Targeting papain-like protease for broad-spectrum coronavirus inhibition.

The emergence of SARS-CoV-2 variants of concern and repeated outbreaks of coronavirus epidemics in the past two decades emphasize the need for next-generation pan-coronaviral therapeutics. Drugging the multi-functional papain-like protease (PLpro) domain of the viral nsp3 holds promise. However, none of the known coronavirus PLpro inhibitors has been shown to be in vivo active. Herein, we screened a structurally diverse library of 50,080 compounds for potential coronavirus PLpro inhibitors and identified a noncovalent lead inhibitor F0213 that has broad-spectrum anti-coronaviral activity, including against the Sarbecoviruses (SARS-CoV-1 and SARS-CoV-2), Merbecovirus (MERS-CoV), as well as the Alphacoronavirus (hCoV-229E and hCoV-OC43). Importantly, F0213 confers protection in both SARS-CoV-2-infected hamsters and MERS-CoV-infected human DPP4-knockin mice. F0213 possesses a dual therapeutic functionality that suppresses coronavirus replication via blocking viral polyprotein cleavage, as well as promoting antiviral immunity by antagonizing the PLpro deubiquitinase activity. Despite the significant difference of substrate recognition, mode of inhibition studies suggest that F0213 is a competitive inhibitor against SARS2-PLpro via binding with the 157K amino acid residue, whereas an allosteric inhibitor of MERS-PLpro interacting with its 271E position. Our proof-of-concept findings demonstrated that PLpro is a valid target for the development of broad-spectrum anti-coronavirus agents. The orally administered F0213 may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and future coronavirus outbreaks.

Low Environmental Temperature Exacerbates Severe Acute Respiratory Syndrome Coronavirus 2 Infection in Golden Syrian Hamsters.

BACKGROUND: The effect of low environmental temperature on viral shedding and disease severity of Coronavirus Disease 2019 (COVID-19) is uncertain. METHODS: We investigated the virological, clinical, pathological, and immunological changes in hamsters housed at room (21°C), low (12-15°C), and high (30-33°C) temperature after challenge by 105 plaque-forming units of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RESULTS: The nasal turbinate, trachea, and lung viral load and live virus titer were significantly higher (~0.5-log10 gene copies/ß-actin, P < .05) in the low-temperature group at 7 days postinfection (dpi). The low-temperature group also demonstrated significantly higher level of tumor necrosis factor-α, interferon-γ (IFN-γ), interleukin-1ß, and C-C motif chemokine ligand 3, and lower level of the antiviral IFN-α in lung tissues at 4 dpi than the other 2 groups. Their lungs were grossly and diffusely hemorrhagic, with more severe and diffuse alveolar and peribronchiolar inflammatory infiltration, bronchial epithelial cell death, and significantly higher mean total lung histology scores. By 7 dpi, the low-temperature group still showed persistent and severe alveolar inflammation and hemorrhage, and little alveolar cell proliferative changes of recovery. The viral loads in the oral swabs of the low-temperature group were significantly higher than those of the other two groups from 10 to 17 dpi by about 0.5-1.0 log10 gene copies/ß-actin. The mean neutralizing antibody titer of the low-temperature group was significantly (P < .05) lower than that of the room temperature group at 7 dpi and 30 dpi. CONCLUSIONS: This study provided in vivo evidence that low environmental temperature exacerbated the degree of virus shedding, disease severity, and tissue proinflammatory cytokines/chemokines expression, and suppressed the neutralizing antibody response of SARS-CoV-2-infected hamsters. Keeping warm in winter may reduce the severity of COVID-19.

In Silico Structure-Based Design of Antiviral Peptides Targeting the Severe Fever with Thrombocytopenia Syndrome Virus Glycoprotein Gn.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus in Asia that causes severe disease. Despite its clinical importance, treatment options for SFTSV infection remains limited. The SFTSV glycoprotein Gn plays a major role in mediating virus entry into host cells and is therefore a potential antiviral target. In this study, we employed an in silico structure-based strategy to design novel cyclic antiviral peptides that target the SFTSV glycoprotein Gn. Among the cyclic peptides, HKU-P1 potently neutralizes the SFTSV virion. Combinatorial treatment with HKU-P1 and the broad-spectrum viral RNA-dependent RNA polymerase inhibitor favipiravir exhibited synergistic antiviral effects in vitro. The in silico peptide design platform in this study may facilitate the generation of novel antiviral peptides for other emerging viruses.

In silico structure-based discovery of a SARS-CoV-2 main protease inhibitor.

The Coronavirus Disease 2019 (COVID-19) pandemic caused by the novel lineage B betacoroanvirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant mortality, morbidity, and socioeconomic disruptions worldwide. Effective antivirals are urgently needed for COVID-19. The main protease (Mpro) of SARS-CoV-2 is an attractive antiviral target because of its essential role in the cleavage of the viral polypeptide. In this study, we performed an in silico structure-based screening of a large chemical library to identify potential SARS-CoV-2 Mpro inhibitors. Among 8,820 compounds in the library, our screening identified trichostatin A, a histone deacetylase inhibitor and an antifungal compound, as an inhibitor of SARS-CoV-2 Mpro activity and replication. The half maximal effective concentration of trichostatin A against SARS-CoV-2 replication was 1.5 to 2.7µM, which was markedly below its 50% effective cytotoxic concentration (75.7µM) and peak serum concentration (132µM). Further drug compound optimization to develop more stable analogues with longer half-lives should be performed. This structure-based drug discovery platform should facilitate the identification of additional enzyme inhibitors of SARS-CoV-2.

Clofazimine broadly inhibits coronaviruses including SARS-CoV-2.

The COVID-19 pandemic is the third outbreak this century of a zoonotic disease caused by a coronavirus, following the emergence of severe acute respiratory syndrome (SARS) in 20031 and Middle East respiratory syndrome (MERS) in 20122. Treatment options for coronaviruses are limited. Here we show that clofazimine-an anti-leprosy drug with a favourable safety profile3-possesses inhibitory activity against several coronaviruses, and can antagonize the replication of SARS-CoV-2 and MERS-CoV in a range of in vitro systems. We found that this molecule, which has been approved by the US Food and Drug Administration, inhibits cell fusion mediated by the viral spike glycoprotein, as well as activity of the viral helicase. Prophylactic or therapeutic administration of clofazimine in a hamster model of SARS-CoV-2 pathogenesis led to reduced viral loads in the lung and viral shedding in faeces, and also alleviated the inflammation associated with viral infection. Combinations of clofazimine and remdesivir exhibited antiviral synergy in vitro and in vivo, and restricted viral shedding from the upper respiratory tract. Clofazimine, which is orally bioavailable and comparatively cheap to manufacture, is an attractive clinical candidate for the treatment of outpatients and-when combined with remdesivir-in therapy for hospitalized patients with COVID-19, particularly in contexts in which costs are an important factor or specialized medical facilities are limited. Our data provide evidence that clofazimine may have a role in the control of the current pandemic of COVID-19 and-possibly more importantly-in dealing with coronavirus diseases that may emerge in the future.

Beneficial effect of combinational methylprednisolone and remdesivir in hamster model of SARS-CoV-2 infection.

Effective treatments for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Dexamethasone has been shown to confer survival benefits to certain groups of hospitalized patients, but whether glucocorticoids such as dexamethasone and methylprednisolone should be used together with antivirals to prevent a boost of SARS-CoV-2 replication remains to be determined. Here, we show the beneficial effect of methylprednisolone alone and in combination with remdesivir in the hamster model of SARS-CoV-2 infection. Treatment with methylprednisolone boosted RNA replication of SARS-CoV-2 but suppressed viral induction of proinflammatory cytokines in human monocyte-derived macrophages. Although methylprednisolone monotherapy alleviated body weight loss as well as nasal and pulmonary inflammation, viral loads increased and antibody response against the receptor-binding domain of spike protein attenuated. In contrast, a combination of methylprednisolone with remdesivir not only prevented body weight loss and inflammation, but also dampened viral protein expression and viral loads. In addition, the suppressive effect of methylprednisolone on antibody response was alleviated in the presence of remdesivir. Thus, combinational anti-inflammatory and antiviral therapy might be an effective, safer and more versatile treatment option for COVID-19. These data support testing of the efficacy of a combination of methylprednisolone and remdesivir for the treatment of COVID-19 in randomized controlled clinical trials.

Viruses harness YxxØ motif to interact with host AP2M1 for replication: A vulnerable broad-spectrum antiviral target.

Targeting a universal host protein exploited by most viruses would be a game-changing strategy that offers broad-spectrum solution and rapid pandemic control including the current COVID-19. Here, we found a common YxxØ-motif of multiple viruses that exploits host AP2M1 for intracellular trafficking. A library chemical, N-(p-amylcinnamoyl)anthranilic acid (ACA), was identified to interrupt AP2M1-virus interaction and exhibit potent antiviral efficacy against a number of viruses in vitro and in vivo, including the influenza A viruses (IAVs), Zika virus (ZIKV), human immunodeficiency virus, and coronaviruses including MERS-CoV and SARS-CoV-2. YxxØ mutation, AP2M1 depletion, or disruption by ACA causes incorrect localization of viral proteins, which is exemplified by the failure of nuclear import of IAV nucleoprotein and diminished endoplasmic reticulum localization of ZIKV-NS3 and enterovirus-A71-2C proteins, thereby suppressing viral replication. Our study reveals an evolutionarily conserved mechanism of protein-protein interaction between host and virus that can serve as a broad-spectrum antiviral target.

Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to take at least 12-18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose-response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2-4 and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.

A Large-scale Drug Repositioning Survey for SARS-CoV-2 Antivirals.

The emergence of novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, more than 2.1 million confirmed cases and 139,500 deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. As the development of a vaccine could require at least 12-18 months, and the typical timeline from hit finding to drug registration of an antiviral is >10 years, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. Here, we report the identification of 30 known drugs that inhibit viral replication. Of these, six were characterized for cellular dose-activity relationships, and showed effective concentrations likely to be commensurate with therapeutic doses in patients. These include the PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334, and the CCR1 antagonist MLN-3897. Since many of these molecules have advanced into the clinic, the known pharmacological and human safety profiles of these compounds will accelerate their preclinical and clinical evaluation for COVID-19 treatment.

Discovery of the FDA-approved drugs bexarotene, cetilistat, diiodohydroxyquinoline, and abiraterone as potential COVID-19 treatments with a robust two-tier screening system.

Coronavirus Disease 2019 (COVID-19) caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with a crude case fatality rate of about 0.5-10 % depending on locality. A few clinically approved drugs, such as remdesivir, chloroquine, hydroxychloroquine, nafamostat, camostat, and ivermectin, exhibited anti-SARS-CoV-2 activity in vitro and/or in a small number of patients. However, their clinical use may be limited by anti-SARS-CoV-2 50 % maximal effective concentrations (EC50) that exceeded their achievable peak serum concentrations (Cmax), side effects, and/or availability. To find more immediately available COVID-19 antivirals, we established a two-tier drug screening system that combines SARS-CoV-2 enzyme-linked immunosorbent assay and cell viability assay, and applied it to screen a library consisting 1528 FDA-approved drugs. Cetilistat (anti-pancreatic lipase), diiodohydroxyquinoline (anti-parasitic), abiraterone acetate (synthetic androstane steroid), and bexarotene (antineoplastic retinoid) exhibited potent in vitro anti-SARS-CoV-2 activity (EC50 1.13-2.01 µM). Bexarotene demonstrated the highest Cmax:EC50 ratio (1.69) which was higher than those of chloroquine, hydroxychloroquine, and ivermectin. These results demonstrated the efficacy of the two-tier screening system and identified potential COVID-19 treatments which can achieve effective levels if given by inhalation or systemically depending on their pharmacokinetics.

An optimized high-throughput fluorescence plate reader-based RSV neutralization assay.

A licensed vaccine for respiratory syncytial virus (RSV) has yet to be developed, and a reliable and repeatable neutralizing assay is indispensable for vaccine development. Here, we demonstrated an optimized high-throughput RSV neutralization assay that utilizes a fluorescence plate reader (reader) as a substitute for flow cytometry to detect fluorescent signals in RSV-A2 mKate-infected cells. Furthermore, this study tested the influence of virus input and infectivity on the neutralizing assay and highlighted critical factors (together with a suggested protocol) for obtaining stable data using this assay.

Discovery of a Prefusion Respiratory Syncytial Virus F-Specific Monoclonal Antibody That Provides Greater In Vivo Protection than the Murine Precursor of Palivizumab.

Palivizumab, a humanized murine monoclonal antibody that recognizes antigenic site II on both the prefusion (pre-F) and postfusion (post-F) conformations of the respiratory syncytial virus (RSV) F glycoprotein, is the only prophylactic agent approved for use for the treatment of RSV infection. However, its relatively low neutralizing potency and high cost have limited its use to a restricted population of infants at high risk of severe disease. Previously, we isolated a high-potency neutralizing antibody, 5C4, that specifically recognizes antigenic site Ø at the apex of the pre-F protein trimer. We compared in vitro and in vivo the potency and protective efficacy of 5C4 and the murine precursor of palivizumab, antibody 1129. Both antibodies were synthesized on identical murine backbones as either an IgG1 or IgG2a subclass and evaluated for binding to multiple F protein conformations, in vitro inhibition of RSV infection and propagation, and protective efficacy in mice. Although 1129 and 5C4 had similar pre-F protein binding affinities, the 5C4 neutralizing activity was nearly 50-fold greater than that of 1129 in vitro In BALB/c mice, 5C4 reduced the peak titers of RSV 1,000-fold more than 1129 did in both the upper and lower respiratory tracts. These data indicate that antibodies specific for antigenic site Ø are more efficacious at preventing RSV infection than antibodies specific for antigenic site II. Our data also suggest that site Ø-specific antibodies may be useful for the prevention or treatment of RSV infection and support the use of the pre-F protein as a vaccine antigen.IMPORTANCE There is no vaccine yet available to prevent RSV infection. The use of the licensed antibody palivizumab, which recognizes site II on both the pre-F and post-F proteins, is restricted to prophylaxis in neonates at high risk of severe RSV disease. Recommendations for using passive immunization in the general population or for therapy in immunocompromised persons with persistent infection is limited because of cost, determined from the high doses needed to compensate for its relatively low neutralizing potency. Prior efforts to improve the in vitro potency of site II-specific antibodies did not translate to significant in vivo dose sparing. We isolated a pre-F protein-specific, high-potency neutralizing antibody (5C4) that recognizes antigenic site Ø and compared its efficacy to that of the murine precursor of palivizumab (antibody 1129) matched for isotype and pre-F protein binding affinities. Our findings demonstrate that epitope specificity is an important determinant of antibody neutralizing potency, and defining the mechanisms of neutralization has the potential to identify improved products for the prevention and treatment of RSV infection.

Analytical performance of a PCR assay for the detection of KRAS mutations (codons 12/13 and 61) in formalin-fixed paraffin-embedded tissue samples of colorectal carcinoma.

KRAS mutation testing is mandatory before prescribing anti-epidermal growth factor monoclonal antibodies in the treatment of advanced colorectal cancer. We describe the performance of a TaqMelt polymerase chain reaction (PCR) assay-the cobas® KRAS Mutation Test-designed to detect 19 mutations in codons 12, 13, and 61. The limit of detection was determined using DNA blends from cell lines, plasmids, and formalin-fixed paraffin-embedded tissue specimens. Assay performance was compared to Sanger sequencing using a panel of 188 specimens. Discordant specimens were subjected to next generation pyrosequencing (454). Assay repeatability was assessed using a panel of six specimens. A >95% correct mutation call rate was obtained in all specimen types with ~5% mutant alleles at DNA inputs of 0.8-6.3 ng per PCR reaction; 100% detection rate was observed at the recommended DNA input of 50 ng. The positive percent agreement with Sanger was 97.5% (79/81) for codons 12/13 and 85.7% (6/7) for codon 61. Negative percent agreement was 94.4% (101/107) for codon 12/13 and 99.4% (180/181) for codon 61. Nine of 10 discordant specimens yielded 454 results consistent with the cobas® results. With repeated testing, the assay showed a correct call rate of 100% (192/192) for all operators, instruments, reagent lots, and days tested. The cobas® test detects KRAS mutations in codons 12, 13, and 61 at a limit of detection of <5%. The PCR assay was more sensitive and specific than Sanger sequencing, and performance was highly reproducible. Test performance was not influenced by various endogenous interfering substances or common gut microbes.