key: cord-0817088-06lddk87 authors: Musarrat, Farhana; Chouljenko, Vladimir; Dahal, Achyut; Nabi, Rafiq; Chouljenko, Tamara; Jois, Seetharama D.; Kousoulas, Konstantin G. title: The anti‐HIV drug nelfinavir mesylate (Viracept) is a potent inhibitor of cell fusion caused by the SARSCoV‐2 spike (S) glycoprotein warranting further evaluation as an antiviral against COVID‐19 infections date: 2020-05-17 journal: J Med Virol DOI: 10.1002/jmv.25985 sha: 4ab17b6f4531936db16afbff83fce6661a039809 doc_id: 817088 cord_uid: 06lddk87 Severe acute respiratory syndrome coronavirus‐2 (SARS CoV‐2) is the causative agent of the coronavirus disease‐2019 (COVID‐19) pandemic. Coronaviruses enter cells via fusion of the viral envelope with the plasma membrane and/or via fusion of the viral envelope with endosomal membranes after virion endocytosis. The spike (S) glycoprotein is a major determinant of virus infectivity. Herein, we show that the transient expression of the SARS CoV‐2 S glycoprotein in Vero cells caused extensive cell fusion (formation of syncytia) in comparison to limited cell fusion caused by the SARS S glycoprotein. Both S glycoproteins were detected intracellularly and on transfected Vero cell surfaces. These results are in agreement with published pathology observations of extensive syncytia formation in lung tissues of patients with COVID‐19. These results suggest that SARS CoV‐2 is able to spread from cell‐to‐cell much more efficiently than SARS effectively avoiding extracellular neutralizing antibodies. A systematic screening of several drugs including cardiac glycosides and kinase inhibitors and inhibitors of human immunodeficiency virus (HIV) entry revealed that only the FDA‐approved HIV protease inhibitor, nelfinavir mesylate (Viracept) drastically inhibited S‐n‐ and S‐o‐mediated cell fusion with complete inhibition at a 10‐μM concentration. In‐silico docking experiments suggested the possibility that nelfinavir may bind inside the S trimer structure, proximal to the S2 amino terminus directly inhibiting S‐n‐ and S‐o‐mediated membrane fusion. Also, it is possible that nelfinavir may act to inhibit S proteolytic processing within cells. These results warrant further investigations of the potential of nelfinavir mesylate to inhibit virus spread at early times after SARS CoV‐2 symptoms appear. The severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) is currently associated with a global pandemic causing coronavirus disease, first noted in December 2019 in Wuhan province of China. The resultant disease is termed COVID-19 (coronavirus disease-2019) and is characterized by acute respiratory disease and pneumonia. SARS CoV-2 has infected nearly 4 million people and caused nearly 300 000 deaths worldwide with a predilection of older people and/or people having other health issues including, hypertension, diabetes, obesity, and other comorbidities. [1] [2] [3] SARSCoV-2 is the third human coronavirus that appeared for the first time in the 21st century. One of the other two coronaviruses is SARS, which appeared in November 2002 in China and caused nearly 100 000 infections worldwide and more than 800 deaths. SARS was effectively contained because the virus, although causing high degree of mortality in infected people, was apparently not effectively transmitted from one person to the other. 4, 5 The second human coronavirus, Middle East Respiratory Syndrome Coronavirus (MERS CoV) appeared in 2013 and caused a limited epidemic of few thousand people, but high death rates of approximately 36% predominantly in the Middle East (Saudi Arabia). The primary source of infection was found to be dromedary camels, although the virus was transmitted from person to person in close contact in hospital settings. [6] [7] [8] [9] All coronaviruses specify a spike (S) glycoprotein, which is embedded in viral envelopes in trimeric forms giving them their characteristic corona structures. The S glycoprotein is a major antigen responsible for both receptor-binding and membrane fusion properties. 9 Angiotensin-converting enzyme 2 (ACE2) has been identified as the cell receptor for SARS, 10 and also SARS CoV-2, while other unknown human receptors may be responsible for its wider infectious spread than SARS. Spike is cleaved into two major components S1 and S2 by cellular proteases. Virus entry into cells is mediated after binding of a receptorbinding domain (RBD) located within the S1 ectodomain. Cleavage of the S glycoprotein to produce S1 and S2 proteins is mediated by cellular proteases at the S1/S2 junction as well as at S2′ site located downstream of the S1/S2 proteolytic cleavage. The fusion of the viral envelope with cellular membranes is mediated by the S2 protein that contains a putative fusion peptide region. The mechanism of membrane pore formation that leads to membrane fusion involves the formation of a six-helix bundle fusion core by two heptad repeats HR1 and HR2 domains found in each S monomer forming the initial pore that results in membrane fusion. 11 The cellular serine protease TMPRSS2 has been implicated in priming S2′ cleavage, as well as ACE2 cleavage both required for initiation of the membrane fusion event. [12] [13] [14] Also, the SARS Spike (S) glycoprotein can be cleaved by the cellular protease cathepsin L at the low pH of endosomes, thereby exposing the S2 domain of the spike protein for membrane fusion. [15] [16] [17] [18] [19] [20] Cell surface expression of S mediates S-induced cell fusion and the formation of syncytia, which is a phenomenon similar to virus entry, requiring the presence of ACE2. Virus-induced cell fusion is a mechanism by which the virus can spread from cell-to-cell by a pH-independent mechanism avoiding the extracellular space and potentially evading neutralizing antibody. 21, 22 It has been demonstrated that the RBD domain of SARS CoV-2 Spike (S-new; Sn) has a higher binding affinity for the ACE2 receptor than that of SARS Spike (S-old; So), while the S2 proteins of these two viruses are nearly 90% identical. 3, 22, 23 Nelfinavir mesylate was developed as an anti-human immunodeficiency virus (HIV) protease inhibitor. 24, 25 Also, it was reported that nelfinavir mesylate inhibited SARS replication and cytopathic effects in cell culture. 26 In addition to its potent activity against the HIV protease, nelfinavir mesylate was found to produce multiple effects on cellular processes including the induction of apoptosis and necrosis as well as induction of cell-protective mechanisms, including cell cycle retardation and the unfolded protein response. [27] [28] [29] These nelfinavir mesylate effects have been exploited for anticancer purposes. [30] [31] [32] Previously, we investigated the structure and function of the SARS S glycoprotein in transient transfection-membrane fusion assays. 33, 34 Based on these initial studies, we undertook screening of a number of compounds that may inhibit S-mediated fusion after transient expression in African green monkey kidney cells (Vero). We report herein that the SARS CoV-2 S (Sn) causes extreme S-mediated membrane fusion in comparison to cell fusion caused by transient expression of SARS S (So). Importantly, we report that nelfinavir mesylate inhibited S-mediated fusion at micromolar ranges. In-silico docking experiments, revealed the possibility that nelfinavir binds to the S2 amino terminus within the S trimer and thus, may directly inhibit the formation of the heptad-repeat complex that causes Smediated membrane fusion. Based on these results, further research of nelfinavir's effect in human COVID-19 patients is warranted. African green monkey kidney (Vero) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 2% primocin (Invitrogen, Inc., Carlsbad, CA). parental vector plasmids, respectively. The S1 subunit of the S-n expression construct contained the same amino terminus up to aa700 (Gly). The N-terminal domain of the S-n S2 subunit was engineered to be exactly as in S1 containing the N-MYC tag at its amino terminus and encompassing the S2 S-n amino acid sequence 701-1273. Nelfinavir mesylate was dissolved in DMSO at a 10 mM concentration (stock) and a series of dilutions was made in serum-free DMEM. Following transfection, 500 μL of nelfinavir mesylate solution was added to each well. Vero cells transfected with either S-o or S-n and incubated with the drug for 48 hours at 37°C with 5% CO 2 . The tissue culture plates were observed for fused cells, and then, phase contrast and fluorescent images were taken under either formalin or methanol fixed conditions. Docking of the nelfinavir mesylate to the spike protein of SARS CoV-2 was performed using Autodock. 35 Crystal structure of nelfinavir was obtained from the complex of HIV protease nelfinavir crystal structure from the protein data bank (PDB ID: 2Q64). 36 Structure of the S protein of SARS CoV-2 was reported by Wrapp et al 23 (PDB ID: 6VSB). The trimer structure of the spike protein was used for docking as protein structure of the spike protein exists under dynamic condition while binding to the receptor and fusion to host cell. Grid for docking was created on the spike protein structure at particular docking site as the center but covering a grid box of 102 or 126Å in X, Y, Z directions from the center of the grid. One grid site was created around protease cleavage site S1/S2 and another covering the HR1 region of the protein in the trimer ( Figure S1 ). Docking calculations were performed using the Lamarckian genetic algorithm with 150 starting conformations and 10 million energy evaluations. Olympus IX71 fluorescent microscope was used for live and phase contrast images using Cellsens software. Zeiss Axio Observer Z1 fluorescent microscope was used for fluorescent images using Zen software. Figure 1A ,B,E,F). In addition, the S1 and S2 domains of S-n were cloned independently into the transient expression vector pCMV3, encompassing amino acid domains for S1 (aa16-aa700) and S2 (aa701-aa1273). Both S1 and S2 domains were expressed with an MYC epitope tag at their amino termini ( Figure 1C,D) . The S1 domain included the S1/S2 cleavage site ( Figure 1C Figure 2B ). Co-expression of S1 and S2 was performed to test whether the Sn-mediated cell fusion could be reconstituted by coexpression of both domains. Expression of either S1, S2, or S1 + S2 domains of S-n was readily detected by immunohistochemistry with the anti-N-MYC antibody; however, there F I G U R E 1 Schematics of spike glycoproteins and recombinant gene constructs. (A) Structure of SARS-CoV-2 spike (1273aa) glycoprotein, showing S1 and S2 domains and the cleavage sites S1/S2 and S2′. (B) Structure of pCMV3-SP-N-MYC (Sn). SARS-CoV-2 spike (aa16-aa1273) was cloned into plasmid expression vector at Kpnl and Xbal restriction sites. The N-terminal 15 amino acids were replaced with signal peptide (SP′) and N-MYC sequence. (C) Structure of pCMV3-S1-N-MYC (S1-n). The S1 domain (aa16-aa700) was cloned into the plasmid expression vector at Kpnl and Xbal restriction sites. The N-terminal 15 amino acids were replaced with signal peptide (SP′) and N-MYC sequence. (D) Structure of pCMV3-S2-N-MYC (S2-n). The S2 domain (aa701-aa1273) was cloned at Kpnl and Xbal restriction sites. The N-terminal contains signal peptide (SP′) and N-MYC sequence. (E) Structure of SARS spike (1255aa) glycoprotein, showing S1 and S2 domains and the cleavage sites S1/S2 and S2′. (F) Structure of p3XFLAG-CMV-S (So). SARS spike was cloned into plasmid expression vector as previously described. FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; NTD, nontranslated domain; RBD, receptor-binding domain; SARS CoV, severe acute respiratory syndrome coronavirus; SP, SARS signal peptide; SP′, signal peptide was no cell fusion observed at 48 hpt as evidenced by only well-defined single cells that were stained with the anti-MYC antibody (Figure 3 ), as well as at later times (not shown), suggesting that the S1 and S2 domains have to be part of the entire molecule to be processed correctly for induction of S-mediated cell fusion. F I G U R E 3 Expression of SARS CoV-2 spike (Sn) domains. Vero cells were transfected with pCMV3-SP-N-MYC plasmid expressing either the S1, S2, or S1 + S2 domains of S-n tagged with the N-MYC epitopes at their amino termini. Expression was detected with mAbs against the epitope tags at 48 hours posttransfection and compared to vehicle containing equivalent amount of lipofectamine 2000 reagent. Methanol fixed cells were incubated with mouse anti-MYC antibody and stained with HRP staining followed by goat anti-mouse secondary antibody incubation. Images were taken at ×10 magnification. HRP, horseradish peroxidase; SARS CoV, severe acute respiratory syndrome coronavirus; Sn, S-new Recently, it was shown that a peptide that targeted the S-n HR1 domain S inhibited SARS-CoV-2 virus replication, virus entry, and virus-induced cell fusion. 37 Therefore, we performed in-silico docking experiments to investigate the possibility that nelfinavir may directly bind near this S region. These theoretical docking experiments revealed that nelfinavir may bind near the HR1 helix and in between the HR1 and HR2 helices (Figures S1 and S2 ). 1000) . Cellular tubulin was stained with rabbit anti-alpha tubulin (Abcam; 1:200) and anti-rabbit secondary antibody conjugated with Alexa fluorophore 488. DAPI was used to stain nuclei of cells. Fluorescent images were taken at ×40 magnification. DAPI, 4′,6-diamidino-2phenylindole; DMSO, dimethyl sulfoxide; Sn, S-new; So, S-old uncleaved form that may be proteolytically processed either within endosomes or at cell surfaces by proteases such as TMPRSS2, which is known to be required for Spike activation during virus entry. 12 We utilized the S-n and S-o transient expression system to screen for currently available drugs that may inhibit S-mediated cell fusion and the formation of syncytia. These drugs included cardiac glycosides such as ouabain and digoxin, the anti-HIV fusion inhibitor Fuzeon (enfuvirtide) and kinase inhibitors including Gleevec (imatinib mesylate). These drugs did not substantially inhibit S-mediated cell fusion even at concentrations of 100 μM. However, we found that ID:2910197). Therefore, it may be possible that nelfinavir can be used at even lower concentrations than those prescribed for patients with HIV. These results are significant because nelfinavir did not appear to inhibit overall S-n or S-o synthesis and cell surface expression. We considered the possibility that nelfinavir may act not as a protease inhibitor but as a direct inhibitor of spike-mediated membrane fusion. Computational modeling revealed that nelfinavir may directly bind to the trimeric form of S-n and S-o near the putative fusogenic domain, and thus, it may directly inhibit S-mediated cell fusion (Figures S1 and S2 ). Nelfinavir has been reported to have pleotropic effects on multiple cellular processes including inducing apoptosis an ER stress under certain conditions and has been investigated for anticancer purposes. 32, 41, 42 Thus, it is possible that cellular signaling processes are affected that alter to posttranslational processing of S-n and S-o, without affecting their cell surface expression. It is also possible that nelfinavir may inhibit cellular proteases including TMPRSS2 that may be required for S-n and S-o fusion activation. Other protease inhibitors are currently being investigated for their ability to inhibit SARS CoV-2 replication and spread. However, there are no concrete results that have been obtained in clinical trials yet. 43 Preliminary experiments indicate that S-n and S-o may be cleaved in Vero cells in the presence of nelfinavir, although it is not currently known whether this cleavage occurs efficiently. In addition, transfected cells expressing S-n or S-o in the presence of nelfinavir did not appear to exhibit morphologies indicating of cellular cytotoxicity, suggesting that nelfinavir is not cytotoxic at the concentrations used in this study. Overall, these experiments suggest that nelfinavir should be used to combat SARS CoV-2 infections early during the first symptoms exhibited by infected patients to minimize virus spread and give sufficient time to infected patients to mount a protective immune response. Another decade, another coronavirus A new coronavirus associated with human respiratory disease in China A novel coronavirus from patients with pneumonia in China Severe acute respiratory syndrome (SARS): a review of the history, epidemiology, prevention, and concerns for the future Severe acute respiratory syndrome (SARS): a review Narrative review of Middle East respiratory syndrome coronavirus (MERS-CoV) infection: updates and implications for practice Prevalence of comorbidities in the Middle East respiratory syndrome coronavirus (MERS-CoV): a systematic review and meta-analysis Middle East respiratory syndrome coronavirus (MERS-CoV): A review From SARS to MERS, thrusting coronaviruses into the spotlight Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity Severe acute respiratory syndrome coronavirus (SARS-CoV) infection inhibition using spike protein heptad repeat-derived peptides SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2 A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry Structural characterization of the SARS-coronavirus spike S fusion protein core A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensinconverting enzyme 2 pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN The secret life of ACE2 as a receptor for the SARS virus Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Nelfinavir mesylate: a protease inhibitor Nelfinavir mesylate HIV protease inhibitor nelfinavir inhibits replication of SARS-associated coronavirus The HIV protease inhibitor nelfinavir downregulates Akt phosphorylation by inhibiting proteasomal activity and inducing the unfolded protein response The human immunodeficiency virus (HIV)-1 protease inhibitor saquinavir inhibits proteasome function and causes apoptosis and radiosensitization in non-HIV-associated human cancer cells HIV-1 protease inhibitor induces growth arrest and apoptosis of human prostate cancer LNCaP cells in vitro and in vivo in conjunction with blockade of androgen receptor STAT3 and AKT signaling Nelfinavir induces the unfolded protein response in ovarian cancer cells, resulting in ER vacuolization, cell cycle retardation and apoptosis Nelfinavir and nelfinavir analogs block site-2 protease cleavage to inhibit castration-resistant prostate cancer HIV-1 protease inhibitors nelfinavir and atazanavir induce malignant glioma death by triggering endoplasmic reticulum stress Palmitoylation of the cysteinerich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility Molecular analysis of the HIV-1 resistance development: enzymatic activities, crystal structures, and thermodynamics of nelfinavir-resistant HIV protease mutants 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 Virus and cell fusion mechanisms Virus cell-to-cell transmission Nelfinavir: an update on its use in HIV infection Nelfinavir, a new anti-cancer drug with pleiotropic effects and many paths to autophagy Nelfinavir, a lead HIV protease inhibitor, is a broad-spectrum, anticancer agent that induces endoplasmic reticulum stress, autophagy, and apoptosis in vitro and in vivo Coronavirus puts drug repurposing on the fast track Additional supporting information may be found online in the Supporting Information section.