Characterization of ACTN4 as a novel antiviral target against SARS-CoV-2.

The various mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pose a substantial challenge in mitigating the viral infectivity. The identification of novel host factors influencing SARS-CoV-2 replication holds potential for discovering new targets for broad-spectrum antiviral drugs that can combat future viral mutations. In this study, potential host factors regulated by SARS-CoV-2 infection were screened through different high-throughput sequencing techniques and further identified in cells. Subsequent analysis and experiments showed that the reduction of m6A modification level on ACTN4 (Alpha-actinin-4) mRNA leads to a decrease in mRNA stability and translation efficiency, ultimately inhibiting ACTN4 expression. In addition, ACTN4 was demonstrated to target nsp12 for binding and characterized as a competitor for SARS-CoV-2 RNA and the RNA-dependent RNA polymerase complex, thereby impeding viral replication. Furthermore, two ACTN4 agonists, YS-49 and demethyl-coclaurine, were found to dose-dependently inhibit SARS-CoV-2 infection in both Huh7 cells and K18-hACE2 transgenic mice. Collectively, this study unveils the pivotal role of ACTN4 in SARS-CoV-2 infection, offering novel insights into the intricate interplay between the virus and host cells, and reveals two potential candidates for future anti-SARS-CoV-2 drug development.

Emergence of transmissible SARS-CoV-2 variants with decreased sensitivity to antivirals in immunocompromised patients with persistent infections.

We investigated the impact of antiviral treatment on the emergence of SARS-CoV-2 resistance during persistent infections in immunocompromised patients (n = 15). All patients received remdesivir and some also received nirmatrelvir-ritonavir (n = 3) or therapeutic monoclonal antibodies (n = 4). Sequence analysis showed that nine patients carried viruses with mutations in the nsp12 (RNA dependent RNA polymerase), while four had viruses with nsp5 (3C protease) mutations. Infectious SARS-CoV-2 with a double mutation in nsp5 (T169I) and nsp12 (V792I) was recovered from respiratory secretions 77 days after initial COVID-19 diagnosis from a patient sequentially treated with nirmatrelvir-ritonavir and remdesivir. In vitro characterization confirmed its decreased sensitivity to remdesivir and nirmatrelvir, which was overcome by combined antiviral treatment. Studies in golden Syrian hamsters demonstrated efficient transmission to contact animals. This study documents the isolation of SARS-CoV-2 carrying resistance mutations to both nirmatrelvir and remdesivir from a patient and demonstrates its transmissibility in vivo.

Structural review of SARS-CoV-2 antiviral targets.

The coronavirus disease 2019 (COVID-19), the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents the most disastrous infectious disease pandemic of the past century. As a member of the Betacoronavirus genus, the SARS-CoV-2 genome encodes a total of 29 proteins. The spike protein, RNA-dependent RNA polymerase, and proteases play crucial roles in the virus replication process and are promising targets for drug development. In recent years, structural studies of these viral proteins and of their complexes with antibodies and inhibitors have provided valuable insights into their functions and laid a solid foundation for drug development. In this review, we summarize the structural features of these proteins and discuss recent progress in research regarding therapeutic development, highlighting mechanistically representative molecules and those that have already been approved or are under clinical investigation.

Biochemical characterization of naturally occurring mutations in SARS-CoV-2 RNA-dependent RNA polymerase.

Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13°C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants.

Point mutations at specific sites of the nsp12-nsp8 interface dramatically affect the RNA polymerization activity of SARS-CoV-2.

In a recent characterization of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variability present in 30 diagnostic samples from patients of the first COVID-19 pandemic wave, 41 amino acid substitutions were documented in the RNA-dependent RNA polymerase (RdRp) nsp12. Eight substitutions were selected in this work to determine whether they had an impact on the RdRp activity of the SARS-CoV-2 nsp12-nsp8-nsp7 replication complex. Three of these substitutions were found around the polymerase central cavity, in the template entry channel (D499G and M668V), and within the motif B (V560A), and they showed polymerization rates similar to the wild type RdRp. The remaining five mutations (P323L, L372F, L372P, V373A, and L527H) were placed near the nsp12-nsp8F contact surface; residues L372, V373, and L527 participated in a large hydrophobic cluster involving contacts between two helices in the nsp12 fingers and the long α-helix of nsp8F. The presence of any of these five amino acid substitutions resulted in important alterations in the RNA polymerization activity. Comparative primer elongation assays showed different behavior depending on the hydrophobicity of their side chains. The substitution of L by the bulkier F side chain at position 372 slightly promoted RdRp activity. However, this activity was dramatically reduced with the L372P, and L527H mutations, and to a lesser extent with V373A, all of which weaken the hydrophobic interactions within the cluster. Additional mutations, specifically designed to disrupt the nsp12-nsp8F interactions (nsp12-V330S, nsp12-V341S, and nsp8-R111A/D112A), also resulted in an impaired RdRp activity, further illustrating the importance of this contact interface in the regulation of RNA synthesis.

In silico analysis of non-structural protein 12 sequences from SARS-COV-2 found in Manaus, Amazonas, Brazil, reveals mutations linked to higher transmissibility.

The disease coronavirus COVID-19 has been the cause of millions of deaths worldwide. Among the proteins of SARS-CoV-2, non-structural protein 12 (NSP12) plays a key role during COVID infection and is part of the RNA-dependent RNA polymerase complex. The monitoring of NSP12 polymorphisms is extremely important for the design of new antiviral drugs and monitoring of viral evolution. This study analyzed the NSP12 mutations detected in circulating SARS-CoV-2 during the years 2020 to 2022 in the population of the city of Manaus, Amazonas, Brazil. The most frequent mutations found were P323L and G671S. Reports in the literature indicate that these mutations are related to transmissibility efficiency, which may have contributed to the extremely high numbers of cases in this location. In addition, two mutations described here (E796D and R914K) are close and have RMSD that is similar to the mutations M794V and N911K, which have been described in the literature as influential on the performance of the NSP12 enzyme. These data demonstrate the need to monitor the emergence of new mutations in NSP12 in order to better understand their consequences for the treatments currently used and in the design of new drugs.

Dynamic diversity of SARS-CoV-2 genetic mutations in a lung transplantation patient with persistent COVID-19.

Numerous SARS-CoV-2 variant strains with altered characteristics have emerged since the onset of the COVID-19 pandemic. Remdesivir (RDV), a ribonucleotide analogue inhibitor of viral RNA polymerase, has become a valuable therapeutic agent. However, immunosuppressed hosts may respond inadequately to RDV and develop chronic persistent infections. A patient with respiratory failure caused by interstitial pneumonia, who had undergone transplantation of the left lung, developed COVID-19 caused by Omicron BA.5 strain with persistent chronic viral shedding, showing viral fusogenicity. Genome-wide sequencing analyses revealed the occurrence of several viral mutations after RDV treatment, followed by dynamic changes in the viral populations. The C799F mutation in nsp12 was found to play a pivotal role in conferring RDV resistance, preventing RDV-triphosphate from entering the active site of RNA-dependent RNA polymerase. The occurrence of diverse mutations is a characteristic of SARS-CoV-2, which mutates frequently. Herein, we describe the clinical case of an immunosuppressed host in whom inadequate treatment resulted in highly diverse SARS-CoV-2 mutations that threatened the patient's health due to the development of drug-resistant variants.

Interface design of SARS-CoV-2 symmetrical nsp7 dimer and machine learning-guided nsp7 sequence prediction reveals physicochemical properties and hotspots for nsp7 stability, adaptation, and therapeutic design.

The COVID-19 pandemic, driven by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates a profound understanding of the virus and its lifecycle. As an RNA virus with high mutation rates, SARS-CoV-2 exhibits genetic variability leading to the emergence of variants with potential implications. Among its key proteins, the RNA-dependent RNA polymerase (RdRp) is pivotal for viral replication. Notably, RdRp forms dimers via non-structural protein (nsp) subunits, particularly nsp7, crucial for efficient viral RNA copying. Similar to the main protease (Mpro) of SARS-CoV-2, there is a possibility that the nsp7 might also undergo mutational selection events to generate more stable and adaptable versions of nsp7 dimer during virus evolution. However, efforts to obtain such cohesive and comprehensive information are lacking. To address this, we performed this study focused on deciphering the molecular intricacies of nsp7 dimerization using a multifaceted approach. Leveraging computational protein design (CPD), machine learning (ML), AlphaFold v2.0-based structural analysis, and several related computational approaches, we aimed to identify critical residues and mutations influencing nsp7 dimer stability and adaptation. Our methodology involved identifying potential hotspot residues within the dimeric nsp7 interface using an interface-based CPD approach. Through Rosetta-based symmetrical protein design, we designed and modulated nsp7 dimerization, considering selected interface residues. Analysis of physicochemical features revealed acceptable structural changes and several structural and residue-specific insights emphasizing the intricate nature of such protein-protein complexes. Our ML models, particularly the random forest regressor (RFR), accurately predicted binding affinities and ML-guided sequence predictions corroborated CPD findings, elucidating potential nsp7 mutations and their impact on binding affinity. Validation against clinical sequencing data demonstrated the predictive accuracy of our approach. Moreover, AlphaFold v2.0 structural analyses validated optimal dimeric configurations of affinity-enhancing designs, affirming methodological precision. Affinity-enhancing designs exhibited favourable energetics and higher binding affinity as compared to their counterparts. The obtained physicochemical properties, molecular interactions, and sequence predictions advance our understanding of SARS-CoV-2 evolution and inform potential avenues for therapeutic intervention against COVID-19.

Discovery of C-Linked Nucleoside Analogues with Antiviral Activity against SARS-CoV-2.

The recent COVID-19 pandemic underscored the limitations of currently available direct-acting antiviral treatments against acute respiratory RNA-viral infections and stimulated major research initiatives targeting anticoronavirus agents. Two novel nsp5 protease (MPro) inhibitors have been approved, nirmatrelvir and ensitrelvir, along with two existing nucleos(t)ide analogues repurposed as nsp12 polymerase inhibitors, remdesivir and molnupiravir, but a need still exists for therapies with improved potency and systemic exposure with oral dosing, better metabolic stability, and reduced resistance and toxicity risks. Herein, we summarize our research toward identifying nsp12 inhibitors that led to nucleoside analogues 10e and 10n, which showed favorable pan-coronavirus activity in cell-infection screens, were metabolized to active triphosphate nucleotides in cell-incubation studies, and demonstrated target (nsp12) engagement in biochemical assays.

The PKA-CREB1 axis regulates coronavirus proliferation by viral helicase nsp13 association.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a worldwide threat in the past 3 years. Although it has been widely and intensively investigated, the mechanism underlying the coronavirus-host interaction requires further elucidation, which may contribute to the development of new antiviral strategies. Here, we demonstrated that the host cAMP-responsive element-binding protein (CREB1) interacts with the non-structural protein 13 (nsp13) of SARS-CoV-2, a conserved helicase for coronavirus replication, both in cells and in lung tissues subjected to SARS-CoV-2 infection. The ATPase and helicase activity of viral nsp13 were shown to be potentiated by CREB1 association, as well as by Protein kinase A (PKA)-mediated CREB1 activation. SARS-CoV-2 replication is significantly suppressed by PKA Cα, cAMP-activated protein kinase catalytic subunit alpha (PRKACA), and CREB1 knockdown or inhibition. Consistently, the CREB1 inhibitor 666-15 has shown significant antiviral effects against both the WIV04 strain and the Omicron strain of the SARS-CoV-2. Our findings indicate that the PKA-CREB1 signaling axis may serve as a novel therapeutic target against coronavirus infection. IMPORTANCE: In this study, we provide solid evidence that host transcription factor cAMP-responsive element-binding protein (CREB1) interacts directly with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) helicase non-structural protein 13 (nsp13) and potentiate its ATPase and helicase activity. And by live SARS-CoV-2 virus infection, the inhibition of CREB1 dramatically impairs SARS-CoV-2 replication in vivo. Notably, the IC50 of CREB1 inhibitor 666-15 is comparable to that of remdesivir. These results may extend to all highly pathogenic coronaviruses due to the conserved nsp13 sequences in the virus.

An alphacoronavirus polymerase structure reveals conserved replication factor functions.

Coronaviruses are a diverse subfamily of viruses containing pathogens of humans and animals. This subfamily of viruses replicates their RNA genomes using a core polymerase complex composed of viral non-structural proteins: nsp7, nsp8 and nsp12. Most of our understanding of coronavirus molecular biology comes from betacoronaviruses like SARS-CoV and SARS-CoV-2, the latter of which is the causative agent of COVID-19. In contrast, members of the alphacoronavirus genus are relatively understudied despite their importance in human and animal health. Here we have used cryo-electron microscopy to determine structures of the alphacoronavirus porcine epidemic diarrhea virus (PEDV) core polymerase complex bound to RNA. One structure shows an unexpected nsp8 stoichiometry despite remaining bound to RNA. Biochemical analysis shows that the N-terminal extension of one nsp8 is not required for in vitro RNA synthesis for alpha- and betacoronaviruses. Our work demonstrates the importance of studying diverse coronaviruses in revealing aspects of coronavirus replication and identifying areas of conservation to be targeted by antiviral drugs.

Analysis of the SARS-CoV-2 nsp12 P323L/A529V mutations: coeffect in the transiently peaking lineage C.36.3 on protein structure and response to treatment in Egyptian records.

SARS-CoV-2 nsp12, the RNA-dependent RNA-polymerase plays a crucial role in virus replication. Monitoring the effect of its emerging mutants on viral replication and response to antiviral drugs is important. Nsp12 of two Egyptian isolates circulating in 2020 and 2021 were sequenced. Both isolates included P323L, one included the A529V. Tracking A529V mutant frequency, it relates to the transience peaked C.36.3 variant and its parent C.36, both peaked worldwide on February-August 2021, enlisted as high transmissible variants under investigation (VUI) on May 2021. Both Mutants were reported to originate from Egypt and showed an abrupt low frequency upon screening, we analyzed all 1104 nsp12 Egyptian sequences. A529V mutation was in 36 records with an abrupt low frequency on June 2021. As its possible reappearance might obligate actions for a candidate VUI, we analyzed the predicted co-effect of P323L and A529V mutations on protein stability and dynamics through protein structure simulations. Three available structures for drug-nsp12 interaction were used representing remdesivir, suramin and favipiravir drugs. Remdesivir and suramin showed an increase in structure stability and considerable change in flexibility while favipiravir showed an extreme interaction. Results predict a favored efficiency of the drugs except for favipiravir in case of the reported mutations.

An exonuclease-resistant chain-terminating nucleotide analogue targeting the SARS-CoV-2 replicase complex.

Nucleotide analogues (NA) are currently employed for treatment of several viral diseases, including COVID-19. NA prodrugs are intracellularly activated to the 5'-triphosphate form. They are incorporated into the viral RNA by the viral polymerase (SARS-CoV-2 nsp12), terminating or corrupting RNA synthesis. For Coronaviruses, natural resistance to NAs is provided by a viral 3'-to-5' exonuclease heterodimer nsp14/nsp10, which can remove terminal analogues. Here, we show that the replacement of the α-phosphate of Bemnifosbuvir 5'-triphosphate form (AT-9010) by an α-thiophosphate renders it resistant to excision. The resulting α-thiotriphosphate, AT-9052, exists as two epimers (RP/SP). Through co-crystallization and activity assays, we show that the Sp isomer is preferentially used as a substrate by nucleotide diphosphate kinase (NDPK), and by SARS-CoV-2 nsp12, where its incorporation causes immediate chain-termination. The same -Sp isomer, once incorporated by nsp12, is also totally resistant to the excision by nsp10/nsp14 complex. However, unlike AT-9010, AT-9052-RP/SP no longer inhibits the N-terminal nucleotidylation domain of nsp12. We conclude that AT-9052-Sp exhibits a unique mechanism of action against SARS-CoV-2. Moreover, the thio modification provides a general approach to rescue existing NAs whose activity is hampered by coronavirus proofreading capacity.

SARS-CoV-2 NSP12 associates with TRiC and the P323L substitution acts as a host adaption.

IMPORTANCE: SARS-CoV-2 has caused a worldwide health and economic crisis. During the course of the pandemic, genetic changes occurred in the virus, which have resulted in new properties of the virus-particularly around gains in transmission and the ability to partially evade either natural or vaccine-acquired immunity. Some of these viruses have been labeled Variants of Concern (VoCs). At the root of all VoCs are two mutations, one in the viral spike protein that has been very well characterized and the other in the virus polymerase (NSP12). This is the viral protein responsible for replicating the genome. We show that NSP12 associates with host cell proteins that act as a scaffold to facilitate the function of this protein. Furthermore, we found that different variants of NSP12 interact with host cell proteins in subtle and different ways, which affect function.

Biochemical analysis of SARS-CoV-2 Nsp13 helicase implicated in COVID-19 and factors that regulate its catalytic functions.

Replication of the 30-kilobase genome of SARS-CoV-2, responsible for COVID-19, is a key step in the coronavirus life cycle that requires a set of virally encoded nonstructural proteins such as the highly conserved Nsp13 helicase. However, the features that contribute to catalytic properties of Nsp13 are not well established. Here, we biochemically characterized the purified recombinant SARS-CoV-2 Nsp13 helicase protein, focusing on its catalytic functions, nucleic acid substrate specificity, nucleotide/metal cofactor requirements, and displacement of proteins from RNA molecules proposed to be important for its proofreading role during coronavirus replication. We determined that Nsp13 preferentially interacts with single-stranded DNA compared with single-stranded RNA to unwind a partial duplex helicase substrate. We present evidence for functional cooperativity as a function of Nsp13 concentration, which suggests that oligomerization is important for optimal activity. In addition, under single-turnover conditions, Nsp13 unwound partial duplex RNA substrates of increasing double-stranded regions (16-30 base pairs) with similar efficiency, suggesting the enzyme unwinds processively in this range. We also show Nsp13-catalyzed RNA unwinding is abolished by a site-specific neutralizing linkage in the sugar-phosphate backbone, demonstrating continuity in the helicase-translocating strand is essential for unwinding the partial duplex substrate. Taken together, we demonstrate for the first time that coronavirus helicase Nsp13 disrupts a high-affinity RNA-protein interaction in a unidirectional and ATP-dependent manner. Furthermore, sensitivity of Nsp13 catalytic functions to Mg2+ concentration suggests a regulatory mechanism for ATP hydrolysis, duplex unwinding, and RNA protein remodeling, processes implicated in SARS-CoV-2 replication and proofreading.

Coronavirus RNA-dependent RNA polymerase interacts with the p50 regulatory subunit of host DNA polymerase delta and plays a synergistic role with RNA helicase in the induction of DNA damage response and cell cycle arrest in the S phase.

Disruption of the cell cycle is a common strategy shared by many viruses to create a conducible cellular microenvironment for their efficient replication. We have previously shown that infection of cells with gammacoronavirus infectious bronchitis virus (IBV) activated the theataxia-telangiectasia mutated (ATM) Rad3-related (ATR)/checkpoint kinase 1 (Chk1) pathway and induced cell cycle arrest in S and G2/M phases, partially through the interaction of nonstructural protein 13 (nsp13) with the p125 catalytic subunit of DNA polymerase delta (pol δ). In this study, we show, by GST pulldown, co-immunoprecipitation and immunofluorescent staining, that IBV nsp12 directly interacts with the p50 regulatory subunit of pol δ in vitro and in cells overexpressing the two proteins as well as in cells infected with a recombinant IBV harbouring an HA-tagged nsp12. Furthermore, nsp12 from severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 was also able to interact with p50. These interactions play a synergistic role with nsp13 in the induction of S phase arrest. The fact that subunits of an essential cellular DNA replication machinery physically associate with two core replication enzymes from three different coronaviruses highlights the importance of these associations in coronavirus replication and virus-host interaction, and reveals the potential of targeting these subunits for antiviral intervention.

Structural basis for substrate selection by the SARS-CoV-2 replicase.

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate3,4. Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.

Oral GS-441524 derivatives: Next-generation inhibitors of SARS-CoV-2 RNA-dependent RNA polymerase.

GS-441524, an RNA-dependent RNA polymerase (RdRp) inhibitor, is a 1'-CN-substituted adenine C-nucleoside analog with broad-spectrum antiviral activity. However, the low oral bioavailability of GS-441524 poses a challenge to its anti-SARS-CoV-2 efficacy. Remdesivir, the intravenously administered version (version 1.0) of GS-441524, is the first FDA-approved agent for SARS-CoV-2 treatment. However, clinical trials have presented conflicting evidence on the value of remdesivir in COVID-19. Therefore, oral GS-441524 derivatives (VV116, ATV006, and GS-621763; version 2.0, targeting highly conserved viral RdRp) could be considered as game-changers in treating COVID-19 because oral administration has the potential to maximize clinical benefits, including decreased duration of COVID-19 and reduced post-acute sequelae of SARS-CoV-2 infection, as well as limited side effects such as hepatic accumulation. This review summarizes the current research related to the oral derivatives of GS-441524, and provides important insights into the potential factors underlying the controversial observations regarding the clinical efficacy of remdesivir; overall, it offers an effective launching pad for developing an oral version of GS-441524.

Discovering new potential inhibitors to SARS-CoV-2 RNA dependent RNA polymerase (RdRp) using high throughput virtual screening and molecular dynamics simulations.

RNA dependent RNA polymerase (RdRp), is an essential in the RNA replication within the life cycle of the severely acute respiratory coronavirus-2 (SARS-CoV-2), causing the deadly respiratory induced sickness COVID-19. Remdesivir is a prodrug that has seen some success in inhibiting this enzyme, however there is still the pressing need for effective alternatives. In this study, we present the discovery of four non-nucleoside small molecules that bind favorably to SARS-CoV-2 RdRp over the active form of the popular drug remdesivir (RTP) and adenosine triphosphate (ATP) by utilizing high-throughput virtual screening (HTVS) against the vast ZINC compound database coupled with extensive molecular dynamics (MD) simulations. After post-trajectory analysis, we found that the simulations of complexes containing both ATP and RTP remained stable for the duration of their trajectories. Additionally, it was revealed that the phosphate tail of RTP was stabilized by both the positive amino acid pocket and magnesium ions near the entry channel of RdRp which includes residues K551, R553, R555 and K621. It was also found that residues D623, D760, and N691 further stabilized the ribose portion of RTP with U10 on the template RNA strand forming hydrogen pairs with the adenosine motif. Using these models of RdRp, we employed them to screen the ZINC database of ~ 17 million molecules. Using docking and drug properties scoring, we narrowed down our selection to fourteen candidates. These were subjected to 200 ns simulations each underwent free energy calculations. We identified four hit compounds from the ZINC database that have similar binding poses to RTP while possessing lower overall binding free energies, with ZINC097971592 having a binding free energy two times lower than RTP.

Gossypol Broadly Inhibits Coronaviruses by Targeting RNA-Dependent RNA Polymerases.

Outbreaks of coronaviruses (CoVs), especially severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have posed serious threats to humans and animals, which urgently calls for effective broad-spectrum antivirals. RNA-dependent RNA polymerase (RdRp) plays an essential role in viral RNA synthesis and is an ideal pan-coronaviral therapeutic target. Herein, based on cryo-electron microscopy and biochemical approaches, gossypol (GOS) is identified from 881 natural products to directly block SARS-CoV-2 RdRp, thus inhibiting SARS-CoV-2 replication in both cellular and mouse infection models. GOS also acts as a potent inhibitor against the SARS-CoV-2 variant of concern (VOC) and exerts same inhibitory effects toward mutated RdRps of VOCs as the RdRp of the original SARS-CoV-2. Moreover, that the RdRp inhibitor GOS has broad-spectrum anti-coronavirus activity against alphacoronaviruses (porcine epidemic diarrhea virus and swine acute diarrhea syndrome coronavirus), betacoronaviruses (SARS-CoV-2), gammacoronaviruses (avian infectious bronchitis virus), and deltacoronaviruses (porcine deltacoronavirus) is showed. The findings demonstrate that GOS may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and other coronavirus outbreaks.