Extracellular Disintegration of Viral Proteins as an Innovative Strategy for Developing Broad-Spectrum Antivirals Against Coronavirus

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has claimed millions of lives and caused innumerable economic losses worldwide. Unfortunately, state-of-the-art treatments still lag behind the continual emergence of new variants. Key to resolving this issue is developing antivirals to deactivate coronaviruses regardless of their structural evolution. Here, we report an innovative antiviral strategy involving extracellular disintegration of viral proteins with hyperanion-grafted enediyne (EDY) molecules. The core EDY generates reactive radical species and causes significant damage to the spike protein of coronavirus, while the hyperanion groups ensure negligible cytotoxicity of the molecules. The EDYs exhibit antiviral activity down to nanomolar concentrations, and the selectivity index of up to 20,000 against four kinds of human coronavirus, including the SARS-CoV-2 Omicron variant, suggesting the high potential of this new strategy in combating the COVID-19 pandemic and a future "disease X."

Antiviral properties of trans-δ-viniferin derivatives against enveloped viruses.

Over the last century, the number of epidemics caused by RNA viruses has increased and the current SARS-CoV-2 pandemic has taught us about the compelling need for ready-to-use broad-spectrum antivirals. In this scenario, natural products stand out as a major historical source of drugs. We analyzed the antiviral effect of 4 stilbene dimers [1 (trans-δ-viniferin); 2 (11',13'-di-O-methyl-trans-δ-viniferin), 3 (11,13-di-O-methyl-trans-δ-viniferin); and 4 (11,13,11',13'-tetra-O-methyl-trans-δ-viniferin)] obtained from plant substrates using chemoenzymatic synthesis against a panel of enveloped viruses. We report that compounds 2 and 3 display a broad-spectrum antiviral activity, being able to effectively inhibit several strains of Influenza Viruses (IV), SARS-CoV-2 Delta and, to some extent, Herpes Simplex Virus 2 (HSV-2). Interestingly, the mechanism of action differs for each virus. We observed both a direct virucidal and a cell-mediated effect against IV, with a high barrier to antiviral resistance; a restricted cell-mediated mechanism of action against SARS-CoV-2 Delta and a direct virustatic activity against HSV-2. Of note, while the effect was lost against IV in tissue culture models of human airway epithelia, the antiviral activity was confirmed in this relevant model for SARS-CoV-2 Delta. Our results suggest that stilbene dimer derivatives are good candidate models for the treatment of enveloped virus infections.

Octyl gallate targeting the 3C-like protease exhibits highly efficient antiviral activity against swine enteric coronavirus PEDV.

Infection with porcine epidemic diarrhea virus (PEDV) causes severe watery diarrhea in newborn piglets, leading to substantial financial losses for the swine industry. In this study, we screened small molecule drugs targeting 3 C-like protease (3CLpro) by molecular docking, and further evaluated the antiviral activity of the screened drugs against PEDV. Results showed that octyl gallate (OG), a widely used food additive, exhibited strong binding affinity with the 3CLpro active sites of PEDV. Bio-layer interferometry and fluorescence resonance energy transfer revealed that OG directly interacts with PEDV 3CLpro (KD = 549 nM) and inhibits 3CLpro activity (IC50 = 22.15 µM). OG showed a strong inhibition of PEDV replication in vitro. Virus titers were decreased by 0.58 and 0.71 log10 TCID50/mL for the CV777 and HM2017 strains, respectively. In vivo, all piglets in the PEDV-infected group died at 48 h post-infection (hpi), while 75% of piglets in the OG treatment group showed significant relief from the clinical symptoms, pathological damage, and viral loads in the jejunum and ileum. Moreover, the western blotting results showed that OG also has strong antiviral activity against other swine enteric coronaviruses, including transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). Our findings revealed that OG could be developed as a novel antiviral drug against PEDV. The OG exhibited a potential broad-spectrum antiviral drug for control of other swine enteric coronaviruses.

Therapeutic potential of salicylamide derivatives for combating viral infections.

Since time immemorial human beings have constantly been fighting against viral infections. The ongoing and devastating coronavirus disease 2019 pandemic represents one of the most severe and most significant public health emergencies in human history, highlighting an urgent need to develop broad-spectrum antiviral agents. Salicylamide (2-hydroxybenzamide) derivatives, represented by niclosamide and nitazoxanide, inhibit the replication of a broad range of RNA and DNA viruses such as flavivirus, influenza A virus, and coronavirus. Moreover, nitazoxanide was effective in clinical trials against different viral infections including diarrhea caused by rotavirus and norovirus, uncomplicated influenza A and B, hepatitis B, and hepatitis C. In this review, we summarize the broad antiviral activities of salicylamide derivatives, the clinical progress, and the potential targets or mechanisms against different viral infections and highlight their therapeutic potential in combating the circulating and emerging viral infections in the future.

Targeting the Fusion Process of SARS-CoV-2 Infection by Small Molecule Inhibitors.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a serious threat to global public health, underscoring the urgency of developing effective therapies. Therapeutics and, more specifically, direct-acting antiviral development are still very much in their infancy. Here, we report that two hepatitis C virus (HCV) fusion inhibitors identified in our previous study, dichlorcyclizine and fluoxazolevir, broadly block human coronavirus entry into various cell types. Both compounds were effective against various human-pathogenic CoVs in multiple assays based on vesicular stomatitis virus (VSV) pseudotyped with the spike protein and spike-mediated syncytium formation. The antiviral effects were confirmed in SARS-CoV-2 infection systems. These compounds were equally effective against recently emerged variants, including the delta variant. Cross-linking experiments and structural modeling suggest that the compounds bind to a hydrophobic pocket near the fusion peptide of S protein, consistent with their potential mechanism of action as fusion inhibitors. In summary, these fusion inhibitors have broad-spectrum antiviral activities and may be promising leads for treatment of SARS-CoV-2, its variants, and other pathogenic CoVs. IMPORTANCE SARS-CoV-2 is an enveloped virus that requires membrane fusion for entry into host cells. Since the fusion process is relatively conserved among enveloped viruses, we tested our HCV fusion inhibitors, dichlorcyclizine and fluoxazolevir, against SARS-CoV-2. We performed in vitro assays and demonstrated their effective antiviral activity against SARS-CoV-2 and its variants. Cross-linking experiments and structural modeling suggest that the compounds bind to a hydrophobic pocket in spike protein to exert their inhibitory effect on the fusion step. These data suggest that both dichlorcyclizine and fluoxazolevir are promising candidates for further development as treatment for SARS-CoV-2.

A Synergistic Broad-Spectrum Viral Entry Blocker: In-Silico Approach

Increasing victims of viral attacks is a serious concern of the current time as well as soon. The story of virulence was started with the origin of the Spanish flu pandemic of 1918, later severe other viral infections like SARS, MARS, and many others took the life of many people. Currently, the situation is locked in the world because of the unprecedented arrival of the COVID-19 from the Wuhan, city of China. The current need turned to make suitable candidates with the existing safety data, to get the molecule in a limited period. Because of that, the quinolone 3-carboxy derivatives were docked with many targets enzyme, but the interaction with gp-41 was found interactive, which is represented with interactive binding energy scores. In this regard, the validated target of the virus likes HIV. COVID-19 and other viruses were utilized to see the beneficial interactions. The present research is based on the Quinoline-3carboxy derivatives and their interaction with gp41. The gp41 has been found with the highest similarity with the S2 protein of the Coronavirus;targeting this protein will inhibit the interaction of cells and viruses. The in silico results were found encouraging with the suitable interactions with the amino acid residues. The results give us the hope to develop a lead for the inhibition of viral infection, including HIV, flu, and Coronavirus. The result is summarized with all the in silico docking and residual interaction with the reasonable concept of lead to go further in the drug discovery process. © 2022 by the authors.

In vitro and in vivo effects of 3-indoleacetonitrile-A potential new broad-spectrum therapeutic agent for SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has resulted in significant global morbidity, mortality, and societal disruption. Currently, effective antiviral drugs for the treatment of SARS-CoV-2 infection are limited. Therefore, safe and effective antiviral drugs to combat COVID-19 are urgently required. In previous studies, we showed that 3-indoleacetonitrile, a plant growth hormone produced by cruciferous (Brassica) vegetables, is effective in treating influenza A virus infection. However, the molecular mechanisms underlying these effects remain unclear. Herein, we demonstrated that 3-indoleacetonitrile exhibits broad-spectrum antiviral activity and is effective against HSV-1 and VSV infections in vitro. This phenomenon prompted us to study its role in the anti-SARS-CoV-2 process. Interestingly, 3-indoleacetonitrile exhibited antiviral activity against SARS-CoV-2 in vitro. Importantly, tail vein injection of 3-indoleacetonitrile resulted in good antiviral activity in mouse models infected with WBP-1 (a mouse adaptation of the SARS-CoV-2 strain). Mechanistically, 3-indoleacetonitrile promoted the host interferon signalling pathway response and inhibited autophagic flux. Furthermore, we demonstrated that 3-indoleacetonitrile induced an increase in mitochondrial antiviral-signalling (MAVS) protein levels, which might be attributed to its inhibition of the interaction between MAVS and the selective autophagy receptor SQSTM1. Overall, our results demonstrate that 3-indoleacetonitrile is potently active against SARS-CoV-2 in vitro and in vivo, which may provide a foundation for further clinical testing for the treatment of COVID-19. In addition, considering its broad-spectrum antiviral effect, it should be explored whether it also has an effect on other viruses that threaten human health.

Tackling the Future Pandemics: Broad-Spectrum Antiviral Agents (BSAAs) Based on A-Type Proanthocyanidins.

A-type proanthocyanidins (PAC-As) are plant-derived natural polyphenols that occur as oligomers or polymers of flavan-3-ol monomers, such as (+)-catechin and (-)-epicatechin, connected through an unusual double A linkage. PAC-As are present in leaves, seeds, flowers, bark, and fruits of many plants, and are thought to exert protective natural roles against microbial pathogens, insects, and herbivores. Consequently, when tested in isolation, PAC-As have shown several biological effects, through antioxidant, antibacterial, immunomodulatory, and antiviral activities. PAC-As have been observed in fact to inhibit replication of many different human viruses, and both enveloped and non-enveloped DNA and RNA viruses proved sensible to their inhibitory effect. Mechanistic studies revealed that PAC-As cause reduction of infectivity of viral particles they come in contact with, as a result of their propensity to interact with virion surface capsid proteins or envelope glycoproteins essential for viral attachment and entry. As viral infections and new virus outbreaks are a major public health concern, development of effective Broad-Spectrum Antiviral Agents (BSAAs) that can be rapidly deployable even against future emerging viruses is an urgent priority. This review summarizes the antiviral activities and mechanism of action of PAC-As, and their potential to be deployed as BSAAs against present and future viral infections.

Host-directed therapy with 2-deoxy-D-glucose inhibits human rhinoviruses, endemic coronaviruses, and SARS-CoV-2.

Rhinoviruses (RVs) and coronaviruses (CoVs) upregulate host cell metabolic pathways such as glycolysis to meet their bioenergetic demands for rapid multiplication. Using the glycolysis inhibitor 2-deoxy-d-glucose (2-DG), we assessed the dose-dependent inhibition of viral replication of minor- and major-receptor group RVs in epithelial cells. 2-DG disrupted RV infection cycle by inhibiting template negative-strand as well as genomic positive-strand RNA synthesis, resulting in less progeny virus and RV-mediated cell death. Assessment of 2-DG's intracellular kinetics revealed that after a short-exposure to 2-DG, the active intermediate, 2-DG6P, is stored intracellularly for several hours. Finally, we confirmed the antiviral effect of 2-DG on pandemic SARS-CoV-2 and showed for the first time that it also reduces replication of endemic human coronaviruses. These results provide further evidence that 2-DG could be used as a broad-spectrum antiviral.

Ganoderma microsporum immunomodulatory protein acts as a multifunctional broad-spectrum antiviral against SARS-CoV-2 by interfering virus binding to the host cells and spike-mediated cell fusion.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus that has caused over 6 million fatalities. SARS-CoV-2 variants with spike mutations are frequently endowed with a strong capability to escape vaccine-elicited protection. Due to this characteristic, a broad-spectrum inhibitor against SARS-CoV-2 infection is urgently demanded. Ganoderma microsporum immunomodulatory protein (GMI) was previously reported to alleviate infection of SARS-CoV-2 through ACE2 downregulation whereas the impact of GMI on virus itself was less understood. Our study aims to determine the effects of GMI on SARS-CoV-2 pseudovirus and the more detailed mechanisms of GMI inhibition against SARS-CoV-2 pseudovirus infection. METHODS: ACE2-overexpressing HEK293T cells (HEK293T/ACE2) and SARS-CoV-2 pseudoviruses carrying spike variants were used to study the effects of GMI in vitro. Infectivity was evaluated by fluorescence microscopy and flow cytometry. Fusion rate mediated by SARS-CoV-2 spike protein was examined with split fluorescent protein /luciferase systems. The interactions of GMI with SARS-CoV-2 pseudovirus and ACE2 were investigated by immunoprecipitation and immunoblotting. RESULTS: GMI broadly blocked SARS-CoV-2 infection in various cell lines. GMI effectively inhibited the infection of pseudotyped viruses carrying different emerged spike variants, including Delta and Omicron strains, on HEK293T/hACE2 cells. In cell-free virus infection, GMI dominantly impeded the binding of spike-bearing pseudotyped viruses to ACE2-expressing cells. In cell-to-cell fusion model, GMI could efficiently inhibit spike-mediated syncytium without the requirement of ACE2 downregulation. CONCLUSIONS: GMI, an FDA-approved dietary ingredient, acts as a multifunctional broad-spectrum antiviral against SARS-CoV-2 and could become a promising candidate for preventing or treating SARS-CoV-2 associated diseases.

A Novel Approach of Antiviral Drugs Targeting Viral Genomes.

Outbreaks of viral diseases, which cause morbidity and mortality in animals and humans, are increasing annually worldwide. Vaccines, antiviral drugs, and antibody therapeutics are the most effective tools for combating viral infection. The ongoing coronavirus disease 2019 pandemic, in particular, raises an urgent need for the development of rapid and broad-spectrum therapeutics. Current antiviral drugs and antiviral antibodies, which are mostly specific at protein levels, have encountered difficulties because the rapid evolution of mutant viral strains resulted in drug resistance. Therefore, degrading viral genomes is considered a novel approach for developing antiviral drugs. The current article highlights all potent candidates that exhibit antiviral activity by digesting viral genomes such as RNases, RNA interference, interferon-stimulated genes 20, and CRISPR/Cas systems. Besides that, we introduce a potential single-chain variable fragment (scFv) that presents antiviral activity against various DNA and RNA viruses due to its unique nucleic acid hydrolyzing characteristic, promoting it as a promising candidate for broad-spectrum antiviral therapeutics.

A Synergistic Broad-Spectrum Viral Entry Blocker: In-Silico Approach

Increasing victims of viral attacks is a serious concern of the current time as well as soon. The story of virulence was started with the origin of the Spanish flu pandemic of 1918, later severe other viral infections like SARS, MARS, and many others took the life of many people. Currently, the situation is locked in the world because of the unprecedented arrival of the COVID-19 from the Wuhan, city of China. The current need turned to make suitable candidates with the existing safety data, to get the molecule in a limited period. Because of that, the quinolone 3-carboxy derivatives were docked with many targets enzyme, but the interaction with gp-41 was found interactive, which is represented with interactive binding energy scores. In this regard, the validated target of the virus likes HIV. COVID-19 and other viruses were utilized to see the beneficial interactions. The present research is based on the Quinoline-3carboxy derivatives and their interaction with gp41. The gp41 has been found with the highest similarity with the S2 protein of the Coronavirus;targeting this protein will inhibit the interaction of cells and viruses. The in silico results were found encouraging with the suitable interactions with the amino acid residues. The results give us the hope to develop a lead for the inhibition of viral infection, including HIV, flu, and Coronavirus. The result is summarized with all the in silico docking and residual interaction with the reasonable concept of lead to go further in the drug discovery process. © 2022 by the authors.

Impairment of SARS-CoV-2 spike glycoprotein maturation and fusion activity by nitazoxanide: an effect independent of spike variants emergence.

SARS-CoV-2, the causative agent of COVID-19, has caused an unprecedented global health crisis. The SARS-CoV-2 spike, a surface-anchored trimeric class-I fusion glycoprotein essential for viral entry, represents a key target for developing vaccines and therapeutics capable of blocking virus invasion. The emergence of SARS-CoV-2 spike variants that facilitate virus spread and may affect vaccine efficacy highlights the need to identify novel antiviral strategies for COVID-19 therapy. Here, we demonstrate that nitazoxanide, an antiprotozoal agent with recognized broad-spectrum antiviral activity, interferes with SARS-CoV-2 spike maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Engineering multiple SARS-CoV-2 variant-pseudoviruses and utilizing quantitative cell-cell fusion assays, we show that nitazoxanide-induced spike modifications hinder progeny virion infectivity as well as spike-driven pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. Nitazoxanide, being equally effective against the ancestral SARS-CoV-2 Wuhan-spike and different emerging variants, including the Delta variant of concern, may represent a useful tool in the fight against COVID-19 infections.

Integrated autolysis, DNA hydrolysis and precipitation enables an improved bioprocess for Q-Griffithsin, a broad-spectrum antiviral and clinical-stage anti-COVID-19 candidate.

Across the biomanufacturing industry, innovations are needed to improve efficiency and flexibility, especially in the face of challenges such as the COVID-19 pandemic. Here we report an improved bioprocess for Q-Griffithsin, a broad-spectrum antiviral currently in clinical trials for COVID-19. Q-Griffithsin is produced at high titer in E. coli and purified to anticipated clinical grade without conventional chromatography or the need for any fixed downstream equipment. The process is thus both low-cost and highly flexible, facilitating low sales prices and agile modifications of production capacity, two key features for pandemic response. The simplicity of this process is enabled by a novel unit operation that integrates cellular autolysis, autohydrolysis of nucleic acids, and contaminant precipitation, giving essentially complete removal of host cell DNA as well as reducing host cell proteins and endotoxin by 3.6 and 2.4 log10 units, respectively. This unit operation can be performed rapidly and in the fermentation vessel, such that Q-GRFT is obtained with 100% yield and > 99.9% purity immediately after fermentation and requires only a flow-through membrane chromatography step for further contaminant removal. Using this operation or variations of it may enable improved bioprocesses for a range of other high-value proteins in E. coli.

Nucleosides and emerging viruses: A new story.

With several US Food and Drug Administration (FDA)-approved drugs and high barriers to resistance, nucleoside and nucleotide analogs remain the cornerstone of antiviral therapies for not only herpesviruses, but also HIV and hepatitis viruses (B and C); however, with the exception of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which vaccines have been developed at unprecedented speed, there are no vaccines or small antivirals yet available for (re)emerging viruses, which are primarily RNA viruses. Thus, herein, we present an overview of ribonucleoside analogs recently developed and acting as inhibitors of the viral RNA-dependent RNA polymerase (RdRp). They are new lead structures that will be exploited for the discovery of new antiviral nucleosides.

The New Generation hDHODH Inhibitor MEDS433 Hinders the In Vitro Replication of SARS-CoV-2 and Other Human Coronaviruses.

Although coronaviruses (CoVs) have long been predicted to cause zoonotic diseases and pandemics with high probability, the lack of effective anti-pan-CoVs drugs rapidly usable against the emerging SARS-CoV-2 actually prevented a promptly therapeutic intervention for COVID-19. Development of host-targeting antivirals could be an alternative strategy for the control of emerging CoVs infections, as they could be quickly repositioned from one pandemic event to another. To contribute to these pandemic preparedness efforts, here we report on the broad-spectrum CoVs antiviral activity of MEDS433, a new inhibitor of the human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of the de novo pyrimidine biosynthesis pathway. MEDS433 inhibited the in vitro replication of hCoV-OC43 and hCoV-229E, as well as of SARS-CoV-2, at low nanomolar range. Notably, the anti-SARS-CoV-2 activity of MEDS433 against SARS-CoV-2 was also observed in kidney organoids generated from human embryonic stem cells. Then, the antiviral activity of MEDS433 was reversed by the addition of exogenous uridine or the product of hDHODH, the orotate, thus confirming hDHODH as the specific target of MEDS433 in hCoVs-infected cells. Taken together, these findings suggest MEDS433 as a potential candidate to develop novel drugs for COVID-19, as well as broad-spectrum antiviral agents exploitable for future CoVs threats.

Efficient incorporation and template-dependent polymerase inhibition are major determinants for the broad-spectrum antiviral activity of remdesivir.

Remdesivir (RDV) is a direct-acting antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2. RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, for example, severe acute respiratory syndrome coronavirus and hepatitis C virus, and nonsegmented negative-sense RNA viruses, for example, Nipah virus, whereas segmented negative-sense RNA viruses such as influenza virus or Crimean-Congo hemorrhagic fever virus are not sensitive to the drug. The reasons for this apparent efficacy pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. We found a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Inhibition in primer extension reactions was heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP was seen with all polymerases. Molecular modeling suggests a steric conflict between the 1'-cyano group of the inhibitor and residues of the structurally conserved RNA-dependent RNA polymerase motif F. We conclude that future efforts in the development of nucleotide analogs with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1'-modification.

Safe-in-Man Broad Spectrum Antiviral Agents.

Emerging and re-emerging viral diseases occur with regularity within the human population. The conventional 'one drug, one virus' paradigm for antivirals does not adequately allow for proper preparedness in the face of unknown future epidemics. In addition, drug developers lack the financial incentives to work on antiviral drug discovery, with most pharmaceutical companies choosing to focus on more profitable disease areas. Safe-in-man broad spectrum antiviral agents (BSAAs) can help meet the need for antiviral development by already having passed phase I clinical trials, requiring less time and money to develop, and having the capacity to work against many viruses, allowing for a speedy response when unforeseen epidemics arise. In this chapter, we discuss the benefits of repurposing existing drugs as BSAAs, describe the major steps in safe-in-man BSAA drug development from discovery through clinical trials, and list several database resources that are useful tools for antiviral drug repositioning.

The mechanisms and clinical application of Traditional Chinese Medicine Lianhua-Qingwen capsule.

Lianhua-Qingwen capsule (LQC) is a commonly used Traditional Chinese Medicine (TCM) in China and has 11 herb components. The main active ingredient can target specific molecules and perform many clinic treatment roles. LQC has been authorized by National Medical Products Administration (NMPA) of China to treat severe acute respiratory syndrome (SARS) in 2002-2003, type A influenza virus HIN1 pandemic in 2009, H7N9, H3N2 and coronavirus disease-19 (COVID19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) in 2020. It is also widely used to treat common cold with wind-heat syndrome, chronic rhinosinusitis (CRS), amygdalitis and chronic obstructive pulmonary disease. This article summarizes the advanced research progress of LQC in clinical application, mechanisms and provides new clues in the clinical application of LQC.

NPC1-regulated dynamic of clathrin-coated pits is essential for viral entry.

Viruses utilize cellular lipids and manipulate host lipid metabolism to ensure their replication and spread. Therefore, the identification of lipids and metabolic pathways that are suitable targets for antiviral development is crucial. Using a library of compounds targeting host lipid metabolic factors and testing them for their ability to block pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) infection, we found that U18666A, a specific inhibitor of Niemann-Pick C1 (NPC1), is highly potent in suppressing the entry of diverse viruses including pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). NPC1 deficiency markedly attenuates viral growth by decreasing cholesterol abundance in the plasma membrane, thereby inhibiting the dynamics of clathrin-coated pits (CCPs), which are indispensable for clathrin-mediated endocytosis. Significantly, exogenous cholesterol can complement the dynamics of CCPs, leading to efficient viral entry and infectivity. Administration of U18666A improves the survival and pathology of PRV- and influenza A virus-infected mice. Thus, our studies demonstrate a unique mechanism by which NPC1 inhibition achieves broad antiviral activity, indicating a potential new therapeutic strategy against SARS-CoV-2, as well as other emerging viruses.