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Principal Investigator: JOSHUA ZIMMERBERG
Organization: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
Fiscal Year: 2021
Award: $3,483,111
Funding agency: Eunice Kennedy Shriver National Institute of Child Health and Human Development
1. To combat COVID-19, high throughput screening (HTS) of thousands of candidate compounds that may block or reduce SARS-CoV-2 infection of target tissues is a powerful tool in drug discovery. However, SARS-CoV-2 is a biological safety level 3 (BSL-3) pathogen and most HTS facilities are only BSL-2. To overcome this obstacle and meet the desperate need for HTS of compounds with SARS-CoV-2 antiviral activity, an HTS screening assay was developed using pseudotyped particles (PP). PP serve as mimics for viral binding and entry into target cells, are BSL-2 compatible, and can be used for HTS. PP contain viral envelope proteins such as the spike protein which mediates viral entry into target cells, but carry a fluorescence-based reporter gene instead of the viral genome, and thus display the necessary viral coat proteins for host receptor and membrane interactions without the capacity for replication. Similar PP-based BSL-2 viral entry assays have been successfully applied to HTS campaigns for Ebola virus, influenza, and human immunodeficiency virus (HIV).
In a collaboration with scientists at NCATS (the National Center for Advancing Translational Sciences), we used negative stain electron microscopy and immunogold labeling to evaluate the structure and quantify Spike protein distribution on PP that were used in their HTS assays for compounds with anti-SARS-CoV-2 activity. This technique was used countless times over this year to continuously monitor quality control for the PP for various SARS-CoV-2 S protein variants, as well as control PP.
The PP were created by co-transfection of a plasmid of the Spike (S) protein of a coronavirus and a plasmid for the Gag protein of a murine leukemia virus (Fig 1) For measurement of S-mediated entry luciferase was included into the PP; successful target cell entry was measured as luciferase activity.
In this way, inhibitors of spike (S) mediated cell entry were identified by our colleagues in a high throughput screen of an approved drugs library with SARS-S and MERS-S pseudotyped particle entry assays. Six compounds were discovered (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. After further testing in a SARS-CoV-2 live virus CPE assay and removing cytotoxic compounds, six out of seven entry inhibitors were able to rescue the CPE of SARS-CoV-2 infection, indicating the utility of these PP entry assays.
2. Viral fusion is a critical step in the entry pathway of enveloped viruses and remains a viable target for antiviral exploration. The current approaches for studying fusion mechanisms include ensemble fusion assays, high-resolution cryo-TEM, and single-molecule fluorescence-based methods. While these methods have provided invaluable insights into the dynamic events underlying fusion processes, they come with their own limitations. These often include extensive data and image analysis in addition to experimental time and technical requirements. This work proposes the use of the spinspin T2 relaxation technique as a sensitive bioanalytical method for the rapid quantification of interactions between viral fusion proteins and lipids in real time. In this study, new liposome-coated iron oxide nanosensors (LIONs), which mimic as magnetic-labeled host membranes, are reported to detect minute interactions occurring between the membrane and influenzas fusion glycoprotein, hemagglutinin (HA). The influenza fusion proteins interaction with the LION membrane is detected by measuring changes in the sensitive spinspin T2 magnetic relaxation time using a bench-top NMR instrument. More data is gleaned from including the fluorescent dye DiI into the LION membrane. In addition, the effects of environmental factors on proteinlipid interaction that affect fusion such as pH, time of incubation, trypsin, and cholesterol were also examined. Furthermore, the efficacy and sensitivity of the spinspin T2 relaxation assay in quantifying similar protein/lipid interactions with more native configurations of HA were demonstrated using virus-like particles (VLPs). Shorter domains derived from HA were used to start a reductionist path to identify the parts of HA responsible for the NMR changes observed. Finally, the known fusion inhibitor Arbidol was employed in our spinspin T2 relaxation-based fusion assay to demonstrate the application of LIONs in real-time monitoring of this aspect of fusion for evaluation of potential fusion inhibitors.
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