Efficacy of Licensed Monoclonal Antibodies and Antiviral Agents against the SARS-CoV-2 Omicron Sublineages BA.1 and BA.2.

Newly emerging SARS-CoV-2 variants may escape monoclonal antibodies (mAbs) and antiviral drugs. By using live virus assays, we assessed the ex vivo inhibition of the B.1 wild-type (WT), delta and omicron BA.1 and BA.2 lineages by post-infusion sera from 40 individuals treated with bamlanivimab/etesevimab (BAM/ETE), casirivimab/imdevimab (CAS/IMD), and sotrovimab (SOT) as well as the activity of remdesivir, nirmatrelvir and molnupiravir. mAbs and drug activity were defined as the serum dilution (ID50) and drug concentration (IC50), respectively, showing 50% protection of virus-induced cytopathic effect. All pre-infusion sera were negative for SARS-CoV-2 neutralizing activity. BAM/ETE, CAS/IMD, and SOT showed activity against the WT (ID50 6295 (4355-8075) for BAM/ETE; 18,214 (16,248-21,365) for CAS/IMD; and 456 (265-592) for SOT) and the delta (14,780 (ID50 10,905-21,020) for BAM/ETE; 63,937 (47,211-79,971) for CAS/IMD; and 1103 (843-1334) for SOT). Notably, only SOT was active against BA.1 (ID50 200 (37-233)), whereas BA.2 was neutralized by CAS/IMD (ID50 174 (134-209) ID50) and SOT (ID50 20 (9-31) ID50), but not by BAM/ETE. No significant inter-variant IC50 differences were observed for molnupiravir (1.5 ± 0.1/1.5 ± 0.7/1.0 ± 0.5/0.8 ± 0.01 µM for WT/delta/BA.1/BA.2, respectively), nirmatrelvir (0.05 ± 0.02/0.06 ± 0.01/0.04 ± 0.02/0.04 ± 0.01 µM) or remdesivir (0.08 ± 0.04/0.11 ± 0.08/0.05 ± 0.04/0.08 ± 0.01 µM). Continued evolution of SARS-CoV-2 requires updating the mAbs arsenal, although antivirals have so far remained unaffected.

A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors.

Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for the screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified two molecules which show anti-nsp5 activity, both in our cell-based assay and in vitro on purified nsp5 protein, and inhibit SARS-CoV-2 replication in A549-ACE2 cells with EC50 values in the 4-8 µM range. The here described high-throughput-compatible assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.

SPR-Based Kinetic Analysis of the Early Stages of Infection in Cells Infected with Human Coronavirus and Treated with Hydroxychloroquine.

Cell-based assays are a valuable tool for examination of virus-host cell interactions and drug discovery processes, allowing for a more physiological setting compared to biochemical assays. Despite the fact that cell-based SPR assays are label-free and thus provide all the associated benefits, they have never been used to study viral growth kinetics and to predict drug antiviral response in cells. In this study, we prove the concept that the cell-based SPR assay can be applied in the kinetic analysis of the early stages of viral infection of cells and the antiviral drug activity in the infected cells. For this purpose, cells immobilized on the SPR slides were infected with human coronavirus HCov-229E and treated with hydroxychloroquine. The SPR response was measured at different time intervals within the early stages of infection. Methyl Thiazolyl Tetrazolium (MTT) assay was used to provide the reference data. We found that the results of the SPR and MTT assays were consistent, and SPR is a reliable tool in investigating virus-host cell interaction and the mechanism of action of viral inhibitors. SPR assay was more sensitive and accurate in the first hours of infection within the first replication cycle, whereas the MTT assay was not so effective. After the second replication cycle, noise was generated by the destruction of the cell layer and by the remnants of dead cells, and masks useful SPR signals.

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.

Inhibitors of Coronavirus 3CL Proteases Protect Cells from Protease-Mediated Cytotoxicity.

We describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This assay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, we identified compounds with broad 3CLpro-inhibitory activity. We also adapted the assay for use in compound screening and in doing so uncovered additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CLpro inhibitors. We observed strong concordance between data emerging from this assay and those obtained from live-virus testing. The reported approach democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive biosafety infrastructure. We identified two lead compounds, GC376 and compound 4, with broad activity against all 3CL proteases tested, including 3CLpro enzymes from understudied zoonotic coronaviruses. IMPORTANCE Multiple coronavirus pandemics have occurred over the last 2 decades. This has highlighted a need to be proactive in the development of therapeutics that can be readily deployed in the case of future coronavirus pandemics. We developed and validated a simplified cell-based assay for the identification of chemical inhibitors of 3CL proteases encoded by a wide range of coronaviruses. This assay is reporter free, does not require specialized biocontainment, and is optimized for performance in high-throughput screening. By testing reported 3CL protease inhibitors against a large collection of 3CL proteases with variable sequence similarity, we identified compounds with broad activity against 3CL proteases and uncovered structural insights into features that contribute to their broad activity. Furthermore, we demonstrated that this assay is suitable for identifying chemical inhibitors of proteases from families other than 3CL proteases.

Myasthenia gravis associated with anti-MuSK antibodies developed after SARS-CoV-2 infection.

INTRODUCTION: Since the onset of the novel coronavirus pandemic, several neurological complications secondary to SARS-CoV-2 infection have been reported, affecting central nervous system, peripheral nervous system and neuromuscular junction. CASE REPORT: We present the case of a 77-year-old man who developed bulbar myasthenia gravis (MG) eight weeks after SARS-CoV-2 infection. The search for serum antibodies against the acetylcholine receptor and the muscle-specific tyrosine kinase (MuSK), performed by radioimmunoassay (RIA), and the search of low-density lipoprotein receptor-related protein 4 antibodies, performed by immunohistochemistry, resulted negative, while anti-MuSK antibodies were detected by cell-based assay (CBA). The patient was treated with pyridostigmine (60 mg four times a day) with unsatisfactory clinical response, followed by immunosuppressive therapy (azathioprine 1.5 mg/kg/day) with improvement of MG symptoms after two months of treatment. DISCUSSION: Several viral diseases have been described as associated with the onset of MG, although the underlying mechanisms are not yet fully understood. Similarly, a growing number of scientific reports suggest a correlation between SARS-CoV-2 infection and autoimmune diseases. The interest of our case lies in the timing of the MG onset (after 2 months from infection), together with the unusual late onset of anti-MuSK MG. These elements suggest that coronavirus infection may act as a trigger of the disease. We confirm the importance of CBA in the serological diagnosis of RIA-negative MG.