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The ongoing global threats posed by COVID-19 pandemic, catalyzed by SARS-CoV-2, underscores the pressing need for effective antiviral strategies. The viral non-structural protein 1 (Nsp1) significantly influences pathogenicity by impeding host protein expression and enhancing viral RNA translation through its interaction with the stem-loop 1 (SL1) in the 5' untranslated region (UTR). We have developed a novel dual-luciferase reporter assay, designed to investigate the critical Nsp1-SL1 interaction, and identified P23E02 as a potential inhibitor. Our investigation, combining molecular docking studies and alanine mutagenesis, has unveiled that P23E02 disrupts Nsp1-40S ribosomal subunit interaction, liberating translational inhibition and empowering host antiviral responses. P23E02 exhibits antiviral efficacy against various sarbecoviruses, making it a promising candidate for combatting COVID-19 and related diseases. This study underscores the therapeutic potential of targeting the Nsp1/SL1 axis and lays the foundation for innovative antiviral interventions, ultimately fortifying global preparedness against future viral threats., (© 2024 Wiley‐VCH GmbH.)

The immune escape of Omicron variants significantly subsides by the third dose of an mRNA vaccine. However, it is unclear how Omicron variant-neutralizing antibodies develop under repeated vaccination. We analyze blood samples from 41 BNT162b2 vaccinees following the course of three injections and analyze their B-cell receptor (BCR) repertoires at six time points in total. The concomitant reactivity to both ancestral and Omicron receptor-binding domain (RBD) is achieved by a limited number of BCR clonotypes depending on the accumulation of somatic hypermutation (SHM) after the third dose. Our findings suggest that SHM accumulation in the BCR space to broaden its specificity for unseen antigens is a counterprotective mechanism against virus variant immune escape., (© 2024. The Author(s).)

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.

BACKGROUND: Nafamostat mesylate is a potent in vitro antiviral agent that inhibits the host transmembrane protease serine 2 enzyme used by severe acute respiratory syndrome coronavirus 2 for cell entry. METHODS: This open-label, pragmatic, randomized clinical trial in Australia, New Zealand, and Nepal included noncritically ill hospitalized patients with coronavirus disease 2019 (Covid-19). Participants were randomly assigned to usual care or usual care plus nafamostat. The primary end point was death (any cause) or receipt of new invasive or noninvasive ventilation or vasopressor support within 28 days after randomization. Analysis was with a Bayesian logistic model in which an adjusted odds ratio <1.0 indicates improved outcomes with nafamostat. Enrollment was closed due to falling numbers of eligible patients. RESULTS: We screened 647 patients in 21 hospitals (15 in Australia, 4 in New Zealand, and 2 in Nepal) and enrolled 160 participants from May 2021 to August 2022. In the intention-to-treat population, the primary end point occurred in 8 (11%) of 73 patients with usual care and 4 (5%) of 82 with nafamostat. The median adjusted odds ratio for the primary end point for nafamostat was 0.40 (95% credible interval, 0.12 to 1.34) with a posterior probability of effectiveness (adjusted odds ratio <1.0) of 93%. For usual care compared with nafamostat, hyperkalemia occurred in 1 (1%) of 67 and 7 (9%) of 78 participants, respectively, and clinically relevant bleeding occurred in 1 (1%) of 73 and 7 (8%) of 82 participants. CONCLUSIONS: Among hospitalized patients with Covid-19, there was a 93% posterior probability that nafamostat reduced the odds of death or organ support. Prespecified stopping criteria were not met, precluding definitive conclusions. Hyperkalemia and bleeding were more common with nafamostat. (Funded by ASCOT and others; ClinicalTrials.gov number, NCT04483960.)

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., (© 2023 Wiley Periodicals LLC.)

Here, we describe detailed steps to constitute an in vitro assay for monitoring papain-like protease of coronavirus and a cell-based immunofluorescence infection assay. These assays can be adapted for high-throughput screening to determine the efficacy of novel protease inhibitors of coronaviruses and other viruses. In addition, cell-based immunofluorescence infection assay can be used to visually analyze antiviral efficacy of any novel compounds. For complete details on the use and execution of this protocol, please refer to Jeong et al. (2022). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background/aims: We used next-generation sequencing (NGS) to analyze resistance-associated substitutions (RASs) and retreatment outcomes in patients with chronic hepatitis C virus (HCV) infection who failed direct-acting antiviral agent (DAA) treatment in South Korea., Methods: Using prospectively collected data from the Korean HCV cohort study, we recruited 36 patients who failed DAA treatment in 10 centers between 2007 and 2020; 29 blood samples were available from 24 patients. RASs were analyzed using NGS., Results: RASs were analyzed for 13 patients with genotype 1b, 10 with genotype 2, and one with genotype 3a. The unsuccessful DAA regimens were daclatasvir+asunaprevir (n=11), sofosbuvir+ribavirin (n=9), ledipasvir/sofosbuvir (n=3), and glecaprevir/pibrentasvir (n=1). In the patients with genotype 1b, NS3, NS5A, and NS5B RASs were detected in eight, seven, and seven of 10 patients at baseline and in four, six, and two of six patients after DAA failure, respectively. Among the 10 patients with genotype 2, the only baseline RAS was NS3 Y56F, which was detected in one patient. NS5A F28C was detected after DAA failure in a patient with genotype 2 infection who was erroneously treated with daclatasvir+asunaprevir. After retreatment, 16 patients had a 100% sustained virological response rate., Conclusion: NS3 and NS5A RASs were commonly present at baseline, and there was an increasing trend of NS5A RASs after failed DAA treatment in genotype 1b. However, RASs were rarely present in patients with genotype 2 who were treated with sofosbuvir+ribavirin. Despite baseline or treatment-emergent RASs, retreatment with pan-genotypic DAA was highly successful in Korea, so we encourage active retreatment after unsuccessful DAA treatment.

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 (EC 50  = 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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 IC 50 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 IC 50 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.

Following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, subsequent ChAdOx1 nCoV-19 vaccination induced similar neutralizing antibody levels against the original strain but significantly higher levels against the Omicron variant compared to those who were not vaccinated. Prior SARS-CoV-2 infection exhibited higher neutralization antibody titers than vaccination alone for both original strains and the Omicron variant., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: The views expressed in this publication are those of the authors., (© 2022 The Author(s).)

The molecular manifestations of host cells responding to SARS-CoV-2 and its evolving variants of infection are vastly different across the studied models and conditions, imposing challenges for host-based antiviral drug discovery. Based on the postulation that antiviral drugs tend to reverse the global host gene expression induced by viral infection, we retrospectively evaluated hundreds of signatures derived from 1,700 published host transcriptomic profiles of SARS/MERS/SARS-CoV-2 infection using an iterative data-driven approach. A few of these signatures could be reversed by known anti-SARS-CoV-2 inhibitors, suggesting the potential of extrapolating the biology for new variant research. We discovered IMD-0354 as a promising candidate to reverse the signatures globally with nanomolar IC 50 against SARS-CoV-2 and its five variants. IMD-0354 stimulated type I interferon antiviral response, inhibited viral entry, and down-regulated hijacked proteins. This study demonstrates that the conserved coronavirus signatures and the transcriptomic reversal approach that leverages polypharmacological effects could guide new variant therapeutic discovery., Competing Interests: The authors declare that they have no competing interests., (© 2022 The Author(s).)

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)-β-d-Xyl-(1 → 4)-α-l-Rham-(1 → 2)-β-d-Ara-(1 → ) 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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In the absence of drugs to treat or prevent COVID-19, drug repurposing can be a valuable strategy. Despite a substantial number of clinical trials, drug repurposing did not deliver on its promise. While success was observed with some repurposed drugs (e.g., remdesivir, dexamethasone, tocilizumab, baricitinib), others failed to show clinical efficacy. One reason is the lack of clear translational processes based on adequate preclinical profiling before clinical evaluation. Combined with limitations of existing in vitro and in vivo models, there is a need for a systematic approach to urgent antiviral drug development in the context of a global pandemic. We implemented a methodology to test repurposed and experimental drugs to generate robust preclinical evidence for further clinical development. This translational drug development platform comprises in vitro, ex vivo, and in vivo models of SARS-CoV-2, along with pharmacokinetic modeling and simulation approaches to evaluate exposure levels in plasma and target organs. Here, we provide examples of identified repurposed antiviral drugs tested within our multidisciplinary collaboration to highlight lessons learned in urgent antiviral drug development during the COVID-19 pandemic. Our data confirm the importance of assessing in vitro and in vivo potency in multiple assays to boost the translatability of pre-clinical data. The value of pharmacokinetic modeling and simulations for compound prioritization is also discussed. We advocate the need for a standardized translational drug development platform for mild-to-moderate COVID-19 to generate preclinical evidence in support of clinical trials. We propose clear prerequisites for progression of drug candidates for repurposing into clinical trials. Further research is needed to gain a deeper understanding of the scope and limitations of the presented translational drug development platform.

The COVID-19 pandemic continues to spread around the world, with several new variants emerging, particularly those of concern (VOCs). Omicron (B.1.1.529), a recent VOC with many mutations in the spike protein's receptor-binding domain (RBD), has attracted a great deal of scientific and public interest. We previously developed two D-peptide inhibitors for the infection of the original SARS-CoV-2 and its VOCs, alpha and beta, in vitro . Here, we demonstrated that Covid3 and Covid&#95;extended&#95;1 maintained their high-affinity binding (29.4-31.3 nM) to the omicron RBD. Both D-peptides blocked the omicron variant in vitro infection with IC50s of 3.13 and 5.56 μM, respectively. We predicted that Covid3 shares a larger overlapping binding region with the ACE2 binding motif than different classes of neutralizing monoclonal antibodies. We envisioned the design of D-peptide inhibitors targeting the receptor-binding motif as the most promising approach for inhibiting current and future VOCs of SARS-CoV-2, given that the ACE2 binding interface is more limited to tolerate mutations than most of the RBD's surface.

We previously reported the potent antiviral effect of the 2-aminoquinazolin-4-(3 H )-one 1 , which shows significant activity (IC 50 = 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 (AUC 24h = 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-(3 H )-one 2b is a promising lead compound for developing anti-SARS-CoV-2 agents.

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 IC 50 (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 IC 50 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 IC 50 (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.

Study Design: Experimental study., Objective: In this study, the ambient temperature of a radiofrequency (RF) electrode tip was compared and analyzed in terms of products, mode, flow quantity, and flow rate., Summary of Background Data: Endoscopic spine surgery is a widely used operation for degenerative lumbar stenosis and herniated lumbar disc. To perform endoscopic spine surgery, dedicated instruments like a RF generator and electrode are essential., Methods: An evaluation system capable of measuring temperature under equal conditions at a certain distance from the electrode tip was manufactured. The distance between the electrode tip and the temperature sensor was set to 1, 5, and 10 mm. The flow quantities of 0, 50, 100, and 150 mL/min and the flow rates of 0, 0.20, 0.53, and 0.80 m/s were compared and statistically analyzed., Results: The temperatures measured in the experiments conducted on the four combinations of RF device showed similar values, and showed differences according to the characteristics of each mode of the RF. As the distance between the electrode tip and the temperature sensor increased, the temperature decreased, and as flow quantity or flow rate increased, the temperature decreased. The maximum temperatures differed significantly according to flow quantity, between flow quantities of 0 and 100 mL/min (P  = 0.03) and between 0 and 150 mL/min (P ≤ 0.01). The maximum temperatures also differed significantly between the flow rate of 0 m/s, and the flow rates of 0.20, 0.53, and 0.80 m/s, with P ≤ 0.01 in all three comparisons., Conclusion: This is the first study in which we made a customized RF temperature evaluation system and verified the temperature changes in various environments. When irrigation was performed, we could confirm that the maximum temperature was less than 60°C. Irrigation is considered essential in endoscopic spine surgery., Level of Evidence: 3., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

The emergence of SARS-CoV-2 variants is a significant concern in developing effective therapeutics and vaccines in the middle of the ongoing COVID-19 pandemic. Here, we have identified a novel small molecule that inhibited the interactions between SARS-CoV-2 spike RBDs and ACE2 by modulating ACE2 without impairing its enzymatic activity necessary for normal physiological functions. Furthermore, the identified compounds suppressed viral infection in cultured cells by inhibiting the entry of ancestral and variant SARS-CoV-2. Our study suggests that targeting ACE2 could be a novel therapeutic strategy to inhibit SARS-CoV-2 entry into host cells and prevent the development of COVID-19., (© 2022 Wiley-VCH GmbH.)

Despite the continuing global crisis caused by coronavirus disease 2019 (COVID-19), there is still no effective treatment. Therefore, we designed and synthesized a novel series of 2-benzylaminoquinazolin-4(3 H )-one derivatives and demonstrated that they are effective against SARS-CoV-2. Among the synthesized derivatives, 7-chloro-2-(((4-chlorophenyl)(phenyl)methyl)amino)quinazolin-4(3 H )-one (Compound 39 ) showed highest anti-SARS-CoV-2 activity, with a half-maximal inhibitory concentration value greater than that of remdesivir (IC 50  = 4.2 μM vs. 7.6 μM, respectively), which gained urgent approval from the U.S. Food and Drug Administration. In addition, Compound 39 showed good results in various assays measuring metabolic stability, human ether a-go-go, Cytochromes P450 (CYPs) inhibition, and plasma protein binding (PPB), and showed better solubility and pharmacokinetics than our previous work., (© 2022 Korean Chemical Society, Seoul & Wiley‐VCH GmbH.)

Coronavirus disease 2019 (COVID-19) is an acute respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Other coronaviruses (CoVs) can also infect humans, although the majority cause only mild respiratory symptoms. Because early diagnosis of SARS-CoV-2 is critical for preventing further transmission events and improving clinical outcomes, it is important to be able to distinguish SARS-CoV-2 from other SARS-related CoVs in respiratory samples. Therefore, we developed and evaluated a novel reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting the genes encoding the spike (S) and membrane (M) proteins to enable the rapid identification of SARS-CoV-2, including several new circulating variants and other emerging SARS-like CoVs. By analysis of in vitro-transcribed mRNA, we established multiplex RT-qPCR assays capable of detecting 5 × 10° copies/reaction. Using RNA extracted from cell culture supernatants, our multiple simultaneous SARS-CoV-2 assays had a limit of detection of 1 × 10° TCID 50 /mL and showed no cross-reaction with human CoVs or other respiratory viruses. We also validated our method using human clinical samples from patients with COVID-19 and healthy individuals, including nasal swab and sputum samples. This novel one-step multiplex RT-qPCR assay can be used to improve the laboratory diagnosis of human-pathogenic CoVs, including SARS-CoV-2, and may be useful for the identification of other SARS-like CoVs of zoonotic origin., (© 2022. The Author(s).)

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.

Diagnostics, vaccines, and drugs are indispensable tools and control measures employed to overcome infectious diseases such as coronavirus disease 2019 (COVID-19). Diagnostic tools based on RT-PCR were developed early in the COVID-19 pandemic and were urgently required for quarantine (testing, tracing and isolation). Vaccines such as mRNA vaccines and virus-vectored vaccines were also successfully developed using new platform technologies within one year after identifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of COVID-19. Drug development has been conducted in various ways including drug repurposing, convalescent plasma therapy, and monoclonal antibody development. Among the above efforts, this review examines COVID-19 drug development along with the related and upcoming challenges.

We identified small-molecule enhancers of cellular stress granules by observing molecular crowding of proteins and RNAs in a time-dependent manner. Hit molecules sensitized the IRF3-mediated antiviral mechanism in the presence of poly(I:C) and inhibited the replication of SARS-CoV-2 by inducing stress granule formation. Thus, modulating multimolecular crowding can be a promising strategy against SARS-CoV-2.

The global efforts in the past year have led to the discovery of nearly 200 drug repurposing candidates for COVID-19. Gaining more insights into their mechanisms of action could facilitate a better understanding of infection and the development of therapeutics. Leveraging large-scale drug-induced gene expression profiles, we found 36% of the active compounds regulate genes related to cholesterol homeostasis and microtubule cytoskeleton organization. Following bioinformatics analyses revealed that the expression of these genes is associated with COVID-19 patient severity and has predictive power on anti-SARS-CoV-2 efficacy in vitro. Monensin, a top new compound that regulates these genes, was further confirmed as an inhibitor of SARS-CoV-2 replication in Vero-E6 cells. Interestingly, drugs co-targeting cholesterol homeostasis and microtubule cytoskeleton organization processes more likely present a synergistic effect with antivirals. Therefore, potential therapeutics could be centered around combinations of targeting these processes and viral proteins., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Cardiotonic steroids are steroid-like natural compounds known to inhibit Na + /K + -ATPase pumps. To develop a broad-spectrum antiviral drug against the emerging coronavirus infection, this study assessed the antiviral properties of these compounds. The activity of seven types of cardiotonic steroids against the MERS-CoV, SARS-CoV, and SARS-CoV-2 coronavirus varieties was analyzed using immunofluorescence antiviral assay in virus-infected cells. Bufalin, cinobufagin, and telocinobufagin showed high anti-MERS-CoV activities (IC 50 , 0.017~0.027 μM); bufalin showed the most potent anti-SARS-CoV and SARS-CoV-2 activity (IC 50 , 0.016~0.019 μM); cinobufotalin and resibufogenin showed comparatively low anti-coronavirus activity (IC 50 , 0.231~1.612 μM). Differentially expressed genes in Calu3 cells treated with cinobufagin, telocinobufagin, or bufalin, which had high antiviral activity during MERS-CoV infection were analyzed using QuantSeq 3' mRNA-Seq analysis and data showed similar gene expression patterns. Furthermore, the intraperitoneal administration of 10 mg/kg/day bufalin, cinobufagin, or digitoxin induced 100% death after 1, 2, and 4 days in 5-day repeated dose toxicity studies and it indicated that bufalin had the strongest toxicity. Pharmacokinetic studies suggested that telocinobufagin, which had high anti-coronavirus activity and low toxicity, had better microsomal stability, lower CYP inhibition, and better oral bioavailability than cinobufagin. Therefore, telocinobufagin might be the most promising cardiotonic steroid as a therapeutic for emerging coronavirus infections, including COVID-19.

Blocking the association between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) is an attractive therapeutic approach to prevent the virus from entering human cells. While antibodies and other modalities have been developed to this end, d-amino acid peptides offer unique advantages, including serum stability, low immunogenicity, and low cost of production. Here, we designed potent novel D-peptide inhibitors that mimic the ACE2 α1-binding helix by searching a mirror-image version of the PDB. The two best designs bound the RBD with affinities of 29 and 31 nM and blocked the infection of Vero cells by SARS-CoV-2 with IC 50 values of 5.76 and 6.56 μM, respectively. Notably, both D-peptides neutralized with a similar potency the infection of two variants of concern: B.1.1.7 and B.1.351 in vitro . These potent D-peptide inhibitors are promising lead candidates for developing SARS-CoV-2 prophylactic or therapeutic treatments.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a causative agent of the coronavirus disease 2019 (COVID-19) pandemic, and the development of therapeutic interventions is urgently needed. So far, monoclonal antibodies and drug repositioning are the main methods for drug development, and this effort was partially successful. Since the beginning of the COVID-19 pandemic, the emergence of SARS-CoV-2 variants has been reported in many parts of the world, and the main concern is whether the current vaccines and therapeutics are still effective against these variant viruses. Viral entry and viral RNA-dependent RNA polymerase (RdRp) are the main targets of current drug development; therefore, the inhibitory effects of transmembrane serine protease 2 (TMPRSS2) and RdRp inhibitors were compared among the early SARS-CoV-2 isolate (lineage A) and the two recent variants (lineage B.1.1.7 and lineage B.1.351) identified in the United Kingdom and South Africa, respectively. Our in vitro analysis of viral replication showed that the drugs targeting TMPRSS2 and RdRp are equally effective against the two variants of concern. IMPORTANCE The COVID-19 pandemic is causing unprecedented global problems in both public health and human society. While some vaccines and monoclonal antibodies were successfully developed very quickly and are currently being used, numerous variants of the causative SARS-CoV-2 are emerging and threatening the efficacy of vaccines and monoclonal antibodies. In order to respond to this challenge, we assessed antiviral efficacy of small-molecule inhibitors that are being developed for treatment of COVID-19 and found that they are still very effective against the SARS-CoV-2 variants. Since most small-molecule inhibitors target viral or host factors other than the mutated sequence of the viral spike protein, they are expected to be potent control measures against the COVID-19 pandemic.

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., Competing Interests: Competing interest statement: The compounds described here are patents filed (KR-10-2020-0127319, KR-10-2020-0052509, KR-10-2021-0055736, and PCT/KR2021/005306) for potential commercialization., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Highly effective novel treatments need to be developed to suppress emerging coronavirus (CoV) infections such as COVID-19. The RNA dependent RNA polymerase (RdRp) among the viral proteins is known as an effective antiviral target. Lycorine is a phenanthridine Amaryllidaceae alkaloid isolated from the bulbs of Lycoris radiata (L'Hér.) Herb. and has various pharmacological bioactivities including antiviral function., Purpose: We investigated the direct-inhibiting action of lycorine on CoV's RdRp, as potential treatment for emerging CoV infections., Methods: We examined the inhibitory effect of lycorine on MERS-CoV, SARS-CoV, and SARS-CoV-2 infections, and then quantitatively measured the inhibitory effect of lycorine on MERS-CoV RdRp activity using a cell-based reporter assay. Finally, we performed the docking simulation with lycorine and SARS-CoV-2 RdRp., Results: Lycorine efficiently inhibited these CoVs with IC 50 values of 2.123 ± 0.053, 1.021 ± 0.025, and 0.878 ± 0.022 μM, respectively, comparable with anti-CoV effects of remdesivir. Lycorine directly inhibited MERS-CoV RdRp activity with an IC 50 of 1.406 ± 0.260 μM, compared with remdesivir's IC 50 value of 6.335 ± 0.731 μM. In addition, docking simulation showed that lycorine interacts with SARS-CoV-2 RdRp at the Asp623, Asn691, and Ser759 residues through hydrogen bonding, at which the binding affinities of lycorine (-6.2 kcal/mol) were higher than those of remdesivir (-4.7 kcal/mol)., Conclusions: Lycorine is a potent non-nucleoside direct-acting antiviral against emerging coronavirus infections and acts by inhibiting viral RdRp activity; therefore, lycorine may be a candidate against the current COVID-19 pandemic., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to global public health. Herbal medicines and their derived natural products have drawn much attention in the treatment of COVID-19, but the detailed mechanisms by which natural products inhibit SARS-CoV-2 have not been elucidated. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection routes via lysosome- and transmembrane protease serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by the pharmacological inhibition or gene silencing of NPC1, which is mutated in patients with Niemann-Pick type C (NPC) displaying disrupted membrane cholesterol distribution. Finally, readily available local foods or herbal medicines containing PG root show similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that briefly disrupting the distribution of membrane cholesterol is a potential novel therapeutic strategy for SARS-CoV-2 infection.

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 (IC 50  < 0.25 μM) and anti-MERS-CoV activities (IC 50  < 1.1 μM) with no cytotoxicity (CC 50  > 25 μM). In addition, both compounds showed acceptable results in metabolic stabilities, hERG binding affinities, CYP inhibitions, and preliminary PK studies., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (C T ) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.

Zika virus (ZIKV) infection in both infants and adults is associated with neurological complications including, but not limited to, microcephaly and Guillain-Barre syndrome. Antibody therapy can be effective against virus infection. We isolated ZIKV envelope domain III-specific neutralizing antibodies (nAbs) from two convalescent patients with ZIKV infection. One antibody, 2F-8, exhibited potent in vitro neutralizing activity against Asian and American strains of ZIKV. To prevent FcγR-mediated antibody-dependent enhancement, we prepared IgG 1 with LALA variation. A single dose of 2F-8 in the context of IgG 1 or IgG 1 -LALA prior to or post lethal ZIKV challenge conferred complete protection in mice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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 (IC 50  = 0.0022 µM)., (© 2020 Wiley Periodicals LLC.)

Middle East Respiratory Syndrome (MERS) is a respiratory disease caused by a coronavirus (MERS-CoV). Since its emergence in 2012, nosocomial amplifications have led to its high epidemic potential and mortality rate of 34.5%. To date, there is an unmet need for vaccines and specific therapeutics for this disease. Available treatments are either supportive medications in use for other diseases or those lacking specificity requiring higher doses. The viral infection mode is initiated by the attachment of the viral spike glycoprotein to the human Dipeptidyl Peptidase IV (DPP4). Our attempts to screen antivirals against MERS led us to identify montelukast sodium hydrate (MSH), an FDA-approved anti-asthma drug, as an agent attenuating MERS-CoV infection. We showed that MSH directly binds to MERS-CoV-Receptor-Binding Domain (RBD) and inhibits its molecular interaction with DPP4 in a dose-dependent manner. Our cell-based inhibition assays using MERS pseudovirions demonstrated that viral infection was significantly inhibited by MSH and was further validated using infectious MERS-CoV culture. Thus, we propose MSH as a potential candidate for therapeutic developments against MERS-CoV infections., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 (IC 50  = 0.88 μM) against SARS-CoV-2 without cytotoxicity (CC 50  > 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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

New therapies for treating coronaviruses are urgently needed. A series of 4-anilino-6-aminoquinazoline derivatives were synthesized and evaluated to show high anti-MERS-CoV activities. N 4 -(3-Chloro-4-fluorophenyl)-N 6 -(3-methoxybenzyl)quinazoline-4,6-diamine (1) has been identified in a random screen as a hit compound for inhibiting MERS-CoV infection. Throughout optimization process, compound 20 was found to exhibit high inhibitory effect (IC 50  = 0.157 μM, SI = 25) with no cytotoxicity and moderate in vivo PK properties., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 (IC 50 s), and in particular, two FDA-approved drugs-niclosamide and ciclesonide-were notable in some respects., (Copyright © 2020 American Society for Microbiology.)

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 (IC 50  = 86 nM) and low toxicity (CC 50  > 25 μM). Moreover, it shows good metabolic stability, low hERG binding affinity, no cytotoxicity, and good in vivo PK properties., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Stephania tetrandra and other related species of Menispermaceae are the major sources of the bis-benzylisoquinoline alkaloids tetrandrine (TET), fangchinoline (FAN), and cepharanthine (CEP). Although the pharmacological properties of these compounds include anticancer and anti-inflammatory activities, the antiviral effects of these compounds against human coronavirus (HCoV) remain unclear. Hence, the aims of the current study were to assess the antiviral activities of TET, FAN, and CEP and to elucidate the underlying mechanisms in HCoV-OC43-infected MRC-5 human lung cells. These compounds significantly inhibited virus-induced cell death at the early stage of virus infection. TET, FAN, and CEP treatment dramatically suppressed the replication of HCoV-OC43 as well as inhibited viral S and N protein expression. The virus-induced host response was reduced by compound treatment as compared with the vehicle control. Taken together, these findings demonstrate that TET, FAN, and CEP are potential natural antiviral agents for the prevention and treatment of HCoV-OC43 infection., Competing Interests: Abbreviations: CEP: Cepharanthine; Dpi: Days post-infection; FAN: Fangchinoline; HCoV-OC43: Human coronavirus OC43; IFN: Interferon; IL: Interleukin; TET: Tetrandrine; TNF: Tumor necrosis factor.

Middle East respiratory syndrome coronavirus (MERS-CoV) induces severe aggravating respiratory failure in infected patients, frequently resulting in mechanical ventilation. As limited therapeutic antibody is accumulated in lung tissue following systemic administration, inhalation is newly recognized as an alternative, possibly better, route of therapeutic antibody for pulmonary diseases. The nebulization process, however, generates diverse physiological stresses, and thus, the therapeutic antibody must be resistant to these stresses, remain stable, and form minimal aggregates. We first isolated a MERS-CoV neutralizing antibody that is reactive to the receptor-binding domain (RBD) of spike (S) glycoprotein. To increase stability, we introduced mutations into the complementarity-determining regions (CDRs) of the antibody. In the HCDRs (excluding HCDR3) in this clone, two hydrophobic residues were replaced with Glu, two residues were replaced with Asp, and four residues were replaced with positively charged amino acids. In LCDRs, only two Leu residues were replaced with Val. These modifications successfully generated a clone with significantly greater stability and equivalent reactivity and neutralizing activity following nebulization compared to the original clone. In summary, we generated a MERS-CoV neutralizing human antibody that is reactive to recombinant MERS-CoV S RBD protein for delivery via a pulmonary route by introducing stabilizing mutations into five CDRs., Competing Interests: The authors declare no conflicts of interest.