key: cord-1023459-z7a3mvex authors: Mondal, Samiran; Karmakar, Abhijit; Mallick, Tamanna; Begum, Naznin Ara title: Exploring the efficacy of naturally occurring biflavone based antioxidants towards the inhibition of the SARS-CoV-2 spike glycoprotein mediated membrane fusion date: 2021-02-04 journal: Virology DOI: 10.1016/j.virol.2021.01.015 sha: c7193251658643407d08b3bc7a387d110234f160 doc_id: 1023459 cord_uid: z7a3mvex Molecular docking studies were done to show the inhibitory effect of two naturally occurring biflavone based anti-HIV agents, hinokiflavone and robustaflavone against the SARS-CoV-2 spike (S) protein mediated attack on the human ACE2 receptors via membrane fusion mechanism. Nefamostat, a FDA approved drug, well-known as a serine protease inhibitor for MERS-CoV infection, was used as the reference compound. Both the biflavones, showed potential as inhibitors for SARS-CoV-2 S protein-mediated viral entry. The binding affinities of these naturally occurring biflavones for RBD-S2 subunit protein of SARS-CoV-2 were explored for the first time. Such binding affinities play a critical role in the virus-cell membrane fusion process. These biflavones are able to interact more strongly with the residues of heptad repeat 1 and 2 (HR1 and HR2) regions of S2 protein of SARS-CoV-2 compared to nefamostat, and thus, these biflavones can effectively block the formation of six-helix bundle core fusion structure (6-HB) leading to the inhibition of virus-target cell-membrane fusion. By the end of 2019, scientists came to know about a novel Corona virus, SARS-CoV-2 [Severe Acute Respiratory Syndrome-Corona virus-2] causing COVID-19 (Corona Virus . This initially affected people of Wuhan city of China. Later, this virus became the root cause of deaths and untold sufferings of millions of people around the globe due to the unavailability of specific medicine/vaccine or therapeutic strategies. Corona viruses (CoVs) are a family of RNA viruses, responsible for mild as well as a range of severe respiratory disease outbreaks and epidemics in human in last two decades e.g. Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) [1, 2, 3, 4a, 4b, 4c ]. Like, SARS-CoV and MERS-CoV, the very deadly SARS-CoV-2 belongs to β genus of CoVs containing positive-strand RNA [5] . The size of the genome of SARS-CoV-2 falls in the range of ~30 kb involving 6 to 11 open ring frames (ORFs) [6] . Approximately, 67% of the entire genome is mainly located in the first ORF (ORF1a/ORF1b) which processes two polyproteins, pp1a and pp1ab and also encodes [16] [17] non-structural proteins (NSPs) e.g. 3-chymotrypsin-like protease (3CL pro ), papain-like protease (PL pro ), helicase and RNA-dependent RNA polymerase (RdRp) [7] . The remaining and pathogenesis [4a, 9] . The binding of viral S protein through its receptor-binding domain (RBD) to the host cells instigates various vital steps necessary for viral infections e.g. fusion of viral and host membranes [10] [11] . The S proteins attacks the angiotensin-converting enzyme2 (ACE2) receptors of the host via its RBD and triggers a cascade of inflammation in the lower respiratory tract [12] [13] . Trimeric spike (S) glycoprotein is comprised of two functional subunits proteins, among them S1 subunit is responsible for binding to the host cell ACE2 receptors and induces concomitant changes in the conformation in the S2 subunit; which in turn, facilitates the infection of human cells via membrane fusion mechanism (Scheme 1) [14] . Recent structural and biophysical data showed the evidence of the binding affinity of SARS-CoV-2 S protein with ACE2 receptors of host cells [15] [16] . Furthermore, such effect is much more pronounced in case of SARS-CoV-2 S protein. Because the binding affinity of S1 subunit of SARS-CoV-2 is higher than that of the SARS-CoV. This is attributed to the higher infectivity of novel SARS-CoV-2 compared to SARS-CoV [15] [16] . Comparative analysis of spike (S) glycoprotein by protein sequence alignment of SARS-CoV-2 with SARS-CoV shows 76% of sequence identity [Scheme 1(b)] [17] [18] . Therefore, to develop specific SARS-CoV-2 fusion inhibitors, it is very much necessary to study the fusion capacity of SARS-CoV-2 compared to that of SARS-CoV. As an alternate strategy, various research groups target the viral S protein for the inhibition of the membrane fusion and entry processes of SARS-CoV-2 in host cells with ACE2 receptors [7, 19] . Heptad repeat 1 (HR1) and 2 (HR2) domains of S2 subunit play a crucial task in the SARS-CoV fusion with target cells (Scheme 1). Upon binding of S protein through RBD in S1 to the ACE2 receptor on the target cell, HR1 and HR2 domains combine to form a six-helix bundle core fusion structure (6-HB) and bring the viral envelop and the cellular membranes into close proximity; necessary for effective fusion and infection [20] . Therefore, FDA J o u r n a l P r e -p r o o f approved anti-viral drugs target the HR1 and HR2 regions in the S2 subunit domains and such drugs are now being extensively explored as the potential therapeutic option for COVID-19. Identification of the genome sequence, 3D-structure and mechanism of action/pathogenesis of SARS-CoV-2 is necessary for developing effective treatment strategies to combat COVID-19 [2-4, 8, 21-23] . One of such therapeutic strategies targets the main protease (M pro ) of SARS-CoV-2 i.e. 3CL pro , having high genomic sequence similarity with SARS-CoV and plays a crucial role in COVID-19 pathogenesis. In this direction, a large number of U.S. Food and Drug Administration (FDA) approved protease inhibitors (showing efficacy in case of SERS, MERS and HIV) are put into trials [7, [24] [25] [26] [27] [28] [29] . In this connection, it is worth to mention the efficacy of nefamostat (1), a serine protease inhibitor [ Figure 1 ] which is a FDA approved drug for the treatment of cystic fibrosis and acute pancreatitis [30] . During the invitro screening (with the help of Dual Split Protein (DSP) reporter fusion assay) of more than 1000 FDA-approved drugs to find out effective drug to combat MERS disease, it was observed that compound (1) has the potential to inhibit effectively the MERS-CoV S protein initiated membrane fusion and this study also proposed that (1) could be an effective candidate for inhibiting MERS-CoV infection [30] . However, despite having potentials, such protease inhibitors are not extensively explored for their inhibitory activity towards the SARS-CoV-2 S protein triggered membrane fusion and related infections. In this connection, plants sources and their active components used in traditional Chinese medicine and having antiviral activity are also being extensively explored in China [7, 24, [31] [32] [33] [34] . Medicinal plants are the largest and the best combinational libraries of natural products. Although many drugs are made by synthetic chemistry but most of the core structures or scaffolds for synthetic drugs are based upon the natural products which are used in traditional medicines [35] [36] [37] . showed efficacy as RNA polymerase (DV-NS5 RdRp) inhibitors of dengue 2 virus; in addition to this, the HIV-1 inhibitory activity of hinokiflavone is also reported [49] [50] [51] . On the other hand, robustaflavone (4) showed viral inhibitory activity against various viral species e.g. Hepatitis B virus (HBV) and HIV-1 [47] [48] [49] . However, in spite of the potent viral J o u r n a l P r e -p r o o f inhibitory activity of (3) and (4) towards other viral species, these biflavones were not explored for their inhibitory activity towards the SARS-CoV-2 S protein triggered membrane fusion and related COVID-19 infections. This prompted us to perform the present study involving the biflavones, hinokiflavone (3) and robustaflavone (4) [ Figure 1 ]; both of them showed similar inhibitory activity against HIV-1 reverse transcriptase (RT) and thus, these were identified as anti-HIV agents of natural product origin [49] . The inhibitory effect of these two plant derived anti-HIV or anti-viral biflavones, (3) and (4) against the SARS-CoV-2 S-mediated membrane fusion was explored with the help of molecular docking studies. Binding interactions of these compounds with RBD of S protein of SARS-CoV-2 have been studied. Such interactions are very much necessary to prohibit the exposure of RBD-S of SARS-CoV-2 towards ACE2 receptors of host cells, which is a key step for preventing viral infection initiation. In this study, nefamostat (1), showing potential towards inhibiting the MERS-CoV S protein initiated membrane fusion, was employed as the reference compound so that we can make a comparative assessment of (3) and (4) with a known RNA virus inhibitor, (1). Our molecular docking study suggests that the biflavones, Molecular docking studies of biflavones, hinokiflavone (3) and robustaflavone (4) along with a known RNA virus inhibitor, nefamostat (1) with spike (S) glycoproten of SARS-CoV-2 were done to understand binding interactions of these ligands towards the RBD-S of this deadly virus and showing their potentials as anti-viral therapeutic agents for SARS-CoV-2. As shown in Scheme 1(b), its S1 subunit (14- Table 1 -5. Strength and the affinity of a specific ligand, which binds to the pocket of a target protein, can be expressed in terms of binding energy (ΔG) and the ligand having lower binding energy is preferred as a potential drug candidate. This parameter was compared for each of the test compounds in order to understand their effectiveness as the active antiviral agents for SARS-CoV-2 mediated attack on human ACE2 receptor cells. We have performed molecular docking studies with several FDA approved antiviral drugs of natural product origin using AutoDock Vina tools on the basis of their highest binding affinity with S2 domain of the spike protein of SARS-CoV-2 and these three compounds (1), (3) and (4) were selected for the present work. Nefamostat (1) was considered as the reference compound, which exhibited docked score of binding energy (ΔG) as -8.4 kcal mol -1 with SARS-CoV-2 S2 protein. Figure 2 shows the SARS-CoV-2 S2 protein docked structures of (1) along with the residues of the protein with which the molecule (1) interact. Selected bond lengths of SARS-CoV-2 S2 protein-(1) docked structure are shown in Table 1 . These results reveal that SARS-CoV-2 S2 protein interacts with (1) mostly via its aromatic amino acid residues e.g. Despite of having similar preferences for the S2 domain, the presence of additional -OH group on the chromone ring of (3) and (4) affected the extent of their hydrogen bonding interactions compared to (1) (Figure 1) . This has been reflected in ∆G value of (1) indicating its lowest binding affinity for the binding site of SARS-CoV-2 S2. Moreover, these results strengthen our observation that the selected biflavones, (3) and (4), can interact strongly with the residues of the HR1 and HR2 regions of S2 protein of SARS-CoV-2, thereby they can be as active as to block the formation of six-helix bundle core fusion structure (6-HB). This ultimately prompts the inhibition of the fusion between the virus and target cell membranes. All the naturally occurring biflavones used in this study satisfied the Lipinski's rule of five that mainly determines the prime requisition of a compound to be a potential drug on the basis of its different molecular properties e.g. absorption, distribution, metabolism and excretion (ADME) [58] . The ADME characteristics of our selected drug candidates i.e. (1, 3 and 4) shown in Table 5 , indicates the fulfillment of Lipinski's rule of five by these compounds. Molecular docking based in-silico studies were done to find out naturally occurring biofriendly compounds based novel anti-COVID agents which can bind the SARS-CoV-2 and thus, inhibit its binding affinity towards the ACE2 receptors of human cells. Present study showed that two naturally occurring biflavone based anti-HIV agents, hinokiflavone (3) and robustaflavone (4) can be effective against the SARS-CoV-2 spike (S) protein mediated attack of the human ACE2 receptors via membrane fusion mechanism. Both (3) The authors declare no competing financial interests. Table 3 . Lengths of selected bonds between the interacting active site residues of SARS-CoV-2 S2 subunit (6LXT) and robustaflavone (4) in the docked structure World Health Organization. Coronavirus disease (COVID-19) situation The molecular biology of coronaviruses Coronaviruses as the cause of respiratory infections Bat-to-human: spike features determining 'host jump Complete genome characterisation of a novel coronavirus associated with severe human respiratory disease in From SARS to MERS, thrusting coronaviruses into the spotlight Chemistry and Biology of SARS-CoV-2 Origin and evolution of pathogenic coronaviruses Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein Structure, function, and evolution of coronavirus spike proteins Predicting the receptor-binding domain usage of the coronavirus based on kmer frequency on spike protein A novel coronavirus associated with severe acute respiratory syndrome A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury Phylogenetic analysis and structural modeling of SARS-CoV-2 spike protein reveals an evolutionary distinct and proteolyticallysensitive activation loop SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation A pneumonia outbreak associated with a new coronavirus of probable bat origin Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop Nucleosides for the treatment of respiratory RNA virus infections Severe acute respiratory syndrome coronavirus (SARS-CoV) infection inhibition using spike protein heptad repeat-derived peptides Probable pangolin origin of SARS-CoV-2 associated with the COVID-19 outbreak Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China Middle East respiratory syndrome coronavirus: transmission and phylogenetic evolution Biflavonoids from Torreya nucifera displaying SARS-CoV 3CLpro inhibition Glecaprevir and Maraviroc are high-affinity inhibitors of SARS-CoV-2 main protease: possible implication in COVID-19 therapy Development of antiviral therapy for severe acute respiratory syndrome SARS-CoV 3CLpro inhibitory effects of quinone-methide triterpenes from Tripterygium regelii Characteristics of flavonoids as potent MERS-CoV 3C-like protease inhibitors Inhibition of SARS-CoV 3CL protease by flavonoids Identification of nafamostat as a potent inhibitor of Middle East respiratory syndrome coronavirus S protein-mediated membrane fusion using the split-protein-based cell-cell fusion assay Traditional Chinese herbal medicine for treating novel coronavirus (COVID-19) pneumonia: protocol for a systematic review and meta-analysis Traditional Chinese medicine treatment of COVID-19 Herbal medicine and pattern identification for treating COVID-19: a rapid review of guidelines Traditional Chinese medicine is a resource for drug discovery against 2019 novel coronavirus (SARS-CoV-2) Natural products as sources of new drugs over the last 25 years Natural products as sources of new drugs from 1981 to The re-emergence of natural products for drug discovery in the genomics era An overview of DNA and RNA bindings to antioxidant flavonoids Flavonoids as antioxidants Structure-antioxidant activity relationships of flavonoids: a re-examination Suppression of active oxygen-induced cytotoricity by flavonoids DNA interaction with flavone and hydroxyflavones Rapid Screening and Structural Characterization of Antioxidants from the Extract of Selaginella doederleinii Hieron with DPPH-UPLC-Q-TOF/MS Method Antiviral amentoflavone from Selaginella sinensis Inhibition of fatty acid synthase by amentoflavone reduces coxsackievirus B3 replication Amentoflavone inhibits HSV-1 and ACV-resistant strain infection by suppressing viral early infection Robustaflavone, a potential nonnucleoside anti-hepatitis B agent Robustaflavone, a naturally occurring biflavanoid, is a potent non-nucleoside inhibitor of hepatitis B virus replication in vitro In vitro anti-HIV activity of biflavonoids isolated from Rhus succedanea and Garcinia multiflora Biflavonoids of Dacrydium balansae with potent inhibitory activity on dengue 2 NS5 polymerase Studies on Inhibitors of Skin Tumor Promotion-4-Inhibitory Effects of Flavonoids on Epstein-Barr Virus Activation-1 Anti-influenza virus activity of biflavonoids AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading In vitro DNA binding studies of lenalidomide using spectroscopic in combination with molecular docking techniques Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion Python: A Programming Language for Software Integration and Development Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function Lead-and drug-like compounds: the rule-of-five revolution Neuroprotective effect of apolipoprotein D against human coronavirus OC43-induced encephalitis in mice Apolipoprotein D is involved in the mechanisms regulating protection from oxidative stress Scheme 2. Schematic representation of the plausible route of naturally occurring biflavones, hinokiflavone (3) and robustaflavone