Abstract Title

Measuring Binding Affinity of CoV2 Spike Proteins to ACE2 Receptor Using KinExA

Additional Funding Sources

The project described was supported by Institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant Nos. P20GM103408 and P20GM109095, and National Science Foundation S-STEM Gateway Scholarships in Biological Sciences under Grant Award No. DUE-1644233. We also acknowledge support from the Biomolecular Research Center at Boise State with funding from the National Science Foundation, Grant Nos. 0619793 and 0923535, NSF CAREER Grant No. 1554166, the M.J. Murdock Charitable Trust, the Idaho State Board of Education, and the Boise State University Department of Physics.

Abstract

To infect a cell, viruses specifically interact with receptors present on the surface of target cells. Quantification of this interaction is important not only for a better understanding of infectivity or developing novel diagnosis approaches but also when screening for specific inhibitors to be used as antiviral drugs. In this respect, we describe the use of the Kinetics Exclusion Assay technology for measuring the affinity between the spike protein of the CoV-2 virus and its specific membrane receptor ACE-2. To achieve our scientific goals, we functionalized small plastic beads with the spike protein and utilized them as a solid phase to capture the free ACE-2 present in previously equilibrated solutions containing a fixed amount of ACE-2 receptor (Constant Binding Partner) and a variable amount of spike protein (Titrant). We estimated the fraction of ACE-2 captured by utilizing a biotinylated receptor, which specifically bound fluorescent streptavidin molecules used as label. Our measured binding affinity, Kd, was estimated to be 14.9 nM, which is within the range reported from utilizing different approaches. In conclusion, this approach provides an accurate estimate of the binding affinity of the spike protein to the ACE-2 receptor.

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Measuring Binding Affinity of CoV2 Spike Proteins to ACE2 Receptor Using KinExA

To infect a cell, viruses specifically interact with receptors present on the surface of target cells. Quantification of this interaction is important not only for a better understanding of infectivity or developing novel diagnosis approaches but also when screening for specific inhibitors to be used as antiviral drugs. In this respect, we describe the use of the Kinetics Exclusion Assay technology for measuring the affinity between the spike protein of the CoV-2 virus and its specific membrane receptor ACE-2. To achieve our scientific goals, we functionalized small plastic beads with the spike protein and utilized them as a solid phase to capture the free ACE-2 present in previously equilibrated solutions containing a fixed amount of ACE-2 receptor (Constant Binding Partner) and a variable amount of spike protein (Titrant). We estimated the fraction of ACE-2 captured by utilizing a biotinylated receptor, which specifically bound fluorescent streptavidin molecules used as label. Our measured binding affinity, Kd, was estimated to be 14.9 nM, which is within the range reported from utilizing different approaches. In conclusion, this approach provides an accurate estimate of the binding affinity of the spike protein to the ACE-2 receptor.