Identification of potential targets against SARS-CoV-2 of antiviral drugs based on photoaffinity probes.

Facing the sudden outbreak of coronavirus disease 2019 (COVID-19), it is extremely urgent to develop effective antiviral drugs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Drug repurposing is a promising strategy for the treatment of COVID-19. To identify the precise target protein of marketed medicines, we initiate a chemical biological program to identify precise target of potential antivirus drugs. In this study, two types of recombinant human coronavirus SARS-CoV-2 RdRp protein capturing probes with various photoaffinity labeling units were designed and synthesized based on the structure of FDA-approved drugs stavudine, remdesivir, acyclovir, and aladenosine. Fortunately, it was found that one novel photoaffinity probe, RD-1, could diaplayed good affinity with SARS-CoV-2 RdRp around the residue ARG_553. In addition, RD-1 probe also exhibited potent inhibitory activity against 3CLpro protease. Taken together, our findings will elucidate the structural basis for the efficacy of marketed drugs, and explore a rapid and efficient strategy of drug repurposing based on the identification of new targets. Moreover, these results could also provide a scientific basis for the clinical application of marketed drugs.

What can we learn from the Baduanjin rehabilitation as COVID-19 treatment?: A narrative review.

AIM: To understand Baduanjin rehabilitation therapy in mild COVID-19 patients. DESIGN: A narrative review. METHODS: A literature search for COVID-19 and Baduanjin treatments was conducted on Chinese and English electronic databases: China National Knowledge Infrastructure, Wanfang Data, Embase, PubMed, Scopus, Science Direct, Ebscohost, SPORTDiscus and ProQuest. RESULTS: Twelve studies on the Baduanjin rehabilitation for COVID-19 patients have been included. We acknowledged the considerable published research and current clinical practice using Baduanjin for COVID-19 treatment in the following areas: anxiety, depression, insomnia, lung function rehabilitation, immunity and activity endurance. CONCLUSION: The use of Baduanjin as adjuvant therapy for COVID-19 patients' rehabilitation is still limited, therefore, more clinical studies are needed to confirm its efficacy.

High-throughput screening of SARS-CoV-2 main and papain-like protease inhibitors.

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (Mpro) and papain like protease (PLpro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851Mpro inhibitors and 205 PLpro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both Mpro and PLpro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of Mpro inhibitors and over 20% of PLpro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 Mpro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.

Extracellular vesicles mediate antibody-resistant transmission of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Antibody resistance dampens neutralizing antibody therapy and threatens current global Coronavirus (COVID-19) vaccine campaigns. In addition to the emergence of resistant SARS-CoV-2 variants, little is known about how SARS-CoV-2 evades antibodies. Here, we report a novel mechanism of extracellular vesicle (EV)-mediated cell-to-cell transmission of SARS-CoV-2, which facilitates SARS-CoV-2 to escape from neutralizing antibodies. These EVs, initially observed in SARS-CoV-2 envelope protein-expressing cells, are secreted by various SARS-CoV-2-infected cells, including Vero E6, Calu-3, and HPAEpiC cells, undergoing infection-induced pyroptosis. Various SARS-CoV-2-infected cells produce similar EVs characterized by extra-large sizes (1.6-9.5 µm in diameter, average diameter > 4.2 µm) much larger than previously reported virus-generated vesicles. Transmission electron microscopy analysis and plaque assay reveal that these SARS-CoV-2-induced EVs contain large amounts of live virus particles. In particular, the vesicle-cloaked SARS-CoV-2 virus is resistant to neutralizing antibodies and able to reinfect naïve cells independent of the reported receptors and cofactors. Consistently, the constructed 3D images show that intact EVs could be taken up by recipient cells directly, supporting vesicle-mediated cell-to-cell transmission of SARS-CoV-2. Our findings reveal a novel mechanism of receptor-independent SARS-CoV-2 infection via cell-to-cell transmission, provide new insights into antibody resistance of SARS-CoV-2 and suggest potential targets for future antiviral therapeutics.

Sertraline Is an Effective SARS-CoV-2 Entry Inhibitor Targeting the Spike Protein.

The global spread of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the continuously emerging new variants underscore an urgent need for effective therapeutics for the treatment of coronavirus disease 2019 (COVID-19). Here, we screened several FDA-approved amphiphilic drugs and determined that sertraline (SRT) exhibits potent antiviral activity against infection of SARS-CoV-2 pseudovirus (PsV) and authentic virus in vitro. It effectively inhibits SARS-CoV-2 spike (S)-mediated cell-cell fusion. SRT targets the early stage of viral entry. It can bind to the S1 subunit of the S protein, especially the receptor binding domain (RBD), thus blocking S-hACE2 interaction and interfering with the proteolysis process of S protein. SRT is also effective against infection with SARS-CoV-2 PsV variants, including the newly emerging Omicron. The combination of SRT and other antivirals exhibits a strong synergistic effect against infection of SARS-CoV-2 PsV. The antiviral activity of SRT is independent of serotonin transporter expression. Moreover, SRT effectively inhibits infection of SARS-CoV-2 PsV and alleviates the inflammation process and lung pathological alterations in transduced mice in vivo. Therefore, SRT shows promise as a treatment option for COVID-19. IMPORTANCE The study shows SRT is an effective entry inhibitor against infection of SARS-CoV-2, which is currently prevalent globally. SRT targets the S protein of SARS-CoV-2 and is effective against a panel of SARS-CoV-2 variants. It also could be used in combination to prevent SARS-CoV-2 infection. More importantly, with long history of clinical use and proven safety, SRT might be particularly suitable to treat infection of SARS-CoV-2 in the central nervous system and optimized for treatment in older people, pregnant women, and COVID-19 patients with heart complications, which are associated with severity and mortality of COVID-19.

Identification of phytochemicals in Qingfei Paidu decoction for the treatment of coronavirus disease 2019 by targeting the virus-host interactome.

Qingfei Paidu decoction (QFPDD) has been clinically proven to be effective in the treatment of coronavirus disease 2019 (COVID-19). However, the bioactive components and therapeutic mechanisms remain unclear. This study aimed to explore the effective components and underlying mechanisms of QFPDD in the treatment of COVID-19 by targeting the virus-host interactome and verifying the antiviral activities of its active components in vitro. Key active components and targets were identified by analysing the topological features of a compound-target-pathway-disease regulatory network of QFPDD for the treatment of COVID-19. The antiviral activity of the active components was determined by a live virus infection assay, and possible mechanisms were analysed by pseudotyped virus infection and molecular docking assays. The inhibitory effects of the components tested on the virus-induced release of IL-6, IL-1ß and CXCL-10 were detected by ELISA. Three components of QFPDD, oroxylin A, hesperetin and scutellarin, exhibited potent antiviral activities against live SARS-CoV-2 virus and HCoV-OC43 virus with IC50 values ranging from 18.68 to 63.27 µM. Oroxylin A inhibited the entry of SARS-CoV-2 pseudovirus into target cells and inhibited SARS-CoV-2 S protein-mediated cell-cell fusion by binding with the ACE2 receptor. The active components of QFPDD obviously inhibited the IL-6, IL-1ß and CXCL-10 release induced by the SARS-CoV-2 S protein. This study supports the clinical application of QFPDD and provides an effective analysis method for the in-depth study of the mechanisms of traditional Chinese medicine (TCM) in the prevention and treatment of COVID-19.

Identification, optimization, and biological evaluation of 3-O-ß-chacotriosyl ursolic acid derivatives as novel SARS-CoV-2 entry inhibitors by targeting the prefusion state of spike protein.

The COVID-19 pandemic generates a global threat to public health and continuously emerging SARS-CoV-2 variants bring a great challenge to the development of both vaccines and antiviral agents. In this study, we identified UA-18 and its optimized analog UA-30 via the hit-to-lead strategy as novel SARS-CoV-2 fusion inhibitors. The lead compound UA-30 showed potent antiviral activity against infectious SARS-CoV-2 (wuhan-HU-1 variant) in Vero-E6 cells and was also effective against infection of diverse pseudotyped SARS-CoV-2 variants with mutations in the S protein including the Omicron and Delta variants. More importantly, UA-30 might target the cavity between S1 and S2 subunits to stabilize the prefusion state of the SARS-CoV-2 S protein, thus leading to interfering with virus-cell membrane fusion. This study offers a set of novel SARS-CoV-2 fusion inhibitors against SARS-CoV-2 and its variants based on the 3-O-ß-chacotriosyl UA skeleton.

CoVac501, a self-adjuvanting peptide vaccine conjugated with TLR7 agonists, against SARS-CoV-2 induces protective immunity.

Safe, effective, and economical vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to achieve adequate herd immunity and end the pandemic. We constructed a novel SARS-CoV-2 vaccine, CoVac501, which is a self-adjuvanting peptide vaccine conjugated with Toll-like receptor 7 (TLR7) agonists. The vaccine contains immunodominant peptides screened from the receptor-binding domain (RBD) and is fully chemically synthesized. It has been formulated in an optimized nanoemulsion formulation and is stable at 40 °C for 1 month. In non-human primates (NHPs), CoVac501 elicited high and persistent titers of protective neutralizing antibodies against multiple RBD mutations, SARS-CoV-2 original strain, and variants (B.1.1.7 and B.1.617.2). Specific peptides booster immunization against the B.1.351 variant has also been shown to be effective in improving protection against B.1.351. Meanwhile, CoVac501 elicited the increase of memory T cells, antigen-specific CD8+ T-cell responses, and Th1-biased CD4+ T-cell immune responses in NHPs. Notably, at an extremely high SARS-CoV-2 challenge dose of 1 × 107 TCID50, CoVac501 provided near-complete protection for the upper and lower respiratory tracts of cynomolgus macaques.

A pathogen-like antigen-based vaccine confers immune protection against SARS-CoV-2 in non-human primates.

Activation of nucleic acid sensing Toll-like receptors (TLRs) in B cells is involved in antiviral responses by promoting B cell activation and germinal center responses. In order to take advantage of this natural pathway for vaccine development, synthetic pathogen-like antigens (PLAs) constructed of multivalent antigens with encapsulated TLR ligands can be used to activate B cell antigen receptors and TLRs in a synergistic manner. Here we report a PLA-based coronavirus disease 2019 (COVID-19) vaccine candidate designed by combining a phage-derived virus-like particle carrying bacterial RNA as TLR ligands with the receptor-binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein as the target antigen. This PLA-based vaccine candidate induces robust neutralizing antibodies in both mice and non-human primates (NHPs). Using a NHP infection model, we demonstrate that the viral clearance is accelerated in vaccinated animals. In addition, the PLA-based vaccine induces a T helper 1 (Th1)-oriented response and a durable memory, supporting its potential for further clinical development.

RBD-homodimer, a COVID-19 subunit vaccine candidate, elicits immunogenicity and protection in rodents and nonhuman primates.

The pandemic of COVID-19 caused by SARS-CoV-2 has raised a new challenges to the scientific and industrious fields after over 1-year spread across different countries. The ultimate approach to end the pandemic is the timely application of vaccines to achieve herd immunity. Here, a novel SARS-CoV-2 receptor-binding domain (RBD) homodimer was developed as a SARS-CoV-2 vaccine candidate. Formulated with aluminum adjuvant, RBD dimer elicited strong immune response in both rodents and non-human primates, and protected mice from SARS-CoV-2 challenge with significantly reducing viral load and alleviating pathological injury in the lung. In the non-human primates, the vaccine could prevent majority of the animals from SARS-CoV-2 infection in the respiratory tract and reduce lung damage. In addition, antibodies elicited by this vaccine candidate showed cross-neutralization activities to SARS-CoV-2 variants. Furthermore, with our expression system, we provided a high-yield RBD homodimer vaccine without additional biosafety or special transport device supports. Thus, it may serve as a safe, effective, and low-cost SARS-CoV-2 vaccine candidate.

SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target.

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.

Danshensu alleviates pseudo-typed SARS-CoV-2 induced mouse acute lung inflammation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can induce acute inflammatory response like acute lung inflammation (ALI) or acute respiratory distress syndrome, leading to severe progression and mortality. Therapeutics for treatment of SARS-CoV-2-triggered respiratory inflammation are urgent to be discovered. Our previous study shows that Salvianolic acid C potently inhibits SARS-CoV-2 infection. In this study, we investigated the antiviral effects of a Salvia miltiorrhiza compound, Danshensu, in vitro and in vivo, including the mechanism of S protein-mediated virus attachment and entry into target cells. In authentic and pseudo-typed virus assays in vitro, Danshensu displayed a potent antiviral activity against SARS-CoV-2 with EC50 of 0.97 µM, and potently inhibited the entry of SARS-CoV-2 S protein-pseudo-typed virus (SARS-CoV-2 S) into ACE2-overexpressed HEK-293T cells (IC50 = 0.31 µM) and Vero-E6 cell (IC50 = 4.97 µM). Mice received SARS-CoV-2 S via trachea to induce ALI, while the VSV-G treated mice served as controls. The mice were administered Danshensu (25, 50, 100 mg/kg, i.v., once) or Danshensu (25, 50, 100 mg·kg-1·d-1, oral administration, for 7 days) before SARS-CoV-2 S infection. We showed that SARS-CoV-2 S infection induced severe inflammatory cell infiltration, severely damaged lung tissue structure, highly expressed levels of inflammatory cytokines, and activated TLR4 and hyperphosphorylation of the NF-κB p65; the high expression of angiotensinogen (AGT) and low expression of ACE2 at the mRNA level in the lung tissue were also observed. Both oral and intravenous pretreatment with Danshensu dose-dependently alleviated the pathological alterations in mice infected with SARS-CoV-2 S. This study not only establishes a mouse model of pseudo-typed SARS-CoV-2 (SARS-CoV-2 S) induced ALI, but also demonstrates that Danshensu is a potential treatment for COVID-19 patients to inhibit the lung inflammatory response.

High-throughput screening identifies established drugs as SARS-CoV-2 PLpro inhibitors.

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (Mpro), PLpro is responsible for processing the viral replicase polyprotein into functional units. Therefore, it is an attractive target for antiviral drug development. Here we discovered four compounds, YM155, cryptotanshinone, tanshinone I and GRL0617 that inhibit SARS-CoV-2 PLpro with IC50 values ranging from 1.39 to 5.63 µmol/L. These compounds also exhibit strong antiviral activities in cell-based assays. YM155, an anticancer drug candidate in clinical trials, has the most potent antiviral activity with an EC50 value of 170 nmol/L. In addition, we have determined the crystal structures of this enzyme and its complex with YM155, revealing a unique binding mode. YM155 simultaneously targets three "hot" spots on PLpro, including the substrate-binding pocket, the interferon stimulating gene product 15 (ISG15) binding site and zinc finger motif. Our results demonstrate the efficacy of this screening and repurposing strategy, which has led to the discovery of new drug leads with clinical potential for COVID-19 treatments.

Antiviral Bafilomycins from a Feces-Inhabiting Streptomyces sp.

A new bafilomycin derivative (1) and another seven known bafilomycins (2-8) were isolated from feces-derived Streptomyces sp. HTL16. The structure of 1 was elucidated by 1D and 2D NMR spectroscopic analysis. Biological testing demonstrated that these bafilomycins exhibited potent antiviral activities against the influenza A and SARS-CoV-2 viruses, with IC50 values in the nanomolar range, by inhibiting the activity of endosomal ATP-driven proton pumps.

Drug Repurposing of Itraconazole and Estradiol Benzoate against COVID-19 by Blocking SARS-CoV-2 Spike Protein-Mediated Membrane Fusion.

SARS-CoV-2 caused the emerging epidemic of coronavirus disease in 2019 (COVID-19). To date, there are more than 82.9 million confirmed cases worldwide, there is no clinically effective drug against SARS-CoV-2 infection. The conserved properties of the membrane fusion domain of the spike (S) protein across SARS-CoV-2 make it a promising target to develop pan-CoV therapeutics. Herein, two clinically approved drugs, Itraconazole (ITZ) and Estradiol benzoate (EB), are found to inhibit viral entry by targeting the six-helix (6-HB) fusion core of SARS-CoV-2 S protein. Further studies shed light on the mechanism that ITZ and EB can interact with the heptad repeat 1 (HR1) region of the spike protein, to present anti-SARS-CoV-2 infections in vitro, indicating they are novel potential therapeutic remedies for COVID-19 treatment. Furthermore, ITZ shows broad-spectrum activity targeting 6-HB in the S2 subunit of SARS-CoV and MERS-CoV S protein, inspiring that ITZ have the potential for development as a pan-coronavirus fusion inhibitor.

Axial Chiral Binaphthoquinone and Perylenequinones from the Stromata of Hypocrella bambusae Are SARS-CoV-2 Entry Inhibitors.

A new axial chiral binaphtoquinone, hypocrellone (1), and a new perylenequinone, hypomycin F (2), were isolated from the stromata of Hypocrella bambusae, together with five known compounds, 3-7. The structures of 1 and 2 were assigned by spectroscopic and HRESIMS data analyses. The axial chirality of 1 was determined by electronic circular dichroism data analysis, and the absolute configurations of 2 and 3 were determined by X-ray crystallography. The axial chirality of 7 was determined by UV-induced photooxidation from 4. Compounds 1, 4, and 5 showed inhibitory activity against pseudotyped SARS-CoV-2 infection in 293T-ACE2 cells with IC50 values of 0.17, 0.038, and 0.12 µM. Compounds 4 and 5 were also active against live SARS-CoV-2 infection with EC50 values of 0.22 and 0.21 µM, respectively. Further cell-cell fusion assays, surface plasmon resonance assays, and molecular docking studies revealed that 4 and 5 could bind with the receptor-binding domain of SARS-CoV-2 S protein to prevent its interaction with human angiotensin-converting enzyme II receptor. Our results revealed that 4 and 5 are potential SARS-CoV-2 entry inhibitors.

SARS-CoV-2 ORF9b inhibits RIG-I-MAVS antiviral signaling by interrupting K63-linked ubiquitination of NEMO.

Coronavirus disease 2019 (COVID-19) is a current global health threat caused by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Emerging evidence indicates that SARS-CoV-2 elicits a dysregulated immune response and a delayed interferon (IFN) expression in patients, which contribute largely to the viral pathogenesis and development of COVID-19. However, underlying mechanisms remain to be elucidated. Here, we report the activation and repression of the innate immune response by SARS-CoV-2. We show that SARS-CoV-2 RNA activates the RIG-I-MAVS-dependent IFN signaling pathway. We further uncover that ORF9b immediately accumulates and antagonizes the antiviral type I IFN response during SARS-CoV-2 infection on primary human pulmonary alveolar epithelial cells. ORF9b targets the nuclear factor κB (NF-κB) essential modulator NEMO and interrupts its K63-linked polyubiquitination upon viral stimulation, thereby inhibiting the canonical IκB kinase alpha (IKKα)/ß/γ-NF-κB signaling and subsequent IFN production. Our findings thus unveil the innate immunosuppression by ORF9b and provide insights into the host-virus interplay during the early stage of SARS-CoV-2 infection.

Broad-spectrum antivirals of protoporphyrins inhibit the entry of highly pathogenic emerging viruses.

Severe emerging and re-emerging viral infections such as Lassa fever, Avian influenza (AI), and COVID-19 caused by SARS-CoV-2 urgently call for new strategies for the development of broad-spectrum antivirals targeting conserved components in the virus life cycle. Viral lipids are essential components, and viral-cell membrane fusion is the required entry step for most unrelated enveloped viruses. In this paper, we identified a porphyrin derivative of protoporphyrin IX (PPIX) that showed broad antiviral activities in vitro against a panel of enveloped pathogenic viruses including Lassa virus (LASV), Machupo virus (MACV), and SARS-CoV-2 as well as various subtypes of influenza A viral strains with IC50 values ranging from 0.91 ± 0.25 µM to 1.88 ± 0.34 µM. A mechanistic study using influenza A/Puerto Rico/8/34 (H1N1) as a testing strain showed that PPIX inhibits the infection in the early stage of virus entry through biophysically interacting with the hydrophobic lipids of enveloped virions, thereby inhibiting the entry of enveloped viruses into host cells. In addition, the preliminary antiviral activities of PPIX were further assessed by testing mice infected with the influenza A/Puerto Rico/8/34 (H1N1) virus. The results showed that compared with the control group without drug treatment, the survival rate and mean survival time of the mice treated with PPIX were apparently prolonged. These data encourage us to conduct further investigations using PPIX as a lead compound for the rational design of lipid-targeting antivirals for the treatment of infection with enveloped viruses.