunoDefender," a novel antiviral compound made from a well-known mixture of essential oils, is considered highly effective in treating SARS-CoV-2 infections. The components and doses were chosen based on existing information about their structure and toxicity. Blocking the main protease (Mpro) of SARS-CoV-2 with high affinity and capacity is critical for inhibiting the virus's pathogenesis and transmission. In silico studies were conducted to examine the molecular interactions between the main essential oil components in "ImmunoDefender" and SARS-CoV-2 Mpro. The screening results showed that six key components of ImmunoDefender formed stable complexes with Mpro via its active catalytic site with binding energies ranging from -8.75 to -10.30 kcal/mol, respectively for Cinnamtannin B1, Cinnamtannin B2, Pavetannin C1, Syzyginin B, Procyanidin C1, and Tenuifolin. Furthermore, three essential oil bioactive inhibitors, Cinnamtannin B1, Cinnamtannin B2, and Pavetannin C, had significant ability to bind to the allosteric site of the main protease with binding energies of -11.12, -10.74, and -10.79 kcal/mol; these results suggest that these essential oil bioactive compounds may play a role in preventing the attachment of the translated polyprotein to Mpro, inhibiting the virus's pathogenesis and transmission. These components also had drug-like characteristics similar to approved and effective drugs, suggesting that further pre-clinical and clinical studies are needed to confirm the generated in silico outcomes.

Biochemical and HDX Mass Spectral Characterization of the SARS-CoV-2 Nsp1 Protein.

A major challenge in defining the pathophysiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is to better understand virally encoded multifunctional proteins and their interactions with host factors. Among the many proteins encoded by the positive-sense, single-stranded RNA genome, nonstructural protein 1 (Nsp1) stands out due to its impact on several stages of the viral replication cycle. Nsp1 is the major virulence factor that inhibits mRNA translation. Nsp1 also promotes host mRNA cleavage to modulate host and viral protein expression and to suppress host immune functions. To better define how this multifunctional protein can facilitate distinct functions, we characterize SARS-CoV-2 Nsp1 by using a combination of biophysical techniques, including light scattering, circular dichroism, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and temperature-dependent HDX-MS. Our results reveal that the SARS-CoV-2 Nsp1 N- and C-terminus are unstructured in solution, and in the absence of other proteins, the C-terminus has an increased propensity to adopt a helical conformation. In addition, our data indicate that a short helix exists near the C-terminus and adjoins the region that binds the ribosome. Together, these findings provide insights into the dynamic nature of Nsp1 that impacts its functions during infection. Furthermore, our results will inform efforts to understand SARS-CoV-2 infection and antiviral development.

Conserved Characteristics of NMPylation Activities of Alpha- and Betacoronavirus NiRAN Domains.

Coronavirus genome replication and expression are mediated by the viral replication-transcription complex (RTC) which is assembled from multiple nonstructural proteins (nsp). Among these, nsp12 represents the central functional subunit. It harbors the RNA-directed RNA polymerase (RdRp) domain and contains, at its N terminus, an additional domain called NiRAN which is widely conserved in coronaviruses and other nidoviruses. In this study, we produced bacterially expressed coronavirus nsp12s to investigate and compare NiRAN-mediated NMPylation activities from representative alpha- and betacoronaviruses. We found that the four coronavirus NiRAN domains characterized to date have a number of conserved properties, including (i) robust nsp9-specific NMPylation activities that appear to operate largely independently of the C-terminal RdRp domain, (ii) nucleotide substrate preference for UTP followed by ATP and other nucleotides, (iii) dependence on divalent metal ions, with Mn2+ being preferred over Mg2+, and (iv) a key role of N-terminal residues (particularly Asn2) of nsp9 for efficient formation of a covalent phosphoramidate bond between NMP and the N-terminal amino group of nsp9. In this context, a mutational analysis confirmed the conservation and critical role of Asn2 across different subfamilies of the family Coronaviridae, as shown by studies using chimeric coronavirus nsp9 variants in which six N-terminal residues were replaced with those from other corona-, pito- and letovirus nsp9 homologs. The combined data of this and previous studies reveal a remarkable degree of conservation among coronavirus NiRAN-mediated NMPylation activities, supporting a key role of this enzymatic activity in viral RNA synthesis and processing. IMPORTANCE There is strong evidence that coronaviruses and other large nidoviruses evolved a number of unique enzymatic activities, including an additional RdRp-associated NiRAN domain, that are conserved in nidoviruses but not in most other RNA viruses. Previous studies of the NiRAN domain mainly focused on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and suggested different functions for this domain, such as NMPylation/RNAylation of nsp9, RNA guanylyltransferase activities involved in canonical and/or unconventional RNA capping pathways, and other functions. To help resolve partly conflicting information on substrate specificities and metal ion requirements reported previously for the SARS-CoV-2 NiRAN NMPylation activity, we extended these earlier studies by characterizing representative alpha- and betacoronavirus NiRAN domains. The study revealed that key features of NiRAN-mediated NMPylation activities, such as protein and nucleotide specificity and metal ion requirements, are very well conserved among genetically divergent coronaviruses, suggesting potential avenues for future antiviral drug development targeting this essential viral enzyme.

Structure-guided optimization of adenosine mimetics as selective and potent inhibitors of coronavirus nsp14 N7-methyltransferases.

The COVID-19 pandemic reveals the urgent need to develop new therapeutics targeting the SARS-CoV-2 replication machinery. The first antiviral drugs were nucleoside analogues targeting RdRp and protease inhibitors active on nsp5 Mpro. In addition to these common antiviral targets, SARS-CoV-2 codes for the highly conserved protein nsp14 harbouring N7-methyltransferase (MTase) activity. Nsp14 is involved in cap N7-methylation of viral RNA and its inhibition impairs viral RNA translation and immune evasion, making it an attractive new antiviral target. In this work, we followed a structure-guided drug design approach to design bisubstrates mimicking the S-adenosylmethionine methyl donor and RNA cap. We developed adenosine mimetics with an N-arylsulfonamide moiety in the 5'-position, recently described as a guanine mimicking the cap structure in a potent adenosine-derived nsp14 inhibitor. Here, the adenine moiety was replaced by hypoxanthine, N6-methyladenine, or C7-substituted 7-deaza-adenine. 26 novel adenosine mimetics were synthesized, one of which selectively inhibits nsp14 N7-MTase activity with a subnanomolar IC50 (and seven with a single-digit nanomolar IC50). In the most potent inhibitors, adenine was replaced by two different 7-deaza-adenines bearing either a phenyl or a 3-quinoline group at the C7-position via an ethynyl linker. These more complex compounds are barely active on the cognate human N7-MTase and docking experiments reveal that their selectivity of inhibition might result from the positioning of their C7 substitution in a SAM entry tunnel present in the nsp14 structure and absent in the hN7-MTase. These compounds show moderate antiviral activity against SARS-CoV-2 replication in cell culture, suggesting delivery or stability issue.

SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation.

Viruses evade the innate immune response by suppressing the production or activity of cytokines such as type I interferons (IFNs). Here we report the discovery of a mechanism by which the SARS-CoV-2 virus coopts an intrinsic cellular machinery to suppress the production of the key immunostimulatory cytokine IFN-ß. We reveal that the SARS-CoV-2 encoded nonstructural protein 2 (NSP2) directly interacts with the cellular GIGYF2 protein. This interaction enhances the binding of GIGYF2 to the mRNA cap-binding protein 4EHP, thereby repressing the translation of the Ifnb1 mRNA. Depletion of GIGYF2 or 4EHP significantly enhances IFN-ß production, which inhibits SARS-CoV-2 replication. Our findings reveal a target for rescuing the antiviral innate immune response to SARS-CoV-2 and other RNA viruses.

Inside-out: Antibody-binding reveals potential folding hinge-points within the SARS-CoV-2 replication co-factor nsp9.

Nsp9 is a conserved accessory component of the coronaviral replication and transcription complex. It is the predominant substrate of nsp12's nucleotidylation activity while also serving to recruit proteins required for viral 5'-capping. Anti-nsp9 specific nanobodies have been isolated previously. We confirm that their binding mode is centred upon Trp-53 within SARS-CoV-2 nsp9. Antibody binding at this site surprisingly results in large-scale changes to the overall topology of this coronaviral unique fold. We further characterise the antibody-induced structural dynamism within nsp9, identifying a number of potentially flexible regions. A large expansion of the cavity between the s2-s3 and s4-s5 loops is particularly noteworthy. As is the potential for large-scale movements in the C-terminal GxxxG helix.

Reticulons promote formation of ER-derived double-membrane vesicles that facilitate SARS-CoV-2 replication.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiologic agent for the global COVID-19 pandemic, triggers the formation of endoplasmic reticulum (ER)-derived replication organelles, including double-membrane vesicles (DMVs), in the host cell to support viral replication. Here, we clarify how SARS-CoV-2 hijacks host factors to construct the DMVs. We show that the ER morphogenic proteins reticulon-3 (RTN3) and RTN4 help drive DMV formation, enabling viral replication, which leads to productive infection. Different SARS-CoV-2 variants, including the delta variant, use the RTN-dependent pathway to promote infection. Mechanistically, our results reveal that the membrane-embedded reticulon homology domain (RHD) of the RTNs is sufficient to functionally support viral replication and physically engage NSP3 and NSP4, two viral non-structural membrane proteins known to induce DMV formation. Our findings thus identify the ER morphogenic RTN3 and RTN4 membrane proteins as host factors that help promote the biogenesis of SARS-CoV-2-induced DMVs, which can act as viral replication platforms.

A universal fluorescence polarization high throughput screening assay to target the SAM-binding sites of SARS-CoV-2 and other viral methyltransferases.

SARS-CoV-2 has caused a global pandemic with significant humanity and economic loss since 2020. Currently, only limited options are available to treat SARS-CoV-2 infections for vulnerable populations. In this study, we report a universal fluorescence polarization (FP)-based high throughput screening (HTS) assay for SAM-dependent viral methyltransferases (MTases), using a fluorescent SAM-analogue, FL-NAH. We performed the assay against a reference MTase, NSP14, an essential enzyme for SARS-CoV-2 to methylate the N7 position of viral 5'-RNA guanine cap. The assay is universal and suitable for any SAM-dependent viral MTases such as the SARS-CoV-2 NSP16/NSP10 MTase complex and the NS5 MTase of Zika virus (ZIKV). Pilot screening demonstrated that the HTS assay was very robust and identified two candidate inhibitors, NSC 111552 and 288387. The two compounds inhibited the FL-NAH binding to the NSP14 MTase with low micromolar IC50. We used three functional MTase assays to unambiguously verified the inhibitory potency of these molecules for the NSP14 N7-MTase function. Binding studies indicated that these molecules are bound directly to the NSP14 MTase with similar low micromolar affinity. Moreover, we further demonstrated that these molecules significantly inhibited the SARS-CoV-2 replication in cell-based assays at concentrations not causing cytotoxicity. Furthermore, NSC111552 significantly synergized with known SARS-CoV-2 drugs including nirmatrelvir and remdesivir. Finally, docking suggested that these molecules bind specifically to the SAM-binding site on the NSP14 MTase. Overall, these molecules represent novel and promising candidates to further develop broad-spectrum inhibitors for the management of viral infections.

Targeting the I7L Protease: A Rational Design for Anti-Monkeypox Drugs?

The latest monkeypox virus outbreak in 2022 showcased the potential threat of this viral zoonosis to public health. The lack of specific treatments against this infection and the success of viral protease inhibitors-based treatments against HIV, Hepatitis C, and SARS-CoV-2, brought the monkeypox virus I7L protease under the spotlight as a potential target for the development of specific and compelling drugs against this emerging disease. In the present work, the structure of the monkeypox virus I7L protease was modeled and thoroughly characterized through a dedicated computational study. Furthermore, structural information gathered in the first part of the study was exploited to virtually screen the DrugBank database, consisting of drugs approved by the Food and Drug Administration (FDA) and clinical-stage drug candidates, in search for readily repurposable compounds with similar binding features as TTP-6171, the only non-covalent I7L protease inhibitor reported in the literature. The virtual screening resulted in the identification of 14 potential inhibitors of the monkeypox I7L protease. Finally, based on data collected within the present work, some considerations on developing allosteric modulators of the I7L protease are reported.

All Domains of SARS-CoV-2 nsp1 Determine Translational Shutoff and Cytotoxicity of the Protein.

Replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strongly affects cellular metabolism and results in rapid development of the cytopathic effect (CPE). The hallmarks of virus-induced modifications are inhibition of translation of cellular mRNAs and redirection of the cellular translational machinery to the synthesis of virus-specific proteins. The multifunctional nonstructural protein 1 (nsp1) of SARS-CoV-2 is a major virulence factor and a key contributor to the development of translational shutoff. In this study, we applied a wide range of virological and structural approaches to further analyze nsp1 functions. The expression of this protein alone was found to be sufficient to cause CPE. However, we selected several nsp1 mutants exhibiting noncytopathic phenotypes. The attenuating mutations were detected in three clusters, located in the C-terminal helices, in one of the loops of the structured domain and in the junction of the disordered and structured fragment of nsp1. NMR-based analysis of the wild type nsp1 and its mutants did not confirm the existence of a stable ß5-strand that was proposed by the X-ray structure. In solution, this protein appears to be present in a dynamic conformation, which is required for its functions in CPE development and viral replication. The NMR data also suggest a dynamic interaction between the N-terminal and C-terminal domains. The identified nsp1 mutations make this protein noncytotoxic and incapable of inducing translational shutoff, but they do not result in deleterious effects on viral cytopathogenicity. IMPORTANCE The nsp1 of SARS-CoV-2 is a multifunctional protein that modifies the intracellular environment for the needs of viral replication. It is responsible for the development of translational shutoff, and its expression alone is sufficient to cause a cytopathic effect (CPE). In this study, we selected a wide range of nsp1 mutants exhibiting noncytopathic phenotypes. The attenuating mutations, clustered in three different fragments of nsp1, were extensively characterized via virological and structural methods. Our data strongly suggest interactions between the nsp1 domains, which are required for the protein's functions in CPE development. Most of the mutations made nsp1 noncytotoxic and incapable of inducing translational shutoff. Most of them did not affect the viability of the viruses, but they did decrease the rates of replication in cells competent in type I IFN induction and signaling. These mutations, and their combinations, in particular, can be used for the development of SARS-CoV-2 variants with attenuated phenotypes.

SARS-CoV-2 viral protein Nsp2 stimulates translation under normal and hypoxic conditions.

When viruses like SARS-CoV-2 infect cells, they reprogram the repertoire of cellular and viral transcripts that are being translated to optimize their strategy of replication, often targeting host translation initiation factors, particularly eIF4F complex consisting of eIF4E, eIF4G and eIF4A. A proteomic analysis of SARS-CoV-2/human proteins interaction revealed viral Nsp2 and initiation factor eIF4E2, but a role of Nsp2 in regulating translation is still controversial. HEK293T cells stably expressing Nsp2 were tested for protein synthesis rates of synthetic and endogenous mRNAs known to be translated via cap- or IRES-dependent mechanism under normal and hypoxic conditions. Both cap- and IRES-dependent translation were increased in Nsp2-expressing cells under normal and hypoxic conditions, especially mRNAs that require high levels of eIF4F. This could be exploited by the virus to maintain high translation rates of both viral and cellular proteins, particularly in hypoxic conditions as may arise in SARS-CoV-2 patients with poor lung functioning.

Genetic conservation across SARS-CoV-2 non-structural proteins - Insights into possible targets for treatment of future viral outbreaks.

The majority of SARS-CoV-2 therapeutic development work has focussed on targeting the spike protein, viral polymerase and proteases. As the pandemic progressed, many studies reported that these proteins are prone to high levels of mutation and can become drug resistant. Thus, it is necessary to not only target other viral proteins such as the non-structural proteins (NSPs) but to also target the most conserved residues of these proteins. In order to understand the level of conservation among these viruses, in this review, we have focussed on the conservation across RNA viruses, conservation across the coronaviruses and then narrowed our focus to conservation of NSPs across coronaviruses. We have also discussed the various treatment options for SARS-CoV-2 infection. A synergistic melding of bioinformatics, computer-aided drug-design and in vitro/vivo studies can feed into better understanding of the virus and therefore help in the development of small molecule inhibitors against the viral proteins.

In Silico Binding of 2-Aminocyclobutanones to SARS-CoV-2 Nsp13 Helicase and Demonstration of Antiviral Activity.

The landscape of viral strains and lineages of SARS-CoV-2 keeps changing and is currently dominated by Delta and Omicron variants. Members of the latest Omicron variants, including BA.1, are showing a high level of immune evasion, and Omicron has become a prominent variant circulating globally. In our search for versatile medicinal chemistry scaffolds, we prepared a library of substituted ɑ-aminocyclobutanones from an ɑ-aminocyclobutanone synthon (11). We performed an in silico screen of this actual chemical library as well as other virtual 2-aminocyclobutanone analogs against seven SARS-CoV-2 nonstructural proteins to identify potential drug leads against SARS-CoV-2, and more broadly against coronavirus antiviral targets. Several of these analogs were initially identified as in silico hits against SARS-CoV-2 nonstructural protein 13 (Nsp13) helicase through molecular docking and dynamics simulations. Antiviral activity of the original hits as well as ɑ-aminocyclobutanone analogs that were predicted to bind more tightly to SARS-CoV-2 Nsp13 helicase are reported. We now report cyclobutanone derivatives that exhibit anti-SARS-CoV-2 activity. Furthermore, the Nsp13 helicase enzyme has been the target of relatively few target-based drug discovery efforts, in part due to a very late release of a high-resolution structure accompanied by a limited understanding of its protein biochemistry. In general, antiviral agents initially efficacious against wild-type SARS-CoV-2 strains have lower activities against variants due to heavy viral loads and greater turnover rates, but the inhibitors we are reporting have higher activities against the later variants than the wild-type (10-20X). We speculate this could be due to Nsp13 helicase being a critical bottleneck in faster replication rates of the new variants, so targeting this enzyme affects these variants to an even greater extent. This work calls attention to cyclobutanones as a useful medicinal chemistry scaffold, and the need for additional focus on the discovery of Nsp13 helicase inhibitors to combat the aggressive and immune-evading variants of concern (VOCs).

Alkyne Derivatives of SARS-CoV-2 Main Protease Inhibitors Including Nirmatrelvir Inhibit by Reacting Covalently with the Nucleophilic Cysteine.

Nirmatrelvir (PF-07321332) is a nitrile-bearing small-molecule inhibitor that, in combination with ritonavir, is used to treat infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Nirmatrelvir interrupts the viral life cycle by inhibiting the SARS-CoV-2 main protease (Mpro), which is essential for processing viral polyproteins into functional nonstructural proteins. We report studies which reveal that derivatives of nirmatrelvir and other Mpro inhibitors with a nonactivated terminal alkyne group positioned similarly to the electrophilic nitrile of nirmatrelvir can efficiently inhibit isolated Mpro and SARS-CoV-2 replication in cells. Mass spectrometric and crystallographic evidence shows that the alkyne derivatives inhibit Mpro by apparent irreversible covalent reactions with the active site cysteine (Cys145), while the analogous nitriles react reversibly. The results highlight the potential for irreversible covalent inhibition of Mpro and other nucleophilic cysteine proteases by alkynes, which, in contrast to nitriles, can be functionalized at their terminal position to optimize inhibition and selectivity, as well as pharmacodynamic and pharmacokinetic properties.

Interaction of Laurusides 1 and 2 with the 3C-like Protease (Mpro) from Wild-Type and Omicron Variant of SARS-CoV-2: A Molecular Dynamics Study.

Laurus nobilis (bay laurel) is a natural source of biological compounds, and some of its extracts and phytocompounds are also endowed with antiviral activity toward the family of the severe acute respiratory syndrome (SARS)-associated ß-coronaviruses. Some glycosidic laurel compounds such as laurusides were proposed as inhibitors of important protein targets of SARS-CoV-2, which clearly recalls their potential as anti-COVID-19 drugs. Due to the frequent genomic variations of the ß-coronaviruses and the consequent importance of evaluating a new drug candidate with respect to the variants of the target ß-coronavirus, we decided to investigate at an atomistic level the molecular interactions of the potential laurel-derived drugs laurusides 1 and 2 (L01 and L02, respectively) toward a well-conserved and crucial target, the 3C-like protease (Mpro), using the enzymes of both the wild-type of SARS-CoV-2 and of the more recent Omicron variant. Thus, we performed molecular dynamic (MD) simulations of laurusides-SARS-CoV-2 protease complexes to deepen the knowledge on the stability of the interaction and compare the effects of the targeting among the two genomic variants. We found that the Omicron mutation does not significantly impact the lauruside binding and that L02 connects more stably with respect to L01 in the complexes from both variants, even though both compounds prevalently interact within the same binding pocket. Although purely in silico, the current study highlights the potential role of bay laurel phytocompounds in the antiviral and specifically anti-coronavirus research and shows their potential binding toward Mpro, corroborating the important commitment of bay laurel as functional food and disclosing novel scenarios of lauruside-based antiviral therapies.

Cotton flower metabolites inhibit SARS-CoV-2 main protease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading globally for over 2 years, causing serious contagious disease and incalculable damage. The introduction of vaccines has slowed the spread of SARS-CoV-2 to some extent, but there remains a need for specific and effective treatment. The high chemical diversity and safety profiles of natural products make them a potential source of effective anti-SARS-CoV-2 drugs. Cotton plant is one of the most important economic and medical crops and is the source of a large number of antiviral phytochemicals. In this work, we used SARS-CoV-2 main protein (Mpro ) as the target to identify potential anti-SARS-CoV-2 natural products in cotton. An in vitro assay showed that of all cotton tissues examined, cotton flower extracts (CFs) exhibited optimal inhibitory effects against Mpro . We proceeded to use the CF metabolite database to screen natural Mpro inhibitors by combining virtual screening and biochemical assays. We identified that several CF natural products, including astragalin, myricitrin, and astilbin, significantly inhibited Mpro with half-maximal inhibitory concentrations (IC50s) of 0.13, 10.73, and 7.92 µm, respectively. These findings may serve as a basis for further studies into the suitability of cotton as a source of potential therapeutics for SARS-CoV-2.

Modelling the Transitioning of SARS-CoV-2 nsp3 and nsp4 Lumenal Regions towards a More Stable State on Complex Formation.

During coronavirus infection, three non-structural proteins, nsp3, nsp4, and nsp6, are of great importance as they induce the formation of double-membrane vesicles where the replication and transcription of viral gRNA takes place, and the interaction of nsp3 and nsp4 lumenal regions triggers membrane pairing. However, their structural states are not well-understood. We investigated the interactions between nsp3 and nsp4 by predicting the structures of their lumenal regions individually and in complex using AlphaFold2 as implemented in ColabFold. The ColabFold prediction accuracy of the nsp3-nsp4 complex was increased compared to nsp3 alone and nsp4 alone. All cysteine residues in both lumenal regions were modelled to be involved in intramolecular disulphide bonds. A linker region in the nsp4 lumenal region emerged as crucial for the interaction, transitioning to a structured state when predicted in complex. The key interactions modelled between nsp3 and nsp4 appeared stable when the transmembrane regions of nsp3 and nsp4 were added to the modelling either alone or together. While molecular dynamics simulations (MD) demonstrated that the proposed model of the nsp3 lumenal region on its own is not stable, key interactions between nsp and nsp4 in the proposed complex model appeared stable after MD. Together, these observations suggest that the interaction is robust to different modelling conditions. Understanding the functional importance of the nsp4 linker region may have implications for the targeting of double membrane vesicle formation in controlling coronavirus infection.

Selenoprotein S Interacts with the Replication and Transcription Complex of SARS-CoV-2 by Binding nsp7.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) replicates and evades detection using ER membranes and their associated protein machinery. Among these hijacked human proteins is selenoprotein S (selenos). This selenoprotein takes part in the protein quality control, signaling, and the regulation of cytokine secretion. While the role of selenos in the viral life cycle is not yet known, it has been reported to interact with SARS-CoV-2 nonstructural protein 7 (nsp7), a viral protein essential for the replication of the virus. We set to study whether selenos and nsp7 interact directly and if they can still bind when nsp7 is bound to the replication and transcription complex of the virus. Using biochemical assays, we show that selenos binds directly to nsp7. In addition, we found that selenos can bind to nsp7 when it is in a complex with the coronavirus's minimal replication and transcription complex, comprised of nsp7, nsp8, and the RNA-dependent RNA polymerase nsp12. In addition, through crosslinking experiments, we mapped the interaction sites of selenos and nsp7 in the replication complex and showed that the hydrophobic segment of selenos is essential for binding to nsp7. This arrangement leaves an extended helix and the intrinsically disordered segment of selenos-including the reactive selenocysteine-exposed and free to potentially recruit additional proteins to the replication and transcription complex.

Unravelling lead antiviral phytochemicals for the inhibition of SARS-CoV-2 Mpro enzyme through in silico approach.

A new SARS coronavirus (SARS-CoV-2) belonging to the genus Betacoronavirus has caused a pandemic known as COVID-19. Among coronaviruses, the main protease (Mpro) is an essential drug target which, along with papain-like proteases catalyzes the processing of polyproteins translated from viral RNA and recognizes specific cleavage sites. There are no human proteases with similar cleavage specificity and therefore, inhibitors are highly likely to be nontoxic. Therefore, targeting the SARS-CoV-2 Mpro enzyme with small molecules can block viral replication. The present study is aimed at the identification of promising lead molecules for SARS-CoV-2 Mpro enzyme through virtual screening of antiviral compounds from plants. The binding affinity of selected small drug-like molecules to SARS-CoV-2 Mpro, SARS-CoV Mpro and MERS-CoV Mpro were studied using molecular docking. Bonducellpin D was identified as the best lead molecule which shows higher binding affinity (-9.28 kcal/mol) as compared to the control (-8.24 kcal/mol). The molecular binding was stabilized through four hydrogen bonds with Glu166 and Thr190 as well as hydrophobic interactions via eight residues. The SARS-CoV-2 Mpro shows identities of 96.08% and 50.65% to that of SARS-CoV Mpro and MERS-CoV Mpro respectively at the sequence level. At the structural level, the root mean square deviation (RMSD) between SARS-CoV-2 Mpro and SARS-CoV Mpro was found to be 0.517 Å and 0.817 Å between SARS-CoV-2 Mpro and MERS-CoV Mpro. Bonducellpin D exhibited broad-spectrum inhibition potential against SARS-CoV Mpro and MERS-CoV Mpro and therefore is a promising drug candidate, which needs further validations through in vitro and in vivo studies.