Drugs repurposed for COVID-19 by virtual screening of 6,218 drugs and cell-based assay.

The COVID-19 pandemic caused by SARS-CoV-2 is an unprecedentedly significant health threat, prompting the need for rapidly developing antiviral drugs for the treatment. Drug repurposing is currently one of the most tangible options for rapidly developing drugs for emerging and reemerging viruses. In general, drug repurposing starts with virtual screening of approved drugs employing various computational methods. However, the actual hit rate of virtual screening is very low, and most of the predicted compounds are false positives. Here, we developed a strategy for virtual screening with much reduced false positives through incorporating predocking filtering based on shape similarity and postdocking filtering based on interaction similarity. We applied this advanced virtual screening approach to repurpose 6,218 approved and clinical trial drugs for COVID-19. All 6,218 compounds were screened against main protease and RNA-dependent RNA polymerase of SARS-CoV-2, resulting in 15 and 23 potential repurposed drugs, respectively. Among them, seven compounds can inhibit SARS-CoV-2 replication in Vero cells. Three of these drugs, emodin, omipalisib, and tipifarnib, show anti-SARS-CoV-2 activities in human lung cells, Calu-3. Notably, the activity of omipalisib is 200-fold higher than that of remdesivir in Calu-3. Furthermore, three drug combinations, omipalisib/remdesivir, tipifarnib/omipalisib, and tipifarnib/remdesivir, show strong synergistic effects in inhibiting SARS-CoV-2. Such drug combination therapy improves antiviral efficacy in SARS-CoV-2 infection and reduces the risk of each drug's toxicity. The drug repurposing strategy reported here will be useful for rapidly developing drugs for treating COVID-19 and other viruses.

Novel SARS-CoV-2 entry inhibitors, 2-anilinoquinazolin-4(3H)-one derivatives, show potency as SARS-CoV-2 antivirals in a human ACE2 transgenic mouse model.

The ongoing COVID-19 has not only caused millions of deaths worldwide, but it has also led to economic recession and the collapse of public health systems. The vaccines and antivirals developed in response to the pandemic have improved the situation markedly; however, the pandemic is still not under control with recurring surges. Thus, it is still necessary to develop therapeutic agents. In our previous studies, we designed and synthesized a series of novel 2-anilinoquinazolin-4(3H)-one derivatives, and demonstrated inhibitory activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and MERS-CoV in vitro. We then conducted in vivo studies using modified compounds that are suitable for oral administration. These compounds demonstrated no toxicity in rats and inhibited viral entry. Here, we investigated the in vivo efficacy of these drug candidates against SARS-CoV-2. Three candidate drugs, 7-chloro-2-((3,5-dichlorophenyl)amino)quinazolin-4(3H)-one (1), N-(7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-N-(3,5-dichlorophenyl)acetamide (2), and N-(7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-N-(3,5-difluorophenyl)acetamide (3) were administered orally to hACE2 transgenic mice at a dose of 100 mg/kg. All three drugs improved survival rate and reduced the viral load in the lungs. These results show that the derivatives possess in vivo antiviral efficacy similar to that of molnupiravir, which is currently being used to treat COVID-19. Overall, our data suggest that 2-anilinoquinazolin-4(3H)-one derivatives are promising as potential oral antiviral drug candidates against SARS-CoV-2 infection.

Discovery of 2-aminoquinolone acid derivatives as potent inhibitors of SARS-CoV-2.

The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to threaten human health and create socioeconomic problems worldwide. A library of 200,000 small molecules from the Korea Chemical Bank (KCB) were evaluated for their inhibitory activities against SARS-CoV-2 in a phenotypic-based screening assay to discover new therapeutics to combat COVID-19. A primary hit of this screen was the quinolone structure-containing compound 1. Based on the structure of compound 1 and enoxacin, which is a quinolone-based antibiotic previously reported to have weak activity against SARS-CoV-2, we designed and synthesized 2-aminoquinolone acid derivatives. Among them, compound 9b exhibited potent antiviral activity against SARS-CoV-2 (EC50 = 1.5 µM) without causing toxicity, while having satisfactory in vitro PK profiles. This study shows that 2-aminoquinolone acid 9b provides a promising new template for developing anti-SARS-CoV-2 entry inhibitors.

Feruloyl Esterase (LaFae) from Lactobacillus acidophilus: Structural Insights and Functional Characterization for Application in Ferulic Acid Production.

Ferulic acid and related hydroxycinnamic acids, used as antioxidants and preservatives in the food, cosmetic, pharmaceutical and biotechnology industries, are among the most abundant phenolic compounds present in plant biomass. Identification of novel compounds that can produce ferulic acid and hydroxycinnamic acids, that are safe and can be mass-produced, is critical for the sustainability of these industries. In this study, we aimed to obtain and characterize a feruloyl esterase (LaFae) from Lactobacillus acidophilus. Our results demonstrated that LaFae reacts with ethyl ferulate and can be used to effectively produce ferulic acid from wheat bran, rice bran and corn stalks. In addition, xylanase supplementation was found to enhance LaFae enzymatic hydrolysis, thereby augmenting ferulic acid production. To further investigate the active site configuration of LaFae, crystal structures of unliganded and ethyl ferulate-bound LaFae were determined at 2.3 and 2.19 Å resolutions, respectively. Structural analysis shows that a Phe34 residue, located at the active site entrance, acts as a gatekeeper residue and controls substrate binding. Mutating this Phe34 to Ala produced an approximately 1.6-fold increase in LaFae activity against p-nitrophenyl butyrate. Our results highlight the considerable application potential of LaFae to produce ferulic acid from plant biomass and agricultural by-products.

Lancemaside A from Codonopsis lanceolata: Studies on Antiviral Activity and Mechanism of Action against SARS-CoV-2 and Its Variants of Concern.

Several plant-derived natural products with anti-SARS-CoV-2 activity have been evaluated for the potential to serve as chemotherapeutic agents for the treatment of COVID-19. Codonopsis lanceolata (CL) has long been used as a medicinal herb in East Asian countries to treat inflammatory diseases of the respiratory system but its antiviral activity has not been investigated so far. Here, we showed that CL extract and its active compound lancemaside A (LA) displayed potent inhibitory activity against SARS-CoV-2 infection using a pseudotyped SARS-CoV-2 entry assay system. We demonstrated that this inhibitory effect of LA was due to the alteration of membrane cholesterol and blockade of the membrane fusion between SARS-CoV-2 and host cells by filipin staining and cell-based membrane fusion assays. Our findings also showed that LA, as a membrane fusion blocker, could impede the endosomal entry pathway of SARS-CoV-2 and its variants of concern (VOCs), including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529), in Vero cells with similar of IC50 values ranging from 2.23 to 3.37 µM as well as the TMPRSS2-mediated viral entry pathway in A549 cells overexpressing ACE2 and TMPRSS2 with IC50 value of 3.92 µM. We further demonstrated that LA could prevent the formation of multinucleated syncytia arising from SARS-CoV-2 spike protein-mediated membrane fusion. Altogether, the findings reported here suggested that LA could be a broad-spectrum anti-SARS-CoV-2 therapeutic agent by targeting the fusion of viral envelope with the host cell membrane.

Synthesis and structure-activity relationship study of saponin-based membrane fusion inhibitors against SARS-CoV-2.

We previously discovered that triterpenoid saponin platycodin D inhibits the SARS-CoV-2 entry to the host cell. Herein, we synthesized various saponin derivatives and established a structure-activity relationship of saponin-based antiviral agents against SARS-CoV-2. We discovered that the C3-glucose, the C28-oligosaccharide moiety that consist of (→3)-ß-d-Xyl-(1 â†’ 4)-α-l-Rham-(1 â†’ 2)-ß-d-Ara-(1 â†’ ) as the last three sugar units, and the C16-hydroxyl group were critical components of saponin-based coronavirus cell entry inhibitors. These findings enabled us to develop minimal saponin-based antiviral agents that are equipotent to the originally discovered platycodin D. We found that our saponin-based antiviral agents inhibited both the endosomal and transmembrane protease serine 2-mediated cell surface viral entries. Cell fusion assay experiment revealed that our newly developed compounds inhibit the SARS-CoV-2 entry by blocking the fusion between the viral and host cell membranes. The effectiveness of the newly developed antiviral agents over various SARS-CoV-2 variants hints at the broad-spectrum antiviral efficacy of saponin-based therapeutics against future coronavirus variants.

Comparative analysis of antiviral efficacy of FDA-approved drugs against SARS-CoV-2 in human lung cells.

Drug repositioning represents an effective way to control the current COVID-19 pandemic. Previously, we identified 24 FDA-approved drugs which exhibited substantial antiviral effect against severe acute respiratory syndrome coronavirus 2 in Vero cells. Since antiviral efficacy could be altered in different cell lines, we developed an antiviral screening assay with human lung cells, which is more appropriate than Vero cell. The comparative analysis of antiviral activities revealed that nafamostat is the most potent drug in human lung cells (IC50 = 0.0022 µM).

Design, synthesis and biological evaluation of 2-aminoquinazolin-4(3H)-one derivatives as potential SARS-CoV-2 and MERS-CoV treatments.

Despite the rising threat of fatal coronaviruses, there are no general proven effective antivirals to treat them. 2-Aminoquinazolin-4(3H)-one derivatives were newly designed, synthesized, and investigated to show the inhibitory effects on SARS-CoV-2 and MERS-CoV. Among the synthesized derivatives, 7-chloro-2-((3,5-dichlorophenyl)amino)quinazolin-4(3H)-one (9g) and 2-((3,5-dichlorophenyl)amino)-5-hydroxyquinazolin-4 (3H)-one (11e) showed the most potent anti-SARS-CoV-2 activities (IC50 < 0.25 µM) and anti-MERS-CoV activities (IC50 < 1.1 µM) with no cytotoxicity (CC50 > 25 µM). In addition, both compounds showed acceptable results in metabolic stabilities, hERG binding affinities, CYP inhibitions, and preliminary PK studies.

Discovery of cyclic sulfonamide derivatives as potent inhibitors of SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) continues to spread worldwide, with 25 million confirmed cases and 800 thousand deaths. Effective treatments to target SARS-CoV-2 are urgently needed. In the present study, we have identified a class of cyclic sulfonamide derivatives as novel SARS-CoV-2 inhibitors. Compound 13c of the synthesized compounds exhibited robust inhibitory activity (IC50 = 0.88 µM) against SARS-CoV-2 without cytotoxicity (CC50 > 25 µM), with a selectivity index (SI) of 30.7. In addition, compound 13c exhibited high oral bioavailability (77%) and metabolic stability with good safety profiles in hERG and cytotoxicity studies. The present study identified that cyclic sulfonamide derivatives are a promising new template for the development of anti-SARS-CoV-2 agents.

Identification of Antiviral Drug Candidates against SARS-CoV-2 from FDA-Approved Drugs.

Drug repositioning is the only feasible option to immediately address the COVID-19 global challenge. We screened a panel of 48 FDA-approved drugs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which were preselected by an assay of SARS-CoV. We identified 24 potential antiviral drug candidates against SARS-CoV-2 infection. Some drug candidates showed very low 50% inhibitory concentrations (IC50s), and in particular, two FDA-approved drugs-niclosamide and ciclesonide-were notable in some respects.

Synthesis and biological evaluation of 3-acyl-2-phenylamino-1,4-dihydroquinolin-4(1H)-one derivatives as potential MERS-CoV inhibitors.

3-Acyl-2-phenylamino-1,4-dihydroquinolin-4(1H)-one derivatives were synthesized and evaluated to show high anti-MERS-CoV inhibitory activities. Among them, 6,8-difluoro-3-isobutyryl-2-((2,3,4-trifluorophenyl)amino)quinolin-4(1H)-one (6u) exhibits high inhibitory effect (IC50 = 86 nM) and low toxicity (CC50 > 25 µM). Moreover, it shows good metabolic stability, low hERG binding affinity, no cytotoxicity, and good in vivo PK properties.

Characterization and Immobilization of a Novel SGNH Family Esterase (LaSGNH1) from Lactobacillus acidophilus NCFM.

The SGNH family esterases are highly effective biocatalysts due to their strong catalytic efficiencies, great stabilities, relatively small sizes, and ease of immobilization. Here, a novel SGNH family esterase (LaSGNH1) from Lactobacillus acidophilus NCFM, which has homologues in many Lactobacillus species, was identified, characterized, and immobilized. LaSGNH1 is highly active towards acetate- or butyrate-containing compounds, such as p-nitrophenyl acetate or 1-naphthyl acetate. Enzymatic properties of LaSGNH1, including thermal stability, optimum pH, chemical stability, and urea stability, were investigated. Interestingly, LaSGNH1 displayed a wide range of substrate specificity that included glyceryl tributyrate, tert-butyl acetate, and glucose pentaacetate. Furthermore, immobilization of LaSGNH1 by crosslinked enzyme aggregates (CLEAs) showed enhanced thermal stability and efficient recycling property. In summary, this work paves the way for molecular understandings and industrial applications of a novel SGNH family esterase (LaSGNH1) from Lactobacillus acidophilus.

Rise in broadly cross-reactive adaptive immunity against human ß-coronaviruses in MERS-recovered patients during the COVID-19 pandemic.

To develop a universal coronavirus (CoV) vaccine, long-term immunity against multiple CoVs, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, Middle East respiratory syndrome (MERS)-CoV, and future CoV strains, is crucial. Following the 2015 Korean MERS outbreak, we conducted a long-term follow-up study and found that although neutralizing antibodies and memory T cells against MERS-CoV declined over 5 years, some recovered patients exhibited increased antibody levels during the COVID-19 pandemic. This likely resulted from cross-reactive immunity induced by SARS-CoV-2 vaccines or infections. A significant correlation in antibody responses across various CoVs indicates shared immunogenic epitopes. Two epitopes-the spike protein's stem helix and intracellular domain-were highly immunogenic after MERS-CoV infection and after SARS-CoV-2 vaccination or infection. In addition, memory T cell responses, especially polyfunctional CD4+ T cells, were enhanced during the pandemic, correlating significantly with MERS-CoV spike-specific antibodies and neutralizing activity. Therefore, incorporating these cross-reactive and immunogenic epitopes into pan-CoV vaccine formulations may facilitate effective vaccine development.

Rapid discovery and classification of inhibitors of coronavirus infection by pseudovirus screen and amplified luminescence proximity homogeneous assay.

To identify potent antiviral compounds, we introduced a high-throughput screen platform that can rapidly classify hit compounds according to their target. In our platform, we performed a compound screen using a lentivirus-based pseudovirus presenting a spike protein of coronavirus, and we evaluated the hit compounds using an amplified luminescence proximity homogeneous assay (alpha) test with purified host receptor protein and the receptor binding domain of the viral spike. With our screen platform, we were able to identify both spike-specific compounds (class I) and broad-spectrum antiviral compounds (class II). Among the hit compounds, thiosemicarbazide was identified to be selective to the interaction between the viral spike and its host cell receptor, and we further optimized the binding potency of thiosemicarbazide through modification of the pyridine group. Among the class II compounds, we found raloxifene and amiodarone to be highly potent against human coronaviruses including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. In particular, using analogs of the benzothiophene moiety, which is also present in raloxifene, we have identified benzothiophene as a novel structural scaffold for broad-spectrum antivirals. This work highlights the strong utility of our screen platform using a pseudovirus assay and an alpha test for rapid identification of potential antiviral compounds and their mechanism of action, which can lead to the accelerated development of therapeutics against newly emerging viral infections.

Natural Polyphenols, 1,2,3,4,6-O-Pentagalloyglucose and Proanthocyanidins, as Broad-Spectrum Anticoronaviral Inhibitors Targeting Mpro and RdRp of SARS-CoV-2.

The natural plant dietary polyphenols 1,2,3,4,6-O-Pentagalloylglucose (PGG) and proanthocyanidin (PAC) have potent antioxidant activity and a variety of pharmacological activities, including antiviral activity. In this study, we examined the inhibitory effect of PGG and PAC on SARS-CoV-2 virus infection, and elucidated its mode of action. PGG and PAC have dose-dependent inhibitory activity against SARS-CoV-2 infection in Vero cells. PGG has a lower IC50 (15.02 ± 0.75 µM) than PAC (25.90 ± 0.81 µM), suggesting that PGG has better inhibitory activity against SARS-CoV-2 than PAC. The PGG and PAC inhibit similar Mpro activities in a protease activity assay, with IC50 values of 25-26 µM. The effects of PGG and PAC on the activity of the other essential SARS-CoV-2 viral protein, RdRp, were analyzed using a cell-based activity assay system. The activity of RdRp is inhibited by PGG and PAC, and PGG has a lower IC50 (5.098 ± 1.089 µM) than PAC (21.022 ± 1.202 µM), which is consistent with their inhibitory capacity of SARS-CoV-2 infection. PGG and PAC also inhibit infection by SARS-CoV and MERS-CoV. These data indicate that PGG and PAC may be candidate broad-spectrum anticoronaviral therapeutic agents, simultaneously targeting the Mpro and RdRp proteins of SARS-CoV-2.

Dual functional roles of a novel bifunctional ß-lactamase/esterase from Lactococcus garvieae.

A novel bifunctional ß-lactamase/esterase (LgLacI), which is capable of hydrolyzing ß-lactam-containing antibiotics including ampicillin, oxacillin, and cefotaxime as well as synthesizing biodiesels, was cloned from Lactococcus garvieae. Unlike most bacterial esterases/lipases that have G-x-S-x-G motif, LgLacI, which contains S-x-x-K catalytic motif, has sequence similarities to bacterial family VIII esterase as well as ß-lactamases. The catalytic properties of LgLacI were explored using a wide range of biochemical methods including spectroscopy, assays, structural modeling, mutagenesis, and chromatography. We confirmed the bifunctional property of LgLacI hydrolyzing both esters and ß-lactam antibiotics. This study provides novel perspectives into a bifunctional enzyme from L. garvieae, which can degrade ß-lactam antibiotics with high esterase activity.

Anticoronaviral Activity of the Natural Phloroglucinols, Dryocrassin ABBA and Filixic Acid ABA from the Rhizome of Dryopteris crassirhizoma by Targeting the Main Protease of SARS-CoV-2.

The rhizome of Dryopteris crassirhizoma Nakai. (Dryopteridaceae) has been used in traditional medicine in East Asia and has recently been reported to have anticancer, anti-inflammation, and antibacterial activity as well as antiviral activity. Natural phloroglucinols from D. crassirhizoma, dryocrassin ABBA and filixic acid ABA were reported to inhibit influenza virus infection with an inhibitory activity on neuraminidase. In this study, we found that dryocrassin ABBA and filixic acid ABA have an inhibitory activity against the main protease of SARS-CoV-2. Therefore, dryocrassin ABBA and filixic acid ABA exhibited inhibitory activity against SARS-CoV-2 infection in Vero cells dose-dependently using the immunofluorescence-based antiviral assays. Moreover, these compounds inhibited SARS-CoV and MERS-CoV infection, suggesting their broad-spectrum anticoronaviral activity. In addition, a 5-day repeated-dose toxicity study of dryocrassin ABBA and filixic acid ABA suggested that an approximately lethal dose of these compounds in mice was >10 mg/kg. Pharmacokinetic studies of dryocrassin ABBA showed good microsomal stability, low hERG inhibition, and low CYP450 inhibition. In vivo pharmacokinetic properties of dryocrassin ABBA showed a long half-life (5.5-12.6 h) and high plasma exposure (AUC 19.3-65 µg·h/mL). Therefore, dryocrassin ABBA has therapeutic potential against emerging coronavirus infections, including COVID-19.

Chemical screen uncovers novel structural classes of inhibitors of the papain-like protease of coronaviruses.

The papain-like protease (PLpro) of coronaviruses is an attractive antiviral target to inhibit both viral replication and interference of the host immune response. We have identified and characterized three novel classes of small molecules, thiophene, cyanofuran, and triazoloquinazoline, as PLpro inhibitors. Thiophene inhibited the PLpro of two major coronaviruses, Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV) including SARS-CoV-2, while cyanofuran and triazoloquinazoline more selectively inhibited MERS-CoV PLpro. Unlike GRL0617, a known PLpro inhibitor, all three compounds contain no naphthyl group but like GRL0617 were predicted to fit on the cleft near the BL2 loop. Docking studies further revealed that the location and direction of the binding determined their specificity to different coronaviruses. Together, our work demonstrates that the BL2 loop and nearby regions are outstanding druggable targets, and our three inhibitors can be applicable to the development of therapeutics for coronavirus infection.

Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibits the infection of SARS-CoV-2 variants and syncytium formation.

The continuous emergence of SARS-CoV-2 variants prolongs COVID-19 pandemic. Although SARS-CoV-2 vaccines and therapeutics are currently available, there is still a need for development of safe and effective drugs against SARS-CoV-2 and also for preparedness for the next pandemic. Here, we discover that astersaponin I (AI), a triterpenoid saponin in Aster koraiensis inhibits SARS-CoV-2 entry pathways at the plasma membrane and within the endosomal compartments mainly by increasing cholesterol content in the plasma membrane and interfering with the fusion of SARS-CoV-2 envelope with the host cell membrane. Moreover, we find that this functional property of AI as a fusion blocker enables it to inhibit the infection with SARS-CoV-2 variants including the Alpha, Beta, Delta, and Omicron with a similar efficacy, and the formation of syncytium, a multinucleated cells driven by SARS-CoV-2 spike protein-mediated cell-to-cell fusion. Finally, we claim that the triterpene backbone as well as the attached hydrophilic sugar moieties of AI are structurally important for its inhibitory activity against the membrane fusion event. Overall, this study demonstrates that AI is a natural viral fusion inhibitor and proposes that it can be a broad-spectrum antiviral agent against current COVID-19 pandemic and future outbreaks of novel viral pathogens.

Optimization of 2-Aminoquinazolin-4-(3H)-one Derivatives as Potent Inhibitors of SARS-CoV-2: Improved Synthesis and Pharmacokinetic Properties.

We previously reported the potent antiviral effect of the 2-aminoquinazolin-4-(3H)-one 1, which shows significant activity (IC50 = 0.23 µM) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with no cytotoxicity. However, it is necessary to improve the in vivo pharmacokinetics of compound 1 because its area under the curve (AUC) and maximum plasma concentration are low. Here, we designed and synthesized N-substituted quinazolinone derivatives that had good pharmacokinetics and that retained their inhibitory activity against SARS-CoV-2. These compounds were conveniently prepared on a large scale through a one-pot reaction using Dimroth rearrangement as a key step. The synthesized compounds showed potent inhibitory activity, low binding to hERG channels, and good microsomal stability. In vivo pharmacokinetic studies showed that compound 2b had the highest exposure (AUC24h = 41.57 µg∙h/mL) of the synthesized compounds. An in vivo single-dose toxicity evaluation of compound 2b at 250 and 500 mg/kg in rats resulted in no deaths and an approximate lethal dose greater than 500 mg/kg. This study shows that N-acetyl 2-aminoquinazolin-4-(3H)-one 2b is a promising lead compound for developing anti-SARS-CoV-2 agents.