Identification of FDA-approved bifonazole as a SARS-CoV-2 blocking agent following a bioreporter drug screen
Authors: Zaid Taha, Rozanne Arulanandam, Glib Maznyi, Elena Godbout, Madalina E. Carter-Timofte, Naziia Kurmasheva, Line S. Reinert, Andrew Chen, Mathieu J.F. Crupi, Stephen Boulton, Geneviève Laroche, Alexandra Phan, Reza Rezaei, Nouf Alluqmani, Anna Jirovec, Alexandra Acal, Emily E.F. Fekete, Ragunath Singaravelu, Julia Petryk, Manja Idorn, Kyle G. Potts, Hayley Todesco, Cini John, Douglas J. Mahoney, Carolina S. Ilkow, Patrick Giguère, Tommy Alain, Marceline Côté, Søren R. Paludan, David Olagnier, John C. Bell, Taha Azad, Jean-Simon Diallo
Journal: Molecular Therapy (2022)

We established a split nanoluciferase complementation assay to rapidly screen for inhibitors that interfere with binding of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein with its target receptor, angiotensin-converting enzyme 2 (ACE2). After a screen of 1,200 US Food and Drug Administration (FDA)-approved compounds, we identified bifonazole, an imidazole-based antifungal agent, as a competitive inhibitor of RBD-ACE2 binding. Mechanistically, bifonazole binds ACE2 around residue K353, which prevents association with the RBD, affecting entry and replication of spike-pseudotyped viruses as well as native SARS-CoV-2 and its variants of concern (VOCs). Intranasal administration of bifonazole reduces lethality in K18-hACE2 mice challenged with vesicular stomatitis virus (VSV)-spike by 40%, with a similar benefit after live SARS-CoV-2 challenge. Our screen identified an antiviral agent that is effective against SARS-CoV-2 and VOCs such as Omicron that employ the same receptor to infect cells and therefore has high potential to be repurposed to control, treat, or prevent coronavirus disease 2019 (COVID-19). We established a split nanoluciferase complementation assay to rapidly screen for inhibitors that interfere with binding of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein with its target receptor, angiotensin-converting enzyme 2 (ACE2). After a screen of 1,200 US Food and Drug Administration (FDA)-approved compounds, we identified bifonazole, an imidazole-based antifungal agent, as a competitive inhibitor of RBD-ACE2 binding. Mechanistically, bifonazole binds ACE2 around residue K353, which prevents association with the RBD, affecting entry and replication of spike-pseudotyped viruses as well as native SARS-CoV-2 and its variants of concern (VOCs). Intranasal administration of bifonazole reduces lethality in K18-hACE2 mice challenged with vesicular stomatitis virus (VSV)-spike by 40%, with a similar benefit after live SARS-CoV-2 challenge. Our screen identified an antiviral agent that is effective against SARS-CoV-2 and VOCs such as Omicron that employ the same receptor to infect cells and therefore has high potential to be repurposed to control, treat, or prevent coronavirus disease 2019 (COVID-19). IntroductionCoronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses. Severe acute respiratory syndrome (SARS)-CoV and SARS-CoV-2 infect cells through interaction of the receptor binding domain (RBD) of the viral spike protein with the host receptor, angiotensin-converting enzyme 2 (ACE2).1Hoffmann M. Kleine-Weber H. Schroeder S. Kruger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.H. Nitsche A. et al.SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell. 2020; 181: 271-280.e8https://doi.org/10.1016/j.cell.2020.02.052Abstract Full Text Full Text PDF PubMed Scopus (10317) Google Scholar Since the SARS outbreak in 2002/2003, the current pandemic caused by a genetically related virus has acutely highlighted a missed opportunity and urgent need for development of antiviral agents against SARS-like viruses. Systematic reviews have concluded that no approved antiviral regimens are currently effective against SARS-CoV or SARS-CoV-2.2Mei M. Tan X. Current strategies of antiviral drug discovery for COVID-19.Front. Mol. Biosci. 2021; 8: 671263https://doi.org/10.3389/fmolb.2021.671263Crossref PubMed Scopus (41) Google Scholar Remdesivir (Veklury) has been granted approval or emergency authorization in approximately 50 countries worldwide, but because of controversial clinical effects it remains conditionally recommended by the World Health Organization (WHO). Molnupiravir, a nucleoside analog like remdesivir, has recently become the first oral antiviral agent for the treatment of coronavirus disease 2019 (COVID-19) to be approved because of its effect of reducing hospitalization or death by 50%. Paxlovid, consisting of the protease inhibitors nirmatrelvir and ritonavir, was more recently authorized in December 2021 as an oral treatment for mild to moderate COVID-19 in individuals over 12 years of age at high risk of severe disease progression. Paxlovid has been shown to reduce hospitalization and death by 88% in a clinical trial but has multiple drug interactions. Monoclonal antibody (mAb) treatments such as bamlanivimab have shown promise in individuals at high risk for progression to severe disease. However, mAbs can lead to increased chances of viral escape, are harder to make, require intravenous delivery with medical supervision, are relatively bulky, and may not be broadly effective or capable of dislodging pre-formed virus-receptor interactions. Taking into consideration our current options, there still remains an urgent need for new and effective antiviral drugs to treat and prevent COVID-19.The specific molecular interaction between SARS-CoV-2 and its receptor is a key therapeutic target for the identification of effective antiviral agents that could have a direct contribution to our global response to controlling COVID-19 spread. Small molecules are more versatile in this regard, and their use to prevent viral entry is an approved strategy in the context of HIV (e.g., maraviroc).3Qian K. Morris-Natschke S.L. Lee K.H. HIV entry inhibitors and their potential in HIV therapy.Med. Res. Rev. 2009; 29: 369-393https://doi.org/10.1002/med.20138Crossref PubMed Scopus (99) Google Scholar,4Dorr P. Westby M. Dobbs S. Griffin P. Irvine B. Macartney M. Mori J. Rickett G. Smith-Burchnell C. Napier C. et al.Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity.Antimicrob. Agents Chemother. 2005; 49: 4721-4732https://doi.org/10.1128/AAC.49.11.4721-4732.2005Crossref PubMed Scopus (956) Google Scholar Identification of efficient agents from existing drugs is the most rapid approach for development of a therapy given their proven safety, and it is anticipated that a number of existing small-molecule therapeutic agents could be repurposed to treat COVID-19. Because crystal structures are now available, in silico approaches or artificial intelligence (AI) tools can be implemented to identify potential drug candidates, and some have already used this approach to identify compounds that may block the ACE2-RBD interaction.2Mei M. Tan X. Current strategies of antiviral drug discovery for COVID-19.Front. Mol. Biosci. 2021; 8: 671263https://doi.org/10.3389/fmolb.2021.671263Crossref PubMed Scopus (41) Google Scholar,5Shweta C. Yashpal S.M. Shailly T. Identification of SARS-CoV-2 cell entry inhibitors by drug repurposing using in silico structure-based virtual screening approach.ChemRxiv. 2020; (Preprint at)https://doi.org/10.26434/chemrxiv.12005988.v1Crossref Google Scholar,6Kaushal K. Sarma P. Rana S.V. Medhi B. Naithani M. Emerging role of artificial intelligence in therapeutics for COVID-19: a systematic review.J. Biomol. Struct. Dyn. 2020; : 1-16https://doi.org/10.1080/07391102.2020.1855250Crossref PubMed Scopus (13) Google Scholar However, high-throughput screening can better identify allosteric and non-competitive drugs and remains a gold standard for antiviral drug discovery. We established a split nanoluciferase complementation assay, easily amenable to high-throughput applications, where the RBD-ACE2 interaction facilitates activation of a strong luminescent nanoluciferase signal. We showed that the resulting signal is specific and requires interaction between the spike protein and ACE2.7Azad T. Singaravelu R. Brown E.E.F. Taha Z. Rezaei R. Arulanandam R. Boulton S. Diallo J.S. Ilkow C.S. Bell J.C. SARS-CoV-2 S1 NanoBiT: a nanoluciferase complementation-based biosensor to rapidly probe SARS-CoV-2 receptor recognition.Biosens. Bioelectron. 2021; 180: 113122https://doi.org/10.1016/j.bios.2021.113122Crossref PubMed Scopus (12) Google Scholar Our assay is based on NanoLuc binary technology (NanoBiT), which dissects nanoluciferase into two components, small BiT (SmBiT) and large BiT (LgBiT), that display strong conformational stability, creating an ideal split reporter for investigation of protein-protein interactions.7Azad T. Singaravelu R. Brown E.E.F. Taha Z. Rezaei R. Arulanandam R. Boulton S. Diallo J.S. Ilkow C.S. Bell J.C. SARS-CoV-2 S1 NanoBiT: a nanoluciferase complementation-based biosensor to rapidly probe SARS-CoV-2 receptor recognition.Biosens. Bioelectron. 2021; 180: 113122https://doi.org/10.1016/j.bios.2021.113122Crossref PubMed Scopus (12) Google Scholar, 8Dixon A.S. Schwinn M.K. Hall M.P. Zimmerman K. Otto P. Lubben T.H. Butler B.L. Binkowski B.F. Machleidt T. Kirkland T.A. et al.NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells.ACS Chem. Biol. 2016; 11: 400-408https://doi.org/10.1021/acschembio.5b00753Crossref PubMed Scopus (506) Google Scholar, 9Azad T. Janse van Rensburg H.J. Lightbody E.D. Neveu B. Champagne A. Ghaffari A. Kay V.R. Hao Y. Shen H. Yeung B. et al.A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in angiogenesis.Nat. Commun. 2018; 9: 1061https://doi.org/10.1038/s41467-018-03278-wCrossref PubMed Scopus (90) Google Scholar, 10Azad T. Singaravelu R. Taha Z. Jamieson T.R. Boulton S. Crupi M.J.F. Martin N.T. Brown E.E.F. Poutou J. Ghahremani M. et al.Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry.Mol. Ther. 2021; 29: 1984-2000https://doi.org/10.1016/j.ymthe.2021.02.007Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 11Brown E.E.F. Rezaei R. Jamieson T.R. Dave J. Martin N.T. Singaravelu R. Crupi M.J.F. Boulton S. Tucker S. 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Nikkhah M. et al.Identification of NLRP3(PYD) homo-oligomerization inhibitors with anti-inflammatory activity.Int. J. Mol. Sci. 2022; 23: 1651https://doi.org/10.3390/ijms23031651Crossref PubMed Scopus (2) Google Scholar, 15Azad T. Tashakor A. Rahmati F. Hemmati R. Hosseinkhani S. Oscillation of apoptosome formation through assembly of truncated Apaf-1.Eur. J. Pharmacol. 2015; 760: 64-71https://doi.org/10.1016/j.ejphar.2015.04.008Crossref PubMed Scopus (11) Google Scholar We hypothesized that impairing the interactions between the spike RBD and host ACE2 using small-molecule drugs can disrupt viral attachment and prevent subsequent viral entry, ultimately precluding viral infection and spread. In this study, we adapted our split-luciferase assay for high-throughput screening to identify inhibitors of the RBD-ACE2 interaction from a collection of approved drugs.ResultsA nanoluciferase complementation bioreporter screen identifies potential SARS-CoV-2 blocking agentsThe components of our bioreporter have been described previously,7Azad T. Singaravelu R. Brown E.E.F. Taha Z. Rezaei R. Arulanandam R. Boulton S. Diallo J.S. Ilkow C.S. Bell J.C. SARS-CoV-2 S1 NanoBiT: a nanoluciferase complementation-based biosensor to rapidly probe SARS-CoV-2 receptor recognition.Biosens. Bioelectron. 2021; 180: 113122https://doi.org/10.1016/j.bios.2021.113122Crossref PubMed Scopus (12) Google Scholar LgBiT or SmBiT, fused to the SARS-CoV-2 spike RBD or the extracellular domain of human ACE2, respectively (Figure 1A ). These constructs were cloned into plasmids and transfected into HEK293 cells for their expression in whole-cell lysates or supernatants. We confirmed that the combination of lysates from transfected cells expressing the SmBiT-ACE2 and LgBiT-RBD could lead to complementation of the nanoluciferase by producing a strong and stable luminescence signal upon addition of the substrate native coelenterazine. We demonstrate that treatment of LgBiT-RBD with an antibody against the spike RBD or addition of soluble ACE2 (sACE2) to SmBiT-ACE2 could compete with its counterpart and impair the luminescence signal. However, this effect only occurred under conditions where each construct was transfected separately and the resulting lysates were mixed, in contrast with when the constructs were co-transfected and, thus, the complex pre-formed (Figure 1A, lanes 6 and 7). We therefore employed our bioreporter to screen a library consisting of 1,200 US Food and Drug Administration (FDA)-approved compounds. Two parallel methods were employed (Figure 1B). For method A, library compounds were added to the pre-formed SmBiT-ACE2/LgBiT-RBD complex to identify compounds that could dissociate this complex. Method B involved addition of the compound library to LgBiT-RBD, followed by addition of an equal quantity of SmBiT-ACE2 to identify compounds that could prevent this interaction. Method B also included an RBD-neutralizing sybody as a positive control.16Walter J.D. Hutter C.A.J. Zimmermann I. Wyss M. Egloff P. Sorgenfrei M. Hürlimann L.M. Gonda I. Meier G. Remm S. et al.Sybodies targeting the SARS-CoV-2 receptor-binding domain.bioRxiv. 2020; (Preprint at)https://doi.org/10.1101/2020.04.16.045419Crossref Scopus (0) Google Scholar Both methods were found to retain high signal-to-noise ratios (Figure S1A) and appropriate Z′ factors (Figure S1B), confirming suitability for high-throughput screening (HTS; Figures S1C and S1D).Methods A and B were used to screen the Prestwick library at 4 μM final concentration. Following 1-h incubation with the compounds, coelenterazine (CTZ) was added to each well, and luminescence was measured (n = 3; Figures 1B and 1C). Controls consisting of vehicle (DMSO) alone or RBD alone were added to each 384-well plate (n = 32/plate, 2 per row).A small subset of the library compounds led to a statistically significant reduction in the luminescence signal from the bioreporter using method A or method B (5% false discovery rate [FDR] cutoff; Figures 1D and 1E).Volcano plots and Z scores (Figure S1E) were used to select approximately 45 hit compounds for further validation. Some of these hits were common to method A and method B, whereas some were unique to either method (Figure S1F). Because of the larger number of hits obtained from method A, we eliminated hits affecting bioreporter luminescence by 15% or less to enrich for the most potent compounds. Compound Prestw-1241 (bifonazole) consistently ranked the highest in terms of its ability to disrupt the biosensor through both methods and yielded more than twice the reduction in bioreporter signal using method A (−46%) and B (−51%) compared with the anti-RBD positive control (sybody, −22%). Pharmacological classification of our top 45 hits reveal an equal number (9.62%) of antifungal, antibacterial, anti-inflammatory, and anti-neoplastic agents, which represent 14.3%, 3.03%, 5.21%, and 8.06%, respectively, of each of these classes in the Prestwick library, ultimately revealing an enrichment in antifungal agents such as bifonazole (Figure 1F).Screen validation identifies bifonazole as a top SARS-CoV-2 blocking candidateTo validate the hits from our initial screen, we treated our bioreporter with the top 45 candidates identified at 4 μM final concentration in quadruplicate to confirm their inhibitory activity on the SARS-CoV-2 RBD-ACE2 interaction by methods A and B. In addition, we examined the effect of our top hits on a bioreporter designed to detect the interaction of ACE2 with the larger S1 domain of SARS-CoV-2 spike. As negative controls, we also included an unrelated control bioreporter (the large tumor suppressor kinase or LATS biosensor)9Azad T. Janse van Rensburg H.J. Lightbody E.D. Neveu B. Champagne A. Ghaffari A. Kay V.R. Hao Y. Shen H. Yeung B. et al.A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in angiogenesis.Nat. Commun. 2018; 9: 1061https://doi.org/10.1038/s41467-018-03278-wCrossref PubMed Scopus (90) Google Scholar as well as wild-type nanoluciferase to eliminate compounds that could non-specifically inhibit the enzyme (Figures S2A, S2B, and S2E). Our results confirmed that candidate Prestw-1241 (bifonazole) led to a significant reduction in binding between ACE2 and the RBD with method A or B (Figures 2A and 2B , left, and Z scores in Figure 2C). Similar results were obtained with ACE2 and the S1 domain of spike, where an ∼30% reduction was observed using method A (co-transfected; Figures 2A and 2B, right). The reduction in SARS-CoV-2 bioreporter luminescence generated by some of the other candidates was much lower than Prestw-1241 (<10% inhibition). Prestw-1405 (ethynylestradiol) and Prestw-383 (nicardipine) yielded an 4%–6% reduction in luminescence specific to the RBD/spike:ACE2 interaction. Although Prestw-430 (cisapride) led to a slightly more pronounced (6%–19%) effect (Figure S2E), it also led to a reduction in luminescence with the LATS biosensor (−13%; Figure S2A) but not nanoluciferase on its own, suggesting some non-specific ability to disrupt the molecular complex.Figure 2Screen validation identifies bifonazole as the top SARS-CoV-2 blocking candidateShow full caption(A and B) Validation (n = 4) of the top 45 candidates using the RBD or the S1 domain of SARS-CoV-2 spike protein following method A (A) or method B (B). (C) Table describing the top 4 candidates selected for subsequent validation, including Z scores obtained following method A or method B. (D) SARS-CoV-2 RBD lysates were incubated as in method B with a dose range (0–25 μM) of ethinylestradiol, nicardipine, cisapride, and bifonazole, followed by addition of ACE2 lysates, and luminescence was measured. Anti-RBD was added as a positive control (dashed line), and the RBD alone (LgBiT, dotted line) was used as a negative control (n = 3–6, two-way ANOVA with Dunnett's multiple corrections test compared with mock for each drug; orange asterisks signify that all subsequent values have the same level of significance). (E) VeroE6 or Vero-hACE2-TMPRSS2 (VeroAT) cells were treated with non-toxic concentrations of the indicated drugs for 15 min, followed by infection of cells with SARS-CoV-2-spike-pseudotyped VSV (VSV-GFP-Spike) or WT VSV-GFP (MOI 1.0), followed by high-content fluorescence imaging. The ratio of GFP foci obtained for WT VSV-GFP- over VSV-Spike-GFP-infected cells was graphed across the concentrations tested for all four compounds in both cell lines. Tables on the right indicate concentrations that are able to reduce GFP focus counts from WT VSV- or VSV-Spike-infected cells by 50% (n = 5 per condition; see Figures S2G–S2I for raw data).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Given that the 2002 SARS-CoV also utilizes ACE2 as a receptor for viral entry, we examined the potential effect of bifonazole on this CoV. Alongside the above validation, we tested the top 45 hits from our bioreporter screen with constructs encoding the RBD of SARS-CoV and ACE2. Our data clearly demonstrate a significant reduction in luminescence with 4 μM bifonazole using method A (45% inhibition) and method B (22% inhibition), suggesting that this compound also has the potential to impede SARS-CoV viral entry (Figures S2C and S2D). Ethynylestradiol and nicardipine led to a less pronounced but significant reduction in bioreporter signal using both methods (3%–6% inhibition; Figure S2E).To compare head to head the potency of these lead compounds in impairing our SARS-CoV-2 bioreporter, we treated lysates with a broader range of concentrations (2–25 μM) of newly purchased drugs using method B (Figures 2D and S2F). Although we observed a dose-dependent reduction in bioluminescence with all four compounds, bifonazole led to an 80% signal inhibition at 3.29 μM with half maximal inhibitory concentration (IC50) of 1.3 μM. This inhibition was on par with the positive control anti-RBD antibody. Nicardipine-HCl yielded a similar level of bioreporter impairment as bifonazole (IC50 = 1.9 μM), followed by ethynylestradiol (IC50 = 2.6 μM) and cisapride (IC50 = 16.5 μM), which was noticeably the least potent with minimal effect on the bioreporter.To assess the effectiveness of these compounds in a biological system, we treated VeroE6 cells, which express detectable levels of ACE2, or VeroE6 cells stably overexpressing human ACE2 and TMPRSS2 (VeroAT; Figure S2G) cells with non-toxic concentrations (∼2–50 μM) of bifonazole, nicardipine-HCl, or cisapride for 15 min. Treated cells were then infected with SARS-CoV-2-Spike pseudotyped vesicular stomatitis virus (VSV) expressing green fluorescent protein (VSV-GFP-Spike)17Case J.B. Rothlauf P.W. Chen R.E. Kafai N.M. Fox J.M. Smith B.K. Shrihari S. McCune B.T. Harvey I.B. Keeler S.P. et al.Replication-competent vesicular stomatitis virus vaccine vector protects against SARS-CoV-2-mediated pathogenesis in mice.Cell Host Microbe. 2020; 28: 465-474.e4https://doi.org/10.1016/j.chom.2020.07.018Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar or wild-type VSV-GFP (multiplicity of infection [MOI] 1.0). 12–24 h after infection, high-content fluorescence imaging was performed, and cell viability was assessed 48 h after infection. Our results demonstrate that bifonazole leads to a significant reduction in pseudotyped VSV-Spike-GFP-infected cells (IC50 = 30–40 μM in both model cell lines without any overt toxicity up to 100 μM; Figures 2E and S2E–S2I). Bifonazole also led to a reduction in wild-type (WT) VSV-GFP-infected cells but at much higher concentrations (IC50 = 60–70 μM). In contrast, ethynylestradiol and nicardipine were able to inhibit WT VSV-GFP (IC50 = ∼15–20 μM) more efficiently than VSV-Spike-GFP (IC50 = ∼25–70 μM). Cisapride did not affect VSV infection at all concentrations tested and was toxic at doses of more than 20 μM. To better illustrate the specificity toward spike, the ratio of percent GFP foci obtained for WT VSV over VSV-Spike was graphed across all concentrations for all four compounds in both cell lines. Although the curve for bifonazole trends upward from ∼20–67 μM, indicating greater inhibitory potential toward VSV-Spike, the curves for ethynylestradiol and nicardipine trend downward demonstrating increased inhibition of WT VSV viral growth and less specificity toward spike.Validation attempts using our bioreporter as well as biological assays with spike-pseudotyped VSV identify bifonazole as a promising antiviral candidate for SARS-CoV-2 because of its ability to specifically block the RBD-ACE2 interaction in model cell systems.Imidazole antifungals can impair interaction between ACE2 and the SARS-CoV-2 RBDFurther analysis of the screening data revealed that many other imidazole-category antifungal agents, like bifonazole, have the capacity to block the RBD-ACE2 interaction. These include econazole nitrate (Prestw-304, −36% method A and −23% method B), ketoconazole (Prestw-389, −29% method A), clotrimazole (Prestw-267, −21% method A) and oxiconazole nitrate (Prestw-304, −4% method B) (Figures 3A , S1E, and S1F). Prestw-1019 (tribenoside) is an anti-inflammatory agent also classified as an imidazole with inhibitory potential (−21% method A and −9% method B). The identification of multiple compounds containing an imidazole pharmacophore suggests a potential mechanistic or structural relevance for RBD-ACE2 interaction blockade observed with the biosensor. For these reasons, we further investigated and compared the antiviral potential of the top three imidazole compounds: bifonazole, econazole, and ketoconazole.Figure 3Bifonazole outperforms other imidazole antifungal agents at impairing the interaction between ACE2 and the SARS-CoV-2 RBDShow full caption(A) Table describing the top 3 imidazole antifungal agents identified in the Prestwick library screen, including Z scores for method A and method B. (B) SARS-CoV-2 RBD and ACE2 lysates were treated using method B, as done previously, using bifonazole, econazole, or ketoconazole (n = 3–6, two-way ANOVA with Dunnett's multiple corrections test compared with mock for each drug; orange asterisks signify that all subsequent values have the same level of significance). (C) VeroE6 or VeroAT cells were treated with non-toxic concentrations of the indicated drugs for 15 min, followed by infection of cells with VSV-Spike or WT VSV-GFP (MOI 1.0) and high-content fluorescence imaging. The ratio of GFP foci obtained for WT VSV- over VSV-Spike-GFP-infected cells was graphed across the concentrations tested for all compounds in both cell lines. Tables on the right indicate concentrations that are able to reduce GFP focus counts from WT VSV- or VSV-Spike-infected cells by 50% (n = 5 per condition; see Figures S3B–S3D for raw data).View Large Image Figure ViewerDownload Hi-res image Download (PPT)As shown in Figure 2D, we treated our bioreporter lysates with a broader range of concentrations of newly purchased compounds using method B. Reflecting initial screen results, we observed superior performance by bifonazole (IC50 = 1.6 μM), followed by econazole (IC50 = 2.3 μM) and ketoconazole (IC50 = 14.5 μM) (Figures 3B and S3A).To biologically assess antiviral potency, we subsequently treated VeroE6 and VeroAT cells with increasing concentrations of the three imidazoles up to 100 μM, as shown in Figure 2. Fluorescence imaging was performed 12–24 h after infection, and viability was assessed by resazurin 48 h after infection. The ratio of GFP foci obtained for WT VSV over VSV-Spike was graphed across concentrations tested for all four compounds in both cell lines. Bifonazole led to a spike-specific reduction in viral growth at concentrations of ∼35 μM, whereas econazole and ketoconazole demonstrated similar effects but at higher concentrations of ∼60 and 80 μM, respectively (Figures 3C and S3B–S3D).Our screening data and subsequent validation reveal that members of the imidazole antifungal class of compounds exhibit antiviral activity toward SARS-CoV-2, with bifonazole outperforming all in terms of potency and specificity for disrupting the RBD-ACE2 interaction.Bifonazole blocks the RBD binding site of SARS-CoV-2 on ACE2To investigate the mechanism of action of bifonazole, we first evaluated the effect of the timing of bifonazole addition on spike:ACE2 inhibition via infection of cells with two different strains of VSV-Spike (strain 1, VSV-Spike-GFP; strain 2, VSV-Spike) at MOI 1.0. Briefly, we pre-treated VeroAT cells with bifonazole for 15 min ("pre-treatment") and added the drug at the same time as infection ("co-treatment") or 60 min after infection ("post-treatment"). VSV-Spike spread was quantified by GFP imaging (strain 1; Figure 4A ), and viral output was measured from supernatants 48 h after infection by standard plaque assay (Figure S4A). We found that treatment of ACE2-expressing VeroAT cells for as little as 15 min prior to infection with both strains of VSV-Spike led to the greatest increase in the magnitude of viral blockade by bifonazole.Figure 4Bifonazole impairs viral entry and interacts with ACE2 to block the RBD binding site of SARS-CoV-2Show full caption(A) Bifonazole was added to VeroAT cells prior to infection with VSV-Spike-GFP (pre-treatment), at the time of infection (co-treatment), or after infection (post treatment), and GFP counts were quantified 24 h later (n = 2, two-way ANOVA with Dunnett's multiple comparisons test over mock treated). (B) Vero cells were pre-treated with bifonazole and infected with the indicated viruses at MOI 1, and GFP counts were determined 24 hpi (n = 3, two-way ANOVA with Dunnett's multiple comparison test over mock treated and Sidak's test for counts within concentrations). (C) VeroAT cells were pre-treated with bifonazole, followed by infection with the indicated viruses and immunofluorescence staining (magnification, 63×; scale bar, 20 μm). (D) HEK293T cells expressing hACE2 or hACE2/TMPRSS2 and Zip-Venus-1 were treated with bifonazole for 30 min, and then HEK293T cells expressing SARS-CoV-2 spike and Zip-Venus-2 were added to wells. Cells were imaged for fusion and Venus signal. Data show fold change in relative fluorescent units (RFU) from 3 independent experiments (n = 7–11, two-way ANOVA with Dunnett's multiple comparisons test over mock treated for each condition). (E) Top: bifonazole was added to the RBD, followed by ACE2. Bottom: bifonazole was added to ACE2, followed by the RBD (n = 3 for each). (F) The preferred binding pocket of bifonazole on hACE2 was determined using an automated in silico molecular docking approach (top). The most favorable models are clustered at a pocket of ACE2 that overlaps with RBD binding (bottom). (G) Interacting residues were identified usi