A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike.

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells.

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

SARS-CoV-2 Nucleocapsid Protein TR-FRET Assay Amenable to High Throughput Screening.

Drug development for specific antiviral agents against coronavirus disease 2019 (COVID-19) is still an unmet medical need as the pandemic continues to spread globally. Although huge efforts for drug repurposing and compound screens have been put forth, only a few compounds are in late-stage clinical trials. New approaches and assays are needed to accelerate COVID-19 drug discovery and development. Here, we report a time-resolved fluorescence resonance energy transfer-based assay that detects the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) produced in infected cells. It uses two specific anti-NP monoclonal antibodies conjugated to donor and acceptor fluorophores that produce a robust ratiometric signal for high throughput screening of large compound collections. Using this assay, we measured a half maximal inhibitory concentration (IC50) for remdesivir of 9.3 µM against infection with SARS-CoV-2 USA/WA1/2020 (WA-1). The assay also detected SARS-CoV-2 South African (Beta, ß), Brazilian/Japanese P.1 (Gamma, γ), and Californian (Epsilon, ε) variants of concern (VoC). Therefore, this homogeneous SARS-CoV-2 NP detection assay can be used for accelerating lead compound discovery for drug development and for evaluating drug efficacy against emerging SARS-CoV-2 VoC.

A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry.

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.

Fluorescent quantum dots enable SARS-CoV-2 antiviral drug discovery and development.

INTRODUCTION: SARS-CoV-2 is a highly infectious and deadly coronavirus whose study requires the use of a biosafety level 3 (BSL-3) containment facility to investigate viral biology and pathogenesis, which limits the study of live virus and slows progress toward finding suitable treatments for infection. While vaccines from several companies have proven very effective in combating the virus, few treatments exist for those who do succumb to the viral-induced systemic disease called COVID-19. AREAS COVERED: This short review focuses on fluorescent quantum dot-based modeling of SARS-CoV-2. New BSL-2 viral models are essential for finding small molecules and biologics that may be effective in stopping viral infection, as well as treating already infected individuals. Nanoparticles are invaluable tools for biological research as they can be used to both model pathogens and serve as a platform for developing vaccines. EXPERT OPINION: Visualizing viral activity with fluorescent quantum dots enables both biochemical and cell-based assays to detect virus-host receptor interactions, cellular activity after binding to the cell plasma membrane, screening for interventions using small-molecule drug repurposing, and testing of novel biologics. Quantum dots can also be used for diagnostic assays, vaccine development, and importantly, pan-antiviral drugs to address variants that may escape the immune response.

Inhibiting SARS-CoV-2 infection with lysosomal alkalizers

Understanding the SARS-CoV-2 virus? routes of infection, virus?host?protein interactions, and mechanisms of virus-induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID-19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose-limiting toxicities in patients. To identify an alternative lysosome-based drug repurposing opportunity we evaluated additional lysosomotropic compounds . We found that six of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero E6 cells with half-maximal effective concentration (EC50) values ranging from 2.0 to 13 ?M and selectivity indices (SIs;SI = CC50/EC50) ranging from 1.5- to >10-fold. We demonstrate how the compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) that ROC-325 reduced viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV-NL63 cytopathic effect in LLC-MK2 cells. Moreover, an analysis of SARS-CoV-2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS-CoV-2. Our findings support targeting the lysosome to combat SARS-CoV-2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID-19.

Quantum Dot-Conjugated SARS-CoV-2 Spike Nanoparticles for SARS-CoV-2 infection modeling and drug discovery

The SARS-CoV-2 virus binds to host cell surface ACE2 on the plasma membrane via the spike protein's receptor binding domain. Our work has resulted in the generation of a versatile imaging probe using recombinant Spike receptor binding domain conjugated to fluorescent quantum dots (QDs). This probe is capable of engaging in energy transfer quenching with ACE2-conjugated gold nanoparticles enabling biochemical monitoring of binding. Neutralizing antibodies and recombinant human ACE2 blocked quenching, demonstrating a specific binding interaction. In cell-based assays, we observed immediate binding of the probe on the cell surface of ACE2-expressing cells followed by endocytosis. Neutralizing antibodies and ACE2-Fc fully prevented binding and endocytosis with low nanomolar potency. Importantly, we can use this QD nanoparticle probe to identify and validate inhibitors of the SARS-CoV-2 Spike and ACE2 receptor binding in human cells. This work enables facile, rapid, and high-throughput biochemical- and cell-based screening of inhibitors for coronavirus Spike-mediated cell recognition and entry.

The SARS-CoV-2 Cytopathic Effect Is Blocked by Lysosome Alkalizing Small Molecules.

Understanding the SARS-CoV-2 virus' pathways of infection, virus-host-protein interactions, and mechanisms of virus-induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID-19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose-limiting toxicities in patients. Therefore, we evaluated additional lysosomotropic compounds to identify an alternative lysosome-based drug repurposing opportunity. We found that six of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero E6 cells with half-maximal effective concentration (EC50) values ranging from 2.0 to 13 µM and selectivity indices (SIs; SI = CC50/EC50) ranging from 1.5- to >10-fold. The compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) reduced (ROC-325) viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV-NL63 cytopathic effect in LLC-MK2 cells. Moreover, an analysis of SARS-CoV-2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS-CoV-2. Our findings suggest the lysosome as a potential host cell target to combat SARS-CoV-2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID-19.

Identifying SARS-CoV-2 Entry Inhibitors through Drug Repurposing Screens of SARS-S and MERS-S Pseudotyped Particles.

While vaccine development will hopefully quell the global pandemic of COVID-19 caused by SARS-CoV-2, small molecule drugs that can effectively control SARS-CoV-2 infection are urgently needed. Here, inhibitors of spike (S) mediated cell entry were identified in a high throughput screen of an approved drugs library with SARS-S and MERS-S pseudotyped particle entry assays. We discovered six compounds (cepharanthine, abemaciclib, osimertinib, trimipramine, colforsin, and ingenol) to be broad spectrum inhibitors for spike-mediated entry. This work could contribute to the development of effective treatments against the initial stage of viral infection and provide mechanistic information that might aid the design of new drug combinations for clinical trials for COVID-19 patients.

Development of a High-Throughput Homogeneous AlphaLISA Drug Screening Assay for the Detection of SARS-CoV-2 Nucleocapsid.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is in urgent need of therapeutic options. High-throughput screening (HTS) offers an opportunity to rapidly identify such compounds. In this work, we have developed a homogeneous cell-based HTS system using AlphaLISA detection technology for the SARS-CoV-2 nucleocapsid protein (NP). Our assay measures both recombinant and endogenous NP from viral lysates and tissue culture supernatants (TCS) in a sandwich-based format using two monoclonal antibodies against the NP analyte. Viral NP was detected and quantified in both tissue culture supernatants and cell lysates, with large differences observed between 24 and 48 h of infection. We simulated viral infection by spiking recombinant NP into 384-well plates with live Vero-E6 cells and were able to detect the NP with high sensitivity and a large dynamic range. Antiviral agents that inhibit either viral cell entry or replication decrease the AlphaLISA NP signal. Thus, this assay can be used for high-throughput screening of small molecules and biologics in the fight against the COVID-19 pandemic.

Quantum Dot-Conjugated SARS-CoV-2 Spike Pseudo-Virions Enable Tracking of Angiotensin Converting Enzyme 2 Binding and Endocytosis.

The first step of SARS-CoV-2 infection is binding of the spike protein's receptor binding domain to the host cell's ACE2 receptor on the plasma membrane. Here, we have generated a versatile imaging probe using recombinant Spike receptor binding domain conjugated to fluorescent quantum dots (QDs). This probe is capable of engaging in energy transfer quenching with ACE2-conjugated gold nanoparticles to enable monitoring of the binding event in solution. Neutralizing antibodies and recombinant human ACE2 blocked quenching, demonstrating a specific binding interaction. In cells transfected with ACE2-GFP, we observed immediate binding of the probe on the cell surface followed by endocytosis. Neutralizing antibodies and ACE2-Fc fully prevented binding and endocytosis with low nanomolar potency. Importantly, we will be able to use this QD nanoparticle probe to identify and validate inhibitors of the SARS-CoV-2 Spike and ACE2 receptor binding in human cells. This work enables facile, rapid, and high-throughput cell-based screening of inhibitors for coronavirus Spike-mediated cell recognition and entry.

Drug Discovery Strategies for SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is a novel disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV)-2 virus that was first detected in December of 2019 in Wuhan, China, and has rapidly spread worldwide. The search for a suitable vaccine as well as effective therapeutics for the treatment of COVID-19 is underway. Drug repurposing screens provide a useful and effective solution for identifying potential therapeutics against SARS-CoV-2. For example, the experimental drug remdesivir, originally developed for Ebola virus infections, has been approved by the US Food and Drug Administration as an emergency use treatment of COVID-19. However, the efficacy and toxicity of this drug need further improvements. In this review, we discuss recent findings on the pathology of coronaviruses and the drug targets for the treatment of COVID-19. Both SARS-CoV-2-specific inhibitors and broad-spectrum anticoronavirus drugs against SARS-CoV, Middle East respiratory syndrome coronavirus, and SARS-CoV-2 will be valuable additions to the anti-SARS-CoV-2 armament. A multitarget treatment approach with synergistic drug combinations containing different mechanisms of action may be a practical therapeutic strategy for the treatment of severe COVID-19. SIGNIFICANCE STATEMENT: Understanding the biology and pathology of RNA viruses is critical to accomplish the challenging task of developing vaccines and therapeutics against SARS-CoV-2. This review highlights the anti-SARS-CoV-2 drug targets and therapeutic development strategies for COVID-19 treatment.

RNA-Dependent RNA Polymerase as a Target for COVID-19 Drug Discovery.

COVID-19 respiratory disease caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly become a global health issue since it emerged in December 2019. While great global efforts are underway to develop vaccines and to discover or repurpose therapeutic agents for this disease, as of this writing only the nucleoside drug remdesivir has been approved under Emergency Use Authorization to treat COVID-19. The RNA-dependent RNA polymerase (RdRP), a viral enzyme for viral RNA replication in host cells, is one of the most intriguing and promising drug targets for SARS-CoV-2 drug development. Because RdRP is a viral enzyme with no host cell homologs, selective SARS-CoV-2 RdRP inhibitors can be developed that have improved potency and fewer off-target effects against human host proteins and thus are safer and more effective therapeutics for treating COVID-19. This review focuses on biochemical enzyme and cell-based assays for RdRPs that could be used in high-throughput screening to discover new and repurposed drugs against SARS-CoV-2.