High-Throughput Screening and Identification of Potent Broad-Spectrum Inhibitors of Coronaviruses

Abstract

Coronaviruses (CoVs) act as cross-species viruses and have the potential to spread rapidly into new host species and cause epidemic diseases. Despite the severe public health threat of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome CoV (MERS-CoV), there are currently no drugs available for their treatment; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are urgently needed. To search for effective inhibitory agents, we performed high-throughput screening (HTS) of a 2,000-compound library of approved drugs and pharmacologically active compounds using the established genetically engineered human CoV OC43 (HCoV-OC43) strain expressing Renilla luciferase (rOC43-ns2Del-Rluc) and validated the inhibitors using multiple genetically distinct CoVs in vitro. We screened 56 hits from the HTS data and validated 36 compounds in vitro using wild-type HCoV-OC43. Furthermore, we identified seven compounds (lycorine, emetine, monensin sodium, mycophenolate mofetil, mycophenolic acid, phenazopyridine, and pyrvinium pamoate) as broad-spectrum inhibitors according to their strong inhibition of replication by four CoVs in vitro at low-micromolar concentrations. Additionally, we found that emetine blocked MERS-CoV entry according to pseudovirus entry assays and that lycorine protected BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This represents the first demonstration of in vivo real-time bioluminescence imaging to monitor the effect of lycorine on the spread and distribution of HCoV-OC43 in a mouse model. These results offer critical information supporting the development of an effective therapeutic strategy against CoV infection. IMPORTANCE Currently, there is no approved therapy to treat coronavirus infection; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are needed. Based on our high-throughput screening assay using a compound library, we identified seven compounds with broad-spectrum efficacy against the replication of four CoVs in vitro. Additionally, one compound (lycorine) was found to protect BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This inhibitor might offer promising therapeutic possibilities for combatting novel CoV infections in the future.

In silico screening of Chinese herbal medicines with the potential to directly inhibit 2019 novel coronavirus

“…Through in silico and biological processing, a series of small molecules, including those from natural compounds, have been screened and confirmed to directly inhibit these important proteins in SARS or Middle East respiratory syndrome (MERS) coronavirus [17][18][19][20][21][22][23]. The gene sequence of 2019-nCoV has been released, which suggests high similarities between the main proteins in this virus and those previously identified in SARS-Cov or MERS-Cov [24,25].…”

Discovery and development of safe-in-man broad-spectrum antiviral agents

“…Cell viability assays include MTT, MTS, resazurin or similar assays, mitochondrial membrane potential-dependent dyes-based assays, esterase cleaved dye-based assays, ATP-ADP assays, and assays that measure glycolytic flux and oxygen consumption. Other cell death assays include LDH enzyme leakage assays, membrane impermeable dye-based assays, and apoptosis assays, such as Annexin V, TUNEL, and caspase assays (Shen et al, 2019). For example, the Cell Titer Glo (CTG) assay quantifies ATP, an indicator of metabolically active living cells, whereas Cell Tox Green assay uses fluorescent asymmetric cyanine dye that stains the DNA of dead cells (Bosl et al, 2019;Bulanova et al, 2017;Ianevski et al, 2018;Muller et al, 2014).…”

“…Given that emetine also inhibits ZIKV, EBOV, RABV, CMV, HCoV-OC43 and HIV-1 infections (Chaves Valadao et al, 2015;MacGibeny et al, 2018;Mukhopadhyay et al, 2016;Shen et al, 2019;Yang et al, 2018), and that it is an FDA-approved antiprotozoal drug, it may represent a promising safe-in-man BSAA candidate. (Carter and Shieh, 2015).…”

“…Oritavancin, dalbavancin and monensin are approved antibiotics, whereas emetine is an anti-protozoal drug. These drugs have been shown to inhibit several corona-as well as some other viral infections (drugvirus.info)(Shen et al, 2019;Zhou et al, 2016). Importantly, clinical investigations into the effectiveness of lopinavir, ritonavir and remdesivir have started recently against 2019-nCoV infections (https://en.wikipedia.org/wiki/2019-nCoV_acute_respiratory_disease).…”

Remdesivir, lopinavir, emetine, and homoharringtonine inhibit SARS-CoV-2 replication in vitro

“…The SARS-CoV-2 virus shared 79.5% genetic homology to the SARS-CoV and both are descendants of bat coronaviruses within the Betacoronavirus genus . Antiviral compounds previously reported to show effect against SARS-CoV or other coronaviruses may be effective against SARS-CoV-2 (Chu et al, 2004;de Wilde et al, 2014;Dyall et al, 2014;Shen et al, 2019;Cao et al, 2015). In addition, remdesivir (GS-5734), a prodrug of adenosine analog with a broad-spectrum antiviral activity against filoviruses, paramyxoviruses, and coronaviruses (Brown et al, 2019;Sheahan et al, 2017;de Wit et al, 2020), was recently confirmed to inhibit 2019-nCoV in vitro .…”