Computational and experimental validation of phthalocyanine and hypericin as effective SARS-CoV-2 fusion inhibitors.

Phthalocyanine and hypericin have been previously identified as possible SARS-CoV-2 Spike glycoprotein fusion inhibitors through a virtual screening procedure. In this paper, atomistic simulations of metal-free phthalocyanines and atomistic and coarse-grained simulations of hypericins, placed around a complete model of the Spike embedded in a viral membrane, allowed to further explore their multi-target inhibitory potential, uncovering their binding to key protein functional regions and their propensity to insert in the membrane. Following computational results, pre-treatment of a pseudovirus expressing the SARS-CoV-2 Spike protein with low compounds concentrations resulted in a strong inhibition of its entry into cells, suggesting the activity of these molecules should involve the direct targeting of the viral envelope surface. The combination of computational and in vitro results hence supports the role of hypericin and phthalocyanine as promising SARS-CoV-2 entry inhibitors, further endorsed by literature reporting the efficacy of these compounds in inhibiting SARS-CoV-2 activity and in treating hospitalized COVID-19 patients.Communicated by Ramaswamy H. Sarma.

Antiviral Therapy of COVID-19.

Since the beginning of the COVID-19 pandemic, the scientific community has focused on prophylactic vaccine development. In parallel, the experience of the pharmacotherapy of this disease has increased. Due to the declining protective capacity of vaccines against new strains, as well as increased knowledge about the structure and biology of the pathogen, control of the disease has shifted to the focus of antiviral drug development over the past year. Clinical data on safety and efficacy of antivirals acting at various stages of the virus life cycle has been published. In this review, we summarize mechanisms and clinical efficacy of antiviral therapy of COVID-19 with drugs based on plasma of convalescents, monoclonal antibodies, interferons, fusion inhibitors, nucleoside analogs, and protease inhibitors. The current status of the drugs described is also summarized in relation to the official clinical guidelines for the treatment of COVID-19. In addition, here we describe innovative drugs whose antiviral effect is provided by antisense oligonucleotides targeting the SARS-CoV-2 genome. Analysis of laboratory and clinical data suggests that current antivirals successfully combat broad spectra of emerging strains of SARS-CoV-2 providing reliable defense against COVID-19.

Design and characterization of novel SARS-CoV-2 fusion inhibitors with N-terminally extended HR2 peptides.

Development of potent and broad-spectrum antivirals against SARS-CoV-2 remains one of top priorities, especially in the case of that current vaccines cannot effectively prevent viral transmission. We previously generated a group of fusion-inhibitory lipopeptides, with one formulation being evaluated under clinical trials. In this study, we dedicated to characterize the extended N-terminal motif (residues 1161-1168) of the so-called spike (S) heptad repeat 2 (HR2) region. Alanine scanning analysis of this motif verified its critical roles in S protein-mediated cell-cell fusion. Using a panel of HR2 peptides with the N-terminal extensions, we identified a peptide termed P40, which contained four extended N-terminal residues (VDLG) and exhibited improved binding and antiviral activities, whereas the peptides with further extensions had no such effects. Then, we developed a new lipopeptide P40-LP by modifying P40 with cholesterol, which exhibited dramatically increased activities in inhibiting SARS-CoV-2 variants including divergent Omicron sublineages. Moreover, P40-LP displayed a synergistic effect with IPB24 lipopeptide that was designed containing the C-terminally extended residues, and it could effectively inhibit other human coronaviruses, including SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63. Taken together, our results have provided valuable insights for understanding the structure-function relationship of SARS-CoV-2 fusion protein and offered novel antiviral strategies to fight against the COVID-19 pandemic.

Cyclic lipopeptides as membrane fusion inhibitors against SARS-CoV-2: New tricks for old dogs.

With the resurgence of the coronavirus pandemic, the repositioning of FDA-approved drugs against coronovirus and finding alternative strategies for antiviral therapy are both important. We previously identified the viral lipid envelope as a potential target for the prevention and treatment of SARS-CoV-2 infection with plant alkaloids (Shekunov et al., 2021). Here, we investigated the effects of eleven cyclic lipopeptides (CLPs), including well-known antifungal and antibacterial compounds, on the liposome fusion triggered by calcium, polyethylene glycol 8000, and a fragment of SARS-CoV-2 fusion peptide (816-827) by calcein release assays. Differential scanning microcalorimetry of the gel-to-liquid-crystalline and lamellar-to-inverted hexagonal phase transitions and confocal fluorescence microscopy demonstrated the relation of the fusion inhibitory effects of CLPs to alterations in lipid packing, membrane curvature stress and domain organization. The antiviral effects of CLPs were evaluated in an in vitro Vero-based cell model, and aculeacin A, anidulafugin, iturin A, and mycosubtilin attenuated the cytopathogenicity of SARS-CoV-2 without specific toxicity.

5-(Perylen-3-ylethynyl)uracil as an antiviral scaffold: Potent suppression of enveloped virus reproduction by 3-methyl derivatives in vitro.

Amphipathic nucleoside and non-nucleoside derivatives of pentacyclic aromatic hydrocarbon perylene are known as potent non-cytotoxic broad-spectrum antivirals. Here we report 3-methyl-5-(perylen-3-ylethynyl)-uracil-1-acetic acid and its amides, a new series of compounds based on a 5-(perylen-3-ylethynyl)-uracil scaffold. The compounds demonstrate pronounced in vitro activity against arthropod-borne viruses, namely tick-borne encephalitis virus (TBEV) and yellow fever virus (YFV), in plaque reduction assays with EC50 values below 1.9 and 1.3 nM, respectively, and Chikungunya virus (CHIKV) in cytopathic effect inhibition test with EC50 values below 3.2 µM. The compounds are active against respiratory viruses as well: severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) in cytopathic effect inhibition test and influenza A virus (IAV) in virus titer reduction experiments are inhibited - EC50 values below 51 nM and 2.2 µM, respectively. The activity stems from the presence of a hydrophobic perylene core, and all of the synthesized compounds exhibit comparable 1O2 generation rates. Nonetheless, activity can vary by orders of magnitude depending on the hydrophilic part of the molecule, suggesting a complex mode of action. A time-of-addition experiment and fluorescent imaging indicate that the compounds inhibit viral fusion in a dose-dependent manner. The localization of the compound in the lipid bilayers and visible damage to the viral envelope suggest the membrane as the primary target. Dramatic reduction of antiviral activity with limited irradiation or under treatment with antioxidants further cements the idea of photoinduced ROS-mediated viral envelope damage being the mode of antiviral action.

IN SILICO SCREENING OF THE POTENTIAL SARS-CoV-2 INHIBITORS BLOCKING THE HR1 TRIMER OF THE CORONAVIRUS PROTEIN S

A virtual library of biologically active molecules has been formed and in silico screening has been carried out for identification of small-molecule chemical compounds - potential SARS-CoV-2 inhibitors able to bind to the HR1 trimer of the protein S and to block the formation of a six-helix bundle 6-HB, which is critical for the virus-cell fusion and viral in-fectivity. Molecular modeling methods were used to evaluate the binding affinity of the identified compounds to the HR1 trimer of the protein S. As a result, 12 molecules exhibiting the high binding affinity to this functionally important region of the virus were found. The data obtained indicate the promise of using these compounds in the development of new antiviral drugs presenting SARS-CoV-2 fusion inhibitors that can block the virus entry into the host cell.

Comparison of Cell Fusions Induced by Influenza Virus and SARS-CoV-2.

Virus-cell fusion is the key step for viral infection in host cells. Studies on virus binding and fusion with host cells are important for understanding the virus-host interaction and viral pathogenesis for the discovery of antiviral drugs. In this review, we focus on the virus-cell fusions induced by the two major pandemic viruses, including the influenza virus and SARS-CoV-2. We further compare the cell fusions induced by the influenza virus and SARS-CoV-2, especially the pH-dependent fusion of the influenza virus and the fusion of SARS-CoV-2 in the type-II transmembrane serine protease 2 negative (TMPRSS2-) cells with syncytia formation. Finally, we present the development of drugs used against SARA-CoV-2 and the influenza virus through the discovery of anti-fusion drugs and the prevention of pandemic respiratory viruses.

Sensitivity to Vaccines, Therapeutic Antibodies, and Viral Entry Inhibitors and Advances To Counter the SARS-CoV-2 Omicron Variant.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) keeps evolving and mutating into newer variants over time, which gain higher transmissibility, disease severity, and spread in communities at a faster rate, resulting in multiple waves of surge in Coronavirus Disease 2019 (COVID-19) cases. A highly mutated and transmissible SARS-CoV-2 Omicron variant has recently emerged, driving the extremely high peak of infections in almost all continents at an unprecedented speed and scale. The Omicron variant evades the protection rendered by vaccine-induced antibodies and natural infection, as well as overpowers the antibody-based immunotherapies, raising the concerns of current effectiveness of available vaccines and monoclonal antibody-based therapies. This review outlines the most recent advancements in studying the virology and biology of the Omicron variant, highlighting its increased resistance to current antibody-based therapeutics and its immune escape against vaccines. However, the Omicron variant is highly sensitive to viral fusion inhibitors targeting the HR1 motif in the spike protein, enzyme inhibitors, involving the endosomal fusion pathway, and ACE2-based entry inhibitors. Omicron variant-associated infectivity and entry mechanisms of Omicron variant are essentially distinct from previous characterized variants. Innate sensing and immune evasion of SARS-CoV-2 and T cell immunity to the virus provide new perspectives of vaccine and drug development. These findings are important for understanding SARS-CoV-2 viral biology and advances in developing vaccines, antibody-based therapies, and more effective strategies to mitigate the transmission of the Omicron variant or the next SARS-CoV-2 variant of concern.

Parainfluenza virus entry at the onset of infection.

Parainfluenza viruses, members of the enveloped, negative-sense, single stranded RNA Paramyxoviridae family, impact global child health as the cause of significant lower respiratory tract infections. Parainfluenza viruses enter cells by fusing directly at the cell surface membrane. How this fusion occurs via the coordinated efforts of the two molecules that comprise the viral surface fusion complex, and how these efforts may be blocked, are the subjects of this chapter. The receptor binding protein of parainfluenza forms a complex with the fusion protein of the virus, remaining stably associated until a receptor is reached. At that point, the receptor binding protein actively triggers the fusion protein to undergo a series of transitions that ultimately lead to membrane fusion and viral entry. In recent years it has become possible to examine this remarkable process on the surface of viral particles and to begin to understand the steps in the transition of this molecular machine, using a structural biology approach. Understanding the steps in entry leads to several possible strategies to prevent fusion and inhibit infection.

Discovery of New Potent anti-MERS CoV Fusion Inhibitors.

Middle East respiratory syndrome coronavirus (MERS-CoV), capable of zoonotic transmission, has been associated with emerging viral pneumonia in humans. In this study, a set of highly potent peptides were designed to prevent MERS-CoV fusion through competition with heptad repeat domain 2 (HR2) at its HR1 binding site. We designed eleven peptides with stronger estimated HR1 binding affinities than the wild-type peptide to prevent viral fusion with the cell membrane. Eight peptides showed strong inhibition of spike-mediated MERS-CoV cell-cell fusion with IC50 values in the nanomolar range (0.25-2.3 µM). Peptides #4-6 inhibited 95-98.3% of MERS-CoV plaque formation. Notably, peptide four showed strong inhibition of MERS-CoV plaques formation with EC50 = 0.302 µM. All peptides demonstrated safe profiles without cytotoxicity up to a concentration of 10 µM, and this cellular safety, combined with their anti-MERS-CoV antiviral activity, indicate all peptides can be regarded as potential promising antiviral agents.

Repurposing of anticancer phytochemicals for identifying potential fusion inhibitor for SARS-CoV-2 using molecular docking and molecular dynamics (MD) simulations.

The viral particle, SARS-CoV-2 is responsible for causing the epidemic of Coronavirus disease 2019 (COVID-19). To combat this situation, numerous strategies are being thought for either creating its antidote, vaccine, or agents that can prevent its infection. For enabling research on these strategies, several target proteins are identified where, Spike (S) protein is of great potential. S-protein interacts with human angiotensin-converting-enzyme-2 (ACE2) for entering the cell. S-protein is a large protein and a portion of it designated as a receptor-binding domain (RBD) is the key region that interacts with ACE2, following to which the viral membrane fuses with the alveolar membrane to enter the human cell. The hypothesis is to identify molecules from the pool of anticancer phytochemicals as a lead possessing the ability to interact and mask the amino acids of RBD, making them unavailable to form associations with ACE2. Such a molecule is termed as 'fusion inhibitor'. We hypothesized to identify fusion inhibitors from the NPACT library of anticancer phytochemicals. For this, all the molecules from the NPACT were screened using molecular docking, the five top hits (Theaflavin, Ginkgetin, Ursolic acid, Silymarin and Spirosolane) were analyzed for essential Pharmacophore features and their ADMET profiles were studied following to which the best two hits were further analyzed for their interaction with RBD using Molecular Dynamics (MD) simulation. Binding free energy calculations were performed using MM/GBSA, proving these phytochemicals containing anticancer properties to serve as fusion inhibitors.Communicated by Ramaswamy H. Sarma.

Discovery of New Fusion Inhibitor Peptides against SARS-CoV-2 by Targeting the Spike S2 Subunit.

A novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), caused a worldwide pandemic. Our aim in this study is to produce new fusion inhibitors against SARS-CoV-2, which can be the basis for developing new antiviral drugs. The fusion core comprising the heptad repeat domains (HR1 and HR2) of SARS-CoV-2 spike (S) were used to design the peptides. A total of twelve peptides were generated, comprising a short or truncated 24-mer (peptide #1), a long 36-mer peptide (peptide #2), and ten peptide #2 analogs. In contrast to SARS-CoV, SARS-CoV-2 S-mediated cell-cell fusion cannot be inhibited with a minimal length, 24-mer peptide. Peptide #2 demonstrated potent inhibition of SARS-CoV-2 S-mediated cell-cell fusion at 1 µM concentration. Three peptide #2 analogs showed IC50 values in the low micromolar range (4.7-9.8 µM). Peptide #2 inhibited the SARS-CoV-2 pseudovirus assay at IC50=1.49 µM. Given their potent inhibition of viral activity and safety and lack of cytotoxicity, these peptides provide an attractive avenue for the development of new prophylactic and therapeutic agents against SARS-CoV-2.

Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19.

Coronavirus disease 2019 is a newly emerging infectious disease currently spreading across the world. It is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike (S) protein of SARS-CoV-2, which plays a key role in the receptor recognition and cell membrane fusion process, is composed of two subunits, S1 and S2. The S1 subunit contains a receptor-binding domain that recognizes and binds to the host receptor angiotensin-converting enzyme 2, while the S2 subunit mediates viral cell membrane fusion by forming a six-helical bundle via the two-heptad repeat domain. In this review, we highlight recent research advance in the structure, function and development of antivirus drugs targeting the S protein.

Targeting the SARS-CoV-2 spike glycoprotein prefusion conformation: virtual screening and molecular dynamics simulations applied to the identification of potential fusion inhibitors.

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a renewed interest in studying the role of the spike S glycoprotein in regulating coronavirus infections in the natural host. Taking advantage of the cryo-electron microscopy structure of SARS-CoV-2 S trimer in the prefusion conformation, we performed a virtual screening simulation with the aim to identify novel molecules that could be used as fusion inhibitors. The spike glycoprotein structure has been completed using modeling techniques and its inner cavity, needful for the postfusion transition of the trimer, has been scanned for the identification of strongly interacting available drugs. Finally, the stability of the protein-drug top complexes has been tested using classical molecular dynamics simulations. The free energy of interaction of the molecules to the spike protein has been evaluated through the MM/GBSA method and per-residue decomposition analysis. Results have been critically discussed considering previous scientific knowledge concerning the selected compounds and sequence alignments have been carried out to evaluate the spike glycoprotein similarity among the betacoronavirus family members. Finally, a cocktail of drugs that may be used as SARS-CoV-2 fusion inhibitors has been suggested.